bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigating SARS-CoV-2 ORF3a binding to human TRAF2 and TRAF3
Authors: Busscher Brianna M , Befekadu Henock , Liu Zhonghua , Wang Chuanping , Xiao Tsan Sam 
Published: August 2023

Abstract
Severe COVID-19, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is characterized by widespread inflammation and cytokine storm. A number of viral proteins are capable of inducing inflammatory responses, one of which is open reading frame 3a (ORF3a). ORF3a from SARS-CoV shares 73% homology with SARS-CoV-2 ORF3a and was shown to stimulate NF-κB signaling and inflammasome activation in a manner dependent on TNF receptor-associated factor 3 (TRAF3). TRAF family proteins regulate NF-κB activation and thereby influence inflammatory responses. Both ORF3a homologs contain a TRAF-binding consensus sequence, and SARS-CoV ORF3a has been shown to bind to human TRAFs 2, 3, and 6. We therefore investigated the binding of SARS-CoV-2 ORF3a to TRAF proteins as a potential mechanism through which SARS-CoV-2 may activate inflammatory signaling.
TRAF-C domains from TRAFs 2 and 3 were purified and co-crystallized with a peptide from ORF3a containing the TRAF-binding sequence (PIQAS). Fluorescence polarization (FP) was used to assess binding affinity between TRAF-C domains and ORF3a peptide, and dual-luciferase assays were used to measure NF-κB activation in cells transfected with wildtype ORF3a or ORF3a in which the TRAF-binding sequence was mutated to alanines. X-ray crystallography revealed that the ORF3a peptide is able to bind to the TRAF-C domain of TRAFs 2 and 3, and FP indicated that the binding affinity for these interactions is low. When we compared NF-κB activation in HEK293T cells expressing WT vs mutant ORF3a, there was no significant difference. Therefore, while ORF3a has the potential to bind to TRAFs, this interaction does not significantly contribute to inflammatory signaling through NF-κB in our assay.
This work is supported by NIH grants R01GM127609 and P01AI141350.
CD68+ macrophages are associated with adipogenesis after murine muscle injury and fibrotic material implantation
Authors: Josyula Aditya , Ngo Tran B , Destefano Sabrina , Lokwani Ravi , Fertil Daphna , Faust Mondreakest T , Sadtler Kaitlyn 
Published: August 2023

Abstract
Murphy roths large (MRL) mice are known to regenerate skin and skeletal muscle wounds without scar formation but are also susceptible to metabolic dysfunction and obesity. How biomaterials influence immune cell recruitment and immune-adipocyte interaction after muscle injury in MRL mice is yet to be delineated. To investigate this, we performed volumetric muscle loss surgeries (VML) in C57BL/6 (B6) and MRL mice and evaluated immune responses to injury, regenerative (decellularized extracellular matrix, ECM) and fibrotic (polyethylene, PE) material implants using multiparametric flow cytometry, RNA sequencing, and histopathology. In MRL but not B6 mice, we observed white fat deposition at the site of VML. This was correlated with enrichment of adipogenesis gene sets including Adipoq, Cebpa, Pparg and Acsl1. In addition, F4/80-CD68+ macrophages were significantly more abundant in MRL mice at 3 weeks post-injury in PE and saline groups. The same treatments also highly potentiated expression of the lipid associated macrophage gene signature comprising Trem2, Ly6c2, Cd9, Cd63 and Lyz2. Ly6G+ neutrophils were the second most abundant cell type (~ 25% of CD45+ cells) which was corroborated by increased abundance of Vav1, Ccl2, Adam8 and Rac2. In contrast, ECM implantation produced a largely SiglecF+ eosinophilic response (~ 60% of CD45+ cells) and inhibited CD68+ macrophage prevalence and also potentiated expression of the pro-regenerative Arg1, Chil3, Retnla and Gata3 in both B6 and MRL. Taken together, our results suggest a mechanistic role for CD68+ macrophages in promoting adipogenesis after VML. Moving forward, we will analyze adaptive immune responses to injury and material implants using abovementioned methods.
Intramural Research Program, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health
Injury-induced hair growth response is promoted by tissue macrophages via insulin-like growth factor 1
Authors: Ayush Otgonzaya , Goal Shubham , Viosin Benjamin , Nadella Vinod , Kim Do Young , Sakamoto Keiko , Nagao Keisuke 
Published: August 2023

Abstract
Hair follicles (HFs) produce hair to mediate the first line of defense in mammals. HFs undergo cyclical production of hair (anagen), and while this process is unsynchronized during adulthood, external cues, including shaving, induce synchronized anagen and bolster the surface barrier. Herein, we show that macrophages were crucial in promoting anagen. Shaving triggered the accumulation of macrophages to the secondary hair germs (SHG), which underwent proliferation as early as Day3 post-induction. Constitutive ablation of macrophages markedly delayed anagen, and targeted ablation of macrophages at Day 3, but not at Day 0 or Day 6 post-shaving, impaired SHG activation with delayed anagen. Anagen entry was dependent on Folr2+ tissue macrophages, but not on CCR2+ monocyte-derived macrophages. Single-cell RNA-sequencing and cell-cell communication analyses revealed that Folr2+ macrophages communicated with the SHGs via Igf1 (encoding insulin-like growth factor 1 or IGF1). Ablation of macrophages and deletion of IGF1 from macrophages decreased AKT signaling in SHG and led to delayed anagen, both which were rescued via injection of recombinant IGF1 in vivo. Single-cell ATAC-sequencing revealed that the lack of IGF1-from macrophages resulted in alteration of transcription factor motif enrichment and decreased chromatin accessibility in various genes in SHGs, demonstrating that macrophages primed the SHGs to enter anagen by promoting IGF1R signaling. These results highlight the importance of immune-tissue crosstalk in facilitating hair growth and provide impetus for strategies to promote hair growth.
A novel Fth1-mScarlet reporter mouse supports quantification of monocyte-derived macrophage inflammatory state in vivo
Authors: Michalides Brandon Alan , Shoger Karsen E , Cheemalavagu Neha , Gottschalk Rachel A 
Published: August 2023

Abstract
Monocyte-derived macrophages (mo-macs) acquire a spectrum of pro- and anti-inflammatory functions during infection-associated inflammation and resolution. Quantification of mo-mac functional state is critical to elucidate mechanisms leading to dysregulation of inflammatory control and how these processes can be manipulated therapeutically. Given that binary M1/M2 cell markers often fail to explain macrophage function in vivo, we sought to identify genes predictive of inflammatory gene signatures. We analyzed published single cell RNAseq of macrophages during lung inflammation, using Lasso (L1-penalized linear regression) to weight genes according to their ability to predict all other genes, and identified Fth1 (Ferritin Heavy Chain 1) as the most predictive gene. We generated Fth1-mScarlet reporter mice using CRISPR/Cas9 technology, inserting mScarlet in-frame of Fth1, linked through a T2A self-cleaving peptide. Using live cell imaging in vitro, we found monocyte mScarlet expression peaked 24 hours after stimulation with S. aureus or TLR ligands. We are currently using monocyte adoptive transfer to track mo-mac fate during acute S. aureus lung infection. In this intra-nasal S. aureus model (strain FDA309), pro-inflammatory genes and infiltration of neutrophils and monocytes peak 1–2 days post infection. We transferred Fth1mScarlet bone marrow monocytes 6 hours after infection, analyzed their recruitment into the lungs, and quantified mScarlet expression over time. Increased mScarlet expression was associated with the transition of donor cells from Ly6C+CD11c-monocytes to Ly6C-CD11c+ mo-macs. This model will allow us to quantify the plasticity and persistence of mo-macs during inflammation and resolution.
R35 GM146896 (PI - Gottschalk)
GATA1 controls numbers of hematopoietic progenitors and their response to autoimmune neuroinflammation
Authors: Hwang Daniel , Ishikawa Larissa Lumi Watanabe , Seyedsadr Maryam S , Mari Elisabeth , Kasimoglu Ezgi , Sahin Ziver , Boehm Alexandra , Jang Soohwa , Rasouli Javad , Vaccaro Courtney , Gonzalez Michael , Hakonarson Hakon , Rostami Abdolmohamad , Zhang Guang-Xian , Ciric Bogoljub 
Published: August 2023

Abstract
GATA-binding factor 1 (GATA1) is a transcription factor that governs the development and function of multiple hematopoietic cell lineages. GATA1 is expressed in hematopoietic stem and progenitor cells (HSPCs) and is essential for erythroid lineage commitment; however, whether it plays a role in hematopoietic stem cell (HSC) biology and the development of myeloid cells, and what that role might be, remains unclear. We initially set out to test the role of eosinophils in experimental autoimmune encephalomyelitis (EAE), a model of central nervous system autoimmunity, using mice lacking a double GATA-site (ΔdblGATA), which lacks eosinophils due to the deletion of the dblGATA enhancer to Gata1, which alters its expression. ΔdblGATA mice were resistant to EAE, but not because of a lack of eosinophils, suggesting that these mice have an additional defect. ΔdblGATA mice with EAE had fewer inflammatory myeloid cells than the control mice, suggesting that resistance to EAE is caused by a defect in myeloid cells. Naïve ΔdblGATA mice also showed reduced frequency of CD11b+ myeloid cells in the blood, indicating a defect in myeloid cell production. Examination of HSPCs revealed fewer HSCs and myeloid cell progenitors in the ΔdblGATA bone marrow (BM), and competitive BM chimera experiments showed a reduced capacity of the ΔdblGATA BM to reconstitute immune cells, suggesting that reduced numbers of ΔdblGATA HSPCs cause a functional deficit during inflammation. Taken together, our data show that GATA1 regulates the number of HSPCs and that reduced GATA1 expression due to dblGATA deletion results in a diminished immune response following the inflammatory challenge.
This work was supported by a grant from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (T32 training grant T32AI134646).
Accessory cells precondition naive T cells and regulatory T cells for cytokine-mediated proliferation
Authors: Sato Noriko , Bamford Richard N , Bryant Bonita R , Tagaya Yutaka , Waldmann Thomas A 
Published: August 2023

Abstract
Naïve T cells and regulatory T cells, when purified, do not proliferate to the common cytokine receptor γ-chain family cytokines interleukin (IL)-2, IL-7 or IL-15, despite their expression of cognate cytokine receptors. Cell-to-cell contact with dendritic cells (DCs) enabled proliferation of the T cell to these cytokines, independent of antigen recognition or T cell receptor stimulation. This effect lasted after separation of T cells from DCs, enabling enhanced proliferation of the T cells in mice depleted of DCs. We propose calling this a “preconditioning effect”. Interestingly, IL-2 alone was sufficient to induce phosphorylation and nuclear translocation of STAT5 in T cells, but could not activate MAPK and AKT pathways and failed to induce transcription of IL-2-target genes. Preconditioning was necessary to activate these two pathways, and induced weak Ca2+ mobilization independent of calcium release-activated channels. When preconditioning was combined with IL-2, full activation of downstream mTOR, 4E-BP1 hyperphosphorylation and prolonged S6 phosphorylation occurred, and T cells underwent proliferation. Collectively, accessory cells provide T cell preconditioning, a unique activation mechanism, controlling cytokine-mediated proliferation of T cells.
Treatment of pulmonary fibrosis through normalization of alveolar macrophages
Authors: Baek Jiyeon , Kim Minji , Kwon Byungsuk 
Published: August 2023

Abstract
Idiopathic pulmonary fibrosis (IPF) is a chronically advanced interstitial lung disease with unclear etiology and poor prognosis. Despite numerous studies, the pathogenesis of IPF has not been completely understood. In this study, we searched for a drug that can decrease chronic lung inflammation without deteriorating lung fibrosis. We found that fenofibrate, a PPARα agonist that is clinically used to control dyslipidemia, had therapeutic effects on pulmonary fibrosis through normalization of alveolar macrophages. We first showed that 6 days of oral administration of fenofibrate blocked the progression of bleomycin-induced pulmonary fibrosis and resolved inflammation. In addition, fenofibrate suppressed the LPS-induced pro-inflammatory mediators in bone marrow-derived macrophages in a PPARα-independent manner; however, it promoted the expression of tissue repair and anti-inflammatory genes. Finally, we examined the expression of the pro-inflammatory, tissue-repair, and anti-inflammatory genes in alveolar macrophages at days 2 or 6 after oral administration of fenofibrate in mice with advancing pulmonary fibrosis. Administration of fenofibrate resulted in decreased expression of proinflammatory cytokines and increased expression of tissue repair and anti-inflammatory genes at day 2. However, the gene expression pattern of alveolar macrophages of fenofibrate-treated mice at day 6 became similar to that of naïve alveolar macrophages. Taken together, this study suggests that fenofibrate can rapidly reprogram inflammatory macrophage polarization toward normal state through a transient resolving and anti-inflammatory macrophage polarization stage, which is linked to amelioration of pulmonary fibrosis.
CCL3-deficient HLA-DP2 Transgenic (Tg) mice are protected against Be-induced inflammation in the lungs
Authors: Atif Shaikh M , Falta Michael T , Martin Allison , Tucker Rebecca , Mack Douglas G , Palmer Brent E 
Published: August 2023

Abstract
Chronic beryllium disease (CBD) is an inflammatory lung disorder caused by the recruitment and expansion of pathogenic CD4 T cells. In a murine model of Be-disease, we reported the accumulation of CCL3/Be and CCL4/Be-specific CD4 T cells in the lungs of BeO-exposed HLA-DP2 Tg mice (WT). However, the addition of a single dose of LPS on day 14 (d14) in BeO-exposed WT mice, resulted in a significant increase in the release of chemokines (CCL3 and CCL4), increased accumulation of chemokine/Be-specific CD4 T cells and formation of cellular aggregates in the lungs. In the present study, we explored the loss of chemokine(s) CCL3/4 on the pathogenesis of CBD using HLA-DP2 Tg-CCL3-deficient mice (CCL3-DP2 Tg), which were also found to be deficient in CCL4. CCL3-DP2-Tg mice exposed to BeO+LPS (d14) caused a significant reduction in the accumulation of CCL3/4/Be-specific CD4 T cells in the lungs and the formation of cellular aggregates compared to the WT mice. Also, the ELISPOT assay revealed a significant reduction in the IFNγ and IL-17-producing CD4 T cells in the lungs of BeO-exposed CCL3-DP2-Tg mice. Next, we examined the source of CCL3 in the lungs of BeO-exposed WT mice. CCL3 is majorly contributed by CD11c+ dendritic cells compared to the other cells of the hematopoietic compartment. No or little staining of CCL3 was observed in the stromal compartment. Bone marrow transfer from WT to CCL3-DP2 Tg mice showed the role of the hematopoietic compartment in both the accumulation of CCL3/4-specific CD4 T cells and the formation of cellular aggregates in the lungs after Be-exposure. Overall, we showed that hematopoietic cell-derived CCL3 or CCL4 plays an important role in the pathogenesis of CBD, and the lack of CCL3 and CCL4 protects against Be-induced lung damage.
NIH RO1 HL152756
Post-thymic Thpok-deficiency in CD4 T cells compromises control of Toxoplasma gondii infection
Authors: Vacchio Melanie S , Clark Alex , Bouladoux Nicolas , Belkaid Yasmine , Bosselut Remy 
Published: August 2023

Abstract
Toxoplasma gondii, an intracellular parasite, is estimated to chronically infect &gt;25% of the world-wide human population. While generally kept in check by the host immune system, reactivation of infection can occur when individuals become immunocompromised, particularly with T cell deficiencies. Both CD4+ and CD8+ T cells play significant roles in controlling this parasite. The transcription factor Thpok, is critical for commitment of MHC Class II-restricted thymocytes to the CD4 lineage during thymic differentiation, but also plays a significant post-thymic role in shaping the effector function of mature CD4+ T cells. Based on these findings, we interrogated how mice with post-thymic deletion of Thpok manage a chronic infection using Toxoplasma gondii (“Toxo” hereafter). During acute infection, Thpok-deficient mice have equivalent expansion of Toxo-specific T cells compared to WT littermate controls, but these CD4 T cells have undergone “cytotoxic diversion”, upregulation of a CD8 transcriptional program and loss of CD4 helper function. During the chronic phase of infection Thpok-deficient mice die (~3 weeks p.i. vs &gt;12 weeks for WT) and exhibit increased Toxo cysts in the brain compared to WT littermates, suggesting deficient control of the parasite. This death in the early chronic phase is not simply due to lack of CD4+ T cell help, as Class II-deficient mice survive significantly longer into the chronic phase of Toxo infection (~6 weeks, p.i.). This data indicates that Thpok-deficient CD4+ T cells are far worse at controlling Toxo than complete absence of CD4+ T cells. Studies are currently in progress to address the mechanism by which Thpok-deficiency dysregulates parasite control by CD4+ T cells.
Intramural National Institutes of Health
A pathogenic role for regulatory T cells in chronic liver disease
Authors: Arpaia Nicholas , Savage Thomas M , Fortson Katherine T , de los Santos Alexis Kenia , Alsina Angelica Oliveras , Rouanne Mathieu , Gamarra Jennifer R , Cavero Renzo , Li Fangda , Haeusler Rebecca A , Adam Julien , Schwabe Robert F 
Published: August 2023

Abstract
Current dogma holds that regulatory T (Treg) cells preserve tissue function in settings of inflammation and damage through their anti-inflammatory activity and production of epithelial growth factors – the latter of which is independent from immunosuppression. Consistent with this, we observe that Treg cells – in particular those producing the epidermal growth factor receptor (EGFR)-ligand amphiregulin (Areg) – are enriched in the livers of mice and humans with non-alcoholic steatohepatitis (NASH). Mouse and human liver Treg cells undergo substantial transcriptional changes in chronic liver damage, reflecting their increased activation; however, rather than playing a protective role, we find that Treg cell–derived Areg promotes NASH-induced liver fibrosis through the direct activation of EGFR on hepatic stellate cells. Clinically, NASH is closely linked to insulin resistance, but the mechanistic contributions of NASH-induced disease processes to insulin resistance remain unclear. We further observe that Treg cell–derived Areg promotes glucose intolerance in a NASH-dependent manner – through the induction of hepatic gluconeogenesis – a process also mediated through EGFR signaling on hepatic stellate cells. Mechanistically, Treg cell–derived Areg promotes hepatocyte gluconeogenesis through hepatocyte detection of soluble mediators produced by Areg-activated hepatic stellate cells – offering new insight into how chronic liver disease promotes insulin resistance. Taken together, we provide the first evidence that Treg cells mediate a maladaptive role in tissue injury, finding that their production of a growth factor plays a central role in liver disease and promotes liver fibrosis and glucose intolerance in NASH.
This research was supported by grants from NIH (K22 AI127847, R01 HL148718, R01 CA259634, T32 GM007367, P30 CA013696, P30 DK132710) and the Searle Scholars Program (SSP-2017-2179).
IL-4 acts through aryl hydrocarbon receptor to antagonize TLR7 induced double negative 2 B cells in lupus
Authors: Mountz John D , Lu Changming , Barnes Stephen , Wilson Landon , Gao Min , Chatham Winn W , Hsu Hui-Chen 
Published: August 2023

Abstract
We recently showed that in systemic lupus erythematosus (SLE), IL-4R signaling is a powerful antagonist that can effectively suppress the development of activated naïve (aNAV) and CD11c+T-bet+ IgD−CD27− double negative 2 (DN2) B cells promoted by both type I and type II IFNs. In the present study, we used the BXD2 mouse model of lupus to determine the mechanism of IL-4 in suppressing the development of DN2 B cells in vivo. Administration of IL-4 significantly inhibited the development of anti-Smith, anti-DNA, and anti-histone autoantibodies induced by TLR7 agonist R848 in BXD2 mice. This was associated with a decreased percentages of CD11c+T-bet+ IgD−B cells. Feature-barcoding single cell RNA-sequencing analysis showed that IL-4 modulated B cell development at the transitional stage 2 (T2) and skewed naïve B cells to develop into the CD23+CD21− follicular B cells. IL-4 induced the gene encoding interleukin-4-induced1 (IL4i1), an enzyme that metabolizes aromatic amino acids and this was associated with the upregulation of aryl hydrocarbon receptor (AhR) and downstream genes. Metabolomics analysis revealed IL-4 induced AhR agonistic metabolites in B cells including kynurenine (Kyn), indole-3-acetic acid, and indole-3-lactic acid. In the absence of IL-4, Kyn and a potent AhR agonist, formylindolo[3,2-b]carbazole (FICZ), significantly suppressed TLR7 plus IFNβ-induced DN2 B-cell development in vitro. Our results suggest that IL-4 acts through the IL4i1-AhR pathway to inhibit B-cell regulatory response to TLR7 and type I IFN. Identification of small molecular metabolites that act directly in B cells to induce homeostasis may lead to development of orally dosed metabolome modulating therapeutics efficacious in the treatment of SLE.
This study was supported by grants from VA Merit Review grant (I01BX004049), NIH grants R01 AI134023, and Lupus Research Alliance Distinguished Innovator Award to J.D.M, the LRA Target Identification in Lupus Award to H-C.H., and the P30-AR-048311.
Bacterial catabolism of tryptophan to indole is required to induce collagen induced arthritis via the IL23/Th17 axis
Authors: Seymour Brenda , Allen Brendan , Trent Brandon , Berlinberg Adam , Jubair Widian , Colgan Sean , Kuhn Kristi 
Published: August 2023

Abstract
While intestinal dysbiosis is linked to the pathophysiology of inflammatory arthritides like rheumatoid arthritis (RA) and spondyloarthritis (SpA), the mechanisms by which this occurs are unknown. Therefore, we used the collagen-induced arthritis (CIA) model to connect the microbiome with bacteria-produced metabolites in the development of disease. After initially correlating CIA severity with bacteria-dependent alterations in dietary tryptophan metabolism, and specifically indole, we demonstrate that both bacteria and dietary tryptophan are required for disease, and that indole supplementation fulfills these requirements. When mice with CIA on a low-tryptophan diet were supplemented with indole, we observed significant increases in serum IL-6, TNF, and IL-1β and Th17 cells. These findings were associated with increased anti-CII IgG2b, decreased anti-CII antibody sialyation, and increased complement fixation, suggesting that indole-mediated Th17 immunity may enhance autoantibody pathogenicity. Finally, exposure of human colon lymphocytes to indole increased expression of genes involved in the IL-17 signaling pathway in B and T cells. Altogether, we propose a mechanism by which intestinal dysbiosis during inflammatory arthritis results in altered tryptophan catabolism, leading to indole stimulation of Th17 immunity. Blockade of indole generation may present a novel therapeutic pathway for RA and SpA.
Supported by grants from NIH: NIAMS R01AR075933 (KAK), NIAMS T32AR007534 (WKJ, BT, AJB), NIAID T32AI007405 (BJS), NIAID F30AI174817 (BJS); and the Rheumatology Research Foundation: Future Physician Scientist Award (BJS) and Scientist Development Award (AJB).