TLR Ligands Trigger Marrow-derived Dendritic Cells To Acquire All-trans Retinoic Acids-producing Capacity

Background and ObjectivesMucosal surfaces are the first barrier to defend against infection of pathogenic microbes. It is currently found that more than 90 percent of human infection resulted from mucosa. Mucosal immunity plays a key role in the immune system, it can limit pathogenic bacteria invasion, resist to allergens from diet and breathing and make appropriate immune response to antigens. Meanwhile it tolerates harmless antigens in food and commensal microflora in the gut. Therefore, occurrence of most infectious diseases can be controlled as long as mucosal infections are controlled.Human intestinal mucosa, with a total area of 300m2, is the main communicational interface between body's intestine and external environment, which is likely to suffer from mucosal membrane infection. Intestinal mucosal immune system that consists of gut-associated lymphoid tissue (GALT) is the largest and most complicated net of immune system. It has strict mechanism of immune reaction and perfect mechanism of immune regulation. There are two subsets of GALT:Peyer's patches (PP), mesenteric lymph nodes (MLN) and small isolated lymphoid follicles are inducive sites in intestinal immune system and are responsible for initializing gut immune response; intraepithelial lymphocytes (IEL) and scattered lymphoid tissue inside lamina propria (LP) are effective sites of intestinal immune system. Compared with the systemic immune response, intestinal immune response is highly specialized towards IgA production, with up to 3g of secretory IgA (sIgA) secreted into human intestinal lumen per day. Over 75% of the total immunoglobulins produced in the body are IgA, and most of them are secreted across mucous membranes. Production of sIgA is an important adaptation to keep the intestinal epithelium homeostasis. sIgA can prevent the body from colonization of invasive pathogenic bacteria to epithelial cells, and can neutralize toxins and invasive enzymes generated by pathogenic bacteria. In addition, sIgA has immunological rejection to certain antigens by food intake and aeroaspiration and blocks these antigens in order to make them dissociate from the mucosal surface without entering the body, thereby avoiding causing systemic immune response and reducing local allergic reaction.Dendritic cells (DC), the most potent antigen presenting cells, serve as the bridge between innate and adaptive immunity and regulate the quality and strength of immune response. It has discovered that intestinal DC has different characteristic from DC from spleen, thymus, axillary nodes or inguinal lymph nodes. The former can produce retinoic acid (RA), but the latter doesn't perform this capacity. RA produced by intestinal DC is one of the key regulatory parameter in mucosal immune response. RA induce expressing gut-homing receptors in T and B cells activated in GALT, which are endued with gut-homing characteristics via lymphocyte recirculation; RA involves in promoting B cells to class switching to IgA; RA also has capacity to influence the differentiation of intestinal Treg and Thl7, thereby may contribute to pathgenesis and development of inflammatory bowel disease. However, the mechanisms of RA production by intestinal mucoal DC, namely what signals initiate RA generation by DC hitherto are unknown. Recent experiments have shown the non-intestinal DC such as DC of skin and lung equally can produce RA. It is worth noting that tissues of RA-producing DC interconnected with external environment, which exposed to abundant microflora, while non-RA DC occupies sterile station. Therefore we hypothesize that certain components of microorganisms may affect RA producing ability of DC. In the present study, we show that Toll like receptor 4 and 9 (TLR4/9) ligands, LPS and CpG, can induce bone marrow-derived DC cell line DC2.4 to acquire RA-producing capacity.Methods1. Cell treatment:cultured DC2.4 were collected and washed with PBS, and then resuspended in RPMI-1640 complete medium.106 cells per well were seeded into 24-well plates. Cells were treated by the following three sets for 48h at 37℃and 5%CO2.1.1 Effects of LPS on RA producing:①Control group, namely DC;②DC+LPS(10μg/ml);③DC+LPS (1μg/ml);④DC+LPS(0.1μg/ml);⑤DC+LPS (0.01μg/ml);⑥DC+LPS (0.001μg/ml).1.2 Effects of CpG on RA producing:①Control group, namely DC;②DC+CpG(10μM);③DC+CpG(1μM);④DC+CpG(0.1μM);⑤DC+CpG (0.01μM);⑥DC+CpG(0.001μM).1.3 Effects of inhibitor of NF-κB-SN50 on RA producing:①Control group, namely DC;②DC+LPS(1μg/ml);③DC+CpG(1μM);④DC+LPS(1μg/ml)+ SN50(18μM);⑤DC+CpG(1μM)+SN50(18μM).2. mRNA expression of RA synthesis related enzymes in LPS or CpG treated DC were measured by quantitative real-time PCR. 3. Western blotting analysis of NF-κB:after cells were treated by LPS or CpG in the presence or absence of NF-κB inhibitor (SN50) for 30 and 60 minutes, proteins of cytosol and nucleus were extraxted. NF-κB was detected by Western blotting.4. Co-cultivation of DC and B cells:C57BL/6 murine splenic B cells were isolated and sorted with flow cytometry, and then B cells were cocultured with LPS or CpG-treated DC in combination with IL-5 and/or IL-6 (lOng/ml) for 4 days in the following 10 sets:Control group, namely B cells; B+DC; B+LPS-DC; B+LPS-DC+IL-5; B+LPS-DC+IL-6; B+LPS-DC+IL-5+IL-6; B+CpG-DC; B+CpG-DC+IL-5; B+CpG-DC+IL-6; B+CpG-DC+IL-5+IL-6.5. FACS analysis ofα4β7 and CCR9:Co-cultivative cells were collected and then gut-homing receptorsα4β7 and CCR9 on B cells were analyzed by flow cytometry.6. Chemotaxis:After 4 days coculture, DC and B cells were collected. Migration of B cells to MadCAM-1 or TECK was analyzed in 24-well transwells with 5μm polycarbonate membranes.7. IgA detection:Supernatants of co-cultivative cells were harvested and concentrations of IgA were assessed by ELISA.8. Cytokine measurement:Supernatants of DC treated with different concentrations of LPS or CpG for 48h were harvested and concentrations of IL-6 or TGF-β1 were detemined by ELISA.9. NO detection:Supernatants of DCs treated with different concentrations of LPS or CpG for 48h were harvested and concentrations of NO were detemined by Griess reaction.Results1. LPS or CpG induced DC2.4 to up-regulate expression of Aldhla2 mRNA. When DCs were stimulated with LPS (1μg/ml), higher Aldh1a2 mRNA expression was noted when compared with control DCs. Aldhla2 mRNA levels were enhanced to nearly 10-fold in DCs, ADH4 mRNA expression were up-regulated to 18-fold. CpG (1μM) also stimulated a 12.45-fold increase in expression of Aldh1a2 mRNA, nearly 10-fold in Aldh1a1 mRNA, and 12-fold in ADH4 mRNA. But Aldh1a3 mRNA levels did not have siginifacant change when cells were treated with LPS or CpG.2. NF-κB signaling pathways plays an important role in LPS-or CpG-induced RA production. SN50 can block LPS or CpG induced NF-κB nuclear translocation. NF-κB/Histone H3 in nucleus in cells treated by LPS (CpG) in the presence of SN50 merely increased from 0.34 (0.33) to 0.46 (0.48) respectively. Ratio hardly changes after inhibition for 60min. When NF-κB nuclear translocation was inhibited by SN50, Aldhla2 mRNA expression declined significantly. SN50 down-regulate enhancement of Aldhla2 induced by LPS or CpG from more than 10-fold to only 2-fold.3. LPS- or CpG-DC promoted expression of gut-homing receptorsα4β7 and CCR9 on B cells, MFI of CCR9 increased 1-fold. IL-5 and/or IL-6 increased expression ofα4β7, but hardly influenced on CCR9 expression.4. LPS- or CpG-DCs enhanced chemotactic activity of B cells to TECK. Ratio of migration increased by 6.4 and 13.1 percentage point respectively when B cells were cocultured with LPS- or CpG-DC. But migration of B cells towards MadCAM-1 was only slightly influenced after cocultured with LPS- or CpG-DC, with an increase of 2.5 and 6.25 percentage point respectively.5. DC treated with LPS or CpG were found to promote IgA production. An increase of near 4-fold and 6-fold IgA production was observed after B cells were cocultured with LPS- or CpG-DC respectively when compared to B cells cocultured with DC2.4. But adding IL-5 or IL-6 did not further boost IgA production. 6. LPS or CpG enhanced DC2.4 to produce IL-6, TGF-p and NO. Within a dose range of 0.1-10μg/ml or 0.1-10μM, both LPS and CpG similarly educated DCs to produce IL-6 and TGF-β1 in a dose-dependent manner. DCs treated with 10μg/ml LPS or 10μM CpG increased production of IL-6 up to 1.3-fold or 1.5-fold respectively, increased TGF-βsecretion by 61% or 70% and increased NO production by 47% and 84% as well.ConclusionTLR ligands can trigger bone marrow-derived DC to acquire all-trans retinoic acids-producing capacity, suggesting that microbial population play important roles in RA production by DCs.