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BACKGROUND Airway remodeling is a significant contributor to impaired lung function in chronic allergic airway disease. Currently,no therapy exists that is capable of targeting these structural changes and the consequent loss of function. In the context of chronic allergic inflammation,pericytes have been shown to uncouple from the pulmonary microvasculature,migrate to areas of inflammation,and significantly contribute to airway wall remodeling and lung dysfunction. This study aimed to elucidate the mechanism by which pulmonary pericytes accumulate in the airway wall in a model of chronic allergic airway inflammation. METHODS Mice were subjected to a protocol of chronic airway inflammation driven by the common environmental aeroallergen house dust mite. Phenotypic changes to lung pericytes were assessed by flow cytometry and immunostaining,and the functional capacity of these cells was evaluated using in vitro migration assays. The molecular mechanisms driving these processes were targeted pharmacologically in vivo and in vitro. RESULTS Pericytes demonstrated increased CXCR4 expression in response to chronic allergic inflammation and migrated more readily to its cognate chemokine,CXCL12. This increase in migratory capacity was accompanied by pericyte accumulation in the airway wall,increased smooth muscle thickness,and symptoms of respiratory distress. Pericyte uncoupling from pulmonary vessels and subsequent migration to the airway wall were abrogated following topical treatment with the CXCL12 neutraligand LIT-927. CONCLUSION These results provide new insight into the role of the CXCL12/CXCR4 signaling axis in promoting pulmonary pericyte accumulation and airway remodeling and validate a novel target to address tissue remodeling associated with chronic inflammation. View Publication

N6-methyladenosine (m6A) modification is involved in diverse immunoregulation,while the relationship between m6A modification and immune tolerance post kidney transplantation remains unclear. Expression of Wilms tumor 1-associating protein (WTAP),an m6A writer,was firstly detected in tolerant kidney transplant recipients (TOL). Then the role of WTAP on regulatory T (Treg) cell differentiation and function in CD4+ T cells from kidney transplant recipients with immune rejection (IR) was investigated. The potential target of WTAP and effect of WTAP on immune tolerance in vivo were subsequently verified. WTAP was upregulated in CD4+ T cells of TOL and positively correlated with Treg cell proportion. In vitro,WTAP overexpression promoted Treg cell differentiation and enhanced Treg cell-mediated suppression toward na?ve T cells. Forkhead box other 1 (Foxo1) was identified as a target of WTAP. WTAP enhanced m6A modification of Foxo1 mRNA in coding sequence (CDS) region,leading to up-regulation of Foxo1. Overexpression of m6A demethylase removed the effect of WTAP overexpression,while Foxo1 overexpression reversed these effects. WTAP overexpression alleviated allograft rejection in model mice,as evidenced by reduced inflammatory response and increased Treg population. Our study suggests that WTAP plays a positive role in induction of immune tolerance post kidney transplant by promoting Treg cell differentiation and function. leading to up-regulation of Foxo1. Overexpression of m6A demethylase removed the effect of WTAP overexpression while Foxo1 overexpression reversed these effects. WTAP overexpression alleviated allograft rejection in model mice as evidenced by reduced inflammatory response and increased Treg population. Our study suggests that WTAP plays a positive role in induction of immune tolerance post kidney transplant by promoting Treg cell differentiation and function." View Publication

Myeloid-derived suppressor cells (MDSCs) are known to promote tumor growth in part by their immunosuppressive activities and their angiogenesis support. It has been shown that Bv8 blockade inhibits the recruitment of MDSCs to tumors,thereby delaying tumor relapse associated with resistance to antiangiogenic therapy. However,the impact of Bv8 blockade on tumors resistant to the new immunotherapy drugs based on the blockade of immune checkpoints has not been investigated. Here,we demonstrate that granulocytic-MDSCs (G-MDSCs) are enriched in anti-PD1 resistant tumors. Importantly,resistance to anti-PD1 monotherapy is reversed upon switching to a combined regimen comprised of anti-Bv8 and anti-PD1 antibodies. This effect is associated with a decreased level of G-MDSCs and enrichment of active cytotoxic T cells in tumors. The blockade of anti-Bv8 has shown efficacy also in hyperprogressive phenotype of anti-PD1-treated tumors. In vitro,anti-Bv8 antibodies directly inhibit MDSC-mediated immunosuppression,as evidenced by enhanced tumor cell killing activity of cytotoxic T cells. Lastly,we show that anti-Bv8-treated MDSCs secrete proteins associated with effector immune cell function and T cell activity. Overall,we demonstrate that Bv8 blockade inhibits the immunosuppressive function of MDSCs,thereby enhancing anti-tumor activity of cytotoxic T cells and sensitizing anti-PD1 resistant tumors. Our findings suggest that combining Bv8 blockade with anti-PD1 therapy can be used as a strategy for overcoming therapy resistance. View Publication

Bone disease represents a major cause of morbidity and mortality in Multiple Myeloma (MM); primarily driven by osteoclasts whose differentiation is dependent on expression of RANKL by MM cells. Notably,costimulation by ITAM containing receptors (i.e.,Fc$\gamma$R) can also play a crucial role in osteoclast differentiation. Modeling the pathology of the bone marrow microenvironment with an ex vivo culture system of primary human multiple myeloma cells,we herein demonstrate that Fc$\gamma$R-mediated signaling,via staphylococcal protein A (SpA) IgG immune-complexes,can act as a critical negative regulator of MM-driven osteoclast differentiation. Interrogation of the mode-of-action revealed that Fc$\gamma$R-mediated signaling causes epigenetic modulation of chromosomal 3D architecture at the RANK promoter; with altered spatial orientation of a proximal super enhancer. Combined this leads to substantial down-regulation of RANK at a transcript,protein,and functional level. These observations shed light on a novel mechanism regulating RANK expression and provide a rationale for targeting Fc$\gamma$R-signaling for the amelioration of osteolytic bone pathology in disease. View Publication

The past two decades have witnessed significant strides in leukemia therapies through approval of therapeutic inhibitors targeting oncogene-driving dysregulated tyrosine kinase activities and key epigenetic and apoptosis regulators. Although these drugs have brought about complete remission in the majority of patients,many patients face relapse or have refractory disease. The main factor contributing to relapse is the presence of a small subpopulation of dormant drug-resistant leukemia cells that possess stem cell features (termed as leukemia stem cells or LSCs). Thus,overcoming drug resistance and targeting LSCs remain major challenges for curative treatment of human leukemia. Chronic myeloid leukemia (CML) is a good example,with rare,propagating LSCs and drug-resistant cells that cannot be eradicated by BCR-ABL-directed tyrosine kinase inhibitor (TKI) monotherapy and that are responsible for disease relapse/progression. Therefore,it is imperative to identify key players in regulating BCR-ABL1-dependent and independent drug-resistance mechanisms,and their key pathways,so that CML LSCs can be selectively targeted or sensitized to TKIs. Here,we describe several easily adaptable gene knockdown approaches in CD34+ CML stem/progenitor cells that can be used to investigate the biological properties of LSCs and molecular effects of genes of interest (GOI),which can be further explored as therapeutic modalities against LSCs in the context of human leukemia. View Publication

Oncolytic viruses have been emerging as a promising therapeutic option for cancer patients,including lung cancer. Orf virus (ORFV),a DNA parapoxvirus,can infect its natural ungulate hosts and transmit into humans. Moreover,the ORFV has advantages of low toxicity,high targeted,self-amplification and can induce potent Th1-like immunity. This study explored the therapeutic potential of ORFV infection for human lung cancer therapy and investigated the molecular mechanisms. We used a previously described ORFV NA1/11 strain and tested the oncolysis of ORFV NA1/11 in two lines of lung cancer cells in vitro and in vivo. Treatment of both cell lines with ORFV NA1/11 resulted in a decrease in cell viability by inducing cell cycle arrest in G2/M phase,suppressing cyclin B1 expression and increasing their apoptosis in a caspase-dependent manner. The ORFV NA1/11-infected lung cancer cells were highly immunogenic. Evidently,ORFV NA1/11 infection of lung cancer cells induced oncolysis of tumor cells to release danger-associated molecular patterns,and promoted dendritic cell maturation,and CD8 T cell infiltration in the tumors by enhancing CXCL16 secretion. These findings may help to understand the molecular mechanisms of ORFV oncolysis and aid in the development of novel therapies for lung cancer. View Publication

Dysregulated neutrophilic inflammation can be highly destructive in chronic inflammatory diseases due to prolonged neutrophil lifespan and continual release of histotoxic mediators in inflamed tissues. Therapeutic induction of neutrophil apoptosis,an immunologically silent form of cell death,may be beneficial in these diseases,provided that the apoptotic neutrophils are efficiently cleared from the tissue. Previous research in our group identified ErbB inhibitors as able to induce neutrophil apoptosis and reduce neutrophilic inflammation both in vitro and in vivo. Here,we extend that work using a clinical ErbB inhibitor,neratinib,which has the potential to be repurposed in inflammatory diseases. We show that neratinib reduces neutrophilic migration o an inflammatory site in zebrafish larvae. Neratinib upregulates efferocytosis and reduces the number of persisting neutrophil corpses in mouse models of acute,but not chronic,lung injury,suggesting that the drug may have therapeutic benefits in acute inflammatory settings. Phosphoproteomic analysis of human neutrophils shows that neratinib modifies the phosphorylation of proteins regulating apoptosis,migration,and efferocytosis. This work identifies a potential mechanism for neratinib in treating acute lung inflammation by upregulating the clearance of dead neutrophils and,through examination of the neutrophil phosphoproteome,provides important insights into the mechanisms by which this may be occurring. View Publication

Coronary heart disease (CHD) is a leading cause of death globally,while its current management is limited to reducing the myocardial infarction area without actually replacing dead cardiomyocytes. Direct cell reprogramming is a method of cellular cardiomyoplasty which aims for myocardial tissue regeneration,and CD34+ cells are one of the potential sources due to their shared embryonic origin with cardiomyocytes. However,the isolation and culture of non-adherent CD34+ cells is crucial to obtain adequate cells for high-efficiency genetic modification. This study aimed to investigate the optimal method for isolation and culture of CD34+ peripheral blood cells using certain culture media. A peripheral blood sample was obtained from a healthy subject and underwent pre-enrichment,isolation,and expansion. The culture was subsequently observed for their viability,adherence,and confluence. Day 0 observation of the culture showed a healthy CD34+ cell with a round cell shape,without any adherent cells present yet. Day 4 of observation showed that CD34+ cells within the blood plasma medium became adherent,indicated by their transformations into spindle or oval morphologies. Meanwhile,CD34+ cells in vitronectin and fibronectin media showed no adherent cells and many of them died. Day 7 observation revealed more adherent CD34+ cells in blood plasma medium,and which had 75% of confluence. In conclusion,the CD34+ cells that were isolated using a combination of density and magnetic methods may be viable and adequately adhere in culture using blood plasma medium,but not in cultures using fibronectin and vitronectin. View Publication

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease without effective curative therapy. Recent evidence shows increased circulating myeloid-derived suppressor cells (MDSCs) in cancer,inflammation,and fibrosis,with some of these cells expressing B7H3. We sought to investigate the role of MDSCs in IPF and its potential mediation via B7H3. Here we prospectively collected peripheral blood samples from IPF patients to analyze for circulating MDSCs and B7H3 expression to assess their clinical significance and potential impact on co-cultured lung fibroblasts and T-cell activation. In parallel,we assess MDSC recruitment and potential B7H3 dependence in a mouse model of pulmonary fibrosis. Expansion of MDSCs in IPF patients correlated with disease severity. Co-culture of soluble B7H3 (sB7H3)-treated mouse monocytic MDSCs (M-MDSCs),but not granulocytic MDSCs (G-MDSCs),activated lung fibroblasts and myofibroblast differentiation. Additionally,sB7H3 significantly enhanced MDSC suppression of T-cell proliferation. Activated M-MDSCs displayed elevated TGF$\beta$ and Arg1 expression relative to that in G-MDSCs. Treatment with anti-B7H3 antibodies inhibited bone marrow-derived MDSC recruitment into the bleomycin-injured lung,accompanied by reduced expression of inflammation and fibrosis markers. Selective telomerase reverse transcriptase (TERT) deficiency in myeloid cells also diminished MDSC recruitment associated with the reduced plasma level of sB7H3,lung recruitment of c-Kit+ hematopoietic progenitors,myofibroblast differentiation,and fibrosis. Lung single-cell RNA sequencing (scRNA-seq) revealed fibroblasts as a predominant potential source of sB7H3,and indeed the conditioned medium from activated mouse lung fibroblasts had a chemotactic effect on bone marrow (BM)-MDSC,which was abolished by B7H3 blocking antibody. Thus,in addition to their immunosuppressive activity,TERT and B7H3-dependent MDSC expansion/recruitment from BM could play a paracrine role to activate myofibroblast differentiation during pulmonary fibrosis with potential significance for disease progression mediated by sB7H3. View Publication

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare yet serious adverse effect of the adenoviral vector vaccines ChAdOx1 nCoV-19 (AstraZeneca) and Ad26.COV2.S (Janssen) against COVID-19. The mechanisms involved in clot formation and thrombocytopenia in VITT are yet to be fully determined. Here we show neutrophils undergoing NETosis and confirm expression markers of NETs in VITT patients. VITT antibodies directly stimulate neutrophils to release NETs and induce thrombus formation containing abundant platelets,neutrophils,fibrin,extracellular DNA and citrullinated histone H3 in a flow microfluidics system and in vivo. Inhibition of NETosis prevents VITT-induced thrombosis in mice but not thrombocytopenia. In contrast,in vivo blockage of Fc$\gamma$RIIa abrogates both thrombosis and thrombocytopenia suggesting these are distinct processes. Our findings indicate that anti-PF4 antibodies activate blood cells via Fc$\gamma$RIIa and are responsible for thrombosis and thrombocytopenia in VITT. Future development of NETosis and Fc$\gamma$RIIa inhibitors are needed to treat VITT and similar immune thrombotic thrombocytopenia conditions more effectively,leading to better patient outcomes. View Publication

BACKGROUND Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has become an increasingly vital tool for modifying gene expression in a variety of cell types. Lentiviral transduction and electroporation are the two main approaches used to deliver CRISPR/Cas9 into cells. However,the application of CRISPR/Cas9 in primary hematopoietic cells has been limited due to either low transduction efficiency in terms of viral-based delivery or difficult selection and enrichment of transfected and edited cells with respect to electroporation of CRISPR/Cas9 ribonucleoprotein (RNP). METHODS In this study in vitro transcription was used to synthesize the guide RNA (gRNA),and plasmid pL-CRISPR.EFS.GFP was used as its DNA template. Then the in vitro transcribed gRNA was labeled with pCp-Cy5 via T4 ligase before incubating with Cas9 protein. Furthermore,CRISPR/Cas9 RNP was electroporated into primary CD34+ cells isolated from cord blood,and cell survival rate and transfection efficiency were calculated and compared to that of lentiviral transduction. RESULTS Here,we show that electroporation of CRISPR/Cas9 RNP resulted in higher cell viability compared to electroporation of CRISPR/Cas9 all-in-one plasmid,providing important findings for further studies in hematology via CRISPR/Cas9 technology. Moreover,we established a method for labeling in vitro-transcribed gRNA with fluorophore and the sorted fluorescent cells displayed higher knockout efficiency than nonsorted transfected cells. CONCLUSIONS Electroporation of fluorescence labeled CRISPR/Cas9 RNP is a perspective approach of gene editing. Our study provides an efficient and time-saving approach for genome-editing in hematopoietic cells. View Publication

Ov-ASP-1 (rASP-1),a parasite-derived protein secreted by the helminth Onchocerca volvulus,is an adjuvant which enhances the potency of the influenza trivalent vaccine (IIV3),even when used with 40-fold less IIV3. This study is aimed to provide a deeper insight into the molecular networks that underline the adjuvanticity of rASP-1. Here we show that rASP-1 stimulates mouse CD11c+ bone marrow-derived dendritic (BMDCs) to secrete elevated levels of IL-12p40,TNF-?,IP-10 and IFN-? in a TRIF-dependent but MyD88-independent manner. rASP-1-activated BMDCs promoted the differentiation of na?ve CD4+ T cells into Th1 cells (IFN-?+) that was TRIF- and type I interferon receptor (IFNAR)-dependent,and into Tfh-like cells (IL21+) and Tfh1 (IFN-?+ IL21+) that were TRIF-,MyD88- and IFNAR-dependent. rASP-1-activated BMDCs promoted the differentiation of na?ve CD4+ T cells into Th17 (IL-17+) cells only when the MyD88 pathway was inhibited. Importantly,rASP-1-activated human blood cDCs expressed upregulated genes that are associated with DC maturation,type I IFN and type II IFN signaling,as well as TLR4-TRIF dependent signaling. These activated cDCs promoted the differentiation of na?ve human CD4+ T cells into Th1,Tfh-like and Th17 cells. Our data thus confirms that the rASP-1 is a potent innate adjuvant that polarizes the adaptive T cell responses to Th1/Tfh1 in both mouse and human DCs. Notably,the rASP-1-adjuvanted IIV3 vaccine elicited protection of mice from a lethal H1N1 infection that is also dependent on the TLR4-TRIF axis and IFNAR signaling pathway,as well as on its ability to induce anti-IIV3 antibody production. View Publication