A Study On The Relationship Between The Monocytes/Macrophages And Pathogenesis In The Immune Associated Diffuse Alveolar Hemorrahge

Diffuse alveolar hemorrahge(DAH)is a severe life-threatening clinical syndrome,and can be caused by a wide variety of etiologies[1].The pathogenesis in some cases is associated with the immune disorders[1-6].Until now,the exact pathogenesis of immune associated DAH hasn’t been fully understood.Pristane induced murine systemic lupus erythematosus(SLE)model is a well-known animal model of SLE.During the modeling process,Chella S.David et al found that the mice also developed significant alveolar hemorrahge in the early modeling period[7].Then,they successfully established the murine model of diffuse alveolar hemorrahge in 2007[7].Until now,the pristane induced murine DAH model has been widely recognised as the immune associated DAH animal model internationally.In recent years,nearly all the studies on the pathogenesis of alveolar hemorrahge in animal models have been performed in the pristane induced murine DAH model[8-17].It’s worth nothing that many advances about the pathogenesis of alveolar hemorrahge have been achieved from the studies in the pristane induced murine DAH model.Whereas,there has been very few studies to be performed in the clinical cases with immune associated DAH about the pathogenesis of alveolar hemorrhage.Firstly,immune associated DAH has not received enough attention in clinic,as the disease is relatively rare.Secondly,it’s difficult to identify the study direction,as the disease can be caused by a wide variety of etiologies,and it’s unclear that whether there is a common mechanisum to result in alveolar hemorrahge in the cases with different etiologies.Whereas,to facilitate translation from the basic research towards clinical application need the validation of the conclusions from the animal models in the clinical cases.So,it’s nessessary to perform the study to explore the potential pathogenesis in the clinical cases.Our department is a reginal center for the diagnosis and treatment of pediatric respiratory diseases.Until now,more than 100 cases with immune associated DAH have been treated and followed up in our department,which provided a good platform to perform the study.The studies in the pristane induced murine DAH models have demonstrated that the bone marrow derived monocytes/macrophages played an important role in the pathogenesis of alveolar hemorrhage,and were the critical factors for developing alveolar hemorrhage[8-14].And,the enhances M1 polarization of the monocytes/macrophages was the key immunological mechanisms involved in the pathogenesis of alveolar hemorrhage in the pristane induced murine DAH model[8-14].Besides that,the monocytes from the bone marrow in the pristane induced murine DAH model presented lower expression of IL-10R[12].As the activation of IL-10/IL-10 R signalling pathway was found to be an protective factor for developing DAH in the pristane induced murine DAH model,it demonstrated that systemic inflammation might also participate in the pathogenesis of alveolar hemorrhage[12].Given this,our study focused on the relationship between the monocytes/macrophages and pathogenesis in the patients with immune associated DAH.In clinic,it’s relatively easy to get the peripheral blood sample to perform the study on the peripheral blood monocytes.However,it’s very difficult to get the lung samples,as very few patients with immune associated DAH performed lung biopsy due to the limitations of the disease severity and family factors.Bronchoscopy with bronchoalveolar lavage was relatively safe as well as minimally invasive,and it was accepted by the neally all the children and their family.Until now,it was widely used in the diagonosis of DAH in clinic.BALF is also called “liquid biopsy of the lung”.The supernatant of BALF can reflect the pulmonary immune microenviroment,as there is a large amount of immunologically active substances in the supernatant of BALF.So,we intented to perform the studies to analyze the cytokines in the supuernatant of the BALF in order to learn the pulmonary immune microenviroment.Also,we stimulated the macrophages in vitro using the supernatant of the BALF in order to learn their effect on the macrophage polarization.what’s more,there are amount of alveolar macrophages(AM)in the BALF,which provide a good pathway to study AM in the cases with immune associated DAH.Our study were divided into four parts.Part One A Study of the Peripheral Blood Monocyte Subtypes in the Children with Immune Associated Diffuse Alveolar HemorrahgeObjective To explore the relationship between the peripheral blood monocytes and pathogenesis in the immune associated DAH.Methods Total 37 children with immune associated DAH were included in this study,in which 16 cases were divided into the exacerbation group with acute alveolar hemorrahge(DAH-A group),and 21 cases were divided into the remission group without acute alveolar hemorrahge(DAH-R group).Also,18 healthy children were included as the healthy control group(HC group).The expressions of CD14 and CD16 on the surface of peripheral blood monocytes were detected using flow cytometry(FCM).Then,the percentages of classical monocytes(CD14++/CD16-),intermediate monocytes(CD14++/CD16+)and non-classical monogytes(CD14+/CD16+)were calculated respectively.Serum samples of the included subjects were also obtained.Serum was used to stimulated the monocytes isolated from the healthy volunteers in vitro.And the changes of subtype of the monocytes were detected using FCM.Results 1.The percentage of classical monocytes was significantly increased(P<0.01),and the percentages of intermediate monocytes and non-classical monocytes were both significantly decreased(both P<0.01)in the DAH-A group,when compared to those in the HC group.2.The percentage of non-classical monocytes was significantly increased(P<0.01)in the DAH-R group,when compared to that in the DAH-A group.The percentage of classical monocytes presented a decreased tendency,and the percentage of intermediate monocytes presented an increased tendency in the DAH-R group,when compared to those in the DAH-A group.However,neither of them had significant differences(both P>0.05).3.The percentage of classical monocytes was significantly decreaed(P<0.05),and the percentage of intermediate monocytes was significantly increased(P<0.05)under stimulaiton of the serum from the DAH-A group,when compared to those under stimulation of the serum from the HC group.Also,the percentage of non-classical monocytes presented an increased tendency,but without significant difference(P>0.05)under stimulaiton of the serum from the DAH-A group,when compared to that under stimulation of the serum from the HC group.Conclusions 1.There were significant differences in the peripheral blood monocyte subtypes between the children with immune associated DAH and healthy children,and the abnormalities presented a tendency to normalization with the disease remission,which demonstrated that the peripheral blood monocytes might participate in the pathogenesis of immune associated DAH.2.The soluble immune mediators in the peripheral blood might not mediate the change of the peripheral blood monocyte subtypes in the immune associated DAH.Part Two Estalishment and Assessment of the Cell Model of the Monocyte Derived MacrophagesObjective To estalish and assess the cell model of monocyte derived macrophages(MDMs),and estalish the fundation for the further study of macrophage polarization.Methods Peripheral blood was sampled in the EDTA-coated tubes.Then,the peripheral blood mononuclear cells were isolated using the Ficoll-Paque density gradient centrifugation method,and the monocytes were further isolated using the adherent method.The isolated monocytes were cultured under the stumulaiton of M-CSF for 7 days to differentiate into macrophages,the naive MDMs.The change of the morphology was observed under an inverted microscope.The naive MDMs were futher induced into M0,M1 and M2 cell models under no stimulation,the stimulation of LPS+IFNγ and the stimulation of IL-4,respectively.Twenty-four hours post-stimulation,the m RNA relative expression of IL-1β,TNFα,IL-6,TGM2,CD163 and MRC1 in the different cell models were detected using quantitive real-time PCR method(q RT-PCR).Fourty-eight hours post-stimulation,the expression of CD14,CD80,CD86,CD163 and CD206 in the different cell models were detected using FCM.Results 1.After having been cultured for 7 days in vitro,the monocytes became enlarged,appeared as fusiform or “fried eggs” shape with pseudopod.Also,the cells became tightly adherent.2.The m RNA relative expression of IL-1β and IL-6 were significantly higher(both P < 0.01),and the m RNA relative expression of TGM2 and MRC1 were significantly lower(both P<0.01)in the M1 cell model,when compared to those in the M2 cell model.3.The expression of CD14 and CD80 were significanly higher(both P<0.01),and the expression of CD163 and CD206 were significantly lower(both P<0.01)in the M1 cell model,when compared to those in the M2 cell model.Conclusions We have successfully estalished the cell model of MDMs,and estalished the fundation for the further study of macrophage polarization in vitro.Part Three A Study of the Pulmonary Immune Microenviroment in the Immune Associated Diffuse Alveolar HemorrahgeObjective To study the phenotype of the pulmonary immune microenviroment in the immune associated DAH and its effects on the macrophage polarization.Methods Total 24 children with immune associated DAH were included in this study(DAH group).Also,13 children with acute airway foreingn body or airway stenosis,but without infecion were included as the control group.Bronochoalveolar lavage fluid(BALF)was collected using the bronchoscope.Then,the BALF was centrifuged,and the supernatant was collected.Total 16 kinds of the soluble proteins containing IL-1β,IL-2,IL-5,IL-6,IL-7,IL-8,IL-9,IL-10,IL-13,IL-17 A,MCP-1,RANTES,GM-CSF,VEGF,Granzyme B and TNF were detected using the method of cytometric bread arrays(CBA).The MDMs model was established as mentioned in the previous study.The BALF supertanant was used to stimulated the MDMs cell model.Twenty-four hours post-stimulation,the m RNA relative expression of IL-1β,TNFα,IL-6,TGM2,CD163 and MRC1 in the different cell models were detected using q RT-PCR.Fourty-eight hours post-stimulation,the expression of CD14,CD80,CD86,CD163 and CD206 in the different cell models were detected using FCM.Results 1.The levels of MCP-1,IL-6 and IL-8 were all significantly higher in the BALF supernatants from the DAH group,when compared to those form the control group(all P<0.01).It was noteworthy that the level of MCP-1 in the BALF supertanant rose most sharply,with nearly 30 folds increase.The levels of IL-2 and IL-9 were both significantly lower in the BALF supernatants from the DAH group,when compared to those form the control group(with P<0.05 and<0.01,respectively).Whereas,there were no significant differfences(P>0.05)in the levels of IL-1β,IL-7,IL-10,IL-13,IL-17 A,RANTES,GM-CSF,VEGF,Granzyme B or TNF in the BALF supernatants from the DAH group and those from the control group.The levels of IL-5 were both under the detection threshold in the BALF supernatants from the DAH group and that from control group.2.The m RNA relative expression of IL-1β and IL-6 were significantly higher(both P<0.05),and the m RNA relative expression of MRC1 was significantly lower(P<0.01)under the stimulation of BALF supertanants from the DAH group,when compared to those under the stimulation of BALF supertanants from the control group.Whereas,there was no significant difference in the m RNA relative expression of TNFα,TGM2 or CD163 between the two groups(P>0.05).2.The expression of CD86 was significanly higher(P<0.05),and the expressions of CD163 and CD206 were significantly lower(both P<0.05)under the stimulation of BALF supertanants from the DAH group,when compared to those under the stimulation of BALF supertanants from the control group.Whereas,there was no significant difference in the expressions of CD14 and CD80 between the two groups(P>0.05).Conclusions 1.The pulmomary immune microenviroment in the immune associated DAH preferred a pro-inflammatory phenotype,and strongly favored the recruitment of the peripheral blood monocytes.2.In viro cell experiments,the BALF supernatants from the children with immune associated DAH promoted macrophages into the M1 phenotype,which demonstrated that the recruited monocytes preferred to differentiate into the M1 phenotype under the pulmonary immune microenviroment in the immune associated DAH.3.Taken togather,enhances M1 monocytes/macrophage polarization in the lungs might play a role in the pathogenesis of immune associated DAH.Part Four A Study of Alveolar Macrophage Polarization Phynotype in the Immune Associated Diffuse Alveolar HemorrahgeObjective To explore the relationship between the AM and pathogenesis in the immune associated DAH.Methods Total 15 children with immune associated DAH were included in this study(DAH group).Also,11 children with acute airway foreingn body or airway stenosis,but without infecion were included as the control group.BALF was collected using the bronchoscope.A CD206+ gate was set for AM from the BALF sample using FCM,and the expressions of CD14,CD80,CD86 and CD163 on the surface of AM were further analyzed.Results The expressions of CD80,CD86 were significanly lower(with P<0.01,P<0.05 respectively)in the AM from the DAH group,when compared to those from the control group.Whereas,there was no significant differfence(P>0.05)in the expressions of CD14 or CD163 in the AM from DAH group and those from the control group.Conclusions The AM in the immune associated DAH preferred a M2 phenotype,which demonstrated that AM might play a role in the tissue repair process after the alveolar inflammation and hemorrhage in the pathogenesis of immune associated DAH.