The Study Of Kir In SLE And ReA Pathogenisis And The Effects Of HIC1in SMG Organogenesis

BackgroundSystemic lupus erythematosus (SLE) is a systemic auto-immune disorder,which is characterized by the production of different auto-antibodies and diverse clinical inflammation in multiple organ systems. Although the precise etiology and pathogenesis of SLE still remains unclear, various genetic and environmental factors along with multiple immunological abnormalities may be involved. Previous studies suggested the aberrant activation of T-cells and natural killer (NK) cellsith fewer NK cells and decreased cytotoxicity of NK cells in SLE patients. Furthermore, a lower frequency of CD3+CD56+natural killer T (NKT) cells was found to be associated with higher levels of plasma IgG and IgG anti-dsDNA antibodies in SLE patients. Hence, an imbalance of lymphocyte subsets has been implicated in the pathogenesis of SLE.Killer cell immunoglobulin-like receptors (KIR), which evolved from the Ig superfamily (IgSF) consisting of type I transmembrane glycoproteins, are expressed on NK cells and subsets of T-cells. The KIR genes are polymorphic, whereas the KIR gene complex is polygenic. Recent studies showed that the expression patterns of KIR on NK-and T-cells were regulated by the methylation of KIR gene loci. Fourteen KIR genes and two pseudogenes have been described. The extracellular domains of KIR molecules contain either two (2D) or three (3D) Ig-like domains, while the cytoplasmic tails are either long (L) or short (S). The inhibitory KIRs possess a long cytoplasmic tail, which contains immunoreceptor tyrosine-based inhibitory motif (ITIM), while the stimulatory KIRs possess a relatively shorter cytoplasmic tail, which comprises of tyrosine-based activation motifs containing adaptor DAP12. Human leukocyte antigen (HLA) class I molecules serve as ligands for the KIRs; besides, with different KIR and HLA, it either stimulates or inhibits the killing and secretion of inflammatory cytokines of NK cells.Genetic studies indicated the existence of an association between KIR and infectious diseases such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV), autoimmune and inflammatory disorders such as scleroderma and psoriatic arthritis, cancer such as cervical cancer, and pre-eclampsia. Generally, the genotypes with lower inhibition and higher activation patterns appear to be beneficial in viral infections, but they constitute a risk for susceptibility to auto-immunity. However, the genotypes in which inhibitory KIR predominates seems to be at an increased risk of pre-eclampsia.According to Pellet et al., gene frequency of KIR2DS1in the absence of KIR2DS2increases in SLE patients in comparison to healthy controls. Moreover, patients with vascular arterial events were observed to have a significant increase in KIR2DS2and KIR2DL2gene frequencies when compared with patients without events. We also found that Chinese SLE patients had higher genotypic frequencies of KIR2DL2and KIR2DS1and were frequently observed with two more activating KIR genes than in controls. Stimulatory and inhibitory KIRs of SLE patients were found to be functional on lupus T cells as the DNA of these cells were hypomethylated. However, the mAbs of KIR2DL3/L2/S2and KIR2DL1/S1were used for flow cytometry as the stimulatory and inhibitory receptors could not be distinguished without genotyping nor were they related to any patient characteristics; thus, no conclusion was clearly established.ObjectiveTo investigate the expression pattern of Killer cell immunoglobulinlike receptors (KIR) on natural killer (NK)(CD3-CD56+) cells, natural killer T (NKT)(CD3+CD56+) cells, and subsets of T (CD3+CD56-) cells in SLE. To discussion the correlation between the expressions of CD158a/h, CD158b/i/j on NK-, NKT-, T-cells and SLEDAI score, immunological features of SLE patients. To investigate relative transcription_for KIR2DL1, KIR2DS1and KIR2DL2in NK-, NKT-and T-cells in SLE.Method1. Patients and controlsBoth SLE patients and controls were classified as2DS1+2DL1+groups (SLE patients:n=16; controls:n=12) and2DS2-2DL2+2DL3+groups (SLE patients:n=12; controls:n=8). The clinical laboratory of the hospital offered the immunological indices of SLE patients. Scatter Turbidimeter was used to measure the serum level of IgG, IgA, IgM, C3and C4; IIFT was used to measure ANA, and ELISA was used to measure ds-DNA, ss-DNA, AnuA, AHA, rRNP and SM. The cohort was genotyped for the presence or absence of KIR genes by using PCR-SSP.2. Flow cytometric analysisThe PBMC were isolated and were then analyzed by EPICS XL-4flow cytometry. Monoclonal antibodies as follows:(i) anti-human CD3(PE),(ii) anti-human CD56(Cy5),(iii) FITC-CD158a/h (EB6)(KIR2DL1/KIR2DS1) and (iv) FITC-CD158b/i/j (GL183)(KIR2DL2/KIR2DL3/KIR2DS2).3. We analyzed the correlation between the expressions of CD158a/h, CD158b/i/j on NK-, NKT-, T-cells and SLEDAI score, immunological features of SLE patients by using the Pearson correlation coefficient (r). A two-tailed p-value≤0.05was considered to be statistically significant.4. A CD56multi-sort kit along with CD3magnetic beads were used to isolate the NK-, NKT-and T cells from the PBMCs taken from of SLE patient and healthy control. And RT-PCR analysis was used for quantification of KIR2DL1,2DS1,2DL2, and the control18S gene by the ABI PRISM7500detection system.Results1. In SLE patients, the proportion of NK cells was significantly lower as compared to the controls (p<0.001). Moreover, a reduction in the proportion of NKT cells was observed in the peripheral blood of SLE patients as compared to the controls (p<0.001). However, in the T-cells, no significant difference was detected between the two groups.2. The expression of CD158a/h on NK cells in the SLE patients was significantly increased when compared to the healthy controls (p<0.001). However, the expression of CD158b/i/j on NK cells in the SLE patients was significantly decreased when compared to the healthy controls (p<0.001). And a significant increase of CD158b/i/j MFI (p=0.036) was seen in the SLE group.3. The expression of CD158a/h on NKT cells in the SLE patients was significantly increased when compared to the healthy controls (p<0.001). The expression of CD158b/i/j on NKT cells in the SLE patients was significantly decreased when compared to the healthy controls (p=0.002).4. Expression of CD158a/h on T-cells was also significantly increased in the SLE patients (p=0.026).5. The proportion of CD158a/h+NK cells positively correlated with the serum levels of anti-dsDNA titer in SLE patients (R=0.742, p=0.001).6. The proportion of CD158b1/b2/j+NKT cells in PBMCs and the serum levels of IgG showed a significant negative correlation in SLE patients (R=-0.591, p=0.043).7. The results showed the up regulation of transcription for KIR2DS1(p=0.025) and down regulation of KIR2DL1(p=0.049) in NK cells from SLE patients compared to the controls.8. The transcription of KIR2DS1was up regulated in NKT cells from SLE patients (p=0.041).Conclusion1. Our results verified that SLE patients have increased stimulatory and decreased inhibitory KIRs expression on NK cells. The increased expression of KIR2DS1on NK cells in SLE patients may decrease their cytotoxic effect against the target cells, such as auto-reactive T-cells, thus, resulting in an auto-antibody production (for example, Anti-dsDNA antibodies). So abnormal expression patterns of KIRs on NK cells of SLE patients may influence their differentiation and functional activity, and these may participate in the pathogenesis of SLE.2. The abnormal KIRs expression on the NKT cells maybe functional and contributing to SLE pathogenesis at least partly through influence serum IgG level.3. Stimulatory and inhibitory KIRs are functional on lupus T-cells, and the over expressive stimulatory KIRs may take part in the activation of auto-reactive T-cells in SLE patients.4. The proportion of CD158a/h+NK-, NKT-and T cells appeared to increase in the SLE patients group, while their MFI levels did not show any significant difference. Therefore, the increased KIR2DS1mRNA may be the result of a general expansion of2DS1+lymphocytes, but not a very high expression among the small NK or NKT cell subset. BackgroundReactive arthritis (ReA) is sterile arthritis triggered by bacterial gastrointenstinal or urogenital infections. Although the pathogenesis of ReA remains unclear, genetic factors seem to play an important role. Different killer cell immunoglobulin-like receptors (KIRs) and their corresponding specific histocompatibility leukocyte antigen-C (HLA-C) ligand genotypes have been implicated in susceptibility and resistance to infections and autoimmune diseases but have, thus far, not been investigated in ReA.MethodsThis study was conducted in138ReA patients (65females,73males); aged18to69years (mean,37years) and151randomly selected healthy control individuals matched for ethnicity, age and sex. These subjects were genotyped for KIR genes and HLA-C alleles by polymerase chain reaction with sequence-specific primers.Results The frequencies of inhibitory KIR2DL2and KIR2DL5were significantly lower in the ReA patients than in the controls (P=0.005and P=0.033, respectively). The presence of more than seven inhibitory KIR genes was protective (P=0.016). Moreover, we found that activating KIR2DS1alone or-in combination with the HLA-C1C1genotype (which indicates the absence of the HLA ligands for their homologous inhibitory receptor KIR2DL1) is associated with susceptibility to ReA (P=0.039and P=0.011, respectively), whereas KIR2DL2in combination with the HLA-C1ligand is associated with protection against ReA (P=0.039).ConclusionsThese observations indicate that high levels of activating and low levels of inhibitory KIR signals may affect the functions of NK cells and T cells. This imbalance enables the innate and adaptive immune responses of the host to be easily triggered by pathogens, resulting in the overproduction of local and systemic cytokines that contribute to the pathogenesis of ReA. BackgroundHypermethylated in cancer1(HIC1), which is located at17p13.3, a transcriptional repressor, resides in a CpG island that is hypermethylated in many types of human cancers and its targets genes involved in proliferation, tumour growth and angiogenesis. Now the Underhill group has generated a novel conditional Hicl knockout mice model. Preliminary analysis of these mice indicates that deficiency of this gene leads to a variety of skeletal malformations including, bone morphological anomalies, craniosynostosis and reduced bone mineral density,we found the salivary gland of the HIC1knockout mice embryos have developing disorder, so we try to figure out how does this happen and this would help us know better about HICl.And these studies will give us an opportunity to learn a new animal model for studying the etiology and treatment of salivary gland disorders.Methods1. Dissect different stages embryos out of the uterus in ice cold PBS and place in single wells containing PBS. SMG was isolated and taken images, genotyping of the embryos were done at the same time.2. Preparation of RNA and cDNA and quantitative real-time PCR, RNA was extracted from each pair of salivary glands using RNAeasy micro kit.The transcription levels of BTBD7, FGF10, MMP2, MMP9, Fnl (fibronectin), Inhibin beta A (Activin beta A), Inhibin beta B (Activin beta A) were tested.3. Whole-mount X-gal staining. Dissected E13.5SMGs were grown on filters overnight, prior to processing for X-gal staining..4. Immunohistochemistry and Immunofluorescence. Tissues were fixed in formalin overnight at4℃. Paraffin-embedded sections (7mm) were prepared for H&E staining, Immunofluorescence, and immunohistochemistry (IHC). We stained sections with the following primary antibodies:anti-Fnl (fibronectin), anti-colligenl, anti-Ki67, anti-AQP5. Secondary antibodies, conjugated to either the488Alexa-fluor or the594Alexa-fluor.Results1. HIC1(?)SMG consists primarily of undifferentiated epithelium composed of very few branches surrounded by undifferentiated, condensed mesenchyme.2. AQP5expression was reduced in HIC1(?)SMG.3. HIC1is located in mesenchyme of salivary gland4. Mesenchymal cells proliferation increased in HIC1(?)SMG.5. Fibronectin expressions were decreased in HIC1(?)SMG at later stages.6. Collagen I staining was decreased in order stages of HIC1(?)SMG.7. FGF-10transcripts were decreased in HIC1-/-SMG at E15.5.8. MMP2and MMP9transcripts were increased in HIC1(?)SMG at E14.59. Activin beta A and Activin beta B transcripts were increased in HIC1(?)SMG at stage E14.5while no Smad2staining difference can be found.Conclusions1. Impaired branching and size morphogenesis in HIC1(?)SMGs. Embryonic HIC1(?)SMGs are developmentally-delayed.2. As a functional marker of SMG, reduced AQP5expression means there is a function disorder in HIC1(?)SMGs. HIC1affects epithelial cell differentiation.3. HIC1affects the adjacent mesenchymal cells proliferation 4. HIC1maybe influence the inter lobe formation in the later stages of SMG by affecting Fibronectin’s expression.5. Collagen I,TGF10, MMP2and MMP9maybe also take part in HIC1signaling pathway in mice embryos salivary gland developing.