Effects Of Aflatoxin G₁-induced Inflammation On Differentiation Of Macrophages

Objective:Epidemiological studies suggest that aflatoxin G₁ exposure is related to the development of human respiratory cancer.Our previous studies have reported that oral administration of AFG₁ can induce lung adenocarcinoma in mice.At the pro-tumor stage,AFG₁ causes a chronic pulmonary inflammatory response,evidenced by the increased macrophages infiltration and TNF-?production,and may be involved in AFG₁-induced lung tumorigenesis.Recently,we also reported that TNF-?-dependent lung inflammation regulated macrophage infiltration in inflamed lung tissues.Those studies indicate that macrophage may contribute to AFG₁-induced lung inflammation.It has been shown that Aflatoxins could increase the secretion of pro-inflammatory cytokines from the murine macrophage cell line,J774A.1.However,whether AFG₁ plays a role on the activation and differentiation of macrophage is still unknown.In this study,we treated the mice with a soluble TNF-?receptor s TNFR:Fc and AFG₁ for 3 months,and explored the role of AFG₁-induced TNF-?-mediated lung inflammation on TNF-?and PD-L1 expression in macrophage in inflamed tissues.The bone marrow derived macrophage was isolated,and the role of AFG₁ in the differentiation of M0 to pro-inflammatory M1 and anti-inflammatory M2 were measured.Methods:1. Balb/c mice were orally administered AFG₁?100?g/kg body weight,three times per week?with or without TNF-?neutralizing antibody s TNFR:Fc?10 mg/kg,twice per week?via intraperitoneal?i.p.?injection for three months.AFG₁-induced inflamed lung tissue was observed by immunofluorescence staining.2.Bone marrow mononuclear cells were isolated from the femora of mice,and treated with macrophage colony-stimulating factor?M-CSF?to obtain bone marrow derived M0 macrophages.The M0 macrophages were treated with AFG₁,M1 stimulator lipopolysaccharide?LPS?and interferon?IFN?-?,as well as M2 stimulator interleukin-4?IL-4?and interleukin-13?IL-13?for 24hours.M1 macrophage markers i NOS,TNF-?and IL-6 as well as M2macrophage markers MR,YM-1 and Arg-1 were measured by RT-PCR.In addition,the effect of AFG₁ on PD-L1 expression on M0 macrophages was detected by FCM and RT-PCR.3.Bone marrow mononuclear cells were isolated,and stimulated by AFG₁and M-CSF for 5 days.Then the AFG₁-pretreated M0 macrophages were induced by M1 stimulator LPS and IFN-?or M2 stimulator IL-4 and IL-13 for24 h.The role of AFG₁ in the differentiation of M0 into M1/M2 macrophages was measured.Results:1. AFG₁-induced TNF-?dependent inflammation regulate expression of PD-L1 and TNF-?on CD68⁺macrophagesTo study whether AFG₁-induced TNF-?-dependent lung inflammation regulate macrophage differentiation in pulmonary epithelial cells,weAFG₁-induced inflamed lung tissues treated with the TNF-?inhibitor s TNFR:Fc in vivo.Immunofluorescence results showed that the cell numberinflammation tissues,which was reduced by TNF-?neutralizing antibody s TNFR:Fc.TNF-?-dependent inflammatory response upregulated the PD-L1and TNF-?expression on CD68⁺macrophage,indicating that macrophage activation may be involved in AFG₁-induced chronic inflammatory response.2. The effect of AFG₁on the phenotypic alteration in bone marrow derived macrophagesM0 macrophages were isolated from bone marrow and cultured in vitro,treated with AFG₁for 24 h.RT-PCR showed that AFG₁ up-regulated the expression of TNF-?m RNA,while decreased the expression of i NOS and IL-6 m RNA?P<0.05?.The expression of M2-related phenotype molecules MR,YM-1 and Arg-1 was detected at the m RNA level,and the expression of MR,YM-1 and Arg-1 was up-regulated by AFG₁?P<0.05?.The results suggest that small dose of AFG₁ may induce the bone marrow derived macrophages Mo differentiating towards M2 phenotype.3. Effect of AFG₁ on the expression of PD-L1 in bone marrow-derived M0 macrophagesAfter AFG₁treatment for 24 hours,RT-PCR showed that AFG₁up-regulated PD-L1 m RNA expression on M0?P<0.05?.FCM showed thatincreases PD-L1 expression on macrophage.4. The effect of AFG₁ pretreatment on the expression of PD-L1 in M0macrophagesAfter 5 days induction of low-dose AFG₁+M-CSF,RT-PCR results showed that PD-L1 m RNA expression was up-regulated by AFG₁pretreatment?P<0.05?.FCM showed that the proportion of CD11b⁺PD-L1⁺cells increased by AFG₁ pretreatment?P<0.05?,and the expression of PD-L1in CD11b⁺PD-L1⁺cells was up-regulated by AFG₁ pretreatment?P<0.01?.5. The role of AFG₁ in the differentiation of M0 into M1/M2macrophagesBone marrow mononuclear cells were isolated,and stimulated by AFG₁and M-CSF for 5 days.Then the AFG₁-pretreated M0 macrophages were induced by M1 stimulator LPS and IFN-?or M2 stimulator IL-4 and IL-13 for24 h.AFG₁ promoted the up-regulation of TNF-?expression induced by LPS+IFN-?stimulation,but inhibited the up-regulation of i NOS and IL-6m RNA by LPS+IFN-?stimulation?P<0.05?.AFG₁ also promoted the expression of M2 macrophages phenotype induced by IL-4+IL-13?P<0.05?.The results suggest that AFG₁ may interfere the polarization of M1 and promote the differentiation of M0 to M2 macrophages.Conclusions:1.AFG₁-induced TNF-?-dependent inflammatory response upregulated the PD-L1 and TNF-?expression on CD68⁺macrophage,indicating that macrophage activation may be involved in chronic inflammatory response.2.AFG₁upregulates M2-related phenotype molecules MR,YM-1,Arg-1and PD-L1 expression on bone-marrow-derived macrophage.During the differentiation of bone marrow monocytes to classic M1/M2 macrophages,AFG₁ may promote the M0 differentiating to classic M2 macrophages.