Potential Role And Its Mechanism Of Tumor Necrosis Factor-? Induced Protein 8 Like-2 In Mediated Immune Dysfunction Of Dendritic Cells After Thermal Injury

BACKGROUND AND OBJECTIVE With the development of severe sepsis secondary to major burns/trauma, dendritic cells (DCs) are stimulated to mature and present processed antigens via major costimulatory molecules and antigen presenting molecules. Tumor necrosis factor-a induced protein 8 like-2 (TIPE2) appears to be a critical immunoregulatory molecule involved in the pathophysiological process during septic response. However, the potential role and its regulator mechanism underlying TIPE2 in mediated immune function of DCs remains to be elucidated.METHODS DCs were isolated from mouse spleens with a DC Cell Isolation kit,and laser scanning confocal microscope was performed to observe TIPE2 distribution in DCs. Gene expression and protein levels of TIPE2 in DCs were assessed by RT-PCR and Western blot, respectively. To knock-down or up-regulate TIPE2 expression, DCs were infected with recombinant lentiviruses that carried the TIPE2 siRNA or TIPE2 RNA in vitro. Recombinant lentiviruses that carried the TIPE2 siRNA or TIPE2 RNA were injected intraperitoneally to mice two weeks prior to the in vivo experiment, and then animal scald injury model was made. Expressions of CD80, CD86, and major histocompatibility complex class ? (MHC-?) were determined by flow cytometry. All the samples of T-cell proliferative response were tested with CCK8. In addition,cytokines levels and nuclear factor of activated T cells (NF-AT) activities in T lymphocytes were measured by ELISA.Statistical analysis Data were represented as mean ± standard deviation (SD). Data sets were examined by one-way ANOVA, and individual group means were then compared with Student paired t test. All statistical tests were two sided and a P value of 0.05 or less was considered to indicate statistical significance.RESULTS AND CONCLUSIONS 1. It was found that TIPE2 was a cytoplasmic protein expressed in DCs. 2. Expressions of CD80, CD86, and MHC-II were significantly enhanced in comparison to the normal control group after TIPE2 gene silenced by siRNA, but all these 3 molecules were down-regulated when TIPE2 gene was over-expressed in DCs (P<0.01). Afetr thermal injury, expressions of CD80, CD86,and MHC-II were significantly decreased in TIPE2 over-expressed mice, while they were markedly enhanced in the TIPE2-deficient mice (P<0.01). 3. As shown in ELISA results, production of both IL-12 and TNF-a by siRNA-TIPE2 transfected DCs was obviously increased, while a trend toward lower levels was noted in the TIPE2 over-expressed group in comparison to those in the normal controls or in the scald group (P<0.01). 4. From in viro and in vivo studies, it was noticed that the proliferation of T cells could be enhanced by treatment with TIPE2 gene silence in DCs, but it was lowest in the TIPE2 over-expresed group. 5. IL-2 level and NF-AT activity were significantly lower after TIPE2 over-expressed in DCs than those in the normal group or in the scald group, whereas they were elevated in the TIPE2 knockdown group (P<0.01).6. IL-4 level was elevated in the TIPE2 over-expresed group, while IFN-y levels was down-regulated (P<0.01); however, in vitro or in vivo, treatment with TIPE2 gene silence in DCs significantly elevated IFN-? level and down-regulated IL-4 level of T cells. Taken together, these results suggest that TIPE2 appears to be a critical immunoregulatory molecule involved in T lymphocyte immune function mediated by DCs following major acute insults.