The Regulation Of Innate Immune Responses By MHC Class I Molecule And Interleukin 17

Our human beings live in a world surrounded with various kinds of pathogenic microbes----virus, bacteria, fungus, protozoon and parasites. Our immune system helps us to maintain homeostasis and uninterrupted by those pathogens. Both innate immunity and adaptive immunity play important roles in anti-infectious responses. The innate immune system depends largely on Toll-like receptors (TLRs) to recognize and eliminate pathogens. As the most characterized pattern recognition receptors (PRRs), TLRs are expressed mainly on macrophages and dendritic cells (DC). TLR family is highly conserved among organism, and can recognize certain pathogen associated molecular patterns (PAMPs). These TLRs constitute the first line of host defense to prevent invasion of pathogens, and can also trigger antigen-specific adaptive immune responses against infections. In order to efficiently clear pathogens but not induce excessive inflammatory reaction leading to immunopathgenesis, these TLR signals are under considerable precise regulation. Better understanding of TLR signals and its tight regulation has important theoretical significance and application value.Major Histocompability complex (MHC) class I molecule plays important role in the development and function of the immune system. The classical function of MHC class I is to bind peptide of 9-11 amino acids in the endoplasmic reticulum (ER), and then transport to the cell membrane to present this peptide to T cell receptor (TCR) of CD8~+ T cells. Deficiency of MHC class I would impair the positive selection of CD8~+ T cells in the thymus, and block the export of mature CD8~+ T cells to the periphery. Recently, some studies have demonstrated nonclassical function of MHC class I molecule. Especially when ligated by certain ligands or monoclonal antibodies, MHC class I could transduce certain signals reversely in certain kinds of cells, and induce cellular biological responses, including cell proliferation, apoptosis, activation etc. Whether or not this MHC class I reverse signaling will interfere with the TLR signal pathway remains unkown till now.Overexpression or abnormality of MHC class I always associated with certain autoimmune disorders, such as autoimmune myositis, type I diabetes, inflammatory bowel disease and so on. More recently, Th17 cells are suggested to be the pathogenetic cells related with many kinds of autoimmune diseases. However, there is no report of association between MHC class I and Th17 cells to date. Whether interleukin 17 (IL-17) will induce abnormal expression of MHC I or overexpression of MHC class I leads to exacerbated IL-17 secretion reamin to be addressed.The classical function of MHC class I is to present antigen peptide to CD8~+ T cells to trigger adaptive CTL responses. It can also interact with inhibitory receptors on natural killer (NK) cells to prevent killing cells of itself. If IL-17 can modulate MHC class I expression, it can be presumed that IL-17 may affect CD8~+ T cells responses or regulate NK cell cytotoxity. Previous study of IL-17 has been focused on its recruitment and activation of innate neutrophils. Now, the research of nonclassical function of IL-17 will contribute to better understanding of the precise regulation of the anti-infectious immune response.T cells are major component of the adaptive immune system. Especially CD8~+ T cells help host resistance against intracellular bacterial and viral infections, and are the major effector cells killing tumor cells. The intensity of CD8~+ T cells response relates directly to the health status of the host. To initiate a CD8~+ T cell response, antigen-presenting cells (APCs) presenting certain antigens on its MHC I molecules are needed to provide the first signal; then CD80, CD86 on the surface of these APCs supplies the second co-stimulating signal. Only these two signals existence together can trigger a CTL response. Meanwhile, the activation process is also monitored by numerous other cells and cytokines in the microenvironment, resulting in the variation of CTL immune responses. IL-17 exerts its functions mainly by orchestrating innate immune responses. Recent evidence, however, strongly implicated IL-17 extends its function in adaptive immunity. For example, a study in IL-17-deficient mice demonstrated the importance of IL-17 in delayed type hypersensitivities, suggesting IL-17 may play a role in CD4+ Th cell response. IL-17 has been shown to directly suppress Th1 cell differentiation, and protect the mice from T cell-mediated intestinal inflammation through IL-17RA on CD4+ T cells. However, the influence of IL-17 on CD8~+ cytotoxic T cell responses against L.monocytogenes infection and the cell resources of IL-17 in the process need to be fully understood.NK cells, the major effector of the innate immune system, are the first line of defense against hepatitis viral infection. However, excessive activation of NK cells also leads to the severe change of hepatitis, thus accurate control of NK cell activation is of great significance for understanding the pathogenic mechanism and for clinic treatment of hepatitis. Higher serum IL-17 level was observed in chronic hepatitis B patients, and even higher in severe hepatitis B patients'sera, suggesting IL-17 may participate in the pathogenesis of hepatitis. IL-17 can stimulate various parenchymal cells and myloid immune cells; however, the effect of IL-17 on NK cells remains unknown. By a Poly I:C-induced acute hepatitis model, we studied the cellular source of IL-17 in this model and their function on the severity of hepatitis.Taking together, we integrated four parts of experiments to investigate the cellular and molecular regulation of anti-infectious innate and adaptive immune responses. We focused mainly on four questions: the reverse signal by MHC I and its effect on TLR signal pathway; the regulation of MHC I expression by IL-17 ; the regulation of CTL response by IL-17 through MHC I; the role of IL-17 in the regulation of NK cell function and NK cell-mediated acute hepatitis. Our study will contribute to better understanding of the elaborate crosstalk between innate and acquired systems, and providing clues for the investigation of pathogenic mechanisms and approaches to the therapy of immune disorders.Part I. Reverse signals by MHC I molecules in the regulation of TLR responsesPrevious studies showed that T cell deficient mice are more susceptible to hepatitis, and these mice were died of inflammatory storm generated in the early phase of infection, suggesting adaptive T cells may have suppressive effect on innate immune cells. As MHC molecules are suggested to involved in this regulation, we studied the effect of MHC I on the regulation of TLR responses. Compared with wt mice, MHC I deficient mice exhibited more responses when challenged with TLR ligands like LPS, PolyI:C and CpG. They are also more susceptible to E.coli induced cytokine secretion and bacterial dissemination. When infected with intracellular listeria monocytogenes, MHC I deficient mice showed more resistance in the early phase, though they exhibited more susceptible in the long run compared with wt mice because the lack of cytotoxtitic CD8~+T cells, Bone marrow reconstitution with MHC I deficient stem cells also caused more potent inflammatory responses in recipient mice challenged with TLR ligands, suggesting MHC I on hematopoietic cells was responsible for this regulation. By depleting macrophages in wt mice and then reconstituted with MHC I+/+ or MHC I -/- macrophages, we also suggested a nonredundant role of macrophages in the aggravated inflammatory responses in MHC I deficient mice.Natural MHC I ligands include inhibitory receptors on NK cells and CD8 or TCR on CD8~+ T cells. Mice depleted of CD8~+ T cells or NK cells in vivo showed the enhanced inflammatory responses to TLR ligands, suggesting a negative role of CD8~+ T cells and NK cells in this process. In vitro coculture of macrophages with CD8~+ T cells significantly attenuated TLR ligands-triggered inflammatory cytokine production, and this inhibition was cell-cell contact dependent. However, when CD8~+ T cells were cocultured with MHC I-/- macrophages, no inhibition was observed, suggesting MHC I molecule on macrophages is necessary for the T cell-induced inhibition. Crosslinking of MHC I molecules with MHC I antibodies also attenuated TNF, IL-6 and IFN-βsecretion from TLR-triggered macrophages. Western blot results confirmed impaired NF-κB and IRF3 phosphorylation in crosslinked macrophages. We propose that the MHC I molecules on macrophages could induce reverse suppressive signals when activated with interaction of CD8~+ T cells. But how this reverse signal is transduced and the exact molecule interacting with MHC I remains unknown, which needs to be further investigated.Part II Interleukin 17 induces MHC I expression through activating Stat1Antigen presentation by MHC class I and class II molecules plays important roles in the pathogenesis of many autoimmune diseases. As MHC class I molecule overexpression or abnormality always associated with certain autoimmune diseases like IBD, type I diabetes and myositis etc, and Th17 cells are now recognized as the pathogenic cells of many kinds of autoimmune disorders, we set to find the relationship between these two factors. When macrophages were stimulated with IL-17 in vitro, we observed the upregulation of MHC I expression. IFN-γwas the most studied cytokine to induce MHC I transcription, and phosphorylation of Stat1 is the key step in the procedure. We found that IL-17 could also activate Stat1 signaling in macrophages, and this may explain how it upregulates MHC I expression. IL-17 could also have synergistic effect with LPS to induce more prominent MHC I expression, which suggested us that IL-17 may have some relationship with antigen presentation and CD8~+ T cell proliferation, especially in infectious diseases.Part III. Regulation of anti-infectious CTL responses by interleukin 17 and the role of MHC IBy Q-PCR and ELISA assay, we found that IL-17 expression both in the spleen and serum was increased after infection with listeria monocytogenes (LM). IL-17 production reached the peak on day 5 and then declined, suggesting a certain role of IL-17 in Lm infection. The clearance of bacterial was significantly attenuated in Il17a-/- mice, especially on day 7 post infection when adaptive T cell response dominates the anti-infectious effect. As cytotoxitic T cells (CTL) are necessary in the fully clearance of LM, we compared the CTL responses in these mice. The effector CTLs were observed much reduced in Il17a-/- mice than wt control, while the magnitude of CD4+ T cell responses was not altered. By in vivo CFSE dilution, OT-I cells were suggested to undergo significantly attenuated proliferation when in the absence of IL-17 signal. Meanwhile, in vitro studies revealed IL-17 can't act directly on CD8~+T cells, and APCs are needed in IL-17-induced potent proliferation of CD8~+T cells. IL-17 can stimulate DCs to upregulate its expression of MHC I molecules and secretion of inflammatory cytokines like IL-12, IL-6 etc. DCs isolated from Il17a-/- mice also proved to secret less IL-12 and are less potent to stimulate OT-I cell proliferation than those from wt control. We further identified the cellular sources of this innate IL-17. Not the Th17 cells, a subset of innateγδT cells provides the most IL-17 in the serum after LM infection. Adoptive transfer of wtγδT cells but not Il17a-/-γδT cells or wt CD4+T cells rescued the reduced CTL responses in Il17a-/- mice, confirmed thatγδT cell derived IL-17 are efficient to trigger potent CTL response against Lm infection. Our study demonstrates that, a certain subset ofγδT cells are induced to produce IL-17 quickly post Lm infection. This IL-17 then acts through DCs to increase their ability of cross-presentation, and thus induce stronger anti-infection CTLs. We also revealed that although IL-17 could amplify effector CD8~+T cells responses, IL-17 has no effect on memory T cell generation and function.Part IV. Interleukin 17 aggravates NK-induced hepatitis and the underlying mechanismsPrevious studies revealed the increase of IL-17 in human chronic hepatitis, positively correlating with the severity of the disease, suggesting a pathogenetic role of this cytokine in hepatitis. Il17a-/- mice were found to be more resistant to PolyI:C-induced acute hepatitis, in accordance with the reduction of ALT and AST level in the serum. Pathological analysis also confirmed the reduced hepatocyte apoptosis in Il17a-/- mice. By analyzing liver infiltrating lymphocytes, we found that intraheptic NK cells were less accumulated, and their activation was also attenuated in Il17a-/- mice. In vitro studies suggested IL-17RA expression on NK cells. IL-17, cooperating with PolyI:C, could increase the activation of NF-κB and MAPK pathways in NK cells, and thus help killing YAC-1 target cells and primary heptocytes. However, IL-17 couldn't prolong the survival of NK cells, even together with IL-15. We further found theγδT cells and NKT cells mainly contributed to the production of serum IL-17 after PolyI:C challenge. We also analyzed the role of Kupffer cells in IL-17 aggravated hepatitis. Depleting of Kupffer cells via liposomes or Gdcl3 significantly attenuated the production of serum IL-17 challenged with PolyI:C, suggesting Kupffer cells are involved in IL-17 production fromγδT cells. PolyI:C stimulated production of certain chemokines and cytokines from Kupffers, like IL-1 and IL-23, and these factors could subsequently induce IL-17 secretion fromγδT cells. Adoptive transfer of these cells could aggravate liver damage in some extent.Our results demonstrate that after PolyI:C challenge, innate immune cells likeγδT and NKT cells begin to secret IL-17 in the meantime with NK cell activation through TLR3. Then IL-17 in turn acts on NK cells to enhance its killing ability and thus aggravate liver damage. This will be helpful to explain the mechanisms of hepatitis severity change and design of immune regulation methods for the treatment of the diseases.Taking together, we have investigated the cross-regulation of MHC I and IL-17 both in innate and adaptive immunity. We found that MHC I on macrophages may negatively regulate TLR-triggered innate immune responses; IL-17 could induce MHC I expression through activation of Stat1; and innateγδT cell-derived IL-17 could amplify anti-infectious CTL responses through enhancing DCs cross-presentation; innate IL-17 also acts on NK cells and aggravates NK cell-induced acute hepatitis. Our study will contribute to better understanding of the elaborate crosstalk between innate and acquired systems, and providing clues for the investigation of pathogenic mechanisms and design of approaches to therapy of immune disorders in clinics.