bluuebunny/crossref_metadata_2025 huggingface dataset:
Whole-slide multispectral imaging reveals the immune subtypes of melanoma associated with the tumor microenvironment: An automated 7-color mIF assay
Authors: Mehra Navi , Patel Bhavika , Allen Stephanie , Boldt Brittney , Ramirez Noah , Mammadova Najiba , Haggerty Agnes
Published: January 2023

Immunotherapy and precision medicine are rapidly developing approaches to cancer therapy. Biomarkers that detect the tumor and tumor microenvironment allow for the development of strategies that accelerate the advancement of treatments to enhance a patient’s immune system. Akoya’s MOTiF™ PD-1/PD-L1 Auto Melanoma Kit is a validated, multiplex immunoassay enabling detection of the 6 most clinically relevant immuno-oncology and spatial biomarkers: FoxP3, PD-L1, Sox10/S100, PD-1, CD8 and CD68. In this study, the MOTiF™ PD-1/PD-L1 panel was used to analyze the tumor microenvironment and specifically assess immune phenotypes within melanoma samples from 3 patients. This study demonstrates a fully optimized and end-to-end workflow solution for biomarker discovery in melanoma. We demonstrate the utility of Akoya’s MOTiF™ PD-1/PD-L1 Melanoma panel kit in studying the cellular diversity while retaining spatial context. Stained slides were analyzed using the InForm® and PhenoptrReports image analysis platforms to identify phenotypes and better understand spatial relationships between cell phenotypes. The MOTiF™ PD-1/PD-L1 panel kit provides reproducibility across different patient samples. This data provides insight into the innate and adaptive immune landscape for targeted design of new immunotherapies as well as improved efficacy and reduced toxicity in the treatment of melanoma.
Androgen Receptor Deficiency Eliminates Sex Differences in Mast Cells
Authors: Moeser Adam James , Tang Yu Ping , Thelen Kyan , Fardisi Mahsa
Published: January 2023

A sex bias exists in mast cell (MC)-associated inflammatory diseases with females at increased risk. Our previous studies demonstrated that MCs exhibit a sexually dimorphic phenotype with male MCs exhibiting significantly reduced concentration and release of MC granule mediators (histamine, serotonin, and proteases) which was associated with reduced severity of anaphylaxis in males. Further, we showed that perinatal androgens are important in organizing male MC sex differences. The mechanisms by which androgens organize MC sex differences remain poorly understood. Utilizing male mice with reduced androgen receptor (AR) expression induced by Cre-LoxP technology (induced TFM; iTfm), we tested the hypothesis that ARs mediate sex differences in MC histamine storage and release, and severity of anaphylaxis. Isolated peritoneal MCs (pMCs) from iTFm male mice exhibited a feminized MC phenotype characterized by a greater level of histamine compared with control male pMCs, but similar to female controls. Following induction of IgE-mediate anaphylaxis in vivo, iTFM male and female pMCs contained lower (n=5–6; P<0.01) histamine contents compared with control males, suggesting enhanced release of histamine. Preliminary results also indicate that iTFm male mice exhibit greater IgE-mediated histamine releases and hypothermia. Experiments with bone marrow derived MCs (BMMCs) showed that IgE-mediated histamine release from iTFm male and female BMMCs was greater (n=5–6; P<0.01) relative to control male BMMCs. Together, these studies demonstrate that sex differences in MC phenotype and function are mediated via Ars and that iTFm mice are a valid model to explore AR-mediated sex differences in MC function and disease pathogenesis.

Supported by NIH R01 HD072968, NIH R21 AI140413
Fluoxetine-mediated suppression reveals a role for P2X receptors in mast cell function and allergic disease
Authors: Burchett Jason , Haque Tamara , Kee Sydney Ann , Sebti Said , Martin Rebecca , Ryan John J
Published: January 2023

There is a clinical need for new treatment options addressing allergic disease because current approaches are often suboptimal. Selective serotonin reuptake inhibitors (SSRIs) are a class of antidepressants that have been shown to possess anti-inflammatory properties, but the mechanism of action is unclear. Because mast cells are critical effectors of allergic inflammation, we tested SSRI effects on IgE-induced mast cell function. We show that the SSRI fluoxetine suppresses IgE-mediated degranulation and cytokine production in mouse and human mast cells. Fluoxetine was also effective in a model of allergic airway inflammation, where it reduced bronchoresponsiveness and inflammation. IgE crosslinkage elicited rapid ATP release from mast cells, suggesting ATP-P2X receptor signaling may potentiate IgE-driven responses. We subsequently found that fluoxetine inhibits ATP-induced mast cell function. We propose that fluoxetine acts by blocking an ATP-P2X autocrine or paracrine loop that augments mast cell-mediated inflammation. These data support the potential repurposing of fluoxetine in allergic disease.

Supported by NIH/NIAID R01138495 R01164710
TGF-β promotes glycolysis through PFKL in activated macrophages and exacerbates sepsis by disrupting blood coagulation
Authors: Gauthier Thierry
Published: January 2023

The metabolism of macrophages plays a key role in their immune responses, but the mechanisms by which the metabolism is regulated remain largely unknown. We show here that TGF-β induces a unique phenotype of macrophages and regulates the glycolysis of macrophages independently of inflammatory cytokine production. Specifically, TGF-β increased expression and activity of phosphofructokinase-1 liver type (PFKL) in macrophages and thus promoted their glycolysis during cell activation, yet paradoxically suppressed the production of proinflammatory cytokines in the same macrophages. The upregulation of glycolysis was mediated by an mTORc-MYC dependent pathway, whereas the inhibition of cytokines was ascribed to downregulated activation of the major pro-inflammatory transcription factors, namely AP-1, NFkB and STAT1. Importantly, we found that in LPS-induced sepsis model, TGF-β enhancement of macrophage glycolysis led to a decreased survival in mice by increasing blood coagulation. Analysis of cohorts of patients with sepsis and covid-19 revealed that the expression of PFKL, TGF-b receptors TGFBRI and coagulation factor F13A1 in monocytes positively correlated with the progression of the disease. Thus, TGF-β is emerging as a critical cytokine regulating macrophage metabolism and could serve as a therapeutic target in patients with sepsis and Covid-19.

Supporte by NIH/NIDCR intramural funding
Levels of epigenetic regulators TET2 and DNMT1 correlate with virus-stimulated pDC differentiation from IFN-α producing cells to antigen presenting cells
Authors: Codrington Alicia L , Singh Sukhwinder L , Fitzgerald-Bocarsly Patricia L
Published: January 2023

Plasmacytoid dendritic cells (pDC) are potent producers of IFN-α in response to DNA and RNA viruses and subsequently mature into antigen presenting cells. We investigated the role of DNA methyltransferase 1 (DNMT1) and Ten-eleven translocation methyl-cytosine dioxygenase 2 (TET2), which are generally known as gene silencers and activators, respectively, in pDC activation. PBMC from healthy donors were stimulated with Herpes Simplex Virus (HSV) or Influenza-A virus (IAV) for 6- and 24h. PBMC were surface-stained for pDC markers CD123, HLA-DR and CD11C and co-stimulatory markers CD80 and CD86, then intracellularly for DNMT1, TET2 and IFN-α, and acquired by flow cytometry. mRNA expression of DNMT1 in purified pDC was determined by qRT-PCR. At 6h, DNMT1 protein was expressed at equivalent levels in pDC with and without stimulation and in IFN-α+ and IFN-α-pDC, and in unstimulated controls. In contrast, TET2 proteins were elevated in IFN-α+ but not IFN-α-pDC and controls at 6h. At 24h, DNMT1 increased while TET2 was reduced in stimulated pDC, correlating with high IFN-α levels at 6h and low levels at 24h. There was no change in DNMT1 gene expression in virus-stimulated vs. unstimulated controls. CD80 and CD86 were elevated at 24h compared to 6h in stimulated pDC, confirming pDC differentiation from IFN-α producing to antigen presenting cells. At the point of pDC differentiation, TET2 levels were decreased and DNMT1 increased. These results suggest that TET2 is demethylating and causing activation of genes associated with pDC IFN-α production, while DNMT1 methylates and silences genes in this pathway. This results in an initial induction of TET2 and IFN-α production followed by upregulation of co-stimulatory markers, DNMT1 and loss of IFN-α.

Supported by 1. AAI trainee fellowship Careers in Immunology Fellowship Program AAI EIN #: 52-2317193 2. R01Al106125
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

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

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

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

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

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

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

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

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

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

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<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

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

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.
Ets1 regulates production of dendritic epidermal T cells (DETC) and cooperates with IL17Ra signaling to regulate immune responses to Staphylococcal skin infection
Authors: Battaglia Michael , Sunshine Alex , Luo Wei , Miller Lloyd S , Sinha Satrajit , Wohlfert Elizabeth , Garrett-Sinha Lee Ann
Published: January 2023

Ets1 is a lineage-specific transcription factor regulating lymphocyte differentiation. Mice that lack Ets1 (Ets1 KO) develop autoimmune disease with enhanced B and T cell activation, including increased production of IL-17. To determine if IL-17 contributes to autoimmunity, we crossed Ets1 KO mice to mice lacking the IL17 receptor A subunit (IL17RA KO) to generate double knock out (DKO) mice. We found that loss of IL17RA did not prevent or ameliorate autoimmunity, but that DKO animals exhibited worse symptoms. DKO mice also showed susceptibility to skin infections with Staphylococcal bacteria. To define the molecular mechanisms that might underlie this phenotype, we assessed gene expression in skin of DKO mice and control mice, which showed reduced expression of Skint genes. Skint proteins are involved in γδ T cell development and skin wound healing responses. We examined the presence of subsets of γδ T cells in the skin including dendritic epidermal T cells (DETC), whose role in murine wound healing responses is well described, and dermal γδ T cells that are important for anti-Staphylococcal immunity. Loss of Ets1 leads to a complete absence of DETC in the skin of mice. On the other hand, dermal γδ T cells are present in all genotypes. While DETC are absent from the skin of both Ets1 KO mice and DKO mice, only DKO mice show enhanced susceptibility to skin Staphylococcal infections, indicating that Ets1 and IL17RA regulate additional aspects of skin immunity. We propose that impaired responses to skin Staph infections in DKO mice are driven both by impaired IL17RA signaling and impaired wound healing. We further propose that the chronic bacterial infection in the skin results in enhanced immune activation and enhanced autoimmunity in DKO mice.

Funded by NIH R01 AI122720 and the Lupus Research Alliance.
Lineage analysis defines subpopulations of human lung tissue-resident memory CD8+ T cells
Authors: Williams Mark Elliott , Elliott Jennifer L , Thomas Jeronay K , Kost Kirsten , Laccetti Kristiana , Lobby Jenna L , Mattingly Cameron , Scharer Christopher , Kohlmeier Jacob E
Published: January 2023

Tissue resident memory T cells (TRM) are important for local immunity and recall responses. In contrast to effector and central memory T cells, TRM remain in the tissues and do not circulate throughout the body. CD69 and CD103 expression are distinguishing markers of TRM cells, although TRM cells expressing only one marker, or absent of both markers, have been described, suggesting further heterogeneity beyond these markers. Many of the phenotypic characteristics and transcriptional properties of TRM cells have been defined in animal models, where in vivo labeling can be used to define tissue residency. Therefore, a deeper understanding of what defines human TRM cells and their molecular heterogeneity is necessary. To address this, single-cell multi-omics data of memory CD8+ T cells isolated from human lungs that have been phenotypically subsetted into CD69+, CD69+CD103+, CD69-CD103-, and naive cells were collected and analyzed by scATAC-seq and scRNA-seq approaches. A lineage analysis of these data identified multiple distinct lineages of TRM cells in human lungs. From this, we defined the genes that drive each lineage through their differentiation program, the variable transcription factor motifs that distinguish each lineage terminal state, and estimate the rate of differentiation as the cells transition. In identifying these subpopulations, we can begin to understand the heterogeneity of TRM cells in humans on a molecular level.

Supported by a grant from the NIH/NIAID (75N93019R00028) and a grant from the NIH/NHLBI (R35 HL150803)
Transcriptome and chromatin changes as regulatory T cells expand and contract to IL-2R signaling in vivo
Authors: Moro Alejandro , Gao Zhen , Wang Lily , Yu Aixin , Hsiung Sunny , Ban Yuguang , Yan Aimin , Sologon Corneliu M , Chen Steven , Malek Thomas R
Published: January 2023

Regulatory T cells are essential to maintain peripheral T cell tolerance and require IL-2R signaling for their development and homeostasis. Here, we used mouse IL-2 (mIL-2) and a fusion protein of mouse IL-2 linked to mouse CD25 (mIL-2/CD25) to induce acute, periodic, and persistent IL-2R-dependent signaling in peripheral Tregs in vivo. RNA- and ATAC-seq assessed longitudinal shifts in the transcriptome and chromatin landscape to define the molecular steps associated with IL-2R-dependent Treg activation, expansion, and subsequent contraction in vivo. Analysis of the RNA-seq data revealed that STAT5 and MYC were two critical drivers of IL-2R-dependent signaling in Tregs. Other signaling pathways were also identified that facilitate cell transcription, integration of TCR signaling, cell cycle progression, and growth. Sustained IL-2R signaling by mIL-2/CD25, but not periodic signaling by mIL-2, more effectively supported Treg expansion, oxidative phosphorylation, and development into eTregs. With respect to chromatin remodeling, IL-2R signaling initially preferentially increased accessibility to binding sites and activity of STAT5, whereas more persistent IL-2R signaling favored accessibility and activity of other transcription factors, dominantly CTCF, followed by a genome-wide less accessible chromatin. Our data favor a model whereby persistent IL-2R signaling initially supports more accessible chromatin and active transcription leading to a metabolic shift toward a more energetic type required for Treg proliferation. Subsequently, as IL-2R signaling diminishes, a shift toward less accessible chromatin occurs that facilitates contraction of the expanded Tregs to restore homeostasis.

This research was supported by funding from Bristol Myers Squibb and the NIH (R01AI148675).
Regulatory T cells control Th1 responses to sustain bacterial immunity and skin barrier function
Authors: Singh Tej Pratap , Lovins Victoria , Carvalho Lucas P , Carvalho Edgar M , Grice Elizabeth , Scott Phillip A
Published: January 2023

Commensals actively contribute to homeostasis in the skin, but a perturbation of the skin bacteria leading to a dysbiosis often promotes increased inflammation. How regulatory mechanisms control the degree of inflammation associated with a dysbiosis is unclear. Here we used a Staphylococcus aureus isolate from Leishmania braziliensis patients to study the regulation of the host immune response in the skin and its consequence during cutaneous leishmaniasis. Topical colonization with S. aureus induced type 17 responses with minimal skin inflammation. Analysis of Foxp3+ Treg cells revealed IL-6 dependent accumulation of Blimp-1 expressing RORgt+ Foxp3+ Treg cells in colonized skin, and that may play a protective regulatory role. To test this, we depleted Foxp3+ Treg cells, which resulted in significant inflammation and a higher bacterial burden on the skin. Additionally, there was some decrease in IL-17 responses, but a significant increase in the IFN-γ production from CD4+ T cells. Moreover, blockade of IFN-γ in Foxp3+ Treg depleted mice restored commensal immunity, barrier function and inflammation. In mice infected with L. braziliensis, depletion of Foxp3+ T cells had a minimal effect on lesion development. However, depletion of Foxp3+ T cells in S. aureus colonized and L. braziliensis infected mice resulted in significantly more inflammation and bacterial burden than control mice without altering the parasite burden. Taken together, we identified a role for Foxp3+ Treg cells in limiting inflammatory responses to skin bacteria by controlling production of IFN-γ by CD4+ T cells.
The tuberculosis resistance protein TOLLIP prevents disease progression by regulating the integrated stress response in alveolar macrophages.
Authors: Shah Javeed A , Venkatasubramanian Sambasivan , Plumlee Courtney , Cohen Sarah , Dill-McFarland Kim , Hinderstein Sarah , Altman Matthew , Urdahl Kevin B
Published: January 2023

Genetic variation associated with decreased TOLLIP expression in macrophages is a tuberculosis (TB) risk factor. However, the fundamental mechanisms underlying TOLLIP’s role in TB pathogenesis and macrophage function are unknown. To understand why TOLLIP strongly influences Mtb-macrophage interactions, we infected B6 mice lacking Tollip (Tollip−/−) with Mtb and evaluated lung-resident macrophage function and clinical response. Tollip−/− mice developed worsened Mtb disease with profuse lipid-laden foamy macrophages in their lungs. This phenotype was intrinsic to AM, as Tollip−/− AM, but not other lung-resident macrophage subtypes, from mixed bone marrow chimeras selectively harbored excessive Mtb bacilli and intracellular lipid. Global gene expression analysis of Mtb-infected, Tollip−/− AM demonstrated alteration in dozens of genes consistent with the integrated stress response (ISR), which adapts cells to variable environmental conditions and is associated with TB progression. Administration of mycolic acid, an Mtb cell wall lipid, to Tollip−/− macrophages induced lipid accumulation, ISR, and increased intracellular Mtb burden. Pharmacologic ISR inhibition restored host defense in Tollip−/− mice and improved Mtb outcomes overall. In humans, TOLLIP mRNA expression was increased in caseous TB granulomas, providing translational relevance for our observations. Taken together, TOLLIP deficiency in AMs induces foam cell formation and ISR after Mtb infection, which permits increased replication and drives immunopathology. These findings illustrate a major mechanism by which TOLLIP-deficient individuals are susceptible to TB disease and suggest novel targets for TB host-directed therapy.
Androgen exposure alters the neutrophil response to pyelonephritis
Authors: Hreha Teri , Collins Christina A , Hunstad David A
Published: January 2023

Urinary tract infections (UTIs) in men are uncommon but carry increased risk for severe upper-tract UTI (pyelonephritis) and complications including renal scarring. In preclinical models of Escherichia coli UTI, male C3H/HeN mice uniformly develop high-titer pyelonephritis (most with renal abscesses), while the majority of female C3H/HeN mice resolve renal infection within 7–14 days. We previously showed that this difference in outcomes is testosterone dependent. Here, we find that androgen exposure in female C3H/HeN mice alters the neutrophil response to UTI, increasing the propensity for severe renal inflammation and abscess formation as opposed to bacterial clearance and resolution of infection. Concordant with our prior published results, androgen exposure in C3H female mice given Escherichia coli UTI led to 3-log higher bacterial loads in the kidneys by 14 days post infection. We found that the kidneys of androgen-exposed mice harbored more neutrophils than standard females at baseline and throughout infection, correlated with higher levels of the neutrophil-recruiting chemokine CXCL1 (murine KC) in kidney tissue. In addition, the kidneys of androgen-exposed females were enriched for aged, immature neutrophils (Ly6G+, CD49d+, CD101−). Compared to their mature (CD101+) counterparts, these cells exhibited markers of increased degranulation, altered phagocytic activity, and diminished capacity for efflux from kidney tissue. These data support a model in which androgen-exposed mice, despite an apparently more robust neutrophil presence, fail to control renal bacterial infection due to altered neutrophil functions.

Supported by grants from NIH (R01 DK111541, R01 DK126697, R01 AI158418)
Exposure to Vishniacozyma victoriae elicits airway inflammation and a CD4+ T cell-driven lymphocytic response.
Authors: Rush Rachael E , Blackwood Catherine B , Lemons Angela R , Orandle Marlene S , Croston Tara L
Published: January 2023

Like the pathogenic yeast Cryptococcus neoformans, Vishniacozyma victoriae (syn. Cryptococcus victoriae), a non-pathogenic yeast, has been associated with allergic airway disease. Although V. victoriae is frequently detected in indoor environments, the impact of repeated exposure is currently unknown. In this study, mice were exposed via oropharyngeal aspiration to V. victoriae or C. neoformans every other day for a total of six exposures, and responses were analyzed 1 day and 21 days post final exposure. As expected, repeated exposure to C. neoformans resulted in an increase of myeloid and lymphoid cells in the bronchoalveolar lavage fluid (BALF), granuloma formation with intralesional yeast, and peribronchiolar and perivascular inflammation 1 day post exposure. This response was exacerbated 21 days post exposure as the yeast cells replicated. Repeated exposure to V. victoriae resulted in an increase in eosinophils, Ly6Chi/int monocytes, and CD103+ dendritic cells in the BALF, and mild perivascular and peribronchiolar inflammation 1 day post exposure. Surprisingly, V. victoriae cells were present in the lungs of exposed mice 21 days post exposure, and the response shifted toward a nodular lymphocytic response with an increase of CD4+ T cells in the BALF. Lastly, C. neoformans exposure was predicted to activate antigen presentation, B cell development and Th2 proteomic pathways, whereas V. victoriae was predicted to activate VDR/RXR activation, Granzyme A signaling, and IGF-1 signaling pathways 21 days post exposure. The results of this study suggest that exposure to environmentally ubiquitous V. victoriae may exacerbate allergic airway disease, although not to the extent of exposure to pathogenic C. neoformans.

Interagency Agreement between NIEHS & NIOSH: AES 12007001-1-0-6
Investigating the Three-Way Interaction between Gastric Epithelial Cells, Human Neutrophils, and Helicobacter pylori
Authors: Trujillo Jacqueline , Allen Lee-Ann H
Published: January 2023

Neutrophils are the most abundant white blood cells in humans and are the first line of defense against pathogens with respect to innate immunity. Helicobacter pylori are gram-negative bacteria that infect the gastric epithelium and play an important role in the development of ulcers and gastric cancer. Large numbers of neutrophils are recruited to the gastric epithelium where they become heavily infected. We demonstrated previously that when neutrophils are infected by H. pylori it extends their short lifespan. In addition, these cells display unique characteristic such as nuclear hypersegmentation that are indicative of N1-like subtype differentiation which may increase bacterial virulence. Studies by other groups have examined H. pylori-epithelial cell interactions but notably absent are three-way co-cultures that examine simultaneous interactions of neutrophils with bacteria and gastric epithelial cells. Addressing this knowledge gap is the focus of my project. Specifically, I am investigating the dynamics of infection in these co-cultures and determining whether the infected PMNs alter epithelial cell function or viability. Using immunofluorescence I am determining bacterial localization, analyzing PMN nuclear morphology, and determining the extent of changes in epithelial cell junctions and the actin cytoskeleton over 24 hours. Additionally, I am using flow cytometry, lactate dehydrogenase release assays, western blotting and microscopy to quantify cell death, including apoptosis, to test the hypothesis that infected PMNs are more toxic to the epithelium than the bacteria alone. Ultimately, our studies are expected to advance understanding of the role of neutrophils in H. pylori pathogenesis.