Integrin CD11b Negatively Regulates TLR9-triggered Dendritic Cell Cross-priming By Upregulating MicroRNA-146a And Targeting Notch1

Immune activation or immune tolerance largely depends on the degree of antigen presentation and functional state of the immune cells involved. As specialized antigen-presenting cells and initiator of adaptive immune response, dendritic cells (DCs) can take up, process, and present exogenous antigens with MHC class II molecules to CD4~+ T cells. Apart from this, DCs can also present exogenous antigens to CTLs via MHC class I molecules. This special antigen presentation, known as cross-priming, has been shown to play critical roles in the immune defense against virus, bacteria, and tumor. During this process, IL-12p70, predominantly produced by DCs, not only enhances CTL synapse formation and prolongs CTL-DC interactions, but also promotes CTL proliferation and IFN-γproduction. DC cross-priming of CTL response is mediated by multiple mechanisms, such as activated costimulatory signals and attenuated proapoptotic signals. However, identifying positive and negative regulators in this process is still an ongoing process.β2 integrins (CD11/CD18) play important roles in immune response and inflammation. According to their distinctαsubunits,β2 integrins are divided into four functional heterodimers termed leukocyte function-associated antigen 1 (LFA-1; CD11a/CD18,αLβ2 integrin, ITAL antigen), Mac-1 (CD11b/CD18,αMβ2 integrin, ITAM antigen), p150,95 (CD11c/CD18,αXβ2 integrin, CR4, ITAX antigen), andαDβ2 (CD11d/CD18, ITAD antigen). CD11b is extensively expressed in most immune cells, such as DC, monocytes, macrophages, granulocytes, and NK cells, participating in cell activation, chemotaxis, cytotoxicity, and phagocytosis. Our previous studies have shown that CD11b is able to maintain immunological tolerance and inhibit inflammatory responses via repressing TLR3 signaling in NK cells and TLR4 signaling in macrophages. Analogously, CD11b, abundantly expressed in DCs, can inhibit DC-primed CD4~+ T cell activation and promote peripheral tolerance by breaking Th17 differentiation. These reports have suggested the roles of CD11b in the regulation of T cell adaptive immunity. However, it remains unclear whether CD11b is also involved in DC cross-priming for CTL response. In CTL activation, it is well-accepted that TLR ligands, including LPS and CpG-ODN, are able to improve vaccination aiming at induction of CTLs, but the role of CD11b in this process is still unknown.Although TLR4 and TLR9 both contribute to DC maturation and cytokine production, they depend on different signaling pathways. In DCs, LPS provokes cytokine production mainly through myeloid differentiation factor 88 (MyD88) dependent manner while induces upregulation of costimulatory molecules including CD40, CD80, and CD86 via the TIR domain-containing adaptor inducing IFN-β(TRIF) dependent manner. But TLR9 exclusively initiates a signaling cascade through MyD88, which activates NF-κB and MAPK pathways, and in turn induces proinflammatory cytokine production and DC maturation.Micro-RNAs (miRNAs) are a class of small non-coding regulatory RNAs. They control gene expression at the post-transcriptional level by inhibiting translation or inducing mRNA degradation. miRNAs are emerging as important regulators in immune responses. Among them, miR-146a plays key roles in innate immunity, inflammatory response, virus infection, and human diseases. Both TLR activation and inflammatory stimulation can lead to upregulation of miR-146a, which in turn negatively regulates innate immunity by repressing its targets, such as IL1-R-associated kinase (IRAK) 1, IRAK2, TNFR-associated factor (TRAF) 6, and Tata Binding Protein (TBP). The induction of miR-146a is reported to be dependent on NF-κB activation, but the precise regulatory mechanism remains unclear. Apart from this, as a single miRNA has been thought to target multiple mRNAs, there are still many potential targets of miR-146a remain to be identified.Here, in CD11b-deficient DCs, we found that TLR9-triggered IL-12p70 production was increased, and DC cross-priming for CTL response was elevated, suggesting the negative regulation of CD11b in DC cross-priming. For the mechanisms responsible for its negative regulation, we showed that CD11b participates in TLR9-mediated miR-146a upregulation in DCs. And the upregulated miR-146a in turn targets Notch1 to repress IL-12p70 production. Hence, our results reveal a novel role of CD11b in DC cross-priming for CTL response. 1. CD11b represses DC cross-priming of CTL response by inhibiting IL-12p70 productionTo investigate the involvement of CD11b in DC cross-priming CTL response, we cultured BMDCs from wild-type or CD11b-deficient mice, and stimulated with TLR4 ligand (LPS) or TLR9 ligand (CpG-ODN) for 24 hours. When loaded with antigen OVA and co-cultured with na?ve CD8+ CTLs from OT-I mice, CD11b-deficient BMDCs had significantly enhanced CpG-triggered DC cross-priming of CTL response compared with that of wild-type BMDCs, as shown by more OVA-specific CTLs and higher IFN-γproduction. Unexpectedly, CD11b-deficiency did not affect LPS-triggered DC cross-priming CTL response. These results indicate that CD11b in DCs can negatively regulate TLR9, but not TLR4, -triggered cross-priming CTL response.To further clarify the specific mechanism, we firstly detected the phenotype of CD11b-deficient DCs upon LPS or CpG-ODN stimulation. FACS analysis showed that CD11b-deficiency had little effect on the expression of MHC-I, MHC-II, CD40, CD70, CD80, or CD86 on both immature and mature BMDCs, thus indicating that CD11b mediated negative regulation of DC cross-priming CTL response was independent of cell surface costimulatory molecules. After screening of cytokines production in LPS or CpG-ODN stimulated DCs, we found that CD11b-deficiency had no significant influence on IL-6, TNF-α, or IL-1βproduction. Interestingly, CpG-ODN-induced IL-12p70 production was significantly enhanced in CD11b-deficient BMDCs than that in wild-type BMDCs, furthermore, specific neutralizing antibody to IL-12p70 diminished the differed CTL proliferation and IFN-γproduction between CD11b-deficient and wild-type DCs upon CpG-ODN stimulation. Because IL-12p70 is well-accepted to augment CTL activation, these results demonstrate that CD11b negatively regulates CpG-ODN-induced IL-12p70 production in DCs, and in turn represses DC cross-priming CTL response.2. CD11b participates in CpG-ODN-induced miR-146a upregulation in DCs via sustaining late-phase NF-κB activationIn CpG-ODN-triggered DC cross-priming CTL response, CD11b only negatively regulates CpG-ODN-induced IL-12p70 production, but does not influence DC phenotype maturation or inflammatory cytokines production. This phenomenon indicates that CD11b is less likely to directly influence TLR9 signaling in DCs. So, some other indirect mechanisms may be responsible for the enhanced IL-12p70 production in CD11b-deficient DCs upon CpG-ODN stimulation. miRNAs are important regulators in immune response. Thus, we wondered whether CD11b is involved in the regulation of miRNAs expression induced by CpG-ODN in DCs, which may subsequently contribute to the decreased IL-12p70 production. To further verify this hypothesis, we selected some miRNAs as candidates, especially those identified to be important regulators in TLR response. In details, miR-21 is known to regulate IL-12p35 expression, but CD11b-deficiency does not seem to affect miR-21 expression in LPS or CpG-ODN-stimulated DCs; miR-155 is another TLR signaling induced miRNA, and CD11b-deficiency has no significant effect on its expression either; for miR-146a, known to be upregulated upon TLR agonists stimulation, CD11b-deficiency inhibited CpG-ODN-induced miR-146a upregulation. Apart from this, in wild-type DCs, both LPS and CpG-ODN induced pri-miR-146a expression, and CD11b-deficiency also abolished CpG-ODN, but not LPS, -triggered pri-miR-146a expression, which suggests that miR-146a is regulated by CD11b at the transcriptional level. Since induction of miR-146a has been proved to be NF-κB dependent, we also validated that CpG-ODN-induced miR-146a expression was dependent on NF-κB activation, shown as NF-κB inhibitor inhibited its expression upon CpG-ODN stimulation in DCs. Furthermore, we determined the NF-κB activation by immunoblot, we found that CD11b doesn't influence CpG-ODN-induced early-phase NF-κB activation, thus has no effect on proinflammatory cytokine production; but participates in CpG-ODN-induced late-phase NF-κB activation, and in turn upregulates miR-146a expression in DCs.3. miR-146a represses CpG-ODN-induced IL-12p70 production by targeting Notch1 in DCsAs miRNAs function mainly through repressing their target mRNAs, and the known targets of miR-146a, such as IRAK1, IRAK2, TRAF6, and TBP, seem to be unrelated to IL-12p70 production, we searched new possible targets of miR-146a through TargetScan prediction (http://www.targetscan.org). Among the putative targets of miR-146a, we found that Notch1 had a putative conserved miR-146a target site. Notch signaling has been shown to be induced by TLR activation and participate in TLR activation-induced IL-12p70 production. To certify whether Notch1 3'UTR was directly targeted by miR-146a, we constructed a reporter plasmid containing Notch1 3'UTR and found that its expression was inhibited by miR-146a co-transfection, while the seed region mutated construct failed to be inhibited by miR-146a expression. This result suggests that Nortch1 is directly targeted by miR-146a expression.In addition, in CpG-ODN-stimulated DCs, expression of full length Notch1 and Notch intracellular domain (NICD) 1 were increased by CD11b-deficiency, suggesting that CD11b may repress IL-12p70 production via inhibiting Notch1. To verify the impact of miR-146a on Notch1, we transfected miRNA into DCs, and miR-146a mimic markedly reduced CpG-ODN-induced Notch1 expression at both protein and mRNA levels, whereas miR-146a inhibitor increased their expression. Taken together, these results suggest that Notch1, as a new target of miR-146a, may be responsible for the downregulated IL-12p70 production by CD11b expression.Next we examined the effects of Notch1 knockdown on CpG-ODN-triggered IL-12p70 production. Notch1 specific siRNA inhibited the expression of both full length Notch1 and NICD1, and Notch1 knockdown weakened IL-12p70 production in both wild-type and CD11b-deficient DCs upon CpG-ODN stimulation. These data suggests that Notch1 participates in CpG-ODN-triggered IL-12p70 production. We next determined whether miR-146a was responsible for the upregulated IL-12p70 production in CD11b-deficient DCs upon CpG-ODN stimulation. Transfection of miR-146a inhibitor promoted CpG-ODN induced IL-12p70 production in wild-type DCs but did not influence that in CD11b-deficient DCs. These data further confirm that CD11b represses CpG-ODN-triggered IL-12p70 production via upregulating miR-146a expression and targeting Notch1 in DCs.ConclusionTaken together, we demonstrate that CD11b is a negative regulator of TLR9 triggered DC cross-priming of CTL response via inhibiting IL-12p70 production in DC, which is mediated by upregulating miR-146a and targeting Notch1. Our study presents a new model in the negative regulation of TLR9 agonist-triggered DC cross-priming of CTL response by CD11b. Hence, CD11b and downstream miR-146a may be new regulators in DC licensed cross-priming aimed at induction of CTLs for the treatment of infectious diseases and cancer.