| Objective:Human Immunodeficiency virus-1(HIV-1) infection causes mucosal CD4+T lymphocyte loss, which leads to mucosal immune dysfunction, translocation of microorganisms and chronic immune activation, eventually develop into Acquired immune deficiency syndrome (AIDS). Although during the early stage of infection, a large number of mucosal CD4+T lymphocytes were infected, CD4+T lymphocytes declined significantly, but the peripheral blood CD4+T lymphocytes were maintained at a high level long after infection. The mechanism for the decline of mucosal CD4+T lymphocytes is not fully understood yet. In addition to direct and indirect killing of CD4+T lymphocytes caused by virus infection, the abnormal distribution of CD4+T lymphocytes in some tissues could be another reason. Lymphocyte circulation is a process of lymphocyte migration to a particular tissue, involved various cytokines and adhesion molecules including the selectin family, integrin family, and immunoglobulin superfamily. In this study, SIV/SHIV infected rhesus macaques were used to explore the impact of HIV-1on the genes-associated with gut-mucosa specific immune cell homing.Methods:1) Total tissue RNA for cloning MAdCAM-1was extracted from colon mucosa of a rhesus macaque, and was used for amplifying the MAdCAM-1cDNA nucleotide sequence by5’-and3’-RACE. The PCR products obtained were cloned into pGEM-T Easy vector and sequenced. The MAdCAM-1cDNA was obtained by sequence alignment using DNAMan software. Nucleotide sequences of MAdCAM-1for53animal species were retrieved from NCBI GenBank and analyzed with BioEidt and MEGA software, and a phylogenic tree based on amino acid sequences of47species was constructed using Neighbor-Joining methods.2) Total tissue RNA was extracted using RNAprep Pure Tissue Kit (Tiangen, Beijing) according to the manufacturer’s instructions, while total protein was extracted using Tissue Protein Extraction Kit (Tiangen, Beijing). The semi-quantitative RT-PCR and real-time quantitative RT-PCR were used to detect the expression of MAdCAM-1mRNA in various tissues, while Western Blot was used to detect the expression of MAdCAM-1protein in duodenal biopsies. To examine the splice variant, RT-PCRs to amplify the region flanking the4th exon were performed.3) The sequences with full-length or short variant (without exon4) of the coding region of rhesus macaque MAdCAM-1were sythesized and cloned into pCDNA3.1vector. Recombinant expression plasmids were transfected into293F cells. The surface expression of MAdCAM-1was examined by flow cytometry. The transfected293F cells were stained with CFSE, and the α4β7+Hut-78were stained with PKH26. The adhesion experiments were performed by mixing the two cells in the presence of Mn2+.4) Normal rhesus macaques(GrN), SHIV-SF162p4and SIVmac251infected rhesus macaques(Gr4), SHIV-SF162p4infected rhesus macaques(Grl5) and SHIV-SF162p3infected rhesus macaques(Grl9) were used in this research (frozen tissues of infected animals were mainly the samples collected from virus infected control group used in previous experiments). The total tissue RNA was extracted from frozen tissues according to the manual instructions. The real-time quantitative RT-PCR were established to detect the expression of gene transcription level of MAdCAM-1, FTGA4, ITGB7, NKX2.3, CCL25, CCL9, CCL28, CCR10, CCL19, CCL21, CCR7, RGS1, CCL22, CCR4, CCL20, CCR6, GPR183, FCGRT, PIGR, BCL-6, DDFT3, TNF-a, IFN-y.5) The total protein from four rhesus macaque groups was extracted according to the manufacturer’s instructions. The expression of MAdCAM-1protein was detected by Western Blot, while CCL20, TNF-a, IFN-y, IL-17and IL-6proteins were detected by ELISA.Results:MAdCAM-1play an important role in specific homing to the mucosa. In this research, we cloned the MAdCAM-1cDNA by RACE, and explored its alteration in duodenal mucosa after simian/human immunodeficiency virus (SHIV) infection. The aasembled nucleotide sequence consisted of1503nucleotides, including a14bp5’-untranslated region (UTR) and a403-bp3’-UTR. A canonical polyadenylation signal AATAAA was found at position1463bp. The deduced rhesus macaque MAdCAM-1peptide, similar to that of human, contained a signal peptide,2N-terminal Ig-like domains, a mucin-like domain, a transmembrane domain and a cytoplasmic tail, but no Ig-like domain3, the IgAl-like domain in mice. The conserved motif LDT for α4β7binding was within the first Ig-like domain.In normal rhesus macaques, MAdCAM-1mRNA was detected in all tissues examed and high-level transcripts were observed mainly in gastrointestinal tract, spleen and lymph nodes, especially in mesenteric lymph nodes with the highest level. The expression of MAdCAM-1mRNA in the large intestine was more than that in the small intestine. The expression of MAdCAM-1mRNA was very low in skin, thymus, oral cavity mucosa and liver, with the lowest in the liver. The mRNA levels in SHIV infected duodenal tissues were significantly reduced, and the MAdCAM-1protein expression levels were also decreased in SHIV infected tissues.To further understand the impact of SIV/SHIV on lymphocyte homing related genes, we examed the mRNA levels of MAdCAM-1and its receptor α4β7in normal and infected animals. The expression of MAdCAM-1mRNA was raised in small intestine, while decreased in large intestine after infection. This change regularity was also found in the expression of ITGA4, ITGB7, NKX2.3gene and MAdCAM-1protein. The distribution among MAdCAM-1, ITGA4, and ITGB7in normal rhesue macaques were correlated, while the distribution of MAdCAM-1and NKX2.3in the intestine was not correlated. After infection, the distribution of MAdCAM-1and NKX2.3in the intestine was correlated, which may be related to the regulation of MAdCAM-1expression in the gut.Further studies found that SIV/SHIV infection also affected the transcriptional levels of various chemokines and their receptors in the gastrointestinal mucosa. Among them, the expressions of the gut-associated chemokine CCL25, CCL28and their receptors CCR9and CCR10were decreased in the small intestine and increased in the large intestine after infection, while the effects were different among different viral infections. The expression of chemokine CCL19, CCL21and their receptor CCR7had little change in the lymphoid organs, but significant decreased in the duodenum. The expression of chemokine CCL20mRNA had little change after viral infections, but its receptor CCR6was significant decreased in the duodenum and jejunum. The expression of chemokine CCL22mRNA was decreased in the jejunum and its receptor CCR4was decreased in the inguinal lymph nodes.Also, SIV/SHIV infection affected the transcriptional levels of cell migration regulating gene. GPR183regulates migration of B cells in the lymphoid follicles. The expression of GPR183mRNA was significantly decreased in the spleen, mesenteric lymph nodes and inguinal lymph nodes. The expression of RGS1mRNA was decreased in the fundus and duodenum.To understand the relationship between the changes of lymphocyte homing related genes and the other mucosal immune pathological changes, we studied the changes of inflammatory cytokines TNF-a, LFN-y transcription levels after infection. On the whole, the expression of TNF-a mRNA was decreased in the proximal end of the small intestine, while increased from the ileum to the rectum. The expression of IFN-y mRNA was significantly reduced in the rectum, but had little change in the other intestinal segments after infection. The expression of inflammatory cytokine TNF-a protein was significantly decreased in the jejunum and ileum, with the trend of rising in the small intestine while decreasing in the large intestine. The expression of IFN-γ protein had little significant change after infection. The expression of IL-6protein was significantly increased in the ileum and caecum after SIV/SHIV infection. The expression of IL-17protein was significantly increased in the rectum after infection.Furthermore, we observed the changes in the transcription of immuneglobulin(Ig) transport-related genes. The expression of FCGRT mRNA was increased in the intestine after infection. The expression of PIGR mRNA was decreased in the gastrointestinal tract and lymph nodes. The expression of BCL-6mRNA was decreased in the small intestine while increased in the large intestine after SIV/SHIV infection. The expression of BCL-6mRNA was decreased in the gastrointestinal mucosa (except rectum) after SHIV infection. These results suggested that the SIV/SHIV infection might influence the function of mucosal humoral immunity.Conclusions:1) The nucleotide sequence of macaque MAdCAM-1cDNA was obtained, which is highly similar to that of human;2) SIV/SHIV infection has impact on the expression of mucosal homing related adhesion molecule MAdCAM-1and its receptor integrin α4β7;3) SIV/SHIV infection has impact on the expression of a variety of mucosal homing related chemokines and receptors;4) The expression of mucosal homing related cytokines after SIV/SHIV infection is closely related to the level of inflammation in the mucosa;5) The altered expression of mucosal homing related cytokines after SIV/SHIV infection is accompanied by the changes of the mucosal humoral immune function. The results of this study indicated that HIV-1may affect the mucosal cellular and humoral immune functions by regulating mucosal lymphocyte homing. These results provided a basis for the understanding of HIV/AIDS pathogenesis and searching for new targets for HIV/AIDS treatment. |
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