bluuebunny/crossref_metadata_2025 huggingface dataset:
Hepatocytes engulf and rapidly silence coxsackievirus, protecting the host against systemic viral pathology
Authors: Kimura Taishi , Flynn Claudia T , Whitton J Lindsay
Published: January 2023

Liver has many biological functions, including (but by no means limited to) the removal, from the blood, of various pathogenic organisms. This phenomenon has been best-studied for bacteria, and is thought to be mainly mediated by Kupffer cells and sinusoidal endothelial cells; these cells, too, have been implicated in clearance of some viruses. However, to date, hepatocytes - which constitute ~90% of the liver’s biomass - have been thought to be bystanders in this activity. Herein we show that hepatocytes directly protect the host against type B coxsackieviruses (CVBs) by engulfing, then rapidly silencing, these pathogens. Novel CARHEP KO mice, which carry a hepatocyte-specific deletion of the coxsackievirus-adenovirus receptor (CAR), are unable to capture circulating CVB3, leading to a dramatic early rise in blood-borne virus, followed by markedly-elevated morbidity, mortality, and increased viral load. The enhanced pathogenicity in CARHEP KO mice occurs despite the production of abundant type I interferon (T1IFN), which is derived from plasmacytoid dendritic cells (pDCs). In addition, we show that in genetically-intact (wild type; WT) mice, the rapid hepatocyte silencing of trapped virus is dependent on IRF1; in its absence, CVB3 undergoes efficient productive replication in hepatocytes and, as a result, virus titers in all tested extrahepatic tissues are several logs higher than in WT animals. In summary, hepatocytes act as a trap for CVBs, internalizing the viruses and reducing systemic viral burden, and thereby protecting the host from systemic disease.
ThG cells: a distinct T helper cell subset expressing GM-CSF, TNF, and IL-2, but not Th lineage-specific cytokines
Authors: Rasouli Javad , Casella Giacomo , Zhang Weifeng , Xiao Dan , Zhang Guang-Xian , Ciric Bogoljub , Rostami Abdolmohamad
Published: January 2023

The diversity of human antigen-experienced (CD45RA−) CD4+ T cells in the blood has not been fully elucidated. Using single cells RNA-seq and high-dimensional single-cell mass cytometry (CyTOF), we identified eight distinct populations of CD45RA−CD4+ T cells in peripheral blood of healthy donors. In addition to Tregs and established Th lineages, we also identified a distinct population of T cells that expressed GM-CSF, but lacked expression of master transcription factors and signature cytokines of other Th cell lineages. These cells, which we tentatively designated “ThG”, co-expressed GM-CSF, IL-2, TNF, IL-3 and CCL20. ThG cells had a stable phenotype and were highly encephalitogenic. Similar to Th17 cells, ThG cells switched their phenotype to Th1 in response to IL-12. T-bet was not required for ThG development, but it was required for their encephalitogenicity. Analogous to other Th cells, ThG cells had a tendency to eventually cease GM-CSF expression without other major changes in their phenotype, which clearly opens the possibility that GM-CSF− ThG cells actually exist.
The effects of α-MSH on epidermal γδ T cell activation and function
Authors: Darby Taylor , Jameson Julie M
Published: January 2023

The hair follicle is a site of immune privilege that, when disrupted, results in the autoimmune disorder alopecia areata. Although there are a variety of cell types that reside near the hair follicle and may be involved in the initiation of alopecia areata, few studies have focused on γδ T cells. γδ T cells are resident in epithelial tissues where they normally play roles in inflammation and wound healing. Alpha-melanocyte-stimulating(α-MSH) is an immunoregulator locally produced within the hair follicle. α-MSH ensures the immune system is suppressed during anagen phase (growth phase) and restored during catagen phase (resting phase). The effects of α-MSH on epidermal γδ T cells via MC1R, one of five melanocortin receptors, is unknown. We therefore purified primary epidermal γδ T cells to investigate the immunomodulatory effects of α-MSH. Flow cytometry and the Luminex platform were used to compare changes in MC1R expression levels and cytokine production in response to α-MSH. Here we show that resting epidermal γδ T cells express MC1R, and TCR stimulation increases MC1R expression. Upregulation of MC1R expression is augmented further by stimulation with α-MSH. Epidermal γδ T cells significantly upregulate immunostimulatory cytokines when stimulated with anti-CD3, but cytokine production did not significantly change with the addition of α-MSH. Additional findings suggest that α-MSH instead plays a role in MC1R regulated pathways such as DNA repair and cell survival. This study begins to delineate the mechanisms by which α-MSH impacts γδ T cell function and leads to the disruption of immune privilege that occurs in alopecia areata.
Decoding patterns of macrophage TNF production to model NF-κB dynamics and cell-cell communication
Authors: Cruz J. Agustin , Gottschalk Rachel A. , Lee Robin E.C.
Published: January 2023

Immune and stromal cells communicate through cytokines to initiate intracellular signaling, gene expression, and a multifaceted immune response. Cytokine signaling coordinates the dynamics of intracellular pathways that regulate inflammation. Immune and stromal cells both produce and respond to tumor necrosis factor-alpha (TNF), mediating inflammation and enabling communication between these compartments. The inflammatory role of TNF, activating the nuclear factor-κB (NF-κB) signaling and mitogen-activated protein kinase (MAPK) pathways, is well known. However, the effect of varying the time and amplitude patterns of TNF production in neighboring immune and stromal cells has not been systematically studied. Moreover, little is known about how time and amplitude patterns of an inflammatory signal input impact the nature of inflammatory responses. To study this relationship between production and response to TNF and its role in the dynamics of TNF-dependent NF-κB and MAPK signaling, we are using a systems immunology approach. Specifically, we are experimentally determining the patterns of TNF produced by human macrophages stimulated with heat-killed bacteria and characterizing the effect of these TNF stimulation patterns in the dynamics of NF-κB and MAPK responses in human cell lines. Additionally, we will use these experimental results to calibrate our deterministic 2 feedback with competitor (D2FC) mathematical model and model the dynamics of NF-κB signaling. Findings from this research will likely apply to other cytokine and signaling pathways, contributing to understanding the integration of time varying cytokine stimuli.
De novo T cell development in humans favors the early formation of memory phenotype cells: insights from SCID-X1 patients treated with gene therapy
Authors: Boi Shannon Kathryn , Abdelsamed Hossam Aly , Mamcarz Ewelina ,   LVXSCID-ND Clinical Group, Gottschalk Stephen , Youngblood Benjamin A
Published: January 2023

We recently reported that treatment of SCID-X1 infants (X-linked severe combined immunodeficiency) with autologous bone marrow expressing the corrected IL2RG resulted in reconstitution of T, B, and NK cells (Mamcarz et al., NEJM. 2019). Longitudinal observation of these patients undergoing immune reconstitution affords the unique ability to study human T cell development in real time. Accordingly, we observed that during the first few weeks of reconstitution, T cells egressing early into the peripheral blood were primarily demarcated by CD45RO+ expression, a memory phenotype. After establishing memory-like cells, CCR7+CD45RO− naive T cells appeared, and both populations continued to increase in absolute counts over time. Phenotypic analysis of CD45RO+ cells demonstrated an enrichment of NKG2A+ memory-like phenotype (MP) T cells. Moreover, epigenetic analysis by DNA methylation profiling of CD45RO+ cells exhibited a hybrid state of naïve and effector programs among the memory population. Culture of isolated MP T cells in IL-12/IL-18 from SCID-X1 patients resulted in expression of IFNg in an antigen-independent manner. Collectively, these data suggest that MP T cells develop early in humans and are endowed with the capacity to rapidly elicit effector cytokines in an antigen-independent manner.
Monoallelic and monogenic Tcrb assembly enforced by Vβ recombination signal sequences and physiological roles for Tcrb repertoire and allelic exclusion
Authors: Wu Glendon S , Bassing Craig
Published: January 2023

Monoallelic expression of antigen receptor (AgR) genes is assumed to be critical for the proper selection and function of T and B lymphocytes. V(D)J recombination of Tcrb, Igh, and Igk loci is regulated such that most cells assemble and express functional AgR genes from one allele (allelic exclusion). The V gene segment recombination signal sequences (RSSs) of Tcrb and Igh loci are weak and have been proposed to mediate monoallelic V-to-DJ recombination. To test the role of Vβ RSSs in promoting Tcrb allelic exclusion, we created and studied mice harboring replacements of the DJCβ-distal V2 and -proximal V31 RSSs with the stronger 3′Dβ1 RSS. Here, we show a substantial role for weak Vβ RSSs in limiting: Vβ recombination frequency, biallelic functional Vβ-to-DJβ recombination and biallelic TCRβ expression, and dual TCRβ chain expression encoded by a single Tcrb allele. Our data indicate that weak Vβ RSSs limit Vβ recombination to promote monogenic Tcrb assembly in the time window before feedback inhibition halts Vβ recombination. Given that AgR allelic exclusion is most stringently applied to Tcrb and Igh, whose assembly and expression drives cellular proliferation, we hypothesized that mechanisms directing monoallelic induction of RAG double strand breaks (DSBs) evolved at Tcrb and Igh loci in part to suppress DSBs from entering S phase and forming oncogenic AgR translocations. We find that p53D/D mice with V2 and V31 RSS replacements succumb more rapidly to thymic lymphomas than their p53D/D counterparts, which is consistent with our model. As Vβ RSS replacement mice exhibit dramatic shifts in the Vβ repertoire, we also have begun using them to test relationships between Vβ repertoire and host defense.
Thymic epithelial miR-155 promotes regulatory T cell development through safeguarding medullary thymic epithelial cell maturation
Authors: Dong Jiayi , Warner Lindsey M. , Lin Ling-Li , Chen Mei-Chi , O’Connell Ryan M , Lu Li-Fan
Published: January 2023

During thymocyte development, medullary thymic epithelial cells (mTECs) provide appropriate instructive cues in the thymic microenvironment for not only negative selection but also the generation of regulatory T (Treg) cells. Here, we identify that miR-155, a microRNA whose expression in Treg cells has previously been shown to be crucial for their development and homeostasis, also contributes to thymic Treg (tTreg) cell differentiation by promoting mTEC maturation. Mechanistically, we show that RANKL stimulation induces expression of miR-155 to safeguard the thymic medulla through targeting multiple known and previously uncharacterized molecules within the TGFβ signaling pathway, which is recognized for its role in restricting the maturation and expansion of mTECs. Our work uncovers a miR-155-TGFβ axis in the thymic medulla to determine mTEC maturity and, consequently, the quantity of tTreg cells and suggests that miR-155 ensures proper tTreg cell development in both cell-intrinsic and -extrinsic manners.
In vitro and in vivo modulation of Hematopoietic Stem Cell (HSCs) differentiation into T and B cells by poly dispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNTs)
Authors: MIA MD. BABU , SAXENA RAJIV KRISHNA
Published: January 2023

Background

AF-SWCNTs are known to have significant effect on the immune function. There is not much information available on the effect of carbon nanotubes (CNTs) on the differentiation of the HSCs. In the present study we have focused on studying the modulatory effects of AF-SWCNTs on the differentiation of HSCs into T & B cells.

Methods

Single-walled CNTs were acid-functionalized by treating with concentrated sulfuric & nitric acid; thus, tagged with Alexa Fluor 633 to get fluorescent AF-SWCNTs (FAF-SWCNTs). To study internalization, magnetic beads enriched C57bl/6 mouse bone marrow (BM) HSCs (Lin− Sca1+cKit+) were incubated with FAF-SWCNTs & studied using flow cytometry & confocal microscopy. HSCs were cultured with a cocktail of cytokines to induce differentiation to T & B cells, in (+/−) AF-SWCNTs & monitored by live cell imaging. For in vivo ontogenesis study, mice were lethally irradiated (gamma irradiation) & reconstituted using syngeneic BM cells pretreated (+/−) AF-SWCNTs. At different time points, appearance of T & B cells in the spleen was monitored.

Results

Our results indicate that HSCs internalized significant amounts of FAF-SWCNTs in vitro that remained essentially localized in cytoplasm. Differentiation of cytokines stimulated HSCs to T & B cell was significantly suppressed by AF-SWCNTs. Results of our in vivo experiments indicated that after 17th day of BM transplantation, significant numbers of T & B cells appeared in the reconstituted mouse spleen. However, when AF-SWCNTs pretreated BM cells used to reconstitute irradiated mice, significant decline in both of their percentage & absolute recovery was noted. These results indicate that CNTs may significantly modulate the differentiation of HSCs into lymphocytes.
Redox regulation of autophagy in thymic stromal cell function
Authors: Semwal Manpreet Kaur , Hester Allison K , Xiao Yangming , Udeaja Chioma , Venables Thomas , Griffith Ann V
Published: January 2023

T lymphocytes develop in the thymus, where mutually inductive signaling between lymphoid progenitors and thymic stromal cells (TSCs) directs the progenitors along a well-characterized program of differentiation. However, the biology of the TSCs comprising the lymphopoietic thymic microenvironment remains relatively under-characterized because stromal cells are rare and difficult to isolate. Using a deconvolution technique to study gene expression essentially in situ, we previously identified a deficiency in the H202 quenching enzyme catalase (CAT) in TSCs, and found that CAT deficiency results in high H202 levels in this population, eventually leading to thymic atrophy. Our current studies address the possibility that high H202 levels serve physiological functions in TSCs in the young, steady state thymus. TSCs exhibit high basal levels of autophagy at the steady state, which is critical for self-antigen presentation and T cell selection. The mechanisms governing high basal autophagy in TSCs are unknown, however autophagy is induced by many stressors, including high H202 levels. Our data indicate that catalase overexpression targeted to mitochondria in transgenic mice (mCAT Tg) results in diminished autophagy in TSCs and causes diminished negative selection in the thymus, eventually leading to autoimmunity. The effects on negative selection in mCAT Tg mice are rescued by increased basal autophagy on the beclin 1 knock-in (Becn1F121A/F121A) background. Our results suggest that the high basal autophagy level required in TSCs for T cell negative selection is promoted by physiologically low levels of catalase expression in the steady state thymus.
A transition from MYC to MYCL dominance identifies early specification events during the development of IRF8-dependent dendritic cell subsets
Authors: Anderson David A , Murphy Theresa L , Murphy Kenneth M
Published: January 2023

Early work defined the common dendritic cell progenitor, or CDP, as a clonogenic population that gives rise to pDCs, cDC1s, and cDC2s. It was recently shown that pDC and cDC1 specification can occur within the CDP. Defining phenotypic and transcriptional heterogeneity within the CDP can thus be used to refine our understanding of the gene regulatory network that controls DC development. Using scRNA-Seq and in vivo reporter analysis, we identified populations with mutually exclusive expression of Myc and Mycl. We found that Myc-expressing cells develop into all DC subsets when cultured in Flt3L. Alternatively, Mycl-expressing cells develop into cDC1s and pDCs, but not cDC2s. Therefore, we asked whether Mycl expressing CDPs represent a clonogenic, bipotent progenitor of cDC1s and pDCs. Consistent with work that showed sustained expression of Mycl in differentiated populations of pDCs and cDC1s, we were able to exclude pDC and cDC1-specified populations on the basis of subset-specific marker expression. However, a population of Mycl-expressing CDPs that did not express subset specific markers remained. When purified and cultured in vitro, this rare population gives rise to both pDCs and cDC1s. These results indicate that Mycl expression marks a putative, common progenitor of pDCs and cDC1s that excludes cDC2 potential. These results are consistent with recent work that demonstrated a shared dependence of pDC and cDC1 specification on a distal enhancer of Irf8. Along these lines, we find that the transition from MYC to MYCL dominance in pDCs is disrupted in Irf8−/− mice. We have thus identified Myc and Mycl as targets of the gene regulatory network that controls DC development.
Interleukin-15 bridges astrocyte-microglia crosstalk and exacerbates brain injury following intracerebral hemorrhage
Authors: Shi Samuel , Li Yu-Jing , Shi Kaibin , Wood Kristofer , Ducruet Andrew F , Liu Qiang
Published: January 2023

Background and Purpose

Microglia are among the first cells to respond to intracerebral hemorrhage (ICH), but the mechanisms that underlie their activity following ICH remain unclear. Interleukin (IL)-15 is a proinflammatory cytokine that orchestrates homeostasis and the intensity of the immune response following central nervous system inflammatory events. The goal of this study was to investigate the role of IL-15 in ICH injury.

Methods

Using brain slices of ICH patients, we determined the presence and cellular source of IL-15. A transgenic mouse line with targeted expression of IL-15 in astrocytes was generated to determine the role of astrocytic IL-15 in ICH. The expression of IL-15 was controlled by a glial fibrillary acidic protein promoter (GFAP-IL-15tg). ICH was induced by intraparenchymal injection of collagenase or autologous blood.

Results

In patients with ICH and wild type mice subjected to experimental ICH, we found a significant upregulation of IL-15 in astrocytes. In GFAP-IL-15tg mice, we found that astrocyte-targeted expression of IL-15 exacerbated brain edema and neurological deficits following ICH. This aggravated ICH injury in GFAP-IL-15tg mice is accompanied by increased microglial accumulation in close proximity to astrocytes in perihematomal tissues. Additionally, microglial expression of CD86, IL-1β, and TNF-α is markedly increased in GFAP-IL-15tg mice following ICH. Furthermore, depletion of microglia using a colony stimulating factor 1 receptor inhibitor diminishes the exacerbation of ICH injury in GFAP-IL-15tg mice.

Conclusions

Our findings identify IL-15 as a mediator of the crosstalk between astrocytes and microglia that exacerbates brain injury following ICH.
Mechanisms of anti-NMDAR Antibody Mediated Damage to the Fetal Brain
Authors: Carroll Kaitlin , Kowal Czeslawa , Diamond Betty
Published: January 2023

Mothers with systemic lupus erythematosus (SLE) have an increased frequency of children with learning disorders and autism spectrum disorder (ASD) which associates with maternal anti-DNA antibodies, indicating that in utero exposure to autoantibodies may lead to neurodevelopmental alterations. A subset of anti-dsDNA antibodies present in 40% of SLE patients has been shown to cross-react with the N-methyl D-aspartate receptor (NMDAR) excitatory neuronal receptor. Immunizing mice with these antibodies (DNRAbs) causes morphological neurodevelopmental alterations and persistent behavioral and neurocognitive abnormalities in male offspring, as well as the death of a high proportion (~40%) of female fetuses due to their earlier expression of the GluN2A NMDAR subunit. The brains of the fetuses from DNRAb+ dams display increased neuronal apoptosis and thinned cortical plates, indicating alterations in neuronal migration. Male offspring of DNRAb+ dams exhibit delayed reflex acquisition, cognitive impairments in fear extinction and novel object recognition, and decreased overall cortical volume. These results parallel the substantially higher proportion of male children born to women with SLE, as well as the higher rate of developmental disorders in these children. Current studies are addressing the mechanisms of this antibody-mediated damage and whether these offspring exhibit characteristics of ASD.
A new rapid titration protocol for lamotrigine with reduced risk of skin rash
Authors: Jang Yoonhyuk , Moon Jangsup , Son Hyoshin , Lee Woo-Jin , Sunwoo Jun-Sang , Lee Soon-Tae , Park Kyung-Il , Jeon Daejong , Chu Kon , Lee Sang Kun
Published: January 2023

Introduction

Lamotrigine is one of the most widely used antiepileptic drugs, but it has a critical issue of a skin rash if the starting dose is too high or the escalation rate is too rapid. We investigated the efficacy and safety of a novel and rapid titration protocol for lamotrigine that takes only 11 days to reach a daily dose of 200 mg.

Methods

We prospectively enrolled 33 adult patients (age 18–85) who were diagnosed with epilepsy and started lamotrigine administration for the first time at a single tertiary hospital. Our new protocol starts with a subthreshold dose of the drug and then administer a stepwise-incremental dose until reaching the full therapeutic dose within 11 days.

Results

Of 29 patients analyzed, only two (6.9%) experienced idiosyncratic skin rash before the first follow-up visit at 2 weeks (±3 days). In addition, a therapeutic concentration was reached in more than 75% of studied patients after 2 weeks of lamotrigine administration

Discussion

These findings demonstrate the value of the novel tolerance induction protocol for lamotrigine, which could widen the available application of lamotrigine in various situations.
Effects of human LDLR overexpression on apoE-related tau pathology and brain dysfunction
Authors: wang chao , Li Aimin , Spellman Rebecca , Bao Xin , Scott Nathan , Manis Melissa , Finn Mary Beth , Xiong Monica , Serrano Javier Remolina , Sullivan Patrick , Holtzman David
Published: January 2023

ApoE4 is the strongest genetic risk factor for late-onset Alzheimer Disease. It has been reported that apoE isoforms directly affect tauopathy and tau-mediated neurodegeneration in P301S tau transgenic mice expressing different human apoE isoforms, with apoE4 resulting in markedly increased tau-mediated neurodegeneration and the absence of apoE being marked protective against neurodegeneration. In the brain, low-density lipoprotein receptor (LDLR) is one of the main apoE receptors that regulates apoE levels, but LDLR has very few identified ligands compared to other apoE receptors. LDLR overexpression in the brain can dramatically lower apoE and Aβ levels, as well as decrease Aβ accumulation and deposition. Therefore, we propose to evaluate whether AAV-mediated overexpression of human LDLR (hLDLR) is an efficient way to reduce apoE levels, tau pathology, and neurodegeneration. To study the effect of LDLR overexpression on apoE4-related tau pathology and neurodegeneration, P301S Tau/ApoE4 (TE4) male mice received bilateral intracerebroventricular injection with AAV expressing hLDLR or GFP-control, starting before the onset of tau pathology development. Brains were isolated after 9 month, divided into hemispheres, and analyzed by histological and biochemical techniques. Widespread expression of hLDLR throughout the brain was observed and apoE levels were significantly decreased. Furthermore, mouse nest-building impairment was rescued after hLDLR overexpression. Strikingly, there is a significant decrease in brain atrophy, phosphorylated tau deposition in TE4 mice overexpressing hLDLR. In summary, these data suggest that LDLR may be a promising target to lower apoE levels and decrease neurodegeneration.
RGC-32 regulates astrocyte differentiation during experimental autoimmune encephalomyelitis
Authors: Tatomir Alexandru , Boodhoo Dallas , Nguyen Vinh , Cudrici Cornelia , Badea Tudor Constantin , Rus Violeta , Rus Horea
Published: January 2023

Astrocytes are increasingly recognized as critical contributors to multiple sclerosis pathogenesis. We have previously shown that lack of response gene to complement 32 (RGC-32) alters astrocyte morphology in spinal cords during experimental autoimmune encephalomyelitis (EAE), suggesting a role for RGC-32 in astrocyte differentiation. In addition, we found that RGC-32 regulates TGF-β-induced extracellular matrix production and growth factors expression. We investigated if the lack of RGC-32 alters the expression of the glial fibrillary acidic protein (GFAP) and of astrocytes progenitor markers vimentin and fatty acid binding protein 7 (FABP7) during EAE. Immunohistochemical analysis of spinal cords during the acute phase of EAE (day 14 post EAE induction) showed that the elongated, radial glial cell-like astrocytes from RGC-32 knock-out (KO) mice expressed higher levels of vimentin and FABP7 as compared to wild type (WT) mice, confirming their immature phenotype. In addition, we found that the density of white matter GFAP+ astrocytes was higher in WT as compared to KO mice (p=0.02). Using double staining immunohistochemistry for GFAP and connective tissue growth factor (CTGF), known to be involved in astrocyte differentiation, we found a lower number of CTGF+ astrocytes during EAE in spinal cords of RGC-32 KO when compared with WT mice (p=0.002). We next purified neonatal astrocytes from WT and RGC-32 KO mice, and we found significantly lower levels of GFAP and CTGF mRNA and protein and higher levels of vimentin in RGC-32 KO astrocytes when compared with WT astrocytes. The expression pattern of astrocytic progenitor markers and that of CTGF suggests that RGC-32 is an important regulator of astrocyte differentiation during EAE.
Phenotype-driven screening of 150 strains of mice for allergic lung inflammation identified strains representative of heterogeneous human asthma cohorts
Authors: Howard Emily Diane , Maazi Hadi , Hurrell Benjamin P , Galle-Treger Lauriane , Helou Doumet Georges , Jahani Pedram Shafiei , Painter Jacob , Allayee Hooman , Akbari Omid
Published: January 2023

Asthma is a highly prevalent and heterogeneous disease characterized by airway hyperreactivity (AHR) and an influx of immune cells including eosinophils and neutrophils in the bronchoalveolar lavage (BAL). Furthermore, there are patient subsets, such as severe asthmatics, who are unresponsive to corticosteroids or bronchodilators. Research efforts targeting these cohorts of asthma patients however are severely lacking due to an inadequacy in relevant biological mouse models. For the first time, we immunophenotyped 150+ inbred and RI mouse strains in the Hybrid Mouse Diversity Panel (HMDP) under steady state conditions and after house dust mite (HDM) challenge to assess the lung function, BAL cell composition, serum immunoglobulin levels, as well as quantification of lung and peripheral immune cells. These results revealed at least three unique groups of strains that, when exposed to the same allergen, respond with three distinct systemic and local phenotypes of lung inflammation, specifically a high Th-2 response, a low Th-2 response, and neutrophilia. These murine models of AHR mirror human cohorts of asthma previously underrepresented in ongoing research efforts. We are currently designing mechanistic studies focusing on identifying the genetic factors responsible for these distinct phenotypes. The results of these combined studies may lead to a better understanding of asthma heterogeneity and provide a foundation for further elucidation of the pathogenetic mechanisms of each phenotype of asthma utilizing relevant animal models.
Transdermal sensitization and oral elicitation of anaphylaxis to salt-soluble wheat protein in an adjuvant-free mouse model of food allergy
Authors: Gangur Venugopal , Gao Haoran , Jorgensen Rick , Othman Aqilah , Raghunath Rajsri , Wade Labrawn , Chandra Shivam , Ng Perry KW
Published: January 2023

Occupational exposure to wheat proteins in the baking industry is linked to a variety of allergic diseases including atopic dermatitis, allergic rhinitis, allergic conjunctivitis, and asthma. Mechanisms of occupational hypersensitivity to wheat are incompletely understood at present. For example, the role of route of exposure to wheat proteins in an occupational setting that results in sensitization and wheat allergic disease is largely unknown. Here, we tested the hypothesis that exposure to salt-soluble wheat protein (SSWP) extract via the skin, in the absence of external adjuvant, can cause sensitization for oral wheat-induced anaphylaxis in mice. Balb/c mice were produced and maintained on a wheat-free diet during this study. Groups of mice were exposed to SSWP prepared from a durum wheat, without adjuvant, using a novel transdermal exposure (TDE) method. Mice were sensitized with SSWP by nine weekly exposures. In both males and females, significant and robust wheat-specific IgE antibody responses were observed by the 6th TDE. Anaphylaxis upon oral challenge with SSWP after the 9th TDE was quantified by measuring elevation of plasma MMCP-1, which is a specific immune marker of IgE-mediated anaphylaxis in mice. Sensitized mice showed significant elevation of MMCP-1, confirming IgE-mediated anaphylaxis. These data demonstrate for the first time that skin exposure to SSWP, without an adjuvant, can clinically sensitize mice for oral SSWP-induced anaphylaxis.
Obesity alters the immune landscape of the small intestine and exacerbates food allergy
Authors: Schmiechen Zoe C , Fredrickson Gavin , Robert Sacha , Barrow Fanta , Revelo Xavier
Published: January 2023

In the past few decades, higher rates of obesity have coincided with a rising number of individuals affected by allergic disease. Our study objective was to determine if obesity alters the immune cell landscape of the small intestine towards an inflammatory environment that aggravates the development of food allergy. Obesity was induced by feeding male BALB/c mice a western diet for 8 weeks. To induce food allergy, mice were epicutaneously sensitized and orally-challenged with OVA. Compared with lean controls, sensitized obese mice showed a lower body temperature after challenge and increased OVA-specific IgE in serum, suggesting an exacerbated allergic response. Mass cytometry analysis of the intestinal lamina propria revealed that OVA-sensitized obese mice had decreased T regulatory cells, which are critical for the oral tolerance to food allergens. In the mesenteric lymph node, obese mice showed increased frequencies of T follicular helper cells (Tfh) compared with lean controls. Tfh cells are required for the production of allergen-specific IgE, and implicated in allergic disease pathogenesis. To further define the transcriptional mechanisms underpinning the increased inflammatory and allergic responses during obesity, we performed single-cell RNA sequencing of more than 28,000 barcoded CD45+ enriched lamina propria cells from lean and obese mice, with and without OVA sensitization. We provide a transcriptional map of inflammatory cells and define a module of gene expression that distinguishes immune cell-driven allergic responses in the obese intestine. Overall, our findings highlight obesity-induced inflammatory changes in the intestine that may mediate the increased food allergic sensitization during obesity.
MicroRNA-155 promotes cardiac inflammation, fibrosis, and impairs heart function during Pseudomonas aeruginosa infection
Authors: Rajaram Murugesan , Pestrak Matthew , Murphy Nathaniel , Wu Qian , Lubbers Ellen , Dellos-Nolan Sheri , Saljoughian Noushin , Yu Lianbo , Lafuse William P , Ganesan Latha P. , Mohler Peter , Wozniak Daniel
Published: January 2023

Pneumonia in ICU patients carries a bleak prognosis with high risk for cardiac dysfunction, largely due to cardiac inflammation with subsequent tissue remodeling and fibrosis. Growing evidence indicates that resident macrophages are important regulators of cardiac electrical activity and damage following myocardial infarction. After tissue injury, inflammatory monocytes are recruited to the heart and differentiate into macrophages, which contribute to cardiac dysfunction. MicroRNAs have a key role in the regulation and maintenance of macrophage function and cardiac inflammation. However, it is unclear if microRNAs regulate immune cell recruitment or the function of cardiac resident macrophages. To investigate how bacterial pneumonia promotes cardiac dysfunction, we used P. aeruginosa infection in a murine pulmonary model of infection. Our results show an aberrant cardiac electrical activity, and compromised pump function in P. aeruginosa infected animals. Notably, we found increased cardiac fibrosis mainly due to the accumulation of inflammatory macrophages in the heart. Additionally, we found that miR-155 deficiency reversed the P. aeruginosa mediated cardiac dysfunction by limiting the immune cell migration in to the heart. These findings demonstrate that P. aeruginosa infection induces miR-155 dependent immune cell recruitment to the heart, which changes the phenotype and function of resident macrophages, resulting in elevated cardiac fibrosis and heart function.
A butyrate-producing bacterium Clostridium butyricum prevents Clostridioides difficile infection independent of GPR43/109a
Authors: Hayashi Atsushi , Kamada Nobuhiko
Published: January 2023

Short-chain fatty acids, such as butyrate, are microbial metabolites that harbor bioactivities beneficial to the gastrointestinal health. Clostridium butyricum MIYAIRI588 (CBM) is a bacterium that produces a robust amount of butyrate and therefore used as a live biotherapeutic product in clinical settings. Clostridioides difficile (C. diff) is a bacterium that causes life-threatening diarrhea and colitis. The gut resident microbiota plays a critical role in the prevention of C. diff infection (CDI), as the disruption of the healthy microbiota by antibiotics greatly increases the risk of CDI. In this study, we examined how CBM protects the host from CDI, the mechanism which is not yet completely understood.

Mice were pre-treated with cefoperazone to disrupt the gut microbiota and then infected with the C. diff VPI10463 strain to induce CDI. CBM treatment significantly improved body weight loss and mortality caused by CDI and increased neutrophils in colonic lamina propria (cLP) in the early phase of CDI. The protective effect of CBM was canceled when neutrophils were depleted by an anti-Ly6G antibody, suggesting that neutrophils may play a key role in the process. The administration of tributyrin, which elevates the concentration of butyrate in the colon, increased neutrophils in the cLP, indicating that increased butyrate produced by CBM regulates the neutrophil pool. Since it has been reported that butyrate-GPR43 signaling is crucial for neutrophils migration, we next examined the effect of CBM on CDI in GPR43/109a double knockout mice. Unexpectedly, the protective effect of CBM was retained in these mice. These results indicated that CBM prevents CDI possibly through butyrate but independent of GPR43/109a-mediated signaling.
DDX3X Cellular Valency Allows Stressed Cells to Make a Live or Die Cell Fate Decision by Regulating Stress Granules and the NLRP3 Inflammasome
Authors: Samir Parimal , Kesavardana Sannula , Kanneganti Thirumala-Devi
Published: January 2023

Cells of the immune system have to respond to a variety of stressors to help maintain organismal homeostasis. Stress sensing can lead to the assembly of a cytoplasmic membraneless compartment called stress granules (SGs) that helps the cells survive. Cellular stress can also lead to activation of the NLRP3 inflammasome and induction of pyroptosis – a pro-inflammatory programmed cell death. Thus SGs and the NLRP3 inflammasome provide contrasting cell fate choices to a stressed cell – cell survival or pyroptosis. DDX3X is an integral component of SGs [1]. We show that DDX3X promotes the NLRP3 inflammasome activation and ASC speck assembly[2]. Our data suggests that induction of SGs inhibits the NLRP3 inflammasome activation by sequestering the DDX3X molecules. It also suggests that SGs and the NLRP3 inflammasome compete for DDX3X molecules, which makes DDX3X cellular valency critical for this decision making process[3]. Using LPS mediated peritonitis model, we further show that DDX3X promotes and SGs inhibit the NLRP3 inflammasome activation in vivo. Our data points towards a mechanistic paradigm where cells can exploit the cellular valency of biomolecules and a competition between biological processes for common essential factors to make cell fate decisions.
GITR expressed on macrophages functions as an inflammatory amplifier by promoting NLRP3 inflammasome activation
Authors: Liang Siping , Liu Yiting , Wu Yongjian , Ming Siqi , Wu Minhao
Published: January 2023

Glucocorticoid-induced tumor necrosis factor receptor (GITR) is an important co-stimulatory molecule on lymphocytes, however, its role on innate immunity and inflammatory response remains unclear. Our in vivo and in vitro data showed that bacterial infection up-regulated GITR expression in mouse macrophages. Treatment with agonistic anti-GITR Ab (DTA-1) enhanced, while knockout of GITR reduced the macrophage pyroptosis after bacterial infection. Furthermore, ELISA, Western blot and immune-fluorescence staining data indicated that GITR promoted NLRP3 inflammasome activation by inducing NLRP3 deubiquitination and ASC oligomerization, without affecting the priming stage. More importantly, we found that GITR expression was enhanced on peripheral monocytes/macrophages of sepsis patients vs healthy donors. Using cecal ligation and puncture (CLP) model of sepsis, we demonstrated that GITR triggered NLRP3 inflammasome activation to exacerbate disease progression, while this effect was reversed by treatment with specific inhibitor for NLRP3 or Caspase-1. Overall, our study demonstrated that GITR expressed on macrophages functions as an inflammatory amplifier by promoting NLRP3 inflammasome activation. These findings give a better understanding of GITR in innate immune response, which may also provide potential therapeutic targets of infectious and inflammatory diseases.
Cell-type specific role of non-canonical inflammasome during LPS septic shock
Authors: Kumari Puja , Russo Ashley , Rathinam Vijay
Published: January 2023

Cytosolic sensing of LPS through caspase-11 is the key step of non-canonical inflammasome activation and innate immune defense against Gram-negative bacterial infections. Upon LPS sensing, caspase-11 induces GSDMD-mediated pore formation in the plasma membrane, followed by maturation and secretion of caspase-1 and pro-inflammatory cytokines (IL1band IL18). Excessive pore formation on the membrane leads to pyroptotic death of cell and release of intracellular proteins; many of which exacerbate the immune responses to infection, leading to cytokine storm, tissue damage, organ failure, and death. Caspase-11 sensing of LPS occurs in various cell-types, however the contribution of these cell-types to non-canonical inflammasome mediated pyroptosis, cytokine release, and lethality during LPS septic shock is poorly understood. To understand the cell type-specific role of caspase-11 during LPS septic shock, caspase-11 was conditionally deleted in multiple cell-types including macrophages by utilizing caspase-11 floxed mice and several appropriate Cre strains. The conditional KOs of caspase-11 were subjected to LPS challenge and various responses were assessed. Our in vivostudies showed cell-type specific contribution of caspase-11 to intracellular LPS-induced responses such as gasdermin D activation and lethality. Particularly, myeloid cell-intrinsic expression of caspase-11 is required for pathological manifestations of intracellular LPS sensing. Overall, our findings provide new insights into the cell type-specific role of cytosolic LPS sensing.
Mitochondria-targeted Sulfide Delivery Molecules – New and Novel Players that can Suppress and Reverse Cigarette Smoke-induced Inflammasome Activity
Authors: Hansbro Nicole G , Pacitti Dario , Brown Alexandra , Torregrossa Roberta , Balachandran Lois , Kumar Vinod , Wood Mark , Haw Tatt-Jhong , Scotton Chris , Whiteman Matt , Hansbro Philip
Published: January 2023

RATIONALE

Cigarette smoke (CS) is the major risk factor in development of chronic obstructive pulmonary disease (COPD). Interventions that can prevent and/or reverse disease are urgently needed. Hydrogen sulfide (H2S) is generated in mitochondria (mt) and crucial in maintaining mt respiration, suppressing oxidative stress/inflammation. Lung H2S levels are reduced after CS exposure. Lung inflammation, mitochondrial damage and oxidative injury are exacerbated as a result of inhibition/silencing of H2S enzymes, suggesting impairment of H2S synthesis/loss of bioavailability is detrimental in COPD and negatively impacts mitochondrial health.

METHODS

We have produced novel mt-targeted H2S donors (mtH2SD) AP39 and RT01 to investigate whether these molecules could suppress and/or reverse CS-induced inflammation and lung injury. To investigate suppression, mice were exposed to CS (or air) for 8 wks (with 1.0 mg/kg). To investigate reversal, mice were exposed to CS for 8 wks followed by either 4 wks rest or continued CS exposure, each with mtH2SD (1.0 mg/kg). Airway inflammation (BALF differential cell counts, IL-1β by ELISA) and lung function were assessed.

RESULTS

Lung H2S levels were reduced and inflammasome activity increased in response to CS exposure. mtH2SD significantly suppressed CS-induced alveolar destruction, fibrosis and improved lung function. mtH2SD treatment reversed CS-induced lung neutrophil, eosinophil and macrophage infiltration, loss of lung function, and partially reversed airway resistance in both models.

CONCLUSIONS

Targeting H2S to mitochondria may be a novel therapeutic approach to prevent and/or reverse mitochondria-driven inflammation and lung injury in COPD and related diseases.
Effect of tobacco smoke on respiratory syncytial virus-induced high-mobility group box 1-mediated innate antiviral signaling
Authors: Hosakote Yashoda Madaiah , Kempaiah Kempaiah Rayavara , Kurosky Alexander
Published: January 2023

Exposure to tobacco smoke (TS), either direct or secondhand, is associated with an increase in the severity of lower respiratory tract infections, including bronchiolitis, mainly caused by respiratory syncytial virus (RSV). No vaccine or effective treatment is available for RSV. Although the mechanism of TS-induced RSV severity remains largely unknown, lung immune responses are key. High Mobility Group Box-1 (HMGB1) is a multifunctional nuclear protein that is released to the extracellular space during infection and regulates inflammatory responses as damage-associated molecular pattern. The goal of this study is to dissect the role of HMGB1 in the activation of antiviral signaling in RSV and TS-induced airway inflammation. Studies were conducted in airway epithelial cells, including A549 and normal small alveolar epithelial cells. We show that RSV and TS-induced oxidative stress post-translationally modifies HMGB1 including hyperacetylation, phosphorylation, and oxidation, leading to its enhanced extracellular secretion from lung epithelial cells. Secreted HMGB1 activates signaling cascades to activate p38 MAPK and NF-κB to induce proinflammatory mediator release from immune cells and induce inflammation. We also show that hmgb1 knockdown exacerbates inflammation in RSV-infected and TS-exposed cells and that treatment with recombinant HMGB1 activates immune cells to induce an inflammatory response, promotes the activation of antiviral pathways and inhibits viral replication. Our studies suggest an important role for HMGB1 in TS-induced RSV pathogenesis and guide the development of novel therapeutic strategies against respiratory infections that are caused or exacerbated by environmental toxicants such as TS.
FIP200 is essential for RIG-I-mediated innate immune signaling
Authors: Li Shitao , Wang Lingyan , Song Kun , Patil Girish , Hao Wenzhuo , Wu Yakun
Published: January 2023

Retinoic acid-inducible gene I (RIG-I) is a cytosolic sensor for recognition of viral double-stranded RNA (dsRNA) and 5′ triphosphate RNA, which induces a robust production of type I interferon (IFN). Following RNA recognition, the caspase activation and recruitment domain (CARD) of RIG-I is released from the repressor domain and subsequently oligomerized to activate downstream signal cascades. Although these sequential steps for RIG-I activation are well established; however, the regulatory mechanism is not well elucidated. In this study, we found that the FAK family kinase-interacting protein of 200 kDa (FIP200) interacted with the CARD domain of RIG-I. Ectopic expression of FIP200 activated RIG-I. By contrast, knockout of FIP200 impaired RIG-I signaling, but not other innate immune signaling pathways, in fibroblasts and macrophages. In vivo study showed FIP200 knockout mice were more susceptible to VSV, but not HSV-1, infection due to the reduced innate immune responses, including type I IFN. Mechanistic analyses found that FIP200 promoted the release of the CARD by competing with the repressor domain of RIG-I. Furthermore, FIP200 formed dimers via its C-terminal tail, which facilitated RIG-I oligomerization and subsequent activation. Taken together, our study defines FIP200 as a new innate immune signaling molecule and facilities RIG-I activation.
Regulation of germinal center response by proinflammatory chemokine CCL3 and follicular regulatory T cells
Authors: Grigorova Irina , Ke Fang , Benet Zachary
Published: January 2023

Follicular regulatory T cells (Tfrs) have been reported to play multiple roles in the control of B cell responses. Tfr repress foreign antigen-specific B cells and plasma cells at the peak of germinal center (GC) response. At the same time they promote GC B cell cycling in IL10-dependent fashion and support optimal affinity maturation. Ex vivo Tfrs can act directly on both Tfh and GC B cells. However, relative contribution of direct Tfr-mediated control of Tfh cells vs GC B cells in vivo has been unclear. Our previous work suggested that GC B cell-intrinsic production of proinflammatory chemokine CCL3 promoted direct GC B cell contacts with Tfr cells and was important for modest negative regulation of B cells at the peak of GC response. In this follow up study we have addressed the role of CCL3 in the long-term regulation of GCs. We have also assessed, which subset of GC B cells upregulates production of CCL3 and which receptors on Tfr cells may respond to this chemokine. We found that in addition to the role of CCL3 in the modest repression of foreign antigen-specific B cells at the peak of GC response, it was also important for longer-term participation of B cells in GCs. Both the negative and positive regulation of GC B cells by CCL3 was dependent on the presence of Tregs. We also found that only about 10% of GC centrocytes that were Myc-positive upregulated expression of CCL3 and that Tfr chemotaxis to CCL3 chemokine ex vivo was dependent on both CCR5 and CCR1 chemokine receptors. Further studies are underway to determine the role of Tfr cells in the negative and positive CCL3-mediated regulation of GCs.