The Role Of IRF5 Gene Polymorphisms And Expression In Patients With Community-acquired Pneumonia

Community-acquired pneumonia(CAP),an infection of the terminal airway and the alveolar and pulmonary interstitium,is a common disease with significant morbidity(0.3-0.5%)and mortality(5-15%).Due to the highly infectious nature and drug resistance of the responsible pathogens,as well as the high death rate,CAP has become a medical burden in China,Europe,the USA,and various other parts of the world.The mechanisms and factors regulating the host inflammatory response that lead to the poor outcomes associated with pneumonia and lung injury are still unknown,while innate immune response plays a core role in them.Thus,the exploration of innate immune response in CAP is of great importance.Interferon regulatory factor 5(IRF5),which induces type I interferons(IFNs)and cytokines such as interleukin(IL)-6,tumor necrosis factor(TNF)-?,IL-10,and interferon-gamma-induced protein(IP)10,is a key transcription factor involved in innate immune response.TNF-?and IL-6 act as general markers of inflammation in pneumonia and regulate the expression of acute-phase proteins,and IL-10,one of the most crucial anti-inflammatory cytokines,also act as a key regulator of the degree of lung inflammation.Additionally,type I IFN,the key factor in viral infection,has been shown to inhibit inflammasome activation and upregulate IP10 production.Thus,IRF5 may play a role in regulating immune response of CAP patients.Pathogen species,virulence of microorganism strains,increasing age,and accompanying diseases present important risk factors for CAP,along with genetic variations which affect the host response and influence the susceptibility to CAP as well as its severity and outcome.Previous studies have shown that CAP caused by different pathogens showed correlation with distinct polymorphisms in genes encoding pro-inflammatory cytokines such as TNF-?and IL-6,but no research has yet been conducted on the role of IRF5 gene polymorphisms in CAP.Multiple polymorphisms of the IRF5 gene have been shown to be associated with autoimmune and infectious diseases,including systemic lupus erythematosus,rheumatoid arthritis and HBV infection.Furthermore,upregulation of IFN-?were various in individuals with different IRF5 gene polymorphism.Thus,IRF5 gene polymorphisms may be the reason of distinctions in response to pathogen infection in CAP patients.Based on the above,we compared the expression levels of IRF5 and its-related cytokines in peripheral blood and bronchoalveolar lavage fluid samples between CAP patients and controls,and performed association analysis at the single marker level for SNPs rs77571059,rs2004640,rs10954213 and rs3807306 as well as at the haplotype level,to reveal the role of IRF5 and its-related cytokines in the immune response of CAP patients.The results are as follows:1.The role of IRF5 and IRF5-mediated inflammatory cytokines in CAP1.1 Proportion analysis of immunocytes and lymphocyte subsets in peripheral blood of patients with CAPTo verify whether there are differences in immune function of the patients,we have collected the count of various immune cells in peripheral blood of pneumonia patients,and found that there were significantly differences between CAP patients and healthy controls.The percent of neutrophils in pneumonia patients showed significantly higher in severe CAP patients,and increased as the disease progressed;however,the proportion of lymphocytes and monocytes in patients with pneumonia showed a decreasing trend in severe pneumonia patients.The number of CD4~+and CD8~+T cells decreased as the disease got worse.The percentage of CD4~+CD25~+T cells in patients was significantly higher than that of healthy controls,and the percent of severe patients was significantly higher than that of mild patients.The results showed that the proportion of immune cells were related to the degree of inflammation.1.2 mRNA levels of IRF5 and its mediated inflammatory factors in peripheral WBCs of CAP patientsTo investigate the role of IRF5 in CAP,we analyzed the mRNA expression levels of IRF5 and its downstream factors IFN-?,IL-6,IL-10,IP10,and TNF-?in the peripheral WBCs of 71 CAP patients and 31 healthy controls using qRT-PCR method.Results showed that the mRNA levels of IRF5 and IFN-?were significantly higher in the WBCs of CAP patients compared with healthy controls,but they had little relation with the severity of illness.The IL-10 and IP10 mRNA levels were similarly higher in the WBCs of CAP patients compared with healthy controls,and they were additionally associated with the severity of illness.The overall change in the TNF-?mRNA level was significantly higher in CAP patients compared with healthy controls,and severe CAP patients had a lower level of TNF-?mRNA than mild CAP patients,which may indicate that the excessive production of TNF-?suppressed its own expression as the patient's condition deteriorated.These results revealed that IRF5 may involve in immune response of CAP,and the expression level of IRF5 may associated with species of pathogens.1.3 Protein levels of IRF5 and IRF5-related cytokines increased in the peripheral WBCs of CAP patients1.3.1 Protein level of IRF5 in the WBCs of CAP patientsTo detect the IRF5 protein expression in WBCs,we collected peripheral blood samples from 38 CAP patients(22 cases with severe CAP and 16 cases with mild CAP)and 12 healthy donors,and stained with IRF5-APC,the percent of IRF5 were analyzed by flow cytometry.The results show that the percent of IRF5~+cells in patients with CAP was significantly higher than that in healthy subjects,and the IRF5 protein level in patients with severe CAP was significantly higher than that in patients with mild CAP.Moreover,the IRF5 protein level in patients with a short course(?7 days)of illness was significantly higher than that in patients with a longer course(>7 days)of illness.We monitored the IRF5 protein expression level at different time points in one patient with severe CAP and found that it decreased as the disease progressed.Thus,we speculated that IRF5 might be a key transcription factor in the early phase of CAP and associated with the severity of disease.1.3.2 The expression of IRF5-related cytokines in serum of CAP patientsTo explore the role that IRF5 may play in the development of CAP,we measured the levels of IFN-?,IL-6,IL-10,IP10,and TNF-?,which are all downstream factors of IRF5 in CAP patients and healthy controls by using a CBA flex set.The results showed that the expression levels of these factors were significantly higher in patients with CAP compared with healthy controls,and the expression levels of IL-6,IL-10,IP10,and TNF-?were significantly different in patients with mild CAP compared with the levels in those with severe CAP.The expression levels of these cytokines decreased as the patient's condition improved.These findings indicated that both pro-and anti-inflammatory cytokines increased with the severity of disease,and IL-6,IL-10,and IP10 are indicators of CAP severity and prognosis.1.4 mRNA levels of IRF5 and its related cytokines in bronchoalveolar lavage cells of CAP patientsTo study changes in the levels of IRF5 and IRF5-mediated inflammatory factors in the lung tissue,we collected bronchoalveolar lavage fluid from 20 patients with CAP and from the unaffected lung of 23 patients with lung cancer as the control group,and detected the mRNA levels of IRF5 and its related cytokines by qRT-PCR.We found that,compared with the control group patients,mRNA levels of IRF5 and its related cytokines significantly higher in bronchoalveolar lavage cells of CAP patients.the mRNA levels of IRF5,IL-6,IL-10 and IP10 were significantly higher in severe CAP patients than that of mild patients,whereas the level of TNF-?was significantly lower in mild CAP patients.Thus,IRF5 may participate in the inflammatory response in the lung tissue of patients with CAP and associated with the severity of disease.1.5 IRF5 expression in CAP caused by influenza virus infectionWe compared the IRF5 expression levels between 10 CAP patients infected with influenza A virus who were in the early stage of illness and 10 CAP patients infected with bacteria.The results show that the mRNA and protein levels of IRF5 and IFN-?in the influenza virus-infected group were both significantly higher than those in the bacteria-infected group,and the IRF5 expression level was positively correlated with the IFN-?level.Type I IFN,the key factor in viral infection,play an important role in inflammation caused by influenza virus.Our study results suggest that IRF5 may play an important role in immune response due to influenza virus infection.2.The role of IRF5 gene polymorphisms in CAP patientsTo study possible effects of IRF5 polymorphisms on the susceptibility to CAP in Chinese patients,we genotyped four IRF5 variants,rs77571059,rs2004640,rs10954213,and rs3807306 of 228 CAP patients and 177 healthy donors by using PCR amplification and Sanger sequencing.2.1 Independent IRF5 polymorphism analysis in CAP patientsThe frequencies of the-/GGGGC genotype and the GGGGC allele of rs77571059 were significantly higher in CAP patients compared to healthy controls.The genotype distribution at the IRF5 rs2004640 locus in CAP patients was significantly different from that in the control group,as well as the TT genotype and T allele were significantly more common in CAP patients than in healthy individuals,indicating that IRF5 rs77571059 and rs2004640 were associated with susceptibility of CAP.2.2 The association between IRF5 gene polymorphisms and the severity of CAPThe frequency of the-/GGGGC genotype in the severe CAP group was higher compared to that in the mild CAP group,and this difference was also observed for the GGGGC allele.However,there was no significant difference in genotype or allele frequencies of the rs2004640 locus between the two patient groups.Results indicated that IRF5 rs77571059 was associated with the severity of CAP.2.3 Haplotype analysis of IRF5 variants in CAP patientsHaplotype analysis and calculation of D?and r~2 values were performed using the Haploview 4.2 and SNPstats software.The frequency of IRF5 haplotype GTAA,comprising SNPs rs77571059,rs2004640,rs10594213 and rs3807306,was significantly higher in CAP patients compared to healthy controls,which revealed that IRF5 risk haplotype GTAA was associated with the susceptibility of CAP.2.4 IRF5 risk alleles increased the expression of IRF5 and IFN-? and TNF-? serum levelsThe rs77571059 GGGGC allele,rs2004640 T allele,and rs10954213 A allele were associated with the upregulation of IRF5 mRNA transcription in CAP patients.Moreover,the rs77571059 GGGGC allele was significantly correlated with higher levels of serum IFN-?,and the rs2004640 T allele as well as rs10954213 A allele were associated with higher levels of both IFN-?and TNF-?in CAP patients.However,the TNF-?level of CAP patients showed no significantly difference between the rs77571059-/GGGGC and-/-groups.Still,our results suggest that the identified IRF5 risk haplotypes may affect the expression of multiple downstream genes in the IFN-?signaling pathway and the production of pro-inflammatory cytokines in CAP patients.Conclusions:1.IRF5 and its related inflammatory cytokines associated with the severity,prognosis and pathogen of patients with CAP,IL-6,IL-10,and IP10 are indicators of CAP severity and prognosis2.The levels of IRF5 and IFN-?increased significantly in the early phase of pneumonia caused by influenza virus infection,which may provide new drug targets for the prevention and treatment of severe pneumonia caused by influenza virus.3.IRF5 SNPs are associated with the susceptibility and the severity of CAP,as well as affected inflammatory response of CAP patients.