Sec22b Regulates Inflammatory Responses by Controlling the Nuclear Translocation of NF-κB and the Secretion of Inflammatory Mediators.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) regulate the vesicle transport machinery in phagocytic cells. Within the secretory pathway, Sec22b is an endoplasmic reticulum-Golgi intermediate compartment (ERGIC)-resident SNARE that controls phagosome maturation and function in macrophages and dendritic cells. The secretory pathway controls the release of cytokines and may also impact the secretion of NO, which is synthesized by the Golgi-active inducible NO synthase (iNOS). Whether ERGIC SNARE Sec22b controls NO and cytokine secretion is unknown. Using murine bone marrow-derived dendritic cells, we demonstrated that inducible NO synthase colocalizes with ERGIC/Golgi markers, notably Sec22b and its partner syntaxin 5, in the cytoplasm and at the phagosome. Pharmacological blockade of the secretory pathway hindered NO and cytokine release, and inhibited NF-κB translocation to the nucleus. Importantly, RNA interference-mediated silencing of Sec22b revealed that NO and cytokine production were abrogated at the protein and mRNA levels. This correlated with reduced nuclear translocation of NF-κB. We also found that Sec22b co-occurs with NF-κB in both the cytoplasm and nucleus, pointing to a role for this SNARE in the shuttling of NF-κB. Collectively, our data unveiled a novel function for the ERGIC/Golgi, and its resident SNARE Sec22b, in the production and release of inflammatory mediators.

VAMP3 and VAMP8 Regulate the Development and Functionality of Parasitophorous Vacuoles Housing Leishmania amazonensis.

To colonize mammalian phagocytic cells, the parasite Leishmania remodels phagosomes into parasitophorous vacuoles that can be either tight-fitting individual or communal. The molecular and cellular bases underlying the biogenesis and functionality of these two types of vacuoles are poorly understood. In this study, we investigated the contribution of host cell soluble N-ethylmaleimide-sensitive-factor attachment protein receptor proteins to the expansion and functionality of communal vacuoles as well as the replication of the parasite. The differential patterns of recruitment of soluble N-ethylmaleimide-sensitive-factor attachment protein receptor to communal vacuoles harboring Leishmania amazonensis and to individual vacuoles housing L. major led us to further investigate the roles of VAMP3 and VAMP8 in the interaction of Leishmania with its host cell. We show that whereas VAMP8 contributes to the optimal expansion of communal vacuoles, VAMP3 negatively regulates L. amazonensis replication, vacuole size, as well as antigen cross-presentation. In contrast, neither protein has an impact on the fate of L. major. Collectively, our data support a role for both VAMP3 and VAMP8 in the development and functionality of L. amazonensis-harboring communal parasitophorous vacuoles.

Bacillus Calmette-Guerin vaccination and multiple sclerosis: A population-based birth cohort study in Quebec, Canada.

BACKGROUND AND PURPOSE: The bacillus Calmette-Guerin (BCG) vaccine could reduce the incidence of multiple sclerosis (MS) through immunomodulation. Previous studies, presenting some limitations, reported no association. We re-examined this association in a large cohort focusing on relapsing-remitting MS (RRMS). METHODS: The cohort included 400,563 individuals, and was linked with the Quebec provincial BCG vaccination registry and administrative health data. Individuals were followed up from 1983 to 2014 and then within Period 1 (1983-1996) and Period 2 (1997-2014), for the occurrence of MS. Incident MS cases were defined as those with ≥3 hospital or physician claims for MS. Subjects with ≥1 drug reimbursement for MS disease-modifying therapies were classified as RRMS. Cox proportional hazards regression was used to estimate hazard ratios (HRs) over the follow-ups, adjusting for potential confounders. Possible effect modification due to sex was assessed. RESULTS: A total of 178,335 (46%) individuals were BCG vaccinated. There were 274 (0.06%) incident MS cases identified in 1983-1996, and 1433 (0.4%) in 1997-2014. No association was found with RRMS, either in Period 1 (adjusted HR [HRadj ] = 0.96, 95% confidence interval [CI] = 0.63-1.45; 96 cases) or in Period 2 (HRadj  = 1.02, 95% CI = 0.85-1.23; 480 cases). The remaining MS cases, for whom the phenotype was unknown, were positively associated with BCG over the entire follow-up (HRadj  = 1.25, 95% CI = 1.10-1.41; 1131 cases) and in Period 2 (HRadj  = 1.33, 95% CI = 1.17-1.52; 953 cases). No interaction with sex was found. CONCLUSIONS: Findings suggest that BCG vaccination does not decrease the risk of RRMS, and that future studies should consider phenotypes of MS.

Association between Bacillus Calmette-Guerin vaccination and type 1 diabetes in adolescence: A population-based birth cohort study in Quebec, Canada.

The Bacillus Calmette-Guerin (BCG) vaccine could reduce the incidence of type 1 diabetes through non-specific immunomodulation. Previous epidemiological studies, presenting some limitations, report no association. We examined this association of early life BCG vaccination and age at vaccination with type 1 diabetes incidence in adolescence in a large representative cohort in Quebec. The cohort included 387,704 individuals born in Quebec between 1970 and 1974 whose BCG vaccination status was determined from a provincial registry. Individuals were followed up from 1985 to their 19th birthday (maximum to 1993) for their use of physician services. Individuals were defined as type 1 diabetes cases if they had ≥4 related physician claims over a 2-year period, with at least 30 days between two claims. Cox proportional hazards regression was used to estimate the association of BCG vaccination and age at vaccination with type 1 diabetes. Covariates were selected based on a directed acyclic graph. Interaction according to sex was evaluated. A total of 178,133 (45.9%) individuals were vaccinated and 442 (0.11%) incident cases of type 1 diabetes were identified. The risk of type 1 diabetes was similar in vaccinated compared with unvaccinated individuals (adjusted hazard ratio = 1.06 [95% CI: 0.88-1.29]). There was no association with age at vaccination, and results did not differ by sex (Interaction, p = 0.60). Our results suggest that BCG vaccination does not prevent type 1 diabetes in adolescence.

Hypergammaglobulinemia sustains the development of regulatory responses during chronic Leishmania donovani infection in mice.

Visceral leishmaniasis, a chronic, potentially fatal disease, is characterized by high production of low-affinity antibodies. In humans, hypergammaglobulinemia is prediction of disease progression. Nevertheless, the contribution of hypermutated and/or class-switched immunoglobulins to disease pathogenesis has never been studied. Using Aicda-/- mice and the experimental model of Leishmania donovani infection, we demonstrate that the absence of hypermutated and/or class-switched antibodies was associated with increased resistance to disease, stronger protective Th1 responses, and a lower frequency of regulatory IFNγ+ IL-10+ CD4 T cells. Interestingly, stronger Th1 responses and the absence of IFNγ+ IL-10+ CD4 T cells during chronic infection in infected Aicda-/- mice were not caused by a T-cell intrinsic effect of AID, but by changes in the cytokine environment during chronic disease. Indeed TNF, IL-10 and IFN-ß expressions were only upregulated in the presence of hypermutated, class-switched antibodies and hypergammaglobulinemia at later stages of infection. Taken together, our results suggest that hypergammaglobulinemia sustains inhibitory responses during chronic visceral leishmaniasis.

eIF4E-Binding Proteins 1 and 2 Limit Macrophage Anti-Inflammatory Responses through Translational Repression of IL-10 and Cyclooxygenase-2.

Macrophages represent one of the first lines of defense during infections and are essential for resolution of inflammation following pathogen clearance. Rapid activation or suppression of protein synthesis via changes in translational efficiency allows cells of the immune system, including macrophages, to quickly respond to external triggers or cues without de novo mRNA synthesis. The translational repressors eIF4E-binding proteins 4E-BP1 and 4E-BP2 (4E-BP1/2) are central regulators of proinflammatory cytokine synthesis during viral and parasitic infections. However, it remains to be established whether 4E-BP1/2 play a role in translational control of anti-inflammatory responses. By comparing translational efficiencies of immune-related transcripts in macrophages from wild-type and 4E-BP1/2 double-knockout mice, we found that translation of mRNAs encoding two major regulators of inflammation, IL-10 and PG-endoperoxide synthase 2/cyclooxygenase-2, is controlled by 4E-BP1/2. Genetic deletion of 4E-BP1/2 in macrophages increased endogenous IL-10 and PGE2 protein synthesis in response to TLR4 stimulation and reduced their bactericidal capacity. The molecular mechanism involves enhanced anti-inflammatory gene expression (sIl1ra, Nfil3, Arg1, Serpinb2) owing to upregulation of IL-10-STAT3 and PGE2-C/EBPß signaling. These data provide evidence that 4E-BP1/2 limit anti-inflammatory responses in macrophages and suggest that dysregulated activity of 4E-BP1/2 might be involved in reprogramming of the translational and downstream transcriptional landscape of macrophages during pathological conditions, such as infections and cancer.

Infection-adapted emergency hematopoiesis promotes visceral leishmaniasis.

Cells of the immune system are derived from hematopoietic stem cells (HSCs) residing in the bone marrow. HSCs become activated in response to stress, such as acute infections, which adapt the bone marrow output to the needs of the immune response. However, the impact of infection-adapted HSC activation and differentiation on the persistence of chronic infections is poorly understood. We have examined here the bone marrow outcome of chronic visceral leishmaniasis and show that the parasite Leishmania donovani induces HSC expansion and skews their differentiation towards non-classical myeloid progenitors with a regulatory phenotype. Our results further suggest that emergency hematopoiesis contributes to the pathogenesis of visceral leishmaniasis, as decreased HSC expansion results in a lower parasite burden. Conversely, monocytes derived in the presence of soluble factors from the infected bone marrow environment are more permissive to infection by Leishmania. Our results demonstrate that L. donovani is able to subvert host bone marrow emergency responses to facilitate parasite persistence, and put forward hematopoiesis as a novel therapeutic target in chronic infections.

HIF-1α is a key regulator in potentiating suppressor activity and limiting the microbicidal capacity of MDSC-like cells during visceral leishmaniasis.

Leishmania donovani is known to induce myelopoiesis and to dramatically increase extramedullary myelopoiesis. This results in splenomegaly, which is then accompanied by disruption of the splenic microarchitecture, a chronic inflammatory environment, and immunosuppression. Chronically inflamed tissues are typically hypoxic. The role of hypoxia on myeloid cell functions during visceral leishmaniasis has not yet been studied. Here we show that L. donovani promotes the output from the bone marrow of monocytes with a regulatory phenotype that function as safe targets for the parasite. We also demonstrate that splenic myeloid cells acquire MDSC-like function in a HIF-1α-dependent manner. HIF-1α is also involved in driving the polarization towards M2-like macrophages and rendering intermediate stage monocytes more susceptible to L. donovani infection. Our results suggest that HIF-1α is a major player in the establishment of chronic Leishmania infection and is crucial for enhancing immunosuppressive functions and lowering leishmanicidal capacity of myeloid cells.

Dok-1 and Dok-2 Regulate the Formation of Memory CD8+ T Cells.

Diverse signals received by CD8+ T cells are integrated to achieve the required magnitude of cell expansion and the appropriate balance of effector/memory CD8+ T cell generation. Notably, the strength and nature of TCR signaling influence the differentiation and functional capacity of effector and memory CD8+ T cells. Dok-1 and Dok-2, the two members of the Dok family expressed in T cells, negatively regulate TCR signaling in vitro. However, the role of Dok proteins in modulating T cell function in vivo has not yet studied. We studied the function of Dok-1 and Dok-2 proteins in the regulation of the CD8+ T cell response to vaccinia virus infection. Comparison of responses to vaccinia virus expressing OVA peptide SIINFEKL by wild-type and Dok-1/2-/- CD8+ OT-I cells showed that the absence of Dok-1 and Dok-2 slightly reduced the magnitude of virus-specific effector CD8+ T cell expansion. This was not due to reduced proliferation or enhanced apoptosis of effector CD8+ T cells. Dok-1/2-deficient effector CD8+ T cells showed increased cell surface TCR expression following virus infection in vivo and increased expression of granzyme B and TNF upon stimulation with peptide Ag ex vivo. Finally, Dok-1/2-deficient effector CD8+ T had a severe defect in survival that resulted in impaired generation of memory CD8+ T cells. These results reveal the critical involvement of Dok-1 and Dok-2 in a negative-feedback loop that prevents overactivation of CD8+ T cells and promotes memory formation.

IRF-5-Mediated Inflammation Limits CD8+ T Cell Expansion by Inducing HIF-1α and Impairing Dendritic Cell Functions during Leishmania Infection.

Inflammation is known to be necessary for promoting, sustaining, and tuning CD8+ T cell responses. Following experimental Leishmania donovani infection, the inflammatory response is mainly induced by the transcription factor IRF-5. IRF-5 is responsible for the activation of several genes encoding key pro-inflammatory cytokines, such as IL-6 and TNF. Here, we investigate the role of IRF-5-mediated inflammation in regulating antigen-specific CD8+ T cell responses during L. donovani infection. Our data demonstrate that the inflammatory response induced by IRF-5 limits CD8+ T cell expansion and induces HIF-1α in dendritic cells. Ablation of HIF-1α in CD11c+ cells resulted into a higher frequency of short-lived effector cells (SLEC), enhanced CD8+ T cell expansion, and increased IL-12 expression by splenic DCs. Moreover, mice with a targeted depletion of HIF-1α in CD11c+ cells had a significantly lower splenic parasite burden, suggesting that induction of HIF-1α may represent an immune evasive mechanism adopted by Leishmania parasites to establish persistent infections.

Posttranscriptional regulation of II10 gene expression allows natural killer cells to express immunoregulatory function.

Natural killer (NK) cells play a well-recognized role in early pathogen containment and in shaping acquired cell-mediated immunity. However, indirect evidence in humans and experimental models has suggested that NK cells also play negative regulatory roles during chronic disease. To formally test this hypothesis, we employed a well-defined experimental model of visceral leishmaniasis. Our data demonstrated that NKp46(+)CD49b(+)CD3(-) NK cells were recruited to the spleen and into hepatic granulomas, where they inhibited host protective immunity in an interleukin-10 (IL-10)-dependent manner. Although IL-10 mRNA could be detected in activated NK cells 24 hr after infection, the inhibitory function of NK cells was only acquired later during infection, coincident with increased IL-10 mRNA stability and an enhanced capacity to secrete IL-10 protein. Our data support a growing body of literature that implicates NK cells as negative regulators of cell-mediated immunity and suggest that NK cells, like CD4(+) T helper 1 cells, may acquire immunoregulatory functions as a consequence of extensive activation.

Hypoxia inducible factor 1α: A critical factor for the immune response to pathogens and Leishmania.

Organisms typically react to foreign pathogens by initiating an inflammatory response. However, in order to limit inflammatory tissue injury, it is essential for the organism to maintain the balance between inflammatory and anti-inflammatory responses. Dysregulation of this process can result in the strong inhibition of protective pro-inflammatory responses, and ultimately in pathogen persistence. Chronic infections are often associated with inflammation and tissue disruption. Inflamed tissues are characterized by low levels of oxygen and glucose, a microenvironment that triggers the stabilization of the hypoxia-inducible transcription factor HIF-1α. HIF-1α is the master regulator of the response to hypoxia. Here, we review the role of HIF-1α in the immune response to various pathogens and we highlight how certain microorganisms, including Leishmania parasites, have evolved to hijack the HIF-1 pathway to their advantage.

Leishmania promastigotes induce cytokine secretion in macrophages through the degradation of synaptotagmin XI.

Synaptotagmins (Syts) are type-I membrane proteins that regulate vesicle docking and fusion in processes such as exocytosis and phagocytosis. We recently discovered that Syt XI is a recycling endosome- and lysosome-associated protein that negatively regulates the secretion of TNF and IL-6. In this study, we show that Syt XI is directly degraded by the zinc metalloprotease GP63 and excluded from Leishmania parasitophorous vacuoles by the promastigotes surface glycolipid lipophosphoglycan. Infected macrophages were found to release TNF and IL-6 in a GP63-dependent manner. To demonstrate that cytokine release was dependent on GP63-mediated degradation of Syt XI, small interfering RNA-mediated knockdown of Syt XI before infection revealed that the effects of small interfering RNA knockdown and GP63 degradation were not cumulative. In mice, i.p. injection of GP63-expressing parasites led to an increase in TNF and IL-6 secretion and to an augmented influx of neutrophils and inflammatory monocytes to the inoculation site. Both of these cell types have been shown to be infection targets and aid in the establishment of infection. In sum, our data revealed that GP63 induces proinflammatory cytokine release and increases infiltration of inflammatory phagocytes. This study provides new insight on how Leishmania exploits the immune response to establish infection.

Marginal zone B cells regulate antigen-specific T cell responses during infection.

Marginal zone B cells (MZB) participate in the early immune response to several pathogens. In this study, we show that in µMT mice infected with Leishmania donovani, CD8 T cells displayed a greater cytotoxic potential and generated more effector memory cells compared with infected wild type mice. The frequency of parasite-specific, IFN-γ(+) CD4 T cells was also increased in µMT mice. B cells were able to capture parasites, which was associated with upregulation of surface IgM and MyD88-dependent IL-10 production. Moreover, MZB presented parasite Ags to CD4 T cells in vitro. Depletion of MZB also enhanced T cell responses and led to a decrease in the parasite burden but did not alter the generation of effector memory T cells. Thus, MZB appear to suppress protective T cell responses during the early stages of L. donovani infection.

LAG-3- and CXCR5-expressing CD4 T cells display progenitor-like properties during chronic visceral leishmaniasis.

Maintenance of CD4 T cells during chronic infections is vital for limiting pathogen burden and disease recrudescence. Although inhibitory receptor expression by CD4 T cells is commonly associated with immune suppression and exhaustion, such cell-intrinsic mechanisms that control activation are also associated with cell survival. Using a mouse model of visceral leishmaniasis (VL), we discovered a subset of lymphocyte activation gene 3 (LAG-3)-expressing CD4 T cells that co-express CXCR5. Although LAG3+CXCR5+ CD4 T cells are present in naive mice, they expand during VL. These cells express gene signatures associated with self-renewal capacity, suggesting progenitor-like properties. When transferred into Rag1-/- mice, these LAG3+CXCR5+ CD4 T cells differentiated into multiple effector types upon Leishmania donovani infection. The transcriptional repressor B cell lymphoma-6 was partially required for their maintenance. Altogether, we propose that the LAG3+CXCR5+ CD4 T cell subset could play a role in maintaining CD4 T cell responses during persistent infections.

Critical role of IRF-5 in the development of T helper 1 responses to Leishmania donovani infection.

The transcription factor Interferon Regulatory Factor 5 (IRF-5) has been shown to be involved in the induction of proinflammatory cytokines in response to viral infections and TLR activation and to play an essential role in the innate inflammatory response. In this study, we used the experimental model of visceral leishmaniasis to investigate the role of IRF-5 in the generation of Th1 responses and in the formation of Th1-type liver granulomas in Leishmania donovani infected mice. We show that TLR7-mediated activation of IRF-5 is essential for the development of Th1 responses to L. donovani in the spleen during chronic infection. We also demonstrate that IRF-5 deficiency leads to the incapacity to control L. donovani infection in the liver and to the formation of smaller granulomas. Granulomas in Irf5⁻/⁻ mice are characterized by an increased IL-4 and IL-10 response and concomitant low iNOS expression. Collectively, these results identify IRF-5 as a critical molecular switch for the development of Th1 immune responses following L. donovani infections and reveal an indirect role of IRF-5 in the regulation of iNOS expression.

Leishmania donovani Exploits Tunneling Nanotubes for Dissemination and Propagation of B Cell Activation.

Polyclonal B cell activation and the resulting hypergammaglobulinemia are a detrimental consequence of visceral leishmaniasis (VL); however, the mechanisms underlying this excessive production of nonprotective antibodies are still poorly understood. Here, we show that a causative agent of VL, Leishmania donovani, induces CD21-dependent formation of tunneling nanotubule (TNT)-like protrusions in B cells. These intercellular connections are used by the parasite to disseminate among cells and propagate B cell activation, and close contact both among the cells and between B cells and parasites is required to achieve this activation. Direct contact between cells and parasites is also observed in vivo, as L. donovani can be detected in the splenic B cell area as early as 14 days postinfection. Interestingly, Leishmania parasites can also glide from macrophages to B cells via TNT-like protrusions. Taken together, our results suggest that, during in vivo infection, B cells may acquire L. donovani from macrophages via TNT-like protrusions, and these connections are subsequently exploited by the parasite to disseminate among B cells, thus propagating B cell activation and ultimately leading to polyclonal B cell activation. IMPORTANCE Leishmania donovani is a causative agent of visceral leishmaniasis, a potentially lethal disease characterized by strong B cell activation and the subsequent excessive production of nonprotective antibodies, which are known to worsen the disease. How Leishmania activates B cells is still unknown, particularly because this parasite mostly resides inside macrophages and would not have access to B cells during infection. In this study, we describe for the first time how the protozoan parasite Leishmania donovani induces and exploits the formation of protrusions that connect B lymphocytes with each other or with macrophages and glides on these structures from one cell to another. In this way, B cells can acquire Leishmania from macrophages and become activated upon contact with the parasites. This activation will then lead to antibody production. These findings provide an explanation for how the parasite may propagate B cell activation during infection.

The TLR7/IRF-5 axis sensitizes memory CD4+ T cells to Fas-mediated apoptosis during HIV-1 infection.

HIV-1 infection is characterized by inflammation and a progressive decline in CD4+ T cell count. Despite treatment with antiretroviral therapy (ART), the majority of people living with HIV (PLWH) maintain residual levels of inflammation, a low degree of immune activation, and higher sensitivity to cell death in their memory CD4+ T cell compartment. To date, the mechanisms responsible for this high sensitivity remain elusive. We have identified the transcription factor IRF-5 to be involved in impairing the maintenance of murine CD4+ T cells during chronic infection. Here, we investigate whether IRF-5 also contributes to memory CD4+ T cell loss during HIV-1 infection. We show that TLR7 and IRF-5 were upregulated in memory CD4+ T cells from PLWH, when compared with naturally protected elite controllers and HIVfree participants. TLR7 was upstream of IRF-5, promoting Caspase 8 expression in CD4+ T cells from ART HIV-1+ but not from HIVfree donors. Interestingly, the TLR7/IRF-5 axis acted synergistically with the Fas/FasL pathway, suggesting that TLR7 and IRF-5 expression in ART HIV-1+ memory CD4+ T cells represents an imprint that predisposes cells to Fas-mediated apoptosis. This predisposition could be blocked using IRF-5 inhibitory peptides, suggesting IRF-5 blockade as a possible therapy to prevent memory CD4+ T cell loss in PLWH.

Bcl-6 expression by CD4+ T cells determines concomitant immunity and host resistance across distinct parasitic infections.

Cluster of differentiation (CD4+) T cells consist of multiple subtypes, defined by expression of lineage-specific transcription factors, that contribute to the control of infectious diseases by providing help to immune and nonimmune target cells. In the current study, we examined the role of B cell lymphoma (Bcl)-6, a transcriptional repressor and master regulator of T follicular helper cell differentiation, in T cell-mediated host defense against intestinal and systemic parasitic infections. We demonstrate that while Bcl-6 expression by CD4+ T cells is critical for antibody-mediated protective immunity against secondary infection with the nematode Heligmosoides polygyrus bakeri, it paradoxically compromises worm expulsion during primary infection by limiting the generation of interleukin-10 (IL-10)-producing Gata3+ T helper 2 cells. Enhanced worm expulsion in the absence of Bcl-6 expressing T cells was associated with amplified intestinal goblet cell differentiation and increased generation of alternatively activated macrophages, effects that were reversed by neutralization of IL-10 signals. An increase in IL-10 production by Bcl-6-deficient CD4+ T cells was also evident in the context of systemic Leishmania donovani infection, but in contrast to Heligmosoides polygyrus bakeri infection, compromised T helper 1-mediated liver macrophage activation and increased susceptibility to this distinct parasitic challenge. Collectively, our studies suggest that host defense pathways that protect against parasite superinfection and lethal systemic protozoal infections can be engaged at the cost of compromised primary resistance to well-tolerated helminths.

A human IL10 BAC transgene reveals tissue-specific control of IL-10 expression and alters disease outcome.

Interleukin (IL)-10 is an immunoregulatory cytokine that is produced by diverse cell populations. Studies in mice suggest that the cellular source of IL-10 is a key determinant in various disease pathologies, yet little is known regarding the control of tissue-specific human IL-10 expression. To assess cell type-specific human IL-10 regulation, we created a human IL-10 transgenic mouse with a bacterial artificial chromosome (hIL10BAC) in which the IL10 gene is positioned centrally. Since human IL-10 is biologically active in the mouse, we could examine the in vivo capacity of tissue-specific human IL-10 expression to recapitulate IL-10-dependent phenotypes by reconstituting Il10(-/-) mice (Il10(-/-)/hIL10BAC). In response to LPS, Il10(-/-)/hIL10BAC mice proficiently regulate IL-10-target genes and normalize sensitivity to LPS toxicity via faithful human IL-10 expression from macrophages and dendritic cells. However, in the Leishmania donovani model of pathogen persistence, Il10(-/-)/hIL10BAC mice did not develop the characteristic IL-10(+)IFN-gamma(+)CD4 T cell subset thought to mediate persistence and, like Il10(-/-) mice, cleared the parasites. Furthermore, the IL-10-promoting cytokine IL-27 failed to regulate transgenic human IL-10 production in CD4(+) T cells in vitro which together suggests that the hIL10BAC encodes for weak T cell-specific IL-10 expression. Thus, the hIL10BAC mouse is a model of human gene structure and function revealing tissue-specific regulatory requirements for IL-10 expression which impacts disease outcomes.