Effects And Mechanisms Of Microcystin-LR On The Innate Immune Function Of Zebrafish

In recent decades,the frequent occurrence of harmful cyanobacterial blooms caused by water eutrophication has becoming a worldwide environmental problem.Microcystins(MCs)are cyclic-peptide toxins produced mainly by Microcystis cyanobacteria.More than 200 analogs of MCs have been identified,among which microcystin-LR(MC-LR)is the most widely distributed,toxic,and extensively studied variant.Overwhelming pieces of evidence show that MC-LR mainly accumulated in the liver and exert hepatotoxicity.Recently,the potential impact of MC-LR on the function of immune system,especially on the inflammatory response has become a red-hot topic.The present study uses zebrafish(Danio rerio),a model organism,as the research object.First of all,splenic pathological damages and serum immune parameters as well as transcription levels of splenic innate immune-related genes were studied after chronic exposure to MC-LR.Then,transcriptional levels of key genes in the TLR/MyD88 signaling pathway as well as the protein expression and immunohistochemical observation of MyD88 in fish spleen to elucidate the molecular mechanisms behind chronic inflammatory effects induced by environmental levels of MCLR.At last,a cross-generational study was conducted to explore whether MC-LR and its toxic effects could transfer to the F1 offspring and then disturb the offspring's immune function.The present study makes contributions to experimental evidence for the comprehensive knowledge of inflammatory response and innate immune function when exposure to environmentally relevant concentrations of MC-LR.The main results were as follows,1.In a chronic study,male zebrafish were exposed to 0,0.3,1,3,10 and 30 ?g/L MCLR for 30 d.In the low concentration groups(0.3,1 and 3 ?g/L),zebrafish displayed splenic inflammatory changes including the formation of melano-macrophage centers and the increase of macrophage pseudopodia,remarkable elevation of serum C3 levels,and significantly upregulated expression of innate immune-related genes(c3b,lyz,il1?,tnf? and ifn?).However,high concentrations of MC-LR(10 and 30 ?g/L)resulted in the degeneration of splenic macrophages and lymphocytes,and down-regulation of immunerelated genes as well as significant decreases in the levels of serum C3.Our findings illustrated that chronic exposure of MC-LR has dualistic influences on the fish innate immune system with inflammatory activation at low exposure concentration but turned to immune inhibition with the increases of exposure concentrations.2.Male zebrafish were exposed 0,0.4,2 and 10 ?g/L MC-LR for 30 d.The results showed that MC-LR exposure caused splenic inflammatory changes including the formation of melano-macrophage centers,the remarkable elevation of serum TNF? and IL1? levels as well as significant upregulated expression of MyD88-dependent toll-like receptor(TLR/MyD88)signaling pathway genes(tlr4a,myd88,erk2,p38 a,il1? and tnf?).The immunohistochemical and western blot results further validated that higher MC-LR concentrations tended to enhance the MyD88 signal.Moreover,a significant decrease of serum C3 levels along with splenic c3 b expression in the 10 ?g/L exposure group proved that chronic MC-LR exposure could decrease the innate immunity of fish.Our findings revealed that chronic exposure of MC-LR could cause chronic inflammation through TLR/MyD88 signaling pathway and subsequently induce immune disorders in male zebrafish,which also urge us to pay more attention to the potential immunotoxicity of longterm exposure to low concentrations of MC-LR.3.In a cross-generational study,adult zebrafish pairs were exposed to 0,0.4,2 and 10 ?g/L MC-LR for 60 d and the embryo(F1 generation)were hatched without or with continued MC-LR exposures at the same concentrations until 5 d postfertilization(5 dpf).The results showed the existence of MC-LR both in F0 gonads and in F1 embryos and indicated that MC-LR could be transferred directly from the F0 adult fish to F1 offspring.The adverse effects on sex hormone levels,sexual development,and fecundity in F0 generation along with abnormal development in F1 offspring were observed.These results suggested MC-LR could exert transgenerational toxic effects by interfering the reproductive function of F0 adult fish.In addition,downregulation of antioxidant genes(cat,mn-sod,gpx1a)and upregulations of innate immune-related genes(tlr4a,myd88,tnf?,il1?)as well as the increasing proinflammation cytokine contents(TNF?,IL1?,IL6)were noticed in F1 offspring without/with continued MC-LR exposures.These results indicated that parental exposure of MC-LR inhibited the antioxidant function and induced inflammatory response through TLR/MyD88 signaling pathway,which ultimatly decrease the immune function of the F1 offspring.Furthermore,significant differences between the two F1 embryo treatments demonstrated that continuous MC-LR exposure could result in a higher degree of inflammatory response compared to those without MC-LR exposure.