bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pulmonary infection following skin injury delays wound healing through suppression of IL-1 and chemokine production
Authors: Crane Meredith , Xu Yun , Monaghan Sean F , Hall Benjamin M , Albina Jorge , Henry William L , Tran Holly L , Chhabria Karisma , Jordon Alexander , Carlsen Lindsey , Jamieson Amanda M 
Published: January 2023

Abstract
Individuals who are hospitalized with traumatic injuries are susceptible to pneumonia. The effect of lung infection on the course of wound healing is not well understood in this setting. To begin to understand this, we examined data from patients who had received laparotomies and found that those who developed pneumonia experienced higher rates wound dehiscence compared to those without pneumonia. Based on this finding and the known role for the innate immune system in responding to injuries and infections, we hypothesized that the immune system cannot meet the demands of competing inflammatory insults in the skin and lungs, leading to impairment of one or both responses. To test this, we established two murine models of post-injury lung infection in which mice are wounded by the dorsal subcutaneous implantation of polyvinyl alcohol (PVA) sponges or by tail skin excision, then infected intranasally with Klebsiella oxytoca. In these models, the presence of a wound did not alter the response to pulmonary infection. In contrast, pulmonary infection delayed the closure tail skin wounds. Pulmonary infection also led to rapid suppression of IL-1beta and chemokine levels, as well as decreased leukocyte accumulation, in PVA sponge wounds. The impaired wound healing observed in infected mice could be rescued by the addition of IL-1beta or the chemokines CCL2 and CXCL1 to the wound bed; however, these treatments caused a delay in bacterial clearance in the lungs. These data suggest that the immune response is not fully equipped to respond to disparate and competing inflammatory insults, which could have implications beyond the setting of post-injury pneumonia.
Supported by grants from NIH (NIES T32-ES7272, NIGMS COBRE Award P20GM10935, and NHLBI R01HL126887), Defense Advanced Research Projects Agency (DARPA), and Brown University (Dean’s Areas of Emerging New Science Award and Carney Institute Innovation Awards)
The extracellular matrix maintained by hypodermal macrophages via IGF1 is a niche for Staphylococcus aureus infection
Authors: Nadella Vinod , Voisin Benjamin , Doebel Thomas , Sakamoto Keiko , Goel Shubham , Jo Jay-Hyun , Kelly Micheal , Kobayashi Tetsuro , Jiang Jean X , Hu Ying , Yan Chunhua , Nagao Keisuke 
Published: January 2023

Abstract
Macrophages (MΦ) play a vital role in tissue homeostasis, but their functions in skin hypodermis, a common site of Staphylococcus aureus (S. aureus) infection, remains largely unexplored. Using flow-cytometry, bulk RNA-seq and single-cell RNA-seq, two MΦ subsets with distinct transcriptomes were uncovered. Both MΦ subsets relied on colony stimulating factor 1 (CSF1) receptor signaling for their survival. Single-cell RNA-seq of non-immune cells identified fibroblasts (FB) as a major source of dermal and hypodermal CSF1. Analysis of dermal and hypodermal stromal cells transcriptomes revealed Tek (encoding Tie2) in hypodermal FB as a potential driver for skin layer-specific ablation of CSF1. Tek-cre x Csf1-floxed mice (Csf1ΔTek) displayed a striking post-natal ablation of hypodermal MΦ associated with an alteration of the collagen network and increased accumulation of hyaluronic acid (HA). Generation of a ligand-receptor map identified candidate genes involved in hypodermal MΦ-FB crosstalk. Among which, depletion of insulin-like growth factor 1 (IGF1) from MΦ in Csf1r-cre x Igf1-floxed (Igf1ΔCsf1r) recapitulated the alteration of the ECM observed in absence of hypodermal MΦ. Interestingly, upon injecting S. aureus into the hypodermis, Csf1ΔTek and Igf1ΔCsf1r mice displayed striking reduction of the cellulitis phenotype as compared to WT mice. Pre-injection of HA attenuated cellulitis in WT mice, suggesting that deposition of HA in the absence of MΦ or MΦ-derived IGF1 conferred host protection against S. aureus. Thus, while hypodermal MΦ critically maintained ECM homeostasis, this provided a niche for S. aureus infection, highlighting implications for novel therapeutic strategies in S. aureus soft-tissue infections.
Mechanistic Investigation of Granzyme A inhibitory effects on intracellular Mycobacterium tuberculosis
Authors: Rasi Valerio , Eickhoff Christopher , Wood David , Shakya Shubha , Carlson Haley , Ford David , Hoft Daniel 
Published: January 2023

Abstract
One fourth of the world population is infected with Tuberculosis (TB). Our lab has identified γ9δ2 T cells that secrete Granzyme A (GzmA) with TB protective effects. In this study, we investigated the mechanism(s) by which human GzmA inhibits the intracellular replication of mycobacteria within infected human primary monocytes.
GzmA was added to mycobacteria-infected monocytes for downstream analyses using 2D-DIGE and shotgun proteomics. We generated WT, enzymatically inactive (S195A), and monomeric only (C93S) recombinant GzmA and performed: flow cytometry studies using viable mycobacteria; intracellular inhibition assays neutralizing CD14, TLR4 and TLR2; and GzmA immunoprecipitation experiments.
The 2D-DIGE proteomic analyses found the ER-stress response and ATP metabolism pathways as important for GzmA-mediated inhibition. Separately, shotgun proteomics uncovered the upregulation of Rab11FIP1 (important for phagocytosis). Both GzmA-WT and S195A proteins inhibited intracellular mycobacteria, but C93S did not. Neutralization of CD14 and TLR4, but not TLR2, reversed GzmA-inhibitory activity. GzmA-WT, S195A, and C93S all bound mycobacteria. However, GzmA-WT and S195A, but not GzmA-C93S, stably bound to TLR4 and CD14.
Collectively, these studies demonstrate key structural, functional, and inter-/intra-molecular features required for GzmA-mediated inhibition of intracellular mycobacteria including interactions between GzmA, mycobacteria, TLR4 and CD14. These interactions result in the ER stress response, altered ATP metabolism, enhanced phagocytosis, and inhibition of mycobacteria. Thus, GzmA’s potential role as opsonin could lead to novel host-directed therapeutics for TB infections.
Supported by grants from NIH (F30HL151136-01, R01AI048391-12)
Nucleotide Receptors Mediate Protection Against Neonatal Sepsis and Meningitis Caused by Alpha-Hemolysin Expressing Escherichia coli K1
Authors: Skyberg Jerod A , Chambers Catherine A , Dadelahi Alexis S , Moley Charles R , Olson Rachel M , Logue Catherine M 
Published: January 2023

Abstract
Neonatal meningitis-associated Escherichia coli (NMEC) are among the leading causes of bacterial meningitis and sepsis in newborn infants. Several virulence factors have been identified as common among NMEC, and have been shown to play an important role in the development of bacteremia and/or meningitis. However, there is significant variability in virulence factor expression between NMEC isolates, and relatively little research has been done to assess the impact of variable virulence factor expression on immune cell activation and the outcome of infection. Here we investigated the role of NMEC strain-dependent P2X receptor (P2XR) signaling on the outcome of infection in neonatal mice. We found that alpha hemolysin (HlyA)-expressing NMEC (HlyA+) induced robust P2XR-dependent macrophage cell death in vitro, while HlyA− NMEC did not. P2XR-dependent cell death was inflammasome independent, suggesting an uncoupling of P2XR and inflammasome activation in the context of NMEC infection. In vivo inhibition of P2XRs was associated with increased mortality in neonatal mice infected with HlyA+ NMEC, but had no effect on the survival of neonatal mice infected with HlyA− NMEC. Furthermore, we found that P2XR-dependent protection against HlyA+ NMEC in vivo required macrophages, but not neutrophils or NLRP3. Taken together, these data suggest that HlyA+ NMEC activate P2XRs which in turn confers macrophage dependent protection against infection in neonates. In addition, our findings indicate that strain-dependent virulence factor expression should be taken into account when studying the immune response to NMEC.
Supported by University of Missouri College of Veterinary Medicine Committee on Research.
Primary fallopian tube epithelial cells are resistant to Chlamydia trachomatis infection due to high expression of ido1.
Authors: Yount Kacy S , McQueen Bryan , O’Connell Catherine , Randell Scott H , Nagarajan Uma , Darville Toni 
Published: January 2023

Abstract
Chlamydia trachomatis (Ct) is the most prevalent sexually transmitted bacterial infection in the world. In ~50% of cisgender women, Ct ascends from the cervix to infect the upper genital tract and increased ascension is linked to hormonal therapies. Oviduct infection induces inflammation which can lead to tubal scarring and sequelae of infertility and ectopic pregnancy. Fortunately, these complications occur in a minority of infected women, suggesting that intrinsic factors may protect the fallopian tubes from infection or tissue damage. In vitro inoculation of primary fallopian tube epithelial (FTE) cells with Ct serovar E resulted in a 10-fold reduction in infected cells compared to transformed endocervical HeLa cells. Indoleamine 2,3-dioxygenase (IDO) in the cytoplasm of epithelial cells acts as a key mediator of chlamydial host defense by metabolizing tryptophan, an essential Ct nutrient. We determined that uninfected primary FTE cells expressed on average 9 times higher levels of ido1 transcripts by qPCR compared to HeLa cells, and that in vitro infectivity of FTE cells was rescued by addition of an IDO-inhibitor (9-fold), tryptophan (21-fold), or indole (a metabolite that bypasses tryptophan starvation; 16-fold). Induction of IDO has mostly been attributed to IFNγ, but it can also be induced by Type I IFNs. Detection of high constitutive ido1 transcription in primary FTE cells indicates their ability to produce IDO independently of IFNγ. Ongoing studies are examining the role of Type I IFNs, including IFNɛ, which is constitutively expressed by reproductive tract epithelium and regulated by hormones. This work has implications for defining factors that contribute to protection of women from Ct-induced disease.
The study was funded through a Translational Team Science Award (UNC School of Medicine and NC TRaCS) and also funded by NIAID NIH R01 AI067678 to U.M.N., NIAID NIH R01 AI119164 to T.D., and NIH grant DK065988 and Cystic Fibrosis Foundation grant BOUCHE15R0 to S.H.R.
The Goldilocks Conundrum: The protective and adverse roles of immunoregulation by NOD-like receptors (NLRs) during brucellosis
Authors: Tupik Juselyn D , Benton Angela H , King Kellie A , Ivester Hannah M , Madanik Justin Markov , Coutermarsh-Ott Sheryl L , Caswell Clayton C , Allen Irving C 
Published: January 2023

Abstract
Brucellosis (Brucella spp.) is a bacterial zoonotic disease characterized by immune evasion. By hiding inside of macrophage vacuoles, Brucella often persist in hosts and instigate lifelong chronic inflammatory conditions. Despite mechanisms to combat host immune defense, NOD-like (NLR) pattern recognition receptors play an important role in immunoregulation during brucellosis. We have previously demonstrated that formation of pro-inflammatory NLRs into the multi-protein complex termed the inflammasome plays a protective role against brucellosis, resulting from the initiation of inflammatory cell death following recognition of Brucella genomic (g)DNA. Despite this investigation, the role of anti-inflammatory NLRs such as NLRX1 during brucellosis has not been determined. Here, we infected wildtype (WT) and Nlrx1−/− mice with Brucella abortus to evaluate the effect of an anti-inflammatory NLR during brucellosis. We found that WT mice displayed a stronger phenotypic presentation of infection, exhibiting increased splenomegaly, as well as elevated bacterial load and WBC infiltration in the liver when compared with knockout mice. This indicates that NLRX1 may exacerbate brucellosis presentation in vivo. Through further in vitro studies infecting murine WT and Nlrx1−/− macrophages, we found that NLRX1 attenuated inflammation, particularly in response to Brucella gDNA, and led to increased bacterial load in WT macrophages. Ultimately, these results indicate that NLRX1 may play an adverse role during brucellosis through reducing inflammation. This study further emphasizes the multifaceted role of NLRs during brucellosis and “Goldilocks Conundrum” in establishing immune system homeostasis in NLR-mediated diseases.
Supported by the Virginia-Maryland College of Veterinary Medicine (VT/UMD Joint Seed Grant; DVM/PhD Student Support) and the NIH/NIAID (R03AI151494)
Supported by the Virginia-Maryland College of Veterinary Medicine (VT/UMD Joint Seed Grant; DVM/PhD Student Support) and the NIH/NIAID (R03AI151494)
STK25 functions as an IRF5 kinase to promote TLR7/8-mediated inflammation
Authors: Rice Matthew R , Sherman Cherrie D , Barnes Betsy J 
Published: January 2023

Abstract
Toll-like receptors (TLRs) represent a subset of pathogen-recognition receptors (PRRs) employed by the innate immune system to detect pathogen-associated molecular patterns (PAMPs) and initiate the response to invading microbes. The transcription factor interferon regulatory factor 5 (IRF5) functions as an important mediator of the inflammatory response downstream of myeloid differentiation factor 88 (MyD88)-dependent TLR activation. While the dysregulation of IRF5 activity has been implicated in the development of several autoimmune diseases including systemic lupus erythematosus and rheumatoid arthritis, the factors that modulate TLR-induced IRF5 post-translational modifications (PTMs) are poorly understood. Therefore, the focus of this study is to identify protein kinases involved in the regulation of TLR7/8 signaling. We performed a kinome-based siRNA screen in human THP-1 monocytic cells and human primary myeloid cells to identify important mediators of TLR7/8-induced TNF-α and IL-6 production. We identified serine/threonine protein kinase 25 (STK25) as a positive regulator of proinflammatory cytokine production in response to TLR7/8 activation in human myeloid cells. We determined that STK25 phosphorylates IRF5 in vitro via multiple biochemical assays. Phosphopeptide mapping by mass spectrometry revealed that STK25 phosphorylates IRF5 at a highly conserved threonine residue. Thus, our data implicate STK25 as a regulator of TLR7/8 signaling through the modulation of IRF5 activity.
scRNA-Seq analysis of human CD5+ innate-like B cells identifies AHR expression as a marker of human CD9+ IL-10+ BRegulatory cells
Authors: Blevins Lance K , Crawford Robert B , Karmaus Peer W F , Kaminski Norbert E 
Published: January 2023

Abstract
B lymphocytes are adaptive immune cells responsible for the production of antigen specific antibodies and the release of pro- and anti-inflammatory cytokines. A previous report from our laboratory found that human, primary CD5+ innate-like B cells (ILB) were preferentially sensitive to aryl hydrocarbon receptor (AHR)-mediated immune suppression compared to CD5− adaptive B cells. CD5+ ILB are a heterogenous population of B cells unified by their expression of CD5 and thought to encompass IgM memory B cells, human B1 B cells, marginal zone B cells, and regulatory B cells among others. Specifically, we demonstrated that CD5+ ILB were marked by elevated expression of programmed cell death protein-1 (PD-1) and its ligands. We also identified a putative role for AHR in the expression of PD-1, suggesting AHR may be important for B cell regulatory functions. Unlike TRegulatory cells, BRegulatory cells are heterogenous, unidentifiable by FoxP3 expression, and express a wide range of surface proteins. To elucidate AHR expression in human B cell populations, naïve, circulating CD19+ B cells were isolated directly from PBMC and enriched for CD5 expression. We then used single cell RNA-sequencing (scRNA-seq) to identify different B cell subsets found within human CD5+ ILB. We found that AHR expression was strongly correlated with expression of CD9 and IL10 as well as CYP1B1. Interestingly, CD9 is reported as a marker of IL-10 producing Bregs suggesting a role for AHR in Breg IL-10 production. These findings were confirmed directly with qRT-PCR and flow cytometry. (Supported by NIH grant P42ES004911)
Supported by NIH grant P42ES004911
Expansion of B2 cells in the peritoneal cavity of PD-1−/− mice
Authors: Riggs James Edward , Conti Antonia , Maslanka Jeffrey , Londregan Jennifer , Somerville John 
Published: January 2023

Abstract
The programmed death-1 receptor/ligand pair (PD-1/PD-L1) functions to temper T cell activation. Mice lacking PD-1 have a normal lifespan while exhibiting T cell expansion in their lymphoid tissue. We explored this mutant further focusing on B cell biology. By flow cytometry, C57Bl/6J PD-1−/− spleen (SP) B cells were similar to those of wildtype (WT) mice. However, their peritoneal cavity (PerC) B cells had more IgMlo IgDhi B cells (B2 subset) and fewer CD11blo CD19+ and CD5lo CD19+ B cells (B1b/B1a subsets). Consistent with their B cell expansion, serum IgM, IgG3, IgG1, and IgA levels were 1.5–2.5 fold greater in PD-1−/− mice. Likewise, responses to thymus-dependent (FITC-OVA) and thymus-independent type-1 and −2 antigens (FITC-LPS, FITC-DEX) were greater than those of WT mice. As expected from prior studies, more activated or effector/memory (CD25+ CD62L− CD44+) CD4+ T cells were found in the PD-1−/− SP. However this was not the case in the PerC where CD8+ CD62L− CD44+ T cells were increased relative to WT. The latter likely contributed to the 3-fold greater level of IFNg production found for PD-1−/− PerC cells. PerC B2 cell expansion was less evident in older mice, perhaps reflecting sequelae of thymic involution with aging. These observations will be discussed in the context of T cell modulation impacting B cell biology. Supported by NIH AREA program (R15 AI 060356-01, R15 CA 136901).
Supported by NIH AREA program grants R15 AI 060356-01 and R15 CA 136901.
Alloprimed antibody-suppressor CD8+ T cells preferentially kill alloprimed germinal center B cells
Authors: Zimmerer Jason M , Chaudhari Sachi , Bumgardner Ginny L 
Published: January 2023

Abstract
Antibody-mediated rejection is a significant contributor to transplant rejection. Our prior work identified a novel subset of CXCR5+ CD8+ T cells (CD8+ TAb-supp cells) that suppresses alloantibody production by mediating cytotoxic killing of IgG+ alloantibody-producing B cells. The purpose of these studies was to investigate which B cell subsets are susceptible to CD8+ TAb-supp cell-mediated cytotoxicity. Alloprimed CXCR5+ CD8+ T cells were isolated from splenocytes of C57BL/6 mice on day 7 following allo-lysate stimulation. Various target cells (alloprimed B cell subsets, macrophages, and dendritic cells) were isolated from CD8 KO mice on day 7 following allo-lysate stimulation. Alloprimed CD8 KO mice are known to be high alloantibody producers from our prior studies. Target cells were tested in co-cultures to evaluate their susceptibility to CXCR5+ CD8+ T cell-mediated in vitro cytotoxicity. Alloprimed CXCR5+ CD8+ T cells exhibited significantly higher in vitro cytotoxicity towards alloprimed GC B cells (IgG+ GL-7+ Fas+; 11.4±1.0%) than towards other antibody producing B cell subsets, including alloprimed extrafollicular plasmablasts (IgG+ CD138+; 0.9±0.3%) and plasma cells (IgG− CD138+; 1.6±0.5%; p&lt;0.0001 for both). In addition, CXCR5+ CD8+ T cells did not kill alloprimed dendritic cells (MHCII+ CD11c+; 1.5±0.7) or macrophages (F4/80+; 1.7±0.4) in co-culture. No significant cytotoxicity was observed in control co-cultures using third party-primed CXCR5+ CD8+ T cells (1.1±0.2), naïve CD8+ T cells (1.2±0.3%), or alloprimed CXCR5-negative CD8+ T cells (0.6±0.3%). These data show that alloprimed antibody-suppressor CXCR5+ CD8+ T cells preferentially mediate cytotoxic killing of alloprimed GC B cells.
This work was supported by a National Institutes of Health R01 grant AI083456 (to GLB), CA016058, UL1TR002733, the OSU Division of Transplant Surgery, and the OSU College of Medicine.
Protective Role of Tissue-Resident Regulatory T Cells in a Murine Model of Beryllium-Induced Disease
Authors: Atif Shaikh M , Mack Douglas , Martin Allison , Fontenot Andrew P 
Published: January 2023

Abstract
CD4+ T cells drive the immunopathogenesis of chronic beryllium disease (CBD), and their recruitment to the lung heralds the onset of granulomatous inflammation. We have shown that regulatory CD4+ T cells (Tregs) control granuloma formation in an HLA-DP2 transgenic (Tg) model of CBD. In Be oxide (BeO)-exposed HLA-DP2 Tg mice, the majority of tissue-resident memory cells (CD44+ CD69+ CD103+) expressed FoxP3 while resident effector cells (CD44+ CD69+ CD103−) lacked FoxP3. Increased numbers of tissue-resident effector and regulatory CD4+ T cells accumulated in the lung following BeO exposure. Depletion of Tregs increased the number of IFN-γ-producing CD4+ T cells and enhanced lung injury while mice treated with IL2/αIL-2 complexes had increased Tregs and reduced inflammation and Be-responsive T cells in the lung. BeO-experienced resident Tregs suppressed anti-CD3-induced proliferation of CD4+ T cells in a contact-dependent manner. Addition of LPS to BeO-exposed mice increased the Teff/Treg ratio and enhanced lung injury. Collectively, these data show that the protective role of tissue-resident Tregs is dependent on time and quantity of Be exposure and is overcome by additional environmental insults. Finally, our findings suggest a potential therapeutic role of Treg expansion as an adjunctive therapeutic option in granulomatous lung disease.
This work was supported by the following NIH grants: ES025534, HL062410, HL102245, and HL152756 (to Andrew P. Fontenot).
The signal regulatory protein gamma (SIRPγ) signaling constrains naïve CD8+ T cell activation with T1D risk associated polymorphisms leading to augmented T cell activation
Authors: Sharp Robert C , Brown Matthew , Peters Leeana , Brusko Todd 
Published: January 2023

Abstract
Genome-wide association studies (GWAS) have identified single-nucleotide polymorphisms (SNPs; rs2281808 and rs6043409) in Signal Regulatory Protein Gamma (SIRPG) with increased susceptibility for type 1 diabetes (T1D). SIRPγ is highly expressed on both CD4+/CD8+ T cells, however, its influence on T cell activation state is insufficiently characterized. We hypothesize that SIRPγ restrains T cell activation and T1D-associated risk variants in SIRPγ influence interactions with its binding partner CD47 to augment CD8+ T cell activation leading to a cytotoxic phenotype. Flow cytometric analysis of PBMC identified SIRPγ/CD47 to be highly expressed on CD4+/CD8+ T cells, with a sequential decline to central memory (TCM), effector memory (TEM), and terminal effector memory cell expressing CD45RA (TEMRA). To test the impact of SIRPγ expression, we generated SIRPG and CD47 knockout (KO) of primary human CD8+ T cells by CRISPR-Cas9 gene editing. Following in vitro activation with anti-CD3/CD28, SIRPG/CD47 KO CD8+ T cells exhibited significantly higher surface activation markers (CD25/CD69) and lower co-inhibitory receptors (CTLA-4/PD-1) compared to controls. Moreover, naïve SIRPγ KO CD8+ T cells differentiated at a higher rate into TEMRA cells. In terms of effector function, SIRPG/CD47 KO CD8+ T cell cultures produced higher levels of the effector molecules TNFα, perforin, and granzyme B and exhibited significantly more cytotoxic killing potential when co-cultured with a targeted pancreatic cell line (βLox5). Overall, this study suggests the SIRPγ:CD47 signaling pathway serves as an immune checkpoint for T cell activation and differentiation of T cells, with the potential for therapeutic targeting during T1D pathogenesis.