bluuebunny/crossref_metadata_2025 huggingface dataset:
IRF1 regulates IFN-λ1 expression in MDA5-mediated suppression of the HBV replication
Authors: Kuo Yali , Ng Jenny , Lu Hsin-Lin , Liao Fang
Published: January 2023

MDA5, a member of the RIG-I like receptor family, has been shown to sense HBV DNA and trigger the production of anti-viral molecules to clear the virus. However, the mechanism underlying the suppression of HBV replication mediated by MDA5 signaling remains to be investigated. Our transcriptome analysis using RNA-seq revealed that IFN-λ1, a member of type III interferons, was the most upregulated molecule in Huh7 cells co-transfected with HBV DNA and MDA5 expression plasmid compared to with HBV DNA and RIG-I expression plasmid. Here we aim to investigate the IFN-λ1 expression in MDA5-mediated suppression of HBV replication. Huh7 cells co-transfected with HBV DNA and MDA5 expression plasmid led to increased IFN-λ1 expression associated with decreased HBsAg and HBV RNA. On the other hand, knockdown of MDA5 dramatically impaired IFN-λ1 expression. We next investigated the transcription factors involved in regulating IFN-λ1 expression. IRF1, but not IRF3 and IRF7, was upregulated in Huh7 cells co-transfected with HBV DNA and MDA5 expression plasmid. Knockdown of IRF1 led to reduced IFN-λ1 expression along with reduced inhibitory effect on HBV replication. The promoter region of IFN-λ1 contains ISREs, potential sites for IRF binding. However, ChIP and luciferase reporter assays showed no evidence of direct binding of IRF1 to the ISRE sites of IFN-λ1 promoter. Therefore, it is likely that IRF1 regulates MDA5-mediated IFN-λ1 expression through other mechanism. Mechanism(s) on IRF1 regulating IFN-λ1 expression in MDA5-mediated suppression of HBV replication is currently under investigation.
A novel role for TRIM6 and unanchored ubiquitin in inflammatory disease during virus infection via the PI3K-AKT signaling pathway
Authors: Aguilera-Aguirre Leopoldo , Giraldo Maria I. , Kalveram Birte , Hage Adam , Liang Yuejin , Freiberg Alexander Niclas , Rajsbaum Ricardo
Published: January 2023

The innate immune response is essential to protect the host against virus infections. This response involves activation of signaling pathways leading to inflammatory and antiviral cytokines, which in excess can lead to tissue damage and disease. Therefore, these signaling pathways are regulated by different molecular processes, including the ubiquitin (Ub) system. The TRIM family of E3-Ub ligases has been associated with innate immune signaling. We reported that TRIM6 catalyzes the synthesis of unanchored K48-linked poly-Ub chains, which are not covalently attached to any protein, and promote antiviral type-I Interferon (IFN-I) responses. Here, we generated Trim6−/− mice to study its role in immune responses to two different pathogenic viruses, Influenza virus (IAV) and Ebola virus (EBOV). Surprisingly, Trim6−/− mice have less signs of pathology upon IAV infection even though there are increased IAV titers at early time points post-infection. Next-Generation Sequencing and cytokine multiplex analysis revealed reduced mRNA and protein expression levels of CXCL1, a potent neutrophil chemoattractant. Accordingly, neutrophil infiltration to the lungs was reduced in virus-infected Trim6−/− mice, and blockade of the CXCR1/2 receptors, as well as neutrophil depletion, ameliorated disease caused by both EBOV and IAV in WT but not Trim6−/− mice. Mechanistically, TRIM6 and unanchored Ub form a complex with PI3K-AKT signaling components and their phosphorylation is impaired in Trim6−/− cells leading to reduced CXCL1 expression. Our study uncovers a novel mechanism by which viruses can cause disease and suggest that inhibition of CXCL1 signaling may be used as a therapeutic strategy to highly pathogenic viruses.
Unique MyD88/TRAM signaling post TLR2/6 recognition of conserved viral architectures results in anti-viral immunity and improved clearance of secondary bacterial infection
Authors: Hatton Alexis Alexandria , Shepardson Kelly , Wang Yang , Johns Laura Logan , Goodall Cheri , Douglas Trevor , Rynda-Apple Agnieszka
Published: January 2023

Respiratory virus infections can either reduce or increase host susceptibility to subsequent secondary bacterial infections (SBI). Recently, we discovered that the anti-viral immune response initiated by the recognition of exogenous pattern of virus architectures, namely the repeating protein subunit pattern (RPSP), results in improved clearance of S. aureus during SBI in mice. The RPSP is likely conserved in all viruses, suggesting a generalized mode of viral pattern recognition not specific to any single virus. Thus, we identified RPSP as a new PAMP and found it to be recognized by the TLR2/6 heterodimer on the surface of macrophages. We found that this RPSP recognition occurs prior to particle internalization and independent of virus infection. TLRs are known to signal from either the cell surface, through MyD88/Mal, or from the endosome, through TRAM/TRIF, for the induction of inflammatory responses or type I IFNs, respectively. We found that when compared to WT macrophages, tram−/− macrophages exhibited diminished RPSP internalization by confocal microscopy and flow cytometry. However, blocking endosomal acidification by Bafilomycin A1 did not reduce S. aureus killing by RPSP-treated macrophages. This suggests that a non-canonical endosomal signaling pathway may be induced upon RPSP exposure. Indeed, using knockout mice we found that type I IFN, but also MyD88 signaling are critical for improved S. aureus clearance post RPSP. Our results indicate that the recognition of RPSP by TLR2/6 activates a unique intracellular signaling pathway, specifically a combination of the two known pathways, that results in type I IFN-dependent improvement in S. aureus killing by macrophages.
Defining the role of mucosal-associated invariant T (MAIT) cells in B cell help
Authors: Jensen Owen , Trivedi Shubhanshi , Meier Jeremy D. , Leung Daniel T.
Published: January 2023

Pathogen-associated diarrheal diseases account for over 1.6 million deaths annually, yet the number of effective mucosal vaccines remains limited. An exciting potential target for mucosal vaccine adjuvants are mucosal-associated invariant T (MAIT) cells. MAITs are innate-like T cells which are found in the mucosa, blood, and secondary lymphoid organs (SLO), and can respond rapidly to conserved microbial antigens through cytokine secretion and cytotoxic effects. Recent evidence supports a role for MAITs in B cell help. We aim to improve mucosal vaccines by investigating a novel subset of T follicular helper (Tfh)-like MAIT cells and their ability to aid in B cell help and antibody mediated immunity. We describe Tfh-like MAITs from human blood and tonsil samples using linked TCR and phenotype single cell RNAseq, and in vitro culture assays. We further investigate the sufficiency of MAITs aid in antibody mediated immunity in vivo using a MAIT adoptive transfer model into Tcratm1Mom/J (αβ T cell deficient) mice followed by mucosal bacterial challenge. We identify a subset of CXCR5+ MAITs, enriched in human tonsils and murine SLOs that express the Tfh lineage defining transcription factor, BCL6, co-stimulatory markers and cytokines including IL-10 and IL-21 and can aid in B cell help in vitro. Likewise, in vivo we find that MAIT transfer induces pathogen-specific IgA but not IgG responses following mucosal challenge. Overall our study uncovered a novel subset of Tfh-like MAITs and has demonstrated the capacity of MAITs to provide help in antibody responses. Current work aims to uncover the mechanism of MAIT-B cell help which could critically inform efforts to target this population as mucosal adjuvants.
The protective role of B cell anergy in the development of Atherosclerosis
Authors: Waseem Tayab , Keeter W. Coles , Moriarty Alina , Mussbacher Marion , Edemobi Stefan , Getahun Andrew , Cambier John C , Galkina Elena
Published: January 2023

Anergy is critical for silencing autoreactive B cells and preventing autoimmune diseases. Very little is known about the induction and maintenance of anergic B cells within atherosclerosis. To assess the role of B cell anergy in atherosclerosis, we decided to use the Ars/A1 transgenic model, where anergic B cells express receptors that bind to self-antigen and hapten p-azophenylarsonate (Ars). To further differentiate the role of anergy versus BCR signaling we used MD4 mice, which respond normally to antigen, but have a transgenic BCR specific for hen egg lysosome. Ars/A1, MD4, and BL/6 littermate controls were injected with AAV-PCSK9 to increase circulating cholesterol levels and fed a western diet (WD) for 16 weeks. While there were no significant differences in weight or total plasma cholesterol levels, Ars/A1 mice had a significant decrease in plaque burden compared to littermate controls. Interestingly we observed an increase in B-regulatory cells in Ars/A1 mice compared to BL/6 and MD4 mice. With recent reports suggesting that anergic B cells dampen the overall immune response our data suggests that anergy and not B cell signaling is the important mechanism of controlling the inflammatory environment in atherosclerosis.
Regulatory T cells suppression of antigen-specific CD8+ T cells in vivo is contact-dependent
Authors: Mansoori Mohammad Nizam , Shevach Ethan Menahem
Published: January 2023

T Regulatory cells (Tregs) plays an important role in regulating immune mediated responses against self and foreign antigens by various known and unknown mechanisms. Recent studies have shown that antigen-specific iTreg suppress CD4+ effectors T cells in vitro and in vivo by depleting peptide-MHC-II complexes from the DC surface. These, results raise questions as to how Treg suppress CD8+ T effectors, particularly in vivo. To explore Treg mediated suppression of CD8+ T cells, we generated CD4+ iTregs from OT-II mice specific for Ova323–339 in association with I-Ab and determined their capacity to suppress CD8+ T cells from OT-I mice specific for Ova257–264 in association with H-2Kb. CD4+ OT-II iTreg suppressed the in vitro proliferation of OT-I T cells in the presence of Dendritic Cells (DCs) pulsed with both Ova323–339 and Ova257–264 or with DCs singly pulsed with each peptide. Surprisingly, the expansion of OT-I CD8+ T cells in vivo was not suppressed by OT-II iTreg when the two peptides were presented on separately pulsed DCs but was suppressed when both peptides were presented on the same DCs. These results strongly suggest that suppression of the expansion of CD8+ T cells in vivo requires close contact between Treg and responder CD8+ T cell on the surface of the same DC and raise the possibility that Treg may remove a large molecular complex from the DC surface by a process of trogocytosis.
Mechanistic mathematical representation of CD8+ T cell mediated drug-induced liver injury (DILI) Part 2: translation from experimental antigen, ovalbumin (OVA), to drug, amodiaquine (AQ) response
Authors: Kenz Zackary , Battista Christina , Shoda Lisl
Published: January 2023

Idiosyncratic DILI (iDILI) is poorly understood and costly to patients and drug developers. Some iDILI events appear immune-mediated. This work builds on the DILIsym representation of T cell cytotoxicity against OVA-expressing hepatocytes1, to translate the OVA representation to AQ-induced hepatotoxicity.

AQ-related changes include a physiologically-based pharmacokinetic (PBPK) model for mouse AQ exposure, AQ metabolism to reactive metabolites (RMs), RM-mediated ER stress leading to inflammation, fewer endogenous AQ responsive T cells of reduced affinity and avidity for AQ antigen.

Simulations show no CD8+ T cell mediated ALT elevations in simulated wild-type mice treated with AQ, consistent with data2 and provide a mechanistic basis for the result. With low levels of AQ antigen, low T cell numbers and lower affinity and avidity interactions were insufficient for a cytotoxic T lymphocyte (CTL) response. With high levels of AQ antigen, T cell expansion and differentiation occurred but regulatory pathways, i.e., T cell exhaustion, inhibited an effective CTL response. In PD1−/− mice treated with anti-CTLA4, CD8+ T cell mediated AQ DILI was apparent (ALT >100 U/L)3,4. In DILIsym, blocking coinhibitory activity on proliferation and differentiation was permissive for AQ DILI (ALT >100 U/L) and could be further exacerbated by blocking differentiation to an exhausted phenotype (ALT >200 U/L).

The results provide proof-of-concept for translation of OVA hepatotoxicity to AQ DILI and set the stage for exploration of human AQ DILI5.
Metabolic analysis of tyrosine-kinase inhibitor effect on T cell activation
Authors: Kam Yoonseok , Winer Lisa , Rogers George W , Romero Natalia , Hynes James
Published: January 2023

Tyrosine kinase inhibitors have been actively investigated as potential therapies for various cancers including blood tumors. However, as T cell activation can also be mediated by various tyrosine kinases, relevant inhibitors are also now being considered as pharmacological modulators of CAR-T cells; an example being dasatinib, which has been found to modulate CAR-T activity through Lck inhibition. T cell activation and subsequent differentiation are tightly associated with cellular metabolism, with T cells showing dynamic changes in two primary metabolic activities of glycolysis and mitochondrial respiration throughout the T cell life cycle. This metabolic response begins with an immediate early increase in glycolytic activity which has been shown to be an essential and signature response to activation. In this study, this fundamental biological process is exploited using the Agilent Seahorse XF technology, illustrating how activation can be measured in real-time in response to anti-CD3/CD28 activator injection, and further, how this parameter can be used to conveniently determine the modulatory effect of kinase inhibitors on T cell activation. Consistent with previous observations that kinase inhibitors can suppress naive T cell activation in vitro, here a similar kinase dependency in re-activation of resting (pre-activated) T cells was observed. Several kinase inhibitors also were able to downregulate the glycolytic rate elevated when applied post-activation. Taken together these data suggest that real-time metabolic analysis is a valuable tool in the characterization of putative T cell modulators, including tyrosine kinase inhibitors, and has the potential to inform the design of specific CAR-T modulators.
Metabolic regulation of cytokine production in leishmania resistance and susceptibility
Authors: Bodhale Neelam Prakash , Sarkar Arup , Chattopadhyay Debprasad , Saha Bhaskar , Bhattacharyya Maitree
Published: January 2023

Leishmania, a protozoan parasite, ensures its survival inside its mammalian host by modulating host metabolism and immune response. Recent advances in the field of immune-metabolism have shown that the metabolic enzymes and metabolites have immune-modulatory functions. Leishmania infection of a susceptible host skews immune response towards Th2-type, enhances production of pro-parasitic cytokines IL-10 and IL-4 but reduces IFN-gamma and IL-12 production. By contrast, Leishmania infection of a resistant host sets an immune response bias towards Th1-type, results in high production of pro-inflammatory IL-12 and IFN-gamma, which activates macrophages to kill the intracellular amastigotes. To understand the role of metabolic factors in Leishmania susceptibility or resistance, we used L. donovani susceptible or resistant mouse strains BALB/c or 129/Sv, respectively. The mice were first intravenously infected with L. donovani, followed by intra-peritoneal treatment with different inhibitors of metabolic pathways. The splenocytes from the treated resistant and susceptible mouse strains were studied for in-situ cytokine response and T cell subsets by FACS. Also the mouse serum was used for cytokine estimation by ELISA. The results show that different metabolic pathways regulate cytokine production in L. donovani-infected mouse suggesting their role in resistance and susceptibility to Leishmania infection.
IL-22 Effects on Osteoblast Mineralization and Metabolic Profile
Authors: Holt Vance E , Luo Yongquan , Colbert Robert A
Published: January 2023

Ankylosing spondylitis (AS) is an immune-mediated inflammatory disease characterized by persistent entheseal inflammation and aberrant bone formation that can lead to vertebral fusion. The Type 17 cytokine IL-17 and IL-10 family member IL-22 are involved in pathogenesis. Moreover, IL-22 has been implicated in aberrant bone formation, but its precise role remains unclear. Mesenchymal stem cells (MSCs) differentiate into osteoblasts (OBs) and are believed to reside near entheseal tissues and contribute to the observed bone formation. They express the IL-22 receptor after commitment to the OB lineage. The objective of this study was to determine how IL-22 affects OB development and mineralization in vitro. MSCs were placed in osteogenic media and then treated with IL-22 for up to 21 days after pre-conditioning with IFNg, TNF, or IFNg+TNF (I+T) for 72 hours. Mineralization was measured with Alizarin red staining, gene expression with Nanostring or RNA-seq. IL-22 significantly increases mineralization in cells pre-conditioned with I+T, compared to I+T alone or IL-22 alone, which has no effect. There was no significant acute effect of IL-22 on expression of osteogenic genes during early osteoblast differentiation in I+T treated cells. In contrast, we found that IL-22 induces a glycolytic shift within 24 hours in pre-conditioned early osteoblasts using Seahorse analysis. Given the recently recognized role of metabolic change in osteogenesis, these results suggest a novel effect of IL-22 on osteogenesis and mineralization that may be mediated by increased glycolysis. Further studies are aimed at investigating mechanisms that facilitate these effects and linking the increased matrix deposition with the metabolic shift caused by IL-22.
Progressive Immunologic Dysregulation with Exposure to Tobacco Smoke in a Model of Chronic Obstructive Pulmonary Disease (COPD)
Authors: Wu Ching-Wen , Wang Lei , Bolton Sarah , Peake Janice L. , Pinkerton Kent E.
Published: January 2023

Our research objective is to examine the role of dysregulated immune responses in disease progression and changes in lung physiology in a rat model of COPD. Spontaneously hypertensive rats were exposed to tobacco smoke (TS) (90 mg/m3) or filtered air (FA) for up to 12 weeks. Necropsy timepoints of 4, 6, 8, 10 and 12 weeks were chosen to define the immunological and pathophysiological changes in the lung. Rats exposed to TS showed significant increases in macrophage, neutrophil and lymphocyte number in bronchoalveolar lavage compared to FA at all weeks examined. Proinflammatory cytokines IL-1β, IL-6 and IFN-γ in whole lung homogenates were significantly increased following exposure to TS compared to FA controls. Physiological airway obstruction due to TS exposure was noted by significant elevations in central airway resistance and tissue damping. Central airway resistance was significantly increased at 4 weeks, decreased at 6 weeks, but returned to significantly elevated levels at 10 and 12 weeks following TS exposure. Tissue damping showed a peak at 4 weeks, then a decreasing trend, although remaining significantly elevated through 12 weeks of TS exposure. Strong positive correlations were noted between proinflammatory cytokines IL-1β, IL-6, IFN-γ, TNF-α and the degree of tissue damping. These changes were associated with increases in central airway squamous epithelial metaplasia and distal airway mucous metaplasia. These findings suggest progressive airway obstruction with an abnormal inflammatory response to TS exposure in this rodent model of COPD. We conclude a dysregulated immunologic response, along with airway inflammation are strongly associated with the impairment of physiological lung function in TS-exposed rats.
GPR65, a Potential Therapeutic Target for Th17-mediated Autoimmune Diseases, is Regulated by the CREB/CRTC2 Pathway
Authors: Hernandez Jeniffer B , Cave Si , Decker Carl , Swanson Jack , Yang Qiyuan , Zheng Ye , Montminy Marc
Published: January 2023

The cAMP response element-binding (CREB) protein has emerged as an important regulator of immune function. We have previously shown that CREB, along with its co-activator CRTC2, modulates autoimmune disease by promoting differentiation of the pro-inflammatory T cell, Th17. Th17 cells have been linked to the development of autoimmune diseases including multiple sclerosis, inflammatory bowel disease, psoriasis, rheumatoid arthritis, and asthma. The CREB pathway is induced by a variety of inflammatory signals, growth factors, and hormones that leads to the transcription of genes with cAMP-response elements. Through RNAseq, we identified multiple genes that may be regulated by CREB in Th17 cells. Here we show that the G-Protein-Coupled Receptor (GPCR), GPR65 is highly expressed in Th17 cells and is regulated by the CREB/CRTC2 pathway. The development of GPR65 antagonists may be a novel therapeutic avenue for the treatment of Th17-mediated autoimmune diseases.
Negative regulation of Ifng expression by MLL4 through CNS-28
Authors: REN GANG , CUI KAIRONG , LIU Chengyu , Zhu Jinfang (Jeff) , ZHAO KEJI
Published: January 2023

Interferon-g (IFN-g) is a key cytokine produced by type 1 T helper (Th1) cells in response to viral or intracellular bacterial infections in mammals. While a number of positive regulatory elements have been well studied, no negative regulatory elements at the Ifng locus for its expression have been identified. Here we report a negative regulatory mechanism of Ifng expression by MLL4, a histone methyltransferase responsible for H3K4me1. Deletion of Mll4 resulted in loss of H3K4me1 signals at the previously unrecognized CNS-28 site and led to enhanced IFN-g expression during Th1 differentiation. GATA3 bound to the CNS-28 site and deletion of Gata3 resulted in a similar upregulation of IFN-g with Mll4 deletion. Furthermore, deletion of Mll4 compromised GATA3 binding to CNS-28 and led to a substantial reorganization of the 3D chromatin structure at the Ifng genomic locus in both naïve CD4+ T cells and Th1 cells. Thus, we conclude that MLL4 negatively regulates Ifng expression by facilitating GATA3 binding to the CNS-28 site and maintaining a special local chromatin organization.
Melatonin inhibits Th17 differentiation
Authors: Davis Gwendolyn K. , Madhur Meena S.
Published: January 2023

Hypertension is a leading risk factor for cardiovascular disease, which is a major cause of death worldwide. T cells are required for the development of hypertension. Specifically, we showed that T helper 17 (Th17) cells produce the pro-inflammatory cytokine, IL-17A, which leads to sustained elevations of blood pressure and renal and vascular dysfunction in experimental hypertension. T cell differentiation and trafficking is regulated by circadian clocks. High fat and high salt intake, typical of Western diets, disrupt circadian rhythms and are associated with increased risk for developing cardiovascular disease. These diets are also associated with an increase in Th17 cell differentiation. Melatonin is a major circadian hormone with anti-inflammatory and anti-oxidant actions. To determine the interplay between circadian rhythms and Th17 cell differentiation, we tested the hypothesis that in vitro treatment of Th17 cells with melatonin would inhibit IL-17A cytokine production. Splenic naïve CD4+ T cells from wild type C57Bl/6J male mice were cultured for 72 hours on anti-CD3/anti-CD28 antibody coated plates in the presence or absence of Th17-polarizing cytokines and treated with melatonin (2ng/ml; Sigma Aldrich) or vehicle (0.02% DMSO). IL-17A cytokine levels were analyzed by ELISA. In cells treated with Th17 polarizing cytokines, IL-17A cytokine production was significantly reduced by melatonin. Thus, this study highlights the involvement of circadian control of Th17 cells via the actions of melatonin to suppress Th17 differentiation and IL-17A production. Future studies will determine the effect of melatonin treatment in vivo on development of hypertension and renal/vascular dysfunction.
Opioid-induced protein coding and noncoding transcriptome of human CD8+ T cells reveals opioid receptor subclass specific responses
Authors: Mazahery Claire , Llorens Braulio , Peczak Anna , Valadkhan Saba , Levine Alan D.
Published: January 2023

Opioid peptides are released at sites of injury; their cognate G-protein coupled opioid receptors (OR) are expressed on immune cells. Conflicting reports attribute immunostimulatory and immunosuppressive activity to opioids. From a cohort of methadone patients and controls, we found that chronic opioid use disrupts CD8+ T cell subset balance, via decreased T Effector Memory RA+ cells. Exposure of CD8+ T cells ex vivo to a μ-OR or δ-OR agonist differentially regulates thousands of protein coding genes. Gene set enrichment analysis reveals that μ-OR more strongly regulates cellular processes, including immune response via interferon, IL-2, and mTOR signaling pathways. A striking finding is the linkage of μ-OR, but not δ-OR, signaling to the upregulation of lipid, cholesterol, and steroid biosynthesis. OR-specific gene signatures associate with different transcription factors, supporting our hypothesis that transcriptional regulation underlies cellular outcomes ascribed to individual ORs. The noncoding transcriptome of opioid exposed cells is largely unique to each OR; μ-OR regulates 53 long noncoding RNAs, while δ-OR regulates 59, with only three shared transcripts, one of which is inversely regulated. The noncoding RNA upregulated by both ORs enhances integrin expression and binding; the noncoding RNA downregulated by both ORs represses STAT signaling and apoptosis. Comparison of CD4 vs. CD8 T cell responses to ligation of all three ORs (μ, δ, κ) reveals lineage specific regulation of TNF-signaling and fatty acid metabolism in response to μ-OR, G protein and cytokine receptor signaling in response to δ-OR, and REDOX pathways in response κ-OR, indicating that opioid induced immune regulation is both cell type and OR specific.
Developmental relationships of four exhausted CD8 T cell subsets reveals underlying transcriptional and epigenetic control mechanisms
Authors: Beltra Jean-Christophe , Manne Sasikanth , Hakeem Mohamed S Abdel , Kurachi Makoto , Giles Josephine R , Chen Zeyu , Casella Valentina , Ngiow Shin , Khan Omar , Huang Yinghui Jane , Yan Patrick , Nzingha Kito , Huang Alexander C. , Wherry E. John
Published: January 2023

Exhausted CD8 T cells (TEX) are essential during chronic viral infections and cancer. Two TEX subpopulations including a progenitor and a more terminally exhausted subset cooperate to maintain an active immune response during antigen persistence. However, non-overlapping delineations of these populations have suggested a more complex developmental biology. Here, using the LCMV mouse model of chronic viral infection, we identify four distinct TEX subsets based on Ly108 (Slamf6) and CD69 expression revealing a novel stepwise developmental framework. We reveal the transcriptional and epigenetic control mechanisms and associated biological changes underlying each TEX subset transition. Two TCF1+ progenitors were identified along with a novel TCF1-intermediate subset that re-engaged some aspects of effector biology. This subset depended on T-bet and was re-invigorated upon PD-L1 blockade. Ultimately, Tox coordinated loss of T-bet and differentiation into a fourth, terminally exhausted subset. These data define a new developmental hierarchy of TEX and reveal distinct biological properties with direct relevance to immunotherapy. Defining the control mechanisms of this TEX subset hierarchy provides novel opportunities to manipulate TEX biology for clinical goals.
USP22 controls iNKT immunity partially through MED1-mediated suppression of target-specific histone H2A monoubiquitination
Authors: Zhang Yana , Fang Deyu
Published: January 2023

The development and functions of CD1d-restricted iNKT cells are critical for both innate and adaptive immunity. While the ubiquitin pathway has been shown to regulate iNKT cell development and functions, the deubiquitinase involved in this process has not been identified. Herein we found that the ubiquitin-specific peptidase 22 (USP22) is highly expressed in iNKT cells during their early developmental stage I. USP22-deficiency blocked the transition from stage I to II during iNKT cell development in a cell-intrinsic manner. In addition, genetic USP22 suppression largely diminishes IL-17-producing iNKT17 and IFN-g-producing iNKT1 differentiation but favors IL-4 producing iNKT2 polarization. In contrast, genetic USP22 suppression did not alter conventional CD4 T cell clonal expansion and differentiation. At the molecular level, USP22 interacts with the Mediator of RNA polymerase II transcription subunit 1 (MED1), a transcription co-activator involved in iNKT cell development. Interestingly, while interacting with MED1, USP22 does not function as a deubiquitinase to suppress MED1 ubiquitination for its stabilization. Instead, USP22 enhances MED1 functions for IL-2Rb and T-bet gene expression through deubiquitinating histone H2A but not H2B mono-ubiquitination. Therefore, our study revealed USP22-mediated histone H2A deubiquitination fine-tunes MED1 transcriptional activation as a previously unappreciated molecular mechanism to control iNKT development and functions.
mTORC1 inhibition promotes human Treg differentiation via privileged mRNA translation
Authors: Baos Sandra Perez , Volta Viviana , Ernlund Amanda , Gadi Abhilash , Schneider Robert J
Published: January 2023

Maturation of regulatory T cells (Tregs) in peripheral sites is known to require TGFβ exposure and inhibition of protein kinase mTORC1. It is well known that mTOR inhibition is associated with repression of cap-dependent mRNA translation, which represents the major mechanism for protein synthesis, leaving unanswered how Tregs carry-out essential translation for development and immune suppression activity. To answer this question, we performed genome-wide transcription and translation profiling in CD4+CD127dim/− CD25+Tregs derived from anti-CD3/CD28-activated human naïve CD4+ T cells, treated with the mTORC1 inhibitor RAD001 and/or TGFβ.

We found that TGFβ activated both Treg differentiation and immune suppression genes, while mTORC1 inhibition selectively blocked translation of most T cell mRNAs except those induced by TGFβ, including FOXP3, CTLA-4, CD101 or CD103, locking in Treg lineage commitment and immune suppression function. These canonical Treg fate-determining mRNAs were resistant to mTORC1 inhibition, an effect mediated in part by their 5′-untranslated regions through an alternate form of appears to be cap-dependent, eIF4E-independent mRNA translation.

In conclusion, TGFβ transcriptional reprogramming together with mTORC1-independent translational reprogramming enable a privileged translation mechanism by which activated CD4+ T cells become Tregs.
Targeting c-Rel O-GlcNAcylation to control hyperglycemia-dependent transcription in T lymphocytes
Authors: Ramakrishnan Parameswaran , Centore Joshua , de Jesus Tristan , Church Derek , Pokorski Jonathan
Published: January 2023

The NF-kappaB protein c-Rel plays a critical role in controlling regulating T cell function and T regulatory cell development. We found that hyperglycemia increases O-GlcNAcylation of c-Rel at serine residue 350 and enhances the transcription of c-Rel-induced CD28RE-dependent pro-autoimmune cytokines, interleukin-2 (IL-2), interferon gamma (IFNG) and granulocyte macrophage colony stimulating factor (GM-CSF). Our recent results show that the regulatory effect of c-Rel O-GlcNAcylation is gene dependent and it suppresses the transcription of forkhead box P3 (FOXP3) expression in T cells. Mechanistically, we found that O-GlcNAcylation enhances the DNA binding of c-Rel at CD28RE and decreases it’s binding at the FOXP3 promoter. To specifically block O-GlcNAcylated c-Rel function, we developed a peptoid, called peptoid3 that specifically binds to O-GlcNAcylated c-Rel by molecular modeling and de novo synthesis. We found that peptoid3 treatment significantly decreased T cell receptor-induced, O-GlcNAcylation-dependent expression of proautoimmune cytokines and enhanced FOXP3 expression in T cells. This study reveals c-Rel S350 O-GlcNAcylation as a novel molecular mechanism inversely regulating proautoimmune gene expression and immunosuppressive FOXP3 expression in T cells with potential therapeutic implications in conditions such as type 1 diabetes.
SELENOI is important for maintenance of phosphatidylethanolamine and glycosylphosphatidylinositol anchor protein during T cell activation and immune response
Authors: Ma Chi , Hoffmann Fukun , Hoffmann Peter
Published: January 2023

Selenoproteins are important for both innate and adaptive immune responses. As one of the selenoprotein members, SELENOI is characterized by its ethanolamine phosphotransferase activity in the Kennedy pathway of phosphatidylethanolamine (PE) synthesis and PE is also the donor of phosphoethanolamine moiety in glycosylphosphatidylinositol anchor protein (GPI-AP). To explore how SELENOI is involved in the immune response, we generated three mouse models with different SELENOI expression levels, including defined selenium diets, doxycycline inducible SELENOI knockdown (KD) and T cell specific SELENOI knockout (KO) mice models. We analyzed both the quantity of PE species on activated CD3 T cell membrane by LCMS and GPI-AP expression on activated T cell surface by FACS in all these three models. The SELENOI KO T cells exhibit a lower level of PE species on T cell membrane compared to WT T cells, and reduced expression of GPI-anchored proteins including CD52, CD73, CD48, CD109 and CD44 on cell surface upon activation as well. We also detected the same pattern of reduced PE species and GPI-anchor proteins on defined selenium diets and doxycycline-inducible SELENOI KD mouse models. Furthermore, the SELENOI KO T cells exhibited a reduced proliferation rate upon CD3/CD28 co-stimulation. Vaccination with LCMV peptides in SELENOI deficient mice results in less CD8+ and CD4+ LCMV specific T cells and reduced IFN-γ production, indicating an impaired immune response when SELENOI is absent. These results indicate that SELENOI is crucial for both PE and GPI-AP synthesis during T cell activation and proliferation due to the importance of PE during the activation process.
Sex bias in maternal immune activation-induced neurodevelopmental disease begins at the maternal-fetal interface
Authors: Frost Elizabeth L , Lammert Catherine R. , Johanson David M. , Zunder Eli R. , Lukens John R.
Published: January 2023

Systemic maternal inflammation during pregnancy is increasingly thought to be a risk factor for development of autism spectrum disorder (ASD), which is diagnosed at a rate 4-fold higher in males than in females. Administration of the viral mimic polyI:C to pregnant mice at mid-gestation leads to an ASD-like phenotype in the offspring, consisting of deficits in socialization and communication as well as repetitive behaviors. In this model of maternal immune activation (MIA), elevated production of maternal serum cytokines, specifically IL-6 and IL-17, contributes to alterations in fetal neurodevelopment. Although male and female littermates are exposed to the same maternal inflammation, we show that behavioral deficits manifest only in the male offspring, mirroring the sex bias observed in human ASD. Because the placenta is derived from fetal cells and is the first site of fetal exposure to hematogenous maternal inflammation, we hypothesized that sex-specific reactions to MIA that have deleterious impacts on fetal neurodevelopment originate there. Our preliminary findings show that MIA leads to sexually dimorphic alterations in placental pathology. To further characterize responses to MIA at the maternal-fetal interface over the course of gestation, we conducted bulk RNA-sequencing of the placenta/decidua from polyI:C- and saline-treated embryos at several time points post-injection. Sex of the embryos was determined by PCR genotyping for comparison of male and female placental transcriptomes. We find previously undescribed signatures related to myeloid cells in placentas from MIA males, as well as sex-based differences under homeostasis. This data set will shed light on the immune mechanisms that impact fetal brain health.
Complement initiation and regulation differ by sex in intestinal ischemia reperfusion injury
Authors: Rowe Jennifer M , Wu Miaomiao , Fleming Sherry D
Published: January 2023

Males and females frequently sustain distinct symptoms during ischemic events and subsequent inflammatory events. The inflammatory events include significant complement activation, neutrophil infiltration and significant cytokine secretions. The past literature suggests that complement activation may include a sex-linked protein. Therefore, we hypothesized that complement activation may vary by sex during intestinal ischemia/reperfusion (IR) injury. We subjected male and female mice to 30 min intestinal ischemia followed by 15, 30, 60 or 120 min reperfusion. Additional mice of each sex was subjected to sham surgery. Intestinal sections were stained and complement initiation pathways and time points of deposition determined. Complement regulatory molecules were also examined. Initial results suggest that males expressed more C1q compared to females, while females expressed more MBL than males. In addition, complement regulators varied by sex. Our data demonstrate that complement initiation and regulation differ between sexes during intestinal IR. These response differences in intestinal IR provide insight into the sexual dimorphism of ischemic intestine diseases and may aid in the development of sex-specific therapies that reduce intestinal injury.
Regulation of STING Trafficking and Interferon Response
Authors: Tu Xintao , Chu Ting-ting , Yan Nan
Published: January 2023

STING is an endoplasmic reticulum (ER)-associated transmembrane protein that is essential for the host innate immune response to pathogens. STING is activated by the second messenger cyclic dinucleotides which is produced from the invading pathogen or by the host DNA sensor cGAS. Activation of STING triggers its trafficking from the ER, through ERGIC/Golgi, to vesicles, during which time STING activates and quickly terminates downstream interferon signaling. Despite the importance of STING signaling in infection, autoimmune disease and cancer immunotherapy, the dynamics of STING trafficking and kinetics of signaling are poorly understood. Here, we performed a time-resolved proteomic screen to capture protein interaction networks of STING during its trafficking process. We also established a synchronized live-cell microscopy technique that allows us to define changes in protein trafficking kinetics. We have identified several co-factor candidates that affect various steps of the STING trafficking and signaling. Collectively, our findings revealed the previously unanticipated the regulation of STING trafficking as well as its downstream signaling by the host factors in the secretory pathway.
Cirsium japonicum var. ussuriense extract and cirsimaritin prevents the expressions of IL-17 in high-fat diet induced non-alcoholic steatohepatitis
Authors: Jang Seon Il , Choi Jiwon , Cho Byoung Ok , Kang Hyun Ju , Che Denis Nchang , Shin Jae Young
Published: January 2023

Nonalcoholic fatty liver disease (NAFLD) corresponds to hepatic manifestations of inflammation and accumulations of fatty acids in the liver. Recently, IL-17 signaling pathways have been shown to contribute to the pathogenesis of NAFLD. Cirsium japonicum var. ussuriense is often used in treatment of human diseases such as hemorrhage, blood congestion and inflammation. Despite the known biological activities of C. japonicum var. ussuriense, its effect on NAFLD is still unknown. Here, we investigated the effects of C. japonicum var. ussuriense extract (CJE) and cirsimaritin, its main constituents, on high-fat diet (HFD) or oleic acid (OA)-induced IL-17 expressions associated with steatohepatitis. We found that the levels of IL-17 was reduced in OA-treated HepG2 cells and in mice fed with HFD and administered with CJE and cirsimaritin at the mRNA, protein, and serum expression levels. Further investigation showed that CJE and cirsimaritin suppressed MAPK and NF-κB, the key signaling pathway responsible for IL-17 expression in HFD-fed mice and OA-treated hepatocytes, thus suggesting that the action of CJE and cirsimaritin on signaling molecules might have been responsible for the suppression of IL-17. Here, we also demonstrated that administration of CJE and cirsimaritin reduced triglyceride, aspartate aminotransferase, alanine aminotransferase, and malondialdehyde levels in OA-treated HepG2 cells; and protected mice against HFD-induced triglyceride, aspartate aminotransferase, alanine aminotransferase, and malondialdehyde levels. The results collectively demonstrate that CJE and cirsimaritin may protect against IL-17 exacerbated HFD-induced steatohepatitis.
Polyubiquitin-dependent recruitment of NEMO/IKKγ into T cell receptor signaling microclusters is controlled by IKKβ kinase activity
Authors: Koylass Nicholas Robert , DeRiso Elizabeth , Szymczak-Workman Andrea L , Montecalvo Angela , Murphy Joanne M. , Seminario Maria-Cristina , Bunnell Stephen C , Kane Lawrence P
Published: January 2023

The IkB kinase (IKK) complex coordinates inflammatory responses by activating canonical NFkB downstream of immune receptors. This complex consists of the kinases IKKa/β and the dimeric adaptor subunit NEMO (NFkB essential modulator protein). NEMO mutations cause immunodeficiencies and impair antigen receptor function. Canonical NFkB activation by immunoreceptors, such as the T cell receptor (TCR), requires the assembly of the Carma1/Bcl10/Malt1 (CBM) signalosome. While, functional studies and in vitro interactions, place the IKK complex downstream of the CBM signalosome, the spatial relationship between these complexes has never been observed in intact immune cells.

We observed that NEMO is recruited into TCR microclusters and membrane compartments within ~70 seconds of TCR engagement. Point mutations impacting the K63-linked and linear polyubiquitin ubiquitin-binding domains of NEMO selectively impair NEMO recruitment into TCR microclusters. We verified the involvement of these polyubiquitin chains by showing that inhibitors of either the K63-specific Uev1A/Ubc13 ubiquitin E2 ligase or the linear ubiquitin activating complex (LUBAC) prevent NEMO from entering TCR microclusters. To examine whether the entire IKK complex is recruited to the TCR, we co-expressed IKKb chimeras with NEMO. We observed that wild-type (WT), but not kinase-dead, IKKb caused NEMO microclusters to disappear. Similarly, the treatment of cells expressing WT IKKb with inhibitors of IKKβ or its upstream activator, TAK1, restored the recruitment of IKKβ and NEMO into microclusters.

These results suggest that the IKK complex is recruited to the TCR via linear- and K63-linked polyubiquitin chains and then dissociates from the TCR via IKKβ activity.
Diapedesis and LFA-1 mediate tissue immune cell effector activity via intrinsic complement C3 licensing
Authors: Kemper Claudia , Kolev Martin , West Erin E , Kunz Natalia , Rahman Jubayer , Chauss Daniel , Moutsopoulos Niki , Holland Steven M , Kaplan Mariana J , Wang Luopin , Kazemian Majid , Afzali Behdad
Published: January 2023

Intracellularly generated and autocrine-functioning complement component C3 is a critical integral part of normal human CD4+ Th1 and cytotoxic CD8+ T cells responses. Increased or decreased intracellular C3 result in autoimmunity and infections, respectively. The mechanisms regulating intracellular C3 expression are, however, undefined. By comparing transcriptomes from blood and tissue, we identified the complement system, including C3, as one of the most significantly enriched biological pathways in tissue-occupying cells of human macrophages, CD4+ and CD8+ T cells. By leveraging a novel C3 reporter mouse, we confirmed that C3 gene transcription is a feature of immune cells in tissues, is induced during trans-endothelial diapedesis, and is dependent on the integrin intercellular adhesion molecule (ICAM)-1 liganding lymphocyte function-associated antigen (LFA)-1. Consequently, monocytes and T cells from patients with leukocyte adhesion deficiency (LAD)-1 had reduced C3 and diminished effector activities, which could be rescued proportionally by normalization of intracellular C3. In synovia of patients with rheumatoid arthritis (RA), C3 transcript expression by CD4+ T cells was linearly associated with disease severity and acted as a biomarker distinguishing inflamed versus uninflamed RA. Our study defines the integrin network as a novel and key controller of intracellular complement, demonstrates that perturbations in the LFA-1–C3 axis contribute to primary human immunodeficiency and identifies T cell C3 production as a biomarker of the severity of autoimmunity.
Dynamic Adoption of Anergy by Antigen-Exhausted CD4+ T Cells
Authors: Obst Reinhard , Trefzer Anne , Kadam Pallavi , Wang Shu-Hung , Pennavaria Stefanie , Lober Benedikt , Kranich Jan , Brocker Thomas , Irmler Martin , Beckers Johannes , Straub Tobias
Published: January 2023

Exhausted immune responses to chronic diseases represent a major challenge to global health. We studied CD4+ T cells in a mouse model where presentation of their antigen can be regulated. When the cells are driven through the effector phase, but are then exposed to different levels of persistent antigen, they lose their Th1 functions, upregulate exhaustion and anergy markers, modulate their MAP kinase, mTORC1 and Ca2+/calcineurin signaling pathways with dose and time, lose their capacity to transmit help to B cells and undergo, at the highest dose, apoptosis. Transcriptomic analyses show the dynamic adjustment of gene expression and the accumulation of TCR signals over a period of weeks. The cells also adapt to antigen removal and recover their functionality while losing exhaustion and anergy markers. Our data suggest an adjustable response of CD4+ T cells to different levels of persisting antigen and contribute to a better understanding of chronic disease.
Genome-wide CRISPR screens revealed a reciprocal role of SWI/SNF complexes in controlling regulatory T cell function
Authors: Loo Chin San , Zheng Ye
Published: January 2023

Regulatory T cells (Tregs) are a specialized immune population that act to suppress overactive inflammatory response and maintain immune homeostasis. There is of great interest in harnessing Treg for controlling autoimmune disease or boosting anti-tumor immunity. Since the transcription factor, Forkhead box P3 (Foxp3), is known to control Treg development and function, understanding the regulation of Foxp3 could potentially lead to the development of therapeutics for treating autoimmune diseases and cancer. Here we performed a genome-wide CRISPR/Cas9 knockout screen to identify the regulators of Foxp3 in mouse primary Tregs. The results showed that Foxp3 regulators are highly enriched in genes encoding chromatin regulators, such as SAGA and SWI/SNF complexes. Among the SWI/SNF complexes, the GBAF complex promoted Foxp3 expression, whereas the PBAF complex repressed its expression. Functional genomic studies revealed that both GBAF and PBAF co-localized with Foxp3 in genome-wide binding sites. Targeting of GBAF reduced Foxp3 binding and altered expression a subset of Foxp3 target genes, indicating that GBAF actively participates in the Foxp3-dependent transcriptional program. Importantly, Treg suppressor function can be manipulated by targeting GBAF or PBAF, demonstrating in in vitro suppression assay, as well as mouse in autoimmune diseases and tumor model. Collectively, we provide an unbiased analysis of genes and networks regulating Foxp3, and reveal SWI/SNF complexes, GBAF and PBAF, as novel targets that could be exploited to manipulate Treg function for therapeutics of autoimmune diseases and cancer.