Role Of A Novel C-type Lectin-like Inhibitory Receptor KLRL1 In Immune Tolerance And Its Related Mechanisms

Today with great development on autoimmune disease treatment, bone marrow transplant and organ transplant, more and more attention has been paid to the induction of immune tolerance and the underlying mechanisms which is an important clinical goal in transplantation and autoimmunity. Inhibitory receptors play an important regulatory role in many physiological aspects, including cell growth, differentiation and immune response. Recently, many researches on inhibitory receptors demonstrate that these receptors, which transduce negative signals through their immunoreceptor tyrosine-based inhibitory motifs (ITIM),are also involved and play a vital role in the induction and maintenance of immune tolerance. Therefore, study on the mechanisms of immune protection induced by inhibitory receptors is necessary and could make contributions to the discovery of pathogenesis of some autoimmune diseases.Previously, a new inhibitory receptor from human and mouse DCs has been identified in our lab, designated Killer cell Lectin-like Receptors L1 (KLRL1). Both hKLRL1 and mKLRL1 are type II transmembrane protein with a typical C-type lectin domain and a putative immunoreceptor tyrosine-based inhibitory motif (ITIM). Previous study on KLRL1 indicated that KLRL1 was highly expressed in DCs, NK, CD4+ and CD8+T cells and macrophages. KLRL1 can also recruit the protein-tyrosine phosphatases SHP-1and SHP-2, indicating that it is a novel inhibitory receptor. Previous study also demonstrated that the expression level of KLRL1 was greatly decreased during DCs maturation, and KLRL1 can negatively regulate the function of DCs when exposed to LPS stimulation. All of these results have indicated that KLRL1 is a novel inhibitory receptor and play a negative regulatory role in immune response.The maturation state of DCs may play a role in determining the ending of the immune reaction, which is immune response or immune tolerance. Our previous studies on KLRL1 showed that mKLRL1-modified DCs had stronger phagocytic capacity and less ability to induce T cell proliferation which was very similar with immature DCs. Moreover, the expression level of KLRL1 was greatly decreased during DCs maturation. Therefore, we supposed that the expression level of KLRL1 might have much significance in determining the direction of immune response by DCs. So in the present study, on the basis of the previous work on KLRL1, we observed the protection of mKLRL1-modified DCs from EAE induction which aimed to discover the role of this receptor in the induction and maintenance of immune tolerance. We also explored the underlying mechanisms involved in the negatively regulatory roles of KLRL1 on DCs and the effect of its expression level on TLR4 signaling.Part I KLRL1 plays a role in the induction of antigen-specific immune tolerance in vivoIn this part, we constructed the experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice. By using this EAE model, we then examined the protective effect of KLRL1 in the induction of this kind of autoimmne disease. We used the recombinant adenovirus expressing KLRL1 to tranfect imDCs and stimulated the imDCs with LPS for 18 hours, then pulsed them with MOG35-55 antigen for 4 hours and made pretreatment by peritoneal injection in wide-type C57BL/6 mice. One day after injection, we induced EAE in these mice and observed the disease development. We found that, compared with the control group, the onset of disease was markedly delayed in mKLRL1-modified DCs pretreated group with the longest was 27 days, and the EAE incidence was also decreased. In addition, the clinical nervous system function was also greatly improved with lower clinical score in the mKLRL1-modified DCs pretreated group. To further explore the difference in vivo, we then obtained the lymph node cells from mice of each group in which EAE had been induced, stimulated the cells with MOG35-55 peptide and evaluated their proliferation. Our results showed that MOG35-55 stimulation increased the proliferation of lymph node cells from mice of Ad-ctrl and DCs pretreated group more than those from mice of Ad-KLRL1 pretreated group. All of these above results have demonstrated that the inhibitory receptor KLRL1 probably plays a role in the induction of antigen-specific immune tolerance in vivo.PartⅡunderlying mechanisms involved in the negative regulatory role of KLRL1 on DCsPrevious study showed that mKLRL1-modified DCs produced high level of IL-10 upon exposion to LPS stimulation. IL-10 is an important cytokine involved in immune suppression. Then we supposed that mKLRL1-modified DCs might regulate the function of tolerogenic DCs by secreting high level of IL-10. We found that mKLRL1-modified DCs from wide-type mice had a much stronger phagocytic capacity, while mKLRL1-modified DCs from IL-10 deficient mice had a comparable phagocytic capacity with Ad-ctrl group, indicating an important role of IL-10 in regulating the phagocytic capacity of DCs by KLRL1. Since the expression level of KLRL1 was greatly decreased during DCs maturation, we then want to know whether KLRL1 could regulate the costimulatory molecules on DCs. Our results showed that expression levels of CD80 and CD86 were decreased on mKLRL1-modified DCs upon exposion to LPS, however, IL-10 deficiency could make this condition reversed to a certain extent, which indicated a role of IL-10 in regulating the expression level of costimulatory molecules on mKLRL1- modified DCs. Moreover, we also detected the role of IL-10 in antigen-presentation ability of DCs regulated by KLRL1. We found that, mKLRL1-modified DCs from wide-type mice had less ability to induce peptide-specific CD4+T cells proliferation, while there was no significantly difference between this ability of mKLRL1-modified DCs from IL-10 deficient mice and that of corresponding control group. Taken together, all of these above results have demonstrated an important role of IL-10 for KLRL1 to display its negative regularory role on DCs. IL-10 might be a vital effector molecule of inhibitory receptor KLRL1.Since IL-10 can make the production of most proinflammatory cytokines inhibited, we also detected the secretion of some other important cytokines in mKLRL1-modified DCs upon LPS stimulation, including TNF-αwhich were previously found to be produced at a much lower level in mKLRL1-modified DCs. Our results showed that there were no significantly differences in the produtions of IL-6, IL-1βand IL-12p70 between mKLRL1-modified DCs and Ad-ctrl group, however, the production of TNF-αby mKLRL1-modified DCs was greatly decreased as found in the previous study, suggesting that TNF-αmight be another effector molecule of KLRL1.To further understand the signal pathways involved in the high production of IL-10 by mKLRL1-modified DCs and the role of KLRL1 in TLR4 signaling, we then examined the activation of some important signal pathways. First, we found that the increased secretion of IL-10 by mKLRL1-modified DCs was LPS-stimulation dependant. We then examined the phosphorylation states of some related signaling molecules by western blotting. We found that TLR4 expression level remained unchanged in mKLRL1-modified DCs which indicated that KLRL1-overexpression might not directly influence the TLR4 expression level. It is showed that LPS-induced cytokines production by DCs are mainly through MyD88-dependent pathway, hence we detected the MAP kinase pathways. Our results showed that phosphorylation levels of ERK1/2 and p38 were both increased when stimulated with LPS, but there were no differences between mKLRL1-modified DCs and control group, however, phosphorylation levels of JNK1/2 and its upstream MKK4 kinase were strongly decreased when KLRL1 was overexpressed, suggesting that JNK pathway might be involved in the regulatory function of KLRL1. We also detected the NF-κB signal pathway. Compared with the control, the phosphorylation levels of IKKαand its downstream I-κB were significantly decreased in mKLRL1-modified DCs, indicating that KLRL1 may have negative effect on it. Finally, we examined the activation of PI3K-Akt pathway and found no difference between mKLRL1-modified DCs and control group. To make further confirmation, we also examined the phosphorylation levels of these signal molecules in KLRL1-silenced RAW264.7 cells and found phosphorylation level of JNK1/2 and MKK4 is greatly increased when KLRL1 was silenced. In addition, phosphorylation level of IKKαand I-κB were also increased, further indicating the impact of KLRL1 on NF-κB signal pathway.