Negative Regulation Of Inflammatory Innate Immune Response By LAPF And CD11b

Innate immune response serves as the first line of host defense against invading pathogens. However, inappropriate activation of innate immune response may lead to the damage of host tissues and organs, or even to the inflammatory and autoimmune diseases. So, identification and functional studies of the regulatory cell types or their derived negative molecules attracts much attention in recent years, becoming a frontier field in innate immune research, In this study, the primary aim is to unveil the roles of LAPF protein and CD11b-Src cascade in the regulation of innate immune response.Part I LAPF inhibits TLR-triggered TNF-a production in macrophagesLysosome associated and apoptosis inducing protein containing PH and FYVE domain (LAPF), also called PLEKHF1, was first identified by our lab and reported to be involved in the TNF-a-induced tumor cell apoptosis in 2005. However, the role of LAPF in the innate immunity and inflammation remains unknown. In order to investigate the contribution of LAPF to the regulation of innate immune responses in macrophages, we generated LAPF conditional knock-out (LAPF CKO) mice with impaired LAPF expression in macrophages. Compared with control mice, the LAPF CKO mice produced much more inflammatory cytokines, especially TNF-a, when chanllenged with LPS in vivo.LAPF-deficient macrophages produced more TNF-a after stimulated with TLR ligands. Furthermore, the LAPF-deficiency caused a higher level of LPS-induced phosphorylation of TAK1, IKKα/β and p65, which indicated that LAPF could negatively regulate the TLR-triggered activation of NF-κB signal pathway. Through IP-MS together with in-vitro IP experiment, LAPF was found to interact with TIRAP and Myd88, which are both the important adaptors in TLR-triggered NF-κB signal pathway. These results demonstrated that LAPF could negatively regulate the TLR-triggered TNF-a production by inhibiting the activation of NF-κB pathway.Part Ⅱ CDllb-Src cascade polarizes anti-inflammatory M2 macrophage phenotype and functionIntegrins are involved in the regulation of innate immune response. Integrins are impotant for the development, proliferation, migration and adhesion of innate immune cells. CDllb is the a chain of the integrin Mac-1. The antibody of CD 11b is usually used to separate macrophage and macrophage-like phagocytes. On the basis of the phenotype and function, the myeloid macrophages can be grouped into two subclass:the M1 (classically activated) and M2 (alternatively activated) macrophages. The balance of M1 and M2 macrophages directly associates with the production of pro-inflammatory and anti-inflammatory cytokines, especially TNF-α and IL-10. Our previous work showed that CDllb could promote the ubiquitination-dependent degradation of MyD88 and TRIF through Src-Syk cascade, thus controlling the inflammatory innate response. However, the relationship between the CD11b-Src cascade and the M1/M2 macrophage polarization is still unknown.The CD11b-deficient mice or Dasatinib (Src inhibitor)-treated mice were more susceptible to the DSS-induced coltises, with much more sever inflammation response, more iNOS expression and TNF-a production, and at the same time less Argl expression and IL-10 production in the colon tissue. As the iNOS and TNF-a are important markers of M1 macrophages, while Argl and IL-10 are the important markers of M2 macrophages, the above results indicate that CDllb-Src cascade might polarize anti-inflammatory M2 macrophage generation. What’s more, there were more M1 macrophages but less M2 macrophages in the total intestinal macrophages from the colon of the Dasatinib-treated mice after DSS feeding. Further mechanistic studies showed that 1) CDllb-Src cascade could inhibit the IFN-y induced expression of iNOS in BMDM. To the opposite, CD11b-Src could promote the expression of IL-4 induced expression of Argl and YM1 in BMDM; 2) CDllb-Src enhanced TLR-triggered IL-10 production and STAT6 activation. These data further support our conclusion that CDllb-Src cascade might polarize anti-inflammatory M2 macrophage generation.Further research revealed that Src could positively regulate IL-4-induced activation of Akt and STAT6, which were reported to be necessary for the generation of M2 macrophage phenotype, especially Argl expression. However, only overexpression of STAT6 could rescue the inhibiton of Argl expression by Src inhibitor. Src could associate with STAT6, indicating that Src could activate STAT6. In addition, after stimulation of TLR ligands, CDllb-Src could upregulated the activation of Akt by promoting the ubiquitination and degradation of the regulatory unit p85 of PI3K. The phosphorylated Akt then activated the trascriptor AP-1, which is important for the TLR-triggered IL-10 production.In sum, our results indicate that CD11b-Src cascade polarizes anti-inflammatory M2 macrophage phenotype and function, providing the new mechanistic insight to the polarization of Ml and M2 macrophages, and indicating the importance of CDllb-Src cascade as the targets for the design of anti-inflammatory drugs.