Inflammatory Monocytes Promote Granuloma-Mediated Control of Persistent Salmonella Infection.

Persistent infections generally involve a complex balance between protective immunity and immunopathology. We used a murine model to investigate the role of inflammatory monocytes in immunity and host defense against persistent salmonellosis. Mice exhibit increased susceptibility to persistent infection when inflammatory monocytes cannot be recruited into tissues or when they are depleted at specific stages of persistent infection. Inflammatory monocytes contribute to the pathology of persistent salmonellosis and cluster with other cells in pathogen-containing granulomas. Depletion of inflammatory monocytes during the chronic phase of persistent salmonellosis causes regression of already established granulomas with resultant pathogen growth and spread in tissues. Thus, inflammatory monocytes promote granuloma-mediated control of persistent salmonellosis and may be key to uncovering new therapies for granulomatous diseases.

Mutant p53 suppresses innate immune signaling to promote tumorigenesis.

Mutant p53 (mtp53) proteins can exert cancer-promoting gain-of-function activities. We report a mechanism by which mtp53 suppresses both cell-autonomous and non-cell-autonomous signaling to promote cancer cell survival and evasion of tumor immune surveillance. Mtp53 interferes with the function of the cytoplasmic DNA sensing machinery, cGAS-STING-TBK1-IRF3, that activates the innate immune response. Mtp53, but not wild-type p53, binds to TANK-binding protein kinase 1 (TBK1) and prevents the formation of a trimeric complex between TBK1, STING, and IRF3, which is required for activation, nuclear translocation, and transcriptional activity of IRF3. Inactivation of innate immune signaling by mtp53 alters cytokine production, resulting in immune evasion. Restoring TBK1 signaling is sufficient to bypass mtp53 and lead to restored immune cell function and cancer cell eradication. This work is of translational interest because therapeutic approaches that restore TBK1 function could potentially reactivate immune surveillance and eliminate mtp53 tumors.

CCR2+ Inflammatory Monocytes Are Recruited to Yersinia pseudotuberculosis Pyogranulomas and Dictate Adaptive Responses at the Expense of Innate Immunity during Oral Infection.

Murine Ly6Chi inflammatory monocytes (IMs) require CCR2 to leave the bone marrow and enter mesenteric lymph nodes (MLNs) and other organs in response to Yersinia pseudotuberculosis infection. We are investigating how IMs, which can differentiate into CD11c+ dendritic cells (DCs), contribute to innate and adaptive immunity to Y. pseudotuberculosis Previously, we obtained evidence that IMs are important for a dominant CD8+ T cell response to the epitope YopE69-77 and host survival using intravenous infections with attenuated Y. pseudotuberculosis Here we challenged CCR2+/+ or CCR2-/- mice orally with wild-type Y. pseudotuberculosis to investigate how IMs contribute to immune responses during intestinal infection. Unexpectedly, CCR2-/- mice did not have reduced survival but retained body weight better and their MLNs cleared Y. pseudotuberculosis faster and with reduced lymphadenopathy compared to controls. Enhanced bacterial clearance in CCR2-/- mice correlated with reduced numbers of IMs in spleens and increased numbers of neutrophils in livers. In situ imaging of MLNs and spleens from CCR2-GFP mice showed that green fluorescent protein-positive (GFP+) IMs accumulated at the periphery of neutrophil-rich Yersinia-containing pyogranulomas. GFP+ IMs colocalized with CD11c+ cells and YopE69-77-specific CD8+ T cells in MLNs, suggesting that IM-derived DCs prime adaptive responses in Yersinia pyogranulomas. Consistently, CCR2-/- mice had reduced numbers of splenic DCs, YopE69-77-specific CD8+ T cells, CD4+ T cells, and B cells in organs and lower levels of serum antibodies to Y. pseudotuberculosis antigens. Our data suggest that IMs differentiate into DCs in MLN pyogranulomas and direct adaptive responses in T cells at the expense of innate immunity during oral Y. pseudotuberculosis infection.

Contribution of Asparagine Catabolism to Salmonella Virulence.

Salmonellae are pathogenic bacteria that cause significant morbidity and mortality in humans worldwide. Salmonellae establish infection and avoid clearance by the immune system by mechanisms that are not well understood. We previously showed that l-asparaginase II produced by Salmonella enterica serovar Typhimurium (S Typhimurium) inhibits T cell responses and mediates virulence. In addition, we previously showed that asparagine deprivation such as that mediated by l-asparaginase II of S Typhimurium causes suppression of activation-induced T cell metabolic reprogramming. Here, we report that STM3997, which encodes a homolog of disulfide bond protein A (dsbA) of Escherichia coli, is required for l-asparaginase II stability and function. Furthermore, we report that l-asparaginase II localizes primarily to the periplasm and acts together with l-asparaginase I to provide S Typhimurium the ability to catabolize asparagine and assimilate nitrogen. Importantly, we determined that, in a murine model of infection, S Typhimurium lacking both l-asparaginase I and II genes competes poorly with wild-type S Typhimurium for colonization of target tissues. Collectively, these results indicate that asparagine catabolism contributes to S Typhimurium virulence, providing new insights into the competition for nutrients at the host-pathogen interface.

Asparagine deprivation mediated by Salmonella asparaginase causes suppression of activation-induced T cell metabolic reprogramming.

Salmonellae are pathogenic bacteria that induce immunosuppression by mechanisms that remain largely unknown. Previously, we showed that a putative type II l-asparaginase produced by Salmonella Typhimurium inhibits T cell responses and mediates virulence in a murine model of infection. Here, we report that this putative L-asparaginase exhibits L-asparagine hydrolase activity required for Salmonella Typhimurium to inhibit T cells. We show that L-asparagine is a nutrient important for T cell activation and that L-asparagine deprivation, such as that mediated by the Salmonella Typhimurium L-asparaginase, causes suppression of activation-induced mammalian target of rapamycin signaling, autophagy, Myc expression, and L-lactate secretion. We also show that L-asparagine deprivation mediated by the Salmonella Typhimurium L-asparaginase causes suppression of cellular processes and pathways involved in protein synthesis, metabolism, and immune response. Our results advance knowledge of a mechanism used by Salmonella Typhimurium to inhibit T cell responses and mediate virulence, and provide new insights into the prerequisites of T cell activation. We propose a model in which l-asparagine deprivation inhibits T cell exit from quiescence by causing suppression of activation-induced metabolic reprogramming.

CD11b+ Ly6Chi Ly6G- immature myeloid cells recruited in response to Salmonella enterica serovar Typhimurium infection exhibit protective and immunosuppressive properties.

Immature myeloid cells in bone marrow are a heterogeneous population of cells that, under normal conditions, provide tissues with protective cell types such as granulocytes and macrophages. Under certain pathological conditions, myeloid cell homeostasis is altered and immature forms of these cells appear in tissues. Murine immature myeloid cells that express CD11b and Ly6C or Ly6G (two isoforms of Gr-1) have been associated with immunosuppression in cancer (in the form of myeloid-derived suppressor cells) and, more recently, infection. Here, we found that CD11b(+) Ly6C(hi) Ly6G(-) and CD11b(+) Ly6C(int) Ly6G(+) cells accumulated and persisted in tissues of mice infected with Salmonella enterica serovar Typhimurium (S. Typhimurium). Recruitment of CD11b(+) Ly6C(hi) Ly6G(-) but not CD11b(+) Ly6C(int) Ly6G(+) cells from bone marrow into infected tissues depended on chemokine receptor CCR2. The CD11b(+) Ly6C(hi) Ly6G(-) cells exhibited a mononuclear morphology, whereas the CD11b(+) Ly6C(int) Ly6G(+) cells exhibited a polymorphonuclear or band-shaped nuclear morphology. The CD11b(+) Ly6C(hi) Ly6G(-) cells differentiated into macrophage-like cells following ex vivo culture and could present antigen to T cells in vitro. However, significant proliferation of T cells was observed only when the ability of the CD11b(+) Ly6C(hi) Ly6G(-) cells to produce nitric oxide was blocked. CD11b(+) Ly6C(hi) Ly6G(-) cells recruited in response to S. Typhimurium infection could also present antigen to T cells in vivo, but increasing their numbers by adoptive transfer did not cause a corresponding increase in T cell response. Thus, CD11b(+) Ly6C(hi) Ly6G(-) immature myeloid cells recruited in response to S. Typhimurium infection exhibit protective and immunosuppressive properties that may influence the outcome of infection.

Persistent salmonellosis causes pancreatitis in a murine model of infection.

Pancreatitis, a known risk factor for the development of pancreatic ductal adenocarcinoma, is a serious, widespread medical condition usually caused by alcohol abuse or gallstone-mediated ductal obstruction. However, many cases of pancreatitis are of an unknown etiology. Pancreatitis has been linked to bacterial infection, but causality has yet to be established. Here, we found that persistent infection of mice with the bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) was sufficient to induce pancreatitis reminiscent of the human disease. Specifically, we found that pancreatitis induced by persistent S. Typhimurium infection was characterized by a loss of pancreatic acinar cells, acinar-to-ductal metaplasia, fibrosis and accumulation of inflammatory cells, including CD11b+ F4/80+, CD11b+ Ly6Cint Ly6G+ and CD11b+ Ly6Chi Ly6G- cells. Furthermore, we found that S. Typhimurium colonized and persisted in the pancreas, associated with pancreatic acinar cells in vivo, and could invade cultured pancreatic acinar cells in vitro. Thus, persistent infection of mice with S. Typhimurium may serve as a useful model for the study of pancreatitis as it relates to bacterial infection. Increased knowledge of how pathogenic bacteria can cause pancreatitis will provide a more integrated picture of the etiology of the disease and could lead to the development of new therapeutic approaches for treatment and prevention of pancreatitis and pancreatic ductal adenocarcinoma.

L-asparaginase II produced by Salmonella typhimurium inhibits T cell responses and mediates virulence.

Salmonella enterica serovar Typhimurium avoids clearance by the host immune system by suppressing T cell responses; however, the mechanisms that mediate this immunosuppression remain unknown. We show that S. Typhimurium inhibit T cell responses by producing L-Asparaginase II, which catalyzes the hydrolysis of L-asparagine to aspartic acid and ammonia. L-Asparaginase II is necessary and sufficient to suppress T cell blastogenesis, cytokine production, and proliferation and to downmodulate expression of the T cell receptor. Furthermore, S. Typhimurium-induced inhibition of T cells in vitro is prevented upon addition of L-asparagine. S. Typhimurium lacking the L-Asparaginase II gene (STM3106) are unable to inhibit T cell responses and exhibit attenuated virulence in vivo. L-Asparaginases are used to treat acute lymphoblastic leukemia through mechanisms that likely involve amino acid starvation of leukemic cells, and these findings indicate that pathogens similarly use L-asparagine deprivation to limit T cell responses.

Phenotypic, morphological, and functional heterogeneity of splenic immature myeloid cells in the host response to tularemia.

Recent studies have linked accumulation of the Gr-1⁺ CD11b⁺ cell phenotype with functional immunosuppression in diverse pathological conditions, including bacterial and parasitic infections and cancer. Gr-1⁺ CD11b⁺ cells were the largest population of cells present in the spleens of mice infected with sublethal doses of the Francisella tularensis live vaccine strain (LVS). In contrast, the number of T cells present in the spleens of these mice did not increase during early infection. There was a significant delay in the kinetics of accumulation of Gr-1⁺ CD11b⁺ cells in the spleens of B-cell-deficient mice, indicating that B cells play a role in recruitment and maintenance of this population in the spleens of mice infected with F. tularensis. The splenic Gr-1⁺ CD11b⁺ cells in tularemia were a heterogeneous population that could be further subdivided into monocytic (mononuclear) and granulocytic (polymorphonuclear) cells using the Ly6C and Ly6G markers and differentiated into antigen-presenting cells following ex vivo culture. Monocytic, CD11b⁺ Ly6C(hi) Ly6G⁻ cells but not granulocytic, CD11b⁺ Ly6C(int) Ly6G⁺ cells purified from the spleens of mice infected with F. tularensis suppressed polyclonal T-cell proliferation via a nitric oxide-dependent pathway. Although the monocytic, CD11b⁺ Ly6C(hi) Ly6G⁻ cells were able to suppress the proliferation of T cells, the large presence of Gr-1⁺ CD11b⁺ cells in mice that survived F. tularensis infection also suggests a potential role for these cells in the protective host response to tularemia.

Mechanism-based probe for the analysis of cathepsin cysteine proteases in living cells.

Mechanism-based probes are providing new tools to evaluate the enzymatic activities of protein families in complex mixtures and to assign protein function. The application of these chemical probes for the visualization of protein labeling in cells and proteomic analysis is still challenging. As a consequence, imaging and proteomic analysis often require different sets of chemical probes. Here we describe a mechanism-based probe, azido-E-64, that can be used for both imaging and proteomics. Azido-E-64 covalently modifies active Cathepsin (Cat) B in living cells, an abundant cysteine protease involved in microbial infections, apoptosis, and cancer. Furthermore, azido-E-64 contains an azide chemical handle that can be selectively derivatized with phosphine reagents via the Staudinger ligation, which enables the imaging and proteomic analysis of Cat B. We have utilized azido-E-64 to visualize active Cat B during infection of primary macrophages with Salmonella typhimurium , an facultative intracellular bacterial pathogen. These studies demonstrated that active Cat B is specifically excluded from Salmonella -containing vacuoles, which suggests that inhibition of protease activity within bacteria-containing vacuoles may contribute to bacterial virulence.

Activation of bone marrow-resident memory T cells by circulating, antigen-bearing dendritic cells.

Dendritic cells (DCs) carry antigen from peripheral tissues via lymphatics to lymph nodes. We report here that differentiated DCs can also travel from the periphery into the blood. Circulating DCs migrated to the spleen, liver and lung but not lymph nodes. They also homed to the bone marrow, where they were retained better than in most other tissues. Homing of DCs to the bone marrow depended on constitutively expressed vascular cell adhesion molecule 1 and endothelial selectins in bone marrow microvessels. Two-photon intravital microscopy in bone marrow cavities showed that DCs formed stable antigen-dependent contacts with bone marrow-resident central memory T cells. Moreover, using this previously unknown migratory pathway, antigen-pulsed DCs were able to trigger central memory T cell-mediated recall responses in the bone marrow.