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Platelet factor 4 (PF4) or the CXC chemokine CXCL4 is the most abundant protein within the α-granules of platelets. Previous studies found that PF4 regulates infections of several viruses, including HIV-1, H1N1, hepatitis C virus (HCV), and dengue virus. Here, we show that PF4 is an inhibitor of enterovirus A71 (EV71) and coxsackievirus A16 (CA16) infections. The secreted form of PF4 from transfected cells or soluble purified PF4 from Escherichia coli, even lacking signal peptide affected secretion, obviously inhibited the propagation of EV71 and CA16. Mechanistically, we demonstrated that PF4 blocked the entry of the virus into the host cells by interactions with VP3 proteins of EV71/CA16 and the interaction with SCARB2 receptor-mediated EV71 and CA16 endocytosis. As expected, the incubation of anti-PF4 antibody with PF4 blocked PF4 inhibition on EV71 and CA16 infections further supported the above conclusion. Importantly, pretreatment of EV71 viruses with PF4 significantly protected the neonatal mice from EV71 lethal challenge and promoted the survival rate of infected mice. PF4 derived from natural platelets by EV71/CA16 activation also presented strong inhibition on EV71 and CA16. In summary, our study identified a new host factor against EV71 and CA16 infections, providing a novel strategy for EV71 and CA16 treatment. IMPORTANCE The virus's life cycle starts with binding to cell surface receptors, resulting in receptor-mediated endocytosis. Targeting the entry of the virus into target cells is an effective strategy to develop a novel drug. EV71 and CA16 are the major pathogens that cause hand, foot, and mouth disease (HFMD) outbreaks worldwide since 2008. However, the treatment of EV71 and CA16 infections is mainly symptomatic because there is no approved drug. Therefore, the underlying pathogenesis of EV71/CA16 and the interaction between host-EV71/CA16 need to be further investigated to develop an inhibitor. Here, we identified PF4 as a potent entry inhibitor of EV71 and CA16 via binding to VP3 proteins of EV71 and CA16 or binding to receptor SCARB2. In the EV71 infection model, PF4 protected mice from EV71 lethal challenge and promoted the survival rate of EV71-infected mice. Our study suggests that PF4 represents a potential candidate host factor for anti-EV71 and CA16 infections.

The serine-glycine dipeptide lipid classes, including lipid 430 and lipid 654, are produced by the periodontal pathogen Porphyromonas gingivalis and can be detected in lipid extracts of diseased periodontal tissues and teeth of humans. Both serine-glycine lipid classes were previously shown to engage human and mouse Toll-like receptor 2 (TLR2) and to inhibit mouse osteoblast differentiation and function through engagement of TLR2. It is not clear if other lipids related to serine-glycine lipids are also produced by P. gingivalis The goal of this investigation was to determine whether P. gingivalis produces additional lipid classes similar to the serine-glycine lipids that possess biological properties. P. gingivalis (ATCC 33277) was grown in broth culture, and lipids were extracted and fractionated by high-performance liquid chromatography (HPLC). Lipids were separated using semipreparative HPLC, and specific lipid classes were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-multiple reaction monitoring (LC-MRM) mass spectrometric approaches. Two glycine lipid classes were identified, termed lipid 567 and lipid 342, and these lipid classes are structurally related to the serine-glycine dipeptide lipids. Both glycine lipid classes were shown to promote TLR2-dependent tumor necrosis factor alpha (TNF-α) release from bone marrow macrophages, and both were shown to activate human embryonic kidney (HEK) cells through TLR2 and TLR6 but not TLR1. These results demonstrate that P. gingivalis synthesizes glycine lipids and that these lipids engage TLR2 similarly to the previously reported serine-glycine dipeptide lipids., (Copyright © 2020 American Society for Microbiology.)

Candida albicans is a pervasive commensal fungus that is the most common pathogen responsible for invasive fungal infection (IFI). With incidence of IFI on the rise due to increasing susceptible populations, it is imperative that we investigate how Candida albicans interacts with blood components. When stimulating either human or mouse whole blood with thrombin, we saw a significant decrease in C. albicans survival. We then repeated Candida killing assays with thrombin-stimulated or unstimulated washed platelets and saw a similar decrease in CFU. To investigate whether killing was mediated through surface components or releasable products, platelets were pretreated with an inhibitor of actin polymerization (cytochalasin D [CytoD]). CytoD was able to abrogate C. albicans killing. Moreover, dilution of releasates from thrombin-stimulated platelets showed that the toxicity of the releasates on C. albicans is concentration dependent. We then investigated C. albicans actions on platelet activation, granule release, and aggregation. While C. albicans does not appear to affect alpha or dense granule release, C. albicans exerts a significant attenuation of platelet aggregation to multiple agonists. These results illustrate for the first time that platelets can directly kill C. albicans through release of their granular contents. Additionally, C. albicans can also exert inhibitory effects on platelet aggregation., (Copyright © 2020 American Society for Microbiology.)

Rodents are critical for the transmission of Toxoplasma gondii to the definitive feline host via predation, and this relationship has been extensively studied as a model for immune responses to parasites. Neospora caninum is a closely related coccidian parasite of ruminants and canines but is not naturally transmitted by rodents. We compared mouse innate immune responses to N. caninum and T. gondii and found marked differences in cytokine levels and parasite growth kinetics during the first 24 h postinfection (hpi). N. caninum -infected mice produced significantly higher levels of interleukin-12 (IL-12) and interferon gamma (IFN-γ) by as early as 4 hpi, but the level of IFN-γ was significantly lower or undetectable in T. gondii -infected mice during the first 24 hpi. "Immediate" IFN-γ and IL-12p40 production was not detected in MyD88 -/- mice. However, unlike IL-12p40 -/- and IFN-γ -/- mice, MyD88 -/- mice survived N. caninum infections at the dose used in this study. Serial measures of parasite burden showed that MyD88 -/- mice were more susceptible to N. caninum infections than wild-type (WT) mice, and control of parasite burdens correlated with a pulse of serum IFN-γ at 3 to 4 days postinfection in the absence of detectable IL-12. Immediate IFN-γ was partially dependent on the T. gondii mouse profilin receptor Toll-like receptor 11 (TLR11), but the ectopic expression of N. caninum profilin in T. gondii had no impact on early IFN-γ production or parasite proliferation. Our data indicate that T. gondii is capable of evading host detection during the first hours after infection, while N. caninum is not, and this is likely due to the early MyD88-dependent recognition of ligands other than profilin., (Copyright © 2020 American Society for Microbiology.)

Human studies have shown associations between cryptococcal meningitis and reduced IgM memory B cell levels, and studies in IgM- and/or B cell-deficient mice have demonstrated increased Cryptococcus neoformans dissemination from lungs to brain. Since immunoglobulins are part of the immune milieu that C. neoformans confronts in a human host, and its ability to form titan cells is an important virulence mechanism, we determined the effect of human immunoglobulins on C. neoformans titan cell formation in vitro (i) Fluorescence microscopy showed normal human IgG and IgM bind C. neoformans (ii) C. neoformans grown in titan cell-inducing medium with IgM, not IgG, inhibited titan-like cell formation. (iii) Absorption of IgM with laminarin or curdlan (branched and linear 1-3-beta-d-glucans, respectively) decreased this effect. (iv) Transmission electron microscopy revealed that cells grown with IgM had small capsules and unique features not seen with cells grown with IgG. (v) Comparative transcriptional analysis of cell wall, capsule, and stress response genes showed that C. neoformans grown with IgM, not IgG or phosphate-buffered saline (PBS), had decreased expression of chitin synthetase, CHS1 , CHS2 , and CHS8 , and genes encoding cell wall carbohydrate synthetases α-1-3-glucan ( AGS1 ) and β-1,3-glucan ( FKS1 ). IgM also decreased expression of RIM101 and HOG1 , genes encoding central regulators of C. neoformans stress response pathways and cell morphogenesis. Our data show human IgM affects C. neoformans morphology in vitro and suggest that the hypothesis that human immunoglobulins may affect C. neoformans virulence in vivo warrants further investigation., (Copyright © 2020 American Society for Microbiology.)

Chagas disease, caused by Trypanosoma cruzi , is a major public health issue. Limitations in immune responses to natural T. cruzi infection usually result in parasite persistence with significant complications. A safe, effective, and reliable vaccine would reduce the threat of T. cruzi infections; however, no suitable vaccine is currently available due to a lack of understanding of the requirements for induction of fully protective immunity. We established a T. cruzi strain expressing green fluorescent protein (GFP) under the control of dihydrofolate reductase degradation domain (DDD) with a hemagglutinin (HA) tag, GFP-DDDHA, which was induced by trimethoprim-lactate (TMP-lactate), which results in the death of intracellular parasites. This attenuated strain induces very strong protection against reinfection. Using this GFP-DDDHA strain, we investigated the mechanisms underlying the protective immune response in mice. Immunization with this strain led to a response that included high levels of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α), as well as a rapid expansion of effector and memory T cells in the spleen. More CD8 + T cells differentiate to memory cells following GFP-DDDHA infection than after infection with a wild-type (WT) strain. The GFP-DDDHA strain also provides cross-protection against another T. cruzi isolate. IFN-γ is important in mediating the protection, as IFN-γ knockout (KO) mice failed to acquire protection when infected with the GFP-DDDHA strain. Immune cells demonstrated earlier and stronger protective responses in immunized mice after reinfection with T. cruzi than those in naive mice. Adoptive transfers with several types of immune cells or with serum revealed that several branches of the immune system mediated protection. A combination of serum and natural killer cells provided the most effective protection against infection in these transfer experiments., (Copyright © 2020 American Society for Microbiology.)

Food allergy is a life-threatening response to specific foods, and microbiota imbalance (dysbiosis) in gut is considered a cause of this disease. Meanwhile, the host immune response also plays an important role in the disease. Notably, interleukin 33 (IL-33) released from damaged or necrotic intestinal epithelial cells facilitates IL-2-producing CD4 helper T (Th2) responses. However, causal relationships between the gut and oral dysbiosis and food allergy remain unknown. In this study, we analyzed effects of gut and oral dysbiosis on development of food allergy. A murine model of food allergy was established via ovalbumin (OVA) injection in BALB/c mice. Viable fecal bacteria were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). il33 expression in colon-26 mouse colon cells stimulated by isolated fecal bacteria was quantified by real-time PCR. Intestinal T cells from the mice were analyzed by flow cytometry. Salivary IgA levels were quantified by enzyme-linked immunosorbent assay (ELISA), and IgA-bound oral bacteria were detected by flow cytometry. Among fecal bacteria, the abundance of Citrobacter sp. increased in the feces of allergic mice and induced il33 expression in colon-26 cells. Orally administered Citrobacter koseri JCM1658 exacerbated systemic allergic symptoms and reduced intestinal Th17 cells. Salivary IgA and IgA-bound oral bacteria increased in the allergic mice. Based on the results described above, food allergy induced both gut and oral dysbiosis. Citrobacter sp. aggravated allergy symptoms by inducing IL-33 release from intestinal epithelial cells., (Copyright © 2019 Matsui et al.)

Clostridioides (formerly known as Clostridium ) difficile is the leading cause of hospital-acquired gastrointestinal infections in the United States and one of three urgent health care threats identified by the Centers for Disease Control and Prevention. C. difficile disease is mediated by the production of toxins that disrupt the epithelial barrier and cause a robust host inflammatory response. Studies in humans as well as animal models of disease have shown that the type of immune response generated against the infection dictates the outcome of disease, often irrespective of bacterial burden. Much of the focus on immunity during C. difficile infection (CDI) has been on type 3 immunity because of the established role for this arm of the immune system in other gastrointestinal inflammatory conditions such as inflammatory bowel disease (IBD). For example, interleukin-22 (IL-22) production by group 3 innate lymphoid cells (ILC3s) protects against pathobionts translocating across the epithelium during CDI. On the other hand, interleukin-17 (IL-17) production by Th17 cells increases CDI-associated mortality. Additionally, neutropenia has been associated with increased susceptibility to CDI in humans, but increased neutrophilia in mouse models correlates with host pathology. Taking the data together, these findings suggest dual roles for type 3 immune responses during infection. Here, we review the complex role of type 3 immunity during CDI and delineate what is known about innate and adaptive cellular immunity as well as the downstream effector cytokines known to be important during this infection., (Copyright © 2019 American Society for Microbiology.)

Staphylococcus aureus has evolved different strategies to evade the immune response, which play an important role in its pathogenesis. The bacteria express and shed various cell wall components and toxins during different stages of growth that may affect the protective T cell responses to extracellular and intracellular S. aureus However, if and how the dendritic cell (DC)-mediated T cell response against S. aureus changes during growth of the bacterium remain elusive. In this study, we show that exponential-phase (EP) S. aureus bacteria were endocytosed very efficiently by human DCs, and these DCs strongly promoted production of the T cell polarizing factor interleukin-12 (IL-12). In contrast, stationary-phase (SP) S. aureus bacteria were endocytosed less efficiently by DCs, and these DCs produced small amounts of IL-12. The high level of IL-12 production induced by EP S. aureus led to the development of a T helper 1 (Th1) cell response, which was inhibited after neutralization of IL-12. Furthermore, preincubation with the staphylococcal cell wall component peptidoglycan (PGN), characteristically shed during the exponential growth phase, modulated the DC response to EP S. aureus PGN preincubation appeared to inhibit IL-12p35 expression, leading to downregulation of IL-12 and an increase of IL-23 production by DCs, enhancing Th17 cell development. Taken together, our data indicate that exponential-phase S. aureus bacteria induce a stronger IL-12-dependent Th1 cell response than stationary-phase S. aureus and that this Th1 cell response shifted toward a Th17 cell response in the presence of PGN., (Copyright © 2019 American Society for Microbiology.)

Dendritic cells (DCs) and natural killer (NK) cells are critically involved in the early response against various bacterial microbes. Functional activation of infected DCs and NK cell-mediated gamma interferon (IFN-γ) secretion essentially contribute to the protective immunity against Chlamydia How DCs and NK cells cooperate during the antichlamydial response is not fully understood. Therefore, in the present study, we investigated the functional interplay between Chlamydia -infected DCs and NK cells. Our biochemical and cell biological experiments show that Chlamydia psittaci -infected DCs display enhanced exosome release. We find that such extracellular vesicles (referred to as dexosomes) do not contain infectious bacterial material but strongly induce IFN-γ production by NK cells. This directly affects C. psittaci growth in infected target cells. Furthermore, NK cell-released IFN-γ in cooperation with tumor necrosis factor alpha (TNF-α) and/or dexosomes augments apoptosis of both noninfected and infected epithelial cells. Thus, the combined effect of dexosomes and proinflammatory cytokines restricts C. psittaci growth and attenuates bacterial subversion of apoptotic host cell death. In conclusion, this provides new insights into the functional cooperation between DCs, dexosomes, and NK cells in the early steps of antichlamydial defense., (Copyright © 2019 American Society for Microbiology.)

Infection of the host with Mycobacterium avium subsp. paratuberculosis results in chronic and progressive enteritis that traverses both subclinical and clinical stages. The mechanism(s) for the shift from an asymptomatic subclinical disease state to advanced clinical disease is not fully understood. In the present study, naturally infected dairy cattle were divided into subclinical and clinical infection groups, along with noninfected control cows of similar parity, to study host immune responses in different stages of infection. Both infection groups had higher levels of secretion of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-2 (IL-2) than control cows, whereas only clinical cows had increased secretion of IL-10, IL-12, and IL-18 upon stimulation of peripheral blood mononuclear cells (PBMCs) with antigen. Conversely, secretion of IL-17Α was decreased for clinical cows compared to subclinical and control cows. Proinflammatory cytokine genes were upregulated only for subclinical cows, whereas increased IL-10 and IL-17 gene expression levels were observed for both infection groups. Increased CD4 + , CD8 + , and γδ T cell receptor-positive (TCR + ) T cells were observed for subclinical cows compared to clinical cows. Although clinical cows expressed antigen-specific immune responses, the profile for subclinical cows was one of a dominant proinflammatory response to infection. We reason that a complex coordination of immune responses occurs during M. avium subsp. paratuberculosis infection, with these responses shifting as the host transitions through the different stages of infection and disease (subclinical to clinical). A further understanding of the series of events characterized by Th1/Th2/Th17 responses will provide mechanisms for disease progression and may direct insightful intervention strategies., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

Neisseria meningitidis is an inhabitant of the nasopharynx, from which it is transmitted from person to person or disseminates in blood and becomes a harmful pathogen. In this work, we addressed colonization of the nasopharyngeal niche by focusing on the interplay between meningococci and the airway mucus that lines the mucosa of the host. Using Calu-3 cells grown in air interface culture (cells grown with the apical domain facing air), we studied meningococcal colonization of the mucus and the host response. Our results suggested that N. meningitidis behaved like commensal bacteria in mucus, without interacting with human cells or actively transmigrating through the cell layer. As a result, type IV pili do not play a role in this model, and meningococci did not trigger a strong innate immune response from the Calu-3 cells. Finally, we have shown that this model is suitable for studying interaction of N. meningitidis with other bacteria living in the nasopharynx and that Streptococcus mitis , but not Moraxella catarrhalis , can promote meningococcal growth in this model. IMPORTANCE N. meningitidis is transmitted from person to person by aerosol droplets produced by breathing, talking, or coughing or by direct contact with a contaminated fluid. The natural reservoir of N. meningitidis is the human nasopharynx mucosa, located at the back of the nose and above the oropharynx. The means by which meningococci cross the nasopharyngeal wall is still under debate, due to the lack of a convenient and relevant model mimicking the nasopharyngeal niche. Here, we took advantage of Calu-3 cells grown in air interface culture to study how meningococci colonize the nasopharyngeal niche. We report that the airway mucus is both a niche for meningococcal growth and a protective barrier against N. meningitidis infection. As such, N. meningitidis behaves like commensal bacteria and is unlikely to induce infection without an external trigger., (Copyright © 2019 Audry et al.)

Dysregulated and maladaptive immune responses are at the forefront of human diseases caused by infection with zoonotic viral hemorrhagic fever viruses. Elucidating mechanisms of how the natural animal reservoirs of these viruses coexist with these agents without overt disease, while permitting sufficient replication to allow for transmission and maintenance in a population, is important for understanding the viral ecology and spillover to humans. The Egyptian rousette bat (ERB) has been identified as a reservoir for Marburg virus (MARV), a filovirus and the etiological agent of the highly lethal Marburg virus disease. Little is known regarding how these bats immunologically respond to MARV infection. In humans, macrophages and dendritic cells (DCs) are primary targets of infection, and their dysregulation is thought to play a central role in filovirus diseases, by disturbing their normal functions as innate sensors and adaptive immune response facilitators while serving as amplification and dissemination agents for the virus. The infection status and responses to MARV in bat myeloid-lineage cells are uncharacterized and likely represent an important modulator of the bat's immune response to MARV infection. Here, we generate DCs from the bone marrow of rousette bats. Infection with a bat isolate of MARV resulted in a low level of transcription in these cells and significantly downregulated DC maturation and adaptive immune-stimulatory pathways while simultaneously upregulating interferon-related pathogen-sensing pathways. This study provides a first insight into how the bat immune response is directed toward preventing aberrant inflammatory responses while mounting an antiviral response to defend against MARV infection. IMPORTANCE Marburg viruses (MARVs) cause severe human disease resulting from aberrant immune responses. Dendritic cells (DCs) are primary targets of infection and are dysregulated by MARV. Dysregulation of DCs facilitates MARV replication and virus dissemination and influences downstream immune responses that result in immunopathology. Egyptian rousette bats (ERBs) are natural reservoirs of MARV, and infection results in virus replication and shedding, with asymptomatic control of the virus within weeks. The mechanisms that bats employ to appropriately respond to infection while avoiding disease are unknown. Because DC infection and modulation are important early events in human disease, we measured the transcriptional responses of ERB DCs to MARV. The significance of this work is in identifying cell type-specific coevolved responses between ERBs and MARV, which gives insight into how bat reservoirs are able to harbor MARV and permit viral replication, allowing transmission and maintenance in the population while simultaneously preventing immunopathogenesis.

The release of extracellular vesicles (EVs) by fungi is a fundamental cellular process. EVs carry several biomolecules, including pigments, proteins, enzymes, lipids, nucleic acids, and carbohydrates, and are involved in physiological and pathological processes. EVs may play a pivotal role in the establishment of fungal infections, as they can interact with the host immune system to elicit multiple outcomes. It has been observed that, depending on the fungal pathogen, EVs can exacerbate or attenuate fungal infections. The study of the interaction between fungal EVs and the host immune system and understanding of the mechanisms that regulate those interactions might be useful for the development of new adjuvants as well as the improvement of protective immune responses against infectious or noninfectious diseases. In this review, we describe the immunomodulatory properties of EVs produced by pathogenic fungi and discuss their potential as adjuvants for prophylactic or therapeutic strategies., (Copyright © 2019 Freitas et al.)

Cellular sensing of bacterial RNA is increasingly recognized as a determinant of host-pathogen interactions. The intracellular pathogen Listeria monocytogenes induces high levels of type I interferons (alpha/beta interferons [IFN-α/β]) to create a growth-permissive microenvironment during infection. We previously demonstrated that RNAs secreted by L. monocytogenes (comprising the secRNome) are potent inducers of IFN-β. We determined the composition and diversity of the members of the secRNome and found that they are uniquely enriched for noncoding small RNAs (sRNAs). Testing of individual sRNAs for their ability to induce IFN revealed several sRNAs with this property. We examined ril32, an intracellularly expressed sRNA that is highly conserved for the species L. monocytogenes and that was the most potent inducer of IFN-β expression of all the sRNAs tested in this study, in more detail. The rli32-induced IFN-β response is RIG-I (retinoic acid inducible gene I) dependent, and cells primed with rli32 inhibit influenza virus replication. We determined the rli32 motif required for IFN induction. rli32 overproduction promotes intracellular bacterial growth, and a mutant lacking rli32 is restricted for intracellular growth in macrophages. rli32-overproducing bacteria are resistant to H 2 O 2 and exhibit both increased catalase activity and changes in the cell envelope. Comparative transcriptome sequencing (RNA-Seq) analysis indicated that ril32 regulates expression of the lhrC locus, previously shown to be involved in cell envelope stress. Inhibition of IFN-β signaling by ruxolitinib reduced rli32-dependent intracellular bacterial growth, indicating a link between induction of the interferon system and bacterial physiology. rli32 is, to the best of our knowledge, the first secreted individual bacterial sRNA known to trigger the induction of the type I IFN response. IMPORTANCE Interferons are potent and broadly acting cytokines that stimulate cellular responses to nucleic acids of unusual structures or locations. While protective when induced following viral infections, the induction of interferons is detrimental to the host during L. monocytogenes infection. Here, we identify specific sRNAs, secreted by the bacterium, with the capacity to induce type I IFN. Further analysis of the most potent sRNA, rli32, links the ability to induce RIG-I-dependent induction of the type I IFN response to the intracellular growth properties of the bacterium. Our findings emphasize the significance of released RNA for Listeria infection and shed light on a compartmental strategy used by an intracellular pathogen to modulate host responses to its advantage., (Copyright © 2019 Frantz et al.)

The New World (NW) arenaviruses are a diverse group of zoonotic viruses, including several causative agents of severe hemorrhagic fevers in humans. All known human-pathogenic NW arenaviruses belong to clade B, where they group into sublineages with phylogenetically closely related nonpathogenic viruses, e.g., the highly pathogenic Junin (JUNV) and Machupo viruses with the nonpathogenic Tacaribe virus (TCRV). Considering the close genetic relationship of nonpathogenic and pathogenic NW arenaviruses, the identification of molecular determinants of virulence is of great importance. The host cell's innate antiviral defense represents a major barrier for zoonotic infection. Here, we performed a side-by-side comparison of the innate immune responses against JUNV and TCRV in human cells. Despite similar levels of viral replication, infection with TCRV consistently induced a stronger type I interferon (IFN-I) response than JUNV infection did. Transcriptome profiling revealed upregulation of a largely overlapping set of interferon-stimulated genes in cells infected with TCRV and JUNV. Both viruses were relatively insensitive to IFN-I treatment of human cells and induced similar levels of apoptosis in the presence or absence of an IFN-I response. However, in comparison to JUNV, TCRV induced stronger activation of the innate sensor double-strand RNA-dependent protein kinase R (PKR), resulting in phosphorylation of eukaryotic translation initiation factor eIF2α. Confocal microscopy studies revealed similar subcellular colocalizations of the JUNV and TCRV viral replication-transcription complexes with PKR. However, deletion of PKR by CRISPR/Cas9 hardly affected JUNV but promoted TCRV multiplication, providing the first evidence for differential innate recognition and control of pathogenic and nonpathogenic NW arenaviruses by PKR. IMPORTANCE New World (NW) arenaviruses are a diverse family of emerging zoonotic viruses that merit significant attention as important public health problems. The close genetic relationship of nonpathogenic NW arenaviruses with their highly pathogenic cousins suggests that few mutations may be sufficient to enhance virulence. The identification of molecular determinants of virulence of NW arenaviruses is therefore of great importance. Here we undertook a side-by-side comparison of the innate immune responses against the highly pathogenic Junin virus (JUNV) and the related nonpathogenic Tacaribe virus (TCRV) in human cells. We consistently found that TCRV induces a stronger type I interferon (IFN-I) response than JUNV. Transcriptome profiling revealed an overlapping pattern of IFN-induced gene expression and similar low sensitivities to IFN-I treatment. However, the double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) contributed to the control of TCRV, but not JUNV, providing the first evidence for differential innate recognition and control of JUNV and TCRV., (Copyright © 2019 American Society for Microbiology.)

Rho-associated coiled-coil kinase (ROCK) protein is a central kinase that regulates numerous cellular functions, including cellular polarity, motility, proliferation, and apoptosis. Here, we demonstrate that ROCK has antiviral properties, and inhibition of its activity results in enhanced propagation of human cytomegalovirus (HCMV). We show that during HCMV infection, ROCK1 translocates to the nucleus and concentrates in the nucleolus, where it colocalizes with the stress-related chaperone heat shock cognate 71-kDa protein (Hsc70). Gene expression measurements show that inhibition of ROCK activity does not seem to affect the cellular stress response. We demonstrate that inhibition of myosin, one of the central targets of ROCK, also increases HCMV propagation, implying that the antiviral activity of ROCK might be mediated by activation of the actomyosin network. Finally, we demonstrate that inhibition of ROCK results in increased levels of the tegument protein UL32 and of viral DNA in the cytoplasm, suggesting ROCK activity hinders the efficient egress of HCMV particles out of the nucleus. Altogether, our findings illustrate ROCK activity restricts HCMV propagation and suggest this inhibitory effect may be mediated by suppression of capsid egress out of the nucleus. IMPORTANCE ROCK is a central kinase in cells that regulates numerous cellular functions, including cellular polarity, motility, proliferation, and apoptosis. Here we reveal a novel antiviral activity of ROCK during infection with HCMV, a prevalent pathogen infecting most of the population worldwide. We reveal ROCK1 is translocated to the nucleus, where it mainly localizes to the nucleolus. Our findings suggest that ROCK's antiviral activity may be related to activation of the actomyosin network and inhibition of capsid egress out of the nucleus., (Copyright © 2019 American Society for Microbiology.)

Among the innate immune sentinels, the complement system is a formidable first line of defense against pathogens, including viruses. Chandipura virus (CHPV), a neurotropic vesiculovirus of the family Rhabdoviridae , is a deadly human pathogen known to cause fatal encephalitis, especially among children. The nature of interaction and the effect of human complement on CHPV are unknown. Here, we report that CHPV is a potent activator of complement and, thus, is highly sensitive to complement proteins in normal human serum (NHS). Utilizing a panel of specific complement component depleted/reconstituted human serum, we have demonstrated that CHPV neutralization is C3, C4, and C1q dependent and independent of factor B, suggesting the importance of the classical pathway in limiting CHPV. Employing a range of biochemical approaches, we showed (i) a direct association of C1q to CHPV, (ii) deposition of complement proteins C3b, C4b, and C1q on CHPV, and (iii) virus aggregation. Depletion of C8, an important component of the pore-forming complex of complement, had no effect on CHPV, further supporting the finding that aggregation and not virolysis is the mechanism of virus neutralization. With no approved vaccines or treatment modalities in place against CHPV, insights into such interactions can be exploited to develop potent vaccines or therapeutics targeting CHPV. IMPORTANCE Chandipura virus is a clinically important human pathogen of the Indian subcontinent. The rapidity of death associated with CHPV infection in addition to the absence of an effective vaccine or therapeutics results in poor clinical prognosis. The biology of the virus and its interaction with the host immune system, including the complement system, are understudied. Our investigation reveals the susceptibility of CHPV to fluid phase complement and also dissects the pathway involved and the mechanism of virus neutralization. Direct binding of C1q, an important upstream component of the classical pathway of complement to CHPV, and the strong dependency on C1q for virus neutralization highlight the significance of identifying such interactions to better understand CHPV pathogenesis and devise strategies to target this deadly pathogen., (Copyright © 2019 American Society for Microbiology.)

Vaccinia virus (VACV), the prototypical member of the poxvirus family, was used as a live-virus vaccine to eradicate smallpox worldwide and has recently received considerable attention because of its potential as a prominent vector for the development of vaccines against infectious diseases and as an oncolytic virus for cancer therapy. Studies have demonstrated that VACV exhibits an extremely strong bias for binding to and infection of primary human antigen-presenting cells (APCs), including monocytes, macrophages, and dendritic cells. However, very few studies have assessed the interactions of VACV with primary human B cells, a main type of professional APCs. In this study, we evaluated the susceptibility of primary human peripheral B cells at various differentiation and maturation stages to VACV binding, infection, and replication. We found that plasmablasts were resistant to VACV binding, while other B subsets, including transitional, mature naive, memory, and plasma cells, were highly susceptible to VACV binding. VACV binding preference was likely associated with differential expression of chemokine receptors, particularly CXCR5. Infection studies showed that plasmablast, plasma, transitional, and mature naive B cells were resistant to VACV infection, while memory B cells were preferentially infected. VACV infection in ex vivo B cells was abortive, which occurred at the stage of late viral gene expression. In contrast, activated B cells were permissive to productive VACV infection. Thus, primary human B cells at different differentiation stages exhibit distinct susceptibilities to VACV binding and infection, and the infections are abortive and productive in ex vivo and activated B cells, respectively. IMPORTANCE Our results provide critical information to the field of poxvirus binding and infection tropism. We demonstrate that VACV preferentially infects memory B cells that play an important role in a rapid and vigorous antibody-mediated immune response upon reinfection by a pathogen. Additionally, this work highlights the potential of B cells as natural cellular models to identify VACV receptors or dissect the molecular mechanisms underlying key steps of the VACV life cycle, such as binding, penetration, entry, and replication in primary human cells. The understanding of VACV biology in human primary cells is essential for the development of a safe and effective live-virus vector for oncolytic virus therapy and vaccines against smallpox, other pathogens, and cancer., (Copyright © 2019 American Society for Microbiology.)

Myxovirus resistance 1 ( Mx1 ) is an interferon-induced gene that encodes a GTPase that plays an important role in the defense of mammalian cells against influenza A and other viruses. The Mx1 protein can restrict a number of viruses independently of the expression of other interferon-induced genes. Mx genes are therefore considered to be an important part of the innate antiviral immune response. However, the possible impact of Mx expression in the hematopoietic cellular compartment has not been investigated in detail in the course of a viral infection. To address this, we performed bone marrow chimera experiments using congenic B6.A2G Mx1 +/+ and B6.A2G Mx1 -/- mice to study the effect of Mx1 expression in cells of hematopoietic versus nonhematopoietic origin. Mx1 +/+ mice were protected and Mx1 -/- mice were susceptible to influenza A virus challenge infection, regardless of the type of bone marrow cells ( Mx1 +/+ or Mx1 -/- ) the animals had received. Infection with Thogoto virus, however, revealed that Mx1 -/- mice with a functional Mx1 gene in the bone marrow compartment showed reduced liver pathology compared with Mx1 -/- mice that had been grafted with Mx1 -/- bone marrow. The reduced pathology in these mice was associated with a reduction in Thogoto virus titers in the spleen, lung, and serum. Moreover, Mx1 +/+ mice with Mx1 -/- bone marrow failed to control Thogoto virus replication in the spleen. Mx1 in the hematopoietic cellular compartment thus contributes to protection against Thogoto virus infection. IMPORTANCE Mx proteins are evolutionarily conserved in vertebrates and can restrict a wide range of viruses in a cell-autonomous way. The contribution to antiviral defense of Mx1 expression in hematopoietic cells remains largely unknown. We show that protection against influenza virus infection requires Mx1 expression in the nonhematopoietic cellular compartment. In contrast, Mx1 in bone marrow-derived cells is sufficient to control disease and virus replication following infection with a Thogoto virus. This indicates that, in addition to its well-established antiviral activity in nonhematopoietic cells, Mx1 in hematopoietic cells can also play an important antiviral function. In addition, cells of hematopoietic origin that lack a functional Mx1 gene contribute to Thogoto virus dissemination and associated disease., (Copyright © 2019 American Society for Microbiology.)

Streptococcus agalactiae is not only part of the human intestinal and urogenital microbiota but is also a leading cause of septicemia and meningitis in neonates. Its ability to cause disease depends upon the acquisition of nutrients from its environment, including the transition metal ion zinc. The primary zinc acquisition system of the pathogen is the Adc/Lmb ABC permease, which is essential for viability in zinc-restricted environments. Here, we show that in addition to the AdcCB transporter and the three zinc-binding proteins, Lmb, AdcA, and AdcAII, S. agalactiae zinc homeostasis also involves two streptococcal histidine triad (Sht) proteins. Sht and ShtII are required for zinc uptake via the Lmb and AdcAII proteins with apparent overlapping functionality and specificity. Both Sht-family proteins possess five-histidine triad motifs with similar hierarchies of importance for Zn homeostasis. Independent of its contribution to zinc homeostasis, Sht has previously been reported to bind factor H leading to predictions of a contribution to complement evasion. Here, we investigated ShtII to ascertain whether it had similar properties. Analysis of recombinant Sht and ShtII reveals that both proteins have similar affinities for factor H binding. However, neither protein aided in resistance to complement in human blood. These findings challenge prior inferences regarding the in vivo role of the Sht proteins in resisting complement-mediated clearance. IMPORTANCE This study examined the role of the two streptococcal histidine triad (Sht) proteins of Streptococcus agalactiae in zinc homeostasis and complement resistance. We showed that Sht and ShtII facilitate zinc homeostasis in conjunction with the metal-binding proteins Lmb and AdcAII. Here, we show that the Sht-family proteins are functionally redundant with overlapping roles in zinc uptake. Further, this work reveals that although the Sht-family proteins bind to factor H in vitro this did not influence survival in human blood., (Copyright © 2019 American Society for Microbiology.)

Helminths have coevolved with their hosts, resulting in the development of specialized host immune mechanisms and parasite-specific regulatory products. Identification of new pathways that regulate helminth infection could provide a better understanding of host-helminth interaction and may identify new therapeutic targets for helminth infection. Here we identify the endocannabinoid system as a new mechanism that influences host immunity to helminths. Endocannabinoids are lipid-derived signaling molecules that control important physiologic processes, such as feeding behavior and metabolism. Following murine infection with Nippostrongylus brasiliensis , an intestinal nematode with a life cycle similar to that of hookworms, we observed increased levels of endocannabinoids (2-arachidonoylglycerol [2-AG] or anandamide [AEA]) and the endocannabinoid-like molecule oleoylethanolamine (OEA) in infected lung and intestine. To investigate endocannabinoid function in helminth infection, we employed pharmacological inhibitors of cannabinoid subtype receptors 1 and 2 (CB 1 R and CB 2 R). Compared to findings for vehicle-treated mice, inhibition of CB 1 R but not CB 2 R resulted in increased N. brasiliensis worm burden and egg output, associated with significantly decreased expression of the T helper type 2 cytokine interleukin 5 (IL-5) in intestinal tissue and splenocyte cultures. Strikingly, bioinformatic analysis of genomic and transcriptome sequencing (RNA-seq) data sets identified putative genes encoding endocannabinoid biosynthetic and degradative enzymes in many parasitic nematodes. To test the novel hypothesis that helminth parasites produce their own endocannabinoids, we measured endocannabinoid levels in N. brasiliensis by mass spectrometry and quantitative PCR and found that N. brasiliensis parasites produced endocannabinoids, especially at the infectious larval stage. To our knowledge, this is the first report of helminth- and host-derived endocannabinoids that promote host immune responses and reduce parasite burden., (Copyright © 2018 American Society for Microbiology.)

Type I interferon inhibits viruses through inducing the expression of antiviral proteins, including the myxovirus resistance (Mx) proteins. Compared to the human MxA protein, which inhibits a wide range of viruses, the MxB protein has been reported to specifically inhibit primate lentiviruses, including HIV-1, and herpesviruses. Further, the role of endogenous MxB in alpha interferon-mediated inhibition of HIV-1 infection was questioned by a recent study showing that MxB knockout did not increase the level of infection by HIV-1 which carried the G protein of vesicular stomatitis virus (VSV), allowing infection of CD4-negative HT1080 cells. In order to further examine the anti-HIV-1 activity of endogenous MxB, we have used CRISPR/Cas9 to deplete MxB in different cell lines and observed a substantial restoration of HIV-1 infection in the presence of alpha interferon treatment. However, this rescue effect of MxB knockout became much less pronounced when infection was performed with HIV-1 carrying the VSV G protein. Interestingly, a CRISPR/Cas9 knockout screen of alpha interferon-stimulated genes in U87-MG cells revealed that the genes for interferon-induced transmembrane protein 2 (IFITM2) and IFITM3 inhibited VSV G-pseudotyped HIV-1 much more strongly than the rest of the genes tested, including the gene for MxB. Therefore, our results demonstrate the importance of MxB in alpha interferon-mediated inhibition of HIV-1 infection, which, however, can be underestimated if infection is performed with VSV G protein-pseudotyped HIV-1, due to the high sensitivity of VSV G-mediated infection to inhibition by IFITM proteins. IMPORTANCE The results of this study reconcile the controversial reports regarding the anti-HIV-1 function of alpha interferon-induced MxB protein. In addition to the different cell types that may have contributed to the different observations, our data also suggest that VSV G protein-pseudotyped HIV-1 is much less inhibited by alpha interferon-induced MxB than HIV-1 itself is. Our results clearly demonstrate an important contribution of MxB to alpha interferon-mediated inhibition of HIV-1 in CD4 + T cells, which calls for using HIV-1 target cells and wild-type virus to test the relevance of the anti-HIV-1 activity of endogenous MxB and other restriction factors., (Copyright © 2018 American Society for Microbiology.)

Immune modulators are known to be produced by matured biofilms and during different stages of planktonic growth of Staphylococcus aureus Little is known about immune modulator production during the early stages of biofilm formation, thus raising the following question: how does S. aureus protect itself from the innate immune responses at these stages? Therefore, we determined the production of the following immune modulators: chemotaxis inhibitory protein of staphylococci (CHIPS); staphylococcal complement inhibitor (SCIN); formyl peptide receptor-like 1 inhibitor; gamma-hemolysin component B; leukocidins D, E, and S; staphylococcal superantigen-like proteins 1, 3, 5, and 9; and staphylococcal enterotoxin A. Production was determined during in vitro biofilm formation in Iscove's modified Dulbecco's medium at different time points using a competitive Luminex assay and mass spectrometry. Both methods demonstrated the production of the immune modulators SCIN and CHIPS during the early stages of biofilm formation. The green fluorescence protein promoter fusion technology confirmed scn (SCIN) and, to a lesser extent, chp (CHIPS) transcription during the early stages of biofilm formation. Furthermore, we found that SCIN could inhibit human complement activation induced by early biofilms, indicating that S. aureus is able to modulate the innate immune system already during the early stages of biofilm formation in vitro These results form a stepping stone toward elucidating the role of immune modulators in the establishment of biofilms in vivo and present opportunities to develop preventive strategies., (Copyright © 2018 American Society for Microbiology.)

Pathogenic bacteria that establish chronic infections in immunocompromised patients frequently undergo adaptation or selection for traits that are advantageous for their growth and survival. Clinical isolates of Pseudomonas aeruginosa , a Gram-negative, opportunistic bacterial pathogen, exhibit a temporal transition from a motile to a nonmotile phenotype through loss of flagellar motility during the course of chronic infection. This progressive loss of motility is associated with increased resistance to both antibiotic and immune clearance. We have previously shown that loss of bacterial motility enables P. aeruginosa to evade phagocytic clearance both in vitro and in vivo and fails to activate the phosphatidylinositol 3-kinase (PI3K)/Akt-dependent phagocytic pathway. Therefore, we tested the hypothesis that clearance of phagocytosis-resistant bacteria could be induced by exogenously pretreating innate immune cells with the Akt-activating molecule phosphatidylinositol-(3,4,5)-trisphosphate (PIP 3 ). Here, we demonstrate that PIP 3 induces the uptake of nonmotile P. aeruginosa by primary human neutrophils >25-fold, and this effect is phenocopied with the use of murine phagocytes. However, surprisingly, mechanistic studies revealed that the induction of phagocytosis by PIP 3 occurs because polyphosphoinositides promote bacterial binding by the phagocytes rather than bypassing the requirement for PI3K. Moreover, this induction was selective since the uptake of other nonmotile Gram-negative, but not Gram-positive, bacteria can also be induced by PIP 3 Since there is currently no treatment that effectively eradicates chronic P. aeruginosa infections, these findings provide novel insights into a potential methodology by which to induce clearance of nonmotile pathogenic bacteria and into the endogenous determinants of phagocytic recognition of P. aeruginosa ., (Copyright © 2018 American Society for Microbiology.)

Multidrug-resistant Acinetobacter baumannii is among the most common causes of infectious complications associated with combat-related trauma in military personnel serving overseas. However, little is currently known about its pathogenesis. While the gastrointestinal (GI) tract has been found to be a major reservoir for A. baumannii , as well as to potentially contribute to development of multidrug resistance, no studies have addressed the mechanisms involved in gut colonization. In this study, we address this critical gap in knowledge by first assessing the interaction between secretory IgA (SIgA), the principal humoral immune defense on mucosal surfaces, and the A. baumannii clinical isolate Ci79. Surprisingly, SIgA appeared to enhance A. baumannii GI tract colonization, in a process mediated by bacterial thioredoxin A (TrxA), as evidenced by reduction of bacterial attachment in the presence of TrxA inhibitors. Additionally, a trxA targeted deletion mutant ( ΔtrxA ) showed reduced bacterial burdens within the GI tract 24 h after oral challenge by in vivo live imaging, along with loss of thiol-reductase activity. Surprisingly, not only was GI tract colonization greatly reduced but the associated 50% lethal dose (LD 50 ) of the ΔtrxA mutant was increased nearly 100-fold in an intraperitoneal sepsis model. These data suggest that TrxA not only mediates A. baumannii GI tract colonization but also may contribute to pathogenesis in A. baumannii sepsis following escape from the GI tract under conditions when the intestinal barrier is compromised, as occurs with cases of severe shock and trauma. IMPORTANCE Acinetobacter baumannii is an emerging bacterial pathogen recently classified as a serious threat to U.S. and global health by both the Centers for Disease Control and Prevention and the World Health Organization. It also is one of the leading causes of combat-related infections associated with injured military personnel serving overseas. Little is known regarding mechanisms of gastrointestinal tract colonization despite this site being shown to serve as a reservoir for multidrug-resistant (MDR) A. baumannii isolates. Here, we establish that secretory IgA, the major immunoglobulin of mucosal surfaces, promotes A. baumannii GI tract colonization via bacterial thioredoxin A as evidenced through significant reduction in colonization in IgA-deficient animals. Additionally, bacterial colonization and mortality were significantly reduced in animals challenged with a thioredoxin A-deficient A. baumannii mutant. Combined, these data suggest that thioredoxin A is a novel virulence factor, for which antithioredoxin therapies could be developed, for this important multidrug-resistant pathogen., (Copyright © 2018 Ketter et al.)

Interferon alpha/beta (IFN-α/β) is a critical mediator of protection against most viruses, with host survival frequently impossible in its absence. Many studies have investigated the pathways involved in the induction of IFN-α/β after virus infection and the resultant upregulation of antiviral IFN-stimulated genes (ISGs) through IFN-α/β receptor complex signaling. However, other than examining the effects of genetic deletion of induction or effector pathway components, little is known regarding the functionality of these responses in intact hosts and whether host genetic or environmental factors might influence their potency. Here, we demonstrate that the IFN-α/β response against multiple arthropod-vectored viruses, which replicate over a wide temperature range, is extremely sensitive to fluctuations in temperature, exhibiting reduced antiviral efficacy at subnormal cellular temperatures and increased efficacy at supranormal temperatures. The effect involves both IFN-α/β and ISG upregulation pathways with a major aspect of altered potency reflecting highly temperature-dependent transcription of IFN response genes that leads to altered IFN-α/β and ISG protein levels. Discordantly, signaling steps prior to transcription that were examined showed the opposite effect from gene transcription, with potentiation at low temperature and inhibition at high temperature. Finally, we demonstrate that by lowering the temperature of mice, chikungunya arbovirus replication and disease are exacerbated in an IFN-α/β-dependent manner. This finding raises the potential for use of hyperthermia as a therapeutic modality for viral infections and in other contexts such as antitumor therapy. The increased IFN-α/β efficacy at high temperatures may also reflect an innate immune-relevant aspect of the febrile response. IMPORTANCE The interferon alpha/beta (IFN-α/β) response is a first-line innate defense against arthropod-borne viruses (arboviruses). Arboviruses, such as chikungunya virus (CHIKV), can infect cells and replicate across a wide temperature range due to their replication in both mammalian/avian and arthropod hosts. Accordingly, these viruses can cause human disease in tissues regularly exposed to temperatures below the normal mammalian core temperature, 37°C. We questioned whether temperature variation could affect the efficacy of IFN-α/β responses against these viruses and help to explain some aspects of human disease manifestations. We observed that IFN-α/β efficacy was dramatically lower at subnormal temperatures and modestly enhanced at febrile temperatures, with the effects involving altered IFN-α/β response gene transcription but not IFN-α/β pathway signaling. These results provide insight into the functioning of the IFN-α/β response in vivo and suggest that temperature elevation may represent an immune-enhancing therapeutic modality for a wide variety of IFN-α/β-sensitive infections and pathologies., (Copyright © 2018 Lane et al.)

Gram-positive bacteria, including the major respiratory pathogen Streptococcus pneumoniae , were recently shown to produce extracellular vesicles (EVs) that likely originate from the plasma membrane and are released into the extracellular environment. EVs may function as cargo for many bacterial proteins, however, their involvement in cellular processes and their interactions with the innate immune system are poorly understood. Here, EVs from pneumococci were characterized and their immunomodulatory effects investigated. Pneumococcal EVs were protruding from the bacterial surface and released into the medium as 25 to 250 nm lipid stained vesicles containing a large number of cytosolic, membrane, and surface-associated proteins. The cytosolic pore-forming toxin pneumolysin was significantly enriched in EVs compared to a total bacterial lysate but was not required for EV formation. Pneumococcal EVs were internalized into A549 lung epithelial cells and human monocyte-derived dendritic cells and induced proinflammatory cytokine responses irrespective of pneumolysin content. EVs from encapsulated pneumococci were recognized by serum proteins, resulting in C3b deposition and formation of C5b-9 membrane attack complexes as well as factor H recruitment, depending on the presence of the choline binding protein PspC. Addition of EVs to human serum decreased opsonophagocytic killing of encapsulated pneumococci. Our data suggest that EVs may act in an immunomodulatory manner by allowing delivery of vesicle-associated proteins and other macromolecules into host cells. In addition, EVs expose targets for complement factors in serum, promoting pneumococcal evasion of humoral host defense. IMPORTANCE Streptococcus pneumoniae is a major contributor to morbidity and mortality worldwide, being the major cause of milder respiratory tract infections such as otitis and sinusitis and of severe infections such as community-acquired pneumonia, with or without septicemia, and meningitis. More knowledge is needed on how pneumococci interact with the host, deliver virulence factors, and activate immune defenses. Here we show that pneumococci form extracellular vesicles that emanate from the plasma membrane and contain virulence properties, including enrichment of pneumolysin. We found that pneumococcal vesicles can be internalized into epithelial and dendritic cells and bind complement proteins, thereby promoting pneumococcal evasion of complement-mediated opsonophagocytosis. They also induce pneumolysin-independent proinflammatory responses. We suggest that these vesicles can function as a mechanism for delivery of pneumococcal proteins and other immunomodulatory components into host cells and help pneumococci to avoid complement deposition and phagocytosis-mediated killing, thereby possibly contributing to the symptoms found in pneumococcal infections., (Copyright © 2018 Codemo et al.)

Neutrophils are a major player in host immunity to infection; however, the mechanisms by which human neutrophils respond to the intracellular protozoan parasite Toxoplasma gondii are still poorly understood. In the current study, we found that, whereas primary human monocytes produced interleukin-1beta (IL-1β) in response to T. gondii infection, human neutrophils from the same blood donors did not. Moreover, T. gondii inhibited lipopolysaccharide (LPS)-induced IL-1β synthesis in human peripheral blood neutrophils. IL-1β suppression required active parasite invasion, since heat-killed or mycalolide B-treated parasites did not inhibit IL-1β release. By investigating the mechanisms involved in this process, we found that T. gondii infection of neutrophils treated with LPS resulted in reduced transcript levels of IL-1β and NLRP3 and reduced protein levels of pro-IL-1β, mature IL-1β, and the inflammasome sensor NLRP3. In T. gondii -infected neutrophils stimulated with LPS, the levels of MyD88, TRAF6, IKKα, IKKβ, and phosphorylated IKKα/β were not affected. However, LPS-induced IκBα degradation and p65 phosphorylation were reduced in T. gondii- infected neutrophils, and degradation of IκBα was reversed by treatment with the proteasome inhibitor MG-132. Finally, we observed that T. gondii inhibited the cleavage and activity of caspase-1 in human neutrophils. These results indicate that T. gondii suppression of IL-1β involves a two-pronged strategy whereby T. gondii inhibits both NF-κB signaling and activation of the NLRP3 inflammasome. These findings represent a novel mechanism of T. gondii evasion of human neutrophil-mediated host defense by targeting the production of IL-1β. IMPORTANCE Toxoplasma gondii is an obligate intracellular parasite that infects approximately one-third of humans worldwide and can invade virtually any nucleated cell in the human body. Although it is well documented that neutrophils infiltrate the site of acute T. gondii infection, there is limited understanding of how human neutrophils respond to T. gondii Neutrophils control infectious pathogens by a variety of mechanisms, including the release of the cytokine IL-1β, a major driver of inflammation during infection. This study reveals that T. gondii is able to inhibit IL-1β production in human neutrophils by impairing the activation of the NF-κB signaling pathway and by inhibiting the inflammasome, the protein complex responsible for IL-1β maturation. This two-pronged strategy of targeting the IL-1β pathway may facilitate the survival and spread of T. gondii during acute infection., (Copyright © 2018 Lima et al.)

Hypervirulent group A streptococcus (GAS) can inhibit neutrophil recruitment and cause systemic infection in a mouse model of skin infection. The purpose of this study was to determine whether platelet-activating factor acetylhydrolase Sse and streptolysin S (SLS) have synergistic contributions to inhibition of neutrophil recruitment and systemic infection in subcutaneous infection of mice by MGAS315, a hypervirulent genotype emm3 GAS strain. Deletion of sse and sagA in MGAS315 synergistically reduced the skin lesion size and GAS burden in the liver and spleen. However, the mutants were persistent at skin sites and had similar growth factors in nonimmune blood. Thus, the low numbers of Δ sse Δ sagA mutants in the liver and spleen were likely due to their reduction in the systemic dissemination. Few intact and necrotic neutrophils were detected at MGAS315 infection sites. In contrast, many neutrophils and necrotic cells were present at the edge of Δ sse mutant infection sites on day 1 and at the edge of and inside Δ sse mutant infection sites on day 2. Δ sagA mutant infection sites had massive numbers of and few intact neutrophils at the edge and center of the infection sites, respectively, on day 1 and were full of intact neutrophils or necrotic cells on day 2. Δ sse Δ sagA mutant infection sites had massive numbers of intact neutrophils throughout the whole infection site. These sse and sagA deletion-caused changes in the histological pattern at skin infection sites could be complemented. Thus, the sse and sagA deletions synergistically enhance neutrophil recruitment. These findings indicate that both Sse and SLS are required but that neither is sufficient for inhibition of neutrophil recruitment and systemic infection by hypervirulent GAS., (Copyright © 2017 American Society for Microbiology.)

Maintenance of the structural macromolecule peptidoglycan (PG), which involves regulated cycles of PG synthesis and PG degradation, is pivotal for cellular integrity and survival. PG fragments generated from the degradation process are usually efficiently recycled by Gram-negative bacteria. However, Neisseria gonorrhoeae and a limited number of Gram-negative bacteria release PG fragments in amounts sufficient to induce host tissue inflammation and damage during an infection. Due to limited redundancy in PG-modifying machineries and genetic tractability, N. gonorrhoeae serves as a great model organism for the study of biological processes related to PG. This review summarizes the generation, modification, and release of inflammatory PG molecules by N. gonorrhoeae and related species and discusses these findings in the context of understanding bacterial physiology and pathogenesis., (Copyright © 2017 American Society for Microbiology.)

Epstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus that establishes a latency reservoir in B cells. In this work, we show that ibrutinib, idelalisib, and dasatinib, drugs that block B cell receptor (BCR) signaling and are used in the treatment of hematologic malignancies, block BCR-mediated lytic induction at clinically relevant doses. We confirm that the immunosuppressive drugs cyclosporine and tacrolimus also inhibit BCR-mediated lytic induction but find that rapamycin does not inhibit BCR-mediated lytic induction. Further investigation shows that mammalian target of rapamycin complex 2 (mTORC2) contributes to BCR-mediated lytic induction and that FK506-binding protein 12 (FKBP12) binding alone is not adequate to block activation. Finally, we show that BCR signaling can activate EBV lytic induction in freshly isolated B cells from peripheral blood mononuclear cells (PBMCs) and that activation can be inhibited by ibrutinib or idelalisib. IMPORTANCE EBV establishes viral latency in B cells. Activation of the B cell receptor pathway activates lytic viral expression in cell lines. Here we show that drugs that inhibit important kinases in the BCR signaling pathway inhibit activation of lytic viral expression but do not inhibit several other lytic activation pathways. Immunosuppressant drugs such as cyclosporine and tacrolimus but not rapamycin also inhibit BCR-mediated EBV activation. Finally, we show that BCR activation of lytic infection occurs not only in tumor cell lines but also in freshly isolated B cells from patients and that this activation can be blocked by BCR inhibitors., (Copyright © 2017 American Society for Microbiology.)

Unlabelled: Despite its importance in shaping adaptive immune responses, viral clearance, and immune-based inflammation, tissue-specific innate immunity remains poorly characterized for hepatitis C virus (HCV) infection due to the lack of access to acutely infected tissues. In this study, we evaluated the impact of natural killer (NK) cells and myeloid (mDCs) and plasmacytoid (pDCs) dendritic cells on control of virus replication and virus-induced pathology caused by another, more rapidly resolving hepacivirus, GB virus B (GBV-B), in infections of common marmosets. High plasma and liver viral loads and robust hepatitis characterized acute GBV-B infection, and while viremia was generally cleared by 2 to 3 months postinfection, hepatitis and liver fibrosis persisted after clearance. Coinciding with peak viral loads and liver pathology, the levels of NK cells, mDCs, and pDCs in the liver increased up to 3-fold. Although no obvious numerical changes in peripheral innate cells occurred, circulating NK cells exhibited increased perforin and Ki67 expression levels and increased surface expression of CXCR3. These data suggested that increased NK cell arming and proliferation as well as tissue trafficking may be associated with influx into the liver during acute infection. Indeed, NK cell frequencies in the liver positively correlated with plasma (R = 0.698; P = 0.015) and liver (R = 0.567; P = 0.057) viral loads. Finally, soluble factors associated with NK cells and DCs, including gamma interferon (IFN-γ) and RANTES, were increased in acute infection and also were associated with viral loads and hepatitis. Collectively, the findings showed that mobilization of local and circulating innate immune responses was linked to acute virus-induced hepatitis, and potentially to resolution of GBV-B infection, and our results may provide insight into similar mechanisms in HCV infection., Importance: Hepatitis C virus (HCV) infection has created a global health crisis, and despite new effective antivirals, it is still a leading cause of liver disease and death worldwide. Recent evidence suggests that innate immunity may be a potential therapeutic target for HCV, but it may also be a correlate of increased disease. Due to a lack of access to human tissues with acute HCV infection, in this study we evaluated the role of innate immunity in resolving infection with a hepacivirus, GBV-B, in common marmosets. Collectively, our data suggest that NK cell and DC mobilization in acute hepacivirus infection can dampen virus replication but also regulate acute and chronic liver damage. How these two opposing effects on the host may be modulated in future therapeutic and vaccine approaches warrants further study., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Host cells respond to viral infections by producing type I interferon (IFN), which induces the expression of hundreds of interferon-stimulated genes (ISGs). Although ISGs mediate a protective state against many pathogens, the antiviral functions of the majority of these genes have not been identified. IFITM3 is a small transmembrane ISG that restricts a broad range of viruses, including orthomyxoviruses, flaviviruses, filoviruses, and coronaviruses. Here, we show that alphavirus infection is increased in Ifitm3(-/-) and Ifitm locus deletion (Ifitm-del) fibroblasts and, reciprocally, reduced in fibroblasts transcomplemented with Ifitm3. Mechanistic studies showed that Ifitm3 did not affect viral binding or entry but inhibited pH-dependent fusion. In a murine model of chikungunya virus arthritis, Ifitm3(-/-) mice sustained greater joint swelling in the ipsilateral ankle at days 3 and 7 postinfection, and this correlated with higher levels of proinflammatory cytokines and viral burden. Flow cytometric analysis suggested that Ifitm3(-/-) macrophages from the spleen were infected at greater levels than observed in wild-type (WT) mice, results that were supported by experiments with Ifitm3(-/-) bone marrow-derived macrophages. Ifitm3(-/-) mice also were more susceptible than WT mice to lethal alphavirus infection with Venezuelan equine encephalitis virus, and this was associated with greater viral burden in multiple organs. Collectively, our data define an antiviral role for Ifitm3 in restricting infection of multiple alphaviruses., Importance: The interferon-induced transmembrane protein 3 (IFITM3) inhibits infection of multiple families of viruses in cell culture. Compared to other viruses, much less is known about the antiviral effect of IFITM3 on alphaviruses. In this study, we characterized the antiviral activity of mouse Ifitm3 against arthritogenic and encephalitic alphaviruses using cells and animals with a targeted gene deletion of Ifitm3 as well as deficient cells transcomplemented with Ifitm3. Based on extensive virological analysis, we demonstrate greater levels of alphavirus infection and disease pathogenesis when Ifitm3 expression is absent. Our data establish an inhibitory role for Ifitm3 in controlling infection of alphaviruses., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Rabies virus (RABV) P gene mRNA encodes five in-frame start codons, resulting in expression of full-length P protein (P1) and N-terminally truncated P proteins (tPs), designated P2, P3, P4, and P5. Despite the fact that some tPs are known as interferon (IFN) antagonists, the importance of tPs in the pathogenesis of RABV is still unclear. In this study, to examine whether tPs contribute to pathogenesis, we exploited a reverse genetics approach to generate CE(NiP)ΔP2-5, a mutant of pathogenic CE(NiP) in which the P gene was mutated by replacing all of the start codons (AUG) for tPs with AUA. We confirmed that while CE(NiP) expresses detectable levels of P2 and P3, CE(NiP)ΔP2-5 has an impaired ability to express these tPs. After intramuscular inoculation, CE(NiP)ΔP2-5 caused significantly lower morbidity and mortality rates in mice than did CE(NiP), indicating that tPs play a critical role in RABV neuroinvasiveness. Further examinations revealed that this less neuroinvasive phenotype of CE(NiP)ΔP2-5 correlates with its impaired ability to replicate in muscle cells, indicative of the importance of tPs in viral replication in muscle cells. We also demonstrated that CE(NiP)ΔP2-5 infection induced a higher level of Ifn-β gene expression in muscle cells than did CE(NiP) infection, consistent with the results of an IFN-β promoter reporter assay suggesting that all tPs function to antagonize IFN induction in muscle cells. Taken together, our findings strongly suggest that tPs promote viral replication in muscle cells through their IFN antagonist activities and thereby support infection of peripheral nerves., Importance: Despite the fact that previous studies have demonstrated that P2 and P3 of RABV have IFN antagonist activities, the actual importance of tPs in pathogenesis has remained unclear. Here, we provide the first evidence that tPs contribute to the pathogenesis of RABV, especially its neuroinvasiveness. Our results also show the mechanism underlying the neuroinvasiveness driven by tPs, highlighting the importance of their IFN antagonist activities, which support viral replication in muscle cells., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Type I interferons (IFNs), including IFN-α, upregulate an array of IFN-stimulated genes (ISGs) and potently suppress Human immunodeficiency virus type 1 (HIV-1) infectivity in CD4(+) T cells, monocyte-derived macrophages, and dendritic cells. Recently, we and others identified ISG myxovirus resistance 2 (MX2) as an inhibitor of HIV-1 nuclear entry. However, additional antiviral blocks exist upstream of nuclear import, but the ISGs that suppress infection, e.g., prior to (or during) reverse transcription, remain to be defined. We show here that the HIV-1 CA mutations N74D and A105T, both of which allow escape from inhibition by MX2 and the truncated version of cleavage and polyadenylation specific factor 6 (CPSF6), as well as the cyclophilin A (CypA)-binding loop mutation P90A, all increase sensitivity to IFN-α-mediated inhibition. Using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology, we demonstrate that the IFN-α hypersensitivity of these mutants in THP-1 cells is independent of MX2 or CPSF6. As expected, CypA depletion had no additional effect on the behavior of the P90A mutant but modestly increased the IFN-α sensitivity of wild-type virus. Interestingly, the infectivity of wild-type or P90A virus could be rescued from the MX2-independent IFN-α-induced blocks in THP-1 cells by treatment with cyclosporine (Cs) or its nonimmunosuppressive analogue SDZ-NIM811, indicating that Cs-sensitive host cell cyclophilins other than CypA contribute to the activity of IFN-α-induced blocks. We propose that cellular interactions with incoming HIV-1 capsids help shield the virus from recognition by antiviral effector mechanisms. Thus, the CA protein is a fulcrum for the dynamic interplay between cell-encoded functions that inhibit or promote HIV-1 infection., Importance: HIV-1 is the causative agent of AIDS. During acute HIV-1 infection, numerous proinflammatory cytokines are produced, including type I interferons (IFNs). IFNs can limit HIV-1 replication by inducing the expression of a set of antiviral genes that inhibit HIV-1 at multiple steps in its life cycle, including the postentry steps of reverse transcription and nuclear import. This is observed in cultured cell systems, as well as in clinical trials in HIV-1-infected patients. The identities of the cellular antiviral factors, their viral targets, and the underpinning mechanisms are largely unknown. We show here that the HIV-1 Capsid protein plays a central role in protecting the virus from IFN-induced inhibitors that block early postentry steps of infection. We further show that host cell cyclophilins play an important role in regulating these processes, thus highlighting the complex interplay between antiviral effector mechanisms and viral survival., (Copyright © 2016 Bulli et al.)

Unlabelled: Intestinal homeostasis mechanisms must protect the host intestinal tissue from endogenous lipopolysaccharides (LPSs) produced by the intestinal microbiota. In this report, we demonstrate that murine intestinal fecal lipids effectively block Toll-like receptor 4 (TLR4) responses to naturally occurring Bacteroidetes sp. LPS. Cardiolipin (CL) represents a significant proportion of the total intestinal and fecal lipids and, furthermore, potently antagonizes TLR4 activation by reducing LPS binding at the lipopolysaccharide binding protein (LBP), CD14, and MD-2 steps of the TLR4 signaling pathway. It is further demonstrated that intestinal lipids and CL are less effective at neutralizing more potent Enterobacteriaceae-type LPS, which is enriched in feces obtained from mice with dextran sodium sulfate (DSS)-treated inflammatory bowel disease. The selective inhibition of naturally occurring LPS structures by intestinal lipids may represent a novel homeostasis mechanism that blocks LPS activation in response to symbiotic but not dysbiotic microbial communities., Importance: The guts of animals harbor a variety of Gram-negative bacteria associated with both states of intestinal health and states of disease. Environmental factors, such as dietary habits, can drive the microbial composition of the host animal's intestinal bacterial community toward a more pathogenic state. Both beneficial and harmful Gram-negative bacteria are capable of eliciting potentially damaging inflammatory responses from the host intestinal tissues via a lipopolysaccharide (LPS)-dependent pathway. Physical mucosal barriers and antibodies produced by the intestinal immune system protect against the undesired inflammatory effects of LPS, although it is unknown why some bacteria are more effective at overcoming the protective barriers than others. This report describes the discovery of a lipid-type protective barrier in the intestine that reduces the deleterious effects of LPSs from beneficial bacteria but is less effective in dampening the inflammatory effects of LPSs from harmful bacteria, providing a novel mechanistic insight into inflammatory intestinal disorders., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Lactobacilli are found in diverse environments and are widely applied as probiotic, health-promoting food supplements. Polysaccharides are ubiquitously present on the cell surface of lactobacilli and are considered to contribute to the species- and strain-specific probiotic effects that are typically observed. Two Lactobacillus plantarum strains, SF2A35B and Lp90, have an obvious ropy phenotype, implying high extracellular polysaccharide (EPS) production levels. In this work, we set out to identify the genes involved in EPS production in these L. plantarum strains and to demonstrate their role in EPS production by gene deletion analysis. A model L. plantarum strain, WCFS1, and its previously constructed derivative that produced reduced levels of EPS were included as reference strains. The constructed EPS-reduced derivatives were analyzed for the abundance and sugar compositions of their EPS, revealing cps2-like gene clusters in SF2A35B and Lp90 responsible for major EPS production. Moreover, these mutant strains were tested for phenotypic characteristics that are of relevance for their capacity to interact with the host epithelium in the intestinal tract, including bacterial surface properties as well as survival under the stress conditions encountered in the gastrointestinal tract (acid and bile stress). In addition, the Toll-like receptor 2 (TLR2) signaling and immunomodulatory capacities of the EPS-negative derivatives and their respective wild-type strains were compared, revealing strain-specific impacts of EPS on the immunomodulatory properties. Taken together, these experiments illustrate the importance of EPS in L. plantarum strains as a strain-specific determinant in host interaction., Importance: This study evaluates the role of extracellular polysaccharides that are produced by different strains of Lactobacillus plantarum in the determination of the cell surface properties of these bacteria and their capacity to interact with their environment, including their signaling to human host cells. The results clearly show that the consequences of removal of these polysaccharides are very strain specific, illustrating the diverse and unpredictable roles of these polysaccharides in the environmental interactions of these bacterial strains. In the context of the use of lactobacilli as health-promoting probiotic organisms, this study exemplifies the importance of strain specificity., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Molluscum contagiosum virus (MCV) gene MC159 encodes a viral FLICE inhibitory protein (vFLIP) that inhibits caspase-8-mediated apoptosis. The MC159 protein was also reported to inhibit programmed necrosis (necroptosis) and modulate NF-κB activation by interacting with RIP1 and NEMO. The importance of MC159 during MCV infection has remained unknown, as there is no system for propagation and genetic manipulation of this virus. Here we investigated the functions of MC159 during viral infection using murine cytomegalovirus (MCMV) as a surrogate virus. MC159 was inserted into the MCMV genome, replacing M36 or M45, two MCMV genes with functions similar to those reported for MC159. M36 encodes a viral inhibitor of caspase-8-induced apoptosis (vICA) and M45 a viral inhibitor of RIP activation (vIRA), which inhibits RIP1/RIP3-mediated necroptosis. The M45 protein also blocks NF-κB activation by interacting with NEMO. When expressed by MCMV, MC159 blocked tumor necrosis factor alpha (TNF-α)-induced apoptosis of infected cells and partially restored MCMV replication in macrophages. However, MC159 did not fully replace M45, as it did not inhibit necroptosis in murine cells, but it reduced TNF-α-induced necroptosis in MCMV-infected human HT-29 cells. MC159 also differed from M45 in its effect on NF-κB. While MCMV-encoded M45 blocked NF-κB activation by TNF-α and interleukin-1β (IL-1β), MC159 inhibited TNF-α- but not IL-1β-induced NF-κB activation in infected mouse fibroblasts. These results indicate that the spectrum of MC159's functions differs depending on cell type and expression system and that a cell culture system for the propagation of MCV is needed to determine the biological relevance of presumed viral gene functions., Importance: MCV is a human-pathogenic poxvirus that cannot be propagated in cell culture or laboratory animals. Therefore, MCV gene products have been studied predominantly in cells expressing individual viral genes. In this study, we analyzed the function of the MCV gene MC159 by expressing it from a different virus and comparing its functions to those of two well-characterized MCMV genes. In this system, MC159 displayed some but not all of the previously described functions, suggesting that the functions of a viral gene depend on the conditions under which it is expressed. Until a cell culture system for the analysis of MCV becomes available, it might be necessary to analyze MCV genes in several different systems to extrapolate their biological importance., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: The elongation factor Tu GTP binding domain-containing protein 2 (EFTUD2) was identified as an anti-hepatitis C virus (HCV) host factor in our recent genome-wide small interfering RNA (siRNA) screen. In this study, we sought to further determine EFTUD2's role in HCV infection and investigate the interaction between EFTUD2 and other regulators involved in HCV innate immune (RIG-I, MDA5, TBK1, and IRF3) and JAK-STAT1 pathways. We found that HCV infection decreased the expression of EFTUD2 and the viral RNA sensors RIG-I and MDA5 in HCV-infected Huh7 and Huh7.5.1 cells and in liver tissue from in HCV-infected patients, suggesting that HCV infection downregulated EFTUD2 expression to circumvent the innate immune response. EFTUD2 inhibited HCV infection by inducing expression of the interferon (IFN)-stimulated genes (ISGs) in Huh7 cells. However, its impact on HCV infection was absent in both RIG-I knockdown Huh7 cells and RIG-I-defective Huh7.5.1 cells, indicating that the antiviral effect of EFTUD2 is dependent on RIG-I. Furthermore, EFTUD2 upregulated the expression of the RIG-I-like receptors (RLRs) RIG-I and MDA5 to enhance the innate immune response by gene splicing. Functional experiments revealed that EFTUD2-induced expression of ISGs was mediated through interaction of the EFTUD2 downstream regulators RIG-I, MDA5, TBK1, and IRF3. Interestingly, the EFTUD2-induced antiviral effect was independent of the classical IFN-induced JAK-STAT pathway. Our data demonstrate that EFTUD2 restricts HCV infection mainly through an RIG-I/MDA5-mediated, JAK-STAT-independent pathway, thereby revealing the participation of EFTUD2 as a novel innate immune regulator and suggesting a potentially targetable antiviral pathway., Importance: Innate immunity is the first line defense against HCV and determines the outcome of HCV infection. Based on a recent high-throughput whole-genome siRNA library screen revealing a network of host factors mediating antiviral effects against HCV, we identified EFTUD2 as a novel innate immune regulator against HCV in the infectious HCV cell culture model and confirmed that its expression in HCV-infected liver tissue is inversely related to HCV infection. Furthermore, we determined that EFTUD2 exerts its antiviral activity mainly through governing its downstream regulators RIG-I and MDA5 by gene splicing to activate IRF3 and induce classical ISG expression independent of the JAT-STAT signaling pathway. This study broadens our understanding of the HCV innate immune response and provides a possible new antiviral strategy targeting this novel regulator of the innate response., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Recent studies demonstrated that transgenic mice expressing key human hepatitis C virus (HCV) receptors are susceptible to HCV infection, albeit at very low efficiency. Robust mouse models of HCV infection and replication are needed to determine the importance of host factors in HCV replication, pathogenesis, and carcinogenesis as well as to facilitate the development of antiviral agents and vaccines. The low efficiency of HCV replication in the humanized mouse models is likely due to either the lack of essential host factors or the presence of restriction factors for HCV infection and/or replication in mouse hepatocytes. To determine whether HCV infection is affected by restriction factors present in serum, we examined the effects of mouse and human sera on HCV infectivity. Strikingly, we found that mouse and human sera potently inhibited HCV infection. Mechanistic studies demonstrated that mouse serum blocked HCV cell attachment without significant effect on HCV replication. Fractionation analysis of mouse serum in conjunction with targeted mass spectrometric analysis suggested that serum very-low-density lipoprotein (VLDL) was responsible for the blockade of HCV cell attachment, as VLDL-depleted mouse serum lost HCV-inhibitory activity. Both purified mouse and human VLDL could efficiently inhibit HCV infection. Collectively, these findings suggest that serum VLDL serves as a major restriction factor of HCV infection in vivo. The results also imply that reduction or elimination of VLDL production will likely enhance HCV infection in the humanized mouse model of HCV infection and replication., Importance: HCV is a major cause of liver diseases, such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Recently, several studies suggested that humanized mouse or transgenic mouse expressing key HCV human receptors became susceptible to HCV infection. However, HCV infection and replication in the humanized animals were very inefficient, suggesting either the lack of cellular genes important for HCV replication or the presence of restriction factors inhibiting HCV infection and replication in the mouse. In this study, we found that both mouse and human sera effectively inhibited HCV infection. Mechanistic studies demonstrated that VLDL is the major restriction factor that blocks HCV infection. These findings suggest that VLDL is beneficial to patients by restricting HCV infection. More importantly, our findings suggest that elimination of VLDL will lead to the development of more robust mouse models for the study of HCV pathogenesis, host response to HCV infection, and evaluation of HCV vaccines., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Hepatitis C virus (HCV), a single-stranded positive-sense RNA virus of the Flaviviridae family, causes chronic liver diseases, including hepatitis, cirrhosis, and cancer. HCV infection is critically dependent on host lipid metabolism, which contributes to all stages of the viral life cycle, including virus entry, replication, assembly, and release. 25-Hydroxycholesterol (25HC) plays a critical role in regulating lipid metabolism, modulating immune responses, and suppressing viral pathogens. In this study, we showed that 25HC and its synthesizing enzyme cholesterol 25-hydroxylase (CH25H) efficiently inhibit HCV infection at a postentry stage. CH25H inhibits HCV infection by suppressing the maturation of SREBPs, critical transcription factors for host lipid biosynthesis. Interestingly, CH25H is upregulated upon poly(I · C) treatment or HCV infection in hepatocytes, which triggers type I and III interferon responses, suggesting that the CH25H induction constitutes a part of host innate immune response. To our surprise, in contrast to studies in mice, CH25H is not induced by interferons in human cells and knockdown of STAT-1 has no effect on the induction of CH25H, suggesting CH25H is not an interferon-stimulated gene in humans but rather represents a primary and direct host response to viral infection. Finally, knockdown of CH25H in human hepatocytes significantly increases HCV infection. In summary, our results demonstrate that CH25H constitutes a primary innate response against HCV infection through regulating host lipid metabolism. Manipulation of CH25H expression and function should provide a new strategy for anti-HCV therapeutics., Importance: Recent studies have expanded the critical roles of oxysterols in regulating immune response and antagonizing viral pathogens. Here, we showed that one of the oxysterols, 25HC and its synthesizing enzyme CH25H efficiently inhibit HCV infection at a postentry stage via suppressing the maturation of transcription factor SREBPs that regulate lipid biosynthesis. Furthermore, we found that CH25H expression is upregulated upon poly(I·C) stimulation or HCV infection, suggesting CH25H induction constitutes a part of host innate immune response. Interestingly, in contrast to studies in mice showing that ch25h is an interferon-stimulated gene, CH25H cannot be induced by interferons in human cells but rather represents a primary and direct host response to viral infection. Our studies demonstrate that the induction of CH25H represents an important host innate response against virus infection and highlight the role of lipid effectors in host antiviral strategy., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Constant cross talk between Candida albicans yeast cells and their human host determines the outcome of fungal colonization and, eventually, the progress of infectious disease (candidiasis). An effective weapon used by C. albicans to cope with the host defense system is the release of 10 distinct secreted aspartic proteases (SAPs). Here, we validate a hypothesis that neutrophils and epithelial cells use the antimicrobial peptide LL-37 to inactivate C. albicans at sites of candidal infection and that C. albicans uses SAPs to effectively degrade LL-37. LL-37 is cleaved into multiple products by SAP1 to -4, SAP8, and SAP9, and this proteolytic processing is correlated with the gradual decrease in the antifungal activity of LL-37. Moreover, a major intermediate of LL-37 cleavage-the LL-25 peptide-is antifungal but devoid of the immunomodulatory properties of LL-37. In contrast to LL-37, LL-25 did not affect the generation of reactive oxygen species by neutrophils upon treatment with phorbol esters. Stimulating neutrophils with LL-25 (rather than LL-37) significantly decreased calcium flux and interleukin-8 production, resulting in lower chemotactic activity of the peptide against neutrophils, which may decrease the recruitment of neutrophils to infection foci. LL-25 also lost the function of LL-37 as an inhibitor of neutrophil apoptosis, thereby reducing the life span of these defense cells. This study indicates that C. albicans can effectively use aspartic proteases to destroy the antimicrobial and immunomodulatory properties of LL-37, thus enabling the pathogen to survive and propagate., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The trans-sialidases (TSs) from Trypanosoma cruzi, the agent of Chagas disease, are virulence factors shed to the bloodstream that induce strong alterations in the immune system. Here, we report that both enzymatically active TS (aTS) and its lectinlike isoform (iTS) disturb CD4 T cell physiology, inducing downregulation of Th1 cell functionality and in vivo cell expansion. By using ovalbumin-specific DO11.10 cells as tracers of clones developing the Th1 phenotype, we found that the infection induced significant amounts of gamma interferon (IFN-γ) but low levels of interleukin 2 (IL-2) and increased IL-4 production in vivo, in agreement with a mixed T helper response. The production of cytokines associated with the Th2 phenotype was prevented by passive transfer of anti-TS neutralizing antibodies. TSs also reduced the T cell receptor signaling as assayed by Zap-70 phosphorylation. TSs also reduced IL-2 and IFN-γ secretion, with a concomitant increase in IL-4 production and then an unbalancing of the CD4 T cell response toward the Th2 phenotype. This effect was prevented by using anti-IL-10 neutralizing antibodies or IL-10(-/-) antigen-presenting cells, supporting the subversion of this regulatory pathway. In support, TSs stimulated IL-10 secretion by antigen-presenting cells during their interaction with CD4 T cells. When polarized cells were stimulated in the presence of TSs, the secretion of IL-2 and IFN-γ was strongly downregulated in Th1 cells, while IL-2 production was upregulated in Th2 cells. Although the Th1 response is associated with host survival, it may simultaneously induce extensive damage to infected tissues. Thus, by delaying the elicitation of the Th1 response and limiting its effector properties, TSs restrain the cell response, supporting T. cruzi colonization and persistence while favoring host survival., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Human brucellosis is a protean disease with a diversity of clinical signs and symptoms resulting from infection with Brucella species. Recent reports suggest a cross-regulation between adrenal steroids (cortisol and dehydroepiandrosterone [DHEA]) and the immune system. Monocytes and macrophages are the main replication niche for Brucella. Therefore, we investigated the role of adrenal hormones on the modulation of the immune response mediated by macrophages in B. abortus infection. Cortisol treatment during B. abortus infection significantly inhibits cytokine, chemokine, and MMP-9 secretion. In contrast, DHEA treatment had no effect. However, DHEA treatment increases the expression of costimulatory molecules (CD40, CD86), the adhesion molecule CD54, and major histocompatibility complex class I (MHC-I) and MHC-II expression on the surface of B. abortus-infected monocytes. It is known that B. abortus infection inhibits MHC-I and MHC-II expression induced by gamma interferon (IFN-γ) treatment. DHEA reverses B. abortus downmodulation of the MHC-I and -II expression induced by IFN-γ. Taken together, our data indicate that DHEA immune intervention may positively affect monocyte activity during B. abortus infection., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Farnesol, produced by the polymorphic fungus Candida albicans, is the first quorum-sensing molecule discovered in eukaryotes. Its main function is control of C. albicans filamentation, a process closely linked to pathogenesis. In this study, we analyzed the effects of farnesol on innate immune cells known to be important for fungal clearance and protective immunity. Farnesol enhanced the expression of activation markers on monocytes (CD86 and HLA-DR) and neutrophils (CD66b and CD11b) and promoted oxidative burst and the release of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α] and macrophage inflammatory protein 1 alpha [MIP-1α]). However, this activation did not result in enhanced fungal uptake or killing. Furthermore, the differentiation of monocytes to immature dendritic cells (iDC) was significantly affected by farnesol. Several markers important for maturation and antigen presentation like CD1a, CD83, CD86, and CD80 were significantly reduced in the presence of farnesol. Furthermore, farnesol modulated migrational behavior and cytokine release and impaired the ability of DC to induce T cell proliferation. Of major importance was the absence of interleukin 12 (IL-12) induction in iDC generated in the presence of farnesol. Transcriptome analyses revealed a farnesol-induced shift in effector molecule expression and a down-regulation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor during monocytes to iDC differentiation. Taken together, our data unveil the ability of farnesol to act as a virulence factor of C. albicans by influencing innate immune cells to promote inflammation and mitigating the Th1 response, which is essential for fungal clearance., Importance: Farnesol is a quorum-sensing molecule which controls morphological plasticity of the pathogenic yeast Candida albicans. As such, it is a major mediator of intraspecies communication. Here, we investigated the impact of farnesol on human innate immune cells known to be important for fungal clearance and protective immunity. We show that farnesol is able to enhance inflammation by inducing activation of neutrophils and monocytes. At the same time, farnesol impairs differentiation of monocytes into immature dendritic cells (iDC) by modulating surface phenotype, cytokine release and migrational behavior. Consequently, iDC generated in the presence of farnesol are unable to induce proper T cell responses and fail to secrete Th1 promoting interleukin 12 (IL-12). As farnesol induced down-regulation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, desensitization to GM-CSF could potentially explain transcriptional reprofiling of iDC effector molecules. Taken together, our data show that farnesol can also mediate Candida-host communication and is able to act as a virulence factor., (Copyright © 2015 Leonhardt et al.)

The aims of this study were to develop strains of lactic acid bacteria (LAB) having both immunomodulatory and antioxidant properties and to evaluate their anti-inflammatory effects both in vitro, in different cellular models, and in vivo, in a mouse model of colitis. Different Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains were cocultured with primary cultures of mononuclear cells. Analysis of the pro- and anti-inflammatory cytokines secreted by these cells after coincubation with candidate bacteria revealed that L. delbrueckii subsp. bulgaricus CRL 864 and S. thermophilus CRL 807 display the highest anti-inflammatory profiles in vitro. Moreover, these results were confirmed in vivo by the determination of the cytokine profiles in large intestine samples of mice fed with these strains. S. thermophilus CRL 807 was then transformed with two different plasmids harboring the genes encoding catalase (CAT) or superoxide dismutase (SOD) antioxidant enzymes, and the anti-inflammatory effects of recombinant streptococci were evaluated in a mouse model of colitis induced by trinitrobenzenesulfonic acid (TNBS). Our results showed a decrease in weight loss, lower liver microbial translocation, lower macroscopic and microscopic damage scores, and modulation of the cytokine production in the large intestines of mice treated with either CAT- or SOD-producing streptococci compared to those in mice treated with the wild-type strain or control mice without any treatment. Furthermore, the greatest anti-inflammatory activity was observed in mice receiving a mixture of both CAT- and SOD-producing streptococci. The addition of L. delbrueckii subsp. bulgaricus CRL 864 to this mixture did not improve their beneficial effects. These findings show that genetically engineering a candidate bacterium (e.g., S. thermophilus CRL 807) with intrinsic immunomodulatory properties by introducing a gene expressing an antioxidant enzyme enhances its anti-inflammatory activities.

Leishmania donovani causes visceral leishmaniasis (VL) by infecting the monocyte/macrophage lineage and residing inside specialized structures known as parasitophorous vacuoles. The protozoan parasite has adopted several means of escaping the host immune response, with one of the major methods being deactivation of host macrophages. Previous reports highlight dampened macrophage signaling, defective antigen presentation due to increased membrane fluidity, and the downregulation of several genes associated with L. donovani infection. We have reported previously that the defective antigen presentation in infected hamsters could be corrected by a single injection of a cholesterol-containing liposome. Here we show that cholesterol in the form of a liposomal formulation can stimulate the innate immune arm and reactivate macrophage function. Augmented levels of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI), along with proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), corroborate intracellular parasite killing. Cholesterol incorporation kinetics is favored in infected macrophages more than in normal macrophages. Such an enhanced cholesterol uptake is associated with preferential apoptosis of infected macrophages in an endoplasmic reticulum (ER) stress-dependent manner. All these events are coupled with mitogen-activated protein (MAP) kinase activation, while inhibition of such pathways resulted in increased parasite loads. Hence, liposomal cholesterol is a potential facilitator of the macrophage effector function in favor of the host, independently of the T-cell arm.

Marine macroalgae are rich in bioactive compounds that can, when consumed, impart beneficial effects on animal and human health. The red seaweed Chondrus crispus has been reported to have a wide range of health-promoting activities, such as antitumor and antiviral activities. Using a Caenorhabditis elegans infection model, we show that C. crispus water extract (CCWE) enhances host immunity and suppresses the expression of quorum sensing (QS) and the virulence factors of Pseudomonas aeruginosa (strain PA14). Supplementation of nematode growth medium with CCWE induced the expression of C. elegans innate immune genes, such as irg-1, irg-2, F49F1.6, hsf-1, K05D8.5, F56D6.2, C29F3.7, F28D1.3, F38A1.5 ZK6.7, lys-1, spp-1, and abf-1, by more than 2-fold, while T20G5.7 was not affected. Additionally, CCWE suppressed the expression of PA14 QS genes and virulence factors, although it did not affect the growth of the bacteria. These effects correlated with a 28% reduction in the PA14-inflicted killing of C. elegans. Kappa-carrageenan (K-CGN), a major component of CCWE, was shown to play an important role in the enhancement of host immunity. Using C. elegans mutants, we identified that pmk-1, daf-2/daf-16, and skn-1 are essential in the K-CGN-induced host immune response. In view of the conservation of innate immune pathways between C. elegans and humans, the results of this study suggest that water-soluble components of C. crispus may also play a health-promoting role in higher animals and humans.

The suppressor of cytokine signaling 1 (SOCS1) has emerged as a critical inhibitory molecule for controlling the cytokine response and antigen presentation by dendritic cells (DCs), thereby regulating the magnitude of both innate and adaptive immunity. The aim of this study was to investigate whether the SOCS1 antagonist pJAK2(1001-1013) peptide can weaken or block the inhibition function of SOCS1 in DCs by evaluating the phenotype and cytokine production, antigen-presenting, and specific T-cell-activating capacities of DCs electroporated with human gastric cancer cell total RNA. Furthermore, STAT1 activation of the JAK/STAT signal pathway mediated by SOCS1 was analyzed by Western blotting. The results demonstrate that the SOCS1 antagonist pJAK2(1001-1013) peptide upregulated the expression of the maturation marker (CD83) and costimulatory molecule (CD86) of RNA-electroporated human monocyte-derived mature DCs (mDCs), potentiated the capacity of mDCs to induce T-cell proliferation, stimulated the secretion of proinflammatory cytokines, and enhanced the cytotoxicity of tumor cell antigen-specific CTLs activated by human gastric cancer cell total RNA-electroporated mDCs. Data from Western blot analysis indicate that STAT1 was further activated in pJAK2(1001-1013) peptide-loaded mDCs. These results imply that the SOCS1 antagonist pJAK2(1001-1013) peptide is an effective reagent for the enhancement of antigen-specific antitumor immunity by DCs.