C5a-C5aR1 Regulation Of Neutrophil Accumulation Mediates SWCNT-Induced Pulmonary Inflammation And Fibrosis

Background As the proliferation of manufacturing and use of nanomaterials,people pay more attention to those potential health hazards associated with prolonged exposure of occupational populations to single-walled carbon nanotubes(SWCNT).Studies have shown that SWCNT inhalation induces inflammatory responses and widespread pulmonary toxicity in animals,and that SWCNT exposure can lead to significant inflammatory cellular inward flow to sites of lung injury.Polymorphonuclear neutrophils(PMNs),as the first inflammatory response cells in inflammation,have long been thought to be limited to fighting acute infections and clearing pathogens.It has been,however,shown that pulmonary fibrosis is closely associated with PMNs,yet there remain uncertainties on the role of PMNs in mediating pulmonary inflammation and fibrosis due to SWCNT exposure.Objective1.To elucidate the role of complement activation,PMNs accumulation in lung inflammation,and lung fibrosis induced by SWCNT exposure.2.To investigate the mechanism by which complement C5a-C5aR1 mediates SWCNTinduced pneumonia and pulmonary fibrosis through regulation of PMNs accumulation using in vivo antagonists to intervene in C5aR1 production.Methods1.Observe and evaluate the effects of SWCNT exposure on lung injury,complement activation and PMNs accumulation.1.1 To observe and assess the effects of different times of SWCNT exposure on pulmonary inflammation and pulmonary fibrosis: 8-week-old C57BL/6J female mice were randomly divided into control and SWCNT groups,6-8 mice in each group.Each mouse was treated with single tracheal drip administration of sterile PBS and 40 μg SWCNT,respectively.Mice were continued to be fed for 1,3,7 and 28 days after exposure.Bronchoalveolar lavage fluid(BALF)and lung tissues were taken from mice after sacrifice.Our study assessed the pathological changes of lung injury and the lung inflammatory factors interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)expression levels.The protein expression of epithelial mesenchymal transition(EMT)and fibroblastto-myofibroblast transdifferentiation(FMT)related molecules(Vimentine,α-SMA and Collagen I)were also evaluated.1.2 To observe and assess the effects of different times of SWCNT exposure on complement activation,C5a-C5aR1 binding and PMNs accumulation: We used mouse BALF and lung tissue obtained in Method 1.1 to evaluated the expression of complement component 5a(C5a),C5 a receptor 1(C5aR1),and the end product C5b-9.We further assessed the mutual binding of C5 a and C5aR1 and evaluated the changes in the proportion of PMNs in BALF.2.Further to verify that C5a-C5aR1 mediates SWCNT-induced pneumonia and pulmonary fibrosis through the regulation of PMNs accumulation: Since we have found that the expression levels of C5 a,C5aR1 and PMNs were significantly increased after 1day of SWCNT exposure and promoted early acute inflammation,we chose two time points,1 day and 28 days,to investigate the in vivo antagonistic intervention.Eight-weekold C57BL/6J female mice were randomly divided into Control,SWCNT,PMX205 and SWCNT+ PMX205 groups(6-8 mice in each group),and mice in the Control and SWCNT groups were each intratracheally instilled with sterile PBS and 40 μg SWCNT,respectively.While mice in the SWCNT+PMX205 group were intratracheally instillated40 μg/SWCNT,followed by subcutaneous injection(s.c)of 1 mg/kg PMX205(C5aR1antagonist)daily at 24-hour intervals.and mice in PMX205 group were administered 1mg/kg of PMX205 s.c.(per 24-hour)only.Results1.SWCNT exposure induces lung injury,early pulmonary inflammation and late pulmonary fibrosis in miceSWCNT exposure induced lung injury in mice and showed different time-course characteristics.A large number of inflammatory cells infiltrated in lung tissues on days 1-7 after SWCNT exposure,inflammatory factor content increased,and lung bronchial walls gradually thickened.Large amount of striated collagen fibers deposited around lung bronchi and fine bronchi on day 28 after SWCNT exposure,and HYP content increased in lung tissues.Meanwhile,the expression of characteristic molecules of EMT and FMT in the lung was significantly upregulated.2.SWCNT exposure induces complement activation and C5a-C5aR1 co-expression in mice.SWCNT exposure induced complement activation and showed a time-effect relationship.The expression of complement component C5 a and complement activation product C5b-9 were significantly up-regulated on days 1 and 3 after SWCNT exposure,most significantly on day 1,and gradually decreased from day 7 to day 28.The strongest fluorescence intensity of C5a-C5aR1 co-expression was on day 1,and gradually decreased on days 3,7 and 28.3.SWCNT exposure induces the accumulation of PMNs in mice.SWCNT exposure induced the accumulation of PMNs in the lungs and also showed a time-effect relationship.The results of flow cytometry showed that the proportion of PMNs increased significantly on day 1 after SWCNT exposure,remained high on day 3,and gradually decreased on days 7 and 28.In the meantime,the chemokine interleukin-8(IL-8)of PMNs was significantly increased on day 1 after SWCNT exposure and decreased to lower levels on days 3,7 and 28.4.PMX205 treatment blocks complement C5a-C5aR1 binding induced early by SWCNT exposure.The specific antagonist PMX205 downregulates C5aR1 production early in SWCNT exposure and reduces C5a-C5aR1 binding.PMX205 treatment given subcutaneously for1 day had no significant effect on C5 a and C5b-9 protein expression induced by SWCNT exposure,but down-regulated C5aR1 protein expression induced by SWCNT.Meanwhile,the fluorescence intensity of C5a-C5aR1 co-expression was significantly attenuated by immunofluorescence detection.However,PMX205 had no significant effect on complement C5 a,C5b-9 levels and C5a-C5aR1 co-expression after 28 days of SWCNT exposure.5.C5a-C5aR1 axis regulates the accumulation of PMNs in the lung induced by SWCNT exposure.PMX205 specifically blocked C5a-C5aR1 binding in mice,which prevented C5 a from recruiting PMNs at the early stage of SWCNT exposure.1 day after subcutaneous administration of antagonist treatment,PMX205 reduced PMNs accumulation induced by SWCNT exposure.However,there was no significant effect on PMNs accumulation after 28 days of treatment.6.C5a-C5aR1 axis regulates PMNs accumulation to further alleviate SWCNT exposure-induced pneumonia and pulmonary fibrosis.PMX205 blocked C5a-C5aR1 binding and reduced PMNs accumulation,and we found that SWCNT exposure-induced inflammatory cell infiltration,inflammatory factors and mRNA expression levels decreased after PMX205 treatment for 1 day.Further,collagen deposition in lung tissue induced by SWCNT exposure and downregulation of lung EMT and FMT-related molecular protein expression were partially alleviated after day 28 of PMX205 treatment.Conclusions1.SWCNT exposure induces complement activation,increases C5a-C5aR1 binding and regulates PMNs accumulation,which promotes early lung inflammation and mediates late lung fibrosis2.The potent inhibitor of C5aR1 inhibits C5a-C5aR1 binding and reduces PMNs accumulation,thereby reducing early lung inflammation in SWCNT exposure and thus alleviating advanced pulmonary fibrosis.