| [Purpose] To explore whether S100A9(MRP14,Myeloid related protein 14)maintained the M2 polarization of testicular macrophages(TMs)and its mechanism.And also,to explore whether it was involved in the regulation of immune cell infiltration and testicular fibrosis during orchitis.[Methods] Immunofluorescence staining was used to detect the localization of S100A9 in testicular macrophages of C57BL/6J mice.Macrophages derived from bone marrow of mouse were stimulated with exogenous S100A9 in vitro.Tasquinimod(S100A9 inhibitor)or LY294002(PI3K/Akt pathway inhibitor)were used for in vivo intervention experiment,and the phenotypes of macrophages were detected by flow cytometry.S100a9 was knocked down or overexpressed in Raw 264.7 macrophage cell line,and Western blot was used to further verify whether S100A9 activated the PI3K/Akt pathway.UPEC induced orchitis mouse model was established.Testicular macrophages were isolated and enriched by F4/80+ magnetic beads,and the expression of S100a9 was analyzed by q RT-PCR and Western blot.Tasquinimod or LY294002 were used for in vivo experiment and the number of testicular immune cells and macrophage phenotype were detected by flow cytometry.The m RNA expression of Tgfβ and various pro-fibrotic immune factors in testis was analyzed by q RT-PCR.Morphological changes of testis were observed by HE staining.Masson staining and Western blot were used to observe the expression of collagen fiber and fibronectin,and evaluate the degree of fibrosis.The m RNA expression of S100A9,CD45 and CD68 in testicular biopsy tissue of patients with orchitis was detected by q RT-PCR,and the infiltration of immune cells in testis was determined by immunofluorescence.Masson staining and Hydroxyproline kit were used to detect the content of collagen fiber and total collagen.The correlation between S100A9 and immune cell expression or fibrosis of testis was determined by correlation analysis.[Results] Adult mouse testis macrophages were dominated by M2-type macrophages with high CD206 expression.S100A9 was expressed in TMs at all stages of testicular development.Inhibition of S100A9 or PI3K/Akt pathway can reduce the proportion of M2-type macrophages in testis.Exogenous addition of S100A9 promoted the polarization of bone marrow derived macrophages from M0 to M2 in vitro.Overexpression of S100a9 activated PI3K/Akt pathway in Raw 264.7 macrophages.S100A9 was significantly upregulated in UPEC induced orchitis,and UPEC infection led to infiltration of immune cells in testis,damage of spermatogenesis and gradual aggravation of fibrosis.S100A9 promoted the aggregation of immune cells in testis and the m RNA expression of multiple immune factors,up-regulated M2/M1 ratio and promoted testicular fibrosis.Inhibition of S100A9 or PI3K/Akt pathway reduced the accumulation of immune cells in testis and the m RNA expression of multiple immune factors,down-regulated M2/M1 ratio and inhibited fibrosis.In the testicular biopsy tissue of patient with orchitis,the spermatogenesis function of testis was impaired,a large number of immune cells were infiltrated in the interstitial,and fibrosis appeared around the seminiferous tubules or interstitial of the testis.The m RNA expression of S100A9 was significantly up-regulated in testicular biopsy tissue of patient with orchitis(p = 0.020).The collagen content in testicular biopsy tissue of orchitis group was 0.998 ± 0.295 μg/mg protein,significantly higher than that in control group(0.429 ± 0.116 μg/mg protein)(p = 0.038).The m RNA expression of S100A9 was significantly positively correlated with collagen content and the m RNA expression of CD45 or CD68(r = 0.806,p = 0.003;r = 0.849,p = 0.008;r = 0.805,p = 0.016).[Conclusions] S100A9 can maintain the M2 polarization of testicular macrophages by activating the PI3K/Akt pathway,and exert immunosuppressive function.Inhibition of S100A9 or PI3K/Akt pathway can inhibit the accumulation of immune cells in testis and inhibit testicular fibrosis.It provides a new idea for the treatment of testicular fibrosis caused by orchitis. |
