bluuebunny/crossref_metadata_2025 huggingface dataset:
Enrichement of type I interferon signalling in the colonic ILC2 under inflammatory condition
Authors: Mikami Yohei , Emi Irie , Rino Ishihara , Ichiro Mizushima , Kanai Takanori
Published: August 2023

Background

Group2 innate lymphoid cells (ILC2s) are known to contribute to a frontline defence against parasites and formation of allergic diseases, but little is known about the roles of ILC2s under intestinal inflammation including intestinal bowel diseases (IBD). In this study, we investigate the characteristics of ILC2s in health and colitis.

Methods

ILC2s were analysed in the mice with or without treatment of dextran sulphate sodium (DSS)-induced colitis, where surface markers and cytokine production was assessed by flow cytometry. The global gene expression of ILC2 in DSS-induced colitis and control mice were analysed by bulk RNA-sequencing (RNA-seq). Lastly, the mice was treated with anti-IFN-alpha/beta receptor1 (IFNAR1) antibody and then challenged to DSS-induced colitis.

Results

The number of ILC2s in the gastrointestinal tract was larger than that of spleen and liver in the healthy mice. At day 7 of DSS-induced colitis, IL-5 production from ILC2s was significantly reduced in flow cytometry data, and this was accompanied by enrichment of type I interferon (T1IFN) signalling in colitic ILC2 in RNA-seq data. Neutralisation of T1IFN signalling by anti-IFNAR antibody significantly increased severity of colitis.

Conclusion

Our data suggest that downregulation of Th2 cytokines and enrichment of T1IFN signatures in colitic ILC2s. T1IFN may stimulate ILC2 to induce colitis suppressive functions.

This study was funded by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Transformative Research Areas(B): 21H05123; Advanced Research and Development Programs for Medical Innovation (AMED-CREST: 21gm1510002h0001, and 20gm1210001h0001; the Practical Research Project for Rare/Intractable Disease: 21ek0109556h0001)
Intestinal epithelial expression of neutral ceramidase determines severity of chemical colitis in mice
Authors: Deng Zhongbin , Xu Zhishan , Lei Chao , Sun Rui , Sriwastva Mukesh , Tan Yi
Published: August 2023

Sphingolipids are important eukaryotic signaling molecules and regulate inflammation and immunity. Elevated levels of sphingolipid metabolites, e.g. S1p, are found in many chronic inflammatory disorders, including inflammatory bowel disease (IBD). Neutral ceramidase (NcDase) is a key regulator of ceramide, the central intermediate in the sphingolipid metabolism. The contribution of intestinal epithelial cells (IEC) NcDase to IBD is not well understood. Here we demonstrate that mice with IEC-specific loss of NcDase (NcDaseΔIEC) developed less severe DSS-induced colitis than control mice. Deletion of IEC NcDase leads to increase of antimicrobial peptides, anti-inflammatory cytokines and regulatory T (Treg) cells; and reduction of dysbiosis and gut inflammation. Mechanistically, IEC NcDase is required for the generation of ganglioside GD3, which controls the enteric immune response by activating Treg cells and preventing activation of T-helper (Th)17 cells. Administration of dietary GD3 reduces mucosal inflammation. Thus, GD3 is a key epithelial-derived glycosphingolipid that regulates distinct subsets of intestinal CD4+ T cells during inflammatory conditions, a finding with potential implications for treatment of chronic inflammatory disorders.

Supported by grants from NIH R21AI159194, R01 DK115406, and R01DK131442 (Z.D.).
Investigation of the aryl hydrocarbon receptor’s role in colitis as it relates to observed disease-specific sex differences
Authors: Mitchell Chandani , Saxena Archana , Staley Shanieka Caroline , Hailey Michelle , Bogdon Raymond K , Roark Kasie , Nagarkatti Mitzi , Nagarkatti Prakash S , Busbee Phillip B
Published: August 2023

Colitis is an inflammatory bowel disease (IBD) characterized by chronic inflammation in the colon. Studies suggest that there are differences between males and females regarding colitis disease progression, severity, and response to treatment. However, there lacks clear understanding, and some inconsistencies, on what these differences are attributed to. Previously, our reports showed that activation of the aryl hydrocarbon receptor (AhR) ligand was able to reduce colitis-induced disease severity, mainly through innate lymphoid type 3 cells (ILC3), in an interleukin-22 (IL-22) dependent manner. However, noted sex differences in disease severity and treatment response were observed in the animal models of colitis. In the current study, we generated cell-specific conditional knockout (KO) mice that had AhR deficiency in Rorc-expressing immune cells (ILC3s). We induced colitis using the dextran sodium sulfate (DSS) model in wildtype (WT) control mice and conditional mouse strains with AhR deficiency in Rorc-expressing immune cells (AR mice). Weight data and serum inflammatory biomarker assays showed that male mice developed a more severe colitis compared to females, with the male AR mice even worse. In addition, while control WT DSS female mice had less severe colitis than male counterparts, AR DSS female mice had just as severe disease as males, suggesting AhR, in part, was necessary to help promote less disease severity in females. Collectively, these results show that there are sex differences in colitis induction between male and female mice, and also that these differences might in part be due to some intrinsic sex-linked expression of AhR in different cell types.

The studies were supported in part by NIH grants P20GM103641.
IL10 producing regulatory T cells support fecal microbiota transplant (FMT)-mediated clearance of Clostridioides difficile Infection
Authors: Alam Md Zahidul , Maslanka Jeff R , Denny Joshua E , Bittinger Kyle , Abt Michael C
Published: August 2023

Fecal microbiota transplantation (FMT) is an effective therapy for treating recurrent C. difficile infection. However, the FMT mechanism of action is poorly understood, and the implementation of FMT therapy is limited. A previous study from our lab identified a critical role of CD4+ FoxP3+ regulatory T cells (Treg) in enabling FMT engraftment and subsequent resolution of C. difficile infection. Here, we investigated the cellular and cytokine network regulated by Treg cells to support FMT-mediated resolution of C. difficile infection. On the day of FMT, C. difficile infected mice have increased IL10+ Treg cells in the colon compared to uninfected mice. Further, mice depleted of IL10 and its signaling pathway are defective in resolving C. difficile following FMT, mirroring our observation in Treg-depleted FoxP3-DTR mice. The depletion of Treg cells in C. difficile-infected FoxP3-DTR mice induces a robust type 1 immune response in the colon characterized by IFNγ producing Th1 and type 1 innate lymphoid cells. This immune response drives an increased infiltration of iNOS+ neutrophils into the large intestine. Furthermore, neutralization of IFNγ following Tregs depletion decreases iNOS+ neutrophils in the large intestine, enabling the FMT to resolve C. difficile infection. Last, the depletion of neutrophils in Treg-depleted mice restores the efficacy of FMT to resolve C. difficile. Combined, we provide a mechanism by which IL10+ Treg cells serve as a negative regulator of an IFNγ-neutrophil intestinal inflammatory circuit, thereby supporting the colonic environment to be receptive to FMT and resolution of C. difficile. These results provide a mechanistic example of how immunity can dictate the efficacy of microbiome-based therapies.

Supported by grants from NIH (R01 AI158830, R21 AI164385)
Prostaglandin E2 signaling via its receptor EP4 on macrophages protects against acute colitis by preserving intestinal barrier function
Authors: Murray Page , Kelsall Brian
Published: August 2023

The inflammatory bowel diseases are increasing in incidence and cause significant morbidity; however, disease pathogenesis is poorly understood. Prostaglandin E2 (PGE2) may protect against intestinal inflammation and its receptor EP4 is highly expressed on colon macrophages, cells essential for immune homeostasis, suggesting PGE2 signaling on macrophages may prevent gut inflammation.

To address this, we induced DSS colitis in CX3CR1CRE/+EP4fl/fl (Cre+) mice, which lack EP4 on macrophages, and in their Cre- wildtype littermates. Cre+ mice developed worse colitis than the Cre- based on clinical scores, including increased rectal bleeding that implies major intestinal barrier disruption, a key mechanism of colitis. Cre+ mice have higher intestinal permeability early during inflammation, further supporting intestinal barrier disruption as a mechanism of increased disease severity in Cre+ mice.

To address the protective mechanisms of PGE2, we performed scmRNA sequencing on colon cells of the Cre+ and Cre- mice following DSS treatment. We identified monocyte and macrophage populations, with macrophages (MPs) clustering into CD11c+ MPs expressing inflammatory cytokines, and CD169+ MPs associated with inflammation resolution and wound-healing. During colitis there was a progressive increase in the proportions of inflammatory monocytes and a loss of CD169+ MPs that was accelerated in Cre+ mice. Further, the proportion of CD11c+ MPs was increased in Cre+ mice. These data are the first detailed scmRNA analysis of immune cell populations in DSS colitis and suggest that PGE2 signaling through EP4 may protect non-inflammatory macrophage populations during inflammation, contributing to maintenance of the intestinal barrier.
High salt diet impairs the protective immunity against mucosal HSV-2 infection
Authors: Kwon Myoung Seung , Park Jang Hyun , Lee Heung Kyu
Published: August 2023

Herpes simplex virus type2 (HSV-2) is the main cause of genital ulcer disease. Because of asymptomatic viral shedding, HSV-2 is highly prevalent and infected nearly 500 million people worldwide. Recently, excessive sodium intake has threatened global health. It is well known that excessive sodium intake affects various diseases by altering gut microbiome. However, the effect of excessive sodium intake on vaginal microbiome and vaginal immune system is unknown. In this study, we investigate the effects of high salt diet on vaginal microbiome and HSV-2 disease progression. We found that high salt diet compromises the anti-viral immunity in a mouse HSV-2 model. In 16S ribosomal RNA sequencing of vaginal microbiome, high salt diet results vaginal dysbiosis. High salt diet induced vaginal dysbiosis promotes neutrophil infiltration in vaginal tissue and vaginal lumen. In addition, Single-cell RNA analysis of vaginal neutrophil reveals dominant increase in neutrophil expressing MMPs that could damage vaginal tissue. Our findings suggest that high salt diet related vaginal dysbiosis impairs antiviral protection via neutrophil recruitment.
Death of Tonsillar B Cells by NETosis
Authors: Bukhari Ameera M , Knight Katherine L
Published: August 2023

Regulating B cell death is essential for generating antibodies and maintaining immune tolerance. B cells can die by apoptosis, and we report that human tonsil B cells, but not peripheral blood B cells also die by NETosis. This cell death is density-dependent, characterized by the loss of cell and nuclear membrane integrity, release of reactive oxygen species, and chromatin decondensation. Tonsil B cells secrete high levels of TNF, and inhibiting TNF prevented chromatin decondensation. By in situ fluorescence microscopy, B cell NETosis, as identified by the hyper citrullination of Histone 3, was localized to the light zone (LZ) of germinal centers in normal tonsil and overlapped with the B cell marker CD19/IgM. We propose a model in which stimulation of B cells in the LZ induces NETosis, driven in part by TNF. We also provide evidence that NETosis of tonsil B cells may be inhibited by an unidentified factor in tonsil. The results describe a previously unidentified form of B cell death and suggest a new mechanism to maintain B cell homeostasis during immune responses.
Role of MR1-driven signals and amphiregulin on the recruitment and repair function of skin MAIT cells
Authors: de Poulpiquet du Halgouet Anastasia , Darbois Aurélie , Alkobtawi Mansour , Mestdagh Martin , Premel Virginie , Colombeau Ludovic , Rodriguez Raphaël , Zaiss Dietmar , Aractingi Selim , Golub Rachel , Lantz Olivier J , Salou Marion
Published: August 2023

Epithelial repair is crucial to reestablish adequate barrier integrity and function after tissue injury. In both mice and humans, MAIT (Mucosal associated invariant T) cells involvement in tissue repair is substantiated by their upregulation of tissue repair mediators following TCR-mediated activation. Additionally, in the absence of ɣδT cells, MAIT cells were shown to promote wound healing. By using both our congenic B6-MAITCAST strain (10× more MAIT cells than B6 mice) and a skin excision mouse model preventing epithelial contraction (mimicking the human repair process), we sought to uncover the underlying mechanisms of MAIT cell tissue repair function.

Firstly, our scRNAseq analysis revealed that steady-state skin MAIT cells readily express a repair program. Additionally, we show that MAIT cells have a pro-repair function as their presence promotes keratinocyte proliferation, thereby accelerating healing. Using parabiosis, skin graft and the Kaede models, we found that, contrary to the resident ɣδT cells, MAIT cells are rapidly recruited into the inflamed skin. Parabiosis of MR1+ and MR1− mice and MR1− skin grafted onto MR1+ mice, showed that this recruitment was MR1-independent (MR1: antigen presenting molecule of MAIT cells). Instead, this recruitment relied on the chemokine receptor CXCR6 and its ligand CXCL16. Lastly, using conditional knock-out mice we demonstrated that the secretion by MAIT cells of the amphiregulin growth factor promotes skin wound healing.

Overall, our study provides mechanistic insights into MAIT cell biology in the context of injury. Comprehension of these processes provides clues into the biology of related innate subsets and highlights potential new therapeutic avenues for wound healing.

Institut National de la Santé et de la Recherche Médicale (OL)

Institut Curie (OL)

Agence Nationale de la Recherche Grant MAIT ANR-16-CE15-0020-01 (OL)

Agence Nationale de la Recherche Grant MAITrepair ANR-20-CE15-0028-01 (OL)

Agence Nationale de la Recherche ANR-10-IDEX-0001-02 PSL (OL)

European Research Council (ERC-2019-AdG-885435) (OL)

Fondation pour la Recherche Médicale, grant number FDT202106013036 (AdH)
Peripheral viral detection induces an antimicrobial program in olfactory bulb immature neurons
Authors: Garcia Benjamin J , Kraus Aurora , Salinas Irene
Published: August 2023

The immune and nervous systems have co-evolved to sense and eliminate danger. Neuroimmune interactions occur bidirectionally between the periphery and the central nervous system (CNS) in physiological and disease states. In the olfactory periphery, olfactory sensory neurons (OSNs) are exposed to environmental pathogens and project their axons onto the olfactory bulb. Previous studies have shown that the olfactory bulb participates in the immune response when a pathogen directly invades the CNS. We hypothesize that immune responses occur in the olfactory bulb in response to peripheral pathogen detection by OSNs. We have leveraged zebrafish as a model organism to study neuronal signaling in response to peripheral viral stimuli without infection of the olfactory bulb. Using single cell RNA sequencing we demonstrate that microglia respond to peripheral viral stimuli within 15 min, while a population of immature neurons expressing the neurotransmitter adcyap1 (PACAP-38) and scg5 (secretogranin V) expands within one day. PACAP-38 is a highly conserved neuropeptide with immunomodulatory and antimicrobial functions in mammals, suggesting that immature neurons participate in the antiviral state of the olfactory bulb. Neuronal transcriptional responses to olfactory viral stimuli were coupled to neuronal activation in the olfactory bulb. Furthermore, neuronal changes are translated into behavioral responses, as shown by a reduced mean velocity in an open field test after one day in virally treated compared to control animals. Taken together, these results indicating that peripheral viral stimuli induce a neuropeptide-based antimicrobial program in the olfactory bulb of adult zebrafish.
Premature Death of Sleep Fragmented Males is Associated With An Accumulation of Reactive Oxygen Species In The Gut
Authors: Moriarty Alina K , Keeter W Coles , Kirk Cassandra Lee , Stahr Natalie Ann , Sanford Larry , Galkina Elena
Published: August 2023

70 million Americans report insufficient sleep due to work or lifestyle choices. Decreased duration and poor sleep quality are associated with high rates of CVD and is strongly associated with increased risk of all-cause mortality. The effects of gender have been studied in CVD, but the impact of sexual dimorphism in Sleep Fragmented (SF)-associated atherogenesis is not known. Recently, we found that high fat diet (HFD) fed male Apoe−/− mice on SF experienced premature death. 80% of males did not survive past week 2 of SF, while 80% of females survived past week 8 under same conditions. We sought to identify a cause of premature death in HFD fed male Apoe−/− mice following sleep fragmentation. Interestingly, HFD fed male Ldlr−/− mouse did not experience premature death when SF, suggesting that ApoE absence, not hyperlipidemia, plays an essential function in the induction of death. Furthermore, oral 17β-Estradiol was able to rescue HFD fed male Apoe−/− mice from SF induced premature death. Compared to controls, SF, HFD fed male Apoe−/− mice had decreased body weight and increased circulating LPS, ROS and cell-free dsDNA at death. Finally, the small intestines SF, HFD fed male Apoe−/− mice had increased ROS and decreased tight junction protein (Tjp1) expression, suggesting compromised intestinal barrier function. Our data suggests that a combination of known risk factors for CVD (male sex, ApoE genotype, and HFD) results in premature death when combined with inadequate sleep. Furthermore, our data suggests that this specific combination of co-factors might result in premature death due to a systemic increase in oxidative stress, possibly caused by leakage of LPS from the gut, which may be alleviated through the administration of 17β-Estradiol.

Supported by project–initiation funds from the Center for Integrative Neuroscience and Inflammatory Diseases and AHA Predoctoral (20PRE35180156)
Using a CRISPR based screen to identify genes dysregulated in AML by T-cells
Authors: Pandita Ravina , Kosaka Yoko , Burns Faith , Lind Evan F
Published: August 2023

Acute Myeloid Leukemia (AML) is the most common hematological malignancy in adults and has a 35% survival rate at 5 years. Genetic heterogeneity makes it a highly lethal disease and patients develop resistance to therapy. Our lab has previously shown that in AML, the percentage of T-cells in the bone marrow at the time of diagnosis has a correlation with overall survival, and we have shown there is a dysregulation of T-cells in AML, suggesting their involvement in immune evasion. However, the mechanism by which T-cell activity contributes to leukemia is not well described. Currently, CRISPR screens are widely used to identify sensitivity and resistance genes involved in T-cell killing of cancer cells. Based on this strategy, we used three AML human cell lines (MOLM 14, OCI-AML2 and HL-60) at several effector to target ratios with T-cells and different treatment conditions and found that a majority of AML cells were killed at the higher T-cell ratio. Additionally, stimulating the T-cells using a bispecific antibody that conjoins T-cells with their targets improved target cell killing, particularly the MOLM 14 cell line. All these cell lines achieved differential killing by T-cells, suggesting depending on mutational background, AML immune evasion gene networks challenge T-cells by different mechanisms. Therefore, our future goal is to use a CRISPR/CAS9 lentiviral library genome-wide screen on the tumor cells followed by T-cell killing to identify genes involved in immune evasion in AML. The results of these experiments will help us to better understand T-cell dysregulation mechanisms in AML, thus providing a roadmap for immune based therapies combined with immune checkpoint blockade.

Supported by grant- NIH 1R01CA262145-01A1
Single-cell landscape analysis of tumor infiltrated immune cell population in FcγRIIB deficiency against glioblastoma
Authors: Ku Keun Bon , Kim Chae Won , Lee Heung Kyu
Published: August 2023

Glioblastoma (GBM) is the most common and deadliest cancer known in the central nervous system. Immune checkpoint inhibitors (ICIs) are effective in clinical trials for hematological tumors. However, the application of ICIs is for GBM patients has not proven to be completely successful. The limitations of the treatments can be attributed to the lack of an understanding of the GBM microenvironment (TME), which consists of heterogeneous tumor stem cells and various types of immune cells. Thus, investigating these tumor-infiltrating immune cells and their functions can provide a better insight to the TME of GBM. In order to accomplish this, we performed single-cell RNA sequencing analysis using the GBM mouse model. From the results, the TME up-regulated the inhibitory FcγR gene, Fcgr2b, on infiltrating immune cells, and this inhibitory receptor suppressed the antitumor immune response against GBM. FcγRIIB is known to mediate immune homeostasis through the binding of the immune complex, and blocking the receptor induces a proinflammatory response. We were able to use the FcγRIIB knockout mouse (KO) and analyze the landscape of tumor-infiltrating immune cells. Tumor-associated microglia portrayed anti-tumoral characteristics in KO mice and we were able to see an upregulation of adaptive immune cell recruiting chemokines. In addition, CD8 T cells showed an enrichment of precursor genes related to T cell exhaustion. With tumor progression, the total immune cell number had increased in KO mouse and CD8 T cells enhanced cytotoxic related genes. We can interpret from these results that FcγRIIB plays a critical role in GBM infiltrating immune cells, and targeting of this receptor could be a possible treatment option for treatment of GBM.
Fast and efficient isolation of human EGFR-positive cells using EasySep&[trade]
Authors: Poon Grace F T , Liang Alice , Chehal Manreet , Eaves Allen C , Louis Sharon A , Antignano Frann
Published: August 2023

Epidermal growth factor receptor (EGFR) is a cell surface receptor normally expressed on most epithelial cells and some hematopoietic cells. Mutation and overexpression of EGFR is a key driver of many human cancers, including lung, breast, as well as colorectal cancers. Circulating tumour cells (CTCs) can also express EGFR, where enrichment of EGFR+ CTCs can provide promising liquid biopsy and prognostic values. EGFR-positive cells can be difficult to isolate because they are present in many tissue types at a wide range of frequencies. We have developed a simple method to isolate human EGFR-positive cells to address this challenge. A549 human lung adenocarcinoma cells expressing EGFR were spiked into human PBMCs at defined frequencies as a model system. Starting with a single-cell suspension with 10% EGFR+ cells, the EGFR-expressing cells were labeled with an antibody complex that linked the EGFR-expressing cells to magnetic particles, then separated using an EasySep™ magnet. Using this method, EGFR+ cells were enriched from 9.9 ± 3.3% to 95.0 ± 2.8% (mean ± SD; n = 14). Protocols have been optimized for different sample sizes and different EGFR+ cell starting frequencies. EasySep™-isolated EGFR+ lung adenocarcinoma cells were viable and proliferative, with fold expansion and Ki67 expression comparable to untreated cells. EasySep™ Human EGFR Positive Selection Kit enables simple and easy isolation of EGFR-positive cells in 15 minutes, facilitating EGFR and cancer research.
Analyzing the immunological variability in T cell/tumor interactions to identify a subpopulation of CD8+ T cells (Spark T cells) that drives succesful cancer immunotherapy
Authors: Cavazos Emanuel Salazar , Yeh Michael , Krishnan Anagha Yogam , Achar Sooraj , Jia Dongya , DeVoe Don , Altan-Bonnet Grégoire
Published: August 2023

Thousands of patients have benefited from the growing use of cancer immunotherapies. However, the success of these therapies can be highly variable. Strikingly, some murine tumor models show large variability in the outcome of cancer immunotherapies, even when the mice, tumor cells and anti-tumor immune cells injected into mice are all genetically identical. Here, we sought to analyze this variability in adoptive cell therapies, in order to identify the immune population driving this variability.

To search for the occurrence of large variability ex vivo, we extracted mouse TCR-transgenic CD8+ T cells, and co-cultured them with antigen-expressing tumor cells. By using multiplexed in vitro assays and single-cell analysis of thousands of samples, we identified conditions (e.g. cell numbers, antigen quality, tumor cell types) where large variations in the immune activation against cancer cells is observed even between technical replicates. We then developed a quantitative framework that uses statistical modeling and machine learning to extract useful information from this immunotherapeutic variability. Our framework allowed the prediction and identification of a rare population of naïve CD8+ T cells (“Spark T cells”) that is necessary and sufficient to spark massive anti-tumor immune reactions.

We are currently performing experiments to test the efficacy and functional significance of the identified immune population in vivo. We are also applying this framework in human TCR-engineered T cell blasts to identify the equivalent of the Spark T cells in humans. We envision this framework being applied to identify other relevant immune cell types that act as catalysts for successful cancer immunotherapies.

Supported by the Intramural Research Program of the National Institutes of Health, the National Cancer Institute
Salt-induced maladaptive innate immune memory impedes stroke recovery
Authors: Lin Tze-Yen , Chen Wan-Ru , Lin Jhih Syuan , Chen Chih-Hung , Chang Che-Feng
Published: August 2023

A high salt diet (HSD) is a top risk factor for human mortality. Beyond its heavily implicated role in hypertension, it is now linked to the development of inflammatory diseases before the onset of cardiovascular events. Even after salt reduction, HSD can induce prolonged tissue damage, yet the mechanisms by which HSD induces persistent negative effects on immune system are largely unexplored. While HSD is an established risk factor for stroke, its impact on long-term stroke recovery is unknown. Here, we model two major stroke types, intracerebral hemorrhage, and cerebral ischemia, to investigate how HSD induces unfavorable immune responses and subsequently impacts long-term tissue repair. We found HSD exclusively hampers alternative activation in monocytes-derived macrophages (MDMs) while sparing the major parenchymal macrophage—microglia—in the stroke brain. HSD induces innate immune memory in the hematopoietic stem and progenitor cells through downregulating mitochondrial oxidative phosphorylation and NR4a orphan nuclear receptors expression. This immunological memory was retained in the brain-infiltrating MDMs and restrained their reparative functions. Consequently lead to impaired tissue repair and neurobehavioral recovery during stroke recovery phase. Pharmacological treatment with NR4a1 activator and genetic overexpression of NR4a1 restored MDMs reparative phenotype suppressed by HSD and enabled stroke recovery. These findings establish a new mechanism linking HSD-induced maladaptive innate immune memory to a hindered macrophage alternative activation and delayed tissue recovery. Our data unveil innate immune memory and NR4a1 as potential therapeutic targets for treating HSD-associated diseases.
Dysregulation of circulating MAIT cell subsets in patients with hidradenitis suppurativa
Authors: Toor Jugmohit , Dimitrion Peter Michael , Hans Aakash , Hamzavi Iltefat , Zhou Li , Mi Qing-Sheng
Published: August 2023

Hidradenitis suppurativa (HS) is a chronic inflammatory condition characterized by painful and debilitating skin lesions. Recent reports purport HS as an autoinflammatory keratinization disease with numerous pathways and features of systemic inflammation however few studies have been conducting to characterize peripheral immune dysregulation. Mucosal associated invariant T (MAIT) cells are an invariant T cell subset responding directly to bacterial threats and their dysregulation contributes to the pathogenesis of autoimmune disorders, cancers and inflammatory skin conditions. Here, we analyzed MAIT cells in the blood of 16 HS patients and 12 healthy controls (HC) by flow cytometry. We found that the frequency of MAIT cells did not change in HS patients, but their function changed drastically. We observed a significant reduction in the frequency of IFNy+ and TNFα+ MAIT cells in the periphery of HS patients. Furthermore, we analyzed MAIT cell subsets by their CD4 surface expression. Here, we found that the frequency of CD4+MAIT cells in the periphery of HS patients is significantly increased, while the frequency of CD4-MAIT cells is significantly decreased. Furthermore, HS patients have a reduction in CD4− IFNg+ and TNFa+ MAIT cells. Overall, these findings suggest that peripheral MAIT cells are dysregulated in HS patients which may contribute to the pathogenesis of the disease.

NIH/NIAMS (1R21AR079089 and 1R01AR078688) for Dr. Mi and Henry Ford Immunology Program grant (T71017 for Dr. Mi and T71016 for Di Zhou).
Peripheral T helper CD4+ cells mediate autoimmunity in Aire-deficient model of chronic inflammatory demyelinating polyneuropathy
Authors: Seyedsadr Maryamsadat , Su Maureen
Published: August 2023

Background

Chronic inflammatory demyelinating polyneuropathy (CIDP) occurs spontaneously in NOD mice with Aire deficiency (NOD.AireGW/+). The disease is mainly driven by CD4+ cells. However, the contribution of different CD4+ T cell subsets to the pathogenicity of neuropathy is not well studied.

Results

scRNA-seq of the immune cells infiltrating the sciatic nerves show that the majority of the CD4+ cells express genes associated with T follicular helper (Tfh) and T peripheral helper (Tph) cells, a newly described T helper type. Tph cells are similar to Tfh cells as they have prominent expression of ICOS, PD1, and IL-21. These IL-21+ Tph cells are also IFN-γ, IL-10 and CXCR6 positive, suggesting an effector phenotype. We verified high levels of IL-21 expression in Tfh and Tph cells in spleen and nerves by flow cytometry using IL-21 reporter mice. Transfer of CD4+ IL-21+ cells from NOD.AireGW/+ donor mice to immunodeficient mice resulted in higher neuropathy incidence than transfer of CD4+ IL21− cells. In adoptive transfer model, inhibition of Tfh specific transcription factor BCL6 decreased the frequency of Tfh and Tph cells in the peripheral nerves and protected against neuropathy development.

Conclusion

IL-21 producing Tfh and Tph cells may play a major pathogenic role in neuropathy development and suggests new therapeutic targets for CIDP.

Supported by grants from NIH
Triplication of HSA21-encoded genes modulates Alzheimer’s Disease-related phenotypes in vivo
Authors: Araya Paula , Schade Kyndal A , Waugh Katherine A , Busquet Nicolas , Brindley Connie , Winkler Caitlin , Dunn Lauren N , Siegenthaler Julie , Lyford Hannah R , Britton Eleanor C , Brown Chrisstopher , Ludwig Michael , Kinning Kohl , Galbraith Matthew D , Espinosa Joaquin M , Sullivan Kelly D
Published: August 2023

People with Down syndrome (DS), the genetic condition caused by Trisomy 21 (T21), exhibit early appearance of Alzheimer’s disease (AD), likely due to the triplication of the Amyloid Precursor Protein (APP) gene. However, the impact of many chromosome 21 (HSA21) genes on AD phenotypes in the context of DS remains unknown. To test this, we conducted a detailed phenotypic characterization of how AD-related neuropathology is modulated by an extra copy of genes orthologous to HSA21 by creating a novel DS-AD mouse strain, by crossing Dp16 with 5xFAD mice (models for DS and AD, respectively), generating: WT, Dp16, 5xFAD, and 5xFAD-Dp16 mice. Here, we performed a battery of behavioral assessments longitudinally and paired these analyses with endpoint transcriptional profiling and high-content imaging. We identified that DS-AD mice exacerbate cognitive deficits and show increased neuronal cell loss in multiple brain regions compared to DS and AD mice. Although, DS-AD mice do not present altered amyloid-β (Aβ) pathology compared to AD mice at 9-months of age, an increased number of activated microglia associated to Aβ plaques are present with a distinct transcriptional signature indicative of disease associated and interferon-activated microglia cells in these animals. These results indicate that DS contributes to the neuroinflammation network of AD, causing changes in cellular states consistent with alterations in memory and cognitive decline. However, deeper characterization is needed to understand the mechanisms by which T21 contributes to AD-pathogenesis to determine potential therapeutic intervention to significantly improve neurological health in people with DS. Supported by grants from NIH (R01AI145988 and R01AI150305).

Supported by grants from NIH (R01AI145988 and R01AI150305)
Characterization of the transcriptome and TCR of brain and cerebrospinal fluid infiltrated CD8+ T cells in an Alzheimer’s disease mouse model
Authors: Chen Joseph Ruixin
Published: August 2023

Alzheimer’s disease (AD) is a common form of dementia characterized by the accumulation of protein aggregates in the brain of older adults. It has been observed that CD8+ T cells, particularly CD8+ TEMRA cells, infiltrate the brain and cerebrospinal fluid (CSF) of AD patients. However, the precise role of these cells in the development and progression of AD is not well understood. In this study, we used single-cell RNA sequencing (scRNAseq) and single-cell T cell receptor sequencing (scTCRseq) to examine the CD8+ T cells in the brain and CSF of a mouse model of AD (5xFAD) and its wild-type (WT) littermate controls. The AD mice (age 40–70 weeks) used in our experiments have developed memory deficits. Our analysis revealed a significant increase in the number of CD8+ T cells in the brain and CSF of AD mice compared to WT controls (n=4, p<0.05). Furthermore, these cells displayed a distinct transcriptome profile characterized by increased expression of inflammatory pathways, compared to WT mice. Through TCR sequence analysis, we also observed significant clonal expansion of CD8+ T cells in AD mice compared to WT mice. These findings provide a starting point for further investigation into the role of infiltrating CD8+ T cells in the pathogenesis of AD in this mouse model.
Aiolos restrains acquisition of a cytotoxic program by CD4+ T cells
Authors: Jones Devin M , Read Kaitlin A , Pokhrel Srijana , Tuazon Jasmine , Leonard Melissa R , Salem Miriam , Varkey Aditi , Warren Robert , Collins Patrick L , Oestreich Kenneth
Published: August 2023

CD4+ cytotoxic T lymphocytes, or CD4-CTLs, comprise a CD4+ subset capable of performing functions normally observed in CD8+ T and Natural Killer cells. CD4-CTLs play critical roles in many immune contexts, including protective anti-viral responses to viruses such as SARS-CoV-2 and influenza. Despite their well-documented importance, the complete mechanisms that underlie their formation remain unclear. Here, we define the transcription factor Aiolos as a novel repressor of CD4-CTL differentiation. We find that Aiolos deficiency results in increased CD4-CTL responses in lungs of influenza-infected mice, as assessed by elevated expression of Granzyme B and Perforin, as well as the CTL marker NKG2A/C/E. We further find that Aiolos-deficient CD4-CTLs exhibit increased expression of transcription factors associated with cytotoxic programming, including Eomes and Blimp-1. Mechanistically, we find that Aiolos-deficient cells have a heightened sensitivity to IL-2/STAT5 signaling due to enhanced expression of IL-2 receptor subunits. This translates into increased chromatin accessibility and STAT5 association at regulatory regions of hallmark CD4-CTL genes in the absence of Aiolos. Consistent with these data, in silico analyses demonstrate that STAT5 DNA-binding motifs are significantly enriched at these same regions. Intriguingly, the STAT5 motif partially overlaps with that of the core Aiolos DNA binding motif, suggesting that Aiolos may function to broadly antagonize STAT5 activity throughout the genome. Collectively, this work establishes Aiolos as a novel repressor of cytotoxic programming in CD4+ T cells and highlights its potential as a therapeutic target for enhancing anti-viral immunity.

This work was supported by grants from The National Institutes of Health AI134972 and AI127800 to K.J.O, AI156411 to P.L.C. K.A.R. is supported by funding through The Ohio State University College of Medicine Advancing Research in Infection and Immunity Fellowship Program. J.A.T. and S.P. are supported through funding from the Infectious Disease Institute T32 in Host-Microbe Interactions fellowship.
CD4 T cell intrinsic arginase 1 controls the kinetics of Th1 induction and contraction
Authors: West Erin E , Merle Nicolas S , Kamiński Marcin M , Palacios Gustavo , Kumar Dhaneshwar , Wang Luopin , Overdahl Kristen , Jarmusch Alan K , Freeley Simon , Taylor Naomi , Sitbon Marc , Green Douglas R , Bohrer Andrea , Mayer-Barber Katrin , Afzali Behdad , Kazemian Majid , Scholl-Buergi Sabine , Karall Daniela , Huemer Martina , Kemper Claudia
Published: August 2023

Arginase 1 (ARG1), the enzyme catalyzing the conversion of arginine to ornithine and urea, is a hallmark of IL-10 producing immunosuppressive M2 macrophages, however ARG1 activity in T cells is disputed. Here we demonstrate that Arg1, but not Arg2, expression induction is a key feature of lung CD4 T cells during mouse in vivo influenza infection. Ablation of CD4 T cell-intrinsic Arg1 unexpectedly accelerated both the virus-specific Th1 effector response and its IL-10-associated contraction. Biologically, this led to efficient viral clearance, yet significantly reduced lung pathology. Surprisingly, loss of Arg1 in CD4 T cells did not result in disturbed intracellular ornithine or polyamine levels. Instead, by employing unbiased transcriptomic and metabolomic approaches, we found that Arg1 deficiency triggered altered glutamine metabolism, and rebalancing the glutamine flux normalized the Arg1 deficient T cell response. Further, the role of Arg1 in CD4 T cells was distinct from that of its’ isoenzyme Arg2, as ablation of CD4 T cell intrinsic Arg2 resulted in normal Th1 responses, and instead altered Th2 and Th17 responses. Finally, CD4 T cells from rare patients with a deficiency in ARG1, or from healthy donors with CRISPR-Cas9-mediated ARG1 deletion, recapitulated the mouse data, demonstrating that ARG1 also plays a CD4 T cell intrinsic role in human Th1 responses. Collectively, CD4 T cell-intrinsic ARG1, functions as an unexpected pace-keeper of human and mouse T helper 1 (Th1) responses with implications for Th1-associated tissue pathologies.

Supported by grants from NIH (5K22HL125593 to M.K.) the Intramural Research Program of the NIH (NIDDK ZIA/DK075149 to B.A. and NHLBI ZIA/HL006223 to C.K.)
Direct and Indirect type I Interferon signaling regulates Eomes-driven Th1-responses against Influenza Virus
Authors: Dhume Kunal , Finn Caroline M , Baffoe Eugene , McKinstry Kai
Published: August 2023

In the absence of Th1 master regulator, the T-box transcription factor, T-bet, CD4 T cells primed by Influenza A Virus (IAV) infection, gain Th17 attributes while still retaining robust Th1 functionality. We recently found the transcription factor Eomesodermin (Eomes), to regulate this residual Th1 identity of T-bet−/− CD4 T cells. However, it is unclear how Eomes is regulated in CD4 T cells, especially during antiviral responses. Here we find type I interferons to be potent inducers of Eomes but not of T-bet in naïve antigen specific CD4 T cells stimulated in vitro. Furthermore, blocking type I IFN signaling to T-bet−/− CD4 T cells in IAV infected WT host mice, reduces Eomes expression and drives functional shifts mirroring T-bet−/− Eomes−/− cells, including significantly reduced IFNγ production and stronger Th17 identity. To test the impact of direct type I IFN signals on Eomes-driven Th1 identity of T-bet−/− CD4 T cells, we compared the responses of T-bet−/− CD4 T cells versus T-bet−/− cells also deficient for type I IFN receptor. While the Th1 attributes of the double-deficient cells were compromised compared to T-bet−/− cells, T-bet−/− cells responding in hosts treated to block systemic type I IFN signaling were the most impacted. These results indicate a strong role for indirect type I IFN signals in regulating Eomes-dependent functions during antiviral CD4 T cells responses. Finally, we find depletion of plasmacytoid dendritic cells in IAV infected host mice, a major source of type I IFN, does not impact T-bet−/− CD4 T cell response. Together, these results identify an important axis controlling the expression of Eomes in antiviral CD4 T cells that may in the future be harnessed to fine-tune effector and memory attributes primed by vaccination

Supported by grants from NIH (R01 AI67994 and R21 AI146647)
Rare Factor VIII-reactive CD4 T cells in non-hemophilic blood characterized by ex vivo expansion and ELISPOTs with added costimulation
Authors: Vir Pooja , Pratt Kathleen P
Published: August 2023

Background

Autoimmune responses to self-FVIII can cause a rare but potentially life-threatening autoimmune bleeding disorder referred to as “acquired hemophilia A”.

Objectives

(1) To confirm the frequency and specificity of CD4 T cells recognizing factor (F)VIII in the periphery of non-hemophilic individuals; (2) to improve methodology for detecting rare self-reactive CD4 T cells that apparently escaped thymic editing.

Methods

CD4 T cells were isolated from healthy non-hemophilic blood donors, incubated with autologous irradiated PBMCs, and stimulated with FVIII 15-mer peptides, anti-CD28 and anti-CD49D. Positive interferon-gamma ELISPOT responses were detected, but they were poorly reproducible due to the low frequency of these autoreactive cells. Therefore, pre-expansion of FVIII-specific T-cell lines, followed by ELISPOT assays with added co-stimulation, was carried out to increase sensitivity and accuracy.

Results

Several immunodominant epitopes in FVIII restricted to H LA-DRB1* 01:01, 07:01 and 15:01 were identified. FVIII-specific T-cell lines generated from individual blood donors showed robust interferon-gamma secretion in response to FVIII, compared to ELISPOT assays with co-stimulation but no FVIII pre-expansion.

Conclusions

Our optimized protocol, involving generation of antigen-specific T-cell lines followed by ELISPOTs with added co-stimulation, shows promise for detecting, quantifying and characterizing rare autoreactive T cells in the periphery.

Intramural funds, Uniformed Services University
TIGIT limits anti-bacterial immunity and is a potential target for immunotherapy
Authors: Souza-Fonseca-Guimaraes Fernando , Rossi Gustavo Rodrigues , Wells Timothy , McCulloch Timothy Ryan
Published: August 2023

Inhibitory receptors called “immune checkpoints” restrict overzealous immune responses during inflammatory events such as infection or cancer. Blocking the function of these immune checkpoints using monoclonal antibodies can tip the balance towards immune activation and disease clearance, which has been a revelation in the treatment of various cancers. Considering recent advances have shown that immune dysregulation and evasion are shared hallmarks of cancer and persistent bacterial infection, a better understanding of immune checkpoints during bacterial infection may uncover novel therapeutic targets to help direct host immune responses towards clearing pathogens.

In this study, we use a murine model of Salmonella Typhimurium infection to investigate how immune checkpoints regulate immunity to bacterial infection. We found lymphocytes from infected mice exhibit a phenotype suggestive of dysfunction, characterised by upregulation of multiple co-inhibitory checkpoints. Most strikingly, T cell immunoglobulin and ITIM domain (TIGIT) was upregulated on the majority of CD4+ T cells. We used RNA-sequencing and ex vivo stimulation of CD4+ T cells to show that ligation of TIGIT leads to significant functional deficiencies, including reductions in proliferation and pro-inflammatory cytokine production. Importantly, blockade of this immune checkpoint in vivo using anti-TIGIT antibodies was able to enhance functional immunity leading to improved bacterial clearance. Taken together, this research suggests targeting immune checkpoints such as TIGIT has potential to restore host immune responses towards clearing bacteria and managing serious infection.

F.S.F.G is funded by a UQ Frazer Institute Laboratory Start-Up Package, and T.R.M. is supported by a UQ HDR PhD Scholarship.
T cell-intrinsic C5L2 activation protects against uncontrolled inflammatory Th responses and autoimmunity
Authors: Merle Nicolas S , Freeley Simon , Bibby Jack , Singh Parul , Rahman Jubayer , Kolev Martin , Niyonzima Nathalie , Morgensen Trine Hyrup , Woodruff Trent M , Monk Peter , Lappegard Knut , Espevik Terje , Mollness Tom Eirik , Christiansen Mette , West Erin E , Kemper Claudia
Published: August 2023

Intracellular/autocrine complement proteins have emerged as critical regulators of human Th1 induction and contraction. T cells contain both intracellular C3 and C5 activation systems, with intracellular C3aR1 and C5aR1 stimulation driving T cell homeostatic survival and normal Th1 IFNg production, respectively. Here we demonstrate how the intracellular/autocrine C5 system is regulated by using T cells from the first described patient with C5aR2 deficiency. This patient suffers from an autoinflammatory syndrome, with enhanced inflammatory Th responses and a profound loss of naïve CD4 T cells in the blood (95% have a memory phenotype). Thus, in contrast to intracellular C5aR1 stimulation, cell surface expressed C5aR2 is an important negative regulator of inflammatory Th induction. While both C5aR1 and C5aR2 can bind C5a, we found that the carboxypeptidase-processed form of C5a, C5a-desArg, was twice as potent as C5a in reducing Th1 induction. In addition, carboxypeptidase M (CPM) expression was highly induced upon T cell activation indicating that CPM may be mediating T cell-derived C5a-desArg generation and C5aR2 stimulation. In this vein, activation of CRISPR/Cas9 generated CPMKO T cells, or of T cells in the presence of a CPM inhibitor, induced Th1 hyper-induction, which was rescued by addition of a C5aR2 agonist and reduced by adding C5a-desArg, but not C5a. The in vivo importance of T cell-expressed CPM and autocrine C5aR2 activation is demonstrated by the enhanced inflammatory Th responses in Cpm−/− mice and increased pathology caused by Cpm−/− T cells in a T cell transfer colitis model. These data highlight the importance of intracellular/autocrine C5 system regulation by CPM through C5aR2 signaling in T cell responses.