Histamine’s Effects On HIV-1Infection Of Intestinal Mucosa

ObjectiveTo find the relationship between mast-cell degranulated mediators and HIV-1infection of mucosa:1. We establish the technique for generation or isolation of human primary mast cells;2.We used monocyte-derived immature dendritic cells as target cells. After treatments of histamine and HIV-1, we tested the activation and functional modulation of DCs, as promoting CD4+T-cell proliferation or driving regulatory T-cell polarization, which were benefit for understanding the pathogenesis of HIV-1infection and the potential contribution of allergic mediators in disease progression.Methods1.CD34-hemopoietic stem cells were cultured in presence of SCF, IL-6, and IL-3to generate primary mast cells; Mast cells allocated in human intestinal tissues were isolated through digestion, density gradient centrifugation and purification with specific antibodies-coated magnetic beads. Cell phenotype was monitored by immuostaining of surface markers of CD117and FcεR Ⅰ and intracellular tryptase, or cells were identified with Toluidine blue staining.2.Monocytes were isolated by using specific antibodies-coated magnetic beads, then cultured in vitro for5days to generate immature dendritic cells in presence of GM-CSF and IL-4. Stimulated by histamine and/or JRFL, DCs were tested for phenotype alteration, using flow cytometry. We detected the cytokines production from differently-treated DCs at mRNA level (real-time PCR) and quantified them via flow cytometry. After that, differently-treated DCs were cocultured with naive CD4+T cells or resting CD4+T cells, FCM was taken to measure the activities of DCs in promoting CD4+T-cell proliferation or driving regulatory T-cell polarization. IDO expression of differently-treated DCs was subsequently detected at mRNA level and protein level,1-MT was added to confirm the functional modulation of DCs caused by IDO. A variety of histamine receptor antagonists, pyrilamine (HIR antagonist), cimetidine (H2R antagonist), and thioperamide (antagonist of H3R and H4R), was used to find out histamine receptor responsible for IDO-expression on DCs.Results1.Both of the CD34+cell-derived and intestinal mast cells express CD117and FcεR I, the specific markers for mast cells, and contain intracellular tryptase.2.Monocyte-derived immature dendritic cells showed expression of HIR, H2R, and H4R, but no detectable H3R. Compared with medium-treatment, the treatment of HIV-1/JRFL or histamine alone slightly or did not increase the expression of CD83, CD86, or HLA-DR, but the co-treatment with histamine and HIV-1increased much expression of these three markers, suggesting an activation or enhanced maturation status of MDDCs. Both histamine and HIV-1could significantly induce IL-10production, histamine and HIV-1showed synergetic role in IL-10-induction. In addition, the co-stimulation of MDDCs with histamine and HIV-1benefited in TGF-β production, and histamine did not show much effect on HIV-1induced IL-6expression, While, histamine-treatment hampered HIV-1-induced up-regulation of IL-12. Histamine or HIV-1alone-treated MDDCs did not or slightly drive the proliferation of CD4+CD45RA+naive T cells, however, co-stimulated-MDDCs with histamine and HIV-1drive much of naive T cell proliferation. When resting CD4+T cells were used for mixted-culture with treated-MDDCs, significant T cells proliferation-driven by histamine and HIV-1co-stimulated-MDDCs were also observed. Compared with medium-treated MDDCs group, the addition of histamine during HIV-1treatment of MDDCs benefit MDDC-stimulated Treg-cell generation. Both histamine and HIV-1could induce IDO expression in MDDCs, as detected at increased mRNA level and protein production; notably, a synergetic role of histamine and HIV-1on IDO induction was observed. The treatment of MDDCs with1-MT compromised the capacity of HIV-1/histamine co-treated-MDDCs for driving naive CD4-T cells differentiation into Treg cells, the generation of Treg cells was demonstrated in proliferation suppression assay of allogeneic resting CD4+T cells. Lastly, we found that histamine receptor-2(H2R)-mediated signaling appeared to be required for histamine induced IDO production.Conclusions1.Both methods could provide sufficient and purified primary mast cells for further study in HIV-1mucosal infection.2.Histamine participates in HIV-1infection of immature dendritic cells, leading to decreased production of proinflammatory cytokines, enhanced expression of anti-inflammatory cytokines. Histamine assists HIV-1-induced functional modulation of dendritic cells in skewing differentiation of naive T cells toward regulatory T cells, through activation of H2R. All of those may help to establish the immune suppressive environment.