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Iron is an essential nutrient for microbes, and many pathogenic bacteria depend on siderophores to obtain iron. The mammalian innate immunity protein lipocalin 2 (Lcn2; also known as neutrophil gelatinase-associated lipocalin, 24p3, or siderocalin) binds the siderophore carboxymycobactin, an essential component of the iron acquisition apparatus of mycobacteria. Here we show that Lcn2 suppressed growth of Mycobacterium avium in culture, and M. avium induced Lcn2 production from mouse macrophages. Lcn2 also had elevated levels and initially limited the growth of M. avium in the blood of infected mice but did not impede growth in tissues and during long-term infections. M. avium is an intracellular pathogen. Subcellular imaging of infected macrophages revealed that Lcn2 trafficked to lysosomes separate from M. avium, whereas transferrin was efficiently transported to the mycobacteria. Thus, mycobacteria seem to reside in the Rab11(+) endocytic recycling pathway, thereby retaining access to nutrition and avoiding endocytosed immunoproteins like Lcn2. [ABSTRACT FROM AUTHOR]

Background: Current tuberculosis treatment regimens could be improved by adjunct host-directed therapies (HDT) targeting host responses. We investigated the antimycobacterial capacity of macrophages from patients with tuberculosis in a phase 1/2 randomized clinical trial (TBCOX2) of the cyclooxygenase-2 inhibitor etoricoxib., Methods: Peripheral blood mononuclear cells from 15 patients with tuberculosis treated with adjunctive COX-2i and 18 controls (standard therapy) were collected on day 56 after treatment initiation. The ex vivo capacity of macrophages to control mycobacterial infection was assessed by challenge with Mycobacterium avium, using an in vitro culture model. Macrophage inflammatory responses were analyzed by gene expression signatures, and concentrations of cytokines were analyzed in supernatants by multiplex., Results: Macrophages from patients receiving adjunctive COX-2i treatment had higher M. avium loads than controls after 6 days, suggesting an impaired capacity to control mycobacterial infection compared to macrophages from the control group. Macrophages from the COX-2i group had lower gene expression of TNF, IL-1B, CCL4, CXCL9, and CXCL10 and lowered production of cytokines IFN-β and S100A8/A9 than controls., Conclusions: Our data suggest potential unfavorable effects with impaired macrophage capacity to control mycobacterial growth in patients with tuberculosis receiving COX-2i treatment. Larger clinical trials are required to analyze the safety of COX-2i as HDT in patients with tuberculosis., Clinical Trials Registration: NCT02503839., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Nontuberculous mycobacteria are opportunistic bacteria pulmonary and extra-pulmonary infections in humans that closely resemble Mycobacterium tuberculosis . Although genome sequencing strategies helped determine NTMs, a common assay for the detection of coinfection by multiple NTMs with M. tuberculosis in the primary attempt of diagnosis is still elusive. Such a lack of efficiency leads to delayed therapy, an inappropriate choice of drugs, drug resistance, disease complications, morbidity, and mortality. Although a high-resolution LC-MS/MS-based multiprotein panel assay can be developed due to its specificity and sensitivity, it needs a library of species-specific peptides as a platform. Toward this, we performed an analysis of proteomes of 9 NTM species with more than 20 million peptide spectrum matches gathered from 26 proteome data sets. Our metaproteomic analyses determined 48,172 species-specific proteotypic peptides across 9 NTMs. Notably, M. smegmatis (26,008), M. abscessus (12,442), M. vaccae (6487), M. fortuitum (1623), M. avium subsp. paratuberculosis (844), M. avium subsp. hominissuis (580), and M. marinum (112) displayed >100 species-specific proteotypic peptides. Finally, these peptides and corresponding spectra have been compiled into a spectral library, FASTA, and JSON formats for future reference and validation in clinical cohorts by the biomedical community for further translation.

Mycobacterium avium is one of the prominent disease-causing bacteria in humans. It causes lymphadenitis, chronic and extrapulmonary, and disseminated infections in adults, children, and immunocompromised patients. M. avium has ∼4500 predicted protein-coding regions on average, which can help discover several variants at the proteome level. Many of them are potentially associated with virulence; thus, identifying such proteins can be a helpful feature in developing panel-based theranostics. In line with such a long-term goal, we carried out an in-depth proteomic analysis of M. avium with both data-dependent and data-independent acquisition methods. Further, a set of proteogenomic investigations were carried out using (i) a protein database for Mycobacterium tuberculosis, (ii) an M. avium genome six-frame-translated database, and (iii) a variant protein database of M. avium. A search of mass spectrometry data against M. avium protein database resulted in identifying 2954 proteins. Further, proteogenomic analyses aided in identifying 1301 novel peptide sequences and correcting translation start sites for 15 proteins. Ultimately, we created a spectral library of M. avium proteins, including novel genome search-specific peptides and variant peptides detected in this study. We validated the spectral library by a data-independent acquisition of the M. avium proteome. Thus, we present an M. avium spectral library of 29,033 peptide precursors supported by 0.4 million fragment ions for further use by the biomedical community., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2023. Published by Elsevier Inc.)

First recognized more than 30 years ago, glycine protects cells against rupture from diverse types of injury. This robust and widely observed effect has been speculated to target a late downstream process common to multiple modes of tissue injury. The molecular target of glycine that mediates cytoprotection, however, remains elusive. Here, we show that glycine works at the level of NINJ1, a newly identified executioner of plasma membrane rupture in pyroptosis, necrosis, and post-apoptosis lysis. NINJ1 is thought to cluster within the plasma membrane to cause cell rupture. We demonstrate that the execution of pyroptotic cell rupture is similar for human and mouse NINJ1 and that NINJ1 knockout functionally and morphologically phenocopies glycine cytoprotection in macrophages undergoing lytic cell death. Next, we show that glycine prevents NINJ1 clustering by either direct or indirect mechanisms. In pyroptosis, glycine preserves cellular integrity but does not affect upstream inflammasome activities or accompanying energetic cell death. By positioning NINJ1 clustering as a glycine target, our data resolve a long-standing mechanism for glycine-mediated cytoprotection. This new understanding will inform the development of cell preservation strategies to counter pathologic lytic cell death., Competing Interests: JB, RS, AV, MB, PD, BS, BK, CE, JK, NG, TF, BS No competing interests declared, (© 2022, Borges, Sætra et al.)

Macrophages deploy a variety of antimicrobial programs to contain mycobacterial infection. Upon activation, they undergo extensive metabolic reprogramming to meet an increase in energy demand, but also to support immune effector functions such as secretion of cytokines and antimicrobial activities. Here, we report that mitochondrial import of pyruvate is linked to production of mitochondrial ROS and control of Mycobacterium avium ( M. avium ) infection in human primary macrophages. Using chemical inhibition, targeted mass spectrometry and single cell image analysis, we showed that macrophages infected with M. avium switch to aerobic glycolysis without any major imbalances in the tricarboxylic acid cycle volume or changes in the energy charge. Instead, we found that pyruvate import contributes to hyperpolarization of mitochondria in infected cells and increases production of mitochondrial reactive oxygen species by the complex I via reverse electron transport, which reduces the macrophage burden of M. avium . While mycobacterial infections are extremely difficult to treat and notoriously resistant to antibiotics, this work stresses out that compounds specifically inducing mitochondrial reactive oxygen species could present themself as valuable adjunct treatments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Røst, Louet, Bruheim, Flo and Gidon.)

Bradykinin, a member of the kallikrein-kinin system (KKS), is associated with an inflammatory response pathway with diverse vascular permeability functions, including thrombosis and blood coagulation. In majority, bradykinin signals through Bradykinin Receptor B2 (B2R). B2R is a G protein-coupled receptor (GPCR) coupled to G protein family such as Gα qs , Gα q /Gα 11, Gα i1, and Gβ1 γ 2 . B2R stimulation leads to the activation of a signaling cascade of downstream molecules such as phospholipases, protein kinase C, Ras/Raf-1/MAPK, and PI3K/AKT and secondary messengers such as inositol-1,4,5-trisphosphate, diacylglycerol and Ca 2+ ions. These secondary messengers modulate the production of nitric oxide or prostaglandins. Bradykinin-mediated signaling is implicated in inflammation, chronic pain, vasculopathy, neuropathy, obesity, diabetes, and cancer. Despite the biomedical importance of bradykinin, a resource of bradykinin-mediated signaling pathway is currently not available. Here, we developed a pathway resource of signaling events mediated by bradykinin. By employing data mining strategies in the published literature, we describe an integrated pathway reaction map of bradykinin consisting of 233 reactions. Bradykinin signaling pathway events included 25 enzyme catalysis reactions, 12 translocations, 83 activation/inhibition reactions, 11 molecular associations, 45 protein expression and 57 gene regulation events. The pathway map is made publicly available on the WikiPathways Database with the ID URL: https://www.wikipathways.org/index.php/Pathway:WP5132 . The bradykinin-mediated signaling pathway map will facilitate the identification of novel candidates as therapeutic targets for diseases associated with dysregulated bradykinin signaling., (© 2021. The International CCN Society.)

Macrophages sense and respond to pathogens by induction of antimicrobial and inflammatory programs to alert other immune cells and eliminate the infectious threat. We have previously identified the transcription factor IRF1 to be consistently activated in macrophages during Mycobacterium avium infection, but its precise role during infection is not clear. Here, we show that tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) autocrine/paracrine signaling contributes to controlling the intracellular growth of M. avium in human primary macrophages through activation of IRF1 nuclear translocation and expression of IRG1, a mitochondrial enzyme that produces the antimicrobial metabolite itaconate. Small interfering RNA (siRNA)-mediated knockdown of IRF1 or IRG1 increased the mycobacterial load, whereas exogenously provided itaconate was bacteriostatic at high concentrations. While the overall level of endogenous itaconate was low in M. avium-infected macrophages, the repositioning of mitochondria to M. avium phagosomes suggests a mechanism by which itaconate can be delivered directly to M. avium phagosomes in sufficient quantities to inhibit growth. Using mRNA hybridization, we further show that uninfected bystander cells actively contribute to the resolution of infection by producing IL-6 and TNF-α, which, via paracrine signaling, activate IRF1/IRG1 and strengthen the antimicrobial activity of infected macrophages. This mechanism contributes to the understanding of why patients on anti-inflammatory treatment, e.g., with tocilizumab or infliximab, can be more susceptible to mycobacterial disease. IMPORTANCE The prevalence of lung diseases caused by nontuberculous mycobacteria, such as Mycobacterium avium, is increasing in countries where tuberculosis is not endemic, most likely because of an aging population that is immunocompromised from underlying disease or immunosuppressive therapy. Our study contributes to the understanding of mycobacterial survival and killing in human macrophages and, more broadly, to the impact of immunometabolism during infection. We show evidence of an antimicrobial program in human primary macrophages where activation of the transcription factor IRF1 and expression of the mitochondrial enzyme IRG1 restrict the intracellular growth of M. avium, possibly by directed delivery of itaconate to M. avium phagosomes. The study also sheds light on why patients on immunosuppressive therapy are more susceptible to mycobacterial infections, since TNF-α and IL-6 contribute to driving the described antimycobacterial program.

Background: Preclinical models of Alzheimer's disease (AD) can provide valuable insights into the onset and progression of the disease, such as changes in concentrations of amyloid-β (Aβ) and tau in cerebrospinal fluid (CSF). However, such models are currently underutilized due to limited advancement in techniques that allow for longitudinal CSF monitoring., Objective: An elegant way to understand the biochemical environment in the diseased brain is intracerebral microdialysis, a method that has until now been limited to short-term observations, or snapshots, of the brain microenvironment. Here we draw upon patient-based findings to characterize CSF biomarkers in a commonly used preclinical mouse model for AD., Methods: Our modified push-pull microdialysis method was first validated ex vivo with human CSF samples, and then in vivo in an AD mouse model, permitting assessment of dynamic changes of CSF Aβ and tau and allowing for better translational understanding of CSF biomarkers., Results: We demonstrate that CSF biomarker changes in preclinical models capture what is observed in the brain; with a decrease in CSF Aβ observed when plaques are deposited, and an increase in CSF tau once tau pathology is present in the brain parenchyma. We found that a high molecular weight cut-off membrane allowed for simultaneous sampling of Aβ and tau, comparable to CSF collection by lumbar puncture in patients., Conclusion: Our approach can further advance AD and other neurodegenerative research by following evolving neuropathology along the disease cascade via consecutive sampling from the same animal and can additionally be used to administer pharmaceutical compounds and assess their efficacy.

Mycobacterium avium (Mav) causes chronic infections in immunocompromised patients that require long-term antibiotic treatment. We have previously shown that Mav takes residence in host Mϕs and establishes a compartment (MavC) in which it is hidden from host defenses. Failure to establish the MavC traps Mav in Lamp1 + phagolysosomes where growth is prevented, and inflammatory signaling activated through TLRs 7/8. To elucidate how antibiotic treatment affects mycobacterial trafficking and host defenses, we infected human primary Mϕs with Mav for 4 days prior to treatment with a macrolide, aminoglycoside, and ethambutol. We show that Mav is killed and the MavC fuses with Lamp1 + lysosomes following antibiotic treatment. However, this does not result in nuclear translocation of NF-κB or production of inflammatory cytokines, suggesting different Lamp1 + lysosomal compartments can form that differ in their innate signaling capabilities. Thus, we show that upon antibiotic treatment of a chronic infection, Mav is quietly disposed of by Mϕs., (© 2020 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals, Inc. on behalf of Society for Leukocyte Biology.)

CD4+ T cells (T helper cells) are cytokine-producing adaptive immune cells that activate or regulate the responses of various immune cells. The activation and functional status of CD4+ T cells is important for adequate responses to pathogen infections but has also been associated with auto-immune disorders and survival in several cancers. In the current study, we carried out a label-free high-resolution FTMS-based proteomic profiling of resting and T cell receptor-activated (72 h) primary human CD4+ T cells from peripheral blood of healthy donors as well as SUP-T1 cells. We identified 5237 proteins, of which significant alterations in the levels of 1119 proteins were observed between resting and activated CD4+ T cells. In addition to identifying several known T-cell activation-related processes altered expression of several stimulatory/inhibitory immune checkpoint markers between resting and activated CD4+ T cells were observed. Network analysis further revealed several known and novel regulatory hubs of CD4+ T cell activation, including IFNG, IRF1, FOXP3, AURKA, and RIOK2. Comparison of primary CD4+ T cell proteomic profiles with human lymphoblastic cell lines revealed a substantial overlap, while comparison with mouse CD+ T cell data suggested interspecies proteomic differences. The current dataset will serve as a valuable resource to the scientific community to compare and analyze the CD4+ proteome.

Mycobacterium avium (Mav) complex is increasingly reported to cause non-tuberculous infections in individuals with a compromised immune system. Treatment is complicated and no vaccines are available. Previous studies have shown some potential of using genetically modified Mycobacterium smegmatis (Msm) as a vaccine vector to tuberculosis since it is non-pathogenic and thus would be tolerated by immunocompromised individuals. In this study, we used a mutant strain of Msm disrupted in EspG 3 , a component of the ESX-3 secretion system. Infection of macrophages and dendritic cells with Msm Δ espG 3 showed increased antigen presentation compared to cells infected with wild-type Msm. Vaccination of mice with Msm Δ espG 3 , expressing the Mav antigen MPT64, provided equal protection against Mav infection as the tuberculosis vaccine, Mycobacterium bovis BCG. However, upon challenge with Mav, we observed a high frequency of IL-17-producing CD4+ (Th17 cells) and CD8+ (Tc17 cells) T cells in mice vaccinated with Msm Δ espG 3 :: mpt64 that was not seen in BCG-vaccinated mice. Adoptive transfer of cells from Msm Δ espG 3 -vaccinated mice showed that cells from the T cell compartment contributed to protection from Mav infection. Further experiments revealed Tc17-enriched T cells did not provide prophylactic protection against subsequent Mav infection, but a therapeutic effect was observed when Tc17-enriched cells were transferred to mice already infected with Mav. These initial findings are important, as they suggest a previously unknown role of Tc17 cells in mycobacterial infections. Taken together, Msm Δ espG 3 shows promise as a vaccine vector against Mav and possibly other (myco)bacterial infections., (Copyright © 2020 Kannan, Haug, Steigedal and Flo.)

Mycobacterium tuberculosis is a global health problem in part as a result of extensive cytotoxicity caused by the infection. Here, we show how M. tuberculosis causes caspase-1/NLRP3/gasdermin D-mediated pyroptosis of human monocytes and macrophages. A type VII secretion system (ESX-1) mediated, contact-induced plasma membrane damage response occurs during phagocytosis of bacteria. Alternatively, this can occur from the cytosolic side of the plasma membrane after phagosomal rupture in infected macrophages. This damage causes K + efflux and activation of NLRP3-dependent IL-1β release and pyroptosis, facilitating the spread of bacteria to neighbouring cells. A dynamic interplay of pyroptosis with ESCRT-mediated plasma membrane repair also occurs. This dual plasma membrane damage seems to be a common mechanism for NLRP3 activators that function through lysosomal damage.

During HIV infection, cell-to-cell transmission results in endosomal uptake of the virus by target CD4+ T cells and potential exposure of the viral ssRNA genome to endosomal Toll-like receptors (TLRs). TLRs are instrumental in activating inflammatory responses in innate immune cells, but their function in adaptive immune cells is less well understood. Here we show that synthetic ligands of TLR8 boosted T cell receptor signaling, resulting in increased cytokine production and upregulation of surface activation markers. Adjuvant TLR8 stimulation, but not TLR7 or TLR9, further promoted T helper cell differentiation towards Th1 and Th17. In addition, we found that endosomal HIV induced cytokine secretion from CD4+ T cells in a TLR8-specific manner. TLR8 engagement also enhanced HIV-1 replication and potentiated the reversal of latency in patient-derived T cells. The adjuvant TLR8 activity in T cells can contribute to viral dissemination in the lymph node and low-grade inflammation in HIV patients. In addition, it can potentially be exploited for therapeutic targeting and vaccine development.

Nontuberculous mycobacterial infections caused by the opportunistic pathogen Mycobacterium avium subsp. hominissuis (MAH) are currently receiving renewed attention due to increased incidence combined with difficult treatment. Insights into the disease-causing mechanisms of this species have been hampered by difficulties in genetic manipulation of the bacteria. Here, we identified and sequenced a highly transformable, virulent MAH clinical isolate susceptible to high-density transposon mutagenesis, facilitating global gene disruption and subsequent investigation of MAH gene function. By transposon insertion sequencing (TnSeq) of this strain, we defined the MAH genome-wide genetic requirement for virulence and in vitro growth and organized ∼3,500 identified transposon mutants for hypothesis-driven research. The majority (96%) of the genes we identified as essential for MAH in vitro had a mutual ortholog in the related and highly virulent Mycobacterium tuberculosis ( Mtb ). However, passaging our library through a mouse model of infection revealed a substantial number (54% of total hits) of novel virulence genes. More than 97% of the MAH virulence genes had a mutual ortholog in Mtb Finally, we validated novel genes required for successful MAH infection: one encoding a probable major facilitator superfamily (MFS) transporter and another encoding a hypothetical protein located in the immediate vicinity of six other identified virulence genes. In summary, we provide new, fundamental insights into the underlying genetic requirement of MAH for growth and host infection. IMPORTANCE Pulmonary disease caused by nontuberculous mycobacteria is increasing worldwide. The majority of these infections are caused by the Mycobacterium avium complex (MAC), whereof >90% are due to Mycobacterium avium subsp. hominissuis (MAH). Treatment of MAH infections is currently difficult, with a combination of antibiotics given for at least 12 months. To control MAH by improved therapy, prevention, and diagnostics, we need to understand the underlying mechanisms of infection. Here, we provide crucial insights into MAH's global genetic requirements for growth and infection. We find that the vast majority of genes required for MAH growth and virulence (96% and 97%, respectively) have mutual orthologs in the tuberculosis-causing pathogen M. tuberculosis ( Mtb ). However, we also find growth and virulence genes specific to MAC species. Finally, we validate novel mycobacterial virulence factors that might serve as future drug targets for MAH-specific treatment or translate to broader treatment of related mycobacterial diseases., (Copyright © 2019 Dragset et al.)

Iron is vital for nearly all living organisms, but during infection, not readily available to pathogens. Infectious bacteria therefore depend on specialized mechanisms to survive when iron is limited. These mechanisms make attractive targets for new drugs. Here, by genome-wide phenotypic profiling, we identify and categorize mycobacterial genes required for low iron fitness. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), can scavenge host-sequestered iron by high-affinity iron chelators called siderophores. We take advantage of siderophore redundancy within the non-pathogenic mycobacterial model organism M. smegmatis (Msmeg), to identify genes required for siderophore dependent and independent fitness when iron is low. In addition to genes with a potential function in recognition, transport or utilization of mycobacterial siderophores, we identify novel putative low iron survival strategies that are separate from siderophore systems. We also identify the Msmeg in vitro essential gene set, and find that 96% of all growth-required Msmeg genes have a mutual ortholog in Mtb. Of these again, nearly 90% are defined as required for growth in Mtb as well. Finally, we show that a novel, putative ferric iron ABC transporter contributes to low iron fitness in Msmeg, in a siderophore independent manner.

Neutrophil gelatinase-associated lipocalin (NGAL), also known as Lipocalin 2, is an antimicrobial protein, encoded by the gene LCN2, strongly upregulated in inflammatory bowel disease (IBD) and a promising biomarker for IBD. Here we demonstrate that NGAL is highly expressed in all parts of pyloric metaplasia, also known as the ulcer-associated cell lineage (UACL), a metaplastic cell lineage suggested to play a role in wound healing in Crohn's disease (CD). We further show NGAL expression in regenerative intestinal crypts and in undifferentiated patient-derived colonoids. This indicates that NGAL is important in the tissue regeneration process. The remarkable overexpression of NGAL in UACL led us to explore the pathobiology of these cells by transcriptome-wide RNA sequencing. This study is, to our knowledge, the first to characterize the UACL at this level. Biopsies with UACL and inflamed non-UACL epithelium from the terminal ileum of CD patients and epithelium from healthy controls were laser capture microdissected for RNA sequencing. Among the 180 genes differentially expressed between UACL and control epithelium, the ten most-upregulated genes specific for UACL were MUC5AC, PGC, MUC6, MUC5B, LCN2, POU2AF1, MUC1, SDC3, IGFBP5, and SLC7A5. PDX1 was among the most upregulated in both UACL and inflamed non-UACL epithelium. Immunohistochemistry and iDisco 3D visualization was used to characterize UACL histo-morphologically, and to validate protein expression of 11 selected differentially expressed genes. Among these genes, LCN2, NOTCH2, PHLDA1, IGFBP5, SDC3, BPIFB1, and RCN1 have previously not been linked to UACL. Gene expression results were analyzed for functional implications using MetaCore, showing that differentially expressed genes are enriched for genes involved in cell migration and motility, and for biomarkers of gastrointestinal neoplasia. These results support a role for UACL as part of the reepithelialization process during and after destructive intestinal inflammation. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

Members of the Mycobacterium avium complex (MAC) are characterized as nontuberculosis mycobacteria and are pathogenic mainly in immunocompromised individuals. MAC strains show a wide genetic variability, and there is growing evidence suggesting that genetic differences may contribute to a varied immune response that may impact the infection outcome. The current study aimed to characterize the genomic changes within M. avium isolates collected from single patients over time and test the host immune responses to these clinical isolates. Pulsed-field gel electrophoresis and whole-genome sequencing were performed on 40 MAC isolates isolated from 15 patients at the Department of Medical Microbiology at St. Olavs Hospital in Trondheim, Norway. Isolates from patients (patients 4, 9, and 13) for whom more than two isolates were available were selected for further analysis. These isolates exhibited extensive sequence variation in the form of single-nucleotide polymorphisms (SNPs), suggesting that M. avium accumulates mutations at higher rates during persistent infections than other mycobacteria. Infection of murine macrophages and mice with sequential isolates from patients showed a tendency toward increased persistence and the downregulation of inflammatory cytokines by host-adapted M. avium strains. The study revealed the rapid genetic evolution of M. avium in chronically infected patients, accompanied by changes in the virulence properties of the sequential mycobacterial isolates., (Copyright © 2019 Kannan et al.)

Early detection of microbial patterns is a hallmark of innate immunity and essential for clearance of invading pathogens. A recent Nature publication by Zhou et al. (2018) has uncovered ALPK1 as a pattern recognition receptor for Gram-negative bacteria triggering NF-κB activation and identified the bacterial sugar ADP-Hep as its ligand., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Effective priming and activation of tumor-specific CD8+ cytotoxic T lymphocytes (CTLs) is crucial for realizing the potential of therapeutic cancer vaccination. This requires cytosolic antigens that feed into the MHC class I presentation pathway, which is not efficiently achieved with most current vaccination technologies. Photochemical internalization (PCI) provides an emerging technology to route endocytosed material to the cytosol of cells, based on light-induced disruption of endosomal membranes using a photosensitizing compound. Here, we investigated the potential of PCI as a novel, minimally invasive, and well-tolerated vaccination technology to induce priming of cancer-specific CTL responses to peptide antigens. We show that PCI effectively promotes delivery of peptide antigens to the cytosol of antigen-presenting cells (APCs) in vitro . This resulted in a 30-fold increase in MHC class I/peptide complex formation and surface presentation, and a subsequent 30- to 100-fold more efficient activation of antigen-specific CTLs compared to using the peptide alone. The effect was found to be highly dependent on the dose of the PCI treatment, where optimal doses promoted maturation of immature dendritic cells, thus also providing an adjuvant effect. The effect of PCI was confirmed in vivo by the successful induction of antigen-specific CTL responses to cancer antigens in C57BL/6 mice following intradermal peptide vaccination using PCI technology. We thus show new and strong evidence that PCI technology holds great potential as a novel strategy for improving the outcome of peptide vaccines aimed at triggering cancer-specific CD8+ CTL responses.

One of the author affiliations was missed to include in the original publication. The correct information is given below.

[This corrects the article DOI: 10.1371/journal.ppat.1006551.].

Inflammation is crucial in the defense against infections but must be tightly controlled to limit detrimental hyperactivation. Our diet influences inflammatory processes and omega-3 polyunsaturated fatty acids (n-3 PUFAs) have known anti-inflammatory effects. The balance of pro- and anti-inflammatory processes is coordinated by macrophages and macroautophagy/autophagy has recently emerged as a cellular process that dampens inflammation. Here we report that the n-3 PUFA docosahexaenoic acid (DHA) transiently induces cytosolic speckles of the autophagic receptor SQSTM1/p62 (sequestosome 1) (described as SQSTM1/p62-bodies) in macrophages. We suggest that the formation of SQSTM1/p62-bodies represents a fast mechanism of NFE2L2/Nrf2 (nuclear factor, erythroid 2 like 2) activation by recruitment of KEAP1 (kelch like ECH associated protein 1). Further, the autophagy receptor TAX1BP1 (Tax1 binding protein 1) and ubiquitin-editing enzyme TNFAIP3/A20 (TNF α induced protein 3) could be identified in DHA-induced SQSTM1/p62-bodies. Simultaneously, DHA strongly dampened the induction of pro-inflammatory genes including CXCL10 (C-X-C motif chemokine ligand 10) and we suggest that formation of SQSTM1/p62-bodies and activation of NFE2L2 leads to tolerance towards selective inflammatory stimuli. Finally, reduced CXCL10 levels were related to the improved clinical outcome in n-3 PUFA-supplemented heart-transplant patients and we propose CXCL10 as a robust marker for the clinical benefits mobilized by n-3 PUFA supplementation.

Pathogenic mycobacteria reside in macrophages where they avoid lysosomal targeting and degradation through poorly understood mechanisms proposed to involve arrest of phagosomal maturation at an early endosomal stage. A clear understanding of how this relates to host defenses elicited from various intracellular compartments is also missing and can only be studied using techniques allowing single cell and subcellular analyses. Using confocal imaging of human primary macrophages infected with Mycobacterium avium (Mav) we show evidence that Mav phagosomes are not arrested at an early endosomal stage, but mature to a (LAMP1+/LAMP2+/CD63+) late endosomal/phagolysosomal stage where inflammatory signaling and Mav growth restriction is initiated through a mechanism involving Toll-like receptors (TLR) 7 and 8, the adaptor MyD88 and transcription factors NF-κB and IRF-1. Furthermore, a fraction of the mycobacteria re-establish in a less hostile compartment (LAMP1-/LAMP2-/CD63-) where they not only evade destruction, but also recognition by TLRs, growth restriction and inflammatory host responses that could be detrimental for intracellular survival and establishment of chronic infections.

Macrophages play an essential role in the immune system by ingesting and degrading invading pathogens, initiating an inflammatory response and instructing adaptive immune cells, and resolving inflammation to restore homeostasis. More interesting is the fact that some bacteria have evolved to use macrophages as a natural habitat and tools of spread in the host, e.g., Mycobacterium tuberculosis (Mtb) and some non-tuberculous mycobacteria (NTM). Mtb is considered one of humanity's most successful pathogens and is the causal agent of tuberculosis, while NTMs cause opportunistic infections all of which are of significant public health concern. Here, we describe mechanisms by which intracellular pathogens, with an emphasis on mycobacteria, manipulate macrophage functions to circumvent killing and live inside these cells even under considerable immunological pressure. Such macrophage functions include the selective evasion or engagement of pattern recognition receptors, production of cytokines, reactive oxygen and nitrogen species, phagosome maturation, as well as other killing mechanisms like autophagy and cell death. A clear understanding of host responses elicited by a specific pathogen and strategies employed by the microbe to evade or exploit these is of significant importance for the development of effective vaccines and targeted immunotherapy against persistent intracellular infections like tuberculosis.

Background and Aim: Accurate, noninvasive biomarkers are needed to diagnose and monitor inflammatory bowel disease (IBD). Neutrophil gelatinase-associated lipocalin (NGAL), also known as lipocalin 2, is expressed in inflamed colonic epithelium and neutrophilic granulocytes. This study explores its properties as a biomarker in feces and plasma and, for the first time, compares fecal NGAL systematically with the existing fecal biomarker calprotectin., Methods: Neutrophil gelatinase-associated lipocalin was measured in feces from 73 patients with IBD, 21 patients with infectious enterocolitis, 21 patients with irritable bowel syndrome, and 23 healthy subjects using ELISA. The results were correlated to calprotectin, clinical score, endoscopic score, and high-sensitive C-reactive protein. Plasma from 119 patients with IBD and 28 healthy controls was analyzed for NGAL., Results: Fecal NGAL levels (median and interquartile range) were significantly elevated in active ulcerative colitis (UC) 6.05 (3.6-15.1) mg/kg and Crohn's disease (CD) 4.9 (1.5-7.7) mg/kg, compared with patients with inactive UC 1.3 (0.4-2.6) mg/kg, inactive CD 1.5 (0.5-1.7) mg/kg, irritable bowel syndrome 0.4 (0.2-0.6) mg/kg, and healthy controls (HC) 0.3 (0.1-0.4) mg/kg. Patients with infectious enterocolitis had significantly higher fecal-NGAL levels, 2.7 (1.4-5.6) mg/kg than HC. Sensitivity and specificity was 94.7% and 95.7%, respectively, for distinguishing between active IBD and HC. Stability of NGAL in stool was excellent for 7 days in room temperature. Plasma NGAL was significantly elevated in UC and CD compared with HC., Conclusions: Fecal NGAL is a promising biomarker for IBD. As existing biomarkers are expressed mainly in granulocytes, NGAL's epithelial localization may give supplementary diagnostic information., (© 2016 The Authors Journal of Gastroenterology and Hepatology published by Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Background: Carnitine plays an essential role in fatty acid metabolism, exerts substantial antioxidant action and regulates immune functions. We hypothesized that a disturbed carnitine metabolism could be involved in progression of HIV infection., Materials and Methods: Plasma levels of L-carnitine, its precursors, and short-, medium- and long-chain acylcarnitines were analysed with HPLC/mass spectrometry in HIV-infected patients with various disease severities including patients who acquired Mycobacterium avium complex (MAC) infection. In vitro, we examined the MAC-purified protein derivate (PPD)-induced release of TNF-α and IFN-γ in peripheral blood mononuclear cells (PBMCs) from patients with either high or low plasma levels of acylcarnitines., Results: Plasma levels of the short-chain (e.g. propionyl-carnitine) and medium-chain (e.g. octanoyl-carnitine) acylcarnitines were reduced in patients with advanced HIV infection. These acylcarnitines gradually decreased in rapid progressors, while minimal changes were observed in the nonprogressors. Plasma levels of propionyl-carnitine and octanoyl-carnitine significantly increased during antiretroviral therapy (ART). However, ART did not restore levels to those observed in healthy controls. Depletion of propionyl-carnitine and octanoyl-carnitine was observed prior to MAC infection, and the release of TNF-α and IFN-γ from PBMC was decreased after stimulation with MAC-PPD in samples from HIV-infected patients with low levels of propionyl-carnitine or octanoyl-carnitine., Conclusions: Our findings suggest an association between disturbed acylcarnitine metabolism, immune dysregulation and disease progression in HIV-infected patients. Low levels of propionyl-carnitine and octanoyl-carnitine were associated with increased susceptibility to MAC infection in HIV patients with advanced disease., (© 2016 Stichting European Society for Clinical Investigation Journal Foundation.)

Mycobacteria pose a threat to the world health today, with pathogenic and opportunistic bacteria causing tuberculosis and non-tuberculous disease in large parts of the population. Much is still unknown about the interplay between bacteria and host during infection and disease, and more research is needed to meet the challenge of drug resistance and inefficient vaccines. This work establishes a reliable and reproducible method for performing correlative imaging of human macrophages infected with mycobacteria at an ultra-high resolution and in 3D. Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) tomography is applied, together with confocal fluorescence microscopy for localization of appropriately infected cells. The method is based on an Aclar poly(chloro-tri-fluoro)ethylene substrate, micropatterned into an advantageous geometry by a simple thermomoulding process. The platform increases the throughput and quality of FIB/SEM tomography analyses, and was successfully applied to detail the intracellular environment of a whole mycobacterium-infected macrophage in 3D.

Conditional expression is a powerful tool to investigate the role of bacterial genes. Here, we adapt the Pseudomonas putida-derived positively regulated XylS/Pm expression system to control inducible gene expression in Mycobacterium smegmatis and Mycobacterium tuberculosis, the causative agent of human tuberculosis. By making simple changes to a Gram-negative broad-host-range XylS/Pm-regulated gene expression vector, we prove that it is possible to adapt this well-studied expression system to non-Gram-negative species. With the benzoic acid-derived inducer m-toluate, we achieve a robust, time- and dose-dependent reversible induction of Pm-mediated expression in mycobacteria, with low background expression levels. XylS/Pm is thus an important addition to existing mycobacterial expression tools, especially when low basal expression is of particular importance.

Several mechanisms are involved in controlling intracellular survival of pathogenic mycobacteria in host macrophages, but how these mechanisms are regulated remains poorly understood. We report a role for Kelch-like ECH-associated protein 1 (Keap1), an oxidative stress sensor, in regulating inflammation induced by infection with Mycobacterium avium in human primary macrophages. By using confocal microscopy, we found that Keap1 associated with mycobacterial phagosomes in a time-dependent manner, whereas siRNA-mediated knockdown of Keap1 increased M. avium-induced expression of inflammatory cytokines and type I interferons (IFNs). We show evidence of a mechanism whereby Keap1, as part of an E3 ubiquitin ligase complex with Cul3 and Rbx1, facilitates ubiquitination and degradation of IκB kinase (IKK)-β thus terminating IKK activity. Keap1 knockdown led to increased nuclear translocation of transcription factors NF-κB, IFN regulatory factor (IRF) 1, and IRF5 driving the expression of inflammatory cytokines and IFN-β. Furthermore, knockdown of other members of the Cul3 ubiquitin ligase complex also led to increased cytokine expression, further implicating this ligase complex in the regulation of the IKK family. Finally, increased inflammatory responses in Keap1-silenced cells contributed to decreased intracellular growth of M. avium in primary human macrophages that was reconstituted with inhibitors of IKKβ or TANK-binding kinase 1 (TBK1). Taken together, we propose that Keap1 acts as a negative regulator for the control of inflammatory signaling in M. avium-infected human primary macrophages. Although this might be important to avoid sustained or overwhelming inflammation, our data suggest that a negative consequence could be facilitated growth of pathogens like M. avium inside macrophages.

Staphylococcus aureus may cause serious infections and is one of the most lethal and common causes of sepsis. TLR2 has been described as the main pattern recognition receptor that senses S. aureus and elicits production of proinflammatory cytokines via MyD88 -: NF-κB signaling. S. aureus can also induce the production of IFN-β, a cytokine that requires IFN regulatory factors (IRFs) for its transcription, but the signaling mechanism for IFN-β induction by S. aureus are unclear. Surprisingly, we demonstrate that activation of TLR2 by lipoproteins does not contribute to IFN-β production but instead can suppress the induction of IFN-β in human primary monocytes and monocyte-derived macrophages. The production of IFN-β was induced by TLR8-mediated sensing of S. aureus RNA, which triggered IRF5 nuclear accumulation, and this could be antagonized by concomitant TLR2 signaling. The TLR8-mediated activation of IRF5 was dependent on TAK1 and IκB kinase (IKK)β, which thus reveals a physiological role of the recently described IRF5-activating function of IKKβ. TLR8 -: IRF5 signaling was necessary for induction of IFN-β and IL-12 by S. aureus, and it also contributed to the induction of TNF. In conclusion, our study demonstrates a physiological role of TLR8 in the sensing of entire S. aureus in human primary phagocytes, including the induction of IFN-β and IL-12 production via a TAK1 -: IKKβ -: IRF5 pathway that can be inhibited by TLR2 signaling., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Competition for iron is a critical component of successful bacterial infections, but the underlying in vivo mechanisms are poorly understood. We have previously demonstrated that lipocalin 2 (LCN2) is an innate immunity protein that binds to bacterial siderophores and starves them for iron, thus representing a novel host defense mechanism to infection. In the present study we show that LCN2 is secreted by the urinary tract mucosa and protects against urinary tract infection (UTI). We found that LCN2 was expressed in the bladder, ureters, and kidneys of mice subject to UTI. LCN2 was protective with higher bacterial numbers retrieved from bladders of Lcn2-deficient mice than from wild-type mice infected with the LCN2-sensitive Escherichia coli strain H9049. Uropathogenic E. coli mutants in siderophore receptors for salmochelin, aerobactin, or yersiniabactin displayed reduced fitness in wild-type mice, but not in mice deficient of LCN2, demonstrating that LCN2 imparts a selective pressure on bacterial growth in the bladder. In a human cohort of women with recurrent E. coli UTIs, urine LCN2 levels were associated with UTI episodes and with levels of bacteriuria. The number of siderophore systems was associated with increasing bacteriuria during cystitis. Our data demonstrate that LCN2 is secreted by the urinary tract mucosa in response to uropathogenic E. coli challenge and acts in innate immune defenses as a colonization barrier that pathogens must overcome to establish infection., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Opportunistic infections with non-tuberculous mycobacteria such as Mycobacterium avium are receiving renewed attention because of increased incidence and difficulties in treatment. As for other mycobacterial infections, a still poorly understood collaboration of different immune effector mechanisms is required to confer protective immunity. Here we have characterized the interplay of innate and adaptive immune effector mechanisms contributing to containment in a mouse infection model using virulent M. avium strain 104 in C57BL/6 mice. M. avium caused chronic infection in mice, as shown by sustained organ bacterial load. In the liver, bacteria were contained in granuloma-like structures that could be defined morphologically by expression of the antibacterial innate effector protein Lipocalin 2 in the adjoining hepatocytes and infiltrating neutrophils, possibly contributing to containment. Circulatory anti-mycobacterial antibodies steadily increased throughout infection and were primarily of the IgM isotype. Highest levels of interferon-γ were found in infected liver, spleen and serum of mice approximately 2 weeks post infection and coincided with a halt in organ bacterial growth. In contrast, expression of tumour necrosis factor was surprisingly low in spleen compared with liver. We did not detect interleukin-17 in infected organs or M. avium-specific T helper 17 cells, suggesting a minor role for T helper 17 cells in this model. A transient and relative decrease in regulatory T cell numbers was seen in spleens. This detailed characterization of M. avium infection in C57BL/6 mice may provide a basis for future studies aimed at gaining better insight into mechanisms leading to containment of infections with non-tuberculous mycobacteria., (© 2013 John Wiley & Sons Ltd.)

Anti-microbial peptides might influence the pathogenesis and course of inflammatory bowel disease (IBD). We sought to clarify the role of the anti-microbial glycoprotein lipocalin 2 (LCN2) in the colon by determining its localization and regulation in IBD. Following a microarray gene expression study of colonic biopsies from a large IBD population (n = 133), LCN2 was localized using immunohistochemistry and in-situ hybridization. Moreover, we examined the regulation of LCN2 in HT-29 cells with a panel of pattern recognition receptors (PRRs) and sought evidence by immunohistochemistry that the most relevant PRR, the Toll-like receptor (TLR)-3, was indeed expressed in colonic epithelium in IBD. LCN2 was among the 10 most up-regulated genes in both active ulcerative colitis (UCa) and active Crohn's disease (CDa) versus healthy controls. LCN2 protein was found in both epithelial cells and infiltrating neutrophils, while mRNA synthesis was located solely to epithelial cells, indicating that de-novo synthesis and thus regulation of LCN2 as measured in the gene expression analysis takes place in the mucosal epithelial cells. LCN2 is a putative biomarker in faeces for intestinal inflammation, different from calprotectin due to its epithelial site of synthesis. LCN2 release from the colonic epithelial cell line HT-29 was enhanced by both interleukin (IL)-1β and the TLR-3 ligand poly(I:C), and TLR-3 was shown to be expressed constitutively in colonic epithelial cells and markedly increased during inflammation., (© 2013 British Society for Immunology.)

Objective: To study the prognostic value of neutrophil gelatinase-associated lipocalin (NGAL) in chronic heart failure (HF) of ischaemic aetiology., Background: Neutrophil gelatinase-associated lipocalin is a marker of kidney injury as well as matrix degradation and inflammation and has previously been shown to be increased in HF. We investigated whether serum NGAL levels could provide prognostic information in chronic HF., Methods: We assessed NGAL as a predictor of primary outcomes (cardiovascular death, nonfatal stroke and nonfatal myocardial infarction, n = 307) and all-cause mortality (n = 321), cardiovascular mortality (n = 259) and hospitalization (n = 647) as well as the number of hospitalizations during follow-up for all (n = 1934) and CV causes (n = 1204) in 1415 patients with chronic HF (≥60 years, New York Heart Association class II-IV, ischaemic systolic HF) in the CORONA population, randomly assigned to 10 mg rosuvastatin or placebo. Results.  Multivariate analysis revealed that NGAL added significant information when adjusting for clinical variables, but was no longer significant when further adjusting for apolipoprotein A-1 (ApoA-1), glomerular filtration rate (GFR), C-reactive protein (CRP) and N-terminal pro-brain natriuretic peptide (NT-proBNP). However, belonging to the highest NGAL tertile was associated with more frequent hospitalization, even after adjusting for clinical variables, GFR and ApoA-1, but not after adjusting for CRP and NT-proBNP. There was no interaction between rosuvastatin treatment and NGAL. Conclusion.  Neutrophil gelatinase-associated lipocalin added no significant information to NT-proBNP and GFR in a multivariate model for primary and secondary end-points., (© 2012 The Association for the Publication of the Journal of Internal Medicine.)

Based on the ability to recruit lymphocytes and dendritic cells to lymphoid tissue and to promote inflammation, we hypothesized a role for dysregulated CCL19 and CCL21 levels in human immunodeficiency virus (HIV)-infected patients with advanced immunodeficiency, and in particular in those with accompanying Mycobacterium avium complex (MAC) infection. The hypothesis was explored by studies in HIV-infected patients with and without MAC infection, as well as in vitro, examining the ability of proteins from MAC to promote CCL19 and CCL21 responses in peripheral blood mononuclear cells (PBMC) during highly active anti-retroviral therapy (HAART). Our main findings were: (i) raised serum levels of CCL19 in HIV-infected patients with CD4(+) T cell count <50 cells/µl compared with HIV-infected patients with CD4(+) T cell count >500 cells/µl and healthy controls, with particularly high levels in those with MAC infection; (ii) elevated plasma levels of CCL19 predicted a higher mortality in acquired immune deficiency syndrome (AIDS)-patients, independent of ongoing MAC infection; and (iii) marked production of CCL19 in MAC-stimulated peripheral blood mononuclear cells (PBMC) and pronounced disturbances in MAC-induced CCL19 production in PBMC from HIV patients that was partly reversed during HAART. Our findings suggest the involvement of CCL19 in AIDS patients with advanced immunodeficiency, potentially mediating both adaptive and maladaptive responses., (© 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.)

Due to the slow growth rate and pathogenicity of mycobacteria, enumeration by traditional reference methods like colony counting is notoriously time-consuming, inconvenient and biohazardous. Thus, novel methods that rapidly and reliably quantify mycobacteria are warranted in experimental models to facilitate basic research, development of vaccines and anti-mycobacterial drugs. In this study we have developed quantitative polymerase chain reaction (qPCR) assays for simultaneous quantification of mycobacterial and host DNA in infected human macrophage cultures and in mouse tissues. The qPCR method cannot discriminate live from dead bacteria and found a 10- to 100-fold excess of mycobacterial genomes, relative to colony formation. However, good linear correlations were observed between viable colony counts and qPCR results from infected macrophage cultures (Pearson correlation coefficient [r] for M. tuberculosis = 0.82; M. a. avium = 0.95; M. a. paratuberculosis = 0.91). Regression models that predict colony counts from qPCR data in infected macrophages were validated empirically and showed a high degree of agreement with observed counts. Similar correlation results were also obtained in liver and spleen homogenates of M. a. avium infected mice, although the correlations were distinct for the early phase (< day 9 post-infection) and later phase (≥ day 20 post-infection) liver r = 0.94 and r = 0.91; spleen r = 0.91 and r = 0.87, respectively. Interestingly, in the mouse model the number of live bacteria as determined by colony counts constituted a much higher proportion of the total genomic qPCR count in the early phase (geometric mean ratio of 0.37 and 0.34 in spleen and liver, respectively), as compared to later phase of infection (geometric mean ratio of 0.01 in both spleen and liver). Overall, qPCR methods offer advantages in biosafety, time-saving, assay range and reproducibility compared to colony counting. Additionally, the duplex format allows enumeration of bacteria per host cell, an advantage in experiments where variable cell death can give misleading colony counts.

Background: The molecular pathogenesis of chronic skin wounds is complex and not fully understood. Although these wounds are often characterized as being in a state of persistent inflammation, the impact and participation of the innate immune responses in sustaining this inflammation needs further investigation., Objective: We investigated the cytokine profiles, Toll-like receptor (TLR)-stimulating activities and the levels of the antibacterial peptide Lipocalin-2 (Lcn-2) in a series of healing and non-healing chronic venous leg ulcers (CVLUs) through a study time of 8 weeks., Methods: Wound fluids from healing and non-healing CVLUs were run on a Human Cytokine Antibody Array, and Lcn-2 levels measured with ELISA. HEK 293 cells transfected with TLR2 or TLR4 and their respective co-receptors, and human peripheral blood monocytes were then stimulated with the wound fluids from healing and non-healing venous leg ulcers., Results: Healing wounds were associated with decreasing levels of IL-1alpha, IL-1beta and MIP-1delta, whereas in non-healing wounds decreasing levels of IL-8 and MIP-1alpha were found. Accordingly, wound fluid from non-healing CVLUs contained persistent Lcn-2 levels and TLR2- and TLR4-stimulating activities, while, in healing wounds, the TLR-stimulating activities decreased over time with significantly diminished levels of Lcn-2 (p<0.005)., Conclusions: Innate immune responses contribute to the chronic inflammation in non-healing CVLUs through participation of Toll-like receptors. The levels of the antimicrobial peptide Lcn-2 in wound fluids from these ulcers are elevated as a reflection of this contribution., (Copyright 2010 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Aims: Neutrophil gelatinase-associated lipocalin (NGAL or lipocalin-2) is a glycoprotein with bacteriostatic properties. Growing evidence suggests that NGAL may also be involved in cell survival, inflammation, and matrix degradation. We therefore aimed to investigate the role of NGAL in heart failure (HF)., Methods and Results: Our main findings were (i) patients with acute post-myocardial infarction (MI) HF (n = 236) and chronic HF (n = 150) had elevated serum levels of NGAL (determined by enzyme immunoassay), significantly correlated with clinical and neurohormonal deterioration, (ii) in patients with HF following acute MI, elevated NGAL levels of at baseline were associated with adverse outcomes (median of 27 months follow-up), (iii) in a rat model of post-MI HF, NGAL/lipocalin-2 gene expression was increased in the non-ischaemic part of the left ventricle primarily located to cardiomyocytes, (iv) strong NGAL immunostaining was found in cardiomyocytes within the failing myocardium both in experimental and clinical HF, (v) interleukin-1beta and agonists for toll-like receptors 2 and 4, representing components of the innate immune system, were potent inducers of NGAL/lipocalin-2 in isolated neonatal cardiomyocytes., Conclusion: Our demonstration of enhanced systemic and myocardial NGAL expression in clinical and experimental HF further support a role for innate immune responses in the pathogenesis of HF.

Objectives: Mediterranean spotted fever (MSF) caused by Rickettsia conorii (R. conorii) is a potential lethal disease while African tick bite fever (ATBF) caused by Rickettsia africae is a self-limiting flu-like illness. We hypothesized that different inflammatory potential in endothelial cells could contribute to the different clinical features in these rickettsioses., Methods: We analyzed the effect of heat-inactivated R. africae and R. conorii on the mRNA and protein levels of monocyte chemoattractant protein (MCP)-1, interleukin (IL)-8 and adhesion molecules in endothelial cells. Serum samples from patients with MSF (n=16) and ATBF (n=15) were collected before and after therapy., Results: R. conorii induced a marked increase in MCP-1, IL-8, and adhesion molecules in endothelial cells, involving toll-like receptor 4 activation. In contrast, R. africae induced MCP-1 expression, but only modest or no responses were seen on IL-8 and adhesion molecules. Comparable to the in vitro response, levels of IL-8 and adhesion molecules showed no or only a modest increase in ATBF patients while these inflammatory markers were markedly elevated during MSF., Conclusions: Our findings suggest a superior inflammatory potential of R. conorii as compared to R. africae in endothelial cells, potentially related to the more severe inflammation in MSF comparing ATBF.

Although neutrophil gelatinase-associated lipocalin (NGAL) may play a pivotal role in the innate immune response, there are currently no data on NGAL levels in human immunodeficiency virus (HIV)-infected patients. In this study we aimed to examine the regulation of NGAL in HIV infection. The regulation of NGAL in HIV infection was examined by different experimental approaches, including studies in peripheral blood and mononuclear cells (MNC) from bone marrow aspirates before and during highly active anti-retroviral therapy (HAART). We found that: before initiating HAART, HIV-infected patients (n = 37) had significantly decreased serum NGAL levels compared with healthy controls (n = 26); (ii) during HAART, there was a gradual and significant increase in NGAL concentrations reaching levels comparable to those in healthy controls after 12 months; (iii) this increase was seen primarily in virological responders to HAART (HIV RNA level <200 copies/ml after 24 months); (iv) phytohaemagglutinin-stimulated NGAL release in MNC cells from bone marrow aspirates was decreased in untreated HIV-infected patients compared with healthy controls, but increased after 26 weeks on HAART; and (v) there was a significant positive correlation between neutrophil counts and NGAL levels at all time-points during HAART. We have shown decreased NGAL levels in HIV-infected patients, potentially reflecting decreased number and function of neutrophils as well as impaired bone marrow myelopoiesis. These abnormalities were reversed by successful HAART. Our findings underscore further the involvement of neutrophils and innate immunity in HIV-related immunodeficiency.

Although iron is required to sustain life, its free concentration and metabolism have to be tightly regulated. This is achieved through a variety of iron-binding proteins including transferrin and ferritin. During infection, bacteria acquire much of their iron from the host by synthesizing siderophores that scavenge iron and transport it into the pathogen. We recently demonstrated that enterochelin, a bacterial catecholate siderophore, binds to the host protein lipocalin 2 (ref. 5). Here, we show that this event is pivotal in the innate immune response to bacterial infection. Upon encountering invading bacteria the Toll-like receptors on immune cells stimulate the transcription, translation and secretion of lipocalin 2; secreted lipocalin 2 then limits bacterial growth by sequestrating the iron-laden siderophore. Our finding represents a new component of the innate immune system and the acute phase response to infection.

An ideal vaccine for induction of CD4(+) T cell responses should induce local inflammation, maturation of APC, and peptide loading of MHC class II molecules. Ligation of Toll-like receptor (TLR) 2 provides the first two of these three criteria. We have studied whether targeting of TLR2 results in loading of MHC class II molecules and enhancement of CD4(+) T cell responses. To dissociate MHC class II presentation from APC maturation, we have used an antagonistic, mouse anti-human TLR2 mAb (TL2.1) as ligand and measured proliferation of a mouse Ckappa-specific human CD4(+) T cell clone. TL2.1 mAb was 100-1000 times more efficiently presented by APC compared with isotype-matched control mAb. Moreover, TL2.1 mAb was internalized into endosomes and processed by the conventional MHC class II pathway. This novel function of TLR2 represents a link between innate and adaptive immunity and indicates that TLR2 could be a promising target for vaccines.

The alginate capsule produced by the human pathogen Pseudomonas aeruginosa is composed mainly of mannuronic acid polymers (poly-M) that have immunostimulating properties. Poly-M shares with lipopolysaccharide the ability to stimulate cytokine production from human monocytes in a CD14-dependent manner. In the present study we examined the role of Toll-like receptor (TLR) 2 and TLR4 in responses to poly-M. Blocking antibodies to TLR2 and TLR4 partly inhibited tumor necrosis factor production induced by poly-M in human monocytes, and further inhibition was obtained by combining the antibodies. By transiently transfecting HEK293 cells, we found that membrane CD14 together with either TLR2 or TLR4/MD-2 could mediate activation by poly-M. Transfection of HEK293 cells with TLR2 and fluorescently labeled TLR4 followed by co-patching of TLR2 with an antibody revealed no association of these molecules on the plasma membrane. However, macrophages from the Tlr4 mutant C3H/HeJ mice and TLR4 knockout mice were completely non-responsive to poly-M, whereas the tumor necrosis factor release from TLR2 knockout macrophages was half of that seen with wild type cells. Taken together the results suggest that both TLR2 and TLR4 are involved in cell activation by poly-M and that TLR4 may be required in primary murine macrophages.

Background: Coronary artery bypass grafting with the use of cardiopulmonary bypass is known to mediate an inflammatory response. The stress-inducible heat-shock protein (HSP) 70 has been detected in myocardial cells after CABG, and toll-like receptors (TLRs) are suggested as putative signaling receptors for the HSPs, mediating synthesis of inflammatory cytokines. The main aims of our study were to explore the release of HSP70 and the regulation of monocyte TLR-2 and TLR-4 expression after CABG., Methods and Results: Twenty patients referred for elective CABG were included in this study. Using immunoassays, we detected HSP70 in plasma after CABG, with peak concentration immediately after surgery. Interleukin-6 in plasma reached peak concentration 5 hours after surgery. Monocyte CD14, TLR-2, and TLR-4 expression, as analyzed by flow cytometry, was initially downregulated. On day 1, CD14 expression normalized, whereas TLR-2 and TLR-4 expression was upregulated. TLR-4 was significantly upregulated even on postoperative day 2. Additional experiments revealed that peritoneal macrophages from control (C3H/HeN) mice responded to HSP70 with release of tumor necrosis factor, whereas macrophages from mutated TLR-4 (C3H/HeJ) mice were unresponsive. In vitro, human adherent monocytes responded to recombinant HSP70 with interleukin-6 and tumor necrosis factor release. CD14 and TLR-4 monoclonal antibodies inhibited the cytokine response., Conclusions: In this study, we observed an immediate release of HSP70 into the circulation and a modulation of monocyte TLR-2 and TLR-4 expression after CABG. TLR-4 and CD14 appear to be involved in an HSP70-mediated activation of innate immunity.