Cell-mediated Damage Research Articles

Human Vascularized Macrophage-Islet Organoids to Model Immune-Mediated Pancreatic β cell Pyroptosis upon Viral Infection.

There is a paucity of human models to study immune-mediated host damage. Here, we utilized the GeoMx spatial multi-omics platform to analyze immune cell changes in COVID-19 pancreatic autopsy samples, revealing an accumulation of proinflammatory macrophages. Single cell RNA-seq analysis of human islets exposed to SARS-CoV-2 or Coxsackievirus B4 (CVB4) viruses identified activation of proinflammatory macrophages and β cell pyroptosis. To distinguish viral versus proinflammatory macrophage-mediated β cell pyroptosis, we developed human pluripotent stem cell (hPSC)-derived vascularized macrophage-islet (VMI) organoids. VMI organoids exhibited enhanced marker expression and function in both β cells and endothelial cells compared to separately cultured cells. Notably, proinflammatory macrophages within VMI organoids induced β cell pyroptosis. Mechanistic investigations highlighted TNFSF12-TNFRSF12A involvement in proinflammatory macrophage-mediated β cell pyroptosis. This study established hPSC-derived VMI organoids as a valuable tool for studying immune cell-mediated host damage and uncovered mechanism of β cell damage during viral exposure.

Mesenchymal stromal cells protect tissues from Th1 immune responses via IL-11 secretion.

Mesenchymal stromal cells (MSCs) have been shown to modulate the function of various subsets of T cells such as naïve CD4+ T cells and IFNγ+CD4+ Th1 cells; however, mechanisms underlying this regulation have not been fully deciphered. Our invitro culture assays demonstrate that MSCs suppress the activation and function of CD4+ T cells by secreting interleukin 11, and neutralization of IL11 abrogates MSC-mediated suppression of CD4+ T cell function. Moreover, delayed-type, exogenous supplementation of IL11 significantly suppressed IFNγ+ expression by Th1 cells. Th1 and CD8+ cells play central roles in T cell-mediated tissue damage. Using a murine model of hypersensitivity response to study T cell-mediated tissue damage, we show that silencing IL11 in MSCs significantly abates the capacity of MSCs to suppress the generation of IFNγ-secreting CD4+ and CD8+ cells, failing to prevent T cell-mediated tissue inflammation and tissue damage.

Neurons upregulate PD-L1 via IFN/STAT1/IRF1 to alleviate damage by CD8+ T cells in cerebral malaria

BackgroundCerebral malaria (CM) is the most lethal complication of malaria, and survivors usually endure neurological sequelae. Notably, the cytotoxic effect of infiltrating Plasmodium-activated CD8+ T cells on cerebral microvasculature endothelial cells is a prominent feature of the experimental CM (ECM) model with blood–brain barrier disruption. However, the damage effect of CD8+ T cells infiltrating the brain parenchyma on neurons remains unclear. Based on the immunosuppressive effect of the PD-1/PD-L1 pathway on T cells, our previous study demonstrated that the systemic upregulation of PD-L1 to inhibit CD8+ T cell function could effectively alleviate the symptoms of ECM mice. However, it has not been reported whether neurons can suppress the pathogenic effect of CD8+ T cells through the PD-1/PD-L1 negative immunomodulatory pathway. As the important inflammatory factor of CM, interferons can induce the expression of PD-L1 via different molecular mechanisms according to the neuro-immune microenvironment. Therefore, this study aimed to investigate the direct interaction between CD8+ T cells and neurons, as well as the mechanism of neurons to alleviate the pathogenic effect of CD8+ T cells through up-regulating PD-L1 induced by IFNs.MethodsUsing the ECM model of C57BL/6J mice infected with Plasmodium berghei ANKA (PbA), morphological observations were conducted in vivo by electron microscope and IF staining. The interaction between the ECM CD8+ T cells (immune magnetic bead sorting from spleen of ECM mice) and primary cultured cortical neurons in vitro was observed by IF staining and time-lapse photography. RNA-seq was performed to analyze the signaling pathway of PD-L1 upregulation in neurons induced by IFNβ or IFNγ, and verified through q-PCR, WB, IF staining, and flow cytometry both in vitro and in vivo using IFNAR or IFNGR gene knockout mice. The protective effect of adenovirus-mediated PD-L1 IgGFc fusion protein expression was verified in ECM mice with brain stereotaxic injection in vivo and in primary cultured neurons via viral infection in vitro.ResultsIn vivo, ECM mice showed infiltration of activated CD8+ T cells and neuronal injury in the brain parenchyma. In vitro, ECM CD8+ T cells were in direct contact with neurons and induced axonal damage, as an active behavior. The PD-L1 protein level was elevated in neurons of ECM mice and in primary cultured neurons induced by IFNβ, IFNγ, or ECM CD8+ T cells in vitro. Furthermore, the IFNβ or IFNγ induced neuronal expression of PD-L1 was mediated by increasing STAT1/IRF1 pathway via IFN receptors. The increase of PD-L1 expression in neurons during PbA infection was weakened after deleting the IFNAR or IFNGR. Increased PD-L1 expression by adenovirus partially protected neurons from CD8+ T cell-mediated damage both in vitro and in vivo.ConclusionOur study demonstrates that both type I and type II IFNs can induce neurons to upregulate PD-L1 via the STAT1/IRF1 pathway mediated by IFN receptors to protect against activated CD8+ T cell-mediated damage, providing a targeted pathway to alleviate neuroinflammation during ECM.Graphical

Phosphorylated α-synuclein deposited in Schwann cells interacting with TLR2 mediates cell damage and induces Parkinson’s disease autonomic dysfunction

Despite the significant frequency of autonomic dysfunction (AutD) in Parkinson’s disease (PD) patients, its pathogenesis and diagnosis are challenging. Here, we aimed to further explore the mechanism of phosphorylated α-synuclein (p-α-syn) deposited in vagus nerve Schwann cells (SCs) causing SCs damage and PD AutD. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 20 mg/kg) was administrated to C57BL/6 mice twice a week for 35 days. Following the final injection, locomotor functions, gastrointestinal symptoms, urine functions, and cardiovascular system functions were evaluated. Meanwhile, we examined p-α-syn deposited in vagus nerve SCs, Toll-like receptor 2 (TLR2) activation, and SCs loss using immunofluorescence, western blot, and Luxol fast blue staining. In vitro, the rat SCs line RSC96 cells were exposed to α-synuclein preformed fibril (α-syn PFF), and cell viability was detected by CCK8. Co-IP was used to identify the interaction between p-α-syn and TLR2. Furthermore, the role of TLR2 in p-α-syn-mediated SCs damage was confirmed by the administration of CU-CPT22, a specific blocker of TLR2. In vivo, apart from dyskinesia, MPTP mice exhibited constipation, urinary dysfunction, and cardiovascular failure, which were associated with the deposition of p-α-syn in vagus nerve SCs, TLR2 activation, and vagus nerve demyelination. In vitro, stimulation of α-syn PFF induced a time-dependent loss of viability, and p-α-syn deposited in RSC96 cells induced a cellular inflammatory response by interacting with TLR2, resulting in cell dysfunction and apoptosis. However, both SCs inflammatory response and cell viability were alleviated after inhibition of TLR2. Furthermore, 1 h fecal pellets and water content, the frequency of 1 h urine, blood pressure, heart rate, and heart rate variability of mice in the MPTP + CU-CPT22 group were also improved. Our results support the perspective that p-α-syn interacts with TLR2 induced SCs damage and is involved in PD AutD, which sheds fresh light on the mechanism of PD AutD and indicates a promising treatment for PD AutD targeting SCs p-α-syn/ TLR2 signaling pathway.

Preclinical Studies of the GlyT1 Inhibitor Bitopertin in Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) results from the haploinsufficiency of 1 of 20 different ribosomal proteins. While DBA patients have hematopoietic stem/progenitor cell defects which broadly impact hematopoiesis, the dominant phenotype is ineffective erythropoiesis and severe anemia. In ~60% of patients, the anemia responds to corticosteroid therapy, leaving an unmet need for safe and effective treatment. In DBA, the synthesis of heme (a chemical chelate) proceeds at a near normal rate while the ribosomal haploinsufficiency leads to slowed translation and delayed globin protein synthesis, resulting in a heme-globin imbalance. DBA patient colony forming unit-erythroid (CFU-E)/proerythroblasts and murine Rpl11 haploinsufficient CFU-E/proerythroblasts show increased heme- and reactive oxygen species (ROS)-mediated cell damage, apoptosis, and ferroptosis. Single cell RNAseq analyses also suggest that excessive heme is a major factor in DBA erythroid marrow failure. When patient marrow cells are cultured in the presence of succinylacetone to reduce heme synthesis or hemopexin to facilitate heme export, erythroid output significantly increases. Succinylacetone competitively inhibits the 2 nd step in heme synthesis, while hemopexin enhances heme export 100-fold and scavenges the extracellular heme. Neither would be a pragmatic and safe therapy for DBA patients. In the first step of heme biosynthesis, glycine and succinyl-CoA combine to form aminolevulinic acid. Erythroid precursors are unable to synthesize enough glycine to support their robust heme synthesis and must import glycine via glycine transporter 1 (GlyT1) (PMID 28495915). Bitopertin is an investigational oral small-molecule inhibitor of GlyT1. In Phase 3 clinical studies of schizophrenia, it was found to be ineffective but exhibited a favorable safety profile (PMID 27816567). To determine whether bitopertin could rebalance heme and globin synthesis, reduce ineffective erythropoiesis, and improve red cell production in DBA, we cultured marrow cells from patients with DBA without and with 10 nM bitopertin (n=3). Following 7 days of culture, bitopertin increased the mean number of cells (21%) and the number of CD71+ erythroid precursors (31%) without impacting erythroid maturation. We next made human cord blood CD34+ deficient for RPS19 (44±2% reduction) with RPS19 shRNA transduction (or control luciferase shRNA) to model RPS19 haploinsufficient DBA. Treatment with bitopertin improved erythroid expansion of RPS19-deficient CD34+ cells 60% (P<0.01) but not control CD34+ cells. Additionally, to assess erythroid differentiation in vitro in the context of an erythroblastic island (EBI) niche, we cultured the CD34+ cell-derived erythroblasts with cord blood-derived macrophages. Erythroid expansion is 7.9±1.3-fold higher (P<0.02) in EBI cultures compared to cultures without macrophages. Treatment of the EBI cultures with bitopertin increased the expansion of RPS19-deficient CD34+ cell-derived erythroblasts 3.9±0.3-fold (P<0.002) but did not increase the expansion of control CD34+ cell-derived erythroblasts. As a final study, Rpl11 haploinsufficient mice were treated with bitopertin (10 to 150 ppm in chow, 2-30 mg/kg/day) for 8 weeks. Bitopertin treatment resulted in a dose-dependent improvement in anemia. After treatment with an optimal dose of bitopertin (100 ppm in chow, 20 mg/kg/day), there was an 8.8% increase in the hemoglobin values, and there was significant improvement in red cell numbers (7.5±1.1 M/µL to 8.9±1.3 M/µL, p=0.005), hematocrit (41.6±4.9% to 45.5±4.2%, p<0.05), and mean corpuscular volume (55.7±3.5 fL to 51.8±4.2 fL, p=0.01). Additionally, bitopertin treatment diminished the developmental block at the CFU-E/proerythroblast stage and reduced erythroblast ROS. The improved in vitro erythroid differentiation of DBA patient marrow and RPS19-deficient CD34+ cells and the improved anemia of RPL11 haploinsufficient mice suggest that bitopertin might mitigate anemia in DBA patients. These encouraging preclinical data supported the initiation of a Phase 1/2 clinical trial (NCT05828108: Young et al, abstract this meeting).

Upregulation of IL-37 in epithelial cells: A potential new mechanism of T cell inhibition induced by tacrolimus

Oral lichen planus (OLP) is a chronic T cell-mediated mucocutaneous disease characterized by T cell infiltration at the connective tissue–epithelium interface. Traditionally, topical corticosteroids are used as the first-line drugs to treat OLP. However, long-term use of corticosteroids may lead to drug tolerance, secondary candidiasis, and autoimmune adrenal insufficiency. Although topical tacrolimus has often been recommended for short-term use in corticosteroid-refractory OLP, the precise role of tacrolimus in epithelial cells remains elusive. This study showed that tacrolimus could directly upregulate the expression of IL-37 in human gingival epithelial cells by promoting the TGF-βRI/Smad3 pathway independently of calcineurin inhibition and MAPKs. In contrast, dexamethasone, one of the corticosteroids, did not have the same effect. Moreover, IL-37 could inhibit the proliferation of activated T cells and the secretion of effector cytokines and alleviate epithelial cell apoptosis and death caused by activated T cells ina co-culturesystem. Furthermore, compared with healthy controls, IL-37 and p-Smad3 levels significantly increased in the oral mucosa affected by OLP, especially in the epithelium. IL-37 might have mediated a negative feedback mechanism to curb excessive inflammation in OLP. However, the expression of IL-37 was not associated with the infiltration of CD8+ T cells and Tregs in OLP, implying that IL-37 might mostly affect T cell activation rather than T cell differentiation and migration. Overall, this study discovered a potential novel mechanism by which tacrolimus might indirectly inhibit T cell-mediated immune damage by upregulating IL-37 in human gingival epithelial cells.

Development and investigation of metabolism-associated risk assessment models for patients with viral hepatitis.

Dysregulation of metabolism plays an important role in the onset and progression of multiple pathogenic diseases, including viral hepatitis. However, a model to predict viral hepatitis risk by metabolic pathways is still lacking. Thus, we developed two risk assessment models for viral hepatitis based on metabolic pathways identified through univariate and least absolute shrinkage and selection operator (LASSO) Cox regression analysis. The first model is designed to assess the progression of the disease by evaluating changes in the Child-Pugh class, hepatic decompensation, and the development of hepatocellular carcinoma. The second model is focused on determining the prognosis of the illness, taking into account the patient's cancer status. Our models were further validated by Kaplan-Meier plots of survival curves. In addition, we investigated the contribution of immune cells in metabolic processes and identified three distinct subsets of immune cells-CD8+ T cells, macrophages, and NK cells-that have significantly affected metabolic pathways. Specifically, our findings suggest that resting or inactive macrophages and NK cells contribute to maintaining metabolic homeostasis, particularly with regard to lipid and α-amino acid metabolism, thereby potentially reducing the risk of viral hepatitis progression. Moreover, maintaining metabolic homeostasis ensures a balance between killer-proliferative and exhausted CD8+ T cells, which helps in mitigating CD8+ T cell-mediated liver damage while preserving energy reserves. In conclusion, our study offers a useful tool for early disease detection in viral hepatitis patients through metabolic pathway analysis and sheds light on the immunological understanding of the disease through the examination of immune cell metabolic disorders.

Safety and Efficacy of Tyrosine Kinase Inhibitors in Immune Thrombocytopenic Purpura: A Systematic Review of Clinical Trials.

Immune thrombocytopenic purpura (ITP) is an acquired antibody or cell-mediated platelet damage or decreased platelet production. Steroids, IV immunoglobulins (IVIG), and Rho-anti-D antibodies are the commonly used initial treatments for ITP. However, many ITP patients either do not respond or do not maintain a response to initial therapy. Splenectomy, rituximab, and thrombomimetics are the commonly used second-line treatment. More treatment options include tyrosine kinases inhibitors (TKI), including spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (BTK) inhibitors. This review aims to assess the safety and efficacy of TKIs. Methods: Literature was searched on PubMed, Embase, WOS, and clinicaltrials.gov using keywords, "tyrosine kinase" and "idiopathic thrombocytopenic purpura". PRISMA guidelines were followed. Results: In total, 4 clinical trials were included with 255 adult patients with relapsed/refractory ITP. In all, 101 (39.6%) patients were treated with fostamatinib, 60 (23%) patients with rilzabrutinib, and 34 (13%) with HMPL-523. Patients treated with fostamatinib achieved a stable response (SR) and overall response (OR) in 18/101 (17.8%) and 43/101 (42.5%) of the patients, respectively, while SR and OR were achieved in 1/49 (2%) and 7/49 (14%) of the patients, respectively, in the placebo group. Patients treated with HMPL-523 (300 mg dose expansion) achieved an SR and OR in 5/20 (25%) and 11/20 (55%) of the patients, respectively, while SR and OR were achieved in 1/11 (9%) of the patients treated with the placebo. Patients treated with rilzabrutinib achieved an SR in 17/60 (28%) patients. Dizziness (1%), hypertension (2%), diarrhea (1%), and neutropenia (1%) were serious adverse events in fostamatinib patients. Rilzabrutinib or HMPL-523 patients did not require a dose reduction due to drug-related adverse effects. Conclusions: Rilzabrutinib, fostamatinib, and HMPL-523 were safe and effective in the treatment of relapsed/refractory ITP.

A unique cytotoxic CD4+ T cell-signature defines critical COVID-19.

SARS-CoV-2 infection causes a spectrum of clinical disease presentation, ranging from asymptomatic to fatal. While neutralising antibody (NAb) responses correlate with protection against symptomatic and severe infection, the contribution of the T-cell response to disease resolution or progression is still unclear. As newly emerging variants of concern have the capacity to partially escape NAb responses, defining the contribution of individual T-cell subsets to disease outcome is imperative to inform the development of next-generation COVID-19 vaccines. Immunophenotyping of T-cell responses in unvaccinated individuals was performed, representing the full spectrum of COVID-19 clinical presentation. Computational and manual analyses were used to identify T-cell populations associated with distinct disease states. Critical SARS-CoV-2 infection was characterised by an increase in activated and cytotoxic CD4+ lymphocytes (CTL). These CD4+ CTLs were largely absent in asymptomatic to severe disease states. In contrast, non-critical COVID-19 was associated with high frequencies of naïve T cells and lack of activation marker expression. Highly activated and cytotoxic CD4+ T-cell responses may contribute to cell-mediated host tissue damage and progression of COVID-19. Induction of these potentially detrimental T-cell responses should be considered when developing and implementing effective COVID-19 control strategies.

Triptolide leads to hepatic intolerance to exogenous lipopolysaccharide and natural-killer-cell mediated hepatocellular damage by inhibiting MHC class I molecules

BackgroundTripterygium wilfordii Hook. F (TWHF) is used as a traditional Chinese medicine, called thunder god vine, based on its efficacy for treating inflammatory diseases. However, its hepatotoxicity has limited its clinical application. Triptolide (TP) is the major active and toxic component of TWHF. Previous studies reported that a toxic pretreatment dose of TP leads to hepatic intolerance to exogenous lipopolysaccharide (LPS) stimulation, and to acute liver failure, in mice, but the immune mechanisms of TP-sensitised hepatocytes and the TP-induced excessive immune response to LPS stimulation are unknown. PurposeTo identify both the key immune cell population and mechanism involved in TP-induced hepatic intolerance of exogenous LPS. Study DesignIn vitro and in vivo experiments were conducted to investigate the inhibitory signal of natural killer (NK) cells maintained in hepatocytes, and the ability of TP to impair that signal. MethodsFlow cytometry was performed to determine NK cell activity and hepatocyte histocompatibility complex (MHC) class I molecules expression; the severity of liver injury was determined based on blood chemistry values, and drug- or cell-mediated hepatocellular damage, by measuring lactate dehydrogenase (LDH) release. In vivo H-2Kb transduction was carried out using an adeno-associated viral vector. ResultsInterferon (IFN)-γ-mediated necroptosis occurred in C57BL/6N mice treated with 500 μg TP/kg and 0.1 mg LPS/kg to induce fulminant hepatitis. Primary hepatocytes pretreated with TP were more prone to necroptosis when exposed to recombinant murine IFN-γ. In mice administered TP and LPS, the intracellular IFN-γ levels of NK cells increased significantly. Subsequent study confirmed that NK cells were activated and resulted in potent hepatocellular toxicity. In vivo and in vitro TP administration significantly inhibited MHC class I molecules in murine hepatocytes. An in vitro analysis demonstrated the susceptibility of TP-pretreated hepatocytes to NK-cell-mediated cytotoxicity, an effect that was significantly attenuated by the induction of hepatocyte MHC-I molecules by IFN-α. In vivo induction or overexpression of hepatocyte MHC-I also protected mouse liver against TP and LPS-induced injury. ConclusionThe TP-induced inhibition of hepatocyte MHC-I molecules expression leads to hepatic intolerance to exogenous LPS and NK-cell mediated cytotoxicity against self-hepatocytes. These findings shed light on the toxicity of traditional Chinese medicines administered for their immunomodulatory effects

Abstract 9991: Index Case of Prosthetic Mitral Valve Dehiscence Secondary to SARS CoV 2 Virus: Histopathologic Evidence of T-cell Mediated Injury

Introduction: SARS-Cov-2 virus has been shown to inflict damage on native cardiac valvular tissue. We present the index case of prosthetic mitral valve (MV) dehiscence secondary to SARS-Cov-2 virus as evidenced by histopathologic analysis of peri-valvular annular tissue . Case: 63-year-old male with history of MV replacement 2 years ago presented with complaint of dyspnea for 2 weeks. He was asymptomatic on a cardiology appointment a month ago and had an unremarkable transthoracic echocardiography (TTE) at that time. On arrival, he was hypoxic to 80% on room air and chest exam revealed bibasilar crackles. Complete blood count and electrolytes were within normal ranges. He tested positive for SARS-Cov-2 virus RNA and had a recent family history of COVID-19. Chest X-ray revealed bilateral pulmonary vascular congestion. He was given supplemental oxygen through nasal cannula with improvement in pulse oximetry. Repeat TTE revealed mitral paravalvular leak with suspicion for prosthetic dehiscence. Transesophageal echocardiogram showed multiple jets of severe paravalvular regurgitation and disruption of the MV apparatus. No vegetations were detected on echocardiography. He underwent a pre-operative right heart catheterization which confirmed severe MV regurgitation with pulmonary artery (PA) capillary wedge pressure of 45mmHg (Normal: <12) and a PA pressure of 40mmHg (Normal:<20). Intra-operatively, we found near-complete dehiscence of prosthetic MV with surrounding annular calcifications. Our patient underwent surgical MV replacement with a biosynthetic valve. Histopathologic analysis of the annular tissue revealed myxomatous degeneration and an inflammatory infiltrate of CD4 T helper cells consistent with subacute to chronic inflammation. Of note, neutrophils were not detected which usually signify acute bacterial endocarditis. Tissue and blood cultures remained sterile. Our patient improved hemodynamically after MV replacement and was discharged home in stable condition. Conclusion: We propose that SARS-Cov-2 virus may cause prosthetic MV dehiscence through CD4 T cell-mediated damage to annular tissue. Such cases would require surgical valve replacement to manage the resulting hemodynamic compromise.

Excessive IL-15 promotes cytotoxic CD4 + CD28− T cell-mediated renal injury in lupus nephritis

BackgroundPatients with systemic lupus erythematosus (SLE) are highly susceptible to infection and cardiovascular events, suggesting that chronic antigenic stimulation may accelerate premature aging in SLE patients. Premature aging in SLE is often accompanied with the expansion of cytotoxic CD4 + CD28−T cells. Damage caused by CD4 + CD28− T cells enhances the progressive aging of the tissue function and loss of organism’s fitness. The high serum level of IL-15 has been implicated in the pathogenesis of SLE, but its role in CD4 + CD28−T cell-mediated cytotoxicity in nephritic SLE remains unclear. The aim of this study was to investigate the effect of IL-15 on functional properties and associated renal damage of cytotoxic CD4 + CD28− T cell in lupus nephritis (LN).ResultsFlow cytometry showed that the number of circulating innate-like CD4 + CD28− T cells was increased in patients with nephritic SLE. Immunofluorescence showed CD4 + CD28− T cell infiltration in the kidney of LN patients, which was correlated with multiple clinicopathological features including estimated glomerular filtration rate (eGFR), proteinuria, the proportion of glomerulosclerosis and the degree of renal chronicity. In addition, a high level of IL-15 and IL15-expressing macrophage infiltration was detected in the periglomerular and intraglomerular tissues of LN patients, which enhanced the innate features, cytokine secretion and migratory capability of CD4 + CD28− T cells, and finally exerted direct TCR-independent cytotoxicity on glomerular endothelial cells in an IL-15-dependent manner in vitro.ConclusionOur study demonstrated that excessive IL-15 potentially promoted cytotoxic CD4 + CD28− T cell-mediated renal damage in LN. This finding may provide new insights into the potential association of premature aging and tissue damage in LN.

CircRNA-3302 promotes endothelial-to-mesenchymal transition via sponging miR-135b-5p to enhance KIT expression in Kawasaki disease

Endothelial-to-mesenchymal transition (EndMT) is implicated in myofibroblast-like cell-mediated damage to coronary artery wall of Kawasaki disease (KD) patients, which subsequently increases the risk of coronary artery aneurysm. Many circular RNAs (circRNAs) have been reported to be associated with cardiovascular diseases. However, the roles and underlying molecular mechanism of circRNAs in KD-associated EndMT remains indefinite. In this research, we screened out circRNA-3302 from human umbilical vein endothelial cells (HUVECs) treated by sera from healthy controls (HCs) or KD patients via circRNA sequencing (circRNA-seq). In addition, circRNA-3302 upregulation was verified in endothelial cells stimulated by KD serum and pathological KD mice modeled with Candida albicans cell wall extracts (CAWS). Moreover, in vitro experiments demonstrated that overexpression of circRNA-3302 could markedly induce EndMT, and silencing of circRNA-3302 significantly alleviated KD serum-mediated EndMT. To further explore the molecular mechanisms of circRNA-3302 inducing EndMT, RNA sequencing (RNA-seq), a dual-luciferase reporter system, nuclear and extra-nuclear RNA isolation, RT-qPCR and Western blot analyses and so on, were utilized. Our data demonstrated that circRNA-3302 contributed to the KD-associated EndMT via sponging miR-135b-5p to enhance KIT expression. Collectively, our results imply that circRNA-3302 plays an important role in KD-associated EndMT, providing new insights into minimizing the risks of developing coronary artery aneurysms.

Acute GVHD in liver and lungs is mediated by phenotypically-analogous but transcriptionally unique T cells.

Abstract During acute Graft-versus-Host Disease (aGVHD), donor T cells invade host tissues and cause multi-organ damage. However, the organ-specific drivers of pathogenic alloimmunity are still poorly understood. To uncover both the universal and organ-specific mechanisms driving T cell-mediated damage in target tissues during aGVHD, we analyzed the flow cytometric phenotype as well as transcriptomic signatures of T cells in healthy control animals, in autologous hematopoietic cell transplant (HCT) controls, and during aGVHD after allogeneic HCT, using our established non-human primate aGVHD model. In this study we focused on 2 aGVHD target organs: the liver and lungs. Using both supervised and unsupervised flow cytometric analysis, we discovered that, during aGVHD, both liver- and lung-infiltrating T cells demonstrated similar phenotypes dominated by mature effector-memory CD8 T cells. In contrast, transcriptomic analysis revealed organ-specific gene expression programming. Through Weighted Gene Co-expression Network Analysis (WGCNA), differential expression (DE) analysis, and immune pathway analysis, we identified that the lung aGVHD T cell transcriptomic program was specifically associated with extracellular matrix-remodeling and chemotaxis pathways. In contrast, in liver aGVHD, we found enrichment for metabolic stress responses and cytokine signaling pathways. These results suggest that at the transcriptomic level, T cells infiltrating different aGVHD-target organs are uniquely programmed and could be amenable to organ-specific therapeutic targeting. Supported by grants from the NIH (F31HL1562880)

Intestinal epithelial autophagy protects from cytokine-driven mortality and IFNγ-dependent cell death in acute small intestinal injury

Abstract Crohn’s disease (CD) is characterized by a breakdown of the intestinal mucosal barrier. A genetic polymorphism of the autophagy gene ATG16L1 is associated with increased risk of developing CD, and it impairs the protective and regenerative functions of the intestinal epithelium. Autophagy acts as a regulator of cell death and has been implicated in the protection from both necroptosis and apoptosis in the epithelium. Our work showed that mice specifically deleted for Atg16l1 in intestinal epithelial cells (Atg16l1ΔIEC) had exacerbated T cell-mediated intestinal damage in the model of anti-CD3 enteropathy compared to wild-type mice, which was characterized by crypt epithelial cell death and heightened inflammation. Moreover, Atg16l1 deficiency delayed the recovery of the intestinal epithelium, and Atg16l1-deficient IECs were impaired in their proliferative response. Pathology and mortality were largely driven by microbiota-dependent expression of Ifng, Tnf, and type I IFN response genes in Atg16l1ΔIEC mice. Mechanistically, while survival was rescued by blocking these cytokine pathways independently, only anti-IFNγ treatment abrogated intestinal epithelial cell (IEC) death in Atg16l1ΔIEC mice, thereby decoupling IEC death and survival. In summary, our findings suggest differential roles for IFNγ, TNF, and type I IFN in acute enteropathy and IEC death in the context of autophagy deficiency. Supported by grants from CIHR (MOP-12353, FDN-14333) and NSERC (RGPIN-201).

Human organoid models to study SARS-CoV-2 infection.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is one of the deadliest pandemics in history. SARS-CoV-2 not only infects the respiratory tract, but also causes damage to many organs. Organoids, which can self-renew and recapitulate the various physiology of different organs, serve as powerful platforms to model COVID-19. In this Perspective, we overview the current effort to apply both human pluripotent stem cell-derived organoids and adult organoids to study SARS-CoV-2 tropism, host response and immune cell-mediated host damage, and perform drug discovery and vaccine development. We summarize the technologies used in organoid-based COVID-19 research, discuss the remaining challenges and provide future perspectives in the application of organoid models to study SARS-CoV-2 and future emerging viruses.

CTLA4Ig/VISTAIg combination therapy selectively induces CD4+ T cell-mediated immune tolerance by targeting the SOCS1 signaling pathway in porcine islet xenotransplantation.

T cell inhibitory receptors can regulate the proliferation or function of T cells by binding to their ligands and present a unique opportunity to manage destructive immune responses during porcine islet xenotransplantation. We applied ex vivo porcine islet xenotransplantation and in vitro mixed lymphocyte-islet reaction models to assess immune checkpoint receptor expression profiles in recipient T cells, investigated whether CTLA4 or VISTA immunoglobulin (Ig) combination therapy alone could suppress porcine islet xenograft rejection and further analyzed its potential immune tolerance mechanism. Recipient T cells expressed moderate to high levels of CTLA4, PD-1, TIGIT and VISTA, and the frequency of CTLA4+ CD4+ , TIGIT+ CD4+ , VISTA+ CD4+ and VISTA+ CD8+ T cells was positively correlated with porcine islet xenograft survival time in xenotransplant recipients. Combined treatment with CTLA4Ig and VISTAIg selectively inhibited recipient CD4+ T cell hyper-responsiveness and proinflammatory cytokine production and significantly delayed xenograft rejection. SOCS1 deficiency in CD4+ T cells stimulated by xenogeneic islets facilitated hyper-responsiveness and abolished the suppressive effect of combination therapy on recipient T cell-mediated porcine islet damage in vivo and in vitro. Further mechanistic studies revealed that combined treatment significantly induced SOCS1 expression and inhibited the Jak-STAT signalling pathway in wild-type recipient CD4+ T cells stimulated by xenogeneic islets, whereas SOCS1 deficiency resulted in Jak-STAT signalling pathway activation in recipient CD4+ T cells. We demonstrated a major role for CTLA4 and VISTA as key targets in CD4+ T cell hyper-responsiveness and porcine islet xenograft rejection. The selective inhibition of CD4+ T cell immunity by CTLA4Ig/VISTAIg is based on SOCS1-dependent signalling.

Anti-Inflammatory and Pro-Autophagy Effects of the Cannabinoid Receptor CB2R: Possibility of Modulation in Type 1 Diabetes.

Type 1 diabetes mellitus (T1DM) is an autoimmune disease resulting from loss of insulin-secreting β-cells in islets of Langerhans. The loss of β-cells is initiated when self-tolerance to β-cell-derived contents breaks down, which leads to T cell-mediated β-cell damage and, ultimately, β-cell apoptosis. Many investigations have demonstrated the positive effects of antagonizing cannabinoid receptor 1 (CB1R) in metabolic diseases such as fatty liver disease, obesity, and diabetes mellitus, but the role of cannabinoid receptor 2 (CB2R) in such diseases is relatively unknown. Activation of CB2R is known for its immunosuppressive roles in multiple sclerosis, rheumatoid arthritis, Crohn’s, celiac, and lupus diseases, and since autoimmune diseases can share common environmental and genetic factors, we propose CB2R specific agonists may also serve as disease modifiers in diabetes mellitus. The CNR2 gene, which encodes CB2R protein, is the result of a gene duplication of CNR1, which encodes CB1R protein. This ortholog evolved rapidly after transitioning from invertebrates to vertebrate hundreds of million years ago. Human specific CNR2 isoforms are induced by inflammation in pancreatic islets, and a CNR2 nonsynonymous SNP (Q63R) is associated with autoimmune diseases. We collected evidence from the literature and from our own studies demonstrating that CB2R is involved in regulating the inflammasome and especially release of the cytokine interleukin 1B (IL-1β). Furthermore, CB2R activation controls intracellular autophagy and may regulate secretion of extracellular vesicles from adipocytes that participate in recycling of lipid droplets, dysregulation of which induces chronic inflammation and obesity. CB2R activation may play a similar role in islets of Langerhans. Here, we will discuss future strategies to unravel what roles, if any, CB2R modifiers potentially play in T1DM.

Monoclonal Antibody Targeting the HA191/199 Region of H1N1 Influenza Virus Mediates the Damage of Neural Cells.

Vaccination is the most effective mean of preventing influenza virus infections. However, vaccination-induced adverse reactions of the nervous system, the causes of which are unknown, lead to concerns on the safety of influenza A vaccine. In this study, we used flow cytometry, cell ELISA, and immunofluorescence to find that H1-84 monoclonal antibody (mAb) against the191/199 region of the H1N1 influenza virus hemagglutinin (HA) protein binds to neural cells and mediates cell damage. Using molecular simulation software, such as PyMOL and PDB viewer, we demonstrated that the HA191/199 region maintains the overall structure of the HA head. Since the HA191/199 region cannot be removed from the HA structure, it has to be altered via introducing point mutations by site-directed mutagenesis. This will provide an innovative theoretical support for the subsequent modification the influenza A vaccine for increasing its safety.Electronic supplementary materialThe online version contains supplementary material available at 10.1134/S0006297921110109.

230 Preclinical efficacy of CLEC-1 antagonist as novel myeloid immune checkpoint therapy for oncology

BackgroundC-type lectin receptors (CLRs) are powerful pattern recognition receptors shaping immune cell-mediated tissue damage by positively or negatively regulating myeloid cell functions and hence tumor elimination or evasion. We previously reported that the orphan CLR CLEC-1 expressed by dendritic cells (DCs) tempers T cell’s responses in vivo by limiting antigen cross-presentation by cDC1. Furthermore, we observed that CLEC-1 is highly expressed by myeloid cells purified from human tumor microenvironment, in particular tumor-associated macrophages.MethodsMacrophages were generated from monocytes of healthy volunteers for phagocytosis assays. MC38 and Hepa 1.6 murine tumor cells were implanted in Clec1a KO or KI mice for immunotherapeutic treatment evaluation.ResultsUsing newly developed anti-human CLEC-1 monoclonal antibodies (mAbs), we found that antagonist anti-CLEC-1 mAbs with the capacity to block CLEC-1/CLEC-1Ligand interaction, as opposed to non-antagonist CLEC-1 mAbs, increase the phagocytosis of CLEC-1Ligand-positive human tumor cells by human macrophages, in particular when opsonized by tumor-associated antigen mAbs (Rituximab, Cetuximab, Trastuzumab) or with anti-CD47 mAb (Magrolimab). In-vivo, CLEC-1 knock-out (KO) mice (n=19) display significant prolonged survival in monotherapy as compared to wild-type littermates (n=12) in an orthotopic hepatocellular carcinoma (HCC) model and anti-tumor memory responses was demonstrated by tumor rechallenge in cured mice. CLEC1 KO mice also illustrate significant eradication of MC38 colorectal tumors in combination with chemotherapy promoting CLEC-1Ligand expression by tumor cells (n=16 with Gemcitabine or n=11 with Cyclophosphamide). HCC tumor microenvironment analysis after 2 weeks of tumor implantation shows significantly higher number of CD8+ and memory CD8+ T cells with reduced PD1 expression in CLEC1 KO animals (n=16 versus n=12 for KO vs WT mice respectively). Finally, we recently generated human CLEC-1 knock-in mice expressing the extracellular human CLEC1 domain fused to the intracellular mouse CLEC1 tail and confirmed preclinical efficacy in vivo with anti-human CLEC1 antagonist mAb in monotherapy in the orthotopic HCC model.ConclusionsThese data illustrate that CLEC-1 inhibition represents a novel therapeutic target for immuno-oncology modifying T cell immune responses and tumor cell phagocytosis by macrophages.