bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Thrombopoiesis by the Inhibitor of MyoD Family A (I-mfa)
Authors: Houser Jeremy S , Patel Maulin , Onopiuk Marta , Tsiokas Leonidas , Humphrey Mary Beth
Published: January 2023

The differentiation of megakaryocytes (Mk) from hematopoietic stem and progenitor cells (HSPC) is crucial to homeostasis and requires tight regulation. Cell-intrinsic factors skew HSPCs towards the Mk lineage and promote terminal differentiation, but this regulatory network is not yet well understood. The Inhibitor of MyoD Family A (I-mfa) is a protein which interacts with multiple pathways, including β-catenin/TCF/LEF and JNK which have known roles in HSPC differentiation. This led us to examine the role of I-mfa in hematopoiesis, hypothesizing that it promotes Mk differentiation by regulating cellular signaling events.

Mice which completely lack I-mfa expression (I-mfa KO) had reduced percentages of Mk/Erythroid Progenitors (MEPs) in the bone marrow compared to WT animals by flow cytometry, as well as reduced platelet counts in peripheral blood. To examine the potential role for I-mfa in thrombopoiesis, K562 cells (which show PMA-induced Mk differentiation) were transfected with a vector for doxycycline-induced overexpression of I-mfa, and differentiation was assessed by flow cytometry for Mk surface markers. Cells overexpressing I-mfa showed increased differentiation with a higher proportion of CD61+, CD71− cells compared to parent. The mechanism underlying this effect is being examined by analyzing cellular signaling after PMA stimulation and identifying I-mfa binding partners over time.

This work demonstrates a role for I-mfa in the regulatory control of hematopoiesis and lineage commitment through its regulation of thrombopoiesis. These results may also suggest that I-mfa could be an important cellular regulator in hematopoietic disorders including anemia, bone marrow failure, or hematological malignancies.

Supported by NIH T32 Immunology Training Grant AI00763316A1
Covalent heterobivalent inhibitor effectively inhibits anaphylaxis to peanut allergen in a humanized mouse model
Authors: Alakhras Nada Salem , Shin Jaeho , Smith Scott , Bilgicer Basar , Kaplan Mark H
Published: January 2023

Peanut-induced allergy is an IgE-mediated type I hypersensitivity reaction that manifests symptoms ranging from local edema to life-threatening anaphylaxis. While there are treatments for symptoms in allergic patients resulting from allergen exposure, there are few preventive therapies other than avoidance and no allergen specific therapies. We have previously designed a covalent heterobivalent inhibitor (cHBI) that binds in an allergen-specific manner as a preventive for allergic reactions. Building on previous in vitro testing, in this report we have developed a humanized mouse model to test cHBI efficacy in vivo. Humanized mice (NSG-SCF/GM-CSF/IL-3) developed mature functional human mast cells in various tissues and developed robust anaphylactic reactions when passively sensitized with human IgE monoclonal antibodies specific for peanut allergen. We found that the allergic response is IgE dose-dependent and is mediated by human mast cells indicated by elevated tryptase serum levels and the upregulation of mast cell degranulation markers. Using the validated humanized mouse model, we showed that cHBI inhibited IgE-mediated anaphylaxis and human mast cell degranulation. We demonstrated that cHBI effectively inhibits anaphylaxis for up to 14 days, and cHBI inhibition is specific to peanut allergen. Importantly, cHBI rescued the mice from lethal anaphylactic response during oral peanut-induced anaphylaxis. These findings suggest that cHBI has the potential to be an effective preventative for peanut food allergy in patients.
Comparison of sensitizing and disease-eliciting capacities of salt-soluble protein extracts from diploid (einkorn) versus tetraploid (durum) wheats
Authors: Gao Haoran , Jorgensen Rick Dean , Raghunath Rajsri , Ng Perry K W , Gangur Venugopal
Published: January 2023

Hypersensitivity reactions to wheat have reached significant levels of global public health concern for reasons that are incompletely understood. Einkorn wheat (Triticum monococcum, genome AA) is commercially available but is rarely consumed, whereas durum wheat (Triticum durum, genome AABB) is commonly used for pasta making. At present, most mouse models of wheat allergy are adjuvant-based, which fail to reveal the intrinsic allergenic potential of different wheat products. Here we compared einkorn and durum wheats in terms of their sensitizing and disease-eliciting capacities in an adjuvant-free mouse model of anaphylaxis induced by salt-soluble protein extract (SSPE). Balb/cJ female mice were sensitized transdermally with SSPE or saline for nine weeks. Later, systemic or oral anaphylactic reactions were elicited in these mice using SSPE via intraperitoneal or oral route, respectively. We did not observe major differences in sensitizing capacity as suggested by antibody responses (i.e., sIgE and tIgE levels) in SSPE-sensitized mice. However, SSPE from durum appears to be more potent than that from einkorn in eliciting systemic but not oral anaphylaxis as shown by hypothermic shock responses as well as mucosal mast cell degranulation. This is the first report on comparing the intrinsic allergenicity between tetraploid and diploid wheats in an adjuvant-free mouse model.

This research was funded by: the United States Department of Agriculture (USDA)/National Institute of Food and Agriculture (NIFA); Hatch project MICL02486 (Accession Number: 1012322), Hatch project MICL01699; Agricultural and Food Research Initiative Competitive Program, grant number: 2018-67017-27876; and Project GREEEN (Michigan State University); R.J., was supported by the John Harvey Kellogg Graduate Assistantship and the Academic Achievement Graduate Assistantship from the Michigan State University.
Paneth cells regulate allergic sensitization in the gastrointestinal tract
Authors: Kim Eunsoo , Burnett Drew J , Joldrichsen Marisa R , Mori-Akiyama Yuko , Cormet-Boyaka Estelle , Boyaka Prosper N
Published: January 2023

The gut immune homeostasis plays a major role in controlling sensitization to food allergens and the development of allergic responses in the gastrointestinal tract, but also to skin or lung. Epithelial cells sensing of commensal microbes is crucial for the maturation of the gut immune system and antibiotic treatment during early life is believed to have contributed to the increased incidence of food allergy. Paneth cells secrete antimicrobial products and cytokines in the crypts of the small intestine, but their role in food allergy remains elusive. We found that SOX9ΔIEC mice, which lack Paneth cells, display dysbiosis with increased microbial diversity. Interestingly, Paneth cell deficiency increased the of numbers of ILC2 in intestinal lymphoid tissues. Upon oral sensitization with a model food antigen in the presence of cholera toxin, SOX9ΔIEC mice developed significantly higher allergen-specific serum IgE responses, and enhanced signs of allergic responses (i.e., swelling, hypothermia, impairment of lung functions) after nasal allergen challenge. Transplantation of fecal materials from SOX9ΔIEC mice was able to transfer the hyper-allergenic phenotype to control wild-type mice. In addition, bacteria-free fecal extract from SOX9ΔIEC mice increased the number of IgE producing B cells than fecal extract from control mice with treatment of anti-CD40 and IL-4 in vitro. Ongoing studies are assessing the metabolites present in the intestinal lumen of SOX9ΔIEC mice and their effect of intestinal epithelial cells and myeloid cells to elucidate the mechanisms by which impaired or defective anti-microbial functions of Paneth cells regulate allergic sensitization of the GI tract.

Supported by Ohio State Univerisity NIH
PD-1 blockade in T follicular helper cells protects mice from peanut allergy by promoting production of low-affinity antibodies
Authors: Lama Jyoti K , Iijima Koji , Kobayashi Takao , Kita Hirohito
Published: January 2023

Food allergy, such as peanut allergy can cause life-threatening anaphylaxis. While T follicular helper (Tfh) cells play a major role in production of allergen-specific IgE and IgG antibodies, the immunologic mechanisms that regulate the production of antibodies that mediate anaphylaxis are not well understood. While PD-1 is highly expressed on Tfh cells, our knowledge is limited regarding the role of the molecule in Tfh cells. Thus, the objective of this study was to investigate the roles of PD-1 in allergic immune responses using a mouse model of peanut allergy. Naïve wild-type mice were exposed to peanut flour intranasally (i.n.) and then challenged by intraperitoneal (i.p.) injection of peanut extract. The mice exposed i.n. to peanut flour produced peanut-specific IgE and IgG antibodies and developed anaphylaxis when challenged i.p. with peanut extract. PD-1 blockade with anti-PD-1 treatment Tfh and germinal center B cell numbers in mice exposed to peanut flour. Nonetheless, anti-PD-1 or anti-PD-L1 fully protected mice from anaphylaxis. Anti-PD-1 or genetic deficiency of PD-1 in CD4+ T cells reduced and increased levels of peanut-specific IgE and IgG, respectively. Anti-PD-1 promoted production of low-affinity IgE and IgG antibodies with no effect on high-affinity antibodies. By passive cutaneous anaphylaxis, peanut-specific antibodies generated in anti-PD1 treated mice failed to induce anaphylaxis, and plasma from these mice prevented anaphylaxis. Therefore, blockade of the PD-1 pathway is protective against allergic immune responses. The interaction between Tfh cells and B cells may play a pivotal role in controlling antibody quality and clinical manifestation of allergic diseases.
Interleukin-10 enhances IL-33-mediated MC activation and modulates the development of food allergy
Authors: Mathias Clinton B , Krajewski Dylan , Taruselli Marcela T , Rovatti Jeffrey , Mire Mohamed , Schneider Sallie S , Ryan John J
Published: January 2023

We recently demonstrated an unexpected, proinflammatory role for the pleiotropic cytokine, IL-10, in promoting mast cell (MC) responses during food allergy. IL-10 enhanced MC proliferation and survival and promoted IgE-dependent responses to enteric ovalbumin (OVA) challenge. However, whether the effects of IL-10 on MCs extend beyond IgE-mediated signaling is not clear. To determine whether IL-10 can prime MC activation mediated by IgE-independent stimuli, we assessed the effects of rIL-10 on IL-33-stimulated bone marrow-derived MCs (BMMCs) and examined the development of food allergy in IL-10-depleted ST2−/− mice. IL-10 co-culture significantly enhanced the activity and cytokine production of both IgE and IL-33-activated MCs. Furthermore, the production of MC-derived cytokines such as IL-13 was suppressed in IL-33-stimulated IL-10−/− BMMCs, which could be restored by addition of exogenous IL-10. IL-10 enhanced MC FcɛRI and ST2 expression. Further, ST2−/− BMMCs demonstrated that IL-10 enhanced IgE-mediated activation in the absence of autocrine IL-33 signaling. To assess the role of IL-10 in vivo, food allergy was measured in WT and ST2−/− mice subjected to antibody-mediated IL-10 depletion. IL-10-depleted WT mice exhibited a significant attenuation in allergic diarrhea and MC-mediated responses to OVA challenge. Similarly, ST2−/− mice also exhibited a profound suppression of MC-mediated responses, demonstrating the importance of IL-33 in MC regulation. IL-10 depletion had no additional effects in ST2−/− mice. However, IL-10/ST2−/− mice exhibited a further decrease in OVA-IgE and MC activation compared to ST2−/− mice. Our data reveal a novel role for IL-10 in enhancing both IgE and IL-33-mediated MC responses.

This project was supported by funds from the National Institutes of Health grants: NIAID R15AI107668 (CBM)
Loss of isoprenylation significantly reduces IgE mediated mast cell activation
Authors: Dailey Jordan , Ballance William , Kee Sydney Ann , Kotha Aditya , Sebti Said , Ryan John J
Published: January 2023

Allergic disease is the 6th leading cause of chronic illness in the US and accounts for billions of dollars in healthcare costs annually. Mast cells are tissue resident innate immune cells linked to allergic disease and activated by IgE and other ligands. Upon activation they release histamine, cytokines, chemokines, proteases, and lipid mediators evoking allergic symptoms. New ways of targeting mast cells could greatly benefit allergic disease therapy. Previous findings showed repurposing of statin drugs, such as Fluvastatin, reduced allergic symptoms in vitro and in vivo. We found that Fluvastatin suppresses IgE-mediated mast cell activation by inhibiting isoprenylation of proteins. However, statin effects were dependent on genetic background. To avert genetic background dependency, we tested the hypothesis that a dual farnesyl and geranylgeranyl transferase inhibitor (FGTI 2734) would suppress allergic disease withoutgenetic background limitations. We show that FGTI 2734 reduced IgE-mediated degranulation and cytokine production by bone marrow derived mast cells from multiple genetic backgrounds. Similarly, FGTI 2734 also suppressed IgE-mediated anaphylaxis on multiple genetic backgrounds. These in vivo effects extended to an allergic asthma model, in which FGTI 2734 inhibited pulmonary inflammation. Finally, we show that FGTI-2734 can significantly reduce IgE-induced cytokine production from human skin mast cells. These data show that dual farnesyl and geranylgeranyl transferase inhibitors may be an effective therapeutic treatment for mast cell-associated diseases.

Supported by NIH (1RO1AI101153)
The exon-skipping oligonucleotide, KitStop, depletes tissue-resident mast cells in vivo to ameliorate anaphylaxis
Authors: Hedgespeth Barry , Snider Douglas , Bitting Katie , Arthur Greer , Smith Cierra , Cruse Glenn
Published: January 2023

Anaphylaxis represents the most extreme, life-threatening form of allergic disease and is often an emergency requiring immediate intervention. It occurs following cross-linking of the high-affinity IgE receptor (FcɛRI) on the surface of mast cells by allergen-IgE complexes; however, non-IgE-mediated and idiopathic mast cell activation also contribute. Causes include environmental allergens, foods, venoms, and medications. In addition, some people with mastocytosis have repeated anaphylactic episodes during normal daily activities with no exposure to these triggers. While acute therapy consists primarily of epinephrine and supportive care, chronic therapy relies heavily on desensitization to the inciting allergen—a time-consuming and sometimes unsuccessful process. Desensitization also requires identification of the trigger which is not always possible. Here, we report the use of an exon-skipping oligonucleotide, previously termed KitStop, to safely reduce the severity of anaphylaxis via mast cell depopulation in tissues. KitStop administration results in the integration of a premature stop codon within the mRNA transcript of the Kit receptor—a receptor tyrosine kinase found primarily on mast cells and whose gain-of-function mutation can result in systemic mastocytosis. After either local or systemic KitStop treatment, mice had significantly reduced mast cell numbers in the skin, peritoneum, and lungs. In addition, KitStop-treated mice had a significantly diminished anaphylactic response in a model of passive systemic anaphylaxis. These data demonstrate that KitStop has the potential to serve as a powerful treatment option for patients that experience anaphylaxis, including idiopathic anaphylaxis.

Research was supported by the National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID), Award Number R01AI143985 (G.C.), Department of Molecular Biomedical Sciences, College of Veterinary Medicine, start-up funds (G.C.), NCSU Center for Human Health and the Environment NIH P30ES025128 (G.C.), Institutional National Research Service Award from the Office of the Director of National Institutes of Health, NIH T32OD011130 (B.H., D.S.).
Corticosteroids treatment alters lung and gut microbiome communities that correlates to increased pathologic immune responses
Authors: Asai Nobuhiro , Fonseca Wendy , Yagi Kazuma , Ethridge Alexander D , Morris Susan H , Rasky Andrew J , Falkowski Nicole R , Huang Yvonne J , Huffnagle Gary B , Lukacs Nicholas W
Published: January 2023

Inhaled corticosteroid (ICS) are recommended as first-line controller medications for persistent asthma. Microbial interaction among the gut-lung axis has been suggested. The impact of this crosstalk can modify homeostasis and disease development by altering the host’s immune responses and metabolic status. In this work, we investigated the effect of ICS on microbiome during chronic allergic pulmonary disease. Here we demonstrated that the alternations of lung and gut microbiome can be linked to higher immunological response in mice treated with ICS with Respiratory syncytial virus (RSV) infection. We observed that ICS use during chronic allergy protected the mice, as expected, from lung inflammation and mucus deposition. However, the lung and gut microbiome also presented significant alterations in both naïve mice treated with ICS alone or in ICS asthmatic mice. Microbiome analysis revealed that the principal component analysis of lung and cecum samples in the ICS groups are significantly different from other groups suggesting that ICS impacts the lung and gut microbiome directly with specific organisms altered. When we RSV infected naïve mice treated with ICS, compared to naïve without ICS we observed significant worse pathologic responses. qPCR analysis of Th2-cytokines and of mucus gene showed significantly higher expression in naïve mice treated with ICS than control treated RSV infected mice. Altogether, alternations of lung and gut microbiome can be associated with treatment with ICS with higher immunological response in naïve mice with ICS after RSV infection. Our future studies will focus on how the changed microbiome modifies the subsequent immune responses by focusing on local lung and systemic metabolite changes.
Interorgan transfer of intact micron-sized lipid droplets to macrophages during the Drosophila immune response
Authors: Brennan Catherine A , Kaur Ishneet , Myers Amber , Ogundipe Eniola
Published: January 2023

Macrophages are white blood cells of the immune system in both mammals and Drosophila. In addition to their roles in engulfing and killing microbial invaders by phagocytosis, these cells play important roles in embryonic development, tissue repair, metabolism, and maintenance of homeostasis. Under certain inflammatory conditions, mammalian macrophages accumulate lipids in the form of lipid droplets, including in infection, atherosclerosis, and obesity; however, neither the mechanisms nor purpose of this accumulation are well-understood. We have found that fly macrophages also accumulate lipid droplets, with astonishing speed following bacterial infection: filling up to 30% of cell volume within 3 hrs of infection. We have discovered a completely novel mechanism for this inflammatory lipid accumulation: instead of synthesizing lipids from fatty acids or glucose taken up from the blood, fly macrophages obtain intact, micron-sized lipid droplets from the adipose tissue, through a phagocytosis-like mechanism we term “phagoliposis”. By genetically blocking the metabolism of these lipid droplets, we have discovered some important inflammatory mechanisms fueled by these lipids.

Supported by grants from NIH (SC2AI133653)
Batf3-dependent regulation of the antifungal inflammatory response after pulmonary Aspergillus fumigatus challenge
Authors: Rapp Alexander , Obar Joshua J
Published: January 2023

Aspergillus fumigatus is a ubiquitous fungus that causes invasive aspergillosis in immunocompromised individuals. The innate immune system is critical in the maintenance of host resistance against A. fumigatus. While it is well established that monocyte-derived dendritic cells (moDC) are critical in maintaining host resistance against A. fumigatus, the role of other dendritic cells populations has not been thoroughly explored. Here we demonstrate that Batf3-dependent cells, likely classical type 1 dendritic cells (cDC1s), are critical in preventing invasive aspergillosis. Surprisingly, Batf3(−/−) mice display an elevated inflammatory response against A. fumigatus, particularly IL-17A/F. We show that elevated concentrations of IL-17A/F in the lungs of Batf3(−/−) mice following A. fumigatus infection perpetuates the secretion of various inflammatory lung cytokines in vivo. Neutralization of both IL-17A and IL-17F in Batf3(−/−) mice prevent excessive cytokine secretion and mortality due to A. fumigatus challenge of Batf3(−/−) mice. Following A. fumigatus challenge regulatory T cells are known to limit inflammation. Our preliminary antibody-mediated depletion studies demonstrate that CD4+ CD25+ T cells may be involved in preventing excessive IL-17A/F release in immunocompetent mice. In this study we have identified a critical role for Batf3-dependent cells in limiting A. fumigatus induced inflammatory and mortality due to IL-17 driven lung pathology.

Supported by NIH: [R01 AI139133-01A1]
Skin resident macrophage-derived microRNA21 drives susceptibility to Staphylococcus-aureus infection.
Authors: Salina Ana , Medeiros Alexandra Ivo , Serezani C Henrique
Published: January 2023

Staphylococcus aureus skin infection is orchestrated by the actions of resident and recruited immune cells that lead to abscess formation and bacteria contained. Modulation of the immune response can be achieved by microRNAs. MicroRNA21 (miR21) is a homeostatic regulator of macrophage polarization and miR21 could modulate the expression of either pro- or anti-inflammatory mediators. We are hypothesizing that miR21 is a central node between the regulation of the inflammatory response and antimicrobial effectors functions in the skin. Our data show that miR21 increases as early as 12h, peaks at 24h and 48h. The depletion of skin resident macrophages, but not recruited neutrophils, showed a specific role of skin macrophages in miR21 expression during skin infection. Skin infection in miR21Δmyel mice resulted in reduced lesion size and lower bacterial burden when compared to WT animals. We detected increased IL-1ß and TNF-α and decreased IL-6 and IL-1α levels in the skin of miR21 deficient mice. We also observed increased expression of pro-resolution genes, such as CD36 and collagen3a1 at day 1 after infection. The balance between proinflammatory cytokines, along with the presence of collagen 3 and CD36, resulted in a highly organized abscess with ticker capsule when compared to infected WT mice. Topical treatment with a miR21 antagomir induces reduced lesion size and bacterial burden. Nanostring analysis showed increased Myd88 expression in both naïve and S. aureus infected miR21Δmyel mice and blocking MyD88 actions prevented miR21 antagomir-mediated bacterial clearance in vivo. Our results show blocking miR21 expression leads to a well-controlled inflammatory response necessary to control skin bacterial host defense.

Supported by FAPESP 2018/01622-9, HL-103777, R01HL124159-01
Monocyte/macrophage heterogeneity during skin wound healing in mice
Authors: Pang Jingbo , Maienschein-Cline Mark , Koh Timothy
Published: January 2023

Cells of the monocyte/macrophage (Mo/Ma) lineage orchestrate diverse biological processes required for efficient skin wound healing. Current understanding of Mo/Ma heterogeneity during wound healing has been established by traditional experimental approaches such as flow cytometry, qPCR, and IHC, which indicates a phenotype switch from pro-inflammatory to pro-healing cells as wound healing progresses. However, these phenotypes are limited by the relatively small number of markers assessed. Therefore, our study aimed to generate an unbiased atlas of Mo/Ma phenotypes over the course of skin wound healing by scRNAseq.

Cells were dissociated from excisional skin wounds of normal healing (WT) mice on day 3, 6, and 10 post-injury and live CD11b+Ly6G− cells were isolated by FACS sorting. Using 10× Genomics Chromium system, we pooled data from a total of 2,813 high-quality cells and performed clustering using Seurat which resulted in 8 subpopulations of cells. Cluster dissimilarity and differential expression analysis categorized those clusters into three groups: early stage/pro-inflammatory, antigen-presenting, and late-stage/pro-healing phenotypes that changed over the course of healing. Signature genes and gene ontology analysis of each cluster were used to further characterize the cell populations. Moreover, Ly6C+ vs Ly6C− Mo/Ma were FACS sorted and qPCR was used to validate select marker genes from our scRNAseq clusters. Finally, cell trajectory analysis by pseudotime and RNA velocity indicated the transitions between different Mo/Ma clusters over the course of healing. In summary, our data demonstrated the complexity of Mo/Ma phenotypes and their diverse functions during skin wound healing in mice.

Funded by NIH/NIGMS: R35GM136228
A non-canonical role of caspase-1 in regulating bacterial physiology and antimicrobial resistance
Authors: Akhade Ajay Suresh , Mosquera Gabriela Verdezoto , Arrieta-Ortiz Mario L , Kaur Amardeep , Peterson Eliza J R , Baliga Nitin S , Hughes Kelly T , Subramanian Naeha
Published: January 2023

Caspase-1 is a key effector molecule involved in inflammasome activation and has a well-established role in restricting the growth of intracellular pathogens like Salmonella by triggering a form of cell death called pyroptosis. Here we reveal a non-canonical, cell death independent role for caspase-1 in controlling the transcriptional state and drug resistance of intracellular Salmonella. Using Pathogen-sequencing, a method for sensitive transcriptional profiling of miniscule numbers of intracellular bacteria from infected macrophages, we show that that caspase-1 regulates key processes involved in survival and antimicrobial susceptibility of intracellular Salmonella. Host caspase-1 increased susceptibility of Salmonella to endogenous cationic antimicrobial peptides, as well as to a cationic polypeptide antibiotic used as a last-line drug in Gram-negative bacterial infections. These effects of caspase-1 were independent of its enzymatic activity but dependent on its ability to repress activation of a two-component signal transduction system in intracellular bacteria. These effects were also independent of caspase-11. Our data suggest a “backup” role for caspase-1 in dampening antimicrobial resistance of intracellular Salmonella which evade initial innate immune detection and restriction by caspase-1. These findings also reveal the role of a key innate immune effector in altering pathogen physiology, broadening our view of host-pathogen crosstalk with possible implications for targeting caspase-1 in host-directed therapy to combat antimicrobial resistance.

Supported by grant from NIH (R01 AI155685-A1) and AAI Careers in Immunology Fellowship (2018-2019)
N, N-Dimethylacetamide (DMA) improves survival in a lipopolysaccharide (LPS)-induced endotoxemia model
Authors: Xiao Zhihui , Reznik Sandra
Published: January 2023

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. In cases of severe sepsis, the risk of mortality is 40% to 60%. Proinflammatory cytokine levels are elevated in patients causing capillary leakage, coagulation, and organ damage. N, N-dimethylacetamide (DMA) is a widely used drug excipient, which was found to possess anti-inflammatory properties. In this project, we studied the potential effects of DMA in improving sepsis survival and organ damage and in regulating the immune system.

To study the potential therapeutic effect of DMA, we utilized an LPS-induced endotoxemia murine model to simulate sepsis-like symptoms. DMA improved 72-hour survival from 17% to 83% in severe endotoxemia. Improved mobility and HR were also observed in DMA treated LPS groups as compared to the LPS only groups. To explore the mechanism of DMA’s ability to improve endotoxemia outcomes, primary murine neutrophils were used. DMA reduced mTNFa levels in primary neutrophils treated with LPS for 24 and 48 hours at 10 mM, the same concentration previously reported to attenuate cytokine secretion in vitro. However, mIL-6 levels were not affected. To further study the effect of DMA on macrophages in vitro, differentiation of human monocyte U937 was induced by PMA. In line with our in vivo and ex vivo studies, U937-macrophages released hIL-6 and hTNFa in response to LPS stimulation while DMA significantly reduced cytokine release when cells were treated with DMA and LPS concomitantly. Additionally, cells co-treated with DMA and PMA during differentiation released less cytokines when compared to the corresponding group treated with PMA only.
Interferon-induced transmembrane protein 3 (IFITM3) limits lethality of SARS-CoV-2 in mice
Authors: Yount Jacob S , Zani Ashley , Kenney Adam D
Published: January 2023

Interferon-induced transmembrane protein 3 (IFITM3) is a host antiviral protein that alters cell membranes to block fusion of viruses. Published reports have identified conflicting pro- and anti-viral effects of IFITM3 on SARS-CoV-2 in cultured cells, and its impact on viral pathology in vivo remains unclear. Here, we show that IFITM3 knockout (KO) mice infected with mouse-adapted SARS-CoV-2 experienced extreme weight loss and lethality, while wild type (WT) mice lost only 10% of their body weight and recovered. KO mice had higher lung viral titers and increases in lung inflammatory cytokine levels, CD45-positive immune cell infiltration, and histopathology, compared to WT mice. Mechanistically, we observed disseminated viral antigen staining throughout the lung tissue and pulmonary vasculature in KO mice, while staining was observed in confined regions in WT lungs. Global transcriptomic analysis of infected lungs identified upregulation of gene signatures associated with interferons, inflammation, and angiogenesis in KO versus WT animals, highlighting changes in lung gene expression programs that precede severe lung pathology and fatality. Corroborating the protective effect of IFITM3 in vivo, K18-hACE2/IFITM3 KO mice infected with non-adapted SARS-CoV-2 showed enhanced, rapid weight loss and early death compared to control mice. Increased heart infection was observed in both mouse models in the absence of IFITM3, indicating that IFITM3 constrains extrapulmonary dissemination of SARS-CoV-2. Our results establish IFITM3 KO mice as a new animal model for studying severe SARS-CoV-2 infection, and overall demonstrate that IFITM3 is protective in SARS-CoV-2 infections of mice.

Supported by grants from the NIH (R01 AI130110, R21 AI151230, R01 HL154001, R21 AI142256, and U54 CA260582)
Role of c-Rel O-GlcNAcylation in regulatory B cells
Authors: Liu Angela R , Ramakrishnan Parameswaran
Published: January 2023

O-GlcNAcylation is a dynamic posttranslational modification, characterized by the addition of an O-linked-β-N-acetylglucosamine to a serine, threonine or tyrosine leading to the fine-tuning of various protein functions. Previously, we have shown that hyperglycemia induces O-GlcNAcylation of the nuclear factor kappa B (NF-κB) transcription factor c-Rel subunit at serine 350. We have found that c-Rel O-GlcNAcylation enhances the transcription of c-Rel dependent proinflammatory cytokines in T cells and inversely regulates FOXP3 expression in T regulatory cells. Other studies have shown that enhanced O-GlcNAcylation dysregulates IL-10 signal transduction and results in decreased IL-10 production. Regulatory B cells primarily secrete IL-10 to conduct its immunosuppressive function. Here, we show that c-Rel O-GlcNAcylation regulates c-Rel-dependent transcription in regulatory B cells. LPS, CD40L, and TLR stimulation under hyper O-GlcNAcylation condition differentially regulates the generation of regulatory B cells from primary B cells isolated from WT and c-Rel knockout C57BL/6 and NOD mice. Mutation of the O-GlcNAcylation site in c-Rel (serine 350 to alanine) also showed differential binding to the IL-10 promoter. This study reveals c-Rel O-GlcNAcylation as a novel mechanism regulating transcription in B regulatory cells with implications in autoimmunity.

ARL was supported by the Visual Sciences Training Program grant from the National Institutes of Health (T32 EY007157). PR was supported by supported by the National Institute of Allergy and Infectious Diseases at National Institute of Health grants R01 AI116730 and R21 AI144264, and National Cancer Institute at National Institute of Health grant R21 CA246194.
Fibrinogen Depletion Ameliorates Inflammation and Vision Loss in Mouse Models of Diabetes
Authors: Cardona Astrid E , Sarker Borna , Cardona Sandra M , Church Kaira A , Vanegas Difernando , Velazquez Priscila , Rodriguez Derek , Mendiola Andrew , Kern Timothy , Muzzio Isabel , Stephens Robin
Published: January 2023

Microglia-mediated inflammation plays a significant role in neuronal and vascular damage in diabetic retinopathy, but the mechanism linking inflammation, neurodegeneration, and impaired vascular integrity is still unclear. Our previous studies from diabetic mouse models showed accumulation of fibrinogen, at vessel lesions surrounded by perivascular microglial clusters. In this study, we evaluated whether the pathological hallmarks of gliosis and vascular aberrations characterized in diabetic animal models are consistent with those in diabetic human retinas. Postmortem human retinas were analyzed by immunohistochemistry for markers of gliosis, vascular integrity, and fibrinogen deposition. Immunohistochemical and gene expression analyses of human postmortem retinas revealed evidence of an inflammatory microenvironment, with microgliosis and impaired vasculature. To define the therapeutic potential of reducing fibrinogen in DR, the defibrinogenating agent ancrod was administered in a two-hit inflammatory diabetic mouse model, after which retinal pathology and visual acuity were assessed. Histopathological analyses revealed microglial activation, vascular aberrations, and fibrinogen deposition in the diabetic murine retina. Notably, after treatment with ancrod, diabetic mice appeared to improve visual acuity, which was associated with reduced microglia activation and less fibrinogen deposition in the retina. This study shows that fibrinogen-mediated microglial activation, blood-retinal barrier damage, and vision loss, can be ameliorated by reducing fibrinogen levels. Overall, these findings suggest that that fibrinogen contributes to microglia-mediated inflammation in the diabetic retina.

Supported by R01 EY029913
Single-cell multiomics defines tolerogenic extrathymic Aire-expressing cells with unique homology to thymic epithelium
Authors: Wang Jiaxi , Lareau Caleb A , Bautista Jhoanne L , Sun Im Hong , Gupta Alexander R , Gardner James M
Published: January 2023

The autoimmune regulator (Aire) protein, a key transcriptional regulator expressed in medullary thymic epithelial cells (mTECs), is crucial for central tolerance. Aire is also found in extrathymic Aire-expressing cells (eTACs) in secondary lymphoid organs. eTACs are hematopoietic antigen-presenting cells that are capable of inducing immune tolerance, but the precise identity of eTACs has remained unclear. To define eTACs, we utilized a combination of single-cell multiomics, transgenic murine models, and functional approaches. We found that eTACs consist of two similar cell types: CCR7+ Aire-expressing migratory dendritic cells and an Aire-high population co-expressing Aire and retinoic acid receptor–related orphan receptor γt (RORγt) that we termed Janus cells (JCs). eTACs, particularly JCs, have highly accessible chromatin and share remarkable homology with mTECs. Additionally, transgenic self-antigen expression by eTACs is sufficient to induce negative selection of cognate autoreactive T cells and prevent autoimmune diabetes. To better define the function of extrathymic Aire in eTACs and its contribution to peripheral immune tolerance, we have generated a Peripheral Aire KnockOut (PAKO) mouse (Vav1-Cre x Aire-fl/fl). Using intracellular Aire staining by flow cytometry, we validated that PAKO mice lack all eTACs in secondary lymphoid organs while maintaining normal thymic Aire expression in mTECs. Studying the biology of eTACs and peripheral Aire will help further elucidate the function of Aire and define basic peripheral tolerance mechanisms, which may have significance for a range of clinical applications from autoimmunity to tumor immunity to maternal-fetal tolerance.

Supported by ARCS Foundation
Sterile neutrophilia induced via CXCR4 antagonism ameliorates colonic inflammation by increasing immunosuppressive regulatory T cells
Authors: Saha Piu , Yeoh Beng San , Golonka Rachel , Abokor Ahmed , Vijay-Kumar Matam
Published: January 2023

Sterile neutrophilia (SN) is commonly associated with chronic disease, but its role in inflammatory bowel diseases (IBD) is largely unknown. AMD3100 (plerixafor; AMD) is a CXCR4 antagonist that promotes neutrophil egress from bone marrow resulting in SN. To study the role of SN in IBD, we induced chronic intestinal inflammation in C57BL6 mice via 4 weekly injections of IL-10R neutralizing antibody and administered AMD (10 mg/kg bw, i.p., twice weekly) or vehicle. As expected, AMD-treated control and colitic mice had increased peripheral and splenic neutrophils. AMD treatment remarkably reduced epithelial damage and hyperplasia, immune cell infiltration, and disease activity index. To elucidate the protective mechanism, we analyzed immune cells in peripheral circulation, spleen and lamina propria. In circulation, colitic mice with AMD treatment had elevated % CD3+ CD4+ helper T cells (Th) but reduced % CD3+ CD8+ cytotoxic T (Tc) cells. Yet, the % CD3+ CD4+ FoxP3+ cells (Tregs) and % CD3+ CD4+ RORγt+ cells (Th17) remained unchanged. In spleen, AMD treatment significantly increased Tregs, but decreased Th and Tc cells. In lamina propria, AMD treatment markedly elevated Tregs, including RORγt+ Tregs and tolerogenic IL-10+ Tregs. Conversely, the inflammatory IL-17+ Th cells were decreased in AMD-treated colitic mice. In addition to the immunosuppressive milieu, AMD-treated colitic mice had increased ileal expression of the antimicrobial peptide angiogenin 4, which further suggests SN to be important in maintaining gut microbiota homeostasis. Collectively, our results demonstrate that SN could be an adaptive and beneficial immune response during chronic intestinal inflammation.

This research was funded by R01 grant from the National Institutes of Health (NIH) [grant number CA219144] and Crohn’s and Colitis Foundation (CCF) [grant number 855256].
Triggering receptor expressed on myeloid cells (Trem)-1 plays important roles in UVB induced immune suppression and cutaneous carcinogenesis
Authors: Aguilar Carlos Alberto Mier , Sherwani Mohammad Asif , Tsuruta Yuko , Rashid Harunur , Crossman David Keith , Yusuf Nabiha , Xu Hui
Published: January 2023

American Cancer Society acknowledges that most skin cancers result from exposure to ultraviolet (UV) rays in sunlight. UVB radiation induces mutant cells as well as immune suppression. Tumors only occur when mutant cells are in an immune suppressive environment. The mechanisms for UVB induced immune suppression are yet to be fully elucidated. The goal of this work is to explore new mechanisms for UVB induced immune suppression and skin carcinogenesis by using advanced technology. Analysis of UVB exposed mouse (Nanostring) and human (RNAseq) skin shows that triggering receptor expressed on myeloid cells (TREM)-1 signal pathway is the top canonic pathway increased by UVB. TREM-1 has roles in inflammatory diseases and cancers. However, its role in UVB induced immune suppression and skin cancers is not understood. We have found by using multi-color flow cytometry that UVB induces TREM-1 expression in a subset of conventional dendritic cell type 2 (cDC2) in the draining lymph nodes (LN). TREM-1+ cDC2 cells are barely detected in normal LN and express a high level of the co-inhibitory molecule PD-L1. In functional assays, treatment of mice with a specific TREM-1 blocking peptide LP-17 significantly diminishes UVB-induced suppression of contact hypersensitivity responses that are mediated by T cells. Further, mice treated with this peptide show decreased incidence of photocarcinogenesis and reduced number of tumors. In summary, our findings demonstrate that UVB induces the development of a novel TREM-1+ cDC2 cell subset and that TREM-1 has an important role in UVB induced immune suppression and skin carcinogenesis. Importantly, our study implies new strategies for targeting TREM-1 in the prevention and treatment of UVB induced skin cancers.

Supported by grants from NIH (R01AR072213 and R01AI154842)
A role for a DEAD box RNA helicase in natural killer cells
Authors: Mukhopadhyay Eshana , Krishnamurthy Durga , Tuazon Jasmine , Ohayon David , Ali Ayad , Stevens Joseph , Waggoner Stephen N
Published: January 2023

The X chromosome linked RNA helicase DDX3X plays numerous roles in regulation of cellular RNA metabolism and immune responses. Loss of function mutations in this helicase are linked to neurological disease and malignancies, including NK/T-cell lymphoma. To investigate how DDX3X contributes to development of NK cells, we generated mice with a conditional deletion Ddx3x in Ncr1-expressing cells. Female mice with homozygous deletion of NK cells exhibit a complete loss of NK cells. By comparison, the frequency of NK cells was significantly reduced in hemizygous male mice. The reduction in NK cells is associated with increased apoptosis in this lineage early during differentiation in the bone marrow. Our ongoing work is focused on elucidation of the mechanisms by which DDX3X regulates NK cell survival, differentiation, and malignant transformation, as well as on the capacity of DDX3Y to compensate for loss of DDX3X in hemizygous male mice. Our data identify a novel role for DDX3 RNA helicases in the development and survival of NK cells in mice.
ITK signaling regulates IL-10 production by CD8+ T cells and lung immunopathology during influenza infection
Authors: McGee Michael C , Solouki Sabrina , Limper Candice B , Zhang Tianyi , Magazine Nicholas , Ye Kaixiong , Nidetz Natalie , August Avery , Huang Weishan
Published: January 2023

Influenza (flu) infections cause 250,000 deaths and 3–5 million cases of severe illness during the average flu season. Severe influenza infections are associated with a combination of strong pro-inflammatory and weak anti-inflammatory immune responses. Production of the immunomodulatory cytokine IL-10 by T cells restricts immunopathology during flu infections, however knowledge of the signaling pathways regulating IL-10 induction during flu is limited. Using IL-10GFP reporter mouse models, we found Interleukin-2 inducible T cell kinase (ITK), a critical component in T cell receptor (TCR) signaling, regulates the development of IL-10-producing CD8+ T cells during influenza A infection. Compared to wild type (WT) mice, Itk−/− mice displayed increased morbidity and mortality after influenza infection, accompanied by a significant reduction of IL-10-producing CD8+ T cells in the airways. The absence of ITK and chemical inhibition of the PI3K/AKT/mTOR signaling pathway impaired both BLIMP1 expression and IL-10 production in CD8+ T cells in vitro. Introduction of Blimp-1 or a constitutively active AKT mutant via retroviral transduction rescued IL-10 production and Blimp-1 expression in Itk−/− CD8+ T cells. Utilizing a transgenic allele sensitive Itk (ITKas) mouse model to specifically inhibit ITK kinase activity in coculture, we found ITK kinase activity is not required for the suppressive function of IL-10 producing CD8+ T cells. Together, our data suggests that ITK is a critical regulator of the development and suppressive function of IL-10 producing CD8+ T cells and flu-induced pulmonary immunopathology. Modulating ITK signaling may be a strategy for regulating immunopathology due to respiratory viral infections.

This work was supported in part by grants from the National Institutes of Health (R01 AI151139, R56 AI146226 and P20 GM130555). W. Huang also received research support from MegaRobo Technologies Corporation.
Androgen receptor regulation of CD8 T cell immune responses
Authors: Hawkins Reed M , Polesso Fanny , Moran Amy E
Published: January 2023

Sex differences in disease susceptibility, including infection, cancer, and autoimmunity, have been characterized, with males generally more susceptible to infections and malignancies, and females to autoimmune diseases. While many factors underlie such differences, sex differences in T cell differentiation and function likely lead to disparate adaptive immune responses and disease control. Indeed, sex hormones, including androgens, are strong regulators of gene expression, and have been implicated as drivers of inflammation and regulators of T cell differentiation, but the mechanisms of such control remain unknown. We reveal that androgen receptor (AR) activity in CD8 T cells suppresses activation and reduces proliferation and effector function. We show that anti-CD3- and anti-CD28-activated or peptide-pulsed CD8 T cells exhibit enhanced proliferation, effector cytokine production, activation marker expression, and altered histone modifications upon treatment with AR inhibitors or genetic AR deletion. Further, we demonstrate ligand-dependent AR binding to genetic loci of effector and differentiation genes in CD8 T cells via chromatin immunoprecipitation and quantitative PCR. Finally, in vivo murine models of lymphocytic choriomeningitis virus reveal an increase in AR mRNA expression in virus-specific CD8 T cells following infection, which persists in memory cells and during chronic infection. These data suggest that AR regulates epigenetic and transcriptional changes induced during T cell activation and differentiation, thereby dampening the T cell response. Therefore, disruption of AR activity in T cells may be a viable therapeutic approach for altering the T cell response in the context of human diseases.

Supported by grants from NIH (NRSA T32 5T32GM71338-15, NRSA T32 1T32GM142619-01), Knight Cancer Institute (Knight Pilot Award), and OHSU Foundation
Blimp1 regulates the growth and function of human regulatory T cells
Authors: Ding Ying , Vujanac Milos , Nivelo Luis , Villarino Alejandro , Malek Thomas R
Published: January 2023

Blimp1 is a multifunctional transcriptional regulator that has established roles in mouse T cells. However, its function in human Tregs are not well-understood. To directly evaluate the role of Blimp1 in human Tregs, Blimp1α, the active and dominant isoform of Blimp1, was knocked out in human Tregs in vitro by the Crispr-Cas9 system using dual-targeting sgRNAs. High knockout efficiency was verified at the DNA, RNA, and protein levels. Transcriptome studies identified 356 Blimp1-repressed genes and 399 Blimp1-activated genes in Tregs. The significantly enriched gene sets are associated with cell cycle, apoptosis, cytokine-related signaling, and cytokine production. Consistent with normally restraining Treg growth, functional studies showed that knockout of Blimp1 increased Treg numbers, proliferative responses, and Ki67 expression, but decreased early apoptotic cells. Correspondingly, IL2-induced tyrosine phosphorylation of STAT5 (pSTAT5) was enhanced in Blimp1KO Tregs. Blimp-1 is also positively associated with some aspects of Treg function as loss of Blimp1 downregulated the expression of cytokines, including IL10, IL4, IL5, IL9, IL13, IFNγ, CCL3 and CCL4, and immune checkpoints, including LAG3, PD1, and CTLA4. In the absence of Blimp-1, Foxp3 and Helios levels and IL-6-induced pSTAT3 increased, suggesting a role for Blimp1 in regulating Treg stability. Treg suppressive function, in vitro, however, was not affected by the loss of Blimp1. Collectively, our results suggest that Blimp1 is a pivotal regulator of human Treg growth, function, and stability and is consistent with a model where Blimp1 contributes to Treg homeostasis in vivo. (Supported by R01AI131648).

Supported by grant from NIH (R01AI131648).