Elimination of Chlamydia muridarum from the female reproductive tract is IL-12p40 dependent, but independent of Th1 and Th2 cells.

Chlamydia vaccine approaches aspire to induce Th1 cells for optimal protection, despite the fact that there is no direct evidence demonstrating Th1-mediated Chlamydia clearance from the female reproductive tract (FRT). We recently reported that T-bet-deficient mice can resolve primary Chlamydia infection normally, undermining the potentially protective role of Th1 cells in Chlamydia immunity. Here, we show that T-bet-deficient mice develop robust Th17 responses and that mice deficient in Th17 cells exhibit delayed bacterial clearance, demonstrating that Chlamydia-specific Th17 cells represent an underappreciated protective population. Additionally, Th2-deficient mice competently clear cervicovaginal infection. Furthermore, we show that sensing of IFN-γ by non-hematopoietic cells is essential for Chlamydia immunity, yet bacterial clearance in the FRT does not require IFN-γ secretion by CD4 T cells. Despite the fact that Th1 cells are not necessary for Chlamydia clearance, protective immunity to Chlamydia is still dependent on MHC class-II-restricted CD4 T cells and IL-12p40. Together, these data point to IL-12p40-dependent CD4 effector maturation as essential for Chlamydia immunity, and Th17 cells to a lesser extent, yet neither Th1 nor Th2 cell development is critical. Future Chlamydia vaccination efforts will be more effective if they focus on induction of this protective CD4 T cell population.

Optimal generation of hepatic tissue-resident memory CD4 T cells requires IL-1 and IL-2.

Hepatic CD4 tissue-resident memory T cells (TRM) are required for robust protection against Salmonella infection; however, the generation of this T cell population is poorly understood. To interrogate the contribution of inflammation, we developed a simple Salmonella-specific T cell transfer system that allowed direct visualization of hepatic TRM formation. Salmonella-specific (SM1) T cell receptor (TCR) transgenic CD4 T cells were activated in vitro and adoptively transferred into C57BL/6 mice while hepatic inflammation was induced by acetaminophen overdose or L. monocytogenes infection. In both model systems, hepatic CD4 TRM formation was accentuated by local tissue responses. Liver inflammation also enhanced the suboptimal protection provided by a subunit Salmonella vaccine which typically induces circulating memory CD4 T cells. To further elucidate the mechanism of CD4 TRM formation in response to liver inflammation, various cytokines were examined by RNAseq, bone marrow chimeras, and in vivo neutralization. Surprisingly, IL-2 and IL-1 were found to enhance CD4 TRM formation. Thus, local inflammatory mediators enhance CD4 TRM populations and can boost the protective immunity provided by a suboptimal vaccine. This knowledge will be foundational for the development of a more effective vaccine against invasive nontyphoidal salmonellosis (iNTS).

Cutting Edge: Optimal Formation of Hepatic Tissue-Resident Memory CD4 T Cells Requires T-bet Regulation of CD18.

CD4 tissue-resident memory T cells (TRMs) allow robust protection of barrier surfaces against pathogens. We investigated the role of T-bet in the formation of liver CD4 TRMs using mouse models. T-bet-deficient CD4 T cells did not efficiently form liver TRMs when compared with wild-type (WT). In addition, ectopic expression of T-bet enhanced the formation of liver CD4 TRMs, but only when in competition with WT CD4 T cells. Liver TRMs also expressed higher levels of CD18, which was T-bet dependent. The WT competitive advantage was blocked by Ab neutralization of CD18. Taken together, our data show that activated CD4 T cells compete for entry to liver niches via T-bet-induced expression of CD18, allowing TRM precursors to access subsequent hepatic maturation signals. These findings uncover an essential role for T-bet in liver TRM CD4 formation and suggest targeted enhancement of this pathway could increase the efficacy of vaccines that require hepatic TRMs.

Th1 cells are dispensable for primary clearance of Chlamydia from the female reproductive tract of mice.

Protective immune responses to Chlamydia infection within the female reproductive tract (FRT) are incompletely understood. MHC class II-restricted CD4 Th1 responses are believed to be vital for bacterial clearance due to their capacity to secrete IFN-γ, but an essential requirement for T-bet-expressing Th1 cells has yet to be demonstrated in the mouse model of Chlamydia infection. Here, we investigated the role of T-bet and IFN-γ in primary clearance of Chlamydia after FRT infection. Surprisingly, IFN-γ producing CD4 T cells from the FRT expressed low levels of T-bet throughout infection, suggesting that classical T-bet-expressing Th1 cells are inefficiently generated and therefore unlikely to participate in bacteria clearance. Furthermore, mice deficient in T-bet expression or with a CD4-specific T-bet deficiency cleared FRT infection similarly to wild-type controls. T-bet-deficient mice displayed significant skewing of FRT CD4 T cells towards Th17 responses, demonstrating that compensatory effector pathways are generated in the absence of Th1 cells. In marked contrast, IFN-γ-, and IFN-γR-deficient mice were able to reduce FRT bacterial burdens, but suffered systemic bacterial dissemination and 100% mortality. Together, these data demonstrate that IFN-γ signaling is essential to protect mice from fatal systemic disease, but that classical T-bet-expressing Th1 cells are non-essential for primary clearance within the FRT. Exploring the protective contribution of Th1 cells versus other CD4 effector lineages could provide important information for the generation of new Chlamydia vaccines.

The Ets transcription factor Spi-B is essential for the differentiation of intestinal microfold cells.

Intestinal microfold cells (M cells) are an enigmatic lineage of intestinal epithelial cells that initiate mucosal immune responses through the uptake and transcytosis of luminal antigens. The mechanisms of M-cell differentiation are poorly understood, as the rarity of these cells has hampered analysis. Exogenous administration of the cytokine RANKL can synchronously activate M-cell differentiation in mice. Here we show the Ets transcription factor Spi-B was induced early during M-cell differentiation. Absence of Spi-B silenced the expression of various M-cell markers and prevented the differentiation of M cells in mice. The activation of T cells via an oral route was substantially impaired in the intestine of Spi-B-deficient (Spib(-/-)) mice. Our study demonstrates that commitment to the intestinal M-cell lineage requires Spi-B as a candidate master regulator.

Salmonella Infection Drives Promiscuous B Cell Activation Followed by Extrafollicular Affinity Maturation.

The B cell response to Salmonella typhimurium (STm) occurs massively at extrafollicular sites, without notable germinal centers (GCs). Little is known in terms of its specificity. To expand the knowledge of antigen targets, we screened plasmablast (PB)-derived monoclonal antibodies (mAbs) for Salmonella specificity, using ELISA, flow cytometry, and antigen microarray. Only a small fraction (0.5%-2%) of the response appeared to be Salmonella-specific. Yet, infection of mice with limited B cell receptor (BCR) repertoires impaired the response, suggesting that BCR specificity was important. We showed, using laser microdissection, that somatic hypermutation (SHM) occurred efficiently at extrafollicular sites leading to affinity maturation that in turn led to detectable STm Ag-binding. These results suggest a revised vision of how clonal selection and affinity maturation operate in response to Salmonella. Clonal selection initially is promiscuous, activating cells with virtually undetectable affinity, yet SHM and selection occur during the extrafollicular response yielding higher affinity, detectable antibodies.

Circulating immunity protects the female reproductive tract from Chlamydia infection.

Anatomical positioning of memory lymphocytes within barrier tissues accelerates secondary immune responses and is thought to be essential for protection at mucosal surfaces. However, it remains unclear whether resident memory in the female reproductive tract (FRT) is required for Chlamydial immunity. Here, we describe efficient generation of tissue-resident memory CD4 T cells and memory lymphocyte clusters within the FRT after vaginal infection with Chlamydia Despite robust establishment of localized memory lymphocytes within the FRT, naïve mice surgically joined to immune mice, or mice with only circulating immunity following intranasal immunization, were fully capable of resisting Chlamydia infection via the vaginal route. Blocking the rapid mobilization of circulating memory CD4 T cells to the FRT inhibited this protective response. These data demonstrate that secondary protection in the FRT can occur in the complete absence of tissue-resident immune cells. The ability to confer robust protection to barrier tissues via circulating immune memory provides an unexpected opportunity for vaccine development against infections of the FRT.

CD4+ T cell immunity to Salmonella is transient in the circulation.

While Salmonella enterica is seen as an archetypal facultative intracellular bacterial pathogen where protection is mediated by CD4+ T cells, identifying circulating protective cells has proved very difficult, inhibiting steps to identify key antigen specificities. Exploiting a mouse model of vaccination, we show that the spleens of C57BL/6 mice vaccinated with live-attenuated Salmonella serovar Typhimurium (S. Typhimurium) strains carried a pool of IFN-γ+ CD4+ T cells that could adoptively transfer protection, but only transiently. Circulating Salmonella-reactive CD4+ T cells expressed the liver-homing chemokine receptor CXCR6, accumulated over time in the liver and assumed phenotypic characteristics associated with tissue-associated T cells. Liver memory CD4+ T cells showed TCR selection bias and their accumulation in the liver could be inhibited by blocking CXCL16. These data showed that the circulation of CD4+ T cells mediating immunity to Salmonella is limited to a brief window after which Salmonella-specific CD4+ T cells migrate to peripheral tissues. Our observations highlight the importance of triggering tissue-specific immunity against systemic infections.

Toll-like receptor and inflammasome signals converge to amplify the innate bactericidal capacity of T helper 1 cells.

T cell effector functions can be elicited by noncognate stimuli, but the mechanism and contribution of this pathway to the resolution of intracellular macrophage infections have not been defined. Here, we show that CD4(+) T helper 1 (Th1) cells could be rapidly stimulated by microbe-associated molecular patterns during active infection with Salmonella or Chlamydia. Further, maximal stimulation of Th1 cells by lipopolysaccharide (LPS) did not require T-cell-intrinsic expression of toll-like receptor 4 (TLR4), interleukin-1 receptor (IL-1R), or interferon-γ receptor (IFN-γR) but instead required IL-18R, IL-33R, and adaptor protein MyD88. Innate stimulation of Th1 cells also required host expression of TLR4 and inflammasome components that together increased serum concentrations of IL-18. Finally, the elimination of noncognate Th1 cell stimulation hindered the resolution of primary Salmonella infection. Thus, the in vivo bactericidal capacity of Th1 cells is regulated by the response to noncognate stimuli elicited by multiple innate immune receptors.

NOD1 and NOD2 signalling links ER stress with inflammation.

Endoplasmic reticulum (ER) stress is a major contributor to inflammatory diseases, such as Crohn disease and type 2 diabetes. ER stress induces the unfolded protein response, which involves activation of three transmembrane receptors, ATF6, PERK and IRE1α. Once activated, IRE1α recruits TRAF2 to the ER membrane to initiate inflammatory responses via the NF-κB pathway. Inflammation is commonly triggered when pattern recognition receptors (PRRs), such as Toll-like receptors or nucleotide-binding oligomerization domain (NOD)-like receptors, detect tissue damage or microbial infection. However, it is not clear which PRRs have a major role in inducing inflammation during ER stress. Here we show that NOD1 and NOD2, two members of the NOD-like receptor family of PRRs, are important mediators of ER-stress-induced inflammation in mouse and human cells. The ER stress inducers thapsigargin and dithiothreitol trigger production of the pro-inflammatory cytokine IL-6 in a NOD1/2-dependent fashion. Inflammation and IL-6 production triggered by infection with Brucella abortus, which induces ER stress by injecting the type IV secretion system effector protein VceC into host cells, is TRAF2, NOD1/2 and RIP2-dependent and can be reduced by treatment with the ER stress inhibitor tauroursodeoxycholate or an IRE1α kinase inhibitor. The association of NOD1 and NOD2 with pro-inflammatory responses induced by the IRE1α/TRAF2 signalling pathway provides a novel link between innate immunity and ER-stress-induced inflammation.

Antibody, but not B-cell-dependent antigen presentation, plays an essential role in preventing Chlamydia systemic dissemination in mice.

The obligate intracellular bacterium Chlamydia trachomatis causes the most prevalent bacterial sexually transmitted infection worldwide. CD4 T cells play a central role in the protective immunity against Chlamydia female reproductive tract (FRT) infection, while B cells are thought to be dispensable for resolution of primary Chlamydia infection in mouse models. We recently reported an unexpected requirement of B cells in local Chlamydia-specific CD4 T-cell priming and bacterial containment within the FRT. Here, we sought to tackle the precise effector function of B cells during Chlamydia primary infection. Using mixed bone marrow chimeras that lack B-cell-dependent Ag presentation (MHCIIB-/- ) or devoid of circulating antibodies (AID-/- × µS-/- ), we show that Chlamydia-specific CD4 T-cell expansion does not rely on Ag presentation by B cells. Importantly, we demonstrate that antibody, but not B-cell-dependent Ag presentation, is required for preventing systemic bacterial dissemination following Chlamydia FRT infection.

Optimal protection against Salmonella infection requires noncirculating memory.

While CD4 Th1 cells are required for resistance to intramacrophage infections, adoptive transfer of Th1 cells is insufficient to protect against Salmonella infection. Using an epitope-tagged vaccine strain of Salmonella, we found that effective protection correlated with expanded Salmonella-specific memory CD4 T cells in circulation and nonlymphoid tissues. However, naive mice that previously shared a blood supply with vaccinated partners lacked T cell memory with characteristics of tissue residence and did not acquire robust protective immunity. Using a YFP-IFN-γ reporter system, we identified Th1 cells in the liver of immunized mice that displayed markers of tissue residence, including P2X7, ARTC2, LFA-1, and CD101. Adoptive transfer of liver memory cells after ARTC2 blockade increased protection against highly virulent bacteria. Taken together, these data demonstrate that noncirculating memory Th1 cells are a vital component of immunity to Salmonella infection and should be the focus of vaccine strategies.

Unexpected Role of CD8 T Cells in Accelerated Clearance of Salmonella enterica Serovar Typhimurium from H-2 Congenic mice.

Salmonella infection can cause gastroenteritis in healthy individuals or a serious, systemic infection in immunocompromised patients and has a global impact. CD4 Th1 cells represent the main lymphocyte population that participates in bacterial clearance during both primary and secondary infections in mice of the H-2b haplotype. Previous studies have used congenic mice to examine the function of major histocompatibility complex (MHC) molecules in elimination of this pathogen from the host. In this study, we further characterized the ability of H-2b, H-2k, and H-2u molecules to influence adaptive immunity to Salmonella in MHC congenic mice. By depleting different cell populations during infection, we unexpectedly found that CD8 T cells, in addition to CD4 T cells, play a major role in accelerated clearance of bacteria from H-2k congenic hosts. Our data suggest that CD8 T cells accelerate clearance in some MHC congenic mouse strains and could therefore represent an unexpected contributor to the protective efficacy of Salmonella vaccines outside the typical studies in C57BL/6 mice.

T cell expression of IL-18R and DR3 is essential for non-cognate stimulation of Th1 cells and optimal clearance of intracellular bacteria.

Th1 cells can be activated by TCR-independent stimuli, but the importance of this pathway in vivo and the precise mechanisms involved require further investigation. Here, we used a simple model of non-cognate Th1 cell stimulation in Salmonella-infected mice to examine these issues. CD4 Th1 cell expression of both IL-18R and DR3 was required for optimal IFN-γ induction in response to non-cognate stimulation, while IL-15R expression was dispensable. Interestingly, effector Th1 cells generated by immunization rather than live infection had lower non-cognate activity despite comparable IL-18R and DR3 expression. Mice lacking T cell intrinsic expression of MyD88, an important adapter molecule in non-cognate T cell stimulation, exhibited higher bacterial burdens upon infection with Salmonella, Chlamydia or Brucella, suggesting that non-cognate Th1 stimulation is a critical element of efficient bacterial clearance. Thus, IL-18R and DR3 are critical players in non-cognate stimulation of Th1 cells and this response plays an important role in protection against intracellular bacteria.

CCR7 Deficiency Allows Accelerated Clearance of Chlamydia from the Female Reproductive Tract.

Immune mechanisms responsible for pathogen clearance from the female reproductive tract (FRT) are incompletely defined; in particular, the contribution of lymphocyte trafficking to this process is unclear. CCR7-deficient mice have profoundly altered lymphocyte recirculation and display ectopic formation of lymphocyte aggregates within mucosal nonlymphoid tissues, including the FRT. In this study, we investigated how altered lymphocyte distribution in CCR7-deficient mice would affect host responses to Chlamydia muridarum within the reproductive tract. As expected, CCR7-deficient mice exhibited reduced lymphocyte trafficking to lymph nodes and a corresponding increase in T cell populations within the FRT. After intravaginal infection with Chlamydia, CCR7-deficient mice displayed markedly reduced Ag-specific CD4 T cell responses within the local draining iliac lymph nodes, yet robust Th1 and Th17 responses were prominent in the FRT. In addition, Chlamydia-specific Ab responses were dysregulated in CCR7-deficient mice, displaying an unexpected increase in the systemic IgA responses. Importantly, prominent mucosal immune responses in CCR7-deficient mice increased the efficiency of bacteria clearance from the FRT while reducing tissue-associated inflammation and pathology. Thus, increased numbers of lymphocytes within the FRT result in pathogen clearance with reduced immune-mediated pathology.

Dual Immunization with SseB/Flagellin Provides Enhanced Protection against Salmonella Infection Mediated by Circulating Memory Cells.

The development of a subunit Salmonella vaccine has been hindered by the absence of detailed information about antigenic targets of protective Salmonella-specific T and B cells. Recent studies have identified SseB as a modestly protective Ag in susceptible C57BL/6 mice, but the mechanism of protective immunity remains undefined. In this article, we report that simply combining Salmonella SseB with flagellin substantially enhances protective immunity, allowing immunized C57BL/6 mice to survive for up to 30 d following challenge with virulent bacteria. Surprisingly, the enhancing effect of flagellin did not require flagellin Ag targeting during secondary responses or recognition of flagellin by TLR5. Although coimmunization with flagellin did not affect SseB-specific Ab responses, it modestly boosted CD4 responses. In addition, protective immunity was effectively transferred in circulation to parabionts of immunized mice, demonstrating that tissue-resident memory is not required for vaccine-induced protection. Finally, protective immunity required host expression of IFN-γR but was independent of induced NO synthase expression. Taken together, these data indicate that Salmonella flagellin has unique adjuvant properties that improve SseB-mediated protective immunity provided by circulating memory.

Immunity to intestinal pathogens: lessons learned from Salmonella.

Salmonella are a common source of food- or water-borne infection and cause a wide range of clinical disease in human and animal hosts. Salmonella are relatively easy to culture and manipulate in a laboratory setting, and the infection of laboratory animals induces robust innate and adaptive immune responses. Thus, immunologists have frequently turned to Salmonella infection models to expand understanding of host immunity to intestinal pathogens. In this review, I summarize current knowledge of innate and adaptive immunity to Salmonella and highlight features of this response that have emerged from recent studies. These include the heterogeneity of the antigen-specific T-cell response to intestinal infection, the prominence of microbial mechanisms to impede T- and B-cell responses, and the contribution of non-cognate pathways for elicitation of T-cell effector functions. Together, these different issues challenge an overly simplistic view of host-pathogen interaction during mucosal infection, but also allow deeper insight into the real-world dynamic of protective immunity to intestinal pathogens.

Salmonella Infection Enhances Erythropoietin Production by the Kidney and Liver, Which Correlates with Elevated Bacterial Burdens.

Salmonella infection profoundly affects host erythroid development, but the mechanisms responsible for this effect remain poorly understood. We monitored the impact of Salmonella infection on erythroid development and found that systemic infection induced anemia, splenomegaly, elevated erythropoietin (EPO) levels, and extramedullary erythropoiesis in a process independent of Salmonella pathogenicity island 2 (SPI2) or flagellin. The circulating EPO level was also constitutively higher in mice lacking the expression of signal-regulatory protein α (SIRPα). The expression level of EPO mRNA was elevated in the kidney and liver but not increased in the spleens of infected mice despite the presence of extramedullary erythropoiesis in this tissue. In contrast to data from a previous report, mice lacking EPO receptor (EPOR) expression on nonerythroid cells (EPOR rescued) had bacterial loads similar to those of wild-type mice following Salmonella infection. Indeed, treatment to reduce splenic erythroblasts and mature red blood cells correlated with elevated bacterial burdens, implying that extramedullary erythropoiesis benefits the host. Together, these findings emphasize the profound effect of Salmonella infection on erythroid development and suggest that the modulation of erythroid development has both positive and negative consequences for host immunity.