Functional Characterization Of Innate Immune Genes Of TLR2?TLR7?CD94 And Bf Of Cynoglossus Semilaevis

Tongue sole Cynoglossus semilaevis is an important fish cultured in China.Microbial disease is a severe problem of tongue sole aquaculture,and currently there is no effective means of disease control.In this study,in order to understand the immune defense mechanism against pathogens and provide a basis for the development of safe and effective methods of disease control,we examined the functions of four innate immune genes,i.e.,Toll-Like Receptor(TLR)2(named CsTLR2)?TLR7(named CsTLR7)?Cluster of Differentiation 94(named CsCD94),and complement factor B(named CsBf).Toll-like receptor(TLR)2 is a member of the TLR family that plays a pivotal role in innate immunity.In mammals,TLR2 is known to recognize specific microbial structures and trigger MyD88-dependent signaling to induce various cytokine responses.In this study,we examined the expression and function of the tongue sole Cynoglossus semilaevis TLR2,CsTLR2.CsTLR2 is composed of 898 amino acid residues and shares 25.6%�27.3% overall sequence identities with known teleost TLR2.CsTLR2 is a transmembrane protein with a toll/interleukin-1 receptor domain and eight leucine-rich repeats.Expression of CsTLR2 occurred in multiple tissues and was upregulated during bacterial infection.Stimulation of the CsTLR2 pathway led to enhanced expression of MyD88-dependent signaling molecules.Recombinant CsTLR2(rCsTLR2)corresponding to the extracellular region was able to bind to a wide range of bacteria.Under both in vitro and in vivo conditions,rCsTLR2 significantly reduced bacterial infection.These observations add new insights into the signaling and function of teleost TLR2 s.In mammals as well as in teleost,toll-like receptor 7(TLR7)is known to be involved in antiviral immunity by recognizing viral RNA.However,the antibacterial potential of fish TLR7 is unclear.In this study,we analyzed the TLR7 of tongue sole(Cynoglossus semilaevis),CsTLR7,and examined its potential involvement in antibacterial immunity.CsTLR7 is composed of 1052 amino acid residues and shares 64.0%-75.9% overall sequence identities with known teleost TLR7.CsTLR7 possesses a toll/interleukin-1 receptor domain and six leucine-rich repeats.Constitutive expression of CsTLR7 occurred in relatively high levels in kidney,spleen and liver.Bacterial infection upregulated CsTLR7 expression,whereas viral infection downregulated CsTLR7 expression.Knockdown of CsTLR7 significantly enhanced bacterial dissemination in the tissues of tongue sole.Treatment of tongue sole with the imidazoquinoline compound R848(TLR7 activator)and the endosomal acidification inhibitor chloroquine(TLR7 inhibitor)caused enhanced and reduced resistance against bacterial infection respectively.These results indicate that CsTLR7 plays an essential role in the antibacterial immunity of tongue sole.In this study,we examined the expression patterns and the functions of the tongue sole Cynoglossus semilaevis CD94,CsCD94.CsCD94 is composed of 209 amino acid residues and shares 43.0�50.2% overall identities with known teleost CD94 sequence.CsCD94 has a C-type lectin-like domain.Expression of CsCD94 occurred in multiple tissues and was upregulated during bacterial infection.Recombinant CsCD94(rCsCD94)exhibited apparent binding and agglutinating activities against both Grampositive and Gram-negative bacteria in a Ca2+-dependent manner.Treatment of bacteria with rCsCD94 enhanced phagocytosis of the bacteria by peripheral blood leukocytes.Furthermore,incubation of rCsCD94 with bacteria reduced the survival of the bacteria in vitro.Taken together,these results indicate that rCsCD94 is a key factor in the bactericidal and phagocytic effects of tongue sole,and reveal for the first time an essential role of fish CD94 in antibacterial immunity,thereby adding insight into the function of CD94.Complement factor B(Bf)is a component of the complement system.Following activation of the alternative pathway of the complement system,factor B is cleaved into Ba and Bb fragments.In fish,the functions of Bf and Ba are essentially scarce.In this study,we examined the expression patterns of tongue sole Cynoglossus semilaevis Bf(named CsBf)and the biological activity of the Ba fragment of CsBf(named CsBa).CsBf possesses the conserved domains of Bf and shares 40.3%-56.5% sequence identities with other fish Bf.CsBf expression was high in liver,muscle,and heart,and low in intestine,blood,and kidney.Bacterial infection significantly induced CsBf expression in kidney,spleen,and liver in a time-dependent manner.Recombinant CsBa(rCsBa)exhibited apparent binding capacities to bacteria and tongue sole peripheral blood leukocytes,and binding of rCsBa to bacteria inhibited bacterial growth.When overexpressed in tongue sole,CsBa significantly reduced bacterial dissemination in fish tissues.Together these results indicate for the first time that a fish Ba possesses antibacterial effect as well as immune cell-binding capacity,and thus probably plays a role in host immune defense against bacterial infection.