| Object:Acute skeletal muscle injury is a common form of various sports injuries.Neutrophils and macrophages infiltrating the injured site coordinate with immunosuppressive factors such as PD-1 to regulate skeletal muscle repair and regeneration following injury.Studies have found that PD-1 can promote repair and regeneration of contused skeletal muscle by regulating the phenotypic switch of classical(M1)and alternative(M2)macrophages.As a strong activator of M1 macrophages,IFN-γ can polarize macrophages towards M1 type which promoting inflammatory responses,and inhibit proliferation and differentiation of cultured myoblasts.However,the mechanisms by which the interaction of PD-1 and IFN-γregulates regeneration of skeletal muscle following injury remains unclear.Therefore,the aims of this research are to explore the regulatory effects of PD-1 and IFN-γ on neutrophils and macrophages in injured skeletal muscle,and then to explore their effects on skeletal muscle repair and regeneration process following acute injury.Methods:PD-1 knockout and IFN-γ blockade were used to specifically inhibit PD-1 and IFN-γ signal,and cardiotoxin(CTX)injection was performed to induce acute skeletal muscle injury in C57BL/6 mice.Thirty wild-type C57BL/6 mice were randomly divided into wild-type uninjured group(WTsham,n=6),wild-type normal saline injection injured group(WTNS,n=12)and wild-type Anti-IFN-γ antibody injection injured group(WTAnti-IFN-γ,n=12).Thirty PD-1 knockout C57BL/6 mice were randomly divided into PD-1 knockout uninjured group(Psham,n=6),PD-1 knockout normal saline injection injured group(PNS,n=12)and PD-1 knockout Anti-IFN-γantibody injection injured group(PAnti-IFN-γ,n=12).Then,each injured group was randomly divided into the 3-day post-injury group and the 14-day post-injury group,with 6 mice in each group.On the 3rd and 14th days after injury,bilateral tibialis anterior(TA)muscles of mice were harvested and compared with those of uninjured mice.Morphological changes of injured and regenerated skeletal muscle were analysed by HE staining.Fibrosis of skeletal muscle was analysed by Masson staining.Gene expressions of pro-inflammatory factors,anti-inflammatory factors,fibrosisrelated factors and myogenic regulator factors were determined by RT-PCR.Changes of macrophages and their subtype markers were analysed by Western Blot and RTPCR.Total macrophage,M1-type macrophage,M2-type macrophage and neutrophil accumulation was measured in muscle cross sections using immunofluorescence methods.Results:1.Increased expression of PD-1 and IFN-γ after skeletal muscle injuryThe analysis of real-time PCR data showed that PD-1 and IFN-γ mRNA expression of wild-type mice increased substantially following injury,peaking at 3 days post-injury(P<0.01),and returning to control levels by 14 days.However,PD-1 knockout mice demonstrated higher IFN-γ mRNA expression compared with wildtype mice at both 3 days and 14 days after muscle injury(P<0.01).2.PD-1 knockout and IFN-γ blockade aggravate skeletal muscle damageHE staining results displayed abundant inflammatory cells accumulation,muscle fibre swelling and necrosis in all injured groups by day 3 post-injury.The area with inflammatory cells infiltration of the WTNS group was about 40%,which was significantly lower than that of the WTAnti-IFN-γ group and PNS group(about 60%)(P<0.01),and there still exist a large number of intact muscle fibres.The area with inflammatory cells infiltration of the PAnti-IFN-γ group was the largest,close to 80%,which was significantly larger than that of the WT Anti-IFN-γ group and the PNS group(P<0.05).3.PD-1 knockout and IFN-γ blockade impair skeletal muscle regenerationHE staining results displayed robust centrally nucleated regenerating muscle fibres in all injured groups by day 14 post-injury.The diameter of regenerated muscle fibres in WTNS group was significantly larger than that in WTAnti-IFN-γ group(P<0.01)and PNS group(P<0.01).The diameter of regenerated muscle fibres in PAnti-IFN-γ group was significantly smaller than that in PNS group(P<0.01),while there was no significant difference compared with WTAnti-IFN-γ group(P>0.05).At both 3 days and 14 days post-injury,the analysis of real-time PCR data demonstrated that Myogenin mRNA expression in WTNS group was significantly higher than that in WTAnti-IFN-γgroup(P<0.01)and PNS group(P<0.01).At 3 days post-injury,the Myogenin mRNA expression in PAnti-IFN-γ group was significantly lower than that in WTAti-IFN-γ group(P<0.05)and PNS group(P<0.01).At 14 days post-injury,the Myogenin mRNA expression in PAnti-IFN-γ group was significantly lower than that in PNS group(P<0.05),and there was no significant difference compared with the WTAnti-IFN-γ group(P>0.05).4.PD-1 knockout and IFN-γ blockade aggravate fibrosis of injured skeletal musclesAt 14 days post-injury,Masson staining results manifested that the fibrotic area in WTNS group was significantly smaller than that in WTAnti-IFN-γ group(P<0.01)and PNS group(P<0.01).The fibrotic area in PAnti-IFN-γ group was significantly larger than that in PNS group(P<0.01),but there was no significant difference compared with WTAnti-IFN-γ group(P>0.05).At 3 days post-injury,the analysis of real-time PCR data showed that Col3al and TGF-β mRNA levels in WTNS group were significantly lower than those of WTAnti-IFN-γ group(P<0.01)and PNS group(P<0.01).However,the Col3al and TGF-β mRNA levels in PAnti-IFN-γ group were significantly higher than those of PNS group(P<0.01).In addition,the TGF-β mRNA expression in PAnti-IFN-γgroup was significantly higher than that in WTAnti-IFN-γ group(P<0.01).At 14 days post-injury,the Col3al mRNA expression in WTNS group was significantly lower than that in WTAnti-IFN-γ group(P<0.01)and PNS group(P<0.01).Instead,the Col3al and TGF-β mRNA levels in PAnti-IFN-γ group were significantly higher than those of PNS group(P<0.05).Additionally,the TGF-β mRNA expression in PAnti-IFN-γ group was significantly higher than that in WTAnti-IFN-γ group(P<0.01).5.PD-1 knockout and IFN-γ blockade exacerbate injured skeletal muscle inflammationAt 3 days post-injury,the analysis of real-time PCR data revealed that the mRNA levels of proinflammatory cytokines IL-1β,IL-6 and TNF-α in WTNS group were significantly lower than those of WTAnti-IFN-γ group(P<0.01)and PNS group(P<0.05).The mRNA levels of IL-1β,IL-6 and TNF-α in PAnti-IFN-γ group were significantly higher than those of WTAnti-IFN-γ group(P<0.01)and PNS group(P<0.01).At 14 days post-injury,the mRNA levels of IL-1β,IL-6 and TNF-α in PAnti-IFN-γ group were significantly higher than those of WTAnti-IFN-γ group(P<0.05),and the IL-6 mRNA expression was also higher than that in PNS group(P<0.01).As for the results of antiinflammatory cytokine IL-10,the mRNA expression in WTNS group was significantly higher than that in WTAnti-IFN-γ group(P<0.01)and PNS group(P<0.01)at 3 days postinjury.The IL-10 mRNA expression in PAnti-IFN-γ group was significantly lower than that in WTAnti-IFN-γ group(P<0.01),and there was no significant difference compared with PNS group(P>0.05).Furthermore,there was no significant difference in IL-10 mRNA expression among groups at 14 days post-injury(P>0.05).6.IFN-γ blockade inhibits macrophage infiltration after skeletal muscle injuryAt 3 days post-injury,western blotting analysis demonstrated that the protein level of Mac-2 in PAnti-IFN-γ group was significantly lower than that in PNS group(P<0.05).The analysis of real-time PCR data revealed that Mac-2,F4/80 and CD68 mRNA levels in WTAnti-IFN-γ group were significantly lower than those of WTNS group(P<0.01).The Mac-2,F4/80 and CD68 mRNA levels in PAnti-IFN-γ group were significantly lower than those of PNS group(P<0.01),and its CD68 mRNA expression was also higher than that in WTAnti-IFN-γ group(P<0.01).At 14 days post-injury,the F4/80 and CD68 mRNA levels in WTNS group were significantly higher than those of WTAnti-IFN-γ group(P<0.05).The Mac-2,F4/80 and CD68 mRNA levels in PAnti-IFN-γgroup were significantly higher than those of WTAnti-IFN-γ group(P<0.01).7.Effects of PD-1 knockout and IFN-γ blockade on M1-type macrophagesAt 3 days post-injury,immunofluorescence analysis showed that the number of M1-type macrophages in PAnti-IFN-γ group was higher than that in WTAnti-IFN-γ group(P<0.01)and PNS group(P<0.01).The number of M1-type macrophages in WTNS group was lower than that in PNS group(P<0.01).The analysis of real-time PCR data revealed that mRNA expressions of the M1-type macrophage markers,iNOS and CD86,in WTNS group were lower than those of PNS group(P<0.05).Also,iNOS mRNA expression in WTNS group was greater than that of WTAnti-IFN-γ group(P<0.05).Meanwhile,iNOS and CD86 mRNA levels in PAnti-IFN-γ group were significantly higher than those of WTAnti-IFN-γ group(P<0.01)and PNS group(P<0.05).8.Effects of PD-1 knockout and IFN-γ blockade on M2-type macrophagesAt 3 days post-injury,immunofluorescence analysis showed that the number of M2-type macrophages in PAnti-IFN-γ group was lower than those of WTAnti-IFN-γ group(P<0.01)and PNS group(P<0.01).The number in WTNS group was greater than those of WTAnti-IFN-γ group(P<0.05)and PNS group(P<0.01).The analysis of real-time PCR data revealed that mRNA expressions of the M2-type macrophage markers,Arg1 and CD206,in PAnti-IFN-γ group were lower than those of PNS group(P<0.05)at 3 days post-injury.Instead,CD206 mRNA expression in WTNS group was greater than that in WTAnti-IFN-γ group(P<0.01)and PNS group(P<0.01).At 14 days post-injury,the Argl mRNA expression in WTAnti-IFN-γ group and PAnti-IFN-γ group was higher than that in WTNS group(P<0.01)and PNS(P<0.01)group respectively.Moreover,the CD206 mRNA expression in PAnti-IFN-γ group was greater than that in WTAnti-IFN-γ group(P<0.05)and PNS group(P<0.01).8.Effects of PD-1 knockout and IFN-γ blockade on neutrophilsAt 3 days post-injury,immunofluorescence analysis demonstrated that the number of neutrophils in WTNS group was lower than that in WTAnti-IFN-γ group(P<0.01)and PNS group(P<0.05).Instead,the number of neutrophils in PAnti-IFN-γgroup was more than that in PNS group(P<0.05),while there was no significant difference compared with the WTAnti-IFN-γ group(P>0.05).At 14 days post-injury,there was no neutrophil infiltration in each group.Conclusion:Blocking IFN-γ signalling after PD-1 knockout did not alleviate skeletal muscle damage or promote its regeneration.Instead,it exacerbated injured muscle inflammation,impaired muscle regeneration and aggravated muscle fibrosis by inhibition of macrophage infiltration,blockade of macrophage pro-inflammatory to anti-inflammatory switching,and increased infiltration of neutrophils. |
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