Antigen recognition reinforces regulatory T cell mediated Leishmania major persistence.

Cutaneous Leishmania major infection elicits a rapid T cell response that is insufficient to clear residually infected cells, possibly due to the accumulation of regulatory T cells in healed skin. Here, we used Leishmania-specific TCR transgenic mice as a sensitive tool to characterize parasite-specific effector and immunosuppressive responses in vivo using two-photon microscopy. We show that Leishmania-specific Tregs displayed higher suppressive activity compared to polyclonal Tregs, that was mediated through IL-10 and not through disrupting cell-cell contacts or antigen presentation. In vivo expansion of endogenous Leishmania-specific Tregs resulted in disease reactivation that was also IL-10 dependent. Interestingly, lack of Treg expansion that recognized the immunodominant Leishmania peptide PEPCK was sufficient to restore robust effector Th1 responses and resulted in parasite control exclusively in male hosts. Our data suggest a stochastic model of Leishmania major persistence in skin, where cellular factors that control parasite numbers are counterbalanced by Leishmania-specific Tregs that facilitate parasite persistence.

Protective CD4+ Th1 cell-mediated immunity is reliant upon execution of effector function prior to the establishment of the pathogen niche.

Intracellular infection with the parasite Leishmania major features a state of concomitant immunity in which CD4+ T helper 1 (Th1) cell-mediated immunity against reinfection coincides with a chronic but sub-clinical primary infection. In this setting, the rapidity of the Th1 response at a secondary site of challenge in the skin represents the best correlate of parasite elimination and has been associated with a reversal in Leishmania-mediated modulation of monocytic host cells. Remarkably, the degree to which Th1 cells are absolutely reliant upon the time at which they interact with infected monocytes to mediate their protective effect has not been defined. In the present work, we report that CXCR3-dependent recruitment of Ly6C+ Th1 effector (Th1EFF) cells is indispensable for concomitant immunity and acute (<4 days post-infection) Th1EFF cell-phagocyte interactions are critical to prevent the establishment of a permissive pathogen niche, as evidenced by altered recruitment, gene expression and functional capacity of innate and adaptive immune cells at the site of secondary challenge. Surprisingly, provision of Th1EFF cells after establishment of the pathogen niche, even when Th1 cells were provided in large quantities, abrogated protection, Th1EFF cell accumulation and IFN-γ production, and iNOS production by inflammatory monocytes. These findings indicate that protective Th1 immunity is critically dependent on activation of permissive phagocytic host cells by preactivated Th1EFF cells at the time of infection.

The Phosphoenolpyruvate Carboxykinase Is a Key Metabolic Enzyme and Critical Virulence Factor of Leishmania major.

There is currently no effective vaccine against leishmaniasis because of the lack of sufficient knowledge about the Ags that stimulate host-protective and long-lasting T cell-mediated immunity. We previously identified Leishmania phosphoenolpyruvate carboxykinase (PEPCK, a gluconeogenic enzyme) as an immunodominant Ag that is expressed by both the insect (promastigote) and mammalian (amastigote) stages of the parasite. In this study, we investigated the role of PEPCK in metabolism, virulence, and immunopathogenicity of Leishmania major We show that targeted loss of PEPCK results in impaired proliferation of L. major in axenic culture and bone marrow-derived macrophages. Furthermore, the deficiency of PEPCK results in highly attenuated pathology in vivo. BALB/c mice infected with PEPCK-deficient parasites failed to develop any cutaneous lesions despite harboring parasites at the cutaneous site of infection. This was associated with a dramatic reduction in the frequency of cytokine (IFN-γ, IL-4, and IL-10)-producing CD4+ T cells in spleens and lymph nodes draining the infection site. Cells from mice infected with PEPCK-deficient parasites also produced significantly low levels of these cytokines into the culture supernatant following in vitro restimulation with soluble Leishmania Ag. PEPCK-deficient parasites exhibited significantly greater extracellular acidification rate, increased proton leak, and decreased ATP-coupling efficiency and oxygen consumption rates in comparison with their wild-type and addback counterparts. Taken together, these results show that PEPCK is a critical metabolic enzyme for Leishmania, and its deletion results in altered metabolic activity and attenuation of virulence.

Semaphorin 3E Promotes Susceptibility to Leishmania major Infection in Mice by Suppressing CD4+ Th1 Cell Response.

Protective immunity to cutaneous leishmaniasis is mediated by IFN-γ-secreting CD4+ Th1 cells. IFN-γ binds to its receptor on Leishmania-infected macrophages, resulting in their activation, production of NO, and subsequent destruction of parasites. This study investigated the role of Semaphorin 3E (Sema3E) in host immunity to Leishmania major infection in mice. We observed a significant increase in Sema3E expression at the infection site at different timepoints following L. major infection. Sema3E-deficient (Sema3E knockout [KO]) mice were highly resistant to L. major infection, as evidenced by significantly (p < 0.05-0.01) reduced lesion sizes and lower parasite burdens at different times postinfection when compared with their infected wild-type counterpart mice. The enhanced resistance of Sema3E KO mice was associated with significantly (p < 0.05) increased IFN-γ production by CD4+ T cells. CD11c+ cells from Sema3E KO mice displayed increased expression of costimulatory molecules and IL-12p40 production following L. major infection and were more efficient at inducing the differentiation of Leishmania-specific CD4+ T cells to Th1 cells than their wild-type counterpart cells. Furthermore, purified CD4+ T cells from Sema3E KO mice showed increased propensity to differentiate into Th1 cells in vitro, and this was significantly inhibited by the addition of recombinant Sema3E in vitro. These findings collectively show that Sema3E is a negative regulator of protective CD4+ Th1 immunity in mice infected with L. major and suggest that its neutralization may be a potential therapeutic option for treating individuals suffering from cutaneous leishmaniasis.

The Long Pentraxin 3 (PTX3) Suppresses Immunity to Cutaneous Leishmaniasis by Regulating CD4+ T Helper Cell Response.

The long pentraxin 3 (PTX3) plays a critical role in inflammation, tissue repair, and wound healing. Here, we show that PTX3 regulates disease pathogenesis in cutaneous leishmaniasis (CL). PTX3 expression increases in skin lesions in patients and mice during CL, with higher expression correlating with severe disease. PTX3-deficient (PTX3-/-) mice are highly resistant to L. major and L. braziliensis infections. This enhanced resistance is associated with increases in Th17 and IL-17A responses. The neutralization of IL-17A abolishes this enhanced resistance, while rPTX3 treatment results in decrease in Th17 and IL-17A responses and increases susceptibility. PTX3-/- CD4+ T cells display increased differentiation to Th17 and expression of Th17-specific transcription factors. The addition of rPTX3 suppresses the expression of Th17 transcription factors, Th17 differentiation, and IL-17A production by CD4+ T cells from PTX3-/- mice. Collectively, our results show that PTX3 contributes to the pathogenesis of CL by negatively regulating Th17 and IL-17A responses.

Identification of a Protective Leishmania Antigen Dihydrolipoyl Dehydrogenase and Its Responding CD4+ T Cells at Clonal Level.

There is currently no clinically effective vaccine against cutaneous leishmaniasis because of poor understanding of the Ags that elicit protective CD4+ T cell immunity. In this study, we identified a naturally processed peptide (DLD63-79) that is derived from Leishmania dihydrolipoyl dehydrogenase (DLD) protein. DLD is conserved in all pathogenic Leishmania species, is expressed by both the promastigote and amastigote stages of the parasite, and elicits strong CD4+ T cell responses in mice infected with L. major We generated I-Ab-DLD63-79 tetramer and identified DLD-specific CD4+ T cells at clonal level. Following L. major infection, DLD63-79-specific CD4+ T cells massively expanded and produced effector cytokines (IFN-γ and TNF). This was followed by a gradual contraction, stable maintenance following lesion resolution, and display of memory (recall) response following secondary challenge. Vaccination with rDLD protein induced strong protection in mice against virulent L. major challenge. Identification of Ags that elicit protective immunity and their responding Ag-specific T cells are critical steps necessary for developing effective vaccines and vaccination strategies against infectious agents, including protozoan parasites.

Deficiency of Phosphatidylinositol 3-Kinase δ Signaling Leads to Diminished Numbers of Regulatory T Cells and Increased Neutrophil Activity Resulting in Mortality Due to Endotoxic Shock.

Despite decades of clinical and biomedical research, the pathogenesis of sepsis and its spectrum of diseases (severe sepsis and septic shock), which are leading causes of death in intensive care units, are still poorly understood. In this article, we show that signaling via the p110δ isoform of PI3K is critical for survival in experimental sepsis. Mice with an inactive knock-in mutation in the p110δ gene (p110δD910A) succumbed acutely to nonlethal dose LPS challenge. The susceptibility of p110δD910A mice to LPS was associated with increased neutrophil numbers and activities in the tissues, due in part to delayed apoptosis resulting mostly from inherent reduced regulatory T cell (Treg) numbers. Adoptive transfer of wild-type or p110δD910A Tregs abrogated exaggerated neutrophil activity, increased neutrophil apoptosis, and rescued p110δD910A mice from mortality after LPS challenge. We confirmed the clinical relevance of these findings by showing that human Tregs also regulate neutrophil function and survival. Collectively, our results show that PI3K δ is essential for survival during sepsis. In addition, our data highlight the importance of Tregs in regulating the pathogenesis of sepsis and septic shock via their effects on neutrophil survival and function, and provide evidence of regulation of innate immunity by cells of the adaptive immune system.

Pharmacological inhibition of p110δ subunit of PI3K confers protection against experimental leishmaniasis.

OBJECTIVES: This study aimed to evaluate the immuno-prophylactic and -therapeutic effect of p110δ-specific pharmacological inhibitors (CAL-101 and IC87114), either alone or in combination with amphotericin B, against experimental cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). METHODS: Female BALB/c mice were infected intravenously with Leishmania donovani or subcutaneously with Leishmania major Prophylactic treatment was initiated 24 h prior to infection, whereas therapeutic treatments with or without amphotericin B were initiated either 1 week or 2 weeks post-infection. At different times post-infection, mice were sacrificed and parasite burden, regulatory T cell (Treg) numbers and cytokine production were assessed in the liver, spleen, draining lymph nodes and footpads. In addition, direct cytolytic effects of the inhibitors on parasite growth in axenic cultures and inside infected and uninfected macrophages were also assessed. RESULTS: Prophylactic and therapeutic administration of p110δ pharmacological inhibitors significantly reduced cutaneous lesion (in CL) and parasite burdens (in VL and CL) in the spleens, livers and footpads of infected mice. The reduction in parasite burden was associated with a concomitant reduction in Treg numbers and cytokine production by liver, spleen and lymph node cells. Combined low-dose CAL-101 and amphotericin B therapy caused complete clearance of parasites in mice infected with L. donovani CONCLUSIONS: Our studies clearly show a novel therapeutic option for leishmaniasis based on CAL-101 monotherapy or CAL-101 and amphotericin B combination therapy. These observations have important and direct implications for antimicrobial immunotherapy and drug/vaccine development against leishmaniasis.

Sinomenine Attenuates Angiotensin II-Induced Autophagy via Inhibition of P47-Phox Translocation to the Membrane and Influences Reactive Oxygen Species Generation in Podocytes.

BACKGROUND/AIMS: Sinomenine, a pure alkaloid extracted from the Chinese medicinal plant Sinomenium acutum, and sinomenine hydrochloride (SN) has been successfully used for the therapy of rheumatoid arthritis (RA) and kidney diseases. Autophagy is a cytoprotective mechanism used by podocytes and other cells to alleviate the effects of oxidative stress, and angiotensin II (Ang II) significantly promotes podocyte autophagy. However, excessive autophagy may lead to cell death and podocyte depletion. The present study evaluated the effect of SN in podocytes induced by Ang II. METHODS: Podocytes were pretreated with graded concentrations (10(-8) M ∼ 10(-4) M) of SN and then stimulated with Ang II. The LC3B protein and the p47-phox membrane fraction were measured by Western blot. Autolysosomes were assessed by transmission electron microscopy. FACS was used to quantify the ROS produced by podocytes. The translocation of p47-phox to the membrane was investigated by immunofluorescence. RESULTS: The 10(-8) M ∼ 10(-4) M of SN alone did not effect ROS generation or podocyte autophagy. The 10(-8) M and 10(-6) M SN attenuated Ang II-induced autophagy in podocytes. Furthermore, SN decreased the level of ROS generation in Ang II-induced podocytes via inhibition of NOX subunit p47-phox translocation to the membrane. CONCLUSION: The appropriate concentration of SN attenuated Ang II-induced podocyte autophagy through ROS generation, at least in part, by regulating NOX subunit p47-phox translocation to the membrane.

Hepatic stellate cells regulate liver immunity to visceral leishmaniasis through P110δ-dependent induction and expansion of regulatory T cells in mice.

UNLABELLED: Visceral leishmaniasis (VL) is associated with severe immune dysfunction and if untreated leads to death. Because the liver is one of the primary target organs in VL, unraveling the mechanisms governing the local hepatic immune response is important for understanding the immunopathogenesis of VL. We previously reported that mice with inactivating knockin mutation in the p110δ gene (p110δ(D910A) ) are resistant to VL, due in part to impaired regulatory T-cell (Treg) expansion. In this study, we investigated the mechanism of this resistance by focusing on hepatic stellate cells (HSCs), which are known to regulate Treg induction and expansion. We show that HSCs are infected with Leishmania donovani in vivo and in vitro and that this infection leads to the production of interleukin-2, interleukin-6, and transforming growth factor-ß, cytokines known to induce Tregs. We further demonstrate that L. donovani infection leads to expansion of HSCs in a p110δ-dependent manner and that this correlated with proliferation of hepatic Tregs in vivo. In vitro studies clearly show that L. donovani-infected HSCs induce CD4(+) T cells to become Tregs and expand Tregs in a p110δ-dependent manner. Targeted depletion of HSCs during infection caused a dramatic reduction in liver Treg numbers and proliferation, which was associated with a decrease in interleukin-10 production by hepatic T cells and a more efficient parasite control. CONCLUSION: These results demonstrate the critical role of HSCs in the pathogenesis of VL and suggest that the enhanced resistance of p110δ(D910A) mice to L. donovani infection is due in part to impaired expansion and inability of their HSCs to induce and expand Tregs in the liver.

Identification of broadly conserved cross-species protective Leishmania antigen and its responding CD4+ T cells.

There is currently no clinically effective vaccine against leishmaniasis because of poor understanding of the antigens that elicit dominant T cell immunity. Using proteomics and cellular immunology, we identified a dominant naturally processed peptide (PEPCK335-351) derived from Leishmania glycosomal phosphoenolpyruvate carboxykinase (PEPCK). PEPCK was conserved in all pathogenic Leishmania, expressed in glycosomes of promastigotes and amastigotes, and elicited strong CD4(+) T cell responses in infected mice and humans. I-A(b)-PEPCK335-351 tetramer identified protective Leishmania-specific CD4(+) T cells at a clonal level, which comprised ~20% of all Leishmania-reactive CD4(+) T cells at the peak of infection. PEPCK335-351-specific CD4(+) T cells were oligoclonal in their T cell receptor usage, produced polyfunctional cytokines (interleukin-2, interferon-γ, and tumor necrosis factor), and underwent expansion, effector activities, contraction, and stable maintenance after lesion resolution. Vaccination with PEPCK peptide, DNA expressing full-length PEPCK, or rPEPCK induced strong durable cross-species protection in both resistant and susceptible mice. The effectiveness and durability of protection in vaccinated mice support the development of a broadly cross-species protective vaccine against different forms of leishmaniasis by targeting PEPCK.

B7H1 Expression and Epithelial-To-Mesenchymal Transition Phenotypes on Colorectal Cancer Stem-Like Cells.

Cancer stem cells (CSCs) can invade and metastasize by epithelial-to-mesenchymal transition (EMT). However, how they escape immune surveillance is unclear. B7H1 is crucial negative co-stimulatory molecule but little information about whether it works in CSCs. Therefore, we determined the expression of B7H1 and EMT-associated markers in colorectal cancer stem-like cells to investigate a possible immunoevasion way of CSCs. We enriched CD133+ colorectal cancer cells which manifested the CSCs-like properties such as higher levels of other stem cell markers Oct-4 and Sox-2, tumor sphere forming ability and more tumorigenic in NOD/SCID mice. These CD133+ cells possess EMT gene expression profile including higher level of Snail, Twist, vimentin, fibronectin and lower level of E-cadherin. Moreover, CD133+ cells in both cell line and colorectal cancer tissues expressed high level of negative co-stimulate molecule B7H1. Furthermore, some B7H1+ cancer cells also showed the characteristic of EMT, indicating EMT cells could escape immune attack during metastasis. B7H1 expression and EMT phenotypes on CSCs indicates a possible immunoevasion way.

Deficiency of prolactin-inducible protein leads to impaired Th1 immune response and susceptibility to Leishmania major in mice.

Although the strategic production of prolactin-inducible protein (PIP) at several ports of pathogen entry into the body suggests it might play a role in host defense, no study has directly implicated it in immunity against any infectious agent. Here, we show for the first time that PIP deficiency is associated with reduced numbers of CD4(+) T cells in peripheral lymphoid tissues and impaired CD4(+) Th1-cell differentiation in vitro. In vivo, CD4(+) T cells from OVA-immunized, PIP-deficient mice showed significantly impaired proliferation and IFN-γ production following in vitro restimulation. Furthermore, PIP-deficient mice were highly susceptible to Leishmani major infection and failed to control lesion progression and parasite proliferation. This susceptibility was associated with impaired NO production and leishmanicidal activity of PIP KO macrophages following IFN-γ and LPS stimulation. Collectively, our findings implicate PIP as an important regulator of CD4(+) Th1-cell-mediated immunity.

CD8+ T cells are preferentially activated during primary low dose leishmania major infection but are completely dispensable during secondary anti-Leishmania immunity.

We previously showed that CD8+ T cells are required for optimal primary immunity to low dose Leishmania major infection. However, it is not known whether immunity induced by low dose infection is durable and whether CD8+ T cells contribute to secondary immunity following recovery from low dose infection. Here, we compared primary and secondary immunity to low and high dose L. major infections and assessed the influence of infectious dose on the quality and magnitude of secondary anti-Leishmania immunity. In addition, we investigated the contribution of CD8+ T cells in secondary anti-Leishmania immunity following recovery from low and high dose infections. We found that the early immune response to low and high dose infections were strikingly different: while low dose infection preferentially induced proliferation and effector cytokine production by CD8+ T cells, high dose infection predominantly induced proliferation and cytokine production by CD4+ T cells. This differential activation of CD4+ and CD8+ T cells by high and low dose infections respectively, was imprinted during in vitro and in vivo recall responses in healed mice. Both low and high dose-infected mice displayed strong infection-induced immunity and were protected against secondary L. major challenge. While depletion of CD4+ cells in mice that healed low and high dose infections abolished resistance to secondary challenge, depletion of CD8+ cells had no effect. Collectively, our results show that although CD8+ T cells are preferentially activated and may contribute to optimal primary anti-Leishmania immunity following low dose infection, they are completely dispensable during secondary immunity.

MHC class II restricted innate-like double negative T cells contribute to optimal primary and secondary immunity to Leishmania major.

Although it is generally believed that CD4(+) T cells play important roles in anti-Leishmania immunity, some studies suggest that they may be dispensable, and that MHC II-restricted CD3(+)CD4(-)CD8(-) (double negative, DN) T cells may be more important in regulating primary anti-Leishmania immunity. In addition, while there are reports of increased numbers of DN T cells in Leishmania-infected patients, dogs and mice, concrete evidence implicating these cells in secondary anti-Leishmania immunity has not yet been documented. Here, we report that DN T cells extensively proliferate and produce effector cytokines (IFN-γ, TNF and IL-17) and granzyme B (GrzB) in the draining lymph nodes and spleens of mice following primary and secondary L. major infections. DN T cells from healed mice display functional characteristics of protective anti-Leishmania memory-like cells: rapid and extensive proliferation and effector cytokines production following L. major challenge in vitro and in vivo. DN T cells express predominantly (> 95%) alpha-beta T cell receptor (αß TCR), are Leishmania-specific, restricted mostly by MHC class II molecules and display transcriptional profile of innate-like genes. Using in vivo depletion and adoptive transfer studies, we show that DN T cells contribute to optimal primary and secondary anti-Leishmania immunity in mice. These results directly identify DN T cells as important players in effective and protective primary and secondary anti-L. major immunity in experimental cutaneous leishmaniasis.

Identification of differential proteins in colorectal cancer cells treated with caffeic acid phenethyl ester.

AIM: To investigate the molecular mechanisms of the anti-cancer activity of caffeic acid phenethyl ester (CAPE). METHODS: Protein profiles of human colorectal cancer SW480 cells treated with or without CAPE were analysed using a two-dimensional (2D) electrophoresis gel-based proteomics approach. After electrophoresis, the gels were stained with Coomassie brilliant blue R-250. Digital images were taken with a GS-800 Calibrated Densitometer, and image analysis was performed using PDQuest 2-D Analysis software. The altered proteins following CAPE treatment were further identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry following a database search. The identified proteins were validated by Western blot and immunofluorescence assay. RESULTS: CAPE induced human colorectal cancer cell apoptosis. Four up-regulated proteins and seven down-regulated proteins in colorectal cancer cells treated with CAPE were found. The identified down-regulated proteins in CAPE-treated colorectal cancer cells were Triosephosphate Isomerase (Tim), Proteasome subunit alpha 4 (PSMA4) protein, Guanine nucleotide binding protein beta, Phosphoserine aminotransferase 1 (PSAT1), PSMA1, Myosin XVIIIB and Tryptophanyl-tRNA synthetase. Notably, CAPE treatment led to the down-regulation of PSAT1 and PSMA1, two proteins that have been implicated in tumorigenesis. The identified up-regulated proteins were Annexin A4, glyceraldehyde-3-phosphate dehydrogenase, Glucosamine-6-phosphate deaminase 1 (GNPDA1), and Glutathione peroxidase (GPX-1). Based on high match scores and potential role in cell growth control, PSMA1, PSAT1, GNPDA1 and GPX-1 were further validated by Western blotting and immunofluorescence assay. PSMA1 and PSAT1 were down-regulated, while GNPDA1 and GPX-1 were up-regulated in CAPE-treated colorectal cancer cells. CONCLUSION: These differentiated proteins in colorectal cancer cells following CAPE treatment, may be potential molecular targets of CAPE and involved in the anti-cancer effect of CAPE.

Deficiency of p110δ isoform of the phosphoinositide 3 kinase leads to enhanced resistance to Leishmania donovani.

BACKGROUND: Visceral leishmaniasis is the most clinically relevant and dangerous form of human leishmaniasis. Most traditional drugs for treatment of leishmaniasis are toxic, possess many adverse reactions and drug resistance is emerging. Therefore, there is urgent need for identification of new therapeutic targets. Recently, we found that mice with an inactivating knock-in mutation in the p110δ isoform of pi3k, (p110δ(d910a)) are hyper resistant to L. major, develop minimal cutaneous lesion and rapidly clear their parasite. Here, we investigated whether pi3k signaling also regulates resistance to L. donovani, one of the causative agents of visceral leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: WT and p110δ(D910A) mice (on a BALB/c background) were infected with L. donovani. At different time points, parasite burden and granuloma formation were assessed. T and B cell responses in the liver and spleen were determined. In addition, Tregs were expanded in vivo and its impact on resistance was assessed. We found that p110δ(D910A) mice had significantly reduced splenomegaly and hepatomegaly and these organs harbored significantly fewer parasites than those of WT mice. Interestingly, infected p110δ(D910A) mice liver contains fewer and less organized granulomas than their infected WT counterparts. Cells from p110δ(D910A) mice were significantly impaired in their ability to produce cytokines compared to WT mice. The percentage and absolute numbers of Tregs in infected p110δ(D910A) mice were lower than those in WT mice throughout the course of infection. In vivo expansion of Tregs in infected p110δ(D910A) mice abolished their enhanced resistance to L. donovani infection. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the enhanced resistance of p110δ(D910A) mice to L. donovani infection is due to impaired activities of Tregs. They further show that resistance to Leishmania in the absence of p110δ signaling is independent of parasite species, suggesting that targeting the PI3K signaling pathway may be useful for treatment of both visceral and cutaneous leishmaniasis.

Tryptase is a candidate autoantigen in rheumatoid arthritis.

Autoimmune processes have been implicated in the development of rheumatoid arthritis (RA); however, specific autoantigens that play a role in the aetiology of RA have been lacking. In this study, we found that sera from RA patients were particularly immunoreactive against the protein tryptase. Compared with osteoarthritis (OA) patients and healthy controls, RA patients had relatively higher levels of tryptase and concomitant anti-tryptase antibodies in their synovial tissues and sera. Similarly, synovial fluid from RA patients, but not from OA patients, contained antibodies that recognized tryptase in vitro. In addition, serum tryptase levels in both early and late RA patients significantly correlated with clinical indices usually used to diagnose RA, such as rheumatoid factor, Disease Activity Score using 28 joint counts and autoantibodies against cyclic citrullinated peptide. Our results identify tryptase as a candidate autoantigen involved in the pathogenesis of RA and monitoring its levels may have diagnostic and prognostic value.

Deficiency of Leishmania phosphoglycans influences the magnitude but does not affect the quality of secondary (memory) anti-Leishmania immunity.

Despite inducing very low IFN-γ response and highly attenuated in vivo, infection of mice with phosphoglycan (PG) deficient Leishmania major (lpg2-) induces protection against virulent L. major challenge. Here, we show that mice infected with lpg2- L. major generate Leishmania-specific memory T cells. However, in vitro and in vivo proliferation, IL-10 and IFN-γ production by lpg2- induced memory cells were impaired in comparison to those induced by wild type (WT) parasites. Interestingly, TNF recall response was comparable to WT infected mice. Despite the impaired proliferation and IFN-γ response, lpg2- infected mice were protected against virulent L. major challenge and their T cells mediated efficient infection-induced immunity. In vivo depletion and neutralization studies with mAbs demonstrated that lpg2- L. major-induced resistance was strongly dependent on IFN-γ, but independent of TNF and CD8(+) T cells. Collectively, these data show that the effectiveness of secondary anti-Leishmania immunity depends on the quality (and not the magnitude) of IFN-γ response. These observations provide further support for consideration of lpg2- L. major as a live-attenuated candidate for leishmanization in humans since it protects strongly against virulent challenge, without inducing pathology in infected animals.

CD4+CD25+ regulatory T cells attenuate lipopolysaccharide-induced systemic inflammatory responses and promotes survival in murine Escherichia coli infection.

It is well established that CD4CD25 regulatory T cells (Tregs) downregulate inflammatory immune responses and help to maintain immune homeostasis. Recent reports have shown that ligation of germline encoded pattern recognition receptors such as Toll-like receptors can stimulate Tregs and therefore implicate Tregs in the pathophysiology of sepsis and other inflammatory diseases. In this report, we show that injection of lipopolysaccharide (LPS) leads to expansion of CD4CD25FoxP3 Tregs, suggesting that these cells may play an important role in immune regulation in LPS-induced acute inflammation. Indeed, genetic or immunological inhibition of Treg function using mice lacking functional Tregs (CD25 KO mice) or anti-CD25 monoclonal antibody (anti-CD25 mAb), respectively, led to acute death in an otherwise nonlethal LPS challenge. This was accompanied by exaggerated production of proinflammatory cytokines. Strikingly, adoptive transfer of CD4CD25 Tregs to CD25 KO mice before LPS challenge rescues mice from death. Unlike LPS, depletion of Tregs followed by concanavalin A (Con A) challenge does not result in mortality, suggesting that Treg depletion does not globally influence all models of acute inflammation. We authenticate our findings by showing that depletion of Tregs leads to mortality in a nonlethal Escherichia coli challenge accompanied by elevated serum levels of proinflammatory cytokines. Collectively, our results indicate that in addition to regulation of LPS-induced acute inflammation, Tregs help to improve bacterial clearance and promote survival in an acute model of bacterial infection.