bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular mechanisms of gasdermin D autoinhibition and recognition by inflammatory caspases
Authors: Xiao Tsan Sam , Liu Zhonghua , Yang Jie , Wang Chuanping , Yang Rui 
Published: January 2023

Abstract
Pyroptosis is an inflammatory form of programmed cell death that plays important roles in immune protection against infections and in inflammatory disorders. Gasdermin D (GSDMD) is an executor of pyroptosis upon cleavage by caspases-1/4/5/11 following canonical and noncanonical inflammasome activation. GSDMD N-terminal domain assembles membrane pores to induce cytolysis, whereas its C-terminal domain inhibits cell death through intramolecular association with the N domain. The crystal structures of the human and murine GSDMD C-terminal domains differ from those of the full-length murine GSDMA3 and the human GSDMB C-terminal domain. Mutations of GSDMD C-domain residues predicted to locate at its interface with the N-domain enhanced pyroptosis, in agreement with the role of the GSDMD C-terminal domain as an autoinhibition domain. We further demonstrate that the full-length GSDMD and its cleavage site peptide, FLTD, can directly bind the catalytic domains of inflammatory caspases. A GSDMD-derived inhibitor, N-acetyl-Phe-Leu-Thr-Asp-chloromethylketone (Ac-FLTD-CMK), inhibits GSDMD cleavage by caspases-1, -4, -5, and -11 in vitro, as well as suppresses pyroptosis downstream of both canonical and noncanonical inflammasomes. By contrast, the inhibitor does not target caspase-3 or apoptotic cell death, suggesting that Ac-FLTD-CMK is a specific inhibitor for inflammatory caspases. The present study not only contributes to our understanding of the distinct mode of GSDMD autoinhibition and recognition by inflammatory caspases, but also reports a specific inhibitor for these caspases that can serve as a tool for investigating inflammasome signaling.
Bond tension on neutrophil LFA-1 regulates membrane calcium flux from the outside-in
Authors: Morikis Vasilios Aris , Jo Myung Hyun , Ha Taekjip , Simon Scott I 
Published: January 2023

Abstract
Recruitment of neutrophils in appropriate numbers at vascular sites of inflammation is critical for innate immune surveillance. LFA-1 adhesive bonds that support neutrophil deceleration and arrest under fluid shear, also function to mechanotransduce signals that spatially localize calcium flux necessary to guide transendothelial migration. We set out to characterize how bond tension acting on high-affinity LFA-1/ICAM-1 bonds transduce intracellular signaling in a manner proportional to the shear force acting on adherent cells. A sequential set of events beginning with a shift to high affinity of LFA-1 that binds ICAM-1, a buildup of tension, and assembly of adaptor proteins that terminate in spatial linkage to the CRAC channel Orai-1. Utilizing microfluidic vascular mimetic flow chamber, we observed that force catalyzed integrin clustering and calcium flux in a Kindlin-3 dependent manner. Kindlin-3 has a dual role as a mechanical anchor that links to adaptors that assemble Orai-1 mediated calcium influx and as a support for LFA-1 membrane clustering. RACK1 deficient neutrophils retained the capacity for integrin clustering but lacked calcium signaling. Utilizing a molecular platform that regulates the force acting on LFA-1 bonds we determined the characteristic bond tension necessary to transduce signaling. Forces between 33 and 54 pN were sufficient to support LFA-1 binding to Kindlin-3 and promote integrin clustering. As this characteristic force completed a macromolecular complex constituted of RACK1/Orai1 signaling complex competent to transduce calcium influx. We identify a molecular circuit catalyzed by a well-defined force that may represent the necessity to assemble Kindlin-3 to intracellular LFA-1 domains.
Siglec-1 negatively regulates TLR4-mediated inflammatory response by uniquely controlling Src phosphorylation at Ser17
Authors: Yang Darong , Wu Yin , Chen Guo-Yun 
Published: January 2023

Abstract

Introduction
Sepsis are mainly caused by Gram-negative bacteria, and Toll-like receptor 4 (TLR4) sensing of LPS plays pivotal roles in the pathogenesis of sepsis. Identifying key regulators that govern LPS-induced TLR4 activation of inflammatory pathway will facilitate the understanding of the regulatory mechanisms of inflammation and provide potential targets for sepsis therapy.

Results
LPS induced robust Ser17-phosphorylation (dispensable for kinase activity) of Src kinase in RAW 264.7 and THP1 cells. Silencing of Siglec-1, a membrane-bound lectin, dramatically abrogated Src phosphorylation at Ser17 induced by LPS in RAW 264.7 cells, whereas did not affect Src phosphorylation at Tyr416 and Tyr527, two critical sites that determine Src kinase activity. Furthermore, the phosphorylated levels of P38, NF-kB, JNK, ERK, Syk and PKC triggered by LPS remained comparable between scramble shRNA-transduced and Siglec-1 shRNA-transduced Raw 264.7 cells. Strikingly, LPS failed to induce Ser17-phosphorylation of Src kinase in Siglec-1−/− BMDMs compared with WT BMDMs; no defects on Tyr416 and Tyr527 sites were observed. RAW 264.7 cells with Src depletion showed enhanced cytokine production by LPS treatment. Reconstitution of WT Src and Src-S17D mutant, but not Src-S17A mutant, rescued the elevated inflammatory cytokines in Src knockdown cells with LPS challenge. Co-IP identified a novel interaction between Siglec-1 and Src, and over-expression of DAP12 enhanced the interaction in 293T cells.

Conclusion
Siglec-1 negatively regulates TLR4-mediated inflammatory response by uniquely controlling Src phosphorylation at Ser17. Pharmacological targeting Ser17 in Src may provide promising strategies to fend off sepsis.
H3K4me3 Profiles of LPS and Immune Complex-Activated Macrophages
Authors: Palaga Tanapat , Ruenjaiman Vichaya , Leu Yu-Wei 
Published: January 2023

Abstract
Macrophages play an important role in immune response such as inflammation, phagocytosis, metabolism and tissue homeostasis. Lipopolysaccharide (LPS)-stimulated macrophages produces pro-inflammatory cytokines such as TNFα, IL-6 and IL-12 while repeated stimulation with LPS can lead to unresponsive state called LPS tolerance. Stimulation with LPS in the presence of immune complex (IC) drastically changes gene expression profiles of macrophages. LPS/IC-stimulated macrophages produce high level of IL-10 and low level of IL-12. In this study, we investigated the plasticity between LPS-stimulated and LPS/IC-stimulated macrophages and compared active histone marks (trimethylation on lysine 4 on histone 3 (H3K4me3)) in LPS-stimulated macrophages and LPS/IC-stimulated macrophages using bone marrow derived murine macrophages by the ChIP-seq approach. Stimulation with LPS that was followed immediately by LPS/IC did not result in IL-10 production. Defects in activation of p38, p44/42 Erk and NF-kB p65 were observed. In contrast, the resting of 2 days between LPS and LPS/IC yielded IL-10hiIL-12lo phenotype. Our ChIP-seq data suggested that the global H3K4me3 profiles differed between these LPS- and LPS/IC-stimulated macrophages. Some key genes that are uniquely upregulated in LPS/IC-stimulated macrophages were found to contain H3K4me3 in the regulatory region. Taken together, LPS/IC-stimulated macrophages exhibits unique H3K4me3 profiles and our results may help explain the plasticity of LPS vs. LPS/IC-stimulated macrophages.
Cross-talk between MDP/NOD2 and GPIbα/Mac-1 pathways controls production of PGE2 and of IL-1b and IL-6 cytokines in human monocytes activated with MDP adjuvant
Authors: Zaitseva Marina , Liu Fengjie , Endo Yukinori , Romantseva Tatiana , Wu Wells W , Shen Rongfong , Golding Hana 
Published: January 2023

Abstract
Adjuvants are included in vaccine formulations to activate antigen presenting cells (APC). In some cases, strong activation of APC by adjuvants may induce excessive release of inflammatory substances causing adverse reactions in vaccine recipients. Here we investigate the mechanism of production of prostaglandin E2 (PGE2), a proximal mediator of fever, and of pyrogenic cytokines IL-1β and IL-6 in human monocytes activated with muramyl dipeptide (MDP) adjuvant. MDP was associated with fever and reactogenicity in rabbits and humans.
In monocytes, MDP alone induced nuclear NFkB but did not induce PGE2. Addition of conditioned medium obtained from CD3 bead-purified human T cells (TCM) to MDP resulted in production of PGE2, IL-1β and IL-6, suggesting a need for a second signal. The co-stimulatory factor in TCM was identified as Glycoprotein Ib alpha (GPIbα) protein by mass spectrometry. Western blotting confirmed increased GPIbα in TCM due to shedding of GPIbα from CD3 bead-purified T cells. Antibodies against GPIbα and against the GPIbα receptor, Mac-1 integrin (aMβ2), inhibited secretion of PGE2 and IL-1β/IL-6 in monocytes activated with MDP and TCM. The contribution of Mac-1 to inflammatory response to MDP in vivo was further confirmed by showing reduced expression of COX-2 mRNA in the liver and spleen in β2 KO and aM KO mice following administration of MDP compared with wild type mice. In summary, we describe for the first-time production of GPIbα by T cells and a two-signal model of PGE2 production by monocytes: MDP-induced activation of NFkB represent Signal 1 and Signal 2 is transmitted from GPIbα-activated Mac-1. Further study of T cell-monocyte nexus might help in the assessment/prediction of inflammatory potential of novel adjuvants.
Identification of age-related changes in chromatin accessibility and gene expression in T cells from thymus to periphery
Authors: achour achouak , Chen Guobing , Sharov Alexei , Nguyen Thomas , Li Xiang , Patrick Michael , Wood William , De Supriyo , Becker Kevin , Peng Weiqun , Weng Nan-Ping 
Published: January 2023

Abstract
Aging of immune system is characterized by progressive decline of physiological and cellular function of T cells, leading to a reduced immune function in the old. Recent studies of naïve T cells from young and old mice have identified age-related altered gene expressions, but the mechanisms underlying age associated changes of naïve T cells remain poorly understood. Here, we compared chromatin accessibility and the transcriptome in mature thymocytes and in naïve T cells of spleen between young (6–8 weeks) and old (95–114 weeks) C57BL/6 mice, using Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) and Agilent microarray methods, respectively. We identified age-related changes of chromatin accessibility that are correlated with the gene expression changes, providing a chromatin basis of age-related changes in gene expression. Interestingly, some age-related changes of CD4 and CD8 T cells in thymus did not retain in the spleen, and others appeared later only in the spleen, suggesting different tissue environment may contribute to the age-related changes of T cells. To directly address environmental effects, we currently examine changes of naïve T cells from old mouse before and after hosted in young mouse for 21 days in chromatin accessibility, transcriptomes, and protein expression. Collectively, these approaches will allow us to dissect the time and environmental influences in naïve T cell function in old mice. Our findings will have important implications both for better understanding the mechanisms underlying these age-related changes as well as opening potential new targets to mitigate the age-related reduced T cell functions.
Dose-Dependent Requirements for ETS1 in Invariant Natural Killer T (iNKT) Cell Development
Authors: Morgan Roxroy C , Kee Barbara L 
Published: January 2023

Abstract
iNKT cells are innate-like T cells that use an invariant T cell receptor to recognize glycolipids presented by the non-classical major histocompatibility protein CD1d. Upon recognition, iNKTs rapidly produce cytokines that stimulate innate cells to produce cytokines and prime adaptive cells for activation upon antigenic stimulation. Studies in Ets1 germline-deficient mice showed that the transcription factor ETS1 is required for the development of CD4+ NK1.1+ NKT cells. Development of CD1d tetramers and the recent elucidation of multiple effector iNKT cell subsets has improved our understanding of NKT cell development and function, and revealed that CD4+ NK1.1+ iNKT cells are a minor subset of total NKTs. Using conditional deletion for Ets1 in thymocytes, we show that Ets1 is a dose dependent regulator of all iNKT cells and appears to be particularly important for development of interferon-g-producing iNKT1 cells. Deletion of Ets1 in developing iNKT1 cells using Tbx21Cre demonstrated the critical role of ETS1 in this iNKT cell effector subset. Our findings set the stage for future studies focused on the molecular mechanism by which ETS1 controls iNKT cell development and function.
Peroxisome Proliferator-activated Receptor gamma (PPARγ) suppresses inflammation and bacterial control during influenza bacterial super-infection
Authors: Gopal Radha , Mendy Angelico , Marinelli Michael A , Richwalls Lacee J , Seger Philip J , Patel Shivani , McHugh Kevin J , Rich Helen E , Grousd Jennifer A , Forno Erick , Alcorn John F 
Published: January 2023

Abstract
Influenza is one of the most common causes of respiratory illness worldwide and can be complicated by secondary bacterial pneumonia, which is a common cause of mortality. Influenza-induced “cytokine storm” is thought to play a role in lung pathogenesis during influenza infection. PPARγ is a member of nuclear hormone receptor superfamily that is known to enhance lipid and glucose metabolism, cellular differentiation and suppress the inflammatory immune response. Synthetic PPARγ agonists (thiazolidinedione, rosiglitazone) have been used clinically in the treatment of type II diabetes. In this study, we determined the effect of rosiglitazone in mice infected with influenza or influenza, methicillin-resistant Staphylococcus aureus (MRSA) super-infection. We found decreased influenza viral burden, decreased numbers of neutrophils and macrophages in bronchoalveolar lavage (BAL), and decreased production of IL-6, IL-12, CCL2, and CXCL10 in influenza-infected, rosiglitazone-treated mice when compared to influenza-infected control mice. However, rosiglitazone treatment compromised bacterial control during influenza-bacterial super-infection. Further, we found decreased numbers of neutrophils and decreased gene expression of neutrophil elastase and cathepsin G during super-infection in rosiglitazone-treated mice. Similarly, using data from the National Health and Nutrition Examination Survey (NHANES), we found increased influenza/pneumonia mortality in rosiglitazone-treated patients compared to other diabetic patients (adjusted hazard ratio=3.4, 95% confidence interval=1.1–10.7). Both human and mouse data suggest that rosiglitazone treatment worsens the outcome of influenza-associated pneumonia.
T-cell derived acetylcholine aids host defenses during enteric bacterial infection with Citrobacter rodentium
Authors: Ramirez Valerie Trinidad , Brust-Mascher Ingrid , Gardner Mariana Barboza , Sladek Jessica A. , Gareau Melanie G. , Reardon Colin 
Published: January 2023

Abstract
The regulation of mucosal immune function is critical to host protection from enteric pathogens but is incompletely understood. The nervous system and the neurotransmitter acetylcholine play an integral part in host defense against enteric bacterial pathogens. Here we report that acetylcholine producing-T-cells, as a non-neuronal source of ACh, were recruited to the colon during infection with the mouse pathogen Citrobacter rodentium. These ChAT+ T-cells did not exclusively belong to one Th subset and were able to produce IFNγ, IL-17A and IL-22. To interrogate the possible protective effect of acetylcholine released from these cells during enteric infection, T-cells were rendered deficient in their ability to produce acetylcholine through a conditional gene knockout approach. Significantly increased C. rodentium burden was observed in the colon from conditional KO (cKO) compared to WT mice at 10 days post-infection. This increased bacterial burden in cKO mice was associated with increased expression of the cytokines IL-1β, IL-6, and TNFα, but without significant changes in T-cell and ILC associated IL-17A, IL-22, and IFNγ compared to WT mice. Despite the increased expression of pro-inflammatory cytokines during C. rodentium infection, inducible nitric oxide synthase (Nos2) expression was significantly reduced in intestinal epithelial cells of ChAT T-cell cKO mice 10 days post-infection. Additionally, a cholinergic agonist enhanced IFNγ-induced Nos2 expression in intestinal epithelial cell in vitro. These findings demonstrated that acetylcholine, produced by specialized T-cells that are recruited during C. rodentium infection, are a key mediator in host-microbe interactions and mucosal defenses.
Analysis of cholinergic pathway-induced alterations at the intestinal mucosa and their association with enhanced resistance to lethal bacterial infection
Authors: Fernandez-Cabezudo Maria J , Al-Mansouri Alreem A , Bashir Ghada , Al-Sbiei Achraf , Venkatachalam Karthikkumar , al-Ramadi Basel K 
Published: January 2023

Abstract
The mucosal innate immune system represents the first line of defense against microorganisms invading the intestinal tract. Increasing evidence suggests that mucosal immune defenses are tightly regulated by immunological and neural pathways. We previously demonstrated that activation of the cholinergic pathway through the administration of a highly specific acetylcholinesterase inhibitor (AChEI), paraoxon, confers resistance to an oral infection by virulent S. typhimurium. This protection is mediated by enhanced innate anti-microbial mucosal defenses in the GI tract. In the present study, we investigated the involvement of different immune cell populations in the GI tract in the modulation of AChEI-mediated anti-Salmonella resistance. Morphological evaluation of intestinal tissue from control- or AChEI-treated mice revealed a significant thickening of the mucin layer and increased B and T lymphocytic infiltration into intestinal mucosa in the AChEI group. Multi-color flow cytometric analysis of iliac intraepithelial lymphocytes (IELs) showed that cholinergic stimulation increased the percentage of CD3+CD8+gamma/delta TCR+ cells. No significant alterations were observed in lamina propria cells. Our findings indicate that cholinergic pathway is able to modulate not only mucin-secreting cells but more importantly gamma/delta T cells, the cell population known to be involved in tissue homeostasis and repair as well as surveillance of the epithelial barrier.
Intestinal permeability and IgA provoke immune vasculitis linked to cardiovascular inflammation
Authors: Rivas Magali Noval , Wakita Daiko , Franklin Michael , Chen Shuang , Yamashita Michifumi , Crother Timothy R. , Shimada Kenichi , Arditi Moshe 
Published: January 2023

Abstract
Kawasaki Disease (KD), a systemic vasculitis of unknown etiology is the leading cause of acquired heart disease among children in the US. Subsets of KD children display gastrointestinal abnormalities during the acute phase of the disease and may have increased serum levels of secretory IgA (SIgA), suggestive of a “leaky gut” phenotype. Additionally, KD patients have higher frequencies of IgA producing B cells in the inflamed coronary artery and in the respiratory and gastrointestinal tracts. How intestinal permeability participates in cardiovascular lesions of KD remains unknown. We observed increased intestinal permeability and elevated circulating sIgA in KD patients as well as elevated sIgA and IgA deposition in vascular tissues in the Lactobacillus casei-cell wall extract murine model of KD vasculitis. Pharmacological blocking of intestinal permeability prevented gut permeability, vascular tissues IgA deposition, and ameliorated cardiovascular pathology. By using genetic and pharmacologic inhibition of IL-1β signaling, we demonstrated that IL-1β lies upstream of disrupted intestinal barrier function, subsequent IgA vasculitis development, and cardiac inflammation. Thus, targeting mucosal barrier dysfunction and the IL-1β pathway may be applicable to other IgA-related diseases including IgA vasculitis and IgA nephropathy. These observations enhance our current understanding of KD pathogenesis and may provide new diagnostic and therapeutic strategies for affected patients.
Vitamin A signaling regulates pathological epithelial cell shedding via IL-18
Authors: Iyer Namrata R , Vaishnava Shipra 
Published: January 2023

Abstract
The intestinal epithelium is under continuous renewal via a tightly controlled process of cell proliferation and cell shedding. During infection and sepsis, the rate of cell shedding increases dramatically as a protective response. However, the mechanisms through which epithelial turnover rate is controlled in the gut remain elusive. Here we identify diet-mediated control of epithelial cell shedding as an important mechanism in defense against infectious disease. We show that abrogating vitamin A signaling by expressing a dominant negative retinoic acid receptor exclusively in intestinal epithelial cells (dnRARVillin-Cre) makes mice resistant to pathological epithelial cell shedding and cell death in an LPS model of sepsis. In a Salmonella infection model, dnRARVillin-Cremice had increased intracellular loads of the pathogen in the intestine as compared to control mice. This trend was observed even at early time points of the infection and was associated with drastically reduced epithelial cell shedding compared to dnRARfl/fl mice. Intestinal epithelium RNA-seq and protein analysis uncovered that levels of the pro-inflammatory cytokine IL-18 are severely reduced in epithelia of dnRARVillin-Cre mice. LPS and pathogen induced cell death were both rescued upon IL-18 reconstitution in dnRARVillin-Cre mice. Furthermore, even dietary vitamin A could modulate epithelial IL-18 expression. Our results thus reveal a novel mechanism whereby dietary vitamin A modulates cell shedding response in the epithelium via IL-18, affecting barrier function and infection susceptibility in the gut.
The Cd8 locus Controls the Functional Conversion of CD4 Th cells to CD4 CTL in the Intestine
Authors: Iwaya Hitoshi , Larange Alexandre , Kakugawa Kiyokazu , Cheroutre Hilde 
Published: January 2023

Abstract
CD4 T cells adapt to their surroundings by differentiating into various T helper (Th) subsets. At steady state, regulatory CD4 T cells (Treg) and interleukin (IL)-17 producing CD4 T cells (Th17 cells) are present in the intestine and localize mainly to the lamina propria, whereas CD4 cytotoxic T lymphocytes (CTL) reside in the epithelium. A hallmark of CD4 CTL is the reactivation of the Cd8 locus and the re-expression of CD8a controlled by the Cd8 enhancer I (E8I). We showed before that CD4 Th cells can reprogram to CTL. Here, we elucidated a mechanism that drives the CTL reprogramming of CD4 Th cells. We identified a long noncoding RNA transcribed from the Cd8 locus, as the critical regulator of the reciprocal expression of T-BET and RUNX combined with the suppression of Foxp3 and Rorc in CD4 CTL precursors. We further found that the E8I region functions as is central coordinator of the RUNX3-controlled cytotoxic gene expression program marked by the re-expression of CD8a. Adoptive transfer of naïve E8I deletion mutant CD4 T cells to Rag1−/− recipient mice led to accelerated weight loss, and severe small and large intestine inflammation with excessive accumulation of pathogenic IL-17A and IL-17A/IFNg double producing CD4 T cells. Altogether, these findings indicate that the Cd8 locus controls the functional polarization of CD4 Th cells and furthermore, that the conversion to CTL not only enhances the direct protective capacity but also regulate the immune response and prevents excessive inflammation and immune pathology at the mucosal border of the intestine.