Tmod1 Affects the Immune Functions and Biophysical Properties of Dendritic Cells Through TLR4 Signaling Pathway and Cytoskeleton Remodeling

Background Dendritic cells(DCs)are the most important antigen-presenting cells due to their professional and extremely efficient antigen-presenting function.The dynamics of cytoskeleton plays crucial regulated roles on DCs’immune functions and biophysical properties.Several evidences show that tumor-derived suppressive cytokines deteriorate DCs’immune functions through remodeling their F-actin cytoskeleton.But the underlying mechanism is still elusive.Tropomodulin1(Tmod1),a cytoskeleton-binding protein,regulates and stabilizes actin filaments lengths and cytoskeleton architecture,which involves in the regulations of the morphology,formation of neural dendrites and biophysical properties of cells.Our previous studies found that mature DCs(mDCs)had a higher expression of Tmod1 than immature DCs(imDCs). Therefore,it’s hypothesized that Tmod1 maybe involve in the modification of DCs’functions.Objective The aim of the study is to investigate the effects of Tmodl on the immune functions and biophysical properties of DCs and the underlying mechanisms in order to further understand the biological behaviors of DCs.Methods Bone marrow-derived cells were harvested from wild type(C57BL/6 J)mice and Tmod1 knockout mice(Tmod1 overexpressing transgenic(TOT)/Tmod1-/-)and differentiated to immature dendritic cells(imDCs)by rmGM-CSF and rmIL-4.imDCs were then matured by lipopolysaccharides(LPS)treatment.The expressions of the surface markers in DCs,including CD80,CD86,CD40,MHC-Ⅱand CCR7,were detected by flow cytometry,Western blot and qRT-PCR.The inflammation cytokines such as IL-6,IFN-γ,IFN-βand IL-10 were also detected by flow cytometry.The immune functions and the biophysical properties of DCs were compared between the wild type and Tmod1 knockout mice.The F-actin content and dendritic pseudopodia of these two kinds of DCs were detected by flow cytometry and laser scanning confocal microscope respectively.Finally,we detected the MyD88 dependent and independent signaling pathway to discover the molecular mechanisms.Results We found that Tmod1-deficient mDCs showed deficient antigen-presenting ability and they failed to express enough MHC-Ⅱ,co-stimulated molecules(CD80/86,CD40)and CCR7 on their cell surface.The secretions of the inflammatory cytokines IL-6 and IFN-γwere decreased while the anti-inflammatory cytokines IFN-βand IL-10 were increased in the supernatant of Tmod1-deficient mDCs.As compared to DCs of wild type mice,the migration ability of DCs from Tmod1 knockout mice were dramatically damaged including their free migration and CCL19 mediated chemotaxis migration.However,we found that Tmod1 knockout had no effects on the imDCs’endocytosis ability.Furthermore,Tmod1 knockout DCs showed higher osmotic fragility,lower Young’s modulus,less F-actin content and shorter dendritic pseudopodia.Under LPS stimulation,the phosphorylation level of p65 and p38 were significantly downregulated in Tmod1 knockout mice while the expression of p-IRF3 was upregulated.Conclusions These results indicated that Tmodl knockout leads to deficient antigen-presenting ability and impaired migration of DCs as well as their biophysical properties.The underlying mechanisms are due to the inhibitions of the TLR4-mediated NF-κB and p38 MAPK singling pathway and the activation of the IRF3 signaling pathway,as well as the disturbed reorganization of the F-actin cytoskeleton.Our results provide a new insight on the functions of Tmod1 which can affect the DCs’immune functions and biophysical properties through regulating the TLR4-mediated singling pathways and cytoskeleton remodeling.