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Propolis extracts have gained the attention of consumers and researchers due to their unique chemical compositions and functional properties such as its anti-inflammatory activity. Recently, it was described a complex that is also important in inflammatory processes, named inflammasome. The inflammasomes are a large molecular platform formed in the cell cytosol in response to stress signals, toxins, and microbial infections. Once activated, the inflammasome induces caspase-1, which in turn induces the processing of inflammatory cytokines such as IL-1βand IL-18. So, to understand inflammasomes regulation becomes crucial to treat several disorders including autoinflammatory diseases. Since green propolis extracts are able to regulate inflammatory pathways, this work purpose was to investigate if this extract could also act on inflammasomes regulation. First, the extract was characterized and it demonstrated the presence of important compounds, especially Artepillin C. This extract was effective in reducing the IL-1βsecretion in mouse macrophages and this reduction was correlated with a decrease in activation of the protease caspase-1. Furthermore, we found that the extract at a concentration of 30 μg/mL was not toxic to the cells even after a 18-hour treatment. Altogether, these data indicate that Brazilian green propolis (EPP-AF) extract has a role in regulating the inflammasomes.

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Pecuaria e Abastecimento Instituto Nacional de Ciencia e Tecnologia de Vacinas Pathogens are detected by innate immune receptors that, upon activation, orchestrate an appropriate immune response. Recent studies revealed the intracellular signaling cascades involved in the TLR-initiated immune response to Brucella abortus infection. However, no report has elucidated the role of inflammasome receptors in Brucella recognition. Therefore, we decided to investigate the function of NLRC4, NLRP3, and AIM2 in sensing Brucella. in this study, we showed that NLRC4 is not required to induce caspase-1 activation and further secretion of IL-1 beta by B. abortus in macrophages. in contrast, we determined that AIM2, which senses Brucella DNA, and NLRP3 are partially required for caspase-1 activation and IL-1 beta secretion. Additionally, mitochondrial reactive oxygen species induced by Brucella were implicated in IL-1 beta production. Furthermore, AIM2, NLRP3, ASC, and caspase-1 knockout mice were more susceptible to B. abortus infection than were wild-type animals, suggesting that multiple ASC-dependent inflammasomes contribute to host protection against infection. This protective effect is due to the inflammatory response caused by IL-1 beta and IL-18 rather than pyroptosis, because we observed augmented bacterial burden in IL-1R and IL-18 knockout mice. Finally, we determined that bacterial type IV secretion system VirB and live, but not heat-killed, Brucella are required for full inflammasome activation in macrophages during infection. Taken together, our results indicate that Brucella is sensed by ASC inflammasomes that collectively orchestrate a robust caspase-1 activation and proinflammatory response. the Journal of Immunology, 2013, 190: 3629-3638. Univ Fed Minas Gerais, Inst Biol Sci, Dept Biochem & Immunol, BR-31270901 Belo Horizonte, MG, Brazil Universidade Federal de São Paulo, Ctr Mol & Cellular Therapy, Dept Biol Sci, BR-04044010 São Paulo, Brazil Univ São Paulo, Sch Med Ribeirao Preto São Paulo, Dept Cell Biol, BR-14049900 Ribeirao Preto, Brazil Universidade Federal de São Paulo, Ctr Mol & Cellular Therapy, Dept Biol Sci, BR-04044010 São Paulo, Brazil Web of Science

Activation of the inflammasome occurs in response to a notably high number of pathogenic microbes and is a broad innate immune response that effectively contributes to restriction of pathogen replication and generation of adaptive immunity. Activation of these platforms leads to caspase-1- and/or caspase-11-dependent secretion of proteins, including cytokines, and induction of a specific form of cell death called pyroptosis, which directly or indirectly contribute for restriction of pathogen replication. Not surprisingly, bona fide intracellular pathogens developed strategies for manipulation of cell death to guarantee intracellular replication. In this sense, the remarkable advances in the knowledge of the inflammasome field have been accompanied by several reports characterizing the inhibition of this platform by several pathogenic bacteria. Herein, we review some processes used by pathogenic bacteria, including Yersinia spp., Pseudomonas aeruginosa, Vibrio parahaemolyticus, Chlamydia trachomatis, Francisella tularensis, Shigella flexneri, Legionella pneumophila, and Coxiella burnetii to evade the activation of the inflammasome and the induction of pyroptosis.

Legionella pneumophila is an intracellular bacterium that was evolutionarily selected to survive in freshwater environments by infecting free-living unicellular protozoa. Once humans inhale contaminated water droplets, the bacteria reach the pulmonary alveoli where they are phagocytized by resident alveolar macrophages. Depending on host immunity and bacterial virulence genes, the infection may progress to an acute pneumonia called Legionnaires' disease, which can be fatal. Of note, an effective immune response is critical to the outcome of the human infection. These clinical observations highlight the importance of animal models of pulmonary infection for in vivo investigation of bacterial pathogenesis and host responses. In this chapter we provide detailed protocols for intranasal infection of mouse with L. pneumophila.

Sensing intracellular pathogens is a process mediated by innate immune cells that is crucial for the induction of inflammatory processes and effective adaptive immune responses against pathogenic microbes. NOD-like receptors (NLRs) comprise a family of intracellular pattern recognition receptors that are important for the recognition of damage and microbial-associated molecular patterns. NOD1 and NOD2 are specialized NLRs that participate in the recognition of a subset of pathogenic microorganisms that are able to invade and multiply intracellularly. Once activated, these molecules trigger intracellular signaling pathways that lead to the activation of transcriptional responses culminating in the expression of a subset of inflammatory genes. In this review, we will focus on the role of NOD1 and NOD2 in the recognition and response to intracellular pathogens, including Gram-positive and Gram-negative bacteria, and on their ability to signal in response to non-peptidoglycan-containing pathogens, such as viruses and protozoan parasites.

Legionella pneumophila is an intracellular bacterium that was evolutionarily selected to survive in freshwater environments by infecting free-living unicellular protozoa. Once humans inhale contaminated water droplets, the bacteria reach the pulmonary alveoli where they are phagocytized by resident alveolar macrophages. Depending on host immunity and bacterial virulence genes, the infection may progress to an acute pneumonia called Legionnaires' disease, which can be fatal. Of note, an effective immune response is critical to the outcome of the human infection. These clinical observations highlight the importance of animal models of pulmonary infection for in vivo investigation of bacterial pathogenesis and host responses. In this chapter we provide detailed protocols for intranasal infection of mouse with L. pneumophila.

Caspase-1 (casp-1) regulates key steps in microbial and sterile inflammatory responses. Hypertension is associated with inflammation of the vascular wall, but it is not known whether casp-1 mediates this phenomenon. We hypothesized that casp-1 contributes to vascular dysfunction in angiotensin II (AngII)-induced hypertension. Male C57BL/6 wild type (WT) and casp-1 knockout (casp-1 -/- ) mice were infused with AngII (90 ng/min, 14 days, osmotic mini-pumps). Control mice were sham-operated. Systolic blood pressure (SBP, mmHg) was significantly increased in AngII-WT mice (162±7.9 vs. 138±2.9 WT-sham, p-/- mice (148±13 vs. 140±6.2 casp-1 -/- -sham, p>0.05). AngII-WT mice exhibited cardiac hypertrophy (mg/g: 10±0.36 vs. 4.9±0.19 WT-sham, p-/- mice (mg/g: 7.6±0.70 vs. 5.6±0.68 casp-1 -/- -sham, p>0.05). Severe endothelial dysfunction [acetylcholine (ACh)-dependent dilation, second-order mesenteric arteries] was observed in AngII-WT mice, but this was attenuated in AngII-casp-1 -/- mice (inserted Figure). Vascular superoxide anion production (dihydroethidium fluorescence, arbitrary units) augmented in AngII-WT (6.2±0.60 vs. 4.9±0.48 WT-sham, p-/- (4.9±0.48 vs. 4.0±0.64 casp-1 -/- -sham, p>0.05) mice. Preliminary data also show that AngII in vivo and in vitro induces casp-1 activation (western blotting for cleaved casp-1). These data provide evidence that activation of casp-1 plays a role in vascular dysfunction associated with AngII-hypertension.

Protozoan parasites cause several diseases, such as Malaria, Leishmaniasis, and Trypanosomiasis, hampering human development worldwide. Many protozoa cause infections that often follow chronic courses, owing to coevolution between parasites and host immune system. The survival and transmission of pathogenic protozoa depends on their ability to evade or subvert host’s innate and adaptive immune responses. A great challenge to research in immunology and parasitology is the development of strategies that favor immunity against protozoan parasites and prevent their evasion, chronic, or recurrent infections and associated pathologies. This special issue includes original papers and reviews that summarize current advances in our understanding on the mechanisms of immunity to protozoan parasites in humans and experimental animal models., Fil: Lopes, Marcela F. Universidade Federal do Rio de Janeiro; Brazil, Fil: Zamboni, Dario S. Faculdade de Medicina de Ribeirao Preto. Universidade de Sao Paulo; Brazil, Fil: Luján, Hugo Daniel. Universidad Católica de Córdoba. Facultad de Ciencias de la Salud; Argentina, Fil: Rodrigues, Mauricio M. , Escola Paulista de Medicina; Brazil

Objective Deposition of monosodium urate monohydrate (MSU) crystals in the joints promotes an intense inflammatory response and joint dysfunction. This study evaluated the role of the NLRP3 inflammasome and 5-lipoxygenase (5-LOX)–derived leukotriene B4 (LTB4) in driving tissue inflammation and hypernociception in a murine model of gout. Methods Gout was induced by injecting MSU crystals into the joints of mice. Wild-type mice and mice deficient in NLRP3, ASC, caspase 1, interleukin-1β (IL-1β), IL-1 receptor type I (IL-1RI), IL-18R, myeloid differentiation factor 88 (MyD88), or 5-LOX were used. Evaluations were performed to assess neutrophil influx, LTB4 activity, cytokine (IL-1β, CXCL1) production (by enzyme-linked immunosorbent assay), synovial microvasculature cell adhesion (by intravital microscopy), and hypernociception. Cleaved caspase 1 and production of reactive oxygen species (ROS) were analyzed in macrophages by Western blotting and fluorometric assay, respectively. Results Injection of MSU crystals into the knee joints of mice induced neutrophil influx and neutrophil-dependent hypernociception. MSU crystal–induced neutrophil influx was CXCR2-dependent and relied on the induction of CXCL1 in an NLRP3/ASC/caspase 1/IL-1β/MyD88–dependent manner. LTB4 was produced rapidly after injection of MSU crystals, and this was necessary for caspase 1–dependent IL-1β production and consequent release of CXCR2-acting chemokines in vivo. In vitro, macrophages produced LTB4 after MSU crystal injection, and LTB4 was relevant in the MSU crystal–induced maturation of IL-1β. Mechanistically, LTB4 drove MSU crystal–induced production of ROS and ROS-dependent activation of the NLRP3 inflammasome. Conclusion These results reveal the role of the NLRP3 inflammasome in mediating MSU crystal–induced inflammation and dysfunction of the joints, and highlight a previously unrecognized role of LTB4 in driving NLRP3 inflammasome activation in response to MSU crystals, both in vitro and in vivo.

Intracellular pattern recognition receptors such as the nucleotide-binding oligomerization domain (NOD)-like receptors family members are key for innate immune recognition of microbial infection and may play important roles in the development of inflammatory diseases, including rheumatic diseases. In this study, we evaluated the role of NOD1 and NOD2 on development of experimental arthritis. Ag-induced arthritis was generated in wild-type, NOD1 -/-, NOD2 -/-, or receptor-interacting serine-threonine kinase 2 -/- (RIPK2 -/-) immunized mice challenged intra-articularly with methylated BSA. Nociception was determined by electronic Von Frey test. Neutrophil recruitment and histopathological analysis of proteoglycan lost was evaluated in inflamed joints. Joint levels of inflammatory cytokine/chemokine were measured by ELISA. Cytokine (IL-6 and IL-23) and NOD2 expressions were determined in mice synovial tissue by RT-PCR. The NOD2 -/- and RIPK2 -/-, but not NOD1 -/-, mice are protected from Ag-induced arthritis, which was characterized by a reduction in neutrophil recruitment, nociception, and cartilage degradation. NOD2/RIPK2 signaling impairment was associated with a reduction in proinflammatory cytokines and chemokines (TNF, IL-1b, and CXCL1/KC). IL-17 and IL-17 triggering cytokines (IL-6 and IL-23) were also reduced in the joint, but there is no difference in the percentage of CD4 + IL-17 + cells in the lymph node between arthritic wild-type and NOD2 -/- mice. Altogether, these findings point to a pivotal role of the NOD2/RIPK2 signaling in the onset of experimental arthritis by triggering an IL-17-dependent joint immune response. Therefore, we could propose that NOD2 signaling is a target for the development of new therapies for the control of rheumatoid arthritis. Copyright © 2012 by The American Association of Immunologists, Inc.

Although NLRC4/IPAF activation by flagellin has been extensively investigated, the downstream signaling pathways and the mechanisms responsible for infection clearance remain unclear. In this study, we used mice deficient for the inflammasome components in addition to wild-type (WT) Legionella pneumophila or bacteria deficient for flagellin (flaA) or motility (fliI) to assess the pathways responsible for NLRC4-dependent growth restriction in vivo and ex vivo. By comparing infections with WT L. pneumophila, fliI, and flaA, we found that flagellin and motility are important for the colonization of the protozoan host Acanthamoeba castellanii. However, in macrophages and mammalian lungs, flagellin expression abrogated bacterial replication. The flagellin-mediated growth restriction was dependent on NLRC4, and although it was recently demonstrated that NLRC4 is able to recognize bacteria independent of flagellin, we found that the NLRC4-dependent restriction of L. pneumophila multiplication was fully dependent on flagellin. By examining infected caspase-1−/− mice and macrophages with flaA, fliI, and WT L. pneumophila, we could detect greater replication of flaA, which suggests that caspase-1 only partially accounted for flagellin-dependent growth restriction. Conversely, WT L. pneumophila multiplied better in macrophages and mice deficient for NLRC4 compared with that in macrophages and mice deficient for caspase-1, supporting the existence of a novel caspase-1–independent response downstream of NLRC4. This response operated early after macrophage infection and accounted for the restriction of bacterial replication within bacteria-containing vacuoles. Collectively, our data indicate that flagellin is required for NLRC4-dependent responses to L. pneumophila and that NLRC4 triggers caspase-1–dependent and –independent responses for bacterial growth restriction in macrophages and in vivo.

Nod2 belongs to the nucleotide-binding domain leucine-rich repeat family of proteins and senses bacterial cell wall components to initiate innate immune responses against various pathogens. Recently, it has been reported that T-cell-intrinsic expression of Nod2 promotes host defense against Toxoplasma gondii infection by inducing type 1 immunity. Here, we present results that demonstrate that Nod2 does not play a role in the defense against T. gondii infection. Nod2-deficient mice were fully capable of inducing Th1 immune responses and did not show enhanced susceptibility to infection. Upon TCR stimulation in vitro, Nod2-deficient CD4(+) T cells showed normal activation, IL-2 production, proliferation, and Th1/2 differentiation. Nod2 mRNA and protein were expressed in CD4(+) T and CD8(+) T cells at substantial levels. Therefore, Nod2, although expressed in CD4(+) T cells, does not have an intrinsic function in T-cell activation and differentiation.

The Nlrc4 inflammasome is triggered in response to contamination of the host cell cytoplasm with bacterial flagellin, which induces pyroptosis, a form of cell death that accounts for restriction of bacterial infections. Although induction of pyroptosis has been extensively investigated in response to Salmonella typhimurium and Legionella pneumophila, little is known regarding the role of the inflammasome for restriction of non-pneumophila Legionella species. Here, we used five species of the Legionella genus to investigate the importance of the inflammasome for restriction of bacterial infection in vivo. By infecting mice deficient for inflammasome components, we demonstrated that caspase-1 and Nlrc4, but not Asc, contribute to restriction of pulmonary infection with L. micdadei, L. bozemanii, L. gratiana, and L. rubrilucens. L. longbeachae, a non-flagellated bacterium that fails to trigger pyroptosis, was not restricted by the inflammasome and induced death in the infected mice. In contrast to L. longbeachae, flagellin mutants of L. pneumophila did not induce mice death; therefore, besides bypassing the Nlrc4 inflammasome, L. longbeachae may employ additional virulence strategies to replicate in mammalian hosts. Collectively, our data indicate that the Nlrc4 inflammasome plays an important role in host protection against opportunistic pathogenic bacteria that express flagellin.

Tubule-interstitial nephritis (TIN) results in decreased renal function and interstitial inflammation, which ultimately leads to fibrosis. Excessive adenine intake can cause TIN because xanthine dehydrogenase (XDH) can convert this purine into an insoluble compound, which precipitates in the tubuli. Innate immune sensors, such as Toll-like receptors (TLR) and inflammasome complex, play a crucial role in the initiation of inflammation. The aim of this study was to evaluate the roles of TLR-2 and -4, Myd88 and inflammasome complex in an experimental model of TIN. Here, we show that wild-type (WT) mice fed adenine-enriched food exhibited significant renal dysfunction and enhanced cellular infiltration accompanied by collagen deposition. They also presented higher gene and protein expression of pro-inflammatory cytokines. In contrast, TLR-2, -4, MyD88, ASC and Caspase-1 KO mice showed renoprotection associated with expression of inflammatory molecules at levels comparable to controls. Furthermore, treatment of WT animals with allopurinol, an XDH inhibitor, led to reduced levels of uric acid, oxidative stress, collagen deposition and a downregulation of the NF-kB signaling pathway. We concluded that MyD88 signaling and inflammasome participate in the development of TIN. Furthermore, inhibition of XDH seems to be a promising way to therapeutically target the developing inflammatory process.

Background: Caspase-1 is a cysteine protease responsible for the processing and secretion of IL-1β and IL-18, which are closely related to the induction of inflammation. However, limited evidence addresses the participation of caspase-1 in inflammatory pain. Here, we investigated the role of caspase-1 in inflammatory hypernociception (a decrease in the nociceptive threshold) using caspase-1 deficient mice (casp1−/−). Results: Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. The production of cytokines, PGE2 and neutrophil migration were evaluated by ELISA, radioimmunoassay and myeloperoxidase activity, respectively. The interleukin (IL)-1β and cyclooxygenase (COX)-2 protein expression were evaluated by western blotting. The mechanical hypernociception induced by intraplantar injection of carrageenin, tumour necrosis factor (TNF)α and CXCL1/KC was reduced in casp1−/− mice compared with WT mice. However, the hypernociception induced by IL-1β and PGE2 did not differ in WT and casp1−/− mice. Carrageenin-induced TNF-α and CXCL1/KC production and neutrophil recruitment in the paws of WT mice were not different from casp1−/−mice, while the maturation of IL-1β was reduced in casp1−/− mice. Furthermore, carrageenin induced an increase in the expression of COX-2 and PGE2 production in the paw of WT mice, but was reduced in casp1−/− mice. Conclusion: These results suggest that caspase-1 plays a critical role in the cascade of events involved in the genesis of inflammatory hypernociception by promoting IL-1β maturation. Because caspase-1 is involved in the induction of COX-2 expression and PGE2 production, our data support the assertion that caspase-1 is a key target to control inflammatory pain.

Benznidazole (Bz) is the therapy currently available for clinical treatment of Chagas' disease. However, many strains of Trypanosoma cruzi parasites are naturally resistant. Nitric oxide (NO) produced by activated macrophages is crucial to the intracellular killing of parasites. Here, we investigate the in vitro and in vivo activities against T. cruzi, of the NO donor, trans-[RuCl([15]aneN(4))NO](2+).Trans-[RuCl([15]aneN(4))NO](2+)was incubated with a partially drug-resistant T. cruzi Y strain and the anti-proliferative (epimastigote form) and trypanocidal activities (trypomastigote and amastigote) evaluated. Mice were treated during the acute phase of Chagas' disease. The anti-T. cruzi activity was evaluated by parasitaemia, survival rate, cardiac parasitism, myocarditis and the curative rate.Trans-[RuCl([15]aneN(4))NO](2+) was 10- and 100-fold more active than Bz against amastigotes and trypomastigotes respectively. Further, trans-[RuCl([15]aneN(4))NO](2+) (0.1 mM) induced 100% of trypanocidal activity (trypomastigotes forms) in vitro. Trans-[RuCl([15]aneN(4))NO](2+) induced permanent suppression of parasitaemia and 100% survival in a murine model of acute Chagas' disease. When the drugs were given alone, parasitological cures were confirmed in only 30 and 40% of the animals treated with the NO donor (3.33 micromol.kg(-1).day(-1)) and Bz (385 micromol.kg(-1).day(-1)), respectively, but when given together, 80% of the animals were parasitologically cured. The cured animals showed an absence of myocarditis and a normalisation of cytokine production in the sera. In addition, no in vitro toxicity was observed at the tested doses.These findings indicate that trans-[RuCl([15]aneN(4))NO](2+)is a promising lead compound for the treatment of human Chagas' disease.

An effective innate immune recognition of the intracellular protozoan parasite Trypanosoma cruzi is critical for host resistance against Chagas disease, a severe and chronic illness that affects millions of people in Latin America. In this study, we evaluated the participation of nucleotide-binding oligomerization domain (Nod)-like receptor proteins in host response to T. cruzi infection and found that Nod1-dependent, but not Nod2-dependent, responses are required for host resistance against infection. Bone marrow-derived macrophages from Nod1−/− mice showed an impaired induction of NF-κB–dependent products in response to infection and failed to restrict T. cruzi infection in presence of IFN-γ. Despite normal cytokine production in the sera, Nod1−/− mice were highly susceptible to T. cruzi infection, in a similar manner to MyD88−/− and NO synthase 2−/− mice. These studies indicate that Nod1-dependent responses account for host resistance against T. cruzi infection by mechanisms independent of cytokine production.

The immune system must discriminate between pathogenic and nonpathogenic microbes in order to initiate an appropriate response. Toll-like receptors (TLRs) detect microbial components common to both pathogenic and nonpathogenic bacteria, whereas Nod-like receptors (NLRs) sense microbial components introduced into the host cytosol by the specialized secretion systems or pore-forming toxins of bacterial pathogens. The host signaling pathways that respond to bacterial secretion systems remain poorly understood. Infection with the pathogen Legionella pneumophila, which utilizes a type IV secretion system (T4SS), induced an increased proinflammatory cytokine response compared to avirulent bacteria in which the T4SS was inactivated. This enhanced response involved NF-κB activation by TLR signaling as well as Nod1 and Nod2 detection of type IV secretion. Furthermore, a TLR- and RIP2-independent pathway leading to p38 and SAPK/JNK MAPK activation was found to play an equally important role in the host response to virulent L. pneumophila. Activation of this MAPK pathway was T4SS-dependent and coordinated with TLR signaling to mount a robust proinflammatory cytokine response to virulent L. pneumophila. These findings define a previously uncharacterized host response to bacterial type IV secretion that activates MAPK signaling and demonstrate that coincident detection of multiple bacterial components enables immune discrimination between virulent and avirulent bacteria., Author Summary The host immune system senses bacterial infection by recognizing conserved bacterial components. The host can differentiate between virulent and avirulent bacteria by detecting the activity of bacterial secretion systems that inject effector proteins into cells. How the host responds to such bacterial secretion systems is not fully understood. Using the bacterial pathogen Legionella pneumophila and isogenic mutants that differ in defined virulence properties, a robust immune response to L. pneumophila and its type IV secretion system was identified. Using macrophages lacking various aspects of the innate immune system, it was found that this host response is comprised of signaling by extracellular and intracellular immune receptors, as well as host signaling triggered by the type IV secretion system. Through genomic and biochemical analyses of L. pneumophila–infected macrophages, type IV secretion was found to activate additional host signaling pathways distinct from known immune pathways. Our data indicate that coincident detection of multiple bacterial components is required for a robust immune response to bacterial infection and highlights a key host pathway triggered by bacterial type IV secretion that contributes to this immune response.

A mononuclear phagocyte derived from B1b cells (B1CDP) has been described. As these cells migrate from the peritoneal cavity to non-specific inflammatory lesion sites and are highly phagocytic via Fc and mannose receptors, their microbicidal ability of these cells was investigated using the Coxiella burnetii cell infection model in vitro. In this report, the pattern of infection and C. burnetii phase II survival in B1CDP phagosomes was compared with the pattern of infection of peritoneal macrophages from Xid mice (PMphi) and bone marrow derived macrophages (BMMphi). Infection was assessed by determining the large parasitophorous vacuole formation, the relative focus forming units and the quantification of DAPI (4',6-diamino-2-phenylindole) fluorescence images acquired by confocal microscopy. When compared to macrophages, B1CDP are more permissive to the bacterial infection and less effective to kill them. Further, results suggest that IL-10 secreted by B1 cells are involved in their susceptibility to infection by C. burnetti, since B1CDP from IL-10 KO mice are more competent to control C. burnetii infection than cells from wild type mice. These data contribute further to characterize B1CDP as a novel mononuclear phagocyte.

Macrophages from C57BL/6J (B6) mice restrict growth of the intracellular bacterial pathogen Legionella pneumophila. Restriction of bacterial growth requires caspase-1 and the leucine-rich repeat-containing protein Naip5 (Birc1e). We identified mutants of L. pneumophila that evade macrophage innate immunity. All mutants were deficient in expression of flagellin, the primary flagellar subunit, and failed to induce caspase-1-mediated macrophage death. Interestingly, a previously isolated flagellar mutant (fliI) that expresses, but does not assemble, flagellin did not replicate in macrophages, and induced macrophage death. Thus, flagellin itself, not flagella or motility, is required to initiate macrophage innate immunity. Immunity to Legionella did not require MyD88, an essential adaptor for toll-like receptor 5 (TLR5) signaling. Moreover, flagellin of Legionella and Salmonella induced cytotoxicity when delivered to the macrophage cytosol using Escherichia coli as a heterologous host. It thus appears that macrophages sense cytosolic flagellin via a TLR5-independent pathway that leads to rapid caspase-1-dependent cell death and provides defense against intracellular bacterial pathogens., Synopsis Legionella pneumophila is a bacterial pathogen that is the cause of a severe form of pneumonia known as Legionnaires' disease. A crucial aspect of the propensity of Legionella to cause disease lies in its ability to survive and multiply inside host immune cells known as macrophages. The intracellular survival and replication of Legionella can be studied using isolated macrophages grown in culture. Macrophages isolated from different laboratory mouse strains are differentially permissive for intracellular Legionella growth. This difference in permissiveness is genetic, and is conferred by differences in a mouse protein known as Naip5. The authors determined that Legionella strains that are unable to produce a protein called flagellin are able to grow inside normally resistant mouse macrophages. In addition, these flagellin− strains are defective in initiating a cell-death response on the part of infected macrophages. Based on these data, the authors suggest that there is an intracellular mechanism for detecting the presence of bacterial flagellin protein, and that a cell-death response is initiated upon the detection of flagellin.

Baculovirus inhibitor of apoptosis repeat-containing 1 (Birc1) proteins have homology to several germline-encoded receptors of the innate immune system. However, their function in immune surveillance is not clear. Here we describe a Birc1e-dependent signaling pathway that restricted replication of the intracellular pathogen Legionella pneumophila in mouse macrophages. Translocation of bacterial products into host-cell cytosol was essential for Birc1e-mediated control of bacterial replication. Caspase-1 was required for Birc1e-dependent antibacterial responses ex vivo in macrophages and in a mouse model of Legionnaires' disease. The interleukin 1beta converting enzyme-protease-activating factor was necessary for L. pneumophila growth restriction, but interleukin 1beta was not required. These results establish Birc1e as a nucleotide-binding oligomerization-leucine-rich repeat protein involved in the detection and control of intracellular L. pneumophila.

Coxiella burnetii, the agent of Q fever in humans and coxiellosis in other mammals, is an obligate intracellular bacterium which is sheltered and multiplies within typically large phagolysosome-like replicative vacuoles (LRVs). We have previously shown that, compared with fibroblasts, mouse resident peritoneal macrophages control the development of LRVs and bacterial multiplication within these vacuoles. Earlier experiments with the nitric oxide (NO) synthase inhibitor aminoguanidine (AG) revealed that the control is exerted by NO induced by the bacteria. We report here that phagocytosis of apoptotic-like, but not of aldehyde-killed, lymphocytes by the macrophages reduced the production of NO induced by the bacteria and increased the development of LRVs and, therefore, the total bacterial load in the cultures. Experiments with macrophages from mice deficient for inducible NO synthase (iNOS−/−) confirmed the involvement of NO in the control of infection, since neither apoptotic lymphocytes nor AG affected the development of LRVs in these phagocytes. Since macrophages are important for the clearance of apoptotic bodies andC. burnetiiis able to induce apoptosis in human monocytes, the phenomenon shown here may be biologically relevant to the development of Q fever and coxiellosis.

In most primary or continuous cell cultures infected with the Q-fever agent Coxiella burnetii, bacteria are typically sheltered in phagolysosome-like, large replicative vacuoles (LRVs). We recently reported that only a small proportion of mouse peritoneal macrophages (PMPhi) infected with a nonvirulent, phase II strain of C. burnetti developed LRVs and that their relative bacterial load increased only slowly. in the majority of infected PM(D, the bacteria were confined to the small vesicles. We show here that nitric oxide (NO) induced by the bacteria partially accounts for the restricted development of LRVs in primary macrophages. Thus, (i) PMPhi and bone marrow-derived macrophages (BMMPhi) challenged with phase II C. burnetii produced significant amounts of NO; (ii) the NO synthase inhibitors aminoguanidine and N-methyl-L-arginine reduced the production of NO and increased the frequency of LRVs (although the relative bacterial loads of individual LRVs did not change, the estimated loads per well increased appreciably); (iii) gamma interferon (IFN-gamma) or the NO donor sodium nitroprusside, added to BMMPhi prior to or after infection, reduced the development and the relative bacterial loads of LRVs and lowered the yield of viable bacteria recovered from the cultures; and (iv) these effects of IFN-gamma may not be entirely dependent on the production of NO since IFN-gamma also controlled the infection in macrophages from inducible NO synthase knockout mice. It remains to be determined whether NO reduced the development of LRVs by acting directly on the bacteria; by acting on the traffic, fusion, or fission of cell vesicles; or by a combination of these mechanisms. UNIFESP, Escola Paulista Med, Disciplina Parasitol, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, Brazil UNIFESP, Escola Paulista Med, Disciplina Parasitol, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, Brazil Web of Science

Chronic inflammation affects most stages of tumorigenesis, including initiation, promotion, malignant differentiation, invasion and metastasis. Inflammasomes have been described as involved with persistent inflammation and are known to exert both pro and antitumour effects. We evaluated the influence of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase (CASP)-1 in the antitumor immune response using a multistage model of squamous cell carcinoma (SCC) development. Absence of ASC and CASP-1 resulted in an earlier incidence and increased number of papilloma. Loss of inflammassome function in mice resulted in decreased presence of natural killer (NK), dendritic (DC), CD4+, CD8+ and CD45RB+ T cells in the tumor lesions as well as in lymph nodes (LN) compared with WT mice. Increased percentage of CD4+CD25+Foxp3+ T cells was associated with association with inflammasome loss of function. Moreover, significant differences were also found with neutrophils and macrophage infiltrating the lesions. Myeloperoxidase (MPO), but not elastase (ELA), activity oscillated among the groups during the SCC development. Levels of proinflammatory cytokines IL-1β, IL-18, Tumor Necrosis Factor (TNF)-α and Interferon (IFN)-γ were decreased in the tumor microenvironment in the absence of inflammasome proteins. These observations suggest a link between inflammasome function and SCC tumorigenesis, indicating an important role for inflammasome activation in the control of SCC development. Fil: Gasparoto, Thais Helena. Universidad de São Paulo. Faculdade de Odontologia de Bauru. Departamento de Ciencias Biológicas; Brasil Fil: Ervolino de Oliveira, Carine. Universidad de São Paulo. Faculdade de Odontologia de Bauru. Departamento de Ciencias Biológicas; Brasil Fil: Thomazini de Freitas, Luisa. Universidad de São Paulo. Faculdade de Odontologia de Bauru. Departamento de Ciencias Biológicas; Brasil Fil: Ramos Pinheiro, Claudia. Universidad de São Paulo. Faculdade de Odontologia de Bauru. Departamento de Ciencias Biológicas; Brasil Fil: Issa Hori, Juliana. University of São Paulo. Faculdade de Medicina de Ribeirão Preto; Brasil Fil: Pompermaier Garlet, Gustavo. Universidad de São Paulo. Faculdade de Odontologia de Bauru. Departamento de Ciencias Biológicas; Brasil Fil: Cavassani, Karen Angélica. University Of Michigan; Estados Unidos Fil: Schillaci, Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Santana Da Silva, Joao. University of São Paulo. Faculdade de Medicina de Ribeirão Preto; Brasil Fil: Simmões Zamboni, Darío. University of São Paulo. Faculdade de Medicina de Ribeirão Preto; Brasil Fil: Campanelli, Ana Paula. Universidad de São Paulo. Faculdade de Odontologia de Bauru. Departamento de Ciencias Biológicas; Brasil

Background T. cruzi strains have been divided into six discrete typing units (DTUs) according to their genetic background. These groups are designated T. cruzi I to VI. In this context, amastigotes from G strain (T. cruzi I) are highly infective in vitro and show no parasitemia in vivo. Here we aimed to understand why amastigotes from G strain are highly infective in vitro and do not contribute for a patent in vivo infection. Methodology/Principal Findings Our in vitro studies demonstrated the first evidence that IFN-γ would be associated to the low virulence of G strain in vivo. After intraperitoneal amastigotes inoculation in wild-type and knockout mice for TNF-α, Nod2, Myd88, iNOS, IL-12p40, IL-18, CD4, CD8 and IFN-γ we found that the latter is crucial for controlling infection by G strain amastigotes. Conclusions/Significance Our results showed that amastigotes from G strain are highly infective in vitro but did not contribute for a patent infection in vivo due to its susceptibility to IFN-γ production by host immune cells. These data are useful to understand the mechanisms underlying the contrasting behavior of different T. cruzi groups for in vitro and in vivo infection., Author Summary Trypanosoma cruzi, an obligate intracellular protozoan, is the etiological agent of Chagas disease that represents an important public health burden in Latin America. The infection with this parasite can lead to severe complications in cardiac and gastrointestinal tissue depending on the strain of parasite and host genetics. Currently, six genetic groups (T. cruzi I to VI) have been identified in this highly genetic and diverse parasite.The majority of published data concerning host immune response has been obtained from studying T. cruzi II to VI-infected mice, and the genetic differences between T. cruzi II to VI and T. cruzi I strains are large. Here we aimed to understand how amastigotes from T. cruzi I G strain are highly infective in vitro and do not contribute for a patent parasitemia in vivo. Our results showed that amastigotes from G strain are highly susceptible to IFN-γ treatment in vitro and secretion by immune cells in vivo. This information may represent important findings to design novel immune strategies to control pathology that may be caused by different strains in the same host.

The broad use of transgenic and gene-targeted mice has established bone marrow-derived macrophages (BMDM) as important mammalian host cells for investigation of the macrophages biology. Over the last decade, extensive research has been done to determine how to freeze and store viable hematopoietic human cells; however, there is no information regarding generation of BMDM from frozen murine bone marrow (BM) cells. Here, we establish a highly efficient protocol to freeze murine BM cells and further generate BMDM. Cryopreserved murine BM cells maintain their potential for BMDM differentiation for more than 6 years. We compared BMDM obtained from fresh and frozen BM cells and found that both are similarly able to trigger the expression of CD80 and CD86 in response to LPS or infection with the intracellular bacteria Legionella pneumophila. Additionally, BMDM obtained from fresh or frozen BM cells equally restrict or support the intracellular multiplication of pathogens such as L. pneumophila and the protozoan parasite Leishmania (L.) amazonensis. Although further investigation are required to support the use of the method for generation of dendritic cells, preliminary experiments indicate that bone marrow-derived dendritic cells can also be generated from cryopreserved BM cells. Overall, the method described and validated herein represents a technical advance as it allows ready and easy generation of BMDM from a stock of frozen BM cells.

The establishment of a local inflammatory response with production of cytokines/chemokines and the consequent neutrophil recruitment are critical to control an infection. In this context, our group had demonstrated an impairment of neutrophil migration toward the infectious focus in severe sepsis. The failure of neutro-phil migration is markedly associated with increased systemic cytokines levels and high mortality of the severe sepsis, either in experimental models or in patients. Recently, the participation of Toll-like receptors (TLRs) in the failure of neutrophil migration was described. Caspase-1 seems to be important in the activation of TLR signaling pathways. Moreover, it is also critical to the activation of inflammatory cytokines IL-1β, IL-18 and IL-33. The aim of the present study was to evaluate the participation of caspase-1 during severe sepsis. The caspase-1-deficient mice presented increased resistance to severe sepsis induced by CLP. However, IL-18 and ST2 (receptor of IL-33)-deficient mice did not present a reduction of mortality after sepsis. The treatment with antagonist of IL-1 receptor was also unable to modify the survival rate of wild-type mice that underwent severe sepsis. These data indicate that the reduction in levels of these cytokines is not critical for reduction of mortality observed in caspase-1-deficient mice. The reduction in mortality of caspase-1-deficient mice was associated with decreased systemic levels of TNFα and IL-6. Despite the unaltered local levels of cytokines and chemokines, caspase-1-deficient mice that underwent severe sepsis presented a marked increase in neutrophil migration to the peritoneal cavity, which was supported by an increased rolling and adhesion of leukocytes in these mice. As consequence, a reduced bacterial growth in peritoneal exudates and blood was observed in these animals, although neutrophils from caspase-1-deficient and wild-type mice presented similar killing and cellular viability. Thus, in the absence of caspase-1, neutrophil migration to the peritoneal cavity is increased and culminates in a reduction of mortality because of efficient control of the infection.

Due to their secretive habits, very little is known about the ecology of amphisbaenians. Based on a large series of specimens from the Cerrado of central Brazil, the diet composition, reproduction, and sexual dimorphism of Amphisbaena alba are described. Amphisbaena alba displayed a relatively diverse diet, even ingesting vertebrates and plant material. Beetles, ants, and spiders dominated the diet numerically, whereas ants, insect larvae, and beetles were more important volumetrically. No association was observed between prey size and body dimensions. The smallest reproductive male was 422 mm SVL (snout-vent length), and the smallest reproductive female was 457 mm SVL. Clutch size ranged from 8-16, one of the largest known for amphisbaenians, and is possibly influenced by the large body size of A. alba. Reproduction is apparently seasonal and restricted to the dry season. No intersexual differences were observed, either in morphometric or meristic characters, presumably because of functional constraints related to fossoriality.

The aim of this study was to analyse the expression and function of nucleotide-binding oligomerization domain (NOD)1 and NOD2 in isolated cells of patients with rheumatoid arthritis (RA).mRNA expression levels of NOD1, NOD2, and receptor-interacting serine/threonine kinase 2 (RIPK2) genes were determined by quantitative polymerase chain reaction (qPCR) in peripheral blood mononuclear cells (PBMCs) and synovial fluid T cells (SFTCs) isolated from RA and osteoarthritis (OA) patients. Cytokines were measured by enzyme-linked immunosorbent assay (ELISA) in plasma and cell culture supernatants. The stimulatory effect of RA SF was assessed by an in-vitro NOD2 activation assay using nuclear factor kappa B (NF-κB) luciferase-transfected cells.A significantly higher level of NOD2 and RIPK2 mRNA expression, but not NOD1, was observed on PBMCs and SFTCs isolated from RA patients compared to the OA control group. In addition, the NOD2 pathway up-regulation was functional, as stimulation of PBMCs with muramyl dipeptide (MDP) induced the production of higher amounts of tumour necrosis factor (TNF)-α, interleukin (IL)-8, and IL-1β compared with OA PBMCs. Incubation of PBMCs from healthy donors with recombinant TNF-α or RA serum induced the expression of NOD2 mRNA. Finally, SF isolated from RA patients is able to activate the NF-κB signalling pathway in HEK293T-transfected cells in a NOD2-dependent manner.Our findings suggest that NOD2/RIPK2 signalling is up-regulated in immune cells of RA patients. Moreover, it seems that there is a NOD2 agonist in the SF of RA patients. Therefore, NOD2/RIPK2 activation can modulate the innate immune response and may play a role in the perpetuation of the inflammatory response in RA.