Listeria monocytogenes (LM) is a Gram-positive, intracellular bacterium that can induce spontaneous abortion, septicemia, and meningitis. Although it is known that neutrophils are required for elimination of the bacteria and for survival of the host, the mechanisms governing the recruitment of neutrophils to LM-infected tissues are not fully understood. We demonstrate here that IL-23 and the IL-17 receptor A (IL-17RA), which mediates both IL-17A and IL-17F signaling, are necessary for resistance against systemic LM infection. LM-infected IL-23p19 knockout (KO) mice have decreased production of IL-17A and IL-17F, while IFN-γ production is not altered by the lack of IL-23. LM induces the production of IL-17A from γδ T cells, but not CD4, CD8, or NK cells. Furthermore, a lack of efficient neutrophil recruitment to the liver is evident in both IL-23p19 KO and IL-17RA KO mice during LM infection. Immunocytochemical analysis of infected livers revealed that neutrophils were able to localize with LM in IL-23p19 KO and IL-17RA KO mice, indicating that IL-23 and IL-17RA do not regulate the precise localization of neutrophils with LM. The importance of IL-23-induced IL-17A was demonstrated by injecting IL-23p19 KO mice with recombinant IL-17A. These mice had reduced LM bacterial burdens compared with IL-23p19 KO mice that did not receive IL-17A. These results indicate that during LM infection, IL-23 regulates the production of IL-17A and IL-17F from γδ T cells, resulting in optimal liver neutrophil recruitment and enhanced bacterial clearance.

Most studies investigating the function of IL-23 have concluded that it promotes IL-17-secreting T cells. Although some reports have also characterized IL-23 as having redundant pro-inflammatory effects with IL-12, we have instead found that IL-23 antagonizes IL-12-induced secretion of IFN-c. When splenocytes or purified populations of T cells were cultured with IL-23, IFN-c secretion in response to IL-12 was dramatically reduced. The impact of IL-23 was most prominent in CD8 1 T cells, but was also observed in NK and CD81 T cells in IL-23p19-deficient mice as compared with WT mice. This increase in IFN-c production coincided with increased LM clearance at days 2 and 3 post-infection. Our data suggest that IL-23 may be a key factor in determining the responsiveness of lymphocytes to IL-12 and their subsequent secretion of IFN-c.Key words: CD8 1 T cells . Cytokines . Innate immunity . Listeria monocytogenes Introduction IL-23 is an IL-12 family cytokine which is composed of the IL-12p40 subunit and a novel cytokine subunit, p19. The heterodimeric structure of IL-23 binds to a receptor complex containing the IL-12Rb1 and a novel receptor termed IL-23R [1]. Similar to IL-12, IL-23 is secreted by activated macrophages and dendritic cells [2] in response to gram-positive and -negative bacterial, viral, and fungal infections, as well as multiple other stimuli [3][4][5][6][7][8][9][10][11]. The majority of the research investigating the actions of IL-23 has focused on its role in maintaining populations of IL-17-secreting cells as well as the possibility of directly inducing IL-17 and IL-22 secretion from a variety of cell types (for reviews see [2,12]). Initially, however, IL-23 was found to have some overlapping functions with IL-12. In a previous report, stimulation of human CD4 1 T cells with anti-CD3 and anti-CD28 resulted in IFN-g secretion, which could be significantly enhanced by IL-12 and slightly enhanced by . Contrary to this, using the murine system, several studies have suggested that IL-23 does not induce production of IFN-g in CD4 1 T cells [13,14]. In this article, we also provide evidence that murine CD4 1 and CD8 1T cells do not respond to IL-23 by secreting IFN-g. IL-23 has been shown to play a role in infectious diseases, autoimmunity and cancer. Mice deficient in IL-23 show increased susceptibility to Mycobacterium tuberculosis, Klebsiella pneumoniae, Citrobacter rodentium, Toxoplasma gondii, Salmonella enterica, and Cryptococcus neoformans [15][16][17][18][19][20][21][22][23]. Increased susceptibility to these infections was linked to the ability of IL-23 to regulate the production of IL-17 and IL-22, although during certain infections, IL-23 does not regulate both cytokines. Figure 1. IL-12, but not IL-23, induces the differentiation of IFN-g secreting T cells. Purified CD8 1 (A, B, D, and E) or CD4 1 (C and F) T cells from naïve WT B6 mice were cultured 6 days on plates coated with anti-CD3 and soluble anti-CD28 in IL-2-supplemented medium with 5 ng/mL IL-12, 10 ng/ mL IL-23, or no additional ...

Immune responses to pathogens occur within the context of current and previous infections. Cross protection refers to the phenomena where infection with a particular pathogen provides enhanced resistance to a subsequent unrelated pathogen in an antigen-independent manner. Proposed mechanisms of antigen-independent cross protection have involved the secretion of IFN-c, which activates macrophages, thus providing enhanced innate immunity against the secondary viral or bacterial pathogen. Here we provide evidence that a primary infection with the chronic respiratory pathogen, Mycoplasma pulmonis, provides a novel form of cross protection against a secondary infection with Listeria monocytogenes that is not mediated by IFN-c, but instead relies upon IL-17 and mobilization of neutrophils. Mice infected with M. pulmonis have enhanced clearance of L. monocytogenes from the spleen and liver, which is associated with increased numbers of Gr-1 1 CD11b 1 cells and higher levels of IL-17. This enhanced clearance of L. monocytogenes was absent in mice depleted of Gr-1 1 cells or in mice deficient in the IL-17 receptor. Additionally, both the IL-17 receptor and neutrophils were essential for optimal clearance of M. pulmonis. Thus, a natural component of the immune response directed against M. pulmonis was able to enhance clearance of L. monocytogenes.

Listeria monocytogenes (LM) is a gram-positive, intracellular bacterium that is commonly used to investigate the immune response to infection. Pro-inflammatory cytokines, such as IL-12 and IL-18, are secreted during LM infection to promote clearance of the bacteria through the induction of IFN-γ. LM secretes listeriolysin O (LLO) which allows the bacterium to invade the cytoplasm. Neither heat-killed LM (HKLM) nor LLO deficient LM (LLO−LM) actively produce LLO. When bone marrow derived macrophages (macs) and dendritic cells (DCs) are infected with LM, HKLM, or LLO−LM and co-cultured with naïve splenocytes, only macs or DCs infected with LM are able to induce IFN-γ secretion. ELISA data demonstrates that IL-12 is secreted from LM, HKLM, or LLO−LM infected macs and DCs, but IL-18 is only secreted from macs and DCs infected with LM. Therefore, IL-12 can be secreted by macs and DCs regardless of cytoplasmic invasion of LM whereas the secretion of IL-18 requires LM to gain access to the cytoplasm. When mice are infected with HKLM, there is no detectable IFN-γ secretion and this can be attributed to a lack of both IL-12 and IL-18. This implies that IL-12 is induced differently in response to HKLM in vivo versus in vitro. Further studies are ongoing to determine which pattern recognition receptors and cell types are responsible for recognizing and secreting IL-12 and IL-18 during in vivo LM infection. Supported by NIH AI064592.

Listeria monocytogenes (LM) is a gram‐positive, intracellular bacterium that induces the secretion of pro‐inflammatory cytokines, IL‐12 and IL‐18. LM strains either deficient in listeriolysin O (LLO−LM) or deficient in LLO but replaced with an alternate hemolysin, perfringolysin, tagged for proteasome degradation (LLO−PFO+PEST) have enhanced our ability to understand the secretion of IL‐12 and IL‐18 during cytoplasmic or noncytoplasmic invasion of LM. It is unknown which innate immune cells are secreting IL‐12 and IL‐18 during different stages of LM infection; however, we have found that neutrophils are secreting predominantly more IL‐12 one day after infection with LM. Neutrophils are associated with LM in the periarteriolar lymphoid sheath (PALS) by one day post infection. Infection of mice with LLO−LM does not induce migration of infected cells into the PALS at one day post infection whereas this migration is induced during infection with LLO−PFO+PEST LM. Interestingly, neither mutant strain induces IL‐12 or IL‐18 secretion. Studies are currently ongoing to determine how this localization is induced and whether these mutant strains can induce protective immunity.

Listeria monocytogenes (LM) is a gram-positive, intracellular bacterium that can induce spontaneous abortion, septicemia, and meningitis. Recently, it has been demonstrated that interleukin (IL)-17A is necessary for an optimal immune response against LM in the liver. As IL-23-dependent cytokines, IL-17A and IL-17F induce the mobilization of neutrophils to sites of infection. The importance of IL-23 during infection with LM has not been studied. We demonstrate here that IL-23 and the IL-17 receptor A (IL-17RA), which mediates both IL-17A and IL-17F signaling, are necessary for resistance against LM infection. During infection with LM, mice deficient in either IL-23 (IL-23p19 KO) or the IL-17RA (IL-17RA KO) have increased susceptibility to infection and increased bacterial burden in the spleen and liver. Interestingly, IL-17A, IL-17F, and IL-22 are decreased in supernatants from cells of LM infected IL-23p19 KO mice. Furthermore, neutrophils are decreased in IL-23p19 KO and IL-17RA KO mice at early time points. When IL-23p19 KO mice are rescued with the administration of exogenous IL-17A, a protective phenotype similar to that seen during infection of wild-type mice is achieved. Therefore, it is likely that IL-23 regulates the optimal production of IL-17A/F during LM infection which results in early neutrophil recruitment and bacterial clearance.

Most studies have concluded that interleukin-23 (IL-23) plays a unique role in promoting IL-17 secreting T cells. While some reports have also characterized IL-23 as having redundant pro-inflammatory effects with IL-12, we have instead found that IL-23 can regulate the innate immune response in a novel manner, by antagonizing IL-12 induced secretion of interferon-γ (IFN-γ). Our recent data documents the rapid, innate production of IFN-γ from memory CD8 T cells, as well as NK cells, stimulated with IL-12 and IL-18. When splenocytes are cultured with IL-23, IFN-γ secretion in response to IL-12 is dramatically reduced, as measured by ELISA and intracellular cytokine staining. The impact of IL-23 is most prominent in CD8 T cells, but can also be seen in NK, NK-T, and CD4 T cells. We also show that IL-23 can regulate the induction of IFN-γ by endogenously produced IL-12 from Listeria monocytogenes infected macrophages. Furthermore, IL-23 appears to act directly on purified CD8 T cells to negatively regulate IFN-γ induced by IL-12. We are currently investigating the mechanism by which IL-23 inhibits IFN-γ secretion. In addition, experiments to determine if IL-23 can inhibit other effector functions are ongoing. Our data suggest that IL-23 may be a key player in determining the responsiveness of lymphocytes to IL-12 and their subsequent secretion of IFN-γ. This research is supported by NIH AI064592.

Most studies have concluded that interleukin‐23 (IL‐23) promotes IL‐17 secreting T cells. While some reports have also characterized IL‐23 as having redundant pro‐inflammatory effects with IL‐12, we have instead found that IL‐23 can regulate the innate immune response in a novel manner, by antagonizing IL‐12 induced secretion of interferon‐γ (IFN‐γ). Our recent data documents the rapid, innate production of IFN‐γ from memory CD8 T cells, as well as NK cells, stimulated with IL‐12 and IL‐18. When splenocytes are cultured with IL‐23, IFN‐γ secretion in response to IL‐12 is dramatically reduced, as measured by ELISA and intracellular cytokine staining. The impact of IL‐23 is most prominent in CD8 T cells, but can also be seen in NK, NK‐T, and CD4 T cells. We also show that IL‐23 can regulate the induction of IFN‐γ by endogenously produced IL‐12 from Listeria monocytogenes (LM) infected macrophages. In vivo, LM infection induces higher serum IFN‐γ levels and a greater percentage of IFN‐γ + CD8 T cells in IL‐23p19 deficient mice as compared to wild‐type mice. We are currently investigating the mechanism by which IL‐23 inhibits IFN‐γ secretion, and if IL‐23 can inhibit other effector functions. Our data suggest that IL‐23 may be a key player in determining the responsiveness of lymphocytes to IL‐12 and their subsequent secretion of IFN‐γ.