bluuebunny/crossref_metadata_2025 huggingface dataset:
Mucosal correlates associated with natural regression of HPV-associated dysplasia
Authors: Choi Yoojin , Salit Irving , Grech Sarah , Sano Marie , Weiss Edward , Coburn Bryan , Kaul Rupert
Published: January 2023

Background

Anal HPV infection and associated pre-cancerous changes, known as anal intraepithelial neoplasia (AIN), are common among men who have sex with men (MSM). While natural regression of AIN is often observed, there are currently no predictors for disease outcomes. Therefore, we assessed mucosal immune and microbiological correlates of natural AIN regression.

Methods

Fifty-five HIV-infected, ART-treated MSM were recruited, of whom 26 had confirmed AIN. The microbiome was defined via qPCR and 16s sequencing of anal swabs; biopsies were collected from both healthy and AIN-confirmed sites by high-resolution anoscopy, and lymphocyte populations were defined by flow cytometric analysis including CD38 and HLA-DR as activation markers. AIN regressors vs non-regressors were compared by Mann-Whitney (SPSS).

Results

AIN regression was observed in 15/26 (58%) participants. Regression was associated with a higher proportion of CD8+ T cells (p= 0.006), and AIN regressors had significantly higher levels of both activated CD8+ (p= 0.023; 44.5% vs 27.9%) and activated CD4+ T cells (p=0.023; 20.4% vs 9.81%) compared to non-regressors. Interestingly, AIN regressors had reduced microbiome diversity (p=0.026; 241 vs 284 OTUs) and a trend towards a lower bacterial load (p=0.059). While age was similar between groups, AIN regressors had been living with HIV for a shorter time (p=0.023). No differences were observed between groups in T cells derived from AIN-free anal mucosa.

Conclusions

AIN-specific immune activation was associated with subsequent regression of AIN. Given that AIN non-regressors had greater anal microbiome diversity, defining key bacterial species and their role in HPV pathogenesis may merit further investigation.
Single-cell RNA-sequencing of leprosy skin lesions reveals a titin (TTN) expressing T cell subset associated with the progressive disease
Authors: de Andrade Silva Bruno Jorge , Hughes Travis K , Andrade Priscila R , Ma Feiyang , Teles Rosane MB , Shalek Alex K , Bloom Barry , Pellegrini Matteo , Modlin Robert L
Published: January 2023

The immune response to intracellular bacteria is critical for host defense against the invading pathogen. This interaction can be studied in the human disease leprosy caused by the intracellular bacterium Mycobacterium leprae affecting primarily the skin compartments. Patients with tuberculoid leprosy (T-lep) express a Th1 cytokine pattern in skin lesions and develop a protective immunity that eliminates the infection, whereas those with lepromatous leprosy (L-lep) express Th2 cytokines in lesions and sustain a progressive infection. In the reversal reaction (RR) state, patients upgrade, either spontaneously or during chemotherapy, from the L-lep to the T-lep pole. Herein, we used unbiased single-cell RNA-sequencing to explore the breadth of potential immune cell states in leprosy. Unexpectedly, we identified a lymphocyte cluster marked by expression of titin (TTN), which is commonly found in the sarcomere of muscle cells. TTN+ T cells were predominantly enriched in L-lep lesions and RNA-FISH showed a higher number of TTN mRNA dots on CD3+ T cells from L-lep in comparison with RR patients. TTN gene expression decreased in the skin lesion cells of L-lep patients undergoing RR episodes associated with the onset of therapy. Protein expression was confirmed by analysis of T cell clones isolated from leprosy lesions and blood-derived CD3+ T cells from healthy donors. Indeed, immunohistochemical staining of lesions and healthy tonsil showed that TTN was present in the CD3+ T cell zones. Canonical pathways analysis revealed that TTN might be involved with T cell inhibitory RhoA signaling pathway. The role of TTN on T cells remains unknown, however, it could represent a novel mechanism in the skin immune response against cutaneous pathogens.
MISTR: A conserved

MI

tochondrial

ST

ress

R

esponse network revealed by signatures of evolutionary conflict
Authors: Hancks Dustin , Sorouri Mahsa , Chang Tyron , Pinkham Chelsea , Jesudhasan Palmy
Published: January 2023

While the identities of many interferon-stimulated genes (ISGs) are established, the functions of the majority of these gene products as well as their relationship with other cellular factors are unknown. To drive functional characterization, we sought to identify ISGs with genetic hallmarks indicative of conflicts with pathogens and common to key host defense factors: 1) rapid evolution and 2) viral homolog. Using these “molecular scars” as a guide, we discovered a vertebrate-specific MItochondrial STress Response circuit (MISTR). MISTR proteins are associated with electron transport chain factors and activated by stress signals such as interferon-gamma and hypoxia. Upon stress, ultraconserved miRNAs downregulate MISTR1 followed by replacement with paralogs MISTR AntiViral (MISTRAV) or MISTR Hypoxia (MISTRH), depending on the insult. While cells lacking MISTR1 are more sensitive to apoptotic triggers, cells lacking MISTRAV or expressing the poxvirus-encoded vMISTRAV exhibit resistance to the same insults. Rapid evolution signatures across primate genomes for MISTR1 and MISTRAV indicate ancient and ongoing conflicts with pathogens. MISTR proteins are also found in plants, yeasts, and an algal virus indicating ancient origins and suggesting diverse means of altering mitochondrial function under stress. The discovery of MISTR circuitry highlights the use of evolution-guided studies to reveal not only new dimensions of innate defenses but also fundamental biological processes.
VAMP8 mediates TLR4-dependent TNF secretion in RBL-2H3 pro-inflammatory cells
Authors: Segura Deisy Lizbeth , Ibarra-Sánchez Alfredo , Blank Ulrich , González-Espinosa Claudia
Published: January 2023

Mast cells (MCs) participate in allergic diseases by producing pro-inflammatory mediators and regulatory cytokines. After high affinity IgE (FceRI) receptor activation, they release tryptase, b-hexosaminidase and other preformed pro-inflammatory substances by anaphylactic degranulation. Also, upon stimulation through Toll-like receptor 4 (TLR4), they release pro-inflammatory mediators (such as Tumor Necrosis Factor, TNF), by piecemeal degranulation, orchestrating protective reactions against pathogens. Reactions involved in anaphylactic degranulation require specific vesicular-associated membrane proteins (VAMPs) involved in the latest step of exocytosis. However, the particular roles of VAMP proteins on piecemeal secretion observed after TLR4 receptor activation are not known. Utilizing RBL-2H3 cells differentiated to a pro-inflammatory phenotype, we investigated the role of VAMP8 and VAMP3 on lipopolysaccharide (LPS)-induced mediator secretion in MCs. Confocal microscopy images showed that, in non-stimulated cells, TNF localizes in VAMP3 and VAMP8-positive vesicles. After TLR4 stimulation, we observed an increase in TNF and VAMP8 co-localization, without any change on TNF and VAMP3 co-localization. Remarkably, VAMP8 blockade with specific siRNA impaired TNF secretion after LPS-challenge, while VAMP3 siRNA-mediated down regulation did not. Our data indicate that, although TNF is maintained in VAMP3-containing vesicles, TLR4 triggering leads to the secretion of TNF in VAMP8-containing carriers. Our results contribute to the description of the molecular events involved in late steps of TNF secretion induced by innate immune stimuli in that important immune cell type.
Extensive sex-dimorphism in age-related transcriptional remodeling in mouse peritoneal macrophages
Authors: Lu Ryan , Brown Shelby B , Sampathkumar Nirmal Kumar , Shen Courtney , Chae Jisoo , Benayoun Bérénice A
Published: January 2023

Chronic low-level inflammation is one of the hallmarks of aging. In addition, age-related diseases share common inflammatory mechanisms, a phenomenon named “inflamm-aging”. To date, the effect of age and sex on the genomic landscape of macrophages is largely unexplored, especially regarding sex-dimorphic trajectories in aging macrophages. Based on accumulating evidence indicating aging is highly sex-dimorphic, we hypothesize that aging will affect the phenotype of immune cells, such as macrophages, in a sex-dimorphic manner. To test our hypothesis, we used female and male C57BL/6N mice aged 4 and 20 months to investigate resident peritoneal macrophages. We generated RNA-seq data from purified peritoneal macrophages and identified genes modulated with aging as a function of sex. Complementing this ‘omic’ dataset, we performed an array of functional assays for these cells in several cohorts of aging mice, including immunophenotyping, metabolic profiling, lysosomal acidification tracking, phagocytic efficiency, etc. We observed sex-dependent functional decline, such as a male-specific decrease in macrophage ability to acidify lysosomes upon exposure to Zymosan. Interestingly, aspects of age-related functional changes may be mediated by age-related changes in circulating sex hormones. We are currently analyzing our single cell RNA-seq datasets from peritoneal lavages of naïve aging female and male mice, and generating complementary datasets that are profiling chromatin-level changes to identify putative drivers of age-related transcriptional and functional remodeling in macrophages. Ultimately, this project will help delineate sex dimorphic mechanisms modulating “inflamm-aging”.
The G9a methyltransferase inhibitor BIX01294 inhibits IFN-α production and regulates DNMT1 in virus-activated pDC
Authors: Codrington Alicia L , Yin Zhiwei , Singh Sukhwinder , Fitzgerald-Bocarsly Patricia
Published: January 2023

Plasmacytoid dendritic cells (pDC) are innate immune cells that represent 0.2–0.5% of human PBMC and produce IFN-α upon TLR7 or -9 activation by virus to stimulate innate and adaptive responses. Because of its potency and propensity to induce autoimmunity, IFN-α production must be tightly controlled. We investigated the role of histone 3 lysine 9 di-methylation (H3K9me2) and DNA Methyltransferase 1 (DNMT1) in IFN-α gene silencing in virus-activated pDC. DNMT1 recruits histone deacetylases, which are known to regulate cytokines. H3K9me2 is methylated by G9a methyltransferase, which suppresses IFN-α in fibroblasts. However, in virus-activated pDC, H3K9me2 may induce IFN-α. PBMCs were isolated from healthy donors and stimulated with IAV, HIV, or HSV for 24-hrs. We surface stained for pDC markers CD123 and HLA-DR, and CD11c, then intracellular stained for DNMT1. We also treated pDC with G9a methyltransferase inhibitor, BIX01294, and measured IFN-α and DNMT1 6-hr post stimulation with HSV and IAV. qRT-PCR was performed to measure DNMT1, demethylases, and G9a mRNA in purified pDC. In IAV and HSV stimulated pDC, DNMT1 was significantly upregulated at 24-hrs compared to 8-hr stimulated and unstimulated controls. IFN-α production and DNMT1 expression in IAV-activated pDC, but not in HSV, was significantly inhibited by BIX01294. Furthermore, G9a methyltransferase mRNA was elevated in IAV and HIV-1 purified pDC, but absent in HSV-stimulated cells. The elevation of DNMT1 may assist with limiting IFN-α production in pDC at 24-hrs to prevent excessive immune activation. Further, the reduction of DNMT1 in IAV, but not HSV-activated pDC upon treatment with BIX01294 implies a virus or TLR pathway (TLR7 vs -9) specific role of DNMT1.
STING-dependent type-1 interferon restrains schistosome immunopathology via down-regulation of the CD209a lectin receptor
Authors: Kalantari Parisa , Shecter Ilana , Gois Andrea Pilotta , Hopkins Jacob , Morales Yoelkys , Sharma Shruti , Stadecker Miguel
Published: January 2023

Infection with the helminth parasite Schistosoma mansoni causes morbidity and mortality via a pathogenic host CD4 T cell-mediated immune response directed against parasite egg antigens. We now demonstrate that stimulation of dendritic cells (DCs) with schistosome eggs induces robust IFNβ production in a manner dependent on the cyclic GMP-AMP synthase (cGAS)/Stimulator of Interferon genes (STING) cytosolic DNA sensing pathway, resulting in the suppression of proinflammatory IL-1β and IL-23 production and in Th17 cell activation. Consistent with these results, low-pathology BL/6 mice lacking STING exhibited markedly enhanced hepatic granulomatous inflammation associated with significantly increased Th17 and diminished Th2 cytokine responses. Mechanistically, IFNβ acts by suppressing DC expression and function of CD209a, a C-type lectin receptor associated with severe schistosome immunopathology. Importantly, there was an increased baseline CD209a expression in unstimulated DCs from STING−/− mice, suggesting a role for constitutive IFN signaling. Our findings demonstrate that innate, cGAS/STING-dependent sensing of parasite DNA represents a novel pathway inducing type I IFN production, which protects the host from excessive inflammation and immunopathology in schistosomiasis. This work is supported by NIAID grant R01 AI018919 to MJS.
Activation of the cGAS-STING pathway during Trypanosoma cruzi infection induces a type I interferon response that promotes parasite growth at the infection site
Authors: Iyam-Perumal Amanda-Natasha , Tarleton Rick L.
Published: January 2023

Host cell invasion by Trypanosoma cruzi is a markedly silent process that induces a modest, yet reproducible type I interferon (IFN-I) response. In this study, we report that T. cruzi -induced IFNβ production occurs through the cGAS-STING pathway. The induction of IFNβ transcription in primary murine macrophages at 24 hours post-infection was nearly abolished in cGAS−/− or STING−/− macrophages. Infected cGAS−/−, STING−/− and IFNAR−/− macrophages had significantly higher amastigotes compared to WT macrophages, indicating that activation of the STING pathway constrains intracellular parasite growth. To further assess the impact of the STING pathway in vivo, mice were infected in the footpads with tdTomato-expressing T. cruzi and the fluorescence intensity at the infection site was quantified as a measure of parasite growth. Despite an initial increase in parasite growth, we unexpectedly found that STING−/− and IFNAR−/− mice ultimately accumulated fewer parasites in the footpads as compared to WT mice. Surprisingly, contrary to STING−/− mice, we observed increased parasite growth in the footpads in cGAS−/− mice but not in cGAS−/−IFNAR−/− mice. Together, these results indicate that IFN-I response favors parasite growth at the site of infection. T. cruzi -induced IFN-I production results in increased monocyte and decreased neutrophil recruitment to the infection site, but depletion of these cells in mice had a minimal role in affecting parasite growth. Future studies will investigate the mechanism of IFN-I mediated enhancement of parasite growth and the overall impact of cGAS-STING activation in parasite control.
Phagocyte-derived SOCS1 disrupts Staphylococcus aureus skin host defense
Authors: Klopfenstein Nathan , Brandt Stephanie , Serezani C. Henrique
Published: January 2023

Methicillin-resistant Staphylococcus aureus (MRSA) skin infection is controlled by the actions of skin resident macrophages and the formation of a neutrophilic abscess that prevents bacterial spread and tissue damage. We and others have shown that myeloid differentiation factor 88 (MyD88) is required for the clearance of MRSA skin infection in mice. MyD88 expression is controlled by the balance between STAT1 and the suppressor of cytokine signaling 1 (SOCS1). Here, we hypothesized that SOCS1 inhibits antimicrobial effector functions and the inflammatory response, leading to poor abscess formation and tissue injury during MRSA skin infection. Our data show that MRSA skin infection enhances SOCS1 expression. Infection in myeloid-specific SOCS1 deficient mice displays decreased lesion size, lower bacterial loads, and increased abscess thickness when compared to WT mice. When we treated infected mice with a peptide that specifically inhibits the kinase inhibitory region (KIR) of SOCS1, it also improved infection outcome. Examining the mechanisms by which SOCS1 enhanced skin host defense, we observed increased phagocytosis and bacterial killing in SOCS1 deficient macrophages and KIR peptide-treated cells. Increased antimicrobial effector function correlated with enhanced STAT1 activation and increased production of IFNγ in vivo and in vitro. Next we tested whether IFNγ is crucial to improved host defense in KIR-treated mice. Our data show that the beneficial effect of SOCS1 KIR peptide in skin host defense was abrogated in mice treated with an IFNR blocking antibody and in the IFNR deficient mice. Overall, these data show that preventing SOCS1 actions enhances microbial clearance and host defense during MRSA skin infection.
Regulation of the immune response by ST8Sia6
Authors: Crotts Sydney B , Friedman David J , Wang Zheng , Shapiro Michael J , Rajcula Matthew , McCue Shaylene , Sun Jie , Shapiro Virginia Smith
Published: January 2023

Sialic acids are the terminal modification on glycoproteins and glycolipids. The sialic acid transferase ST8Sia6 generates a2,8-linked disialic acids on O-linked glycoproteins, which bind to the inhibitory receptor Siglec-E on innate immune cells and suppress immune activation. Recently, our lab has shown that ST8Sia6 expression on tumor cells inhibits the immune response through Siglec-E, leading to increased tumor growth and decreased survival. Therefore, the products of ST8Sia6 can modulate the immune response to tumors. Our lab has generated ST8Sia6 knockout (KO) mice, and we seek to understand the role of ST8Sia6 in regulating the immune response to pathogens. We find that ST8Sia6 KO mice, when challenged with influenza virus, clear the infection significantly faster than wildtype controls, suggesting that absence of ST8Sia6 reduces inhibition of the innate compartment, thereby increasing immune recruitment to infection. This data suggests an important role for interactions between Siglec-E and ST8Sia6-generated disialic acids in immune regulation and recruitment to infection sites.
Anergy-derived Foxp3+ T regulatory cell differentiation in the neonatal mouse
Authors: Morales Milagros Silva , Mueller Daniel L
Published: January 2023

The neonatal period is a natural scenario to study the effects of peripheral self-antigen (Ag) recognition by autoreactive conventional CD4+ T cells under conditions of low Foxp3+ T regulatory cell (Treg) numbers and function. Moreover, the neonatal environment is an interesting scenario to study the relationship between the induction of CD4+ T cell anergy and the peripheral generation of anergy-derived Tregs, given that anergy induction relies on normal Treg function whereas anergy-derived Treg generation is optimal in the setting of Treg cell deficiency. We have hypothesized that the neonatal period is both necessary and sufficient to allow for the induction of anergy as well as the peripheral differentiation of anergy-derived Foxp3+ Tregs from naive conventional CD4+ T cells during self-Ag recognition.

To test our hypotheses we developed a system of neonatal peripheral tolerance induction. Neonatal pups engineered to express the experimental “self” peptide Ag chicken ovalbumin (OVAp) were given OVAp/I-Ab–specific OT-II CD4+ T cells, and the pups were later examined for anergy induction, Treg differentiation, and development of autoimmunity. Our data demonstrate that in the neonatal period: 1) previously naive CD4+ T cells express markers of anergy following exposure to self-Ag, and a small fraction of these cells can be seen to differentiate to a Foxp3+ Treg phenotype, and 2) previously anergic CD4+ T cells typically retain their anergic phenotype but also give rise to a substantial fraction of Foxp3+ Tregs, and 3) animals remain free of autoimmune disease. These findings suggest that during the neonatal period peripheral Foxp3+ Treg cell differentiation may proceed through a conventional anergic CD4+ T cell intermediate.
TAGLN2 is essential for both dendritic cell migration and dendritic cell-mediated T cell stimulation
Authors: Jun Chang-Duk
Published: January 2023

Actin polymerization and depolymerization are essential for many aspects of cell function including migration, adhesion, and activation. However, the importance of actin stabilization has been relatively underestimated. Here, we show that TAGLN2—a small 22-kDa actin-stabilizing protein—is critical for dendritic cell (DC) functioning in vitro and in vivo. TAGLN2 is minimally expressed in DCs, but highly upregulated in response to lipopolysaccharide (LPS), suggesting that TAGLN2 may play a certain role in DCs under the inflammatory conditions. Knockout of TAGLN2 (TAGLN2−/−) gene revealed normal differentiation and activation in terms of their surface marker expression and cytokine production by LPS. Interestingly, however, TAGLN2−/− DCs showed attenuated ability to prime CD4+ T cells as a result of weak contact between DCs and T cells. In addition to the T cell priming, we further observed drastic reduction of TAGLN2−/− DC homing to draining lymph nodes, suggesting that TAGLN2-mediated stabilization of filamentous actin (F-actin) is also important process for DC migration. Accordingly, knockout of TAGLN2 strongly reduced signals linked to the actin remodeling. Collectively, our findings indicate that TAGLN2 positively regulates DC migration and DC-evoked T cell responses via actin stabilization in processes that heavily require dynamic actin rearrangement.
Distinct roles of programmed cell death ligands 1 and 2, based on the type of immunity
Authors: Tanaka Ryota , Ichimura Yuki , Fujimoto Manabu , Okiyama Naoko
Published: January 2023

Programmed cell death-1 (PD-1) is an immuno-regulatory receptor binding to two ligands, PD-L1 and PD-L2, which are expressed on stimulated antigen presenting cells (APCs). Isolated APCs such as macrophages (Mϕ), dendritic cells (DC), and B cells were cultured with interferon (IFN)-γ, interleukin (IL)-4, and IL-17A, respectively, followed by PD-L1/L2 expression analysis using flow cytometry. Upon stimulation by IFN-γ on all APCs and IL-17A on Mϕ and B cells, respectively, strong and moderate upregulation of PD-L1 expression was observed over unstimulated controls. Only stimulation by IL-4 could upregulate PD-L2 expression on all APCs.

Therefore, experiments were performed on murine models, causing 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity (CHS), fluorescein isothiocyanate (FITC)-induced CHS, and imiquimod (IMQ)-induced psoriasis-like dermatitis, in order to trigger IFN-γ-mediated T helper (Th)1, IL-4-mediated Th2, and IL-17A-mediated Th17-type responses, respectively. In both Th1 and Th17-type immunity models, ear thickness changes in PD-L1 deficient (PD-L1−/−) mice were more severe than in wild-type (WT) and PD-L2 deficient (PD-L2−/−) mice (PD-L1−/−: 26.6±3.0, WT: 12.0±1.8 and PD-L2−/−: 12.3±1.9 [μm] in DNFB-induced CHS; 9.5±0.7, 6.2±0.4 and 5.7±0.6 [μm] in IMQ-induced psoriasis-like dermatitis [P < 0.01 by the U test], respectively). Thickness changes in PD-L2−/− were more severe than those in the WT and PD-L1−/− mice in the Th2-type immunity model (PD-L2−/−: 10.8±1.0, WT: 3.8±0.6 and PD-L1−/−: 3.6±0.6 [μm], [P < 0.01 by the U test], respectively).

Collectively, PD-L1 has predominant roles in Th1 and Th17-type immunity; similarly, PD-L2 for Th2-type immunity.
RORγt-driven TH17 cell differentiation requires epigenetic control by the Swi/Snf chromatin remodeling complex
Authors: Seong Rho H , Lee Sungkyu , Kim Jieun , Min Hyungyu
Published: January 2023

Epigenetic regulation, including chromatin accessibility and posttranslational modifications of histones, is of importance for T cell lineage decision. TH17 cells play a critical role in protective mucosal immunity and pathogenic multiple autoimmune diseases. The differentiation of TH17 cells is dictated by a master transcription factor, RORγt. However, the epigenetic mechanism that controls TH17 cell differentiation remains poorly understood. Here we show that the Swi/Snf complex is required for TH17-mediated cytokine production both in vitro and in vivo. We demonstrate that RORγt recruits and forms a complex with the Swi/Snf complex to cooperate for the RORγt mediated epigenetic modifications of target genes, including both permissive and repressive ones for TH17 cell differentiation. Our findings thus highlight the Swi/Snf complex as an essential epigenetic regulator of TH17 cell differentiation and provide a basis for the understanding of how a master transcription factor RORγt collaborates with the Swi/Snf complex to govern epigenetic regulation.
IFN-γ-induced nitric oxide leads to inhibition of Th17 differentiation via induction of iNOS and nitration of ROR-γ T
Authors: Robinson-Joern Rachel , Dietz Alina , Garza Celeste , Moy Brian , Crisostomo Neil Elijah S. , Vukmirovic Dora , Forsthuber Thomas
Published: January 2023

Elucidating the molecular mechanisms involved in pleotropic cytokine signaling can lead to novel treatment approaches for autoimmune diseases that target proinflammatory functions while maintaining immune-regulatory functions. To that end, we sought to further investigate the immune-regulatory roles of the pleotropic cytokine IFN-γ. Previous studies demonstrated that IFN-γ prevents the differentiation of pathogenic Th17 cells. In addition, nitric oxide generated via inducible nitric oxide synthase (iNOS) prevents differentiation of Th17 cells by nitration of ROR-γT. However, it remains unresolved whether IFN-γ prevents Th17 differentiation directly via inducing nitric oxide (NO) and nitration of ROR-γT. In line with previous observations, we found an increase in the frequency of autoreactive Th17 cells and a decrease in iNOS expression and NO in the absence of IFN-γ in mice with experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Additionally, the increase in Th17 cells in the absence of IFN-γ was reversible both in vitro and in vivo with treatment with a NO donor. This indicates a role for IFN-γ in inhibiting the differentiation of Th17 cells via iNOS-derived NO. We are currently elucidating the underlying mechanisms of NO mediated suppression and the molecular link between ROR-γt-nitration and decreased Th17 differentiation by flow cytometry, single-cell western blotting of key transcription factors, and in vivo adoptive transfer studies. Our studies may lead to a better understanding of the role of IFN-g and NO/iNOS axis in Th17 cell differentiation and autoimmune diseases.
Evidence for a prescribed NK cell Ly49 developmental pathway in mice
Authors: Millan Alberto Jovani , Sindi Suzanne , Manilay Jennifer O.
Published: January 2023

Previous studies of NK cell Ly49 receptors suggest that the inhibitory receptor expression is stochastic. However, similar studies of activating Ly49 receptors have not been an area of focus. We hypothesize that the expression of activating receptors is not stochastic. We analyzed NK cell “clusters” expressing 16 combinations of Ly49I, Ly49G2, Ly49H and Ly49D in B6 mice. We sorted the 16 clusters, and cultured them with IL-15 for 6 days. In parallel, we also transplanted a select group of clusters in vivo. We discovered a prescribed pathway of cluster transitions in vitro and in vivo, and these data suggest that Ly49 activating receptor acquisition is not stochastic. We found that the majority of clusters that express activating receptors maintain their expression. In addition, Ly49D, which is not reported to have an MHC-I ligand in B6 mice, was upregulated on clusters that transition in the prescribed pathway. Previously, other groups have used the product rule to mathematically predict the independent expression of Ly49 inhibitory receptors. However, the product rule has not been applied to predict activating Ly49 receptor frequencies. Our application of the product rule to our data suggest that Ly49H and Ly49D expression is not independent and cannot be predicted by this mathematical model. This suggests that the biological system that controls the Ly49 activating receptors is distinct from inhibitory receptors. Currently we are investigating whether MHC-I is critical in the prescribed pathway via the transplantation of these clusters in MHC-I-deficient mice. Taken together, these data support the idea that NK cell clusters develop in a sequential, non-random manner that is determined by expression of activating Ly49 receptors.
Bile acid metabolites control Th17 and Treg cell differentiation
Authors: Hang Saiyu , Paik Donggi , Yao Lina , Jamma Trinath , Lu Jingping , Ha Soyoung , Nelson Brandon N , Kelly Samantha P , Wu Lin , Zheng Ye , Longman Randy S , Rastinejad Fraydoon , Devlin A Sloan , Krout Michael R , Fischbach Michael A , Littman Dan R , Huh Jun R
Published: January 2023

Bile acids are abundantly present in the mammalian gut, where they undergo bacteria-mediated transformation, generating a large pool of bioactive molecules. While they have been shown to affect host metabolism, cancer progression and innate immunity, it is unknown whether bile acids affect the function of adaptive immune cells such as T cells expressing IL-17a (Th17 cells) and regulatory T cells (Tregs) that mediate inflammatory and anti-inflammatory responses, respectively. By screening a small-molecule library primarily composed of bile acid metabolites, we identified two distinct derivatives of lithocholic acid (LCA), 3-oxoLCA and isoalloLCA, as specific regulators of Th17 and Treg cells. While 3-oxoLCA inhibited Th17 cell differentiation by directly binding to its key transcription factor RORγt (retinoid-related orphan receptor γt), isoalloLCA enhanced Treg differentiation through the production of mitochondrial reactive oxygen species (mitoROS), leading to increased FoxP3 expression. IsoalloLCA-mediated Treg enhancement required an intronic FoxP3 enhancer the conserved noncoding sequence 3 (CNS3), suggesting a distinct mode of action from other previously identified Treg enhancing metabolites that require CNS1. Lastly, oral administration of 3-oxoLCA and isoalloLCA to mice led to reduced Th17 and increased Treg cell differentiation in the intestinal lamina propria. Altogether, our data suggest novel mechanisms by which bile acid metabolites control host immune responses by directly modulating the Th17 and Treg balance.
Pathogen-elicited memory γδ T cells express a broadly reactive canonical TCR or a pathogen-specific non-canonical TCR
Authors: Khairallah Camille F , Bettke Julie , Imperato Jessica Nancy , Zhang Yue , Qiu Zhijuan , Gorbatsevych Oleksandr , Cho Kyungjin , Kim Kwang Soon , Puddington Lynn , Surh Charles D , Bliska James B , Van der Velden Adrianus WM , Sheridan Brian S
Published: January 2023

γδ T cells are critical sentinels protecting barrier surfaces against pathogens. However, our understanding of γδ T cell specificity remains limited. We previously identified a resident memory Vγ4Vδ1 T cell population elicited by foodborne Listeria monocytogenes (Lm) infection. Analysis of the CDR3δ sequences revealed that a large subset of non-canonical clones expanded during primary Lm infection, contracted during memory homeostasis and recalled after secondary Lm infection suggesting antigenic-specificity may be involved in this response. However, most of the response was elicited by cells bearing a canonical Vδ1 TCR suggesting that many Lm-elicited cells may not be Lm-specific. Indeed, stimulation of mesenteric lymph node cells from Lm-immune mice with diverse pathogens induced a functional response by Lm-elicited memory γδ T cells. Similarly, induction of Salmonella colitis (STm-colitis) in Lm-immune mice resulted in a TCR-dependent γδ T cell recall-like response, demonstrating that the majority of these memory cells respond to heterologous infection. Interestingly, most of the non-canonical γδ T cell clones that responded to Lm infection did not expand in response to STm-colitis suggesting some level of specificity is imparted by the priming pathogen. Importantly, other enteropathogens triggered a recall-like response of Lm-elicited memory γδ T cells. Adaptive γδ T cells were also generated after foodborne infection of germ-free mice and in response to diverse bacterial infections of SPF mice, indicating that this population can be induced by a broad range of disseminating pathogens and may provide a broad target for universal mucosal vaccines of the gut.
Characterization of a potential Escherichia coli virulence factor identified in a screen using the social amoeba phagocyte, Dictyostelium discoideum
Authors: Snyder Michelle , Schmidt Katrina , Lane Jorden , Weichseldforfer Matthew
Published: January 2023

Escherichia coli are a typical component of mammalian gut microbiota; however, a number of pathogenic strains remain a health challenge for developing countries. To identify novel bacterial virulence factors, we used the social amoeba, Dictyostelium discoideum, which preys upon soil bacteria, including E. coli, as a model for mammalian phagocytic cells. Using random mutagenesis, screening for mutant E. coli strains resistant to killing upon phagocytosis, and genomic sequencing of these resistant strains, we identified mutations in several potential genes of interest, including an E. coli gene of unknown function, yfaT. Preliminary results suggest that D. discoideum Erk signaling is reduced in response to yfaT-deficient E. coli. Furthermore, mammalian TLR4 signaling in response to yfaT-deficient E. coli also appears attenuated. Our current studies focus on characterizing differences in growth patterns for wild-type and yfaT-deficient E. coli using Biolog screenings, as well as analyzing potential differences in LPS structure in these strains. These studies are aimed at identification and characterization of novel mechanisms that may be involved in bacterial virulence. In addition, given that the development of mechanisms to resist D. discoideum predation may have contributed to the selection and maintenance of bacterial virulence factors, such as yfaT, against mammalian hosts, these studies may provide insight on the evolution of host-pathogen interactions.
Interferon Receptor 2 (IFNAR2) is involved in regulation of host damage responses and fungal clearance during Aspergillus fumigatus pulmonary infection
Authors: Shepardson Kelly , Johns Laura Logan , Malkoc Zeynep , Hernandez Emiliano , Schulein Clyde , Rynda-Apple Agnieszka
Published: January 2023

Three million people globally suffer from infections caused by Aspergillus fumigatus (Af). Over 300,000 of these cases are due to invasive pulmonary aspergillosis (IPA) in patients with suppressed immune systems. The recent increase of aspergillosis in influenza-infected patients suggests that influenza infection creates transiently suppressed immune environments permissive to fungal infection. Consistent with this, we found that influenza infected WT mice had increased Af lung fungal burden and cellular damage compared to Af-only infected mice. We recently discovered that type I interferon (IFN) signaling, via IFNAR2 of the IFNAR1/2 receptor, regulates susceptibility to and damage from influenza. A main factor contributing to IPA pathology and outcome is the level of damage the host incurs. Importantly, we found that IFNAR2 deficiency (Ifnar2−/− mice) resulted in increased cellular damage and morbidity at 24 hrs post-Af compared to WT and Ifnar1−/− mice. Interestingly, Ifnar2−/− mice cleared spores more efficiently than both WT and Ifnar1−/− mice but were unable to control invasive disease and maintain pulmonary architecture, evidenced by hyphal growth and fibrosis-like tissue at 48 hrs post-Af. Although cellular recruitment was unaltered, the levels of inflammatory cytokines in Ifnar2−/− mice was two-fold higher than WT and Ifnar1−/− mice, and Ifnar2−/− neutrophils produced more external ROS in response to Af suggesting altered effector cell function may be involved in the IFNAR2 regulated damage response. Together, our results begin to establish a role for IFNAR2 in regulation of the host damage response to Af and suggest that aberrant type I IFN signaling creates a permissive environment allowing for Af infection to occur.
Controlled human infection with Neisseria lactamica induces B-cell responses that are cross-reactive with Neisseria meningitidis
Authors: Dale Adam P , Theodosiou Anastasia A , Laver Jay R , Roche Eleanor F , Hill Alison R , Gorringe Andrew , Polak Marta E , Vaughan Andrew T , Read Robert C
Published: January 2023

Neisseria lactamica (Nlac) is a commensal of the human oropharynx. Colonisation with Nlac significantly reduces colonisation by N. meningitidis (Nmen). We hypothesised that cross-reactive adaptive cellular immune responses were responsible for this effect.

31 volunteers were inoculated intra-nasally with 105 colony-forming units of Nlac or vehicle control. Nlac/Nmen colonisation status was assessed at baseline and 7-, 14- and 28-days post-inoculation. Nlac/Nmen-specific plasma cells (PCs)/memory B-cells were quantified in PBMCs at all time points using ELISpot assays.

Amongst Nlac-colonised volunteers (n=17), Nlac-specific PC frequencies/2×105 PBMCs increased significantly. Median baseline vs. post-colonisation peak Nlac-specific PC frequencies were 0 (range 0–0.5) vs. 2 (0–31) for IgM, 0 (0–1) vs. 5 (0–20.5) for IgA, and 0 (0–1) vs. 3 (0–27) for IgG (all p<0.0001). Nlac-specific IgG memory B-cell frequencies also increased significantly from baseline to day 28 (0.000% vs. 0.04%, p<0.0001). Interestingly, there was also a significant increase in IgM, IgA and IgG Nmen-specific PCs amongst Nlac-colonised volunteers. Median baseline vs. post-colonisation peak Nmen-specific PC frequencies were 0 (0–0) vs. 0 (0–30) for IgM, 0 (0–1) vs. 0.5 (0–8.5) for IgA, and 0 (0–0.5) vs. 0.5 (0–10) for IgG (all p≤0.01). There was no induction of Nlac-specific PCs/memory B-cells in the control group (n=10).

These data suggest that the previously observed protective effect of Nlac on Nmen colonisation may have an immunological basis. We plan to assess the frequency/effector phenotype of Nlac-specific CD4+ memory T-cells following Nlac colonisation and hypothesise that cross-reactive CD4+ memory T-cells directed towards Nmen will be Th17.
Immune respones against Trypanosoma cruzi infection by oral route in cynomolgus macaques (Macaca fascicularis)
Authors: Nehete Pramod N , Chitta Sriram , Nehete Bharti P , Abee Christian R , Hamer Sarah A , Hodo Carolyn L , Wilkerson Gregory K
Published: January 2023

American Trypanosomiasis is an important neglected reemerging tropical parasitism, infecting about 8 million people worldwide. Its agent, Trypanosoma cruzi, exhibits multiple mechanisms to evade the host immune response and infect host cells. An important immune evasion strategy of T. cruzi infective stages is its capacity to inhibit the complement system activation on the parasite surface, avoiding opsonizing, immune stimulating and lytic effects. Eight male M. fascicularis between 8 and 10 years of age of Chinese origin were infected through oral administration of T. cruzi-infected triatomine bugs and followed over 6 weeks. The purpose of this study was to define cellular immunological responses in T. Cruzi challenged M. fascicularis. We measured immunofluorescent staining and multifunctional T. cruzi antigen specific T-cell responses in experimental infected M. fascicularis. Significant increases in the CD4+ T-cell population and polyfunctional T cell responses were observed in all experimental-infected animals compared with the uninfected time points. We observed a decrease in absolute number of lymphocytes, T cells and T cell subsets, in early phase of infection but recovered in later period. T. cruzi antigen specific IFN-g and IL-12p40 ELISPOT showed no significant infection specific responses during the study period. Circulating cytokines measurement in plasma revealed a significant increase IFN-g and IL-1ra during infection period. In conclusion, our present findings provide the first evidence demonstrating the Trypanosoma cruzi infection by oral route.
The tropic life forms of Pneumocystis alter alveolar macrophage responses to inflammatory stimuli
Authors: Garvy Beth A , Pickett Carol L , Hollifield Melissa L
Published: January 2023

Pneumocystis is an opportunistic fungal pathogen that causes pneumonia in immunosuppressed individuals including patients with HIV. There are two identifiable life forms of Pneumocystis including a cyst or ascus form that has a β-glucan-rich fungal cell wall and a trophic form that lacks the cell wall. In infected lungs the trophic forms can outnumber the asci by as much as 10 to 1. We have previously published that the trophic forms of Pneumocystis are able to inhibit the production of pro-inflammatory cytokines such as TNF induced by β-glucans or LPS in bone marrow-derived dendritic cells. Recently we found that trophic forms also inhibit TNF production by alveolar macrophages when stimulated in vitro. To understand relevance of this observation during Pneumocystis infection, RAG−/− mice were infected with the trophic forms of Pneumocystis, treated with an echinocandin drug to prevent the formation of ascus forms, and challenged with a low dose of LPS given intratracheally. Twenty-four hours later bronchial alveolar lavage fluids were obtained and TNF quantitated. The trophic forms of Pneumocystis significantly inhibited the production of TNF in mice challenged with LPS compared to those given LPS alone or LPS plus echinocandin. Interestingly, there was no difference in the influx of neutrophils into the lungs in LPS-treated mice compared to trophic form infected and LPS-treated mice. It is well known that TNF is an important factor in host defense against Pneumocystis while neutrophils have not been shown to enhance clearance but coincide with lung pathology. Together our data indicates that trophic forms of Pneumocystis alter selected parts of the inflammatory milieu of the lungs.
Temporal patterns in single cell immune profile of tuberculosis lung granulomas
Authors: Gideon Hannah Priyadarshini , Hughes Travis K. , Wadsworth Marc H. , Nyquist Sarah , Lin Philana Ling , Fortune Sarah , Shalek Alex K , Flynn JoAnne L
Published: January 2023

Infection with Mycobacterium tuberculosis results in the formation of tuberculosis lung granulomas, which are complex structures with a network of multiple immune cells closely intertwined and function together. Within a given host, granulomas have unique trajectories and outcomes resulting in bacterial control or persistent bacterial replication and dissemination. The timing of establishment and immune composition of the granuloma in the lung plays a crucial role in the control of bacterial burden. Using high-throughput single-cell mRNA sequencing (SeqWell) on individual TB lung granulomas and computational algorithms, we are generating models to understand infection dynamics that incorporate timing of establishment, immune cell composition, its functional interactions; bacterial burden and trajectories of the granulomas to identify robust drivers of bacterial control. We evaluated original and new lung granulomas identified by PET CT scan from 15 cynomolgus macaques infected with M. tuberculosis (Erdman strain) for 4, 6 or 10 weeks. We identified over 30 functional clusters of cells spanning multiple immune and non-immune cells types including T cells, B cells, plasma cells, dendritic cells, mast cells, macrophages, neutrophils, pneumocytes, fibroblasts and endothelial cells. We observe differential cellular composition associated with temporal patterns (original vs. new) in granulomas that are associated with bacterial burden. By understanding these immune features associated with temporal patterns that limit bacterial replication, we hope to identify novel targets for host-directed immune therapies and prophylactics in M. tuberculosis infection and other granulomatous diseases.
Continuous generation of effector (Teff) and effector memory (Tem) T cells with mouse cytomegalovirus (MCMV) vaccination to prolong malaria immunity
Authors: Gbedande Komi , Ibitokou Samad , Brown Michael G. , Degli-Esposti Mariapia , Stephens Robin
Published: January 2023

Immunity to Plasmodium infection or vaccination decays without continuous exposure. This decay correlates with loss of malaria-specific T cells, not antibody. We recently demonstrated that Teff protect better than memory T cells, and that Ifng+ Teff are responsible for the protection provided by persistent P. chabaudi infection. Based on this data, we have reverse-engineered a vaccine strategy that first induces a long-lived antibody response, and then boosts the Teff and Tem response to prolong protection. We chose live vaccination with “infection and drug cure”, which generates protection that decays over 200 days. The boost uses MCMV as it is an excellent T cell-inducing vector with promise against SIV, tuberculosis and liver-stage malaria. First, we observed that MCMV infection strongly reduced parasite growth, as previously shown for Leishmania, suggesting a useful adjuvant effect. To promote continuous induction of specific T cells, we generated MCMV expressing the P. chabaudi T cell epitope B5. Vaccination with MCMV-B5 activated both adoptively transferred B5 TCR Tg T cells and MCMV-specific CD4 T cells, generating protective Teff and Tem phenotypes for over 2.5 months. Importantly, we show that boosting with the MCMV vector prolongs protection generated by the live vaccine, as shown by better protection from re-infection at day 200. We are currently investigating the contribution of activated Plasmodium-specific T cells in prolonging protective immunity by MCMV-B5, and mechanisms of protection. Our findings suggest that chronic vaccination can redress the problem of short-lived protection to malaria infection and vaccines, which would be beneficial for any efficacious but short-term vaccine strategy.
T cell-derived interferon-γ is required for host defense to Toxoplasma gondii
Authors: Sasai Miwa , Pradipta Ariel , Yamamoto Masahiro
Published: January 2023

Interferon-γ (IFN-γ) is important for host defense against various intracellular organisms including a protozoan pathogen Toxoplasma gondii. Various immune cells are recently shown to produce IFN-γ in T. gondii infection, however, it remains elusive which cell types are important for anti-T. gondii host defense so far. Here we generate a new IFN-γ reporter mouse line in which a fusion protein of Venus and NanoLuc to analyze IFN-γ producing cells during T. gondii infection and find that CD4+, CD8+, γδ T cells and natural killer cells express Venus in a time dependent manner. Furthermore, Lck-Cre/Ifngfl/fl mice are highly susceptible to T. gondii infection. Taken together, our results demonstrate that T cell-derived IFN-γ plays an important role in anti-T. gondii host defense.
The role of Axl in bacterial infection-mediated inflammatory responses
Authors: Kim Minjae , Choi Harim , Kim Su-Man , Kang Hyung-Sik
Published: January 2023

Salmonella Typhimurium (S. Typhimurium), one of the most common infectious bacteria, triggers an inflammatory reaction in epithelial cells of the intestine and migrates into the spleen and liver to colonize. To elucidate the role of Axl in bacterial infection-mediated inflammatory responses, Axl-depleted mice and WT mice were infected with S. Typhimurium, and the systemic dissemination caused by the infection was evaluated. The Axl-depleted mice shows better histological scores and increased intraepithelial T cells. The colony number and CFU of S. typhimurium were also lower in the liver of Axl-depleted mice than in that of WT mice. Thus, lack of Axl results in better resistance to S. Typhimurium, suggesting that downregulation of Axl is essential for bacterial resistance by inducing increased number and activity of intraepithelial T cells.