CC10+Club Cells Derived High Mobility Group Box-1 Protein Promotes Type 2 Response In The Later Stage Of Respiratory Syncytial Virus Infection

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THE ROLE OF CC10+CLUB CELLS IN THE TYPE 2 RESPONSE IN THE LATER STAGE OF RSV INFECTIONObjective: respiratory syncytial virus(RSV)is the most common and important virus causing acute lower respiratory tract infection in infants worldwide.About 30% of infants with severe RSV bronchiolitis still experience recurrent wheezing at age 13.The type 2 immune response,induced by infection of RSV,has been linked to asthma development,but it remains unclear how the response is initiated.Epithelial dysfunction is essential to initiate and maintain the type 2 response in asthma models.Club cells are epithelial cells with self-renewal ability.The infected-club cells can partially survive and participate in airway repair and mediate chronic airway inflammation at the same time.This study intends to explore the role of club cells in the type II response in the later stage of RSV infection.Methods: The mice,aged at 6-8 weeks,were randomly divided into three groups and infected intranasally(i.n.)with 100 ?l of a stock suspension of 6�107 PFU/ml RSV(RSV group),UV-inactivated virus(UV RSV group)and HEp-2 suspension without virus(Mock group)under sodium pentobarbital anesthesia.The changes of weight were recorded.0.1g of lung tissues were homogenized and plaqued in HEp-2 cell to detect viral titers.Total RNA was extracted from the whole lung tissues using the TRIZOL reagent to calculate the number of copies of the RSV N gene.The CC10+club cells were observed by immunofluorescence.Naphthalene(NA)was administered intraperitoneally(i.p.)for CC10+club cells deletion and the effect was verified by flow cytometry.The levels of type 2 cytokines,including IL-4,IL-5 and IL-13 in BALF were detected by ELISA.Lung histopathology and cell counts in BALF were used to evaluate the inflammatory response.Airway resistance was measured to evaluate AHR.Results: Weight loss was most obvious on day 6 and 7,and weight recovery was observed on day 14 after RSV infection in BALB/c mice.On the 4th day of RSV infection,the copy number of RSV N gene reached a peak,while the RSV N gene could not be detected in the UV RSV group from the 3rd day.Plaques were observed when hep-2 cells were stimulated by lung tissue homogenates from RSV infected BALB/c mice.The virus titer was the highest on the third day of infection: 331.30� 58.07� 103 PFU/g,and plaque was not observed on the 7th and 14 th days.Plaque was not observed in the control group and UV RSV group.Type 1 cytokines IFN-? peaked on the 7th day after RSV infection.From day 14 to day 30 after RSV infection,type 2 cytokines IL-4,IL-5 and IL-13 were significantly increased compared with the control group and the UV RSV group.The expression of CC10 decreased on day 7,rising dramatically from day 14 to day 30 when compared to the mock group observing by immunofluorescence.When club cells were depleted by treatment with naphthalene(NA),type 2 cytokines were reduced significantly with morphological changes and the severity scores attenuated and inflammatory cells suppressed.However,the Rrs deteriorated.Conclusion: CC10+club cells are involved in the type 2 response,which mainly reflected in:(1)CC10+club cells proliferate a lot in the late stage of RSV infection,accompanied by a significant increase of type 2 cytokines in the late stage of infection,(2)Type 2 cytokines were reduced significantly after CC10+club cells deletion by NA administration accompanied by the reduced airway inflammation. 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CC10+CLUB CELLS DERIVED HMGB1 IS INVOLVED IN THE TYPE 2 RESPONSE IN THE LATER STAGE OF RSV INFECTION Objective: Epithelial cells are able to recognize the exogenous pathogen-associated Molecular patterns(PAMP)through the Pattern recognition receptor(PRR),and produce the damage-associated Molecular patterns(damps)to mediate the type 2 response.High Mobility Group Protein B1(HMGB1),as an important member of the DAMP family,can be derived from the epithelial cells and is reported to be involved in type 2 response.Based on the first part of the study showing that CC10+club cells participate in the type 2 response in the late stage of RSV infection,this part intends to further clarify whether CC10+club cells participate in type 2 response by generating endogenous HMGB1.Methods: The mice,aged at 6-8 weeks,were randomly divided into three groups and infected intranasally(i.n.)with 100 ?l of a stock suspension of 6�107 PFU/ml RSV(RSV group),UV-inactivated virus(UV RSV group)and HEp-2 suspension without virus(Mock group)under sodium pentobarbital anesthesia.At indicated post infection time points,the colocalization of HMGB1 and CC10+club cells was observed by immunofluorescence.The co-expression of HMGB1 and CC10+club cells was further observed in the RSV+NA group.The protein level of HMGB1 in lung tissues and BALF in the RSV+NA group was observed by Western blot and Elisa to determine whether endogenous HMGB1 could be generated by CC10+club cells.To clarify the role of CC10+club cell derived HMGB1 in type 2 response,anti-HMGB1 neutralizing antibody was administrated to RSV-infected mice at 400 ?g/kg and the levels of IL-4,IL-5 and IL-13 in BALF were detected by ELISA.Type 2 response related transcription factors were detected by PCR.Lung histopathology and cell counts in BALF were used to evaluate the inflammatory response.Airway resistance was measured to evaluate AHR.Results: The co-localization of HMGB1 and CC10+club cells was observed by immunofluorescence.The Pearson's correlation and Mander's overlap values were used to show the colocalization of HMGB1 and CC10.The higher values reflect the greater colocalization.The co-localization was most obvious at the 21 st day,when Pearson's correlation coefficient was 0.73 and Mander's overlap value was 0.81.NA efficiently depleted CC10+ club cells,and depletion clearly attenuated the protein level of HMGB1 in the lung tissues and in the BALF at day 21 after RSV infection.The expression of HMGB1 was examined in the mice on days 7,14,21,30 after RSV infection.Mice,inoculated with RSV,showed a significant increase in the levels of HMGB1 in the lung tissues and in the BALF compared to the mock group.The concentrations of IL-4,IL-5 and IL-13 were dramatically reduced after HMGB1 blockade at day 21 after RSV infection.GATA3,which is a crucial transcription factor in the modulation of the type 2 response,increased at day 21,which was prevented when HMGB1 was blocked.The morphological changes and the severity scores were significantly attenuated in the RSV + anti-HMGB1 antibody group.Treatment with anti-HMGB1 antibody was also found to significantly suppress the infiltration of inflammatory cells.Next,AHR was evaluated by calculation of Rrs,which was also successfully alleviated by anti-HMGB1 antibody administration.Conclusion: CC10+club cells can produce HMGB1,the important member of the family of DAMP,promoting the expression of GATA3,thus mediating type 2 response related airway inflammation and AHR.PART ? THE ROLE OF HMGB1 IN THE EVALUATION OF DISEASE SEVERITY AND PREDICTION OF RECURRENT WHEEZING IN CHILDREN WITH RSV BRONCHIOLITIS Objective: Whether HMGB1 is related to the severity of disease and recurrent wheezing after RSV infection has not been reported.This study intends to observe whether HMGB1 level can be used as a biological marker to evaluate the severity and predictor for recurrent wheezing of hospitalized children with RSV bronchiolitis in clinic.Methods: Hospitalized infants aged<2 years with RSV bronchiolitis(defined as the first episode of wheezing with manifestations of a viral respiratory infection)were recruited from December 2014 to March 2015 in respiratory department in Children's Hospital of Chongqing Medical University.NPA samples were collected on the first day of admission.Seven kinds of respiratory viruses were detected by direct immunofluorescence.Sputum culture and blood tests have been done.Patients with positive RSV detection alone were included in the study,patients infected with other viruses and bacteria were excluded,and children with congenital heart disease and immune deficiency were excluded.At the same time,NPA specimens of children with no respiratory infection were collected in the intensive care unit and gastrointestinal surgery department of Children's Hospital of Chongqing Medical University as the control group.According to the scoring system developed by wang et al,the disease severity of hospitalized children with RSV bronchiolitis was scored.HMGB1 level in NPA was detected by ELISA,and the relationship between HMGB1 and severity of children was analyzed.Further telephone follow-up was conducted to analyze the relationship between HMGB1 and recurrent wheezing.Results: 52 patients of the RSV bronchiolitis and 23 patients in the control group were collected in this study.There were 43 males in RSV group and 18 males in control group.The median age of RSV group was 3 months(IQR,2-6 months),and that of the control group was 2 months(IQR,2-6 months),P>0.05.Further comparison of HMGB1 level showed that the level of HMGB1 in RSV group was 161.20 ng/ml(68.06-221.30)ng/ml,which was significantly higher than 21.94 ng/ml(12.12-59.82)ng/ml which in control patients(P<0.001).According to Wang's severity scoring system,there were 42 cases of moderate to severe and 10 case of mild.In the moderate and severe group,HMGB1 level was 237.50 ng/ml(193.00-283.60)ng/ml,significantly higher than 147.50 ng/ml(54.52-214.40)ng/ml in the mild group(P=0.003).HMGB1 performed well in distinguishing the severe cases from the mild ones(AUC 0.81,cut-off point >203.00 ng/ml,sensitivity 80.00%,and specificity of 69.05%).Follow-up was conducted on the above 52 hospitalized children with RSV bronchiolitis,and a total of 33 cases were followed up by telephone.Among them,26 cases had no recurrent wheezing and 7 cases suffered recurrent wheezing.The level of HMGB1 in infants suffered recurrent wheezing was higher than those did not suffer recurrent wheezing(P=0.06).Conclusion: The level of HMGB1 in NPA is closely related to the severity of disease in hospitalized children with RSV bronchiolitis,and may be a target for RSV infection.Whether it can be used as a biological marker for recurrent wheezing still needs further observation and verification.