bluuebunny/crossref_metadata_2025 huggingface dataset:
Enrichment of Epstein Barr Virus in patients with Multiple Sclerosis
Authors: Kim Ellie , Forney Carmy , Viel Kenyatta , O’Brien Madison , Nelson Catrina , Gecaine Paul , Zabeti Aram , Weirauch Matthew , Kottyan Leah
Published: January 2023

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system affecting more than 2 million people worldwide and has been linked with Epstein-Barr Virus (EBV) infection. Previous studies by the Kottyan and Weirauch labs show that the EBNA2 protein of both EBV type 1 and 2 binds unique MS risk loci potentially causing changes at the transcriptional level contributing to MS disease progress. We hypothesize that type 1 and type 2 EBV will be detectable in the MS patient samples, and we aim to quantify EBV enrichment and identify EBV type in MS patient-derived blood samples. Analysis by qPCR showed that 30% of the control population (n=10) and 66% of the MS patient population (n=41) had detectable levels of EBV whereas the MS patients had a significantly higher frequency of EBV presence (X2 test p=0.0389). Of the samples that tested positive for EBV, 66% showed presence of type 1 EBV while 33% were unknown without a significant difference between MS patients and control group (X2 test p>0.9999). Due to issues with the limit of detection, droplet digital PCR was assessed for higher sensitivity compared to qPCR, but was found to have a comparable limit of detection to qPCR. To overcome the challenge of detecting low copy number viruses, we look toward targeted DNA capture and next generation sequencing to improve the sensitivity of EBV detection and specificity of EBV typing. Understanding the distribution of EBV types among MS patients would elucidate the precise role of EBV in MS development, potentially leading to improved treatment and prevention methods.

Supported by grants from NIH grants R01 HG010730, R01 NS099068, R01 GM055479, and U01 AI130830 to MTW; R01 DK107502, R01 AI148276, U19 AI070235, U01 HG011172, and P30 AR070549 to LCK; R01 AR073228, R01 AI024717, and CCHMC ARC Award 53632 to MTW and LCK.
Cyanobacteria Geitlerinema sp. lipopolysaccharide induces neutrophil-dependent lung inflammation
Authors: Del Toro Rosalinda Monroy , Incrocci Ryan , Williams Philip , Mayer Alejandro M S , Swartzendruber Julie A , Swanson-Mungerson Michelle
Published: January 2023

Cyanobacterial blooms found in freshwater systems are a source of environmental toxins that impact the immune system. Geitlerinema sp., a strain of cyanobacteria, produces toxins including lipopolysaccharide (LPS) which induces fever and potential respiratory disease. One mechanism of respiratory disease and inflammation is through the inhalation of LPS that is associated with neutrophil infiltration of the lungs. Based on our previous data that Geitlerinema sp. induces pro-inflammatory cytokines, we hypothesized that inhalation of Geitlerinema sp. LPS would induce lung inflammation in vivo. To address this hypothesis, c57Bl/6 mice were intranasally exposed to Geitlerinema sp. LPS before lungs and BAL fluid were assessed for inflammation and inflammatory markers 24 hours later. Our data show that inhalation of Geitlerinema sp. LPS induced lung inflammation by recruiting neutrophils into the airways. The data also demonstrate that Geitlerinema sp. LPS induces proinflammatory cytokines and chemokines: IL-6, IL-8, IFN-g, RANTES and TNF-a in the BAL fluid. Additionally, in vitro data using an airway epithelial cell line show that Geitlerinema sp. LPS induced production of pro-inflammatory cytokines is dependent on the NF-κB pathway. These data help to better understand the mechanism of Geitlerinema sp. LPS airway-induced inflammation and implicates the importance of the toxin in promoting human disease near freshwater ecosystems where cyanobacteria bloom.
A new β2 integrin activation reporter mouse reveals localized intra- and extra-vascular neutrophil integrin activation in vivo
Authors: Wen Lai , Marki Alex , Wang Zhihao , Orecchioni Marco , Makings Jeffrey , Kim Kenneth , Kiosses William B , Mikulski Zbigniew , Ley Klaus
Published: January 2023

β2 integrins (LFA-1, Mac-1, CD11c-CD18, and CD11d-CD18) are leukocyte-specific adhesion receptors that play critical roles in leukocyte recruitment, as well as other immunological processes such as phagocytosis and immunological synapse formation. Adhesion of leukocytes to other cells such as endothelial cells are regulated by integrin affinity changes for their ligands (“activation”). Human β2 integrin activation can be detected by reporter antibodies including mAb24 and KIM127. No such activation epitopes are known in mouse β2 integrins. Because of the lack of mouse β2 integrin activation reporter antibodies, nothing is known about β2 integrin activation in vivo. Here, we generated a humanized β2 integrin knockin mouse by targeting the human β2 integrin coding sequence into the mouse Itgb2 locus. We show that this enables imaging of β2 integrin activation using the KIM127 (extension conformation) and mAb24 (high affinity) reporter antibodies. Human β2 pairs with the mouse integrin α chains, yielding normal expression of the β2 integrins LFA-1, Mac-1 and CD11c-CD18 in all major leukocyte populations. Using a CXCL1-induced acute inflammation model, we uncovered the dynamics and subcellular localization of β2 integrin activation in arresting neutrophils in vivo in venules of the mouse cremaster muscle. Activated integrins in arresting neutrophils in vivo are concentrated at the interface of neutrophils and the endothelium at the rear side of neutrophils facing against the blood flow. In a high-dose lipopolysaccharide (LPS) model, we found that β2 integrins are activated in association with elevated neutrophil adhesion in lung and liver. Thus, these mice, for the first time, enable studies into β2 integrin activation in vivo.

This work was supported by grants from the National Institutes of Health, USA (HL078784 to K.L.) and a Postdoctoral Fellowship (19POST34450228 to L.W.) from the American Heart Association, USA.
Isoflurane targets BLT1 to attenuate lung injury
Authors: Yuki Koichi , Koutsogiannaki Sophia
Published: January 2023

Sepsis-associated lung injury is one of major comorbidities in sepsis, largely responsible for its mortality. Lack of specific therapeutic for lung injury is an urgent issue to be addressed. We found that volatile anesthetic isoflurane attenuated lung injury associated with experimental polymicrobial abdominal sepsis. This was associated with a reduction in the number of recruited neutrophils in the lung and bronchoalveolar space. Furthermore, isoflurane attenuated neutrophil chemotaxis induced by major chemoattractant leukotriene B4 (LTB4), not by C5a. Although LTB4 level did not differ by isoflurane exposure, isoflurane was shown to interact with LTB4’s neutrophil receptor BLT1 in silico. The interaction site was predicted to overlap with BLT1 antagonist BIIL260 binding site, suggesting isoflurane as a BLT1 antagonist. Five biotinylated compounds with a range of spacer lengths were developed and subjected to calcium mobilization test using CHO-BLT1 cells to determine their property as BLT1 antagonist. Two compounds showed adequate inhibition and we performed the competition assay for BLT1 between the compound and isoflurane. Isoflurane significantly competed with the BIIL260 biotinylated compound, indicating that isofluane bound to the BIIL260 binding site on BLT1. Isoflurane is considered as an alternative sedative in Europe and Canada, and this result could support the potential use of isoflurane as an ICU sedative in lung injury setting.

Supported by GM118277
Inflammation depletes humoral immunity by limiting plasma cell access to the bone marrow survival niche
Authors: Aaron Tonya , Benet Zachary , Fooksman David
Published: January 2023

The survival of plasma cells (PCs) is directly correlated to the longevity of prophylactic titers against pathogens. The bone marrow contains hematopoietic and non-hematopoietic cells that contribute to the maintenance of PCs. Studies have shown that pathogens contain a variety of mechanisms that result in the depletion of bone marrow PCs and antibody titers. Most, if not all, forms of infection induce inflammation, however, the role of inflammation in bone marrow PC depletion is unclear. Here, we show that acute Tumor Necrosis Factor Alpha (TNFa) treatment can deplete pre-existing antibody titers in a variety of models. We also found that recombinant TNFa treatment limits PC retention in the bone marrow via adoptive transfer experiments. To determine if PC retention in the bone marrow was mediated in a cell-intrinsic or − extrinsic manner, we generated bone marrow chimeras with wild-type and TNFa receptor knockout mice. Results showed that TNFa signaling through TNFa receptor 1 in non-hematopoietic cells regulates PC retention in the bone marrow, indicating that this mechanism is cell-extrinsic. Another cell-extrinsic mechanism that we identified is TNFa regulation of syndecan-1, a cell surface proteoglycan found to regulate PC survival and motility. These findings demonstrate that inflammation limits PC access to the bone marrow survival niche, ultimately leading to the reduction of antibody titers and long-term humoral protection.

Supported by grants from the NIH (R01 HLI141491) and the Eric Heyer M.D. Ph.D Translational Research Grant
XIST is a source of TLR7 ligands underlying the sex bias in Systemic Lupus Erythematosus
Authors: Crawford Jonathan D , Wang Hong , Talbot C Conover , Curran Ashley M , Goldman Daniel W , Petri Michelle , Antiochos Brendan , Darrah Erika
Published: January 2023

Systemic Lupus Erythematosus (SLE) is among the most sex-biased autoimmune diseases identified to date, affecting 9-times more women than men. Recognition of self-RNA by Toll-like receptor 7 (TLR7) is implicated as a central pathogenic process leading to the aberrant production of type-I interferon (IFN) in SLE, but the specific RNA molecules contributing to this process have not been defined. Given the role of self-RNA and biological sex in SLE pathogenesis, we investigated which sex-biased self-RNAs are potentially responsible for aberrant TLR7 activation in SLE. We used recent revelations about TLR7 sequence specificity and publicly available RNA sequencing data to identify sex-biased sources of self-RNA containing TLR7 ligands. We found X-inactive specific transcript (XIST) to be a particularly rich source of TLR7 ligands that is specifically expressed in women. We then investigated the capacity of XIST to act as a TLR7 ligand in vitro and found that multiple fragments of XIST induce IFNα production in a TLR7-dependent manner more than control RNA of equal length. Furthermore, we found that RNA isolated from XIST-knockout cells have significantly reduced capacity to stimulate TLR7 compared to RNA from wild-type cells. Finally, we used flow cytometry and publicly available RNA sequencing data to investigate the connection between XIST and SLE disease variables. We found that higher XIST expression correlated with SLE disease status, higher SLE disease activity index (SLEDAI) scores, and the interferon signature. Our data suggest that XIST is a source of TLR7 ligands that may underlie the sex bias in SLE.

Supported by grants from National Institute of Dental and Craniofacial Research (NIDCR), NIH (R21 DE028391-02), the Jerome L. Greene Foundation and Scleroderma Research Foundation.
T cells dominate peripheral inflammation in obesity-associated diabetes
Authors: Pugh Gabriella , Fouladvand Sajjad , SantaCruz-Calvo Sara , Agrawal Madhur , Zhang Xiaohua D , Chen Jin , Kern Philip , Nikolajczyk Barbara
Published: January 2023

Many studies have examined obese/insulin-resistant mice to conclude that macrophages dominate pathogenic inflammation in metabolic disease, but few studies have ranked chronic sources of peripheral inflammation in obese people across a natural spectrum of metabolic health. We compared the relative contributions of myeloid and T cells to peripheral inflammation in samples from metabolically healthy and unhealthy obese people to address disappointing outcomes from clinical trials of anti-inflammatories in obesity-associated type 2 diabetes (T2D). We stimulated PBMCs from obese subjects with euglycemia, prediabetes, or T2D with T cell-targeting CD3/CD28 or myeloid-targeting LPS, and measured supernatant cytokines over time. Cytokines increased with time following CD3/CD28, but LPS responses were time-independent. A series of surface marker and intracellular staining assays taking into account these temporal differences in cytokine production unexpectedly showed that T cells contribute more TNFα to peripheral inflammation than do myeloid cells from all subject groups. Bioinformatic modeling combining cytokine outcomes from all subjects, stimuli, and time points, indicated IFNγ generated by CD3/CD28 stimulation was most important for differentiating peripheral inflammation in T2D compared to non-T2D samples. This result was consistent with the previously identified Th1/Th17 profile in T2D. We conclude that human T cells dominate peripheral inflammation in obesity-associated T2D, and thus targeting T cells may be an effective approach for prevention/management of obesity-associated metabolic diseases.

Supported by NIH Training Grants T32 DK007778 and TL1 UL1TR001998, R01DK108056, the Shared Resource Facility of the University of Kentucky Markey Cancer Center P30 CA177558, University of Kentucky College of Medicine, Barnstable Brown Diabetes and Obesity Center, The Center for Clinical and Translational Research (UL1TR000117), and NIH National Center for Advancing Translational Sciences UL1TR001998.
Neutrophils control autoreactive Th17 responses in spondyloarthritis
Authors: Asare-Konadu Kofi N , Vance Emily E , Sen Rouhin , Caplan Liron , Rosenzweig Holly L , Napier Ruth J
Published: January 2023

Spondylarthritis (SpA) refers to a group of autoimmune diseases characterized by inflammation of the spine (spondylitis) and joints (arthritis) including ankylosing spondylitis and psoriatic arthritis. Genetic studies suggest that autoreactive Th17 cells drive SpA pathogenesis and in support IL-17 blockade in the clinic moderates symptoms. Although Th17 responses appear to be key in driving symptomatic disease, less is understood of the underlying innate cellular mechanisms that contribute to the initiation of pathogenic T cells. Upon analysis of SpA patient cell blood count data (1,497 events) we discovered a positive correlation between the absolute number of neutrophils and disease severity. Thus, we sought to determine how dysregulated neutrophil responses might control autoreactive Th17 responses using an experimental model of SpA (SKG mice). SKG mice have a point mutation in Zap70 resulting in autoreactive Th17 cells that cause arthritis and spondylitis. When SKG splenocyte cultures were treated with monoclonal antibodies to CD3 and simultaneously co-incubated with autologous neutrophils for 72h we detected increased T cell activation status (CD69 expression) and production of IL-17 but not IFNγ compared to media only (no neutrophils) controls. Neutrophil-deplete SKG mice had reduced autoreactive Th17 responses and delayed onset of arthritis compared to neutrophil-replete controls, supporting a role for neutrophils in promoting autoreactive Th17 responses and the induction of disease. In conclusion our data reveal a role for neutrophils in pathogenesis of SpA, indicating that neutrophil modulating drugs could be considered as a future treatment strategy for SpA.

Supported by grants from VA (CDA-2 IK2BX004523 and Merit I01BX002180) and NIH (R01 EY025250)
GFI1 KO can develop allergic reaction independently from IL5 and ILC2 cells
Authors: Fraszczak Jennifer , Dalloul Zeinab , Moroy Tarik
Published: January 2023

The number of people living with a chronic inflammatory lung disease, such as asthma, is constantly increasing. The development of resistance to existing therapies and the fact that exposure to air pollution and particular matter are continuously increasing have made asthma a major health problem worldwide. We have observed that in mice lacking the transcriptional factor GFI1 (short for “Growth factor independent 1”) where ILC2 are not functional and IL5 is almost absent, a severe asthmatic inflammatory reaction can still be provoked with increased numbers of eosinophils in the airways. This finding strongly suggests that pathways other than those initiated by IL5/IL5R and ILC2 cells exist that play a major role in severe allergic asthma. We hypothesize that the situation in GFI1 KO mice reflects what it is observed in asthmatic patients that are resistant to the recent therapies. We further posit that GFI1 KO mice is a tool to discover new pathways involved in asthmatic reactions different from the known and established mechanisms involving IL5. We are using this unique mouse model (Gfi1 deficient mice) and high throughput genomic sequencing technologies with a specific bioinformatics approach to identify new molecules pathways mediating the inflammatory reaction in asthma. We expect to reveal new molecular signaling pathways that cause lung inflammation during an allergic asthma reaction, specifically those regulating eosinophil recruitment and activities. Our first results show that GFI1 KO seem to be able to support an allergic reaction though IL17 pathway and Th17 cells. We anticipate to identify of molecular targets, which could be used to develop new, more effective therapies for patients with severe allergic asthma.
β-Glucosylceramides and Tocopherols Regulate Development and Function of Dendritic Cells
Authors: Lajiness Jacquelyn D , Amarsaikhan Nansalmaa , Tat Kiet , Tsoggerel Angar , Cook-Mills Joan
Published: January 2023

In humans and mice, offspring of allergic mothers are predisposed to development of allergy. In mice, allergic mothers have elevated β-glucosylceramides (βGlcCers) that are transported to the fetus via the placenta and to offspring via milk. Elevated βGlcCers increase numbers of fetal liver CD11c+CD11b+ dendritic cells (DCs) and offspring allergen-induced lung eosinophilia. These effects are modifiable by maternal dietary supplementation with the plant-derived lipids α-tocopherol and γ-tocopherol. It is not known whether βGlcCers and tocopherols directly regulate development of DCs. In this study, we demonstrated that βGlcCers (C16:0, C18:0, C18:1, and C24:1) increased development of GM-CSF-stimulated bone marrow-derived DCs (BMDCs) in vitro. This increase in BMDC numbers was blocked by α-tocopherol and potentiated by γ-tocopherol. Furthermore, βGlcCer increased PKCα and PKCδ activation in BMDCs that was blocked by α-tocopherol. In contrast, γ-tocopherol increased BMDC PKCα and PKCδ activation and enhanced the βGlcCer-induced increase in PKCδ activation in a DC subset. Antigen processing per DC was minimally enhanced in βGlcCer-treated BMDCs and not altered ex vivo in lung DCs from pups of allergic mothers. However, lung DCs from pups of allergic mothers had an increased proportion of CD11b+CD11c+ DCs and exhibited enhanced stimulation of T cells ex vivo. Thus, βGlcCer, which is both necessary and sufficient for development of allergic predisposition in offspring of allergic mothers, directly increased development and PKC activation in BMDCs. Furthermore, this was modifiable by dietary tocopherols. This may inform design of future studies for approaches in the prevention or intervention in asthma and allergic disease.

Supported by grants from the NIH (U01 AI131337 (Cook-Mills), R01AI127695 (Cook-Mills)) as well as the Pediatric Scientist Development Program (Lajiness) and Marshall Klaus Perinatal Research Award (Lajiness)
Myeloid cell-specific IL4Rα deletion protects against mixed allergen-induced lung injury in mice
Authors: Choudhary Ishita , Vo Thao , Singamsetty Dhruthi , Paudel Kshitiz , Mao Yun , Lamichhane Richa , Patial Sonika , Saini Yogesh
Published: January 2023

Introduction

IL-4 and IL-13 play an essential role in the pathogenicity of allergic asthma via the common receptor, i.e., IL4Rα. However, the myeloid cell-specific role of IL4Rα-mediated signaling in allergic asthma has remained unclear. We hypothesized that myeloid cell-specific IL4Rα signaling is essential for granulocytic inflammation and associated pathological outcomes.

Methods

Myeloid cell-specific IL4Rα-deficient (LysMCRE/CRE/IL4RαFl/Fl) and control (LysMCRE/CRE/IL4RαWT/WT) mice were exposed to mixed allergens, and total and differential cell counts were performed on bronchoalveolar lavage fluid. Unlavaged left lung lobe was fixed in 10% formalin and used for lung pathology assessment.

Results

In contrast to mixed allergen-exposed control mice that had exaggerated immune cell recruitment, myeloid cell-specific IL4Rα-deficient mice had significantly reduced immune cell infiltration into the lung airspaces. The reduction in immune cell infiltration in myeloid cell-specific IL4Rα-deficient mice was attributable to a significant decrease in eosinophils and lymphocytes. Additionally, while mixed allergen-exposed control mice exhibited pronounced lung inflammation and consolidation, myeloid cell-specific IL4Rα deletion significantly ameliorates lung pathology. Mucous cell metaplasia (MCM), another key feature of allergic asthma, was unaltered in myeloid cell-specific IL4Rα-deficient mice suggesting its dispensable role in mixed allergen-induced MCM.

Conclusion

Collectively, our data show that myeloid cell-specific IL4Rα signaling plays an indispensable role in immune cell recruitment specifically eosinophils, and its deletion protects against mixed allergen-induced lung injury.

Supported by grants from NIEHS (R01 ES030125) and NIGMS (P20 GM130555-6611)
Early life exposure to Streptococcus pneumonia has sex-specific long-term impact on inflammatory responses
Authors: Lauzon-Joset Jean-Francois , Lerdu Ophelie , Bouchard Karine , Patoine Dany , Mbareche Hamza , Bissonnette Elyse
Published: January 2023

INTRODUCTION

Lung microbiota plays a central role in health and disease development. Recently, recurrent viral infections in preschool age children were associated with lung microbiota carriage of Streptococcus pneumonia (SP). In addition, immune development and inflammatory responses have sexual dimorphism, but it is still controversial whether there is sex-specific microbiota composition.

OBJECTVE

Given that immune development occurs early in life, the objective of this project is to evaluate the consequences of early life lung microbiota alteration by SP exposure on inflammatory response later in life.

METHODS

Neonatal Brown Norway rats were exposed to SP via intranasal administration (sub inflammatory dose). Lung microbiota was assessed until weaning age (21 days old), using v3–v4 16S rRNA sequencing. At 8 weeks old (post-puberty), rats were infected with LPS and their inflammatory responses were assessed 24h post using bronchoalveolar lavage (BAL).

RESULTS

Early life exposure to SP reduced lung microbiota diversity and impacted its constitution until weaning age. Although control females exposed to LPS had higher inflammatory response than control males, early life exposure to SP significantly reduced BAL inflammatory cell recruitment in females but not in SP-males. Opposingly, in SP-males, the inflammatory response shifted from a neutrophilic response to a more eosinophilic inflammation.

CONCLUSION

Early life exposure to SP modulated both lung microbiota and immune responses later in life. The sex-specific immune modulation by lung microbiota composition in early life could have large implication for many inflammatory diseases presenting sexual bias.

Supported by grants from Quebec Health Research Network and Fondation IUCPQ (Quebec, Canada).
Immunoresponsive gene 1 modulates neuroinflammation through the induction of heme oxygenase-1
Authors: Kuo Ping-Chang , Weng Wen-Tsan , Scofield Barbara A , Furnas Destin , Paraiso Hallel C , Yu I-Chen , Yen Jui-Hung
Published: January 2023

Inflammatory stimuli induce immunoresponsive gene 1 (IRG1) expression that in turn catalyzes the production of itaconate through diverting cis-aconitate away from the tricarboxylic acid cycle. The immunoregulatory effect of IRG1/itaconate axis has been recently documented in lipopolysaccharide-activated mouse and human macrophages. Currently, whether IRG1/itaconate axis exerts a modulatory effect in ischemic stroke remains unexplored. In this study, we investigated whether IRG1 plays a role in modulating ischemic brain injury. Our results showed IRG1 was highly induced in the ischemic brain following ischemic injury. We found that IRG1−/− stroke animals exhibited exacerbated brain injury, displayed with enlarged cerebral infarct, compared to wild type stroke controls. Furthermore, IRG1−/− stroke animals presented aggravated blood-brain barrier disruption, associated with augmented Evans blue leakage of the ischemic brain. Moreover, IRG1−/− stroke animals displayed elevated microglia activation, demonstrated with increased CD68, CD86, and Iba1 expression. Further analysis revealed that IRG1 was induced in microglia after ischemic stroke, and deficiency in IRG1 resulted in repressed microglial heme oxygenase-1 (HO-1) expression. Notably, the administration of dimethyl itaconate, an itaconate derivative, to compensate the deficiency of IRG1/itaconate axis led to enhanced microglial HO-1 expression, alleviated ischemic brain injury, improved motor function, and decreased mortality rate in IRG1−/− stroke animals. In summary, we demonstrate that the induction of IRG1 in microglia following ischemic stroke may serve as a protective mechanism to restrain brain injury through HO-1 upregulation.

This work was supported by Indiana University Startup Fund and in part by the National Institutes of Health (R01NS102449) to J.-H.Y.
MEF2A is a Regulator of Interferon-Mediated Inflammation
Authors: Forero Adriana , Smith Julian R , Dowling Jack W , Savan Ram
Published: January 2023

Interferons (IFN) are antiviral cytokines induced by recognition of viral nucleic acids or cellular replicative stress. In particular, the accumulation of cytosolic nucleic acids or the accumulation of DNA lesions has the potential to activate cGAS/STING-mediated IFN induction if not properly constrained. While these responses can be harnessed as tumor therapies, the excessive induction of IFNs and deleterious IFN-mediated inflammation is associated with tissue damage in non-malignant disease. We sought to identify non-canonical IFN-modulatory transcription factors (TF) by examining factors with known genetic alterations that associate with both malignant and inflammatory diseases. Here, we identify MEF2A as a negative regulator of the production of IFNs at homeostasis. The loss of MEF2A function results in the spontaneous induction of type I and III IFNs and downstream ISG expression. We demonstrate that IFN production following MEF2A depletion was dependent on DNA replication and cell cycle progression with CDK2 inhibition ablating this response. Furthermore, DNA lesions that induced DNA damage responses following MEF2A loss resulted in cGAS/STING pathway activation and genetic deletion of STING maintained homeostatic levels of type I IFN in the absence of this TF factor. Thus, our study is first to connect MEF2A with protection from maladaptive type I IFN responses due to genomic instability across various cell types. Overall, our study reveals that therapeutic modulation of MEF2A or other members of its family could be beneficial for the management of viral, malignant, and autoinflammatory diseases.
Myeloid cells regulate retinal neovascularization via SOCS3
Authors: Sun Ye , Wang Tianxi , Lam Enton
Published: January 2023

Neovascularization is the leading cause of vision loss in retinopathy of prematurity (ROP). Inflammation and myeloid cells are involved in retinal angiogenesis. However, the underlying mechanisms of how myeloid cells control neovascularization in ROP remain unknown. We and others previously reported that suppressor of cytokine signaling 3 (SOCS3) is induced in retinopathy. To investigate whether myeloid cells regulate retinal angiogenesis via SOCS3 in ROP, we used myeloid lineage specific SOCS3 loss-of-function (conditional knockout, cKO) mice and gain-of-function (conditional overexpression, cOE) mice and generated ROP mouse model - mice with oxygen induced retinopathy (OIR) by exposing mice to 75% oxygen from postnatal day (P)7 to P12 for 5 days, and then returned to normal air until P17. Retinas were collected for analysis including quantification of retinal neovascularization, gene expression, immune cell profiling and single cell sequencing. Our results showed that mice with myeloid SOCS3 deficiency had a substantial increase in pathological retinal neovascularization, whereas overexpression of SOCS3 in myeloid cells prevented pathological retinal neovascularization in response to OIR. Our single-cell sequencing data showed that population of myeloid lineage cells including microglia, macrophages and neutrophils was changed in mice with OIR and controlled by SOCS3. Therefore, myeloid cells are involved in retinal neovascularization via SOCS3 in ROP. Manipulating SOCS3 expression in myeloid cells may provide a new way to cure neovascularization in retinas.

Supported by grants from NIH (R01EY030140, R01EY029238) and from BrightFocus Foundation
Targeting TCA cycle metabolites by small molecule inhibitors ameliorate LPS induced immune tolerance in Macrophages through epigenetic mechanisms
Authors: Abhimanyu Abhimanyu , Ladki Malik , Longlax Santiago Carrero , Sheikh Daanish , Nishiguchi Tomoki , Dinardo Andrew
Published: January 2023

Background

Severe and chronic infections, such as sepsis and Tuberculosis, result in macrophage immune tolerance in order to prevent pathologic inflammation. Immune tolerance is believed to be a result of metabolic dysregulation, especially of the TCA cycle. Metabolites play a critical role in regulating epigenetic mechanism, but many aspects remain poorly understood.

Methods

An in-vitro LPS tolerance model was used to evaluate if manipulating the TCA cycle could mitigate immune tolerance. Intracellular signaling, DNA methylation, and nuclear localization were measured using RPPA, Illumina EPIC, and confocal microscopy, respectively.

Results

Metabolic inhibitors of glycolysis (metformin), the TCA cycle (BAY 1436032), and mTOR (rapamycin and everolimus) blocked LPS-induced immune tolerance. LPS treatment changed the methylome with hypermethylation of key genes of the TCA cycle including PDP2, NDUFS8, TRAP1 and PDK2 and lead to increased nuclear localization of the TCA enzymes, which could be ameliorated with inhibitors. Increased levels of key mediators of the TCA by LPS treatment including p-mTOR, pAKT, LDHA, IDH1, LC3A and epigenetic regulator p300 was significantly downregulated by pre-treatment with metformin, everolimus and BAY 1436032. BAY 1436032 also significantly reduced the LPS induced upregulation of epigenetic enzymes including histone deacetylases (HDAC) 3,4, and EZH2.

Conclusion

Previous studies demonstrated that LPS induces epigenetic-mediated immune tolerance. Here we show this can be mitigated by inhibiting glycolysis, mTOR pathway and TCA cycle. This could have clinical implications in treatment of sepsis and Tuberculosis among other diseases.

Supported by NIAID K23 AI141681-02
Targeting intrinsic regulators of antibody and B cell memory to control chronic viral infection
Authors: Good-Jacobson Kim , Di Pietro Andrea , Cooper Lucy
Published: January 2023

The production of antibody and formation of immunological memory underpins vaccine success. Yet, chronic viral infections result in an inadequate humoral response, and little is known about the mechanisms underpinning this dysregulation. We therefore utilised acute or chronic strains of lymphocytic choriomeningitis virus (LCMV) to investigate B cell behaviour in vivo during chronic infection. Our work revealed that chronic infection altered epigenetic regulation of B cell fate, and a key epigenetic target could be therapeutically modulated to correct antibody responses. Deep-sequencing revealed key differences in chromatin accessibility of B cells responding to acute vs chronic infection, leading us to investigate the Polycomb repressive complex 1 component BMI-1. BMI-1 was upregulated in germinal centre B cells of mice infected with chronic LCMV, compared to acute LCMV. B cell-intrinsic deletion of BMI-1 resulted in a restoration of splenic architecture, and an enhanced and rapid ability to clear virus. Deletion of BMI-1 re-established appropriate c-Myc expression in B cells, concomitant with improved quality of antibody with enhanced antibody-dependent effector function and increased neutralising capacity. Together, our data defines BMI-1 as a crucial immune modifier that controls antibody-mediated responses in chronic infection. Further, single-cell RNA-seq and genetic models revealed that excess immune complexes, in concert with dysregulated complement-mediated functions, contribute to ineffective memory formation during chronic viral infection. Understanding the molecular events that may sustain dysfunction lays the foundation for improving therapeutic interventions in chronic infectious diseases.

Supported by Bellberry-Viertel Senior Medical Research Fellowship and an AAI Careers in Immunology Fellowship.
Blimp1 controls GC B cell expansion and exit through regulating cell cycle progression and key transcription factors BCL6 and IRF4
Authors: Luo Wei , Conter Laura , Callahan Derrick , Smita Shuchi , Shlomchik Mark J
Published: January 2023

Blimp1 is required for plasma cell differentiation and antibody production. Blimp1 is also expressed in some dark zone germinal center (GC) B cells (GCBC), however, the exact role of Blimp1 in regulating GC B cell function is not clear. To investigate this, we generated chimeric mice using equal mixtures of bone marrow (BM) from Blimp1+/+ CD19Cre and Blimp1fl/fl CD19Cre mice. In these BM chimeras, Blimp1 sufficient and deficient B cells compete in the same environment, allowing us to examine the cell-intrinsic function of Blimp1 in GCBC, independent of environmental factors such as antibody or antigen levels. We discovered striking B cell-intrinsic roles for Blimp1. Blimp1 deficient B cells quickly dominate GCs and persist for a much longer time compared to the wild type cells. Mechanistically, Blimp1 deficiency increases the percentage of GCBC in the S phase. This difference seems independent of positive selection signals, because Blimp1 deficient GCBC express normal levels of c-Myc, a marker of positive selection. Importantly, Blimp1 deficient GCBC fail to downregulate BCL6 and upregulate IRF4, suggesting that Blimp1 induction in GCBC controls the expression of these key transcription factors, which are required for exit from the GC program. Consistent with this, memory B cells derived from the Blimp1 deficient B cells were only slightly increased, despite the fact that Blimp1 deficient GCBC are highly expanded and persistent. In addition, Blimp1 deficiency also reduced class-switching to IgG1 in response to NP-CGG immunization. These findings revealed unique antibody- and antigen-independent functions of Blimp1 in regulating GCBC responses that impact long-lived immune compartments.
Inverse-agonist (SMM-189) suppresses colitis by inducing endogenous cannabinoids and attenuating Th17, neutrophils, natural killer cells
Authors: Rakib Ahmed , Kiran Sonia , Boddu Geetha , Moore Bob M , Singh Udai P
Published: January 2023

The cause of inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), are multi-factorial and include activated immune cells, chronic inflammation, genetics, and environmental exposure. It is well known that endocannabinoids mediate protection against intestinal inflammation and other autoimmune diseases. However, the effect of cannabinoid receptors induction by inverse-agonist during experimental colitis has not been investigated. Here, we investigate the activation of the cannabinoid receptor-2 (CB2) by SMM-189, a potent CB2 inverse agonist, on the inhibition of dextran sodium sulfate (DSS) induced colitis. In the present study, we found that SMM-189 effectively attenuated the overall clinical score, reversed colitis-associated pathogenesis, increased body weight and colon length while increasing the expression of CB2 receptors and protein kinase A (PKA) in the colon lamina propria (LPs). We also measured a decrease in the percentage and number of Th17 cells in the spleen, mesenteric lymph nodes (MLNs), and LPs treated with SMM-189 compared to DSS controls. Similarly, the percentage and number of natural killer T (NKT) cells and neutrophils are decreased after SMM-189 treatment. SMM-189 also induces the myeloid-derived suppressor cells (MDSCs) during chronic colitis progression as compared to DSS alone. These findings suggest that SMM-189 ameliorates experimental colitis by inducing CB2 receptors and PKA in the LPs, MDSCs and reduces Th17, NKT cells, and neutrophils from the spleen and LPs. Taken together, these data support the idea that the CB2 inverse agonists, such as SMM-189, may be developed as a novel therapeutic target for IBD.

Supported by NIH grant R01 AI140405
MicroRNA-10a negatively controls regulatory T cell suppression of colitis through inhibition of mitochondrial oxidation and Blimp1 expression
Authors: Yang Wenjing , Yu Tianming , Yao Suxia , Cong Yingzi
Published: January 2023

MicroRNAs play critical roles in maintaining intestinal homeostasis, and regulatory T cells (Tregs) restrict intestinal inflammation. However, how Tregs is regulated is still not completely understood. In this study, we investigate the mechanisms by which miR-10a controls Tregs’ suppressive function, thereby regulating intestinal inflammation. We found that miR-10a deficient Tregs expressed higher function markers in the intestine and were more potent in suppressing T cell proliferation and colitis induced by CD4+ CD45Rbhi T cells. Moreover, miR-10a did not affect Treg stability, which was evidenced by no differences in Foxp3 expression and methylation in Regulatory T-Cell-Specific Demethylated Region (TSDR). RNA sequencing data indicated that miR-10a regulates Tregs’ oxidative phosphorylation, which was verified by a higher level of oxygen consumption and mitochondrial ROS in miR-10a deficient Tregs. Pretreatment with a low dose of oligomycin, the ATP inhibitor which reduces mitochondrial respiration, impaired Tregs function in suppressing naïve T cell proliferation and colitis induced by CD4+ CD45Rbhi T cells in Rag−/− mice. Mechanically, miR-10a directly binds to uqcrq, the subunit VII of mitochondrial respiratory chain complex III, and Prdm1, which encodes Blimp1, a transcription factor that regulates Tregs’ functions. Furthermore, Blimp1-deficient Tregs showed decreased expression of function markers with the lower level of OCR and failed to restrict CD45Rbhi T cell-induced colitis. Collectively, miR-10a negatively regulates Tregs’ suppressive function by altering the mitochondrial metabolic process and inhibiting Blimp1, thereby controlling intestinal inflammation.
The autophagy gene Epg5 promotes susceptibility to enteric viral infection
Authors: Kalugotla Gowri , Lee Sanghyun , Schriefer Lawrence , Perez Luis Alberto Casorla , Huang Huiyan , Lu Qun , Orvedahl Anthony , Silverman Gary , Pak Stephen , Wang David , Baldridge Megan T
Published: January 2023

Mutations in the autophagy gene EPG5 are responsible for Vici syndrome, a multisystem human genetic disease with a combined immunodeficiency component. However, the mechanism(s) by which EPG5 regulates immune signaling are incompletely understood. Previously, we found that Epg5−/− mice are resistant to influenza and exhibit hyperinflammation in the lungs including elevated IL1B/IL-1β and TNF/TNFa. Here, we show that the disruption of Epg5 results in protection against multiple enteric viruses including norovirus and rotavirus. RNA sequencing revealed that IFN-λ responses are highly upregulated in the intestines of Epg5−/− mice. Further, mice lacking Epg5 exhibit substantial alterations of the intestinal microbiota. Surprisingly, germ-free Epg5−/− mice showed persistent inflammation of both the intestine and lung, suggesting a microbiota independent mechanism. Epg5−/− Ifnlr1−/− mice, which lack the receptor for IFN-λ, regained susceptibility to viral infection but maintained microbial dysbiosis, indicating that IFN-λ signaling is the primary mediator of resistance to enteric viruses but is dispensable for microbial dysbiosis in Epg5−/− mice. Intriguingly, epg-5-mutant Caenorhabditis elegans animals are also resistant to Orsay virus, which is an intestinal cell-tropic virus and the only known virus that naturally infects C. elegans. This study unveils an important role for autophagy gene Epg5 in protection of mammalian hosts by modulating intestinal IFN-λ responses, which is unexpectedly independent of the microbiota, and raises the possibility of a conserved viral resistance mechanism that is shared between C. elegans and mammals.
Intestinal epithelial autophagy protects from cytokine-driven mortality and IFNγ-dependent cell death in acute small intestinal injury
Authors: Foerster Elisabeth G , Streutker Catherine J , Maughan Heather , Tsang Derek K L , Robert Lukian M , De Sa Oliver , Girardin Stephen E , Philpott Dana J
Published: January 2023

Crohn’s disease (CD) is characterized by a breakdown of the intestinal mucosal barrier. A genetic polymorphism of the autophagy gene ATG16L1 is associated with increased risk of developing CD, and it impairs the protective and regenerative functions of the intestinal epithelium. Autophagy acts as a regulator of cell death and has been implicated in the protection from both necroptosis and apoptosis in the epithelium.

Our work showed that mice specifically deleted for Atg16l1 in intestinal epithelial cells (Atg16l1ΔIEC) had exacerbated T cell-mediated intestinal damage in the model of anti-CD3 enteropathy compared to wild-type mice, which was characterized by crypt epithelial cell death and heightened inflammation. Moreover, Atg16l1 deficiency delayed the recovery of the intestinal epithelium, and Atg16l1-deficient IECs were impaired in their proliferative response. Pathology and mortality were largely driven by microbiota-dependent expression of Ifng, Tnf, and type I IFN response genes in Atg16l1ΔIEC mice. Mechanistically, while survival was rescued by blocking these cytokine pathways independently, only anti-IFNγ treatment abrogated intestinal epithelial cell (IEC) death in Atg16l1ΔIEC mice, thereby decoupling IEC death and survival. In summary, our findings suggest differential roles for IFNγ, TNF, and type I IFN in acute enteropathy and IEC death in the context of autophagy deficiency.

Supported by grants from CIHR (MOP-12353, FDN-14333) and NSERC (RGPIN-201).
NAIP—NLRC4 inflammasome activation in tuft cells contributes to host defense against bacteria
Authors: Churchill Madeline J , Rauch Isabella
Published: January 2023

The small intestinal epithelium is exposed to various microbes, and intestinal epithelial cells (IECs) have developed an array of host defense mechanisms to distinguish commensals from pathogenic microbes and initiate an immune response against the later. NAIP—NLRC4 inflammasome induced-pyroptosis, which is activated when bacterial flagellin or type III secretion systems are detected in the cytosol of the host cell, is one such mechanism. Activation of NAIP—NLRC4 induces extrusion of IECs and release of the inflammatory mediators prostaglandin E2 and IL-18. Work on epithelial inflammasome activation thus far has not delineated if different subtypes of IECs have specific roles in the ensuing immune response. Tuft cells are a rare subset of IEC that is primarily known for its role in sensing parasites, and it is unknown whether they are involved in promoting antibacterial host responses. Here we show that upon activation of the NAIP—NLRC4 inflammasome specifically in tuft cells, they uniquely among IECs release prostaglandin D2, and within the small intestine, tissue IL-22 increases. NLRC4 expression only in tuft cells leads to better control of Salmonella Typhimurium compared to NLRC4 null controls. Taken together, these data suggest that NAIP— NLRC4 inflammasome activation in tuft cells leads to lipid mediator induced downstream inflammatory responses that are complementary to signaling pathways that are activated by other IEC subsets. Understanding how NAIP—NLRC4 activation in different epithelial subsets affects downstream inflammatory responses will inform on the different signaling pathways that synergize together to mount pathogen specific host defense mechanisms.
Contribution of the microbiome, environment, and genetics to mucosal Type 2 immunity and anaphylaxis in a murine food allergy model
Authors: Stark Kelsey , Falkowski Nicole R , Brown Christopher A , McDonald Roderick A , Huffnagle Gary B
Published: January 2023

Murine models of food allergy are useful tools for identifying potential pathways involved in the manifestation of anaphylactic reactions to food in humans. Individual mice exhibit inherent heterogeneity in their immune responses, including anaphylaxis, much like what is seen clinically in food allergic humans. The microbiome (MB) may be one major driver of this difference in individuals. MB dysbiosis by broad spectrum antibiotics and/or yeast colonization can alter systemic immunity and favor the development of mucosal Type 2 immunity to aeroallergens. Our objective was to use a well-characterized murine model of food allergies (chicken egg ovalbumin, OVA) and determine if dysbiosis could enhance the manifestation of food allergies and identify elements of the MB and host response associated with this heterogeneity. In our dataset, the intensity of anaphylaxis was most strongly associated with a disrupted MB that included C. albicans colonization, loss of a specific Lachnoclostridium species, development of a highly polarized Type 2 response in the intestines, and activation of mucosal mast cells. OVA-specific IgE serum levels were not predictive of the response and in germ-free mice the response was not fully recapitulated. Conventionalization of germ-free mice resulted in Akkermansia muciniphila outgrowth and more heterogeneity in the allergic response. We also observed that housing environment could induce MB changes that altered the response. Thus, our data recapitulate the heterogeneity in anaphylactic reactions, ranging from severe to none, seen in patients with circulating levels of food allergen-specific IgE and support the idea that alterations in the MB can be one factor underlying this heterogeneity.

Supported by grants from NIH NIAID R01AI138348, Mary H. Weiser Food Allergy Center (MHWFAC), and the Nina and Jerry D. Liptak Endowment of the MHWFAC
Exploiting commensal bacteria to strengthen host immunity and treat infections
Authors: Ramirez Zeni Elizia , Surana Neeraj K
Published: January 2023

As the rise of microbial resistance to antibiotics continues, there is a pressing need for new therapeutic strategies. One method includes augmenting host immunity and subsequent defense molecules to protect against pathogenic organisms. Antimicrobial peptides (AMPs) represent an ancient arm of the innate immune system that is critical for maintaining host–microbiome interactions. We previously identified that the commensal bacteria Ruminococcus gnavus and Lactobacillus reuteri induce ileal expression of Reg3γ, an AMP with bactericidal activity against gram-positive bacteria. We now find that R. gnavus and L. reuteri induced expression of these AMPs requires Myd88 and group 3 innate lymphoid cells but, interestingly, does not require persistent probiotic colonization. Consistent with this increase in AMP expression, R. gnavus- and L. reuteri-treated mice were protected against intestinal colonization by vancomycin-resistant enterococci (VRE). Finally, patients at high-risk for VRE domination were >4 times less likely to have Enterococcus in their fecal microbiota if they also had Ruminococcus present, a finding that demonstrates the clinical translatability of R. gnavus mediated protection. Taken together, these results spotlight the potential for probiotic based therapies to combat antimicrobial resistant organisms.

Supported by grants from NIH (1F31AI161989-01)