The Protective Effect Of Hypo-phosphorylated IκBβ At Ser-313 Against Lung Injury After Sepsis

BackgroundSepsis is a general immune and inflammation reaction induced by direct or indirect infection.The inflammatory amplification could result in multi-organ function disorder even failure.As the air exchange center,lung is highly sensitive to inflammatory reaction making itself extremely vulnerable to sepsis.Sepsis induced lung damages are acute inflammatory lung damages with obvious infiltration of inflammatory cells in the lung.Due to the dysregulation of immune system caused by sepsis,the inflammatory amplification aggravates the lung damage and we believe the reduction of neutrophil chemotaxis would protect the organ.Thus,exploring a way to reduce inflammation would effectively reduce the lung damage upon sepsis.NF-κB is an important transcription factor.Under normal condition,NF-κB forms a dimer with its inhibitor Ik B as inactive form in cytoplasm.Under the stimulation of LPS,Ik B is degraded and NF-κB is activated and transfers into nucleus to regulate gene expression.The phosphorylation and de-phosphorylation of IκBβ are the basis of this regulatory network.It has been reported that IκBβ knockout mice showed reduced over reaction of inflammation which protects organs from damages indicating that IκBβ down regulation may have a protective role of organ damage caused by over reaction of inflammation.Normal IκBβ contains two casein kinase active phosphorylation sites Ser-313 and Ser-315 at C terminal.Under the stimulation of LPS,the phosphorylated IκBβ are degraded.Although new IκBβ are generated,they are not as phosphorylated.This hypo-phosphorylated IκBβ binds to NF-κB in nucleus and maintains the interaction of NF-κB.Hence,hypo-phosphorylated IκBβ at Ser-313 and Ser-315 may play a role in the immune responses induced by either endogenous or exogenous toxin.The regulatory role of hypo-phosphorylated IκBβ at Ser-313 on chemokines was not clear.Through Ser-313 mutation,we can generate hypo-phosphorylated IκBβ and test the possibilities.We generated a transgenic mice that express IκBβ with Ser-313 mutation and built up a mice mode of sepsis to explore the expression level,distribution,inflammatory regulation of hypo-phosphorylated IκBβ.We also examine the survival,lung damage and neutrophil chemotaxis of sepsis mice to modulate clinical sepsispatients and reveal the protective function and mechanism of hypo-phosphorylated IκBβat Ser-313.Part 1.Construction of transgenic mice and the protective role of hypo-phosphorylated IκBβ at Ser-313 plays in sepsis miceObjective: Explore the expression level and distribution of hypo-phosphorylated IκBβ at Ser-313 and the role hypo-phosphorylated IκBβ at Ser-313 plays in immune response in sepsis mice.We also evaluated the protective role of hypo-phosphorylated IκBβ at Ser-313 on sepsis mice by examine the survival rate.Methods: Mutate the Ser-313 phosphorylation site to generate hypo-phosphorylated IκBβ.Build up the CLP based sepsis model.Examine the expression level and distribution of IκBβ in wild type and transgenic sepsis mice.Use co-IP to identify binding proteins.Study the death rate and examine the circulating inflammatory factors TNF-a and IL-6.Results: We successfully generated hypo-phosphorylated IκBβ transgenic mice that stably express hypo-phosphorylated IκBβ.We found that the survival rate of transgenic mice was better than wild type in CLP model since the survival rate of wild type and transgenic mice was 60% and 100% respectively 24 hours post-surgery.At 64 hours post-surgery,all wild type mice died while 50% of transgenic mice survived.We found in wild type CLP model that the rate of Ser-313 phosphorylated IκBβ in tissue were 0% at 3hours,17.5% at 6hours,54.1% at 12 hours and 73.9% at 24 hours demonstrating that in early sepsis,IκBβ were degraded and newly generated IκBβ were hypo-phosphorylated IκBβ at Ser-313.The IκBβ m RNA increased stably in wild type sepsis mice and reach expression peak at 12 hours post-surgery corresponding to the protein expression levels.We also revealed that hypo-phosphorylated IκBβ interacts with p105/p50,p65(Rel A)and Rel B in the nucleus.The serum level of TNFa and IL-6 were low in transgenic sepsis mice indicating that hypo-phosphorylated IκBβ reduced the expression of inflammatory factors.Conclusions: Hypo-phosphorylated IκBβ at Ser-313 interacts with p105/p50,p65(Rel A)and Rel B in the nucleus and reduced the death rate of sepsis mice effectively and regulates inflammatory reaction.Part 2.Hypo-phosphorylated IκBβ at Ser-313 regulates chemotaxis of sepsis mice and protect lung damage caused by sepsis.Objective: Study the protective function of hypo-phosphorylated IκBβ at Ser-313 on sepsis mice lung tissue and explore the relationship between hypo-phosphorylated IκBβ at Ser-313 and chemotaxis.Methods: Exam and grade the lung damages of wild type and transgenic sepsis mice by HE staining and exam the wet and dry ratio of lung tissues.Compare the expression changes of CD11 b on wild type and transgenic sepsis mice by IHC.Use microarray to detect the expression changes of chemokines and validate with q PCR.Examine CXCL1 and CXCL2 in lung tissues with ELISA and western blotting.Results: Hypo-phosphorylated IκBβ at Ser-313 sepsis mice showed mild lung damages compared to wild type sepsis mice as well as less neutrophile granulocytes.24 hours post CLP surgery,lung damage grading of wild type mice are higher than transgenic mice(10 versus 6,p<0.01)and the wet and dry ratio of lung tissues are3.79±0.12 vs 2.56±0.11(n=3,p< 0.01)in wild type vs transgenic mice.CD11 b staining showed reduced neutrophil infiltration in transgenic mice.Microarray revealed that the enrichment of chemokines of KEGG signaling is lower in transgenic mice compare to wild type mice 12 hours post CLP surgeries.Both CXCL1 and CXCL2 are down regulated in transgenic mice compare to wild type mice.q PCR also validated the down regulation of 9 chemokines in transgenic mice corresponding with microarray results.ELISA and western blotting experiments showed identical results that,the two important chemokines of neutrophil,CXCL1 and CXCL2 are significantly down-regulated in transgenic mice at both 6 hours and 12 hours post CLP surgeries.Conclusions: Hypo-phosphorylated IκBβ at Ser-313 has a protective role on lung damage induced by sepsis by down-regulate the expression of chemokines especially CXCL1 and CXCL2 and in turn inhibit the neutrophil chemotaxis to prohibit the inflammation in the lung tissue.