Cytotoxic CD4+ T Cells Are Induced during Infection with Chlamydia trachomatis.

Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infection in both men and women. Immunity to C. trachomatis involves many cell types, but CD4+ T cells play a key role in protecting the host during natural infection. Specifically, IFN-γ production by CD4+ T cells is the main effector responsible for bacterial clearance, yet the exact mechanism by which IFN-γ confers protection is poorly defined. In our efforts to define the specific mechanisms for bacterial clearance, we now show that IFN-γ upregulates expression of MHC class II (MHCII) on nonhematopoietic cells during C. trachomatis infection in vivo. We also find that MHCII expression on epithelial cells of the upper genital tract contributes to the efficient clearance of bacteria mediated by pathogen-specific CD4+ Th1 cells. As we further cataloged the protective mechanisms of C. trachomatis-specific CD4+ T cells, we found that the T cells also express granzyme B (GzmB) when coincubated with infected cells. In addition, during C. trachomatis infection of mice, primed activated-naive CD4+ Th1 cells displayed elevated granzyme transcripts (GzmA, GzmB, GzmM, GzmK, GzmC) compared with memory CD4+ T cells in vivo. Finally, using intracellular cytokine staining and a GzmB-/- mouse strain, we show that C. trachomatis-specific CD4+ Th1 cells express GzmB upon Ag stimulation, and that this correlates with Chlamydia clearance in vivo. Together these results have led us to conclude that Chlamydia-specific CD4+ Th1 cells develop cytotoxic capacity through engagement with nonhematopoietic MHCII, and this correlates to C. trachomatis clearance.

The PD-1/PD-L1 pathway is induced during Borrelia burgdorferi infection and inhibits T cell joint infiltration without compromising bacterial clearance.

The Lyme disease bacterial pathogen, Borrelia burgdorferi, establishes a long-term infection inside its mammalian hosts. Despite the continued presence of the bacteria in animal models of disease, inflammation is transitory and resolves spontaneously. T cells with limited effector functions and the inability to become activated by antigen, termed exhausted T cells, are present in many long-term infections. These exhausted T cells mediate a balance between pathogen clearance and preventing tissue damage resulting from excess inflammation. Exhausted T cells express a variety of immunoinhibitory molecules, including the molecule PD-1. Following B. burgdorferi infection, we found that PD-1 and its ligand PD-L1 are significantly upregulated on CD4+ T cells and antigen presenting cell subsets, respectively. Using mice deficient in PD-1, we found that the PD-1/PD-L1 pathway did not impact bacterial clearance but did impact T cell expansion and accumulation in the ankle joint and popliteal lymph nodes without affecting B cell populations or antibody production, suggesting that the PD-1/PD-L1 pathway may play a role in shaping the T cell populations present in affected tissues.

T cell responses to Chlamydia.

Chlamydia trachomatis is the most commonly reported sexually transmitted infection in the United States. The high prevalence of infection and lack of a vaccine indicate a critical knowledge gap surrounding the host's response to infection and how to effectively generate protective immunity. The immune response to C. trachomatis is complex, with cells of the adaptive immune system playing a crucial role in bacterial clearance. Here, we discuss the CD4+ and CD8+ T cell response to Chlamydia, the importance of antigen specificity and the role of memory T cells during the recall response. Ultimately, a deeper understanding of protective immune responses is necessary to develop a vaccine that prevents the inflammatory diseases associated with Chlamydia infection.

Sticholysins, pore-forming proteins from a marine anemone can induce maturation of dendritic cells through a TLR4 dependent-pathway.

Sticholysins (Sts) I and II (StI and StII) are pore-forming proteins (PFPs), purified from the Caribbean Sea anemone Stichodactyla helianthus. StII encapsulated into liposomes induces a robust antigen-specific cytotoxic CD8+ T lymphocytes (CTL) response and in its free form the maturation of bone marrow-derived dendritic cells (BM-DCs). It is probable that the latter is partially supporting in part the immunomodulatory effect on the CTL response induced by StII-containing liposomes. In the present work, we demonstrate that the StII's ability of inducing maturation of BM-DCs is also shared by StI, an isoform of StII. Using heat-denatured Sts we observed a significant reduction in the up-regulation of maturation markers indicating that both PFP's ability to promote maturation of BM-DCs is dependent on their conformational characteristics. StII-mediated DC maturation was abrogated in BM-DCs from toll-like receptor (TLR) 4 and myeloid differentiation primary response gene 88 (MyD88)-knockout mice but not in cells from TLR2-knockout mice. Furthermore, the antigen-specific CTL response induced by StII-containing liposomes was reduced in TLR4-knockout mice. These results indicate that StII, and probably by extension StI, has the ability to induce maturation of DCs through a TLR4/MyD88-dependent pathway, and that this activation contributes to the CTL response generated by StII-containing liposomes.

Antigen-specific memory and naïve CD4+ T cells following secondary Chlamydia trachomatis infection.

Memory antigen-specific CD4+ T cells against Chlamydia trachomatis are necessary for protection against secondary genital tract infection. While it is known that naïve antigen-specific CD4+ T cells can traffic to the genital tract in an antigen-specific manner, these T cells are not protective during primary infection. Here, we sought to compare the differences between memory and naïve antigen-specific CD4+ T cells in the same mouse following secondary infection using transgenic CD4+ T cells (NR1 T cells). Using RNA sequencing, we found that there were subtle but distinct differences between these two T cell populations. Naïve NR1 T cells significantly upregulated cell cycle genes and were more proliferative than memory NR1 T cells in the draining lymph node. In contrast, memory NR1 T cells were more activated than naïve NR1 T cells and were enriched in the genital tract. Together, our data provide insight into the differences between memory and naïve antigen-specific CD4+ T cells during C. trachomatis infection.

Gamma Interferon Is Required for Chlamydia Clearance but Is Dispensable for T Cell Homing to the Genital Tract.

While there is no effective vaccine against Chlamydia trachomatis infection, previous work has demonstrated the importance of C. trachomatis-specific CD4+ T cells (NR1 T cells) in pathogen clearance. Specifically, NR1 T cells have been shown to be protective in mice, and this protection depends on the host's ability to sense the cytokine gamma interferon (IFN-γ). However, it is unclear what role NR1 production or sensing of IFN-γ plays in T cell homing to the genital tract or T cell-mediated protection against C. trachomatis Using two-photon microscopy and flow cytometry, we found that naive wild-type (WT), IFN-γ-/-, and IFN-γR-/- NR1 T cells specifically home to sections in the genital tract that contain C. trachomatis We also determined that protection against infection requires production of IFN-γ from either NR1 T cells or endogenous cells, further highlighting the importance of IFN-γ in clearing C. trachomatis infection.IMPORTANCEChlamydia trachomatis is an important mucosal pathogen that is the leading cause of sexually transmitted bacterial infections in the United States. Despite this, there is no vaccine currently available. In order to develop such a vaccine, it is necessary to understand the components of the immune response that can lead to protection against this pathogen. It is well known that antigen-specific CD4+ T cells are critical for Chlamydia clearance, but the contexts in which they are protective or not protective are unknown. Here, we aimed to characterize the importance of gamma interferon production and sensing by T cells and the effects on the immune response to C. trachomatis Our work here helps to define the contexts in which antigen-specific T cells can be protective, which is critical to our ability to design an effective and protective vaccine against C. trachomatis.

The well-evolved pathogen.

Stan Falkow looked at the world with his eyes peering from the outer membrane of a gram-negative bacterium. It was a great vantage point from which to dream about the possible superpowers these organisms might have. He mused about these dreams with his trainees who sometimes found through rigorous scientific exploration that the superpowers really were there! Stan also realized that bacterial pathogenesis by definition was a two-way street, and that masterful understanding of bacterial virulence factors also required a masterful understanding of host cell processes against which the virulence factors were deployed. In my own scientific journey, I have sought to explore bacterial-host interactions that result in subtle alterations of the host's adaptive immune response. Here, as an example, I describe an interaction between Chlamydia trachomatis and host T cells that may contribute to the establishment of persistent infection.

Gut-Innervating Nociceptor Neurons Regulate Peyer's Patch Microfold Cells and SFB Levels to Mediate Salmonella Host Defense.

Gut-innervating nociceptor sensory neurons respond to noxious stimuli by initiating protective responses including pain and inflammation; however, their role in enteric infections is unclear. Here, we find that nociceptor neurons critically mediate host defense against the bacterial pathogen Salmonella enterica serovar Typhimurium (STm). Dorsal root ganglia nociceptors protect against STm colonization, invasion, and dissemination from the gut. Nociceptors regulate the density of microfold (M) cells in ileum Peyer's patch (PP) follicle-associated epithelia (FAE) to limit entry points for STm invasion. Downstream of M cells, nociceptors maintain levels of segmentous filamentous bacteria (SFB), a gut microbe residing on ileum villi and PP FAE that mediates resistance to STm infection. TRPV1+ nociceptors directly respond to STm by releasing calcitonin gene-related peptide (CGRP), a neuropeptide that modulates M cells and SFB levels to protect against Salmonella infection. These findings reveal a major role for nociceptor neurons in sensing and defending against enteric pathogens.

Early Colonization of the Upper Genital Tract by Chlamydia muridarum Is Associated with Enhanced Inflammation Later in Infection.

Chlamydia trachomatis is the most commonly reported bacterial sexually transmitted infection in the United States. Modeling infection in animals can be challenging, as mice naturally clear C. trachomatis when it is deposited in the lower genital tract. However, C. trachomatis can productively infect mice when the lower genital tract is bypassed and bacteria are deposited directly into the upper genital tract via transcervical inoculation. Interestingly, the mouse-adapted Chlamydia species C. muridarum can infect mice both by transcervical inoculation and by natural ascension if introduced into the vaginal vault. In this study, we investigated whether the route of infection plays a role in the downstream immune responses to C. muridarum infection. We found that transcervical infection with C. muridarum results in higher bacterial burdens in the upper genital tract at earlier time points, correlating with levels of innate immune cells. When bacterial burdens were equivalent in intravaginally and transcervically infected mice at later time points, we observed substantially higher levels of adaptive immune cells in transcervically infected mice. Our data suggest that different routes of infection with the same organism can elicit different immune responses in the same tissue.

A Chlamydia trachomatis Strain Expressing Ovalbumin Stimulates an Antigen-Specific CD4+ T Cell Response in Mice.

Antigen-specific CD4+ T cells against Chlamydia are crucial for driving bacterial clearance and mediating protection against reinfection. Although the Chlamydia trachomatis protein Cta1 has been identified to be a dominant murine CD4+ T cell antigen, its level of expression during the bacterial developmental cycle and precise localization within the host cell are unknown. Newly developed tools for Chlamydia genetic manipulation have allowed us to generate a C. trachomatis strain expressing a heterologous CD4+ T cell epitope from ovalbumin (OVA) consisting of OVA residues 323 to 339 (OVA323-339). By tagging proteins expressed in C. trachomatis with OVA323-339, we can begin to understand how protein expression, developmental regulation, and subcellular compartmentalization affect the potential of those proteins to serve as antigens. When OVA323-339 was expressed as a fusion with green fluorescent protein, we found that we were able to elicit an OT-II T cell response in an antigen-dependent manner, but surprisingly, these T cells were unable to reduce bacterial burden in mice. These data suggest that the subcellular localization of antigen, the level of antigen expression, or the timing of expression within the developmental cycle of Chlamydia may play a crucial role in eliciting a protective CD4+ T cell response.

A FACS-Based Genome-wide CRISPR Screen Reveals a Requirement for COPI in Chlamydia trachomatis Invasion.

The invasion of Chlamydia trachomatis, an obligate intracellular bacterium, into epithelial cells is driven by a complex interplay of host and bacterial factors. To comprehensively define the host genes required for pathogen invasion, we undertook a fluorescence-activated cell sorting (FACS)-based CRISPR screen in human cells. A genome-wide loss-of-function library was infected with fluorescent C. trachomatis and then sorted to enrich for invasion-deficient mutants. The screen identified heparan sulfate, a known pathogen receptor, as well as coatomer complex I (COPI). We found that COPI, through a previously unappreciated role, promotes heparan sulfate cell surface presentation, thereby facilitating C. trachomatis attachment. The heparan sulfate defect does not fully account for the resistance of COPI mutants. COPI also promotes the activity of the pathogen's type III secretion system. Together, our findings establish the requirement for COPI in C. trachomatis invasion and the utility of FACS-based CRISPR screening for the elucidation of host factors required for pathogen invasion.

The Vacuolar Pathway in Macrophages Plays a Major Role in Antigen Cross-Presentation Induced by the Pore-Forming Protein Sticholysin II Encapsulated Into Liposomes.

Cross-presentation is an important mechanism for the differentiation of effector cytotoxic T lymphocytes (CTL) from naïve CD8+ T-cells, a key response for the clearance of intracellular pathogens and tumors. The liposomal co-encapsulation of the pore-forming protein sticholysin II (StII) with ovalbumin (OVA) (Lp/OVA/StII) induces a powerful OVA-specific CTL activation and an anti-tumor response in vivo. However, the pathway through which the StII contained in this preparation is able to induce antigen cross-presentation and the type of professional antigen presenting cells (APCs) involved have not been elucidated. Here, the ability of mouse bone marrow-derived dendritic cells (BM-DCs) and macrophages (BM-MΦs) stimulated with Lp/OVA/StII to activate SIINFEKL-specific B3Z CD8+ T cells was evaluated in the presence of selected inhibitors. BM-MΦs, but not BM-DCs were able to induce SIINFEKL-specific B3Z CD8+ T cell activation upon stimulation with Lp/OVA/StII. The cross-presentation of OVA was markedly decreased by the lysosome protease inhibitors, leupeptin and cathepsin general inhibitor, while it was unaffected by the proteasome inhibitor epoxomicin. This process was also significantly reduced by phagocytosis and Golgi apparatus function inhibitors, cytochalasin D and brefeldin A, respectively. These results are consistent with the concept that BM-MΦs internalize these liposomes through a phagocytic mechanism resulting in the cross-presentation of the encapsulated OVA by the vacuolar pathway. The contribution of macrophages to the CTL response induced by Lp/OVA/StII in vivo was determined by depleting macrophages with clodronate-containing liposomes. CTL induction was almost completely abrogated in mice depleted of macrophages, demonstrating the relevance of these APCs in the antigen cross-presentation induced by this formulation.

Action Needed on Chlamydia Vaccines.

Chlamydia trachomatis is the most common infectious disease in the USA for which the Centers for Disease Control (CDC) collects case reports. Its prevalence in young women is a public health crisis given the threat to their reproductive health. Consequently, development of a vaccine to prevent infection should be prioritized.

LysMD3 is a type II membrane protein without an in vivo role in the response to a range of pathogens.

Germline-encoded receptors recognizing common pathogen-associated molecular patterns are a central element of the innate immune system and play an important role in shaping the host response to infection. Many of the innate immune molecules central to these signaling pathways are evolutionarily conserved. LysMD3 is a novel molecule containing a putative peptidoglycan-binding domain that has orthologs in humans, mice, zebrafish, flies, and worms. We found that the lysin motif (LysM) of LysMD3 is likely related to a previously described peptidoglycan-binding LysM found in bacteria. Mouse LysMD3 is a type II integral membrane protein that co-localizes with GM130+ structures, consistent with localization to the Golgi apparatus. We describe here two lines of mLysMD3-deficient mice for in vivo characterization of mLysMD3 function. We found that mLysMD3-deficient mice were born at Mendelian ratios and had no obvious pathological abnormalities. They also exhibited no obvious immune response deficiencies in a number of models of infection and inflammation. mLysMD3-deficient mice exhibited no signs of intestinal dysbiosis by 16S analysis or alterations in intestinal gene expression by RNA sequencing. We conclude that mLysMD3 contains a LysM with cytoplasmic orientation, but we were unable to define a physiological role for the molecule in vivo.

Pathology after Chlamydia trachomatis infection is driven by nonprotective immune cells that are distinct from protective populations.

Infection with Chlamydia trachomatis drives severe mucosal immunopathology; however, the immune responses that are required for mediating pathology vs. protection are not well understood. Here, we employed a mouse model to identify immune responses required for C. trachomatis-induced upper genital tract pathology and to determine whether these responses are also required for bacterial clearance. In mice as in humans, immunopathology was characterized by extravasation of leukocytes into the upper genital tract that occluded luminal spaces in the uterus and ovaries. Flow cytometry identified these cells as neutrophils at early time points and CD4+ and CD8+ T cells at later time points. To determine what draws these cells to C. trachomatis-infected tissue, we measured the expression of 700 inflammation-related genes in the upper genital tract and found an up-regulation of many chemokines, including a node of interaction between CXCL9/10/11 and their common receptor CXCR3. Either depleting neutrophils or reducing T-cell numbers by CXCR3 blockade was sufficient to significantly ameliorate immunopathology but had no effect on bacterial burden, demonstrating that these responses are necessary for mucosal pathology but dispensable for C. trachomatis clearance. Therapies that specifically target these host responses may therefore prove useful in ameliorating C. trachomatis-induced pathology without exacerbating infection or transmission.

TRAPPC13 modulates autophagy and the response to Golgi stress.

Tether complexes play important roles in endocytic and exocytic trafficking of lipids and proteins. In yeast, the multisubunit transport protein particle (TRAPP) tether regulates endoplasmic reticulum (ER)-to-Golgi and intra-Golgi transport and is also implicated in autophagy. In addition, the TRAPP complex acts as a guanine nucleotide exchange factor (GEF) for Ypt1, which is homologous to human Rab1a and Rab1b. Here, we show that human TRAPPC13 and other TRAPP subunits are critically involved in the survival response to several Golgi-disrupting agents. Loss of TRAPPC13 partially preserves the secretory pathway and viability in response to brefeldin A, in a manner that is dependent on ARF1 and the large GEF GBF1, and concomitant with reduced caspase activation and ER stress marker induction. TRAPPC13 depletion reduces Rab1a and Rab1b activity, impairs autophagy and leads to increased infectivity to the pathogenic bacterium Shigella flexneri in response to brefeldin A. Thus, our results lend support for the existence of a mammalian TRAPPIII complex containing TRAPPC13, which is important for autophagic flux under certain stress conditions.

Novel Adjuvant Based on the Pore-Forming Protein Sticholysin II Encapsulated into Liposomes Effectively Enhances the Antigen-Specific CTL-Mediated Immune Response.

Vaccine strategies to enhance CD8+ CTL responses remain a current challenge because they should overcome the plasmatic and endosomal membranes for favoring exogenous Ag access to the cytosol of APCs. As a way to avoid this hurdle, sticholysin (St) II, a pore-forming protein from the Caribbean Sea anemone Stichodactyla helianthus, was encapsulated with OVA into liposomes (Lp/OVA/StII) to assess their efficacy to induce a CTL response. OVA-specific CD8+ T cells transferred to mice immunized with Lp/OVA/StII experienced a greater expansion than when the recipients were injected with the vesicles without St, mostly exhibiting a memory phenotype. Consequently, Lp/OVA/StII induced a more potent effector function, as shown by CTLs, in vivo assays. Furthermore, treatment of E.G7-OVA tumor-bearing mice with Lp/OVA/StII significantly reduced tumor growth being more noticeable in the preventive assay. The contribution of CD4+ and CD8+ T cells to CTL and antitumor activity, respectively, was elucidated. Interestingly, the irreversibly inactive variant of the StI mutant StI W111C, encapsulated with OVA into Lp, elicited a similar OVA-specific CTL response to that observed with Lp/OVA/StII or vesicles encapsulating recombinant StI or the reversibly inactive StI W111C dimer. These findings suggest the relative independence between StII pore-forming activity and its immunomodulatory properties. In addition, StII-induced in vitro maturation of dendritic cells might be supporting these properties. These results are the first evidence, to our knowledge, that StII, a pore-forming protein from a marine eukaryotic organism, encapsulated into Lp functions as an adjuvant to induce a robust specific CTL response.

An Excess of the Proinflammatory Cytokines IFN-γ and IL-12 Impairs the Development of the Memory CD8+ T Cell Response to Chlamydia trachomatis.

The obligate intracellular bacterium Chlamydia trachomatis is the most common cause of bacterial sexually transmitted disease in the United States and the leading cause of preventable blindness worldwide. Transfer of cultured Chlamydia-specific CD8(+) T cells or vaccination with recombinant virus expressing an MHC I-restricted Chlamydia Ag confers protection, yet surprisingly a protective CD8(+) T cell response is not stimulated following natural infection. In this study, we demonstrate that the presence of excess IL-12 and IFN-γ contributes to poor memory CD8(+) T cell development during C. trachomatis infection of mice. IL-12 is required for CD8(+) T cell expansion but drives effector CD8(+) T cells into a short-lived fate, whereas IFN-γ signaling impairs the development of effector memory cells. We show that transient blockade of IL-12 and IFN-γ during priming promotes the development of memory precursor effector CD8(+) T cells and increases the number of memory T cells that participate in the recall protection against subsequent infection. Overall, this study identifies key factors shaping memory development of Chlamydia-specific CD8(+) T cells that will inform future vaccine development against this and other pathogens.

Global Mapping of the Inc-Human Interactome Reveals that Retromer Restricts Chlamydia Infection.

Chlamydia trachomatis is a leading cause of genital and ocular infections for which no vaccine exists. Upon entry into host cells, C. trachomatis resides within a membrane-bound compartment­the inclusion­and secretes inclusion membrane proteins (Incs) that are thought to modulate the host-bacterium interface. To expand our understanding of Inc function(s), we subjected putative C. trachomatis Incs to affinity purification-mass spectroscopy (AP-MS). We identified Inc-human interactions for 38/58 Incs with enrichment in host processes consistent with Chlamydia's intracellular life cycle. There is significant overlap between Inc targets and viral proteins, suggesting common pathogenic mechanisms among obligate intracellular microbes. IncE binds to sorting nexins (SNXs) 5/6, components of the retromer, which relocalizes SNX5/6 to the inclusion membrane and augments inclusion membrane tubulation. Depletion of retromer components enhances progeny production, revealing that retromer restricts Chlamydia infection. This study demonstrates the value of proteomics in unveiling host-pathogen interactions in genetically challenging microbes.

VACCINES. A mucosal vaccine against Chlamydia trachomatis generates two waves of protective memory T cells.

Genital Chlamydia trachomatis (Ct) infection induces protective immunity that depends on interferon-γ-producing CD4 T cells. By contrast, we report that mucosal exposure to ultraviolet light (UV)-inactivated Ct (UV-Ct) generated regulatory T cells that exacerbated subsequent Ct infection. We show that mucosal immunization with UV-Ct complexed with charge-switching synthetic adjuvant particles (cSAPs) elicited long-lived protection in conventional and humanized mice. UV-Ct-cSAP targeted immunogenic uterine CD11b(+)CD103(-) dendritic cells (DCs), whereas UV-Ct accumulated in tolerogenic CD11b(-)CD103(+) DCs. Regardless of vaccination route, UV-Ct-cSAP induced systemic memory T cells, but only mucosal vaccination induced effector T cells that rapidly seeded uterine mucosa with resident memory T cells (T(RM) cells). Optimal Ct clearance required both T(RM) seeding and subsequent infection-induced recruitment of circulating memory T cells. Thus, UV-Ct-cSAP vaccination generated two synergistic memory T cell subsets with distinct migratory properties.