bluuebunny/crossref_metadata_2025 huggingface dataset:
Transient Receptor Potential Vanilloid 4 (TRPV4) Protects the Lung from Bacterial Pneumonia via MAPK Molecular Switching
Authors: Scheraga Rachel Greenberg , Abraham Susamma , Grove Lisa M , Southern Brian D , Crish James , Hasday Jeffrey D , McDonald Christine , Olman Mitchell A
Published: January 2023

Mechanical cell-matrix interactions can drive the innate immune responses to infection, however the molecular underpinnings of these responses remain elusive. We have discovered that the biophysical properties of the matrix in the range of injured fibrotic lung (≥ 25 kPa) conditions the macrophage response to LPS and infection respectively through the mechanosensitive cation channel, TRPV4 in vitro and in vivo. Studies suggest that LPS-induced macrophage activation is controlled in part by the MAPK pathway (i.e. p38, ERK, JNK). Thus, we investigated if TRPV4 plays a role in the macrophage activation response after LPS through alteration of the MAPK pathway. TRPV4 KO mice exhibited reduced lung bacterial clearance by macrophages (6-fold, p=0.012) after intratracheal P. aeruginosa administration and increased lung injury as measured by inflammatory cell infiltration (≥80±3%, p<0.05), vascular permeability (BAL total protein ≥63±6%, p<0.05), and pro-inflammatory cytokine secretion in BALF (IL-6, CCL2, CXCL1 ≥71±4%, p<0.05). LPS-induced p38 activation was decreased (69%, p<0.05) while JNK activation was increased (2-fold, p<0.05) in a stiffness-dependent manner with no change in ERK activation in TRPV4 KO BMDMs. Inhibition of p38 (SB203580, BIRB796) decreased phagocytosis whereas inhibition of JNK (SP600125) decreased cytokine secretion (IL-6, CCL2, and CXCL1) after LPS (2 fold, p<0.05). DUSP1/MKP1 (MAPK phosphatase) protein was reduced by 3-fold in TRPV4 KO and inhibition of DUSP1 decreased phagocytosis and selectively increased activation of JNK. These data are the first to demonstrate new roles for macrophage TRPV4 in regulating innate immunity in a mechanosensitive manner, through MAPK activation switching.
Sts-1 and Sts-2 regulate the anti-fungal immune response
Authors: Frank David , Carpino Nick
Published: January 2023

Mice lacking expression of the phosphatases Sts-1 and Sts-2 (Sts−/−) are significantly resistant to disseminated candidiasis caused by the fungal pathogen Candida albicans. Resistance is associated with diminished levels of many inflammatory molecules, a reduction in kidney leukocyte infiltrates by 48 hours post infection, and an absence of inflammatory lesions. Unlike wild type mice, Sts−/− mice clear the infection from the kidney following inoculation with supra-lethal doses. To better understand parameters underlying the Sts−/− phenotype of enhanced resistance, we examined the kinetics of fungal clearance at early time points. We observed enhanced fungal clearance within Sts−/− kidneys beginning at 12–18 hrs. post infection. This corresponds to the timeframe when innate leukocytes such as Ccr2+ inflammatory monocytes (IMs) begin entering the tissue to counter the infection. To examine the role of IMs in promoting enhanced fungal clearance evident in Sts−/− animals, we infected mice lacking Ccr2 expression. Loss of Ccr2 abrogated both the fungal clearance and the survival advantage of Sts−/− mice. Analysis of innate anti-fungal effector responses revealed enhanced production of reactive oxygen species by phagocytes lacking Sts expression, with no apparent differences in the responses of other effector pathways. Downstream of fungal receptor Dectin-1, the Syk kinase was hyper-activated in Sts−/− cells. Our results identify Syk as a target of Sts activity in phagocytes and highlight a novel mechanism regulating the anti-fungal immune response. Further understanding of this regulatory pathway could aid in the development of therapeutics to protect against invasive candidiasis.
Production of leukotriene signaling mediators is limited by ORMDL3/serine palmitoyltransferase/5-lipoxygenase crosstalk
Authors: Draber Petr , Paulenda Tomas , Utekal Pavol , Halova Ivana , Kuchar Ladislav , Kuda Ondrej , Vavrova Petra , Schuster Bjoern , Potuckova Lucie , Cernohouzova Sara , Draberova Lubica , Bambouskova Monika , Bugajev Viktor
Published: January 2023

Leukotrienes and sphingolipids are critical lipid mediators participating in cellular signal transduction and development of various diseases. Metabolic pathways initiating production of these lipid mediators involve 5-lipoxygenase (5-LO)-mediated conversion of arachidonic acid to leukotrienes and serine palmitoyltransferase (SPT) de novo synthesis of sphingolipids. Previous studies showed that endoplasmic reticulum membrane protein ORMDL3 inhibits the activity of SPT and sphingolipid synthesis. However, the role of ORMDL3 in synthesis of leukotrienes is not known. In this study, we used peritoneal-derived mast cells (PDMCs) isolated from mice with ORMDL3 knockout (KO) or control mice and examined their properties. We found that PDMCs with ORMDL3 KO exhibited increased calcium response and ß-glucuronidase release when activated with antigen. These events were accompanied by increased phosphorylation of IκB-α and TNF-α production. Lipid analysis showed that ORMDL3-deficient cells exhibited not only enhanced production of sphingolipids, but also increased production of leukotriene inflammatory mediators, such as LTB4 and LTC4. These data were supported by the finding that ORMDL3 physically interacts with 5-LO. Further studies showed that 5-LO interacts with the SPT long-chain (LC)1 and SPTLC2 subunits and decreases the ceramide levels. In line with these findings, 5-LO knockdown increased the ceramide levels, and silencing of SPTLC1 decreased transition of arachidonic acid to leukotrienes. These results demonstrate physical and functional crosstalk between leukotriene and sphingolipid metabolism pathways leading to production of lipid signaling mediators.
The Role of Phosphodiesterase (Pde) 3B in the Inflammatory Response of Macrophages to LPS
Authors: Liu Yusen , Meng Xiaomei , Wang Xiantao , Snedden Ali , Kirk Sean G. , Cash Charles J. , Murphy Parker R. , Hafner Markus , Nelin Leif D. , Chen Bernadette
Published: January 2023

Pde3B hydrolyzes both cAMP and cGMP, important second messengers in a variety of cellular processes. Pde3B has been shown to regulate NLRP3 inflammasome activity in adipose tissue. To understand whether Pde3B plays a role in the macrophage response to bacterial infection, we derived macrophages from the bone marrow of wildtype and Pde3B knockout mice. Macrophages were then stimulated with LPS for 6 h or left untreated, and total RNA was prepared for RNA-seq analyses. Our data indicate that Pde3B expression was inhibited by LPS in macrophages. Knockout of Pde3B resulted in significant changes in the expression of a large group of genes: 427 genes were up-regulated by at least 2-fold while 428 genes were down-regulated by more than 50% in LPS stimulated macrophages as the result of Pde3B deficiency. Gene profiling indicates that a number of biological processes are affected by Pde3B knockout, including cellular ion/cation homeostasis, immune system processes, and chemotaxis. Interestingly, Pde3B deficiency disturbed the expression of several chemokines: Cxcl-1, -2, and -3 expression were decreased by 96, 60, and 75%, respectively. In contrast, Ccl-4, -7, -12, -22, Cxcl-9 and -l11 expression were enhanced 1.8, 1.5, 1.9, 2, 3, and 3-fold, respectively. Differential expression of dual specificity phosphatase (Dusp)-1, -2, -3, -4, -5, -6, -10, -11, and -16 were also seen between LPS-stimulated wildtype and Pde3B-deficient macrophages. Since the Dusps are important regulators of MAP kinases that control the expression of chemokines, alterations in the expression of Dusps provides a plausible explanation for the changes in chemokine expression. Our studies suggest that Pde3B may regulate chemokine expression via modulating MAP kinases.
Epigenetic regulation of C-Kitlo FcɛR1a+ mast cells by cannabidiol during staphylococcal enterotoxin B-induced liver injury
Authors: Culpepper Courtney D , Miranda Kathryn , Wilson Kiesha , Nagarkatti Prakash S. , Nagarkatti Mitzi
Published: January 2023

Although recent studies have established cannabidiol (CBD) as a potent anti-inflammatory agent, the mechanisms governing these processes remain unclear. Our previous research demonstrated that CBD treatment induced expansion of CD11b+Gr-1+MDSC in the peritoneum, which suppressed T cell functions leading to attenuation of staphylococcal enterotoxin B (SEB)-mediated liver inflammation in mice. Using the same model, we characterize the effects of CBD administration in the liver. Briefly, mice were injected intraperitoneally (I.P.) with 50mg/kg of CBD for 4 days (d), followed by SEB challenge on d 3. Flow cytometry analysis revealed a significant reduction in Vβ8+ CD8+ T cells. In agreement with our earlier findings, mice treated with CBD displayed robust expansion of Gr-1+ MDSC in the liver, which was primarily comprised of Ly6CHI MHCIILO leukocytes. As mast cells have been found to play a role in CBD-mediated induction of MDSC via increased peroxisome proliferator-activated receptor gamma (PPARγ) signaling, we examined changes to liver mast cell populations. We discovered two distinct subsets of mast cells, C-KithiFcɛR1a+ and C-Kitlo FcɛR1a+present in the liver. CBD treatment promoted expansion of the C-Kitlo FcɛR1a+ subset. Analysis by miRNA microarray displayed enrichment of miRNAs targeting mast cell growth factor, KIT ligand (KITLG), and significant downregulation of miR-217-5p, which is conserved for the co-activator of PPARγ (PPARγC1A). Increased gene expression of PPARγ was confirmed by single-cell RNA sequencing (sc-RNAseq). Taken together, our results identify CBD induced epigenetic reduction of C-Kit expression and KITLG signaling in mast cells to favor PPARγ signaling as a novel anti-inflammatory mechanism.
Differentiation of antibody-secreting cells in the thymus of human neonates
Authors: Cordero Hector , King Rodney G , Dogra Pranay , Dufeu Chloe , See Sarah B. , Chong Alexander M. , Uhlemann Anne-Catrin , Ho Siu-Hong , Farber Donna L , Kearney John F , Zorn Emmanuel
Published: January 2023

Thymic resident B cells were reported for the first time more than 40 years ago. Yet, their phenotype and significance remain unclear. In this study, we investigated the heterogeneity of thymic B cells in neonates as an indication of their possible function. Using thymuses from 21 neonates, we observed two consistent subsets, CD19+CD21+CD35+ and CD19+CD21−CD35−, accounting for virtually all thymic B cells. ELISPOT assays revealed the presence of IgG, IgA, IgM and even IgE antibody-secreting cells within the CD21−CD35− subset but not the CD21+CD35+ subset. To characterize the heterogeneity of CD21−CD35− subset, we used a single-cell RNA-sequencing and identified 4 separate clusters, 3 of which included cells with a phenotype and transcriptome consistent with that of B cell activation and proliferation. The more distant cluster corresponded to plasma cells (PCs) expressing IGHG, IGHA, and IGHE, implying prior class-switch recombination. To assess their specificity, we generated recombinant monoclonal antibodies (rAbs) from 362 PCs using a paired IgH and lgL chain expression cloning strategy. For comparison, rAbs were also generated from 296 undifferentiated CD21+CD35+ thymic B cells. The PC contingent was highly enriched in clones reactive to apoptotic cells when compared to CD21+CD35+ naïve B cells, an essential characteristic of natural antibodies. Remarkably, we identified individual PC clones reactive to some bacterial species also recognized by natural antibodies. Taken together, we demonstrate the differentiation of plasma cells reactive to apoptotic cells and common bacteria within the thymus, suggesting that thymus is a source of natural antibodies, contributing to newborn humoral immunity.
Differential survival requirements for Lck- and CD3e-dependent signaling defines two populations of ATMKO thymic T cell lymphomas
Authors: Hathcock karen S , An Joseph , Xu Max , Hodes Richard J
Published: January 2023

The product of theataxia-telangiectasia mutated gene (ATM) is a serine-threonine kinase thatcommunicates between molecules that detect dangerous DNA double strand breaksand effector mechanisms that maintain genomic integrity. ATM deficiency in bothhumans and mice is associated with genomic instability and increased malignancies. ATMKO mice exclusively develop thymic T cell lymphomas that resemble thymically-derivedT-ALL that occur in human ATM-deficient ataxia telangiectasia patients. Flowcytometry identified two subsets of ATMKO T cell lymphomas based on surfaceexpression of CD3ɛ, TCRβ and pre-Tα:Cells that express surface CD3ɛ, TCRβ and pre-Tα(surface receptor high; sRhi) and cells that do not express detectable surface CD3ɛ, TCRβ orpre-Tα(sRlo). When assayed for CD25 and CD44 expression sRhi lymphomas resemble DN3or DN4 cells while sRlo lymphomas resemble more immature DN1 or DN2 cells. Pre-TCRsignaling in normal DN3 and DN4 thymocytes provides an essential survivalsignal that is proposed to be constitutive and ligand-independent. Therefore, weasked if surface sRhi and sRlo cells are dependent on pre-TCR signaling byincubating cells with either Lck inhibitor, CAS 213743-31-8, or CD3ɛshRNA to inhibit pre-TCR signaling. Both treatments significantly inhibitedsurvival of sRhi lymphomas, while survival of sRlo cells was largely unaffected. These results suggest that ATMKO T celllymphomas arise from cells transformed at different stages of DN development andthat sRhigh cells, like normal DN3 and DN4 thymocytes, retain apre-TCR-dependent survival requirement. These results also suggest that pre-TCRsignaling may be a useful target for clinical intervention in treatment ofhuman T-ALL that express a pre-TCR.
Fine-tuning Notch expression by lymphocytic Granzyme B production
Authors: Tibbs Ellis J , Cao Xuefang
Published: January 2023

Notch signaling has been studied for many years and has been found to play a role in many aspects of cellular development, tissue homeostasis, and progression of disease. Upon interaction with its ligand, Delta-like-4, the Notch1 molecule undergoes two proteolytic cleavage events. This eventually leads to the translocation of the intracellular domain (NICD) into the nucleus. Once there, NICD interacts with transcription factors such as CBF1/RBPJ. Recently, Notch has been found to play a role in the Th1/Th2 imbalance found in many diseases. Using a software to determine possible cleavage target sites for Granzyme B (GzmB), two were found following the start site of NICD. One group found that cleavage of Notch1 by GzmB disables its activity, however lymphocytes were not the target tissue of research. Thus, we hypothesize that Granzyme B production plays a functional role in the translocation of intracellular Notch1 in affecting CD4+ and CD8+ T lymphocyte activation.

We test our hypothesis by stimulating T lymphocytes collected from transgenic mice that are deficient in GzmB and measuring intracellular and extracellular Notch1 expression compared to wildtype following in vitro aCD3-CD28 stimulation. Flow Cytometry was used to measure Notch expression. Preliminary results show that 24hrs after stimulation, CD8+ and CD4+ T cells from GzmB-deficient mice show 12% and 6%, more NICD expression than WT counterparts and 19% and 14% more extracellular Notch1 expression, respectively. These results show that the presence of GzmB can cause an alteration in the fine-tuning expression of Notch1. This work helps us understand the role of Granzyme B in Notch activation and differentiation of lymphocytes.
ABCB7 is required to prevent ferroptosis in peripheral T cells
Authors: Lehrke Michael Jonathan , Belmonte Paul , McCue Shaylene , Rajcula Matthew , Shapiro Virginia
Published: January 2023

ABCB7 exports iron-sulfur (Fe-S) cluster intermediates from the mitochondria to the cytoplasm. Loss of ABCB7 results in mitochondrial iron accumulation in some cell types. We found that ABCB7 is required for peripheral T cell homeostasis, as CD4-cre ABCB7 cKO have normal thymic T cell development but a severe reduction in peripheral T cells, with a majority of peripheral T cells being recent thymic emigrants. The metal-specific dye Phen Green SK indicated iron accumulation was occurring in the absence of ABCB7. We hypothesized that ABCB7-deficient T cells are eliminated by ferroptosis, an iron-dependent form of regulated cell death that is marked by generation of lipid peroxides. Both Bodipy-C11 and Liperfluo, dyes specific for lipid peroxides, were elevated in ABCB7 cKO T cells. Interestingly, expression of GPX4, the master regulator of ferroptosis, was found to be normal in ABCB7-deficient T cells. A high vitamin E diet, which can reduce ferroptosis in GPX4-deficient T cells, did not restore peripheral T cell numbers in CD4-cre ABCB7 cKO mice. Additionally, treatment with the iron chelator deferoxamine did not restore peripheral T cell numbers. Thus, more than one mechanism may be involved in regulating T cell survival in the absence of ABCB7.
Novel roles for Cullin 3 in T cell-mediated immune responses
Authors: Yarosz Emily L , Chang Cheong-Hee
Published: January 2023

The E3 ubiquitin ligase Cullin 3 (Cul3) has been shown to play a key role in several processes in mammalian cells, including antioxidation, cell cycle regulation, and immunity. Published work has shown that T cell-specific deletion of Cul3 leads not only to a severe defect in natural killer T (NKT) cell development but also to enhanced T regulatory cell and T follicular helper cell differentiation. However, the role of Cul3 in controlling NKT and CD4 T cell proliferation, activation, and homeostasis remains unknown. To begin investigating this, we generated a tamoxifen-inducible CD4 ERT-Cre system to delete Cul3 in mature NKT cells. Surprisingly, Cul3 does not seem to play a role in NKT cell maintenance in either the spleen or the liver, yet activated NKT cells lacking Cul3 proliferate better than wild type cells. We also used a CD4-Cre driven Cul3 knockout mouse model to study CD4 T cell responses. In contrast to our NKT cell studies, the loss of Cul3 in this model severely impacted naïve and effector CD4 T cell frequencies the peripheral tissues. Interestingly, similar to NKT cells, deletion of Cul3 caused naïve CD4 T cells to proliferate better than wild type cells after activation. The proliferative advantage observed in Cul3-deficient naïve CD4 T cells appears to be IL-2 dependent, as these cells display increased IL-2 secretion and IL2Rα surface expression. Preliminary data suggests that major metabolic pathways within the cell may be differentially regulated in NKT and CD4 T cells, and we are currently investigating this possibility further. In all, Cul3 may have unique functions during NKT and CD4 T cell-mediated immune responses, and future work will focus on determining the mechanisms by which Cul3 exerts its effects on these T cell subsets.
Iron in infection and immunity in C. elegans
Authors: Rajan Malini , Anderson Cole P , Romney Steven Joshua , Dos Santos Maria Carolina Ferreira , Gertz Jason , Leibold Elizabeth A
Published: January 2023

Iron is essential for survival of most organisms, but is toxic in excess. All organisms have thus developed mechanisms to sense, acquire and sequester iron. In C. elegans, iron uptake and sequestration are regulated by HIF-1. We previously showed that hif-1 loss of function mutants are developmentally delayed when grown under iron limitation. Here we identify nhr-14, encoding a nuclear receptor homologous to vertebrate HNF4, in a genetic screen conducted for mutations that rescue the developmental delay of hif-1 mutants under iron limitation. NHR-14 is highly expressed in intestinal cell nuclei and in cells in the head, and its subcellular localization and expression are not regulated by iron. Loss of nhr-14 leads to the upregulation of the intestinal metal transporter SMF-3 that increases iron uptake in hif-1 mutants, rescuing the low iron-dependent developmental delay. Loss of nhr-14 also promotes the nuclear localization of the zinc-finger transcription factor PQM-1, which activates smf-3 through the interaction with GATA-like DAF-16-associated elements (DAEs) in the smf-3 promoter. In addition to smf-3, RNA-seq analysis revealed upregulation of innate immune response genes as well as DAF-16/FoxO-suppressed Class 2 genes. Consistent with the RNA-seq results, nhr-14 mutants showed enhanced resistance to the human pathogen Pseudomonas aeruginosa that depends in part on iron uptake as well as the upregulation of innate immune response genes. Our data provide insight into how C. elegans utilizes nuclear receptors to regulate innate immunity and iron availability, and show iron sequestration as an important component of the innate immune response.
Specific Cell-Surface Glycans on Phagocytes Mediate Binding of Pseudomonas aeruginosa
Authors: Sanchez Hector Felipe , Hopkins Daniel , Demirdjian Sally , Gutierrez Cecilia , O’Toole George , Berwin Brent
Published: January 2023

Pseudomonas aeruginosa is a Gram-negative bacterium that, as an opportunistic pathogen, substantially contributes to high morbidity and mortality rates in susceptible individuals such as those with cystic fibrosis or neutropenia. Previous studies identified that the downregulation or loss of bacterial flagellar motility, typically observed within chronic infections, enables bacteria to evade interactions with phagocytic cells that would result in phagocytic uptake. Our recent work demonstrated that exogenous addition of a negatively charged lipid, PIP3, induces binding and phagocytosis of non-motile strains of P. aeruginosa. Based on this work, we hypothesized that the engagement of P. aeruginosa by host innate cells, and subsequent phagocytosis, is mediated by motility-dependent interactions with cell-surface polyanions. We now report that endogenous polyanionic N-linked glycans and heparan sulfate mediate bacterial binding of P. aeruginosa by human monocytic cells. These specific cell-surface interactions result in P. aeruginosa phagocytosis, bacterial type 3 secretion system (T3SS)-mediated cellular intoxication and the IL-1β inflammatory response of the host innate immune cells. Concomitantly, inhibition of host cell N-glycan synthesis reduces T3SS-mediated cytotoxicity and the IL-1β response induced by the bacteria. Importantly, the bacterial interactions with the glycans were motility-dependent and could be recapitulated with purified, immobilized glycans. Therefore, this work describes novel interactions of P. aeruginosa with specific phagocyte cell-surface glycans that modulate relevant host innate immune responses to the bacteria, including phagocytosis, inflammation and cytotoxicity.
IL-15 complex modulates the Plasmodium-specific CD4 T cell response in mice
Authors: Burrack Kristina Stoermer , Dileepan Thamotharampillai , Jenkins Marc K , Frosch Anne E.P.
Published: January 2023

Malaria, which results from infection with Plasmodium parasites, remains a major public health problem with over 200 million cases and nearly 500,000 deaths annually. CD4 T helper cells, especially T follicular helper (Tfh) cells, provide help to B cells to induce effective antibody responses. Although long-lived, sterilizing immunity does not develop in humans following repeated exposure to Plasmodium parasites, protection against symptomatic disease correlates with the acquisition of humoral immunity. The study of parasite-specific CD4 T cell responses has been hampered by the lack of defined parasite-derived T cell epitopes. Utilizing newly identified CD4 T cell epitopes, we generated novel peptide:MHC class II tetramers to track Plasmodium-specific CD4 T cells in mice following infection with multiple rodent Plasmodium strains, including Plasmodium berghei ANKA (PbA), P. berghei NK65 and P. yoelii. Infection of susceptible mice with PbA results in a fatal severe malaria disease. We previously showed that treatment of mice with IL-15 complex (IL-15C; IL-15 bound to an IL-15Rα-Fc fusion protein) induces IL-10 expression in NK cells and protects mice from PbA-induced death. IL-15C treatment results in reduced Ag-specific CD4 T cells on day 6 post-infection but an increased ratio of regulatory T cells to Tfh cells. These data suggest that IL-15C treatment, via its induction of IL-10 from NK cells, modulates the Ag-specific CD4 T cell response, with potential downstream effects on the germinal center reaction and antibody responses. We propose that analysis of the Plasmodium-specific CD4 T cell response will inform the design of therapeutics and vaccine strategies for Plasmodium infection.
Distinct host immune programs in lung macrophages elicit cell-type specific Mycobacterium tuberculosis transcriptional responses
Authors: Delahaye Jared L , Cohen Sara B , Peterson Eliza , Adams Kristin , Plumlee Courtney R , Plaisier Christopher , Baliga Nitin , Urdahl Kevin B
Published: January 2023

During Mycobacterium tuberculosis (Mtb) infection, bacteria are inhaled into the lung where they first infect resident, alveolar macrophages (AM). The majority of the infection remains in AM until D14, when other cells, including recruited monocyte-derived macrophages (MDM) begin to become infected. By the peak of infection (D28) MDM represent the major infected cell type. However, even at this time point, a small population of infected AM remain. This led us to ask how these cell types control Mtb. Using microscopy, we observed that AM harbor more bacteria than MDM on a per cell basis at multiple time points, including after the initiation of the T cell response. Using RNA-seq, we identified multiple differentially expressed pathways between the two cell types. While infected MDM upregulate proinflammatory signaling pathways associated with Mtb control, infected AM are enriched for proliferation and fatty acid metabolism pathways. We performed validation studies using dyes to track cell division and mitochondrial metabolism and observed that AM were more proliferative and had sustained engagement of mitochondrial metabolism relative to MDM. In parallel, we analyzed the bacterial transcriptome and found that Mtb in MDM have a signature associated with late hypoxia. These bacterial responses also suggested that Mtb may differ in its drug tolerance in a cell type specific manner, which we are currently investigating. Together, this work is in agreement with other studies demonstrating differences in the response to Mtb by tissue resident and recruited macrophages and suggests that these differences may lead to distinct transcriptional changes in the bacteria, which could have important implications for therapeutic interventions.
Differential immune cell response to Mycobacterium intracellulare colony morphotypes
Authors: Claeys Tiffany , Clary Gillian , Robinson Richard
Published: January 2023

Mycobacterium intracellulare is an opportunistic pathogen which causes disease in immunocompromised individuals. Like several other nontuberculous mycobacteria (NTM) species, a single strain of M. intracellulare forms bacterial colonies with varying morphologies; however, the relative virulence and immunogenicity of differing M. intracellulare morphotypes are unknown. We have obtained a strain of M. intracellulare isolated from an infected patient and further subcultured it to distinguish the component colony morphotypes present within the parent isolate. To determine whether these distinct colony morphotypes have the ability to stimulate differential immune responses in an infected host, C57BL/6J mice were aerosol infected with either the parent clinical strain or each of the isolated component colony morphotypes. Differential immune response and virulence characteristics of the morphotypes were evaluated by tracking host bacterial load, cytokine expression profiles, and histology. In addition, bone marrow-derived macrophages were isolated and infected in vitro with the different morphotypes and parent isolate. From this study, we have found that the component colony morphotypes are able to differentially activate macrophages and affect the host immune response to infection. These results may establish an important immunological basis for patient clinical outcomes.
Increased cervical neutrophil survival during bacterial vaginosis in Canadian women from the THRIVE study
Authors: Farr Christina , Noel-Romas Laura , Birse Kenzie , De Leon Marlon , Kratzer Kateryna , Perner Michelle , Ayele Hossaena , Wiwchar Cobi , Pymar Helen , Berard Alicia R , Poliquin Vanessa , Burgener Adam
Published: January 2023

Introduction

Bacterial vaginosis (BV), characterized by Lactobacillus depletion and replacement by facultative or anaerobic bacteria, impacts up to 30% of women and associates with negative reproductive health outcomes. We used a systems immunology approach to investigate cellular and molecular inflammation associated with BV.

Methods

Matched cervical cytobrush and cervicovaginal lavage were collected from BV− (n=16) and BV+ (n=11) women. Cytobrushes were immunophenotyped by flow cytometry and lavages were analyzed by tandem mass spectrometry. Differences in immune cell levels were evaluated with Mann-Whitney U tests, and assessed against host and bacterial proteins using Spearman’s Rank correlations.

Results

There were no clinical or demographic differences between BV+ and BV− women, including age (range 22–49), birth control use and STI history. Proteomic analysis identified 1085 human proteins and 516 bacterial proteins from 14 genera, including Lactobacillus, Gardnerella, and Mobiluncus. Long living neutrophils (CD49d+) were significantly increased in BV+ women (p=0.0005), and associated with cell adhesion (p=4.04E-5), T cell mediated immunity (p=0.004), and regulation of myeloid differentiation (p=8.63E-16) pathways. CD49+ neutrophils correlated positively with Mobiluncus (p=0.040, R2=0.49) and negatively with Lactobacillus (p=0.035, R2=−0.498).

Discussion

This data indicates that cervical neutrophil survival is increased in women with BV. These cells associated with anaerobic bacteria and molecular pathways of immune activation. Studies are ongoing to delineate the relationship between BV-associated bacteria and neutrophil functionality, which may have implications for BV treatment.
Crosstalk between skin homing innate T cells and epithelial cells via cholesterol byproduct messengers is required for tissue immunity
Authors: Frascoli Michela , Miu Bing , Ferraj Enxhi , Lee Jisu , Esposito Kelsey , Spidale Nicholas , Cowan Jennifer , Bhandoola Avinash , Kang Joonsoo , Reboldi Andrea
Published: January 2023

Innate T cells and innate lymphoid cells (ILCs) in the mucosal tissues are essential for both barrier tissue fitness and immunity against pathogens. γδTCR+ lymphocytes that secrete IL-17 (Tγδ17) localize to dermis and are critical regulators of skin immune responses. Despite their functional importance, how Tγδ17 cells mediate their unique function in the skin is largely unknown. Tγδ17 cells express the two prototypic G-protein coupled receptors (GPCRs) CCR6 and GPR183 (EBI2) that marks most type 3 cytokine (IL-17 and IL-22) producing lymphocytes. Mice lacking both receptors had significantly diminished dermal Tγδ17 cells and were resistant to psoriasis induction. The deficit in dermal Tγδ17 cells in Ccr6−/−Gpr183−/− double knock-out (DKO) mice was attributed to a block in their maturation in the thymus. EBI2 recognizes oxysterols generated by the cholesterol hydroxylase CH25H and functional oxysterols were detected in both the thymus and skin. Analysis of Ch25h-reporter mice showed that a subset of immature medullary thymic epithelial cells (mTEC) is the depot for oxysterols. We confirmed this finding by single cell transcriptome analyses of TECs. These results demonstrate that developing Tγδ17 cells must be conditioned by sensing cholesterol byproducts synthesized by a discrete subset of mTECs. This conditioning is likely to be required to position Tγδ17 cells in specific niches in the skin where oxysterols are generated. Given that CH25H expression can be regulated by infection and inflammation, this sensory circuit directed by cholesterol is predicted to program skin innate T cells’ ability to rapidly survey for perturbations in the skin.
Skin-resident natural killer T cells induce cutaneous allergic inflammation in atopic dermatitis
Authors: Park Chang Ook , Sun ZhengWang , Kim Ji Hye , Lee Kwang Hoon , Kupper Thomas S
Published: January 2023

Natural killer T (NKT) cells are unconventional T cells that bridge innate and adaptive immunity. NKT cells have been implicated in the development of atopic dermatitis (AD). Here, we demonstrate that CXCR4 and its cognate ligand CXCL12 are significantly up-regulated in human AD skin in global transcriptomic analysis; proteomic analysis revealed that CXCR4+ NKT cells are enriched in AD skin and are consistently elevated in our AD mouse model. Adoptive transfer of allergen-specific NKT cells confer antigen-specific cutaneous inflammation, and predominant skin NKT cells are CXCR4+ and CD69+, similar to tissue-resident memory T (TRM) cells. Skin-resident NKT cells uniquely expressed CXCR4, which was confirmed using a parabiosis system. Intravital imaging showed that CXCR4+ NKT cells preferentially trafficked to a CXCL12-rich area, forming an enriched CXCR4+ NKTRM/CXCL12+ cell cluster, which developed acute and chronic allergic inflammation. CXCR4+ NKTRM cells may form a niche to develop AD, where CXCL12 is highly expressed.
Transcriptomic, phenotypical, and functional characterization of human liver sinusoidal CD69+CD4+ T cells
Authors: Koh June-Young , Han Ji Won , Rha Min-Seok , Park Jun Yong , Joo Dong Jin , Park Su-Hyung , Shin Eui-Cheol
Published: January 2023

Tissue-resident memory T (TRM) cells are non-circulating T cells that rapidly respond to the local antigen stimulation. Human liver CD8+ T cells have been reported to have phenotypes of TRM and respond to hepatotropic viruses. However, characteristics of human liver CD4+ T cells are still unclear. Here, we examined transcriptomic, phenotypical, and functional characteristics of human liver CD4+ T cells by analyzing liver sinusoidal mononuclear cells (LSMCs) from liver perfusates of healthy donors obtained during liver transplantation. The liver sinusoidal (LS) CD4+ T cell population had a significantly higher frequency of CD69+ cells than the peripheral blood (PB) counterpart. However, CD103 was barely expressed in the both populations. In transcriptomic analysis, LS CD69+CD4+ T cells were distinctly clustered and showed tissue-resident features. In flow cytometry analysis, we found that the LS CD69+CD4+ T cell population had significantly higher frequencies of CXCR6+, LFA-1+, or CD49a+ cells than the PB CD4+ T cell population and the LS CD69−CD4+ T cell population, confirming that LS CD69+CD4+ T cells have tissue-resident phenotypes. Moreover, LS CD69+CD4+ T cells were mostly PD-1+ effector memory cells, which is consistent with the characteristics of TRM cells. In functional analysis following anti-CD3 stimulation, we found that LS CD69+CD4+ T cells had a higher capacity for IFN-γ production than LS CD69− CD4+ T cells. In addition, TH1-associated gene sets were enriched in the LS CD69+CD4+ T cells. In summary, these findings indicate that LS CD69+CD4+ T cells are TRM-like cells with TH1 effector functions. Roles of LS CD69+CD4+ T cells in various liver diseases need to be further elucidated.
Immunosuppressive therapy of immune-mediated GI damage may impair epithelial regeneration
Authors: Arnhold Viktor , Takashima Shuichiro , Vinci Paola , Kuttiyara Jason , Egorova Anastasiya , Calafiore Marco , Hanash Alan
Published: January 2023

Glucocorticoids (GC) are a standard therapy of immune-mediated gastrointestinal (GI) damage. However, there is little understanding of the effects that GC use may have on intestinal epithelium and its regenerative function. Using ex vivo organoid models of immune-mediated intestinal injury, we sought to investigate direct effects of GC on intestinal epithelium and test the impact of GC on cytokine- and T-cell-mediated effects. GC suppressed proliferation of small intestine (SI) organoids, reducing organoid size and decreasing cell cycle progression of Lgr5+ intestinal stem cells. Consistently, GC treatment of C57BL/6J mice suppressed epithelial proliferation, decreasing crypt height and reducing the percentage of Ki67+ crypt cells in vivo. We next investigated the role of GC-mediated epithelial suppression in the setting of tissue damage, modeling immune-mediated GI pathology by culturing organoids in the presence of IFNγ and by co-culturing organoids with allogeneic T-cells. Culture with GC in addition to IFNγ significantly increased the toxicity of IFNγ leading to reduced efficiency of organoid formation. Consistently, GC pretreated organoids were more susceptible to allogeneic T-cells with reduced formation efficiency. Finally, we studied cytokine-mediated epithelial regeneration in the context of GC treatment. Culturing organoids with both GC and IL-22 indicated that IL-22 could promote regeneration of intestinal epithelium in the presence of GC. Taken together, our findings indicate that GC, which are necessary clinically for suppressing pathologic inflammatory immune responses, may have direct effects on the epithelium potentially impairing protective regeneration responses and augmenting toxicity.
Dissecting the cellular responses of immune cells and Lgr5+ stem cells in the inflamed mouse colon with the RNAscope in situ hybridization technology
Authors: Anderson Courtney M , Li Na M , Todorov Courtney , Dikshit Anushka , Ma Xiao-Jun
Published: January 2023

Due to its exposure to a harsh luminal environment, the intestinal epithelium has a remarkably fast turnover rate that is facilitated by a resident intestinal stem cell (ISC) population present at the base of the intestinal crypt. These ISCs, marked by the GPCR Lgr5, allow the intestinal epithelium to adapt to different types of damage, such as inflammation. Chronic intestinal inflammation is a hallmark of the inflammatory bowel diseases (IBD) yet the interplay between inflammatory immune cells and ISCs remains to be elucidated. Therefore, we utilized the single-molecule RNA in situ hybridization (ISH) technology RNAscope to visualize the expression of multiple immune and ISC markers within the inflamed intestinal tissue environment. To interrogate the expression pattern of inflammatory immune cell and ISC markers within the intestinal crypt, we performed the assay on colons from either control or TNBS-treated mice. We visualized the location of each intestinal cell population, including the resident Lgr5+ ISC population, within the crypt. The impact of inflammation on the Lgr5+ ISC population, as well as the Wnt/β-catenin pathway, was also examined. Using a multiplex assay, we assessed infiltration of regulatory T cells and Th17 cells in the inflamed region. Lastly, we examined the expression of several receptor-ligand pairs for cytokines and ISC markers. Taken together, these results demonstrate the ability of the RNAscope assay to visualize the ISCs within the morphological context of the intestinal crypt and in relationship to inflammatory immune cells. This work may help understand mechanisms behind the pathogenesis of IBD and other inflammatory diseases and also aid in the development of potential therapies.
Immune Activation of the Blood Brain Barrier and Implications for Neuroinflammation in Schizophrenia
Authors: Crockett Alexis Mary , Ryan Sean K. , Vasquez Adriana Hernandez , Canning Caroline , Kanyuch Nickole , Ceja Guadalupe , Gesualdi James , Kebir Hania , Kapoor Richa , Benallegue Nail , Anderson Stewart A. , Alvarez Jorge I.
Published: January 2023

Neuroimmune dysregulation has been implicated in various neuropsychiatric disorders, including schizophrenia (SZ). Multiple studies indicate peripheral inflammation, including increased serum IL-6 and circulating Th17 cells, and neuroinflammation in the context of psychosis. Reports that the blood brain barrier (BBB), the highly specialized CNS endothelial cells, is disrupted in patients suggest that vascular alterations contribute to neuroimmune dysfunction in SZ. Therefore, we investigated whether the BBB is intrinsically compromised in the most common genetic risk factor for SZ, the hemizygous deletion of chromosome 22q11.2 (22qDS). We employed an innovative approach based on the differentiation of human induced pluripotent stem cells (iPSCs) from 22qDS patients with SZ into BBB-like endothelium and further substantiated our findings in vivo in a mouse model of 22qDS. We demonstrate that barrier function and immune privilege properties of the BBB are compromised in 22qDS, as we found compromised expression of the tight junction protein claudin-5 and upregulation of the cell adhesion molecule ICAM-1 (CD54). To assess the immunological consequences of such imbalances, we determined the impact of the BBB on transmigrating immune cells and found that the 22qDS BBB supported leukocyte migration and activation. Furthermore, we observed increased astrocyte activation and IL-6 expression in 22qDS mice and post-mortem 22qDS patients, suggesting that compromised BBB may promote astrocyte-mediated neuroinflammation in 22qDS. These results indicate that 22qDS is associated with changes in the BBB, and suggest that the BBB may play a role in the increased risk for neuropsychiatric disease in 22qDS patients.
CCR9+ dendritic cells promote the differentiation of Foxp3+ regulatory CD4 T cells
Authors: Lal Girdhari , Pathak Manisha , Halder Namrita , Padghan Priyanka
Published: January 2023

Chemokine receptor CCR9 is known to play an important role in the migration of immune cells to the gut. Intestinal epithelial cells under gut inflammation or in inflammatory bowel disease (IBD) produce several folds higher CCL25, the only known ligand for CCR9, and drive the recruitment of CCR9+ immune cells. Due to its gut tropism activity, CCR9 and CCL25 are suggested as a potential therapeutic target. Still, many of the clinical trials targeting CCR9/CCL25 did not give a positive outcome; instead, they increase the severity of the disease. The non-chemotactic function of CCR9 in the gut inflammation and immunity is not known.

Using dextran sodium sulfate (DSS)-induced gut inflammation model in C57BL/6 mice, we showed that CCR9+ DCs, specifically CD11b−CD103+ DCs recruited in high frequency to the gut and gut-associated lymphoid tissues (GALT) in DSS treated mice as compared to control group. These CCR9+ DCs showed lower MHC II and CD86 molecules and had higher regulatory surface markers (FasL and Latency-associated peptide, LAP) in the GALT. Further, we demonstrate that thymic stromal lymphopoietin (TSLP) produced by CCR9+ DCs but not IL-10 or TGF-β, promotes the differentiation of Foxp3+ Treg. Furthermore, adoptive transfer of CCR9+ DCs in C57BL/6 mice promoted Tregs but reduced the Th17 cells in the GALT, and also suppressed the ovalbumin-specific gut allergic immune response. Together, these results suggest that CCR9+ DCs have a regulatory function and can be explored as an adoptive cellular therapy to control the gut inflammation and allergic immune response.
GPR120 regulates neutrophil functions and intestinal homeostasis
Authors: Zhou Zheng , Yang Wenjing , Yu Yanbo , Zhao Xiaojing , Yu Yu , Bilotta Anthony J , Yao Suxia , Cong Yingzi
Published: January 2023

GPR120 (also known as free fatty acid receptor 4, FFAR4) has been identified recently as a bona fide receptor for long-chain fatty acids (LCFA), and has a critical role in various physiological homeostasis mechanisms such as adipogenesis. However, if and how GPR120 regulates intestinal homeostasis is still largely unknown. In this study, we found that GPR120−/− mice showed more severe colitis induced by DSS compared to WT mice, which was evident as increased weight loss and higher histopathology scores. Depletion of neutrophils with anti-Ly6G increased the colitis severity, indicating protective roles of GPR120 and neutrophils in the intestines from inflammation. GPR120 agonist, CpdA, promoted neutrophil production of IL-17 and IL-22, but not TNF-α and TGF-β expression. Consistently, GPR120−/− neutrophils showed impaired IL-17 and IL-22 production compared with WT neutrophils. Additionally, treatment with CpdA and LCFA Omega-3, DHA, enhanced neutrophil production of reactive oxygen species (ROS). Furthermore, CpdA increased antimicrobial peptide S100A9, but not S100A8, production in neutrophils. Mechanically, inhibition of mTOR pathway by mTOR inhibitors, rapamycin and AZD8055, suppressed neutrophil IL-22 and IL-17 production induced by CpdA, while it did not affect the GPR120 agonist induction of ROS. Additionally, inhibition of glycolysis by glycolysis inhibitor 2-DG suppressed GPR120 agonist-induced IL-17 but not IL-22 and ROS production in neutrophils, indicating mTOR and glycolysis differentially regulate GPR120 induction of IL-17, IL-22 and ROS. Taken together, our results highlight a critical role of GPR120 in regulating neutrophil functions, thus contributing to the maintenance of intestinal homeostasis.
Quantitative immune profiling of human tumor tissues with multiplexed ChipCytometry
Authors: Northcutt Adam J , Christians Arne , Forys Jason T , Campbell Thomas D , Winkeler Crystal L
Published: January 2023

Objective

Many immune cell profiling methods have demonstrated limits on the number of biomarkers that can be reliably assayed from the same cell; to remedy this, we present here ChipCytometry, an imaging technology for quantitative immune profiling of cells and sectioned tissues.

Methods

ChipCytometry is a fluorescence-based imaging system that utilizes multiplexed immuno-fluorescence staining in combination with high-dynamic-range (HDR) imaging to facilitate quantitative phenotyping of individual cells within tissue samples. Employing this technology to profile metastatic and primary tumors, multiple biomarkers were stained in iterative multiplex assays to profile single cells with an AI-powered cell segmentation algorithm. This immune profiling was completed on 6 different cryosectioned human tumor tissues (head & neck, liver, lung, breast, colon, and pancreas).

Results

ChipCytometry allowed for the quantification of both tumor and immune cells in all 6 of the human tumor tissues that were analyzed, revealing the diversity of T cells (helper, cytotoxic, regulatory, naïve/effector/memory), B-cells, granulocytes, monocytes, dendritic cells, and natural killer cells present in the tumor microenvironment. The resolution, dynamic range, and AI-powered cell segmentation offered by ChipCytometry allowed for quantification of 15 immune cell types at the single-cell level. The data show exact counts of each specific cell type, relative percentages within each tissue, and the precise spatial distribution of each cell.

Conclusion

These results demonstrate the potential of ChipCytometry to broadly probe a diverse set of human tumor tissue types and acquire a deep immunological cell type profile.
ImmuneSpace enables multicohort immune correlates analysis
Authors: Kim Ju Yeong , Henrich Evan , Miller Helen , Jiang Mike , Wagner Jake , Finak Greg , Gottardo Raphael
Published: January 2023

ImmuneSpace is a data repository and analysis platform of the Human Immunology Project Consortium (HIPC), a multi-center collaborative effort to characterize the status of the immune system in different populations under diverse stimulations and disease states. This ongoing effort has generated large amounts of varied high-throughput, high-dimensional immunological data. ImmuneSpace directly extracts data from ImmPort -- the central NIAID database, and further standardizes it for meta-analysis and reporting, making it ready to be explored using state-of-art visualization and analysis tools. ImmuneSpace will act as a central immunological hub, allowing experimentalists, statisticians, and bioinformaticians to freely retrieve, explore and compare data across assays and across studies generated within and outside of HIPC. We present a selection of multi-cohort analyses using standardized gene expression and flow cytometry data sets across multiple studies identifying novel baseline predictors of vaccine efficacy across a variety of pathogens.