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Background: Differences in DNA methylation have been reported in B and T lymphocyte populations,including CD4+ T cells,isolated from rheumatoid arthritis (RA) patients when compared to healthy controls. CD4+ T cells are a heterogeneous cell type with subpopulations displaying distinct DNA methylation patterns. In this study,we investigated DNA methylation using reduced representation bisulfite sequencing in two CD4+ T cell populations (CD4+ memory and na{\{i}}ve cells) in three groups: newly diagnosed disease modifying antirheumatic drugs (DMARD) na{\"{i}}ve RA patients (N = 11) methotrexate (MTX) treated RA patients (N = 18) and healthy controls (N = 9) matched for age gender and smoking status. Results: Analyses of these data revealed significantly more differentially methylated positions (DMPs) in CD4+ memory than in CD4+ na{\""{i}}ve T cells (904 vs. 19 DMPs) in RA patients compared to controls. The majority of DMPs (72{\%}) identified in newly diagnosed and DMARD na{\""{i}}ve RA patients with active disease showed increased DNA methylation (39 DMPs) whereas most DMPs (80{\%}) identified in the MTX treated RA patients in remission displayed decreased DNA methylation (694 DMPs). Interestingly we also found that about one third of the 101 known RA risk loci overlapped (±500 kb) with the DMPs. Notably introns of the UBASH3A gene harbor both the lead RA risk SNP and two DMPs in CD4+ memory T cells. Conclusion: Our results suggest that RA associated DNA methylation differences vary between the two T cell subsets but are also influenced by RA characteristics such as disease activity disease duration and/or MTX treatment.""" View Publication

Human noroviruses (HuNoVs) are the leading cause of nonbacterial gastroenteritis worldwide. Histo-blood group antigen (HBGA) expression is an important susceptibility factor for HuNoV infection based on controlled human infection models and epidemiologic studies that show an association of secretor status with infection caused by several genotypes. The fucosyltransferase 2 gene (FUT2) affects HBGA expression in intestinal epithelial cells; secretors express a functional FUT2 enzyme,while nonsecretors lack this enzyme and are highly resistant to infection and gastroenteritis caused by many HuNoV strains. These epidemiologic associations are confirmed by infections in stem cell-derived human intestinal enteroid (HIE) cultures. GII.4 HuNoV does not replicate in HIE cultures derived from nonsecretor individuals,while HIEs from secretors are permissive to infection. However,whether FUT2 expression alone is critical for infection remains unproven,since routinely used secretor-positive transformed cell lines are resistant to HuNoV replication. To evaluate the role of FUT2 in HuNoV replication,we used CRISPR or overexpression to genetically manipulate FUT2 gene function to produce isogenic HIE lines with or without FUT2 expression. We show that FUT2 expression alone affects both HuNoV binding to the HIE cell surface and susceptibility to HuNoV infection. These findings indicate that initial binding to a molecule(s) glycosylated by FUT2 is critical for HuNoV infection and that the HuNoV receptor is present in nonsecretor HIEs. In addition to HuNoV studies,these isogenic HIE lines will be useful tools to study other enteric microbes where infection and/or disease outcome is associated with secretor status.IMPORTANCE Several studies have demonstrated that secretor status is associated with susceptibility to human norovirus (HuNoV) infection; however,previous reports found that FUT2 expression is not sufficient to allow infection with HuNoV in a variety of continuous laboratory cell lines. Which cellular factor(s) regulates susceptibility to HuNoV infection remains unknown. We used genetic manipulation of HIE cultures to show that secretor status determined by FUT2 gene expression is necessary and sufficient to support HuNoV replication based on analyses of isogenic lines that lack or express FUT2. Fucosylation of HBGAs is critical for initial binding and for modification of another putative receptor(s) in HIEs needed for virus uptake or uncoating and necessary for successful infection by GI.1 and several GII HuNoV strains. View Publication

Acute graft-versus-host disease causes significant mortality in patients undergoing allogeneic hematopoietic cell transplantation. Immunosuppressive treatment for graft-versus-host disease can impair the beneficial graft-versus-leukemia effect and facilitate malignancy relapse. Therefore,novel approaches that protect and regenerate injured tissues without impeding the donor immune system are needed. Bile acids regulate multiple cellular processes and are in close contact with the intestinal epithelium,a major target of acute graft-versus-host disease. Here,we found that the bile acid pool is reduced following graft-versus-host disease induction in a preclinical model. We evaluated the efficacy of bile acids to protect the intestinal epithelium without reducing anti-tumor immunity. We observed that application of bile acids decreased cytokine-induced cell death in intestinal organoids and cell lines. Systemic prophylactic administration of tauroursodeoxycholic acid,the most potent compound in our in vitro studies,reduced graft-versus-host disease severity in three different murine transplantation models. This effect was mediated by decreased activity of the antigen presentation machinery and subsequent prevention of apoptosis of the intestinal epithelium. Moreover,bile acid administration did not alter the bacterial composition in the intestine suggesting that its effects are cell-specific and independent of the microbiome. Treatment of human and murine leukemic cell lines with tauroursodeoxycholic acid did not interfere with the expression of antigen presentation-related molecules. Systemic T cell expansion and especially their cytotoxic capacity against leukemic cells remained intact. This study establishes a role for bile acids in the prevention of acute graft-versus-host disease without impairing the graft-versus-leukemia effect. In particular,we provide a scientific rationale for the systematic use of tauroursodeoxycholic acid in patients undergoing allogeneic hematopoietic cell transplantation. View Publication

Intestinal epithelial organoids established from gut tissue have become a widely used research tool. However,it remains unclear how environmental cues,divergent microbiota composition and other sources of variation before,during and after establishment confound organoid properties,and how these properties relate to the original tissue. While environmental influences cannot be easily addressed in human organoids,mice offer a controlled assay-system. Here,we probed the effect of donor microbiota differences,previously identified as a confounding factor in murine in vivo studies,on organoids. We analysed the proteomes and transcriptomes of primary organoid cultures established from two colonised and one germ-free mouse colony of C57BL/6J genetic background,and compared them to their tissue of origin and commonly used cell lines. While an imprint of microbiota-exposure was observed on the proteome of epithelial samples,the long-term global impact of donor microbiota on organoid expression patterns was negligible. Instead,stochastic culture-to-culture differences accounted for a moderate variability between independently established organoids. Integration of transcriptome and proteome datasets revealed an organoid-typic expression signature comprising 14 transcripts and 10 proteins that distinguished organoids across all donors from murine epithelial cell lines and fibroblasts and closely mimicked expression patterns in the gut epithelium. This included the inflammasome components ASC,Naip1-6,Nlrc4 and Caspase-1,which were highly expressed in all organoids compared to the reference cell line m-ICc12 or mouse embryonic fibroblasts. Taken together,these results reveal that the donor microbiota has little effect on the organoid phenotype and suggest that organoids represent a more suitable culture model than immortalised cell lines,in particular for studies of intestinal epithelial inflammasomes. View Publication

CD47 deficient mice are resistant to dextran sulfate sodium (DSS)-induced experimental colitis. The underlying mechanism,however,remains incompletely understood. In this study,we characterized the role of CD47 in modulating homeostasis of gastrointestinal tract. We found that CD47 expression in both human and mouse intestinal epithelium was upregulated in colitic condition compared to that under normal condition. In line with this,CD47 deficiency protected mice from DSS-induced colitis. Analysis based on both intestinal organoid and cultured cell assays showed that CD47 deficiency accelerated intestinal epithelial cell proliferation and migration. Mechanistically,western blot and functional assays indicated that CD47 deficiency promoting mouse intestinal epithelial cell proliferation and migration follow cell injury is likely through upregulating expression of four Yamanaka transcriptional factors Oct4,Sox2,Klf4 and c-Myc (OSKM in abbreviation). Our studies thus reveal CD47 as a negative regulator in intestinal epithelial cell renewal during colitis through downregulating OSKM transcriptional factors. View Publication

Overcoming the global health threat of HIV infection requires continuous pipelines of novel drug candidates. We identified the $\gamma$-pyrone polyketides Aureothin/Neoaureothin as potent hits by anti-HIV screening of an extensive natural compound collection. Total synthesis of a structurally diverse group of Aureothin-derivatives successfully identified a lead compound ({\#}7) superior to Aureothin that combines strong anti-HIV activity (IC90{\textless}45 nM),photostability and improved cell safety. Compound {\#}7 inhibited de novo virus production from integrated proviruses by blocking the accumulation of HIV RNAs that encode the structural components of virions and include viral genomic RNAs. Thus,the mode-of-action displayed by compound {\#}7 is different from those of all current clinical drugs. Proteomic analysis indicated that compound {\#}7 does not affect global protein expression in primary blood cells and may modulate cellular pathways linked to HIV infection. Compound {\#}7 inhibited multiple HIV genotypes,including HIV-type 1 and 2 and synergistically inhibited HIV in combination with clinical reverse transcriptase and integrase inhibitors. We conclude that compound {\#}7 represents a promising new class of HIV inhibitors that will facilitate the identification of new virus-host interactions exploitable for antiviral attack and holds promise for further drug development. View Publication

Immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide show remarkable antitumor activity in multiple myeloma (MM) via directly inhibiting MM-cell growth in the bone marrow (BM) microenvironment and promoting immune effector cell function. They are known to bind to the ubiquitin 3 ligase CRBN complex and thereby triggering degradation of IKZF1/3. In this study,we demonstrate that IMiDs also directly bind and activate zeta-chain-associated protein kinase-70 (Zap-70) via its tyrosine residue phosphorylation in T cells. IMiDs also triggered phosphorylation of Zap-70 in natural killer (NK) cells. Importantly,increased granzyme-B (GZM-B) expression and NK-cell activity triggered by IMiDs is associated with Zap-70 activation and inhibited by Zap-70 knockdown (KD),independent of CRBN. We also demonstrate a second mechanism whereby IMiDs trigger GZM-B and NK cytotoxicity which is CRBN and IKZF3 mediated,and inhibited or enhanced by KD of CRBN or IKZF3,respectively,independent of Zap-70. Our studies therefore show that IMiDs can enhance NK and T-cell cytotoxicity in (1) ZAP-70-mediated CRBN independent,as well as (2) CRBN-mediated ZAP-70 independent mechanisms; and provide the framework for developing novel therapeutics to activate Zap-70 and thereby enhance T and NK anti-MM cytotoxicity. View Publication

Circulating tumor cells (CTC) play important roles in various cancers; however,few studies have assessed their clinical utility in neuroendocrine tumors. This study aimed to prospectively evaluate the prognostic value of CTC counts in Asian patients with neuroendocrine tumors before and during anti-cancer therapy. Patients who were diagnosed with unresectable histological neuroendocrine tumors between September 2011 and September 2017 were enrolled. CTC testing was performed before and during anti-cancer therapy using a negative selection protocol. Chromogranin A levels were also assessed. Univariate and multivariate Cox's proportional hazard model with forward LR model was performed to investigate the impact of independent factors on overall survival and progression-free survival. Kaplan-Meier method with log-rank tests were used to determine the difference among different clinicopathological signatures and CTC cutoff. The baseline CTC detection rate was 94.3{\%} (33/35). CTC counts were associated with cancer stages (I-III vs. IV,P = 0.015),liver metastasis (P = 0.026),and neuroendocrine tumor grading (P = 0.03). The median progression-free survival and overall survivals were 12.3 and 30.4 months,respectively. In multivariate Cox regression model,neuroendocrine tumors grading and baseline CTC counts were both independent prognostic factors for progression-free survival (PFS,P = 0.005 and 0.015,respectively) and overall survival (OS,P = 0.018 and 0.023,respectively). In Kaplan-Meier analysis,lower baseline chromogranin A levels were associated with longer PFS (P = 0.024). Baseline CTC counts are associated with the clinicopathologic features of neuroendocrine tumors and are an independent prognostic factor for this malignancy. View Publication

Heterotopic ossification (HO) is defined as abnormal differentiation of local stromal cells of mesenchymal origin,resulting in pathologic cartilage and bone matrix deposition. Cyr61,CTGF,Nov (CCN) family members are matricellular proteins that have diverse regulatory functions on cell proliferation and differentiation,including the regulation of chondrogenesis. However,little is known regarding CCN family member expression or function in HO. Here,a combination of bulk and single-cell RNA sequencing defined the dynamic temporospatial pattern of CCN family member induction within a mouse model of trauma-induced HO. Among CCN family proteins,Wisp1 (also known as Ccn4) was most upregulated during the evolution of HO,and Wisp1 expression corresponded with chondrogenic gene profile. Immunohistochemistry confirmed WISP1 expression across traumatic and genetic HO mouse models as well as in human HO samples. Transgenic Wisp1LacZ/LacZ knockin animals showed an increase in endochondral ossification in HO after trauma. Finally,the transcriptome of Wisp1-null tenocytes revealed enrichment in signaling pathways,such as the STAT3 and PCP signaling pathways,that may explain increased HO in the context of Wisp1 deficiency. In sum,CCN family members,and in particular Wisp1,are spatiotemporally associated with and negatively regulate trauma-induced HO formation. View Publication

3-dimensional (3D) bioprinting technology provides promising strategy in the fabrication of artificial tissues and organs. As the fundamental element in bioprinting process,preparation of bioink with ideal mechanical properties without sacrifice of biocompatibility is a great challenge. In this study,a supramolecular hydrogel-based bioink is prepared by polyethylene glycol (PEG) grafted chitosan,$\alpha$-cyclodextrin ($\alpha$-CD) and gelatin. It has a primary crosslinking structure through the aggregation of the pseudo-polyrotaxane-like side chains,which are formed from the host-guest interactions between $\alpha$-CD and PEG side chain. Apparent viscosity measurement shows the shear-shinning property of this bioink,which might be due to the reversibility of the physical crosslinking. Moreover,with $\beta$-glycerophosphate at different concentrations as the secondary crosslinking agent,the printed constructs demonstrate different Young's modulus (p {\textless} 0.001). They could also maintain the Young's modulus in cell culture condition for at least 21 days (p {\textless} 0.05). By co-culturing each component with fibroblasts,CCK-8 assay demonstrate cellular viability is higher than 80{\%}. After bioprinting and culturing,immunofluorescence staining with quantification indicate the expression of Ki-67,Paxillin,and N-cadherin is higher in day 14 than those in day 3 (p {\textless} 0.05). Oil red O and Nissl body specific staining reflect strength tunable bioink may have impact on the cell fate of mesenchymal stem cells (p {\textless} 0.05). This work might provide new idea for advanced bioink in the application of re-establishing complicated tissues and organs. View Publication

The widely varying therapeutic response of patients with inflammatory bowel disease (IBD) continues to raise questions regarding the unclarified heterogeneity of pathological mechanisms promoting disease progression. While biomarkers for the differentiation of Crohn's disease (CD) versus ulcerative colitis (UC) have been suggested,specific markers for a CD subclassification in ileal CD versus colonic CD are still rare. Since an altered signature of the tryptophan metabolism is associated with chronic inflammatory disease,we sought to characterize potential biomarkers by focusing on the downstream enzymes and metabolites of kynurenine metabolism. Using immunohistochemical stainings,we analyzed and compared the mucosal tryptophan immune metabolism in bioptic samples from patients with active inflammation due to UC or CD versus healthy controls. Localization-specific quantification of immune cell infiltration,tryptophan-metabolizing enzyme expression and mucosal tryptophan downstream metabolite levels was performed. We found generally increased immune cell infiltrates in the tissue of all patients with IBD. However,in patients with CD,significant differences were found between regulatory T cell and neutrophil granulocyte infiltration in the ileum compared with the colon. Furthermore,we observed decreased kynurenine levels as well as strong kynureninase (KYNU) expression specifically in patients with ileal CD. Correspondingly,significantly elevated levels of the kynurenine metabolite 3-hydroxyanthranilic acid were detected in the ileal CD samples. Highlighting the heterogeneity of the different phenotypes of CD,we identified KYNU as a potential mucosal biomarker allowing the localization-specific differentiation of ileal CD versus colonic CD. View Publication

BACKGROUND Longitudinal studies have verified the pivotal role of mesenchymal stem/stromal cells (MSCs) in the bone marrow microenvironment for hematopoiesis and coordinate contribution to leukemia pathogenesis. However,the precise characteristics and alternation of MSCs during acquired aplastic anemia (AA) remain obscure. METHODS In this study,we originally collected samples from both healthy donors (HD) and AA patients to dissect the hematological changes. To systematically evaluate the biological defects of AA-derived MSCs (AA-MSCs),we analyzed alterations in cellular morphology,immunophenotype,multi-lineage differentiation,cell migration,cellular apoptosis,and chromosome karyocyte,together with the immunosuppressive effect on the activation and differentiation of lymphocytes. With the aid of whole genome sequencing and bioinformatic analysis,we try to compare the differences between AA-MSCs and HD-derived MSCs (HD-MSCs) upon the molecular genetics,especially the immune-associated gene expression pattern. In addition,the efficacy of umbilical cord-derived MSC (UC-MSC) transplantation on AA mice was evaluated by utilizing survivorship curve,histologic sections,and blood cell analyses. RESULTS In coincidence with the current reports,AA patients showed abnormal subsets of lymphocytes and higher contents of proinflammatory cytokines. Although with similar immunophenotype and chromosome karyotype to HD-MSCs,AA-MSCs showed distinguishable morphology and multiple distinct characteristics including genetic properties. In addition,the immunosuppressive effect on lymphocytes was significantly impaired in AA-MSCs. What is more,the cardinal symptoms of AA mice were largely rescued by systemic transplantation of UC-MSCs. CONCLUSIONS Herein,we systematically investigated the signatures and efficacy of MSCs to dissect the alterations occurred in AA both at the cellular and molecular levels. Different from HD-MSCs,AA-MSCs exhibited multifaceted defects in biological characteristics and alterative molecular genetics in the whole genome. Our findings have provided systematic and overwhelming new evidence for the defects of AA-MSCs,together with effectiveness assessments of UC-MSCs on AA as well. View Publication