The Role of Monocytes/Macrophages In The Pathogenesis of Immune Associated Diffuse Alveolar Hemorrhage

Backgroud The studies in the immnue associated diffuse alveolar hemorrahge (DAH) animal models showed that monocytes/macrophages played an critical role in the pathogenesis.Whether monocytes/macrophages contribute to the pathogenesis of immune associated DAH in human is still unknow. The aim of this study was to explore the role of monocytes/macrophages in the pathogenesis of immune associated DAH in human. Methods This study was conducted in two parts. In the rst part, 37 children with immune associated DAH were included (DAH group), and 18 healthy children were recruited as the controls (HC group). Peripheral blood monocyte subtype was analyzed using ow cytometry. In the second part, 24 children with immune associated DAH were included (DAH group), and 13 children with acute airway foreingn body or mild benign airway stenosis were included as the controls (HC group). Bronochoalveolar lavage uid (BALF) was collected using bronchoscope. Cytokines in the BALF supernatant were detected using cytometric bread array. BALF supertanant was used to stimulated the macrophages in vitro. The mRNA relative expressions of IL-1β, TNFα, IL-6, TGM2, CD163 and MRC1 were detected using quantitative real-time PCR, and the expressions of CD14, CD80, CD86, CD163 and CD206 were detected using ow cytometry.

Results 1. The percentage of classical monocyte was signi cantly increased, whereas the percentages of intermediate and non-classical monocyte were signi cantly decreased in the DAH group, when compared to those in the HC group. 2. The levels of MCP-1, IL-6 and IL-8 were all signi cantly higher in the BALF supernatant from the DAH group, when compared to those form the HC group. 3. The mRNA relative expressions of IL-1β and IL-6 as well as the expression of CD86 were signi cantly higher, whereas the mRNA relative expression of MRC1 as well as the expressions of CD163 and CD206 were signi cantly lower under the stimulation of BALF supernatant from the DAH group, when compared to that from the HC group.
Conclusions Monocytes/macrophages might participate in the pathogenesis of immune associated DAH in human by enhanced M 1 polarization. Background DAH is a severe life-threatening clinical syndrome, and can be caused by a wide variety of etiologies [1] .
Usually, the cases caused by the immune disorders were named immune associated DAH [1][2][3][4][5][6] . Until now, the exact pathogenesis of immune associated DAH hasn't been fully understood. Pristane induced DAH murine model has been widely recognised as the immune associated DAH animal model internationally [7][8][9][10][11][12] . The studies in the pristane induced murine DAH models have showed that the bone marrow derived monocytes/macrophages in the lungs were the critical factors for developing alveolar hemorrhage, and the enhanced M 1 polarization of the monocytes/macrophages was the key immunological mechanism [7][8][9][10][11][12] . Besides the local in ammation in the lungs, systemic in ammation also participated in the pathogenesis of alveolar hemorrhage, as the monocytes from the bone marrow presented lower expression of IL-10R, whereas the activation of IL-10/IL-10R signalling pathway was found to be a protective factor for developing DAH in the pristane induced murine DAH model [10] .
Whereas, whether monocytes/macrophages contribute to the pathogenesis of immune associated DAH in human remains to be investigated. The purpose of this study was to investigate the role of monocytes/macrophages in the pathogenesis of immune associated DAH in human. In the present study, we found that the peripheral blood classical monocytes were signi cantly increased, and could be strongly recruited to the lungs in the immune associated DAH. Also, we found that the pulmomary immune microenviroment in the immune associated DAH preferred a pro-in ammatory phenotype, and could promote the M 1 polarization. Taken togather, the monocytes/macrophages might be involved in the pathogenesis in the immune associated DAH, and the enhanced M 1 monocytes/macrophage polarization in the lungs might be the key immunological mechanism.

Subjects
The study was conducted in two parts. In the rst part, total 37 children with immune associated DAH were included, in which 16 cases were classi ed as the exacerbation group with acute alveolar hemorrahge (DAH-A group), and 21 cases were classi ed as the remission group without acute alveolar hemorrahge (DAH-R group). And, 18 healthy children were included as the healthy control group (HC group). In the second part, total 24 children with immune associated DAH were included in this study (DAH group). And, 13 children with acute airway foreingn body or mild benign airway stenosis, but without infection were included as the control group (HC group). Acute airway foreingn body was de ned as the time elapsed between aspiration event and foreign body removal within 24 hours. All the subjects with acute airway foreingn body included in the control group were absence of any previous health problems, and all the subjects with mild benign airway stenosis were absence of any other prvious health problems except the airway stenosis. The subjects with immune associated DAH, and those with acute airway foreingn body or mild benign airway stenosis were recruited from the pediatric department of the First A liated Hospital of Guangxi Medical University (Nanning, Guangxi, China). The healthy children were recruited from the preventive care department of the same hospotal. All the subjects were given informed consent prior to their inclusion in the study. The study was approved by the institutional ethics review board of the First A liated Hospital of Guangxi Medical University.
Diagnostic criteria for immune associated DAH and de nition of the disease phase Diagnosis of DAH was based on respiratory symptoms (including dyspnea, hemoptysis and cough), iron de ciency anemia, diffuse pulmonary in ltrates (ground-glass opacities or consolidations) on the chest CT and the presence of hemosiderinladenmacrophages in the BALF or lung tissue specimens [1,3] . The cases satisfying the above DAH diagnostic criteria, and accompanying with at least one of the following conditions: immune disorders which could result in DAH de nitely, pulmonary capillaritis, deposition of immune substances in the basement membranes of alveolar walls, a good response to immunosuppressive therapy (for the cases without a proven immunological origin, and having excluded the non-immune mediated DAH pathogenic factors) were identi ed as immune associated DAH [2-4, 6, 13-16] . A good response to immunosuppressive therapy was de ned as relief of respiratory symptoms (dyspnea, hemoptysis), anemia and reduction of pulmonary in ltrates on the chest image within 4-8 weeks of glucocorticoid (prednisone 1-2mg/kg·d or equivalentdoseofmethylprednisolone) monotherapy or combined with immunosuppressive agents initiation [5,17] . Exacerbation phase with acute alveolar hemorrahge was de ned as presence of anemia and diffuse pulmonary in ltrates on the chest CT [15,[18][19][20] . Remission phase without acute alveolar hemorrahge was de ned as absence of hemoptysis, anemia, or pulmonary in ltrates on the chest CT [15,[18][19][20] .
Bronchoalveolar lavage (BAL) with bronchoscope and BALF processing Via the bronchoscope, BAL was performed by instilling 10-20 ml of saline into the target segment, with a dwell time of 30 seconds, followed by aspiration. For the children with immune associated DAH, the choice of target segment preferentially selected the medial or lateral segment of the right middle lobe, or the left lingular segment, or the segment with the most obvious pulmonary in ltrates on the chest CT. For the controls, the choice of target segment selected the segment with normal appearance on the chest CT, and preferentially selected the medial or lateral segment oftherightmiddlelobe, or the left lingular segment. To reduce the pollution of bronchial secretions, the BALF from the rst lavage was discarded.
To obtained the quali ed BALF, the BALF recovery rate with greater than 40% was required in this study. Then, the BALF was centrifuged at 2000rpm for 10 min. The supernatant was aspirated and stored at -80°C for soluble proteins assays and cell experiment in vitro.

Monocytes isolation and MDMs preparation
Based on the previous studies [21] , peripheral blood was obtained from non-smoking healthy donors. Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA anticoagulant whole blood by FicollPaque (STEMCELL) density gradient centrifugation. PBMCs were suspended in serum-free RPMI 1640 medium (Gibco) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (Solarbio), and were seeded in the 6-well (for qRT-PCR) or 12-well (for FCM) culture plate at a density of 1-1.5×10 7 cells per well for 1-1.5 hours in a humidi ed incubator containing 5% CO 2 at 37℃ to allow monocyte adhesion. Non-adherent cells were removed. The adherent monocytes were further incubated in RPMI 1640 medium (Gibco) supplemented with 10% (v/v) heat-inactivated fetal calf serum (Gibco), 100 U/ml penicillin and 100 mg/ml streptomycin (Solarbio), and M-CSF (20ng/ml, Sigma-Aldrich) for 7 days with media replacement every 2-3 days to obtain MDMs.

RNA puri cation and qRT-PCR analysis
Twenty-four hours post-stimulation, total RNA of the MDMs was extracted using the AxyPrep Multisource Total RNA Miniprep Kit (Axygen) according to the manufacturer's protocol. Up to 500ng of the total RNA was reverse transcribed using the PrimeScript™ RT reagent kit (Takara) according to the manufacturer's instructions. qRT-PCR reactions were performed using the SYBR Premix Ex Taq II (Tli RNaseH Plus) kit (Takara) with speci c primers and run using 7500 Real Time PCR System (Applied Biosystems). All primers were used in the synthesis (Sangon Biotech) and sequences are listed in Table 1. The amount of mRNA was quanti ed using the 2 -∆∆Ct method.

Peripheral blood monocytes subtype in the immune associated DAH
The percentage of classical monocytes was signi cantly increased (P 0.01), and the percentages of intermediate monocytes as well as non-classical monocytes were both signi cantly decreased (both P 0.01) in the DAH-A group, when compared to those in the HC group. The percentage of non-classical monocytes was signi cantly 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 signi cant differences (both P 0.05). Data are detailed in Table 2 and Figure 1.  Table 3. The datas were not normally distributed, and were expressed as median (P 25, P75) and compared using Mann -Whitney U test; The rest datas were expressed as means ± standard deviation, and compared with two -sample t -test; c The levels of IL-5 were both under the detection threshold in the BALF supernatant from the DAH group and that from HC group. d Z value; DAH, immune associated DAH; HC, Controls.

The effect of the supernatant of BALF on the macophage polarization
The mRNA relative expression of IL-1β and IL-6 were signi cantly higher (both P 0.05), and the mRNA relative expression of MRC1 was signi cantly lower (P 0.01) under the stimulation of BALF supernatant from the DAH group, when compared to those under the stimulation of BALF supernatant from the HC group. Whereas, there was no signi cant difference in the mRNA relative expression of TNFα, TGM2 or CD163 between the two groups (P 0.05). The expression of CD86 was signi canly higher (P 0.05), and the expressions of CD163 and CD206 were signi cantly lower (both P 0.05) under the stimulation of BALF supernatant from the DAH group, when compared to those under the stimulation of BALF supernatant from the HC group. Whereas, there was no signi cant difference in the expressions of CD14 and CD80 between the two groups (P 0.05). Data are detailed in Table 4, Table 5 and Figure 2. The datas were expressed as means ± standard deviation; a The datas could not obtain homogeneity of variance, and were compared using Mann -Whitney U test; The rest datas were compared with twosample t -test. b Z value; DAH, Immune associated DAH; HC, Controls.

Discussion
The classi cation of DAH as "immune mediated" and "non-immune mediated" was rst proposed by Tobias Peikert in 2012 [2] . In the Tobias Peikert's classi cation system, the DAH cases accompanying with autoimmune disorders (such as SLE, AAV, Goodpasture disease) or pulmonary capillaritis were de ned as immune mediated DAH [2] . However, this classi cation system had some limitations. In recent, immune disorders caused by gene mutation, such as COPA syndrome and STING-associated vasculopathy with onset in infancy (SAVI) were found to develop DAH [22,23] . SpC dysfunction with the I73T mutation was also found to develop autoimmunity accompanying with DAH [24] . Besides that, the DAH cases who had excluded the non-immune mediated pathogenic factors should be highly suspected for immune mediated either, in which some were found to had pulmonary capillaritis [25] or deposition of immune substances in the basement membranes of alveolar walls in the lung biopsy [26] , some were found to be the autoimmune diaseases or develop autoantibodies during the follow-up [27,28] , and some cases were the idiopathic pulmonary hemosiderosis (IPH). IPH was classi ed as the non-immune mediated DAH in the Tobias Peikert's classi cation system, as there was no pulmonary capillaritis or autoantibody in the IPH. However, a considerable number of relevant studies have found that IPH presented a good response to immunosuppressive therapy, which indicated that IPH was also immune mediated [1,3,6,29] . Refering to the Tobias Peikert's classi cation system and the recent research advances mentioned above, we reconceptualized the immune mediated DAH. To avoid confusion with Tobias Peikert's classi cation system, the DAH cases included in this study were named "immune associated DAH" instead of "immune mediated DAH".
Recently, the studies in the pristane induced murine DAH models have demonstrated that the bone marrow derived monocytes/macrophages in the lungs were the critical factors for developing alveolar hemorrhage, and the enhances M 1 polarization of the monocytes/macrophages was the key immunological mechanism. Besides the local in ammation in the lungs, systemic in ammation also participated in the pathogenesis of alveolar hemorrhage, as the monocytes from the bone marrow in the pristane induced murine DAH model presented lower expression of IL-10R. Whereas, whether monocytes/macrophages contribute to the pathogenesis of immune associated DAH in human remains to be investigated. In view of this, we conducted this study to investigate the role of monocytes/macrophages in the pathogenesis of immune associated DAH in human.
According to the expression of CD14 and CD16, the peripheral blood monocytes were classi ed as classical (CD14 ++ /CD16 − ), intermediate (CD14 ++ /CD16 + ) and non-classical (CD14 + /CD16 ++ ) monocytes [30] . Classical monocytes could migrate into the tissue, and initiate or regulate the in ammatory response [30,31] . Whereas, non-classical monocytes usually displayed a distinct motility and crawling pattern along the vasculature to survey the luminal side of vascular endothelium and maintain the vascular homeostasis [32] . Intermediate monocytes were considered to be the transition state between the classical and non-classical monocytes [31] . In this study, we found that the peripheral blood classical monocytes were signi cantly increased in the children with immune associated DAH, which demonstrated that the peripheral blood monocytes in the immune associated DAH preferred to migrate into the tissue and to be a pro-in ammation phenotype. Also, we found that the abnormalities of the peripheral blood monocytes subtype presented a tendency to normalization with the disease remission, which further suggested that the peripheral blood monocytes participated in the pathogenesis of immune associated DAH.
BALF supernatant contained a large number of soluble immune mediators, and could reprsent the pulmonary immune microenvironment [33] . In this study, we found that the levels of MCP-1, IL-6 and IL-8 were all signi cantly increased in the BALF supernatant from the children with immune associated DAH, in which the level MCP-1 rose most sharply with nearly 30 folds increase. In viro cell experiments, we found that BALF supertanant from the children with immune associated DAH signi cantly enhanced the mRNA relative expression of IL-1β as well as IL-6 and the expression of CD86, whereas reduced the mRNA relative expression of MRC1 and the expression of CD163 as well as CD206. According to the literature, MCP-1, IL-6 and IL-8 were all pro-in ammatory mediators [34][35][36][37] , and MCP-1 was a potent chemoattractant for monocytes [38] . Enhanced trancription of IL-1β as well as IL-6 and expression of CD86, accompanying with reduced trancription of MRC1 and expression of CD163 as well as CD206 were the markers of M 1 polarization [39][40][41][42] . Thus, our results indicated that the pulmomary immune microenviroment in the immune associated DAH preferred a pro-in ammatory phenotype, which could strongly favored the recruitment of the peripheral blood monocytes, and promote the M 1 polarization. It was noteworthy that this result echoed with our previous nding in which the peripheral blood monocytes in the immune associated DAH preferred to migrate into the tissue and to be a pro-in ammation phenotype. Taken togather, the monocytes/macrophages were involved in pathogenesis in the immune associated DAH, and the enhanced M 1 monocytes/macrophage polarization in the lungs might play a role in the pathogenesis of immune associated DAH.
Type 2 immunity was characterized by the production of IL4, IL5, IL9 and IL13, and was mainy involved in the allergic in ammation, tissue repair after injury and brosis [43] . In this study, we found that the level of IL-9 was signi cantly redued, the level of IL-13 revealed no signi cant difference, and the level of IL-5 was under the detection threshold in the BALF supernatant from the children with immune associated DAH. The result suggested that type 2 immunity was in an inactive state in the pulmonary immune microenviroment of the immune associated DAH. And, it also indicated that type 2 immunity might not participate in the pathogenesis of alveolar hemorrahge. Whereas, recurrent episodes of DAH usually resulted in interstitial brosis [1] . Given the important role of type 2 immunity in the brosis, type 2 immunity might participate in the alveolar wall repair process following the damage in the immune associated DAH. A further study on the relation between the type 2 immunity and the interstital brosis in the immune associated DAH should be explored.
Our study had some limitions. Firstly, we didn't enroll the healthy children to perform bronchoalveolar lavage due to ethical restrictions. Whereas, to reduce the selection bias of controls, we had setted a strict inclusion criteria, and chosen the segment with normal appearance on the chest CT to perform bronchoalveolar lavage. Secondly, we didn't perform strati ed analysis by disease staging in the BALF supernatant part of this study, as the majority of the cases included were in the exacerbation stage.
Finally, the number of subjects included in this study was small, and should be expanded in the future.

Conclusions
The monocytes/macrophages might be involved in the pathogenesis in the immune associated DAH, and the enhanced M 1 monocytes/macrophage polarization in the lungs might be the key immunological mechanism.

Declarations
Ethics approval and consent to participate The study was approved by the institutional ethics review board of the First A liated Hospital of Guangxi Medical University. A written informed consent was obtained from each study subject or from his/her guardian.

Consent for publication
Not applicable.

Availability of data and materials
We would like to provide the raw data to support the information presented in this publication.

Competing interests
The authors have no con icts of interest relevant to this article to disclose.

Funding
This study were supported by the grant (principal investigator: Guangmin Nong) from the the First A liated Hospital of Guangxi Medical University (YYZS2020014) and the grant (principal investigator: Qing Wei) from Guangxi Medical University (GXMUYSF202114).
Authors' contributions Q Wei and X Chen took part in designing the study, performed the experiments, carried out the acquisition, analysis as well as the interpretation of the datas, and drafted the manuscript. GM Nong conceptualized and designed the whole study, supervised and instructed data collection and analysis, reviewed and revised the manuscript. J Liu and Y Li contributed to the overall design of the study, perfomed the bronchoalveolar lavage with bronchoscope, and critically reviewed the manuscript. All authors have read and approved the nal manuscript.

Figure 1
Representative ow cytometric pro les of monocyte subtypes from the peripheral blood Representative ow cytometric pro les of CD14, CD80, CD86, CD163 and CD206 expression on the surface of MDMs stimulated by the BALF supernatant