The Mechanism In HLA-? Antibody Activated Endothelial Cell-induced Differentiation Of Human Peripheral Monocytes Into M2a/c-like Macrophages

BackgroundThe donor specific anti-HLA antibody(Ab)(HLA DSA)mediated rejection(ABMR)is still the major cause of the long-term graft failure in kidney transplantation.However,the underlying mechanism is not well known,and the effective treatments is also lacking.In a long time after the first report of ABMR in 1990 s,HLA Abs were thought to induce the graft injuries mainly by activating complement system until the C4d-ABMR were reported in the end of the last decade.The findings of ours and other groups suggest that HLA Ab binding to endothelial cells(ECs)may cause graft injury by multiple concurrent mechanisms,including triggering classical complement cascade,endothelial activation,and recruiting effector cells by eliciting the expression of P-selectin and aggregation of ICAM-1 on EC surface,of which the EC activation was defined as a necessary requirement in the criteria for diagnosis of C4d-ABMR in Banff 2013.The other findings were supported by accumulating clinical and animal evidences that almost all of the allografts undergoing AMR were found with macrophages and/or leukocytes infiltration.Moreover,it was found that the expression of several macrophage-related genes were upregulated in the kidney biopsy specimens of patients with ABMR.Further studies indicated that these graft-infiltrating macrophages were correlated with the graft fibrosis and worse outcomes,and mainly presented a M2 phenotype,indicating an important role of these macrophages in the ABMR injuries.However,it is far from known that whether these graft-infiltrating M2-like macrophages were associated with the EC activation by HLA Ab.Macrophages represent a heterogeneous group of cells with distinct functions.With respect to the concept of T cell polarization to “Th1” and “Th2” in adaptive immune response,macrophages were defined as “classically activated”,also termed as M1,presenting pro-inflammatory functions,and “alternatively activated” M2 subtype with anti-inflammatory characteristics.M2 macrophages can be further divided into M2a(generally referred to as alternatively activated macrophages with function of anti-inflammatory and wound healing),M2b(regarded as having anti-inflammatory and increased antigen presenting functions)and M2c(referred to as deactivated macrophages with roles in tissue repair and remodeling).Although M2 macrophages,especially M2 a and M2 c were considered to have tissue reparative characteristics,sustained activity may lead to fibrosis by continuously producing various wound healing growth factors.Traditionally,it is known that the monocyte differentiation doesn't start until they entire the peripheral tissues.However,recent transcriptional studies revealed that the monocyte differentiation was already initiated as early as in the firm adhesion and transmigration stages.Then the question here is whether and how the HLA DSA modify the monocyte differentiation when enhancing the recruitment? The current study will address on these questions which may help us understand more of the mechanism of ABMR as well as giving some useful cues to the clinical treatment.Part ?: The effect of HLA-? antibody activated EC on the phenotypical changes of the human peripheral monocytesAim: To investigate the effect of HLA-? Ab activated EC on the phenotypical changes of the human peripheral monocytes and the underlying molecular mechanism.Methods: We co-cultured the freshly isolated peripheral monocytes from healthy donors with the HLA-? Ab stimulated EC in an in vitro co-culture model for 5 days.Cells in the lower chamber were collected and assessed for surface markers expression by flow cytometry.The monocytes that stimulated with IFN-?,IL-4,h Ig G+LPS,IL-10 and IL-4+GM-CSF+LPS served as positive controls for M1,M2 a,M2b,M2 c and mature dendritic cell(m DC),respectively.Luminex Multiplex assays were performed to detect the cytokine production in the supernatants from co-cultures on day 2 to determine if it was the drive force for the monocyte differentiation.HLA-? F(ab')2 antibodies were produced from HLA-? Abs using Ides,and used to determine the role of Fc fragment in the differentiation process.PSGL-1 fusion protein(r PSGL-1-Ig)and anti-ICAM-1 Ab were used to block the P-selectin and ICAM-1 on the EC surface to determine the role of these two adhesion molecules in the monocyte differentiation.The transmigration ability of the monocytes in different conditions were also evaluated.Results: The in vitro co-culture system with 3?m-pore transwell inserts met the requirements of the current study.After 5 day co-culture,all of the cells in the lower chamber were mononuclears.Unstimulated ECs drove the monocytes into M2a-like macrophages with increased expression of CD206,M2 a marker,(all p < 0.05),which can be further enhanced by the stimulation of HLA-? Ab with increased expression of CD206(p < 0.01),CD163,M2 c marker,(p < 0.05)and CD68,M2 a and c marker,(p < 0.05),indicating that HLA-? Ab activated EC induced the differentiation of monocytes into M2a/c-like macrophages.However,there was no difference in the cytokine production between the supernatants from unstimulated and HLA-? Ab stimulated ECs(all p > 0.05),suggesting the HLA-? Ab cannot induce the EC to produce cytokines which may drive the monocyte differentiation.Comparing with HLA-? Ab,the HLA-? F(ab')2 can only enhance the expression of CD206(p < 0.01),but not CD163(p > 0.05),implying that the expression of CD163,or the M2 c phenotype,was dependent on the HLA-? Ab Fc fragment.Furthermore,blocking either P-selectin or ICAM-1 on the EC can abolish both increased expressions of CD206(all p < 0.05)and CD163(all p < 0.05),suggesting that the M2 a and M2 c phenotypes were both dependent on the adhesion molecules P-selectin and ICAM-1 which were expressed by HLA-? Ab activated EC.Additionally,the transmigration ability of the monocytes was also increased by the HLA-? antibodies stimulation,which,however,were fully inhibited by the blockages of either P-selectin or ICAM-1.Conclusions: EC can drive the monocyte to differentiate into mainly M2a-like macrophages,which can be further enhanced into M2a/c-like macrophages by HLA-? Ab stimulation.This enhancement was dependent on the P-selectin and ICAM-1 expressed on the HLA-? Ab activated EC,and the antibody Fc fragment,but not the cytokines produced by EC.The inhibitors of P-selectin and ICAM-1,and the antibody Fc fragment modulation by therapeutic enzymes may dampen the HLA antibody mediated graft injury.Part ?: Identification of the functions of the macrophages that induced by HLA-? antibody activated ECAim: To determine the functions of the HLA-? Ab induced macrophages by analyzing the profiles of cytokine production and immunology-related gene expression.Methods: Luminex Multiplex assays were performed to detect the cytokine production in the supernatants from unstimulated or HLA-? antibodies stimulated co-cultures on day 4.The transmigrated cells in the lower chamber were collected on day 5,and the expression of 579 immunology-related genes were assessed by nano String.The data of cytokines and gene expressions were then compared with those in positive controls.Results: Comparing with the unstimulated co-cultures,several cytokines/chemokines,including IL-6,IL-8,IL-10 and MCP-1,were significantly increased in the supernatants from the HLA-? Ab stimulated co-cultures(p < 0.05,< 0.001,< 0.01 and < 0.01,respectively).GRO,MIP-1b,Eotaxin and MDC were also increased,although the differences were not significant(all p > 0.05).Of these upregulated cytokines/chemokines,MDC,MIP-1b and MCP-1 had the same trend as that in M2 a and M2 c positive controls.IL-6 was reported as having both pro-and anti-inflammatory activities and leading M2 polarization.IL-10 was known as a cytokine marker of M2 macrophages.These data suggest that the HLA-? Ab induced macrophages had the cytokine profile of M2 macrophage.However,there was no difference between the stimulation of HLA-? Ab and HLA-? F(ab')2 stimulation(all p > 0.05).At transcriptional level,61 genes were differentially expressed among unstimulated,HLA-? Ab and HLA-? F(ab')2 stimulated macrophages using a criteria of p < 0.01 and |fold-change | > 1.5.Functionally,most of these differentially expressed genes(DEGs)were reportedly mainly expressed by or strongly related to M2 macrophages,including C1 QA,C1QB,CD59,SERPING1,CD209,CR1,EGR1,JAK3,NFKBIA,S1PR1,TNFAIP3,CCL24,HAMP,HFE and TFRC.In the six downregulated genes,three were M1-related,i.e.C3?CLEC5A and KLRC4,indicate a mainly M2 profile of these macrophages.additionally,several adhesion molecules(ICAM-1,ICAM-4 and SELPLG),phagocytosis-related molecules(CD209,CR1,MRC1),and costimulatory molecules(CD274,CD40,CD80 and CD83)were also increased,suggesting macrophage activation and enhanced antigen-presenting function.However,most of these genes were expressed lower in those induced by HLA-? F(ab')2.Notably,several M1-related genes,including C2,PTGS2,SOCS3,CCL3,CCL4,CCRL2,IL1 A and CXCL1,were also upregulated,and more highly expressed in HLA-? Ab induced macropahges.Actually,some of them,including PTGS2,CCL3,CCL4 and CXCL1,were even decreased the macrophages induced by HLA-? F(ab')2.These data indicated that the HLA-? Ab induced macrophages exhibited a stronger M2 profile than those stimulated by HLA-? F(ab')2,and,meanwhile,had some pro-inflammatory activities.By comparing with the positive controls,these HLA-? Ab induced macrophage shared the most of the DEGs with M2 a and M2 c positive controls,and also some with M2 b.Conclusions: The macrophages induced by HLA-? antibodies activated EC had the cytokine production profile of M2 macrophage.The HLA-? Ab induced M2a/c-like macrophages exhibited the typical gene expression profile of M2 a and M2 c macrophages,which had a “dual-face” of anti-and pro-inflammatory activities,while the HLA-? F(ab')2 induced M2a-like macrophages mainly present anti-inflammatory properties,which were much lower than that in HLA-? Ab induced macrophages.These findings indicate a higher pathogenicity of the HLA-? Ab than F(ab')2.Part ?: The triggering signaling pathway in the HLA-? antibody activated EC induced macrophage differentiationAim: To reveal the triggering signaling pathway by which the HLA-? Ab activated EC drove the monocyte differentiation into macrophage.Methods: Phosflow was used to assess the phosphorylation of p AktT308 and p ERK1/2T202/Y204 in the monocytes that co-cultured with HLA-? antibodies activated EC to determine the potentially involved signaling pathways.Blockage of the P-selectin and ICAM-1 on the EC surface using r PSGL-1-Ig and anti-ICAM-1 Ab were performed to investigated the downstream signaling following the EC-monocyte interaction.anti-PSGL-1 Ab,and anti-CD18,CD11 a and CD11 b antibodies were used to block the PSGL-1,CD18,CD11 a and CD11 b on the monocytes to confirm the roles of P-selectin and ICAM-1 in the induction of monocyte differentiation,and determine the receptors that ligated to the ICAM-1.The anti-CD16,CD32 and CD64 antibodies were used to pretreat the monocytes to determine which subtype of Fc?Rs interacted with the HLA-? Ab Fc fragment.Results: Co-culture with the HLA-? Ab and HLA-? F(ab')2 activated EC significantly increased the numbers of monocytes with high phosphorylation of p ERK1/2T202/Y204,comparing with those co-cultured with unstimulated EC(p < 0.01 and < 0.05,respectively),although it was much lower in HLA-? F(ab')2 stimulation(p < 0.05),indicating both the activated EC and the HLA-? Ab Fc fragment can trigger the intra-monocyte ERK signaling.On the contrary,the numbers of the monocytes with high phosphorylation of p AktT308 were significantly decreased in HLA-? Ab stimulated macrophages(p < 0.0001),while no change was found in those stimulated by HLA-? F(ab')2(p > 0.05).In the phenotype assays,U0126,a MEK inhibitor,fully abolished the expression of CD206 and CD163 that increased by HLA-? antibodies,indicating the HLA-? Ab induced monocyte differentiation was dependent on ERK pathway.Blockage of P-selectin or ICAM-1 on EC fully abolished the HLA-? F(ab')2 elicited intra-monocyte ERK phosphorylation(p < 0.001 and < 0.05,respectively)and partly inhibited those induced by HLA-? Ab(p < 0.0001 and < 0.001,respectively).The finding was confirmed by blocking PSGL-1,or CD18 or CD11a(all p < 0.05),which also indicates the LFA-1 was the major receptor for ICAM-1 in the setting of HLA-? Ab induced monocyte differentiation.Additionally,blocking CD64 on monocytes can mostly inhibit the antibody Fc fragment elicited ERK phosphorylation,while blocking both of CD64 and CD32 can fully abolish this signaling,suggesting the major role of Fc?R I and a supplementary role of Fc?R II.Conclusions: The induction of monocyte differentiation by HLA-? Ab activated EC was dependent on the intra-monocyte ERK signaling activation through P-selectin/PSGL-1,ICAM-1/LFA-1 and Fc/Fc?R I and II ligation.Inhibition of MEK/ERK pathway can be a potential way to ameliorate the graft injury in AMR.