bluuebunny/crossref_metadata_2025 huggingface dataset:
Inositol polyphosphates promote T cell-independent humoral immunity via the regulation of Bruton’s tyrosine kinase
Authors: Seong Rho H , Kim Wooseob , Min Hyungyu
Published: January 2023

T cell-independent (TI) B cell response is critical for the early protection against pathogen invasion. The regulation and activation of Bruton’s tyrosine kinase (Btk) is known as a pivotal step of B cell antigen receptor (BCR) signaling in TI humoral immunity, as observed in patients with X-linked agammaglobulinemia (XLA) experiencing a high incidence of encapsulated bacterial infections. However, key questions remain as to whether well-established canonical BCR signaling pathway is sufficient to regulate the activity of Btk. Here, we find that inositol hexakisphosphosphate (InsP6) acts as a physiological regulator of Btk in BCR signaling. Absence of higher order inositol phosphates (InsPs), inositol polyphosphates, leads to an inability to mount immune response against TI antigens. Interestingly, the significance of InsP6-mediated Btk regulation is more prominent in IgM+ plasma cells. Hence, present study identifies higher order InsPs as principal components of B cell activation upon TI antigen stimulation and presents a novel mechanism for InsP-mediated regulation of the BCR signaling.
Integrative analysis of human memory B cell coding and non-coding transcriptomes
Authors: Moroney Justin B , Vasudev Anusha , Taylor Julia R , Zan Hong , Pertsemlidis Alexander , Casali Paolo
Published: January 2023

An effective response against previously experienced pathogens is contingent upon the unique functionality of memory B cells. These display intrinsic differences over naïve B cells, including longer lifespan and rapid response to antigen with production of high-affinity, class-switched antibodies. We have developed an integrated analysis of mRNA, miRNA and long non-coding RNA (lncRNA) profiles to determine how the mRNA transcriptome intersects with epigenetic regulatory non-coding RNAs to shape the functionality and phenotype of human memory B cells (CD27+IgG+ and CD27+IgA+) as compared to their naïve B cell counterparts (CD27−IgD+IgM+). Differential expression analysis revealed a novel gene signature shared by CD27+IgG+ and CD27+IgA+ cells, distinguishing memory from naïve B cells. Additionally, CD27+IgD+ B cells, which are not considered bona-fide memory, revealed a transcriptional identity straddling naïve and switched memory B cells. Signaling pathway analysis implicated the “memory” gene signature in the generation, homing and survival of class-switched memory B cells. Integrated analysis of mRNA and miRNA transcriptomes showed that in memory B cells the expression of select genes was dependent upon miRNA-mediated regulation. lncRNA analysis revealed that some of these RNAs clustered with IgH genes, thereby suggesting a role of such lncRNAs in the regulation of the IgH locus. Additional data suggested that select lncRNAs act as miRNA sponges. Thus, we have identified a novel set of genes that play a role in the generation and maintenance of memory B cells and have provided evidence that these genes are subjected to integrated regulation by distinct sets of miRNAs and lncRNAs.
IL-21 is a major suppressor of IgE production in mice and humans
Authors: Allen Christopher DC , Yang Zhiyong , Wu Chung-An M , Targ Sasha
Published: January 2023

Antibodies of the IgE isotype may elicit potent allergic reactions and their production is tightly controlled. The tendency to develop allergic responses has been historically thought to depend on the balance between T helper type 1 (Th1) responses that suppress IgE, versus T helper type 2 (Th2) responses that promote IgE production, based on the activities of the prototypical cytokines interferon gamma (IFN-γ) and interleukin (IL)-4, respectively. Here we reevaluated this paradigm by a direct cellular analysis, demonstrating that IgE production was not limited to type 2 immune responses, yet was generally constrained in vivo. We determined that the cytokine IL-21 was a critical negative regulator of IgE responses, whereas IFN-γ was dispensable. IL-21 inhibited IgE responses by directly engaging the IL-21 receptor on B cells and triggering STAT3-dependent signaling. We reconcile previous discordant results between mouse and human B cells and reveal that the inhibition of IgE responses by IL-21 is modulated by CD40 signaling. The suppression of IgE responses by IL-21 correlated with reduced IgE germline transcription, but was independent of apoptosis, indicating that IL-21 inhibits IgE class switch recombination. These findings establish key features of the extrinsic regulation of IgE production by cytokines.
APRIL signaling in human antibody secreting cells promotes cellular response via p38 and differential immediate early gene regulation
Authors: Stephenson Sophie J , Care Matthew A , Doody Gina M , Tooze Reuben M
Published: January 2023

Long-lived antibody secreting cells (ASCs) persist as quiescent plasma cells (PCs) in niche microenvironments. However, we know little about how ASCs integrate these signals to drive changes in function. APRIL is an important in vivo niche factor, for which the primary receptor is BCMA, but to our knowledge APRIL-induced signaling and consequent gene regulation in primary human ASCs are poorly defined. In particular, little is known of signals that APRIL generates at the transition from proliferative plasmablast to quiescent plasma cell, an important transition in ASC differentiation.

Using a model system of in vitro PC differentiation, which utilizes APRIL signaling as a key survival promoting factor, we demonstrate that significant levels of multimerized mega-APRIL are needed to deliver signals into ASCs. This in part reflects a process of cell intrinsic shedding of BCMA, the APRIL receptor, as previously reported by others. Blockade of proteolytic shedding by gamma secretase inhibitors enhances surface BCMA expression and mega-APRIL induced PC survival, generating phenotypically mature PCs with prolonged longevity from in vitro and ex vivo plasmablasts. At the plasmablast to plasma cell transition APRIL induces a selective pattern of MAP kinase activation in human ASCs, preferentially activating p38. This contrasts to another niche signal, SDF1, which preferentially targets ERK. Differential gene regulatory responses are evident for immediate early genes, with APRIL producing a preferential activation of FOSB relative to FOS or EGR1. The results provide the basis for systematically exploring the signaling and gene regulatory networks involved in the response of human ASCs to niche factors supporting PC survival.
Sumoylation of RORgt regulates TH17 differentiation and thymocyte development
Authors: Sun Zuoming , He Zhiheng , Zhang Jing , Huang Zhaofeng , Du Qian , Li Ning , Zhang Qiang , Chen Yuan
Published: January 2023

RORgt controls the differentiation of TH17 cells involved in autoimmunity such as experimental autoimmune encephalomyelitis (EAE). RORgt also regulates thymocyte development and lymph node genesis. Here we show sumoylation as a novel mechanism to regulate RORgt function. Loss of Sumo3, but not Sumo1, dampens TH17 differentiation and delays the progression of thymic immature CD8+cells (ISP). RORgt is SUMO3-modified at lysine 31 (K31), and mutation of K31 to arginine (RORgtK31R) in mice to prevent sumoylation impairs RORgt-dependent function including defective TH17 differentiation, resistance to TH17-mediated EAE, accumulation of thymic ISP and lack of Peyer’s patches. Mechanically, RORgt-K31 sumoylation stabilizes the binding of SRC1 through recruiting histone acetyltransferase KAT2A. Lastly, E3 ligase PIAS4 binds and sumoylates RORgt at K31, and regulates RORgt-dependent TH17 differentiation and progression of thymic ISP. This study thus demonstrates sumoylation as a critical mechanism for regulating RORgt function, and reveal new drug targets for preventing TH17-mediated autoimmunity.
Batf promotes and stabilizes Th17 cell development by antagonizing the actions of STAT5
Authors: Pham Duy , Silberger Daniel J , Hatton Robin D , Weaver Casey Todd
Published: January 2023

T helper type 17 (Th17) cells secrete IL-17A, IL-17F, IL-21, and IL-22 cytokines and can provide protection against extracellular pathogens or promote certain immune-mediated diseases. The basic leucine zipper transcription factor ATF-like (Batf) contributes to the transcriptional programming of multiple effector T cells but is indispensable for Th17 cell development. Here, we have interrogated mechanisms by which Batf may act to stabilize the Th17 cell phenotype. We find that in vitro differentiated Th17 cells have increased expression of Th1 and Treg signature genes in the absence of Batf. In addition, Batf-deficient (Batf KO) mice succumb to Citrobacter rodentium infection, which correlates with diminished IL-17A and IL-22 cytokine production and increased Foxp3 and Ifng expression compared to WT mice. We find that Batf acts to maintain the Th17 program in long-term Th17 culture conditions by repressing Th1 and Treg programming, at least in part, by antagonizing STAT5 binding to Th1- and Treg-specific genes. Our findings suggest that a function of Batf in Th17 development and maintenance is the antagonism of the actions of STAT5.
Cellular retinoic acid binding protein 2 (CRABP2) serves as a critical regulator in immune regulation and inflammation
Authors: Takazawa Ikuo , Larange Alexandre , Cheroutre Hilde
Published: January 2023

Retinoic acid (RA) is a metabolite of Vitamin A, which plays anti-inflammatory roles by ligating and transactivating nuclear RA receptors that control gene expression and cell differentiation. Cellular retinoic acid binding protein 2 (CRABP2) binds to and transports RA from the cytoplasm to the nucleus. Because of its direct interaction with RARs bound to retinoic acid responsive elements (RARE) in the promoters of RA target genes, CRABP2 specifically channels RA to chromatin-bound nuclear receptors, which then mediate transcriptional activation and expression of RA target genes. CRABP2 is not expressed in naïve T cells, but strongly induced in response to T cell receptor (TCR) signaling. Consequently, activated T cells from CRABP2 KO animals show reduced RARE-luciferase reporter activity, indicating that TCR-induced CRABP2 controls gene transcription and expression in activated T cells. In CD4 T cells we showed before that RA promotes the generation of Foxp3 Tregs and reciprocally suppresses the generation of Th17 cells. Consistent with that, CRABP2 KO mice show a marked reduction of Tregs in the periphery. In the absence of RA, CRABP2 remains in the cytoplasm where it functions in a non-genomic fashion and enhances expression of inflammatory cytokines. In biopsies from patients with various autoimmune diseases, inflammatory CD4 T cells with enhanced expression of CRABP2 accumulate whereas expression of RA target gene is reduced. These findings point at genomic versus non-genomic CRABP2 as an important regulator of the reciprocal generation of Tregs versus inflammatory Th17 cells. Defects in the regulation by activation-induced CRABP2 may form an important underlying cause in autoimmune and other inflammatory diseases.
IL-1β regulates IL-33 mediated T helper 2 (Th2)-biased immunopathophysiology following respiratory syncytial virus infection in an age-dependent and caspase-1 independent manner
Authors: Vu Luan D. , Siefker David T , You Dahui , DeVincenzo John P. , Phan Anh TQ. , Cormier Stephania A.
Published: January 2023

Respiratory syncytial virus (RSV) infection in children is a substantial clinical and health economic burden worldwide. RSV-induced immunopathophysiology has been related to age-variable IL-33 mediated Th2-biased immune responses. However, the mechanisms of the age-dependent regulation of IL-33 remain unclear. Here, we show for the first time that elevated levels of IL-33 in nasal aspirates from RSV infected human infants are associated with the disease severity. We further demonstrate the correlation of IL-33 levels with IL-1β in RSV-infected infants. IL-1β has been shown to induce expression of IL-33 in lung cells. Indeed, we found that intranasal treatment of neonatal mice with IL-1β prior to RSV infection significantly boosted the production of pulmonary IL-33. IL-33 increases coincided with an increased frequency of pulmonary type 2 innate lymphoid cells (ILC2) at 1 day post-infection (dpi) and Th2 biased immunopathophysiology after reinfection. Conversely, antagonizing IL-1β signaling in adult mice with IL-1βR antagonist (Anakinra) prior to RSV infection significantly enhanced IL-33 mediated Th2 biased immunopathophysiology after reinfection. Although it has been shown that IL-1β and IL-33 were regulated at the post-translational level by inflammasome-mediated caspase-1 activation, in our study active caspase-1 was detected exclusively within IL-33negCD45+ cells in RSV-infected lungs and was independent of age at infection. These findings suggest that IL-1β responses regulate RSV-induced IL-33 production via caspase-1 independent mechanisms in an age-dependent manner. More importantly, our findings provide the first data on respiratory IL-33 responses in RSV-infected infants.
Novel roles for the Ikaros zinc finger transcription factor Eos in regulating TH2 differentiation
Authors: Rasheed Mustafa N , Sreekumar Bharath , Moon Sol C , Powell Michael Duane , Read Kaitlin , Oestreich Kenneth J
Published: January 2023

CD4+ T cells differentiate in response to specific cytokine environments that induce the activation of STAT transcription factors and, ultimately, cell-specific gene profiles. The differentiation of TH2 cells requires the cytokines IL-4 and IL-2, which signal via the activation of STAT6 and STAT5, respectively. Interestingly, signaling via the IL-2/STAT5 axis is also required for the differentiation of the TH1 cell type. Our lab recently found that IL-2 signaling induces expression of the Ikaros Zinc Finger (IkZF) transcription factor Eos (Ikzf4) in TH1 cells, and that Eos positively regulates TH1-specific gene expression. Given the requirement for IL-2 signaling in TH2 development, we hypothesized that Eos may also play a role in TH2 differentiation. Indeed, we found that Eos expression was increased in TH2 cells at both the transcript and protein level as compared to TH0, TH1, and TFH-like cells. We also found that Eos knockdown resulted in a significant decrease in the expression of the canonical TH2 genes Il4 and Il13. When we cultured Eos-deficient cells in TH2-polarizing conditions, we similarly observed a decrease in Il4 and Il13 expression, as well as reduced expression of Il2ra and Il2rb. Eos-deficient TH2 cells also displayed decreased expression of Prdm1, the gene encoding Blimp-1, which represses the expression of alternative T helper cell gene programs in TH2 cells. Taken together, these data support a novel, positive role for Eos in the regulation of the TH2 gene program.
Tbet provides survival advantage to Tregs during Type 1 inflammation
Authors: Akkaya Billur , Akkaya Munir , Al Souz Jafar A , Holstein Amanda H , Maz Mitra , Kabat Juraj , Kamenyeva Olena , Shevach Ethan M
Published: January 2023

Regulatory T-cells (Tregs) comprise subgroups with transcriptional programs similar to helper T-cell lineages. To visualize Tbet expression by Tregs during a T helper 1 (Th1) driven inflammation induced by Complete Freund’s Adjuvant in vivo, we imaged Tregs in the lymph nodes of a Tbet.ZsGreen-Foxp3.RFP mouse. While the majority of conventional Foxp3+ Tregs, resided within the T-cell zone, Tbet+Foxp3+ Tregs localized to the interfollicular zone that also contained the recently activated Th1 cells. To understand whether Tbet serves merely as a marker of activation or plays an actual role in Treg function, we generated bone marrow chimeras from Tbet+/+ and Tbet−/− mice. The peripheral Treg pool in chimeric mice was equally constituted by the Tbet+/+ and Tbet−/− cells. However, when the mice were challenged with Plasmodium chabaudi, a parasite that initiates a strong systemic Th1 response, the Treg compartment was significantly dominated by Tbet+/+ Tregs that displayed increased Tbet expression over time. While Tbet+/+ and Tbet−/− Tregs displayed similar activation status and proliferation, Tbet−/− Treg were more prone to apoptosis. Furthermore, Tbet−/− Tregs expressed lower levels of the ATP ectonucleotidase CD39 that was more pronounced during the peak of infection. Pharmacological inhibition of the activity of CD39 increased apoptosis of Tbet+/+ but not Tbet−/− Tregs in an ATP enriched culture in-vitro, suggesting a positive correlation between the availability of Tbet, CD39 activity and viability. Taken together, Tbet may preserve the ability of Tregs to cope with elevated toxic extracellular ATP at the inflammation site by tuning their CD39 expression or activity.
Co-Stimulation–Induced AP-1 Activity is Required for Chromatin Opening During T Cell Activation
Authors: Barski Artem , Yukawa Masashi , Jagannathan Sajjeev , Kartashov Andrey V , Chen Xiaoting , Weirauch Mathew T
Published: January 2023

Activation of T cells is dependent on organized and timely opening and closing of chromatin. Herein, we identify AP-1 as the transcription factor that directs most of this remodeling. Chromatin accessibility profiling showed quick opening of closed chromatin in naïve T cells within 5 hours of activation. These newly open regions were strongly enriched for the AP-1 motif, and indeed, ChIP-seq demonstrated AP-1 binding at more than 70% of them. Broad inhibition of AP-1 activity prevented chromatin opening at AP-1 sites and reduced expression of nearby genes. Similarly, induction of anergy in the absence of co-stimulation during activation, was associated with reduced induction of AP-1 and a failure of proper chromatin remodeling. The translational relevance of these findings was highlighted by the substantial overlap of AP-1–dependent elements with risk loci for multiple immune diseases, most notably multiple sclerosis. Our findings define AP-1 as the key link between T cell activation and chromatin remodeling.
Bach2 attenuates T cell receptor-dependent transcription to fine-tune regulatory T cell differentiation
Authors: Sidwell Tom , Liao Yang , Vasanthakumar Ajithkumar , Shi Wei , Kallies Axel
Published: January 2023

The differentiation of regulatory T (Treg) cells is strictly controlled by T cell receptor (TCR) signals. However, the downstream regulators of this process are incompletely understood. Here we have found that Bach2 blocks the genomic binding of the TCR-induced transcription factor IRF4, attenuating TCR-dependent transcriptional programs to fine-tune Treg cell development and differentiation.

In the absence of Bach2 we observed an increase in TCR-driven Treg cell outcomes, including the enhanced differentiation of effector Treg cells and thymic Treg precursor cells, and a reduction in peripheral Treg cell differentiation. Additional loss of the TCR-responsive transcription factor IRF4 was sufficient to normalise frequencies of each of these Treg cell populations in the absence of Bach2. Transcriptomic analysis identified significant deregulation of gene expression in Bach2-deficient cells which was dependent upon IRF4 expression. Assessing genome-wide occupancy of these transcription factors, we found Bach2 to restrict access of IRF4 to most of its genomic binding sites. Together, these data indicate that Bach2 inhibits IRF4-dependent transcription by blocking its access to their shared binding sites.

Our work reveals Bach2 and IRF4 to drive opposing programs in Treg cell development and differentiation. Bach2 maintains the functional quiescence of Treg cells, in large part by blocking the genomic binding of IRF4 to attenuate the transcriptional program of TCR signalling.
DDX5 regulates chromatin lncRNAs to direct T cell programs
Authors: Ma Shengyun , Zhou Bing , Abbasi Nazia , Luo Claire , Yeo Gene W. , Fu Xiangdong , Huang Wendy Jia Men
Published: January 2023

Recent studies uncovered the growing role of long non-coding RNAs (lncRNAs) in T cell biology. However, little is known about the lncRNAs that localize on the T cell chromatin (T-chrRNAs) with critical roles in gene expressions at the transcription and post-transcriptional level. Using the GRID-seq approach, we uncovered the top 18 T-chrRNAs and their chromatin occupancy in primary mouse CD4+ T helper cells in both the naïve and differentiated states. The chromatin binding of these T-chrRNAs revealed surprising stage-specific clustering patterns and mobilization during development and T cell differentiation. Our eCLIP assay showed that many of these T-chrRNAs interact directly with the DDX5, a nuclear residing RNA binding protein with ATPase activity.

Th17 are unique CD4+ T-cell subset characterized by production of IL-17 and play an important role in inflammation and autoimmunity. At low TGFβ concentration, DDX5 is required for proper Th17 cytokine expression. Our ATAC-seq results suggest that DDX5 knockout Th17 cells fail to maintain chromatin accessibility at numerous regulatory regions, including the super-enhancer at the Il17a-Il17f locus. Moreover, GRID-seq data on WT and DDX5 knockout Th17 cells identified a subset of T-chrRNAs dependent on DDX5 for their proper chromatin localization. In particular, Malat1 is recruited to key Th17 loci in a DDX5 dependent manner. Similar to the DDX5-knockout Th17 cells, CRISPR-Cas targeting of the DDX5 bound regions of Malat1 results in loss of Th17 effector function. Better understanding of the localizations, functions, and upstream regulatory mechanisms governing T-chrRNAs physiology will provide new opportunities for therapeutic intervention to treating T cell mediated diseases.
Electrophilic nitro-fatty acids suppress psoriasiform dermatitis through the inhibition of STAT3 and NF-κB
Authors: Wang Peng , Killeen Meaghan , Mathers Alicia R
Published: January 2023

Psoriasis is a systemic, immune-mediated disorder, characterized by chronic inflammatory skin and joint manifestations. Although the origin of psoriasis and its underlying pathophysiology remain incompletely understood, inflammation has been identified as a central mediator of disease progression. Oxidative inflammatory conditions support the formation of endogenous electrophilic nitro-fatty acids that exert potent anti-inflammatory effects. To study the therapeutic potential of nitro-fatty acids on psoriasis, we employed various experimental models of psoriasis including direct induction and genetically engineered murine models. Our studies demonstrated that oral treatments of nitro-oleic acid (OA-NO2) prior to imiquimod (IMQ) or IL-23 applications, significantly inhibited the induction of psoriasis-like skin inflammation. Consistently, oral OA-NO2 downregulated the production of inflammatory cytokines (IL-1β, IL-6 and IL-17a) in the skin. Moreover, utilizing the chronic K14-VEGF and K5-IL-17C murine models, we determined that oral OA-NO2 had both a preventative and therapeutic effect on psoriasiform inflammation. Further in vitro experiments demonstrated that OA-NO2 impaired STAT3 phosphorylation and translocation to the nucleus, which resulted in decreased keratinocyte proliferation. Importantly, OA-NO2 decreased the basal expression and IL-17A-induced expression of IL-6 in normal human dermal fibroblasts through the inhibition of NF-κB phosphorylation. Overall, our results confirm the critical role of both STAT3 and NF-κB, in psoriasiform dermatitis, and highlight the potential of nitrofatty acids as therapeutic agents for the treatment of this cutaneous inflammatory disease.
Chromatin interaction maps of active cis-regulatory elements in primary human immune cells reveal extensive overlap with disease-associated genetic variants
Authors: Chandra Vivek , Bhattacharyya Sourya , Schmiedel Benjamin J. , Madrigal Ariel , Ay Ferhat , Vijayanand Pandurangan
Published: January 2023

Surveys of purified human immune cell types have revealed that common genetic variants have profound effects of gene expression in a highly cell-specific manner. However, it has been challenging to determine the mechanisms behind cell-specific regulatory effects as well as to pinpoint the potentially functional variants in dense haplotype blocks. Here, we performed genome-wide ChIP-Seq and proximity ligation-assisted ChIP-seq assays (HiChIP) for the H3K27ac modification to capture physical interactions between active cis-regulatory elements and their target gene promoters for 5 common and diverse immune cell types from 6 donors. The generated interaction maps identified long-range loops (over 20kb) for more than 90% of active cis-regulatory regions. We found that majority of the cis-regulatory interactions were highly cell-specific, which in turn correlated with cell-specific transcription factor binding motifs and gene expression patterns. Nearly 10% of expression quantitative trait loci (eQTLs), including disease-associated eQTLs and GWAS SNPs, were located at active cis-regulatory regions that interacted with the target genes of eQTLs (eGenes), thus allowing us to narrow down potentially functional/causative QTLs. Majority of cis-eQTLs that are specific to only a single cell type overlapped cis-regulatory elements that showed interactions with the promoter of their target eGenes only in that specific cell type. Our study thus provides a comprehensive interaction map of active regulatory elements for each of the 5 immune cell types across multiple donors and highlights the cell type-specificity of potentially functional variants and interactions linking them to genes in human disease.
Lipid droplets in mouse thymus express immune molecules and inhibit antigen-specific CD4+ T cell responses
Authors: Bamezai Anil K , Robinson Sarah , Humrich Sarah
Published: January 2023

Lipid droplets (LDs) are vesicles present inside a wide variety of cells. LDs play a vital role in storing neutral lipids as a source for nutrients. While LDs are readily detected intracellularly, their extracellular presence is not widely reported. The cellular origins of LDs and the functions they serve in the thymus remain largely unknown. We detected presence of LDs in the mouse thymus and lymph nodes. We have investigated the proteins expressed by mouse thymic LDs and their functional role. To facilitate this inquiry, we first developed a novel method to isolate and quantify LDs from the thymus. Isolated LDs were analyzed by flow cytometer after staining with either fluorescent-tagged antibodies against immune cell proteins, or a lipid staining orange dye oil stain scarlet 6G. We report that the thymic LDs are heterogeneous in size (range 5 to 100 μm). LDs, on their membrane, express many immune molecules, including T cell receptor αβ in significant amounts. Thymic LDs do not express MHC proteins and are functionally incompetent to serve as antigen presenters. However, when included in antigen presenting assays using bona fide antigen presenting cells, LDs inhibit CD4+ T cell responses. We conclude that LDs found in the thymus express immune molecules on their membrane. While LDs do not directly present antigens to CD4+ T cells, however, their inclusion in cell cultures inhibit antigen-specific clonal expansion of CD4+ T cells in the presence of APC and an appropriate antigen.
15(S)-HETE plays a regulatory role in the immune inflammatory responses
Authors: Choe Jongseon , Kwon Bongjae
Published: January 2023

Arachidonic acids are subjected to the enzymatic transformation by PGH2 synthase or lipoxygenase upon the liberation from the membrane phospholipid. Using in vitro and in vivo experimental models, we have investigated the cellular and molecular mechanisms in the production and function of arachidonic acid metabolites. Here we report that a downstream product of 15-lipoxygenase, 15-S-hydroxyeicosatetraenoic acid (15-S-HETE) is not produced by human follicular dendritic cell (FDC)-like cells but exerts a regulatory effect on prostaglandin production by this type of cells. 15-S-HETE appears to derive from endothelial cells. FDC-like cells, which were isolated from human tonsil, do not express 15-lipoxyenase or 5-lipoxygenase but metabolize 15-S-HETE into downstream products. Immunoblotting analysis and results with chemical inhibitors imply that 15-S-HETE acts on FDC-like cells via PPAR-γ and leukotriene B4 receptor 2. 15-S-HETE enhances or inhibits the expression of PGH2 synthase and resultant prostaglandin production in FDC-like cells, which depends on the types of co-stimulation. Based on these data, we suggest a novel cellular and molecular pathway of immune regulation mediated by arachidonic acid metabolites.
Basophil-derived tumor necrosis factor can enhance immune responses in a sepsis model in mice
Authors: Piliponsky Adrian Martin , Shubin Nicholas J , Lahiri Asha , Truong Phuong , Clauson Morgan , Niino Kerri , Tsuha Avery , Nedospasov Sergei A , Karasuyama Hajime , Reber Laurent , Tsai Mindy M , Mukai Kaori , Galli Stephen J
Published: January 2023

Basophils are evolutionarily conserved in vertebrates, despite their small numbers and short lifespan, suggesting that basophils have beneficial roles in maintaining health. However, these roles are not fully defined. Here, we demonstrate that basophil-deficient mice exhibited reduced bacterial clearance, and increased morbidity and mortality, in the cecal ligation and puncture (CLP) model of sepsis. Among the several pro-inflammatory mediators we measured, tumor necrosis factor (TNF) was the only cytokine that was significantly reduced in basophil-deficient mice after CLP. TNF is critical for mounting effective immune responses against certain pathogens. Accordingly, we investigated whether basophil-derived TNF can significantly contribute to the innate immune response triggered by CLP. For this purpose, we generated a conditional mouse in which TNF production was specifically ablated in basophils. Serum and intraperitoneal TNF levels were significantly reduced in mice with genetic ablation of Tnf in basophils after LPS administration, indicating that basophils contribute to increased TNF levels during LPS-induced endotoxemia. Furthermore, mice with basophils deficient in TNF exhibited reduced neutrophil and macrophage TNF production and effector functions, reduced bacterial clearance, and increased mortality after CLP. Taken together, our studies in mice show that basophils can enhance the innate immune response against bacterial infection and help to prevent sepsis.
NLRC4 deficiency enhances macrophage and T cell responses in polymicrobial sepsis
Authors: Paudel Sagar , Ghimire Laxman , Jin Liliang , Jeyaseelan Samithamby
Published: January 2023

Rationale

Sepsis, a dysregulated systemic host inflammatory response to infection with organ failure, is leading cause of death in non-coronary intensive care unit worldwide. Recently, the NLRC4 inflammasome has been implicated in sensing pathogens and regulating inflammation during microbial infections. However, the role of NLRC4 in sepsis-induced immunosuppression, specifically macrophage dysfunction and T cell loss, is poorly understood.

Methods

We performed cecal ligation and puncture (CLP) to induce polymicrobial sepsis in wild type (C57BL6) and NLRC4-gene deficient (KO) mice. Cellular influx, bacterial burden, and host survival were measured. Macrophages were depleted using Clodronate liposomes. Depletion of lymphocytes, frequency of IFN-γ producing T cells, expression of costimulatory molecules in peritoneal macrophages were examined using flow cytometry.

Results

NLRC4 KO mice were protected from CLP-induced lethality. Furthermore, NLRC4 KO mice had reduced cellular influx, cytokine storm (IL-1b, TNF-a, MCP-1, CXCL-1, CXCL2), and bacterial burdens following CLP. NLRC4 KO mice were protected from sepsis-induced impairment of macrophage function and loss of CD4 and CD8 T cells. Peritoneal macrophages (CD11b+F4/80+) were protected from sepsis-induced loss of MHCII and CD86. Intriguingly, macrophage depletion prior to polymicrobial sepsis eliminated the NLRC4-mediated survival advantage. NLRC4 KO mice had increased frequency of IFN-γ producing T cells post-CLP.

Conclusions

These findings identify NLRC4 as a negative regulator of macrophage and T cell responses in sepsis and its selective inhibition as a potential therapeutic strategy to augment host immunity in sepsis.
Sphingosine 1-phosphate is a harbinger of S. aureus invasion and activates host defense in epithelial barriers
Authors: Igawa Satomi , Choi Jae Eun , Wang Zhenping , Chang Yu-Ling , Wu Chia-Chi , Werbel Tyler , Ishida-Yamamoto Akemi , Di Nardo Anna
Published: January 2023

Background

Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator generated in the skin when cell membrane or barrier components are damaged. S1P regulates diverse cell activities via S1P receptors (S1PR). Keratinocytes express S1PR1–5. Although it is known that S1PRs control keratinocyte differentiation, apoptosis and wound healing, S1PR functions in keratinocyte infections are not fully elucidated. We hypothesized that S1P and S1PR-axis in keratinocytes works as a biosensor for bacterial invasion.

Methods

The expression of S1PRs were studied by immunofluorescence and qPCR. Cytokine transcription and secretion were detected by qPCR and ELISA, respectively. siRNA and S1PR2 antagonist, JTE013, were used to test the different S1PR functions. The S1P synthesis was tested by ATP depletion assay and ELISA.

Results

We found a high epidermal S1PR1 and 2 expressions in human impetigo skin. In vitro, in normal human epidermal keratinocytes (NHEK), S. aureus bacterial supernatant not only induced S1P production but also increased S1PR2 transcription and TNFα, IL36γ, 6 and 8 transcriptions, confirming our in vivo observation. S1P directly increased NHEK IL36γ, TNFα and IL8 expression, but not IL6. In both, S1P and S. aureus bacterial supernatant treated NHEK, S1PR1 knock down reduced IL36γ, TNFα and IL8 transcription, while S1PR2 antagonist, JTE013, blocked their protein secretion.

Conclusions

We proved that, during infections, keratinocytes communicate the infection by sensing S1P release and respond to it using receptors S1PR1 and 2, releasing pro-inflammatory cytokines.
Perforin-2 dependent intracellular bacterial killing by skin γδ T cells
Authors: O’Neill Katelyn Elizabeth , Romero Laura , Fisher Eva , Garcia Denisse , Wikramanayake Tongyu , Pastar Irena , Tomic-Canic Marjana , Strbo Natasa
Published: January 2023

The skin serves as a first line of defense against physical, chemical insults and a vast array of potentially pathogenic microorganisms. Gamma delta (γδ) T cells are a subset of unconventional T cells residing in the skin that play a critical role in protection against bacterial infections, malignancies and in maintaining keratinocyte homeostasis. To characterize the mechanisms governing γδ T cell cytotoxic responses against intracellular pathogens, such as Methicillin Resistant Staphylococcus Aureus(MRSA), we investigated the role of Perforin-2 (P-2), a novel member of the pore-forming family of innate immune proteins.

Skin cells (epidermis and dermis) were separated enzymatically from C57/Bl6 WT and P2 deficient (−/−) mouse dorsal skin using collagenase D. Cells were stained with anti-TCR γδ antibodies, sorted, infected with MRSA and used for intracellular gentamicin protection assays or electron microscopy analysis. γδ T cell lysates were plated and colony forming unit (CFU) count was determined. Frozen sections of dorsal mouse skin were used for immunofluorescence analysis.

We found that P-2 deficient γδ T cells infected intracellularly with MRSA are unable to kill MRSA. Using electron microscopy we confirmed that MRSA resides within γδ T cells. In P2 −/− mice we observed a lower number of γδ T cells in the skin at steady state. Upon transcutaneous MRSA infection (109 CLP 153), in the absence of P2 (−/−), all mice die. We observed that P2 (−/−) γδ T cells migrate more rapidly in the blood than γδ T cells expressing P2. In summary, we showed for the first time that skin γδ T cells can directly kill an intracellular pathogen (MRSA) through a P-2 dependent mechanism. Our data confirms that Perforin-2 is a novel γδ T cell effector molecule in the skin.
Essential Roles of CCL2-CCR2 and C5a-C5aR1 Pathways in irritant Contact Dermatitis
Authors: Shibuya Rintaro , Kitoh Akihiko , Kabashima Kenji
Published: January 2023

Cutaneous immune response induced by epithelial cell stress induces represents the first line defense against pathogens. We have reported that basophils play a pivotal role in the skin inflammation caused by irritants. However, it has not been fully understood how they accumulate in the skin. Previous studies have shown that epithelial cell damage induces complement activation in organs such as the lung and the kidney, implicating a possible involvement of the complement activation in the immune response caused by epithelial cell stress in the skin. Thus, we investigated the mechanism of basophil recruitment to the skin focusing on the complement system.

Topical application of the surfactant sodium dodecyl sulfate (SDS) onto murine ears induced basophil-dependent skin inflammation. Among chemokines which were up-regulated by SDS application, we found that CCL2-CCR2 axis was critical for basophil recruitment to the skin. Here, CCR2 on basophils was dispensable and CCR2 on radioresistant cells was essential.

Finally, we evaluated the contribution of complement system to SDS-induced basophil recruitment to the skin. SDS application induced C3 and C5a production in the skin, suggesting a complement activation in the skin. We evaluated whether C3 and C5a are involved in SDS-induced basophil recruitment by using C3 deficient mice and C5a receptor 1 deficient mice. The result showed that C3 deficiency or C5aR1 deficiency significantly attenuated SDS-induced basophils accumulation. By contrast, SDS-induced CCL2 production in these mice was comparable to WT mice.

Collectively, our data suggest that both CCL2-CCR2 and C5a-C5aR pathways are essential to the SDS-induced innate immune response in the skin by modulating basophil recruitment.
Noncanonical Inflammasome-Induced release of Alarmins during Sepsis
Authors: Russo Ashley Jean , Menoret Antoine , Duduskar Shivalee , Rabinovich Gabriel , Deshmukh Sachin D , Vella Anthony T , Rathinam Vijay
Published: January 2023

Sepsis is a life-threatening disease caused by the immune system’s response to infection that results in excessive inflammation, organ damage and often death. In fact, sepsis is the leading cause of death in intensive care units and is responsible for more than 250,000 deaths every year in the USA. Activation of inflammatory caspases, such as caspase-11 by lipopolysaccharide (LPS) from Gram-negative bacteria, is a key mechanism of innate immune defense against infection. Intracellular sensing of LPS by caspase-11 results in pyroptosis, maturation of caspase-1, IL-1β and IL-18 and unconventional secretion of intracellular proteins that lack a leader sequence for conventional secretion via endoplasmic reticulum-Golgi route. These proteins can act as alarmins or damage associated molecular patterns (DAMPs) to regulate the inflammatory response and therefore play an important role during infectious diseases. However, caspase-11-dependent release of alarmins and DAMPs following noncanonical inflammasome activation is poorly characterized. Using a proteomics approach involving ProteomeLab’s PF2D fractionation system followed by mass spectrometry, we have profiled several new alarmins released in a caspase-11 dependent manner following Gram-negative bacterial infection. By employing various mutant mice and cells, we have defined the molecular and cellular mechanisms underlying the release of a candidate alarmin as a consequence of noncanonical inflammasome activation in vitro and in vivo. Importantly, our in vivo studies demonstrate a critical role for this alarmin in LPS shock. Overall, these findings provide new insights into potential biomarkers and therapeutic targets for blocking the lethal inflammation in sepsis.
Development of a humanized mouse model for functional testing of biomaterial fibrosis
Authors: Greiner Dale L , Doloff Joshua C , Sadraei Atieh , Farah Shady , Brehm Michael A , Shultz Leonard D , Langer Robert , Anderson daniel G
Published: January 2023

Implanted biomedical devices reside within tens of millions of patients in the United States alone, and are involved in millions of new surgeries every year. They comprise a major component of modern medicine and are essential for many clinical applications ranging from hip/knee replacement, tissue repair/reconstruction, controlled release, sensing, and cell transplantation. Limiting therapeutic performance and lifespan, immune-mediated foreign body rejection results in sequestration behind dense layers of fibrotic scar tissue. Deeper understanding of wildtype rodent and non-human primate foreign body responses was leveraged to generate a variant of humanized mice that is capable of recapitulating fibrosis following implantation. Cellular and cytokine responses were also determined with multiple biomaterials—hydrogel alginate and polymers polystyrene and polydimethylsiloxane (PDMS), as well as across multiple implant sites. Innate immune macrophages were verified as essential in the development of fibrosis in this model, and that they were capable of crosstalk with mouse fibroblasts for collagen matrix deposition. Cytokine and cytokine-receptor array analysis confirmed core signaling in the fibrotic cascade. Lastly, high-resolution microscopy coupled with multiplexed antibody-capture analyses revealed spatial resolution of rejection responses. This model allows us to investigate dynamic human immune cell interactions with implantables, and offers a surrogate pre-clinical diagnostic for screening biomaterial immunogenicity.
Microglial antigen presentation is required for antigen specific CD8 T cell infiltration to the brain parenchyma following CNS viral challenge
Authors: Goddery Emma , Malo Courtney , Jin Fang , Hansen Michael , Fryer John , Pavelko Kevin , Johnson Aaron
Published: January 2023

Cytotoxic T cell infiltration into the central nervous system (CNS) is required for clearance of pathogen or tumor. Whether microglia, the resident innate immune cells of the brain, are involved in orchestrating CD8 T cell infiltration of the brain in an antigen dependent manner remains to be elucidated. We hypothesized that antigen presentation by major histocompatibility complex (MHC) class I molecules expressed by microglia is required to orchestrate antigen specific CD8 T cell infiltration into the brain parenchyma. To test this hypothesis, we generated a novel tamoxifen-inducible microglial MHC class I conditional knockout mouse (CX3CR1-creERT2H-2Kb cKO). In these mice, administration of tamoxifen mediates deletion of the H-2Kbmolecule in cells expressing CX3CR1. Short-lived peripheral CX3CR1+ cells repopulate within 4–6 weeks after tamoxifen treatment and restore expression of H-2Kb, leaving only long-lived microglia deficient for MHC class I. We challenged these mice with intracranial infection of Theiler’s murine encephalomyelitis virus expressing the model antigen ovalbumin (TMEV-OVA) to assess antigen specific CD8 T cell responses within the brain parenchyma. In microglia MHC I cKO mice, the frequency of brain infiltrating OVA-specific CD8 T cells was attenuated while the frequency of intravascular OVA-specific CD8 T cells was increased when compared to cre− littermate controls. These results suggest that microglial antigen presentation is important for antigen specific CD8 T cell infiltration into the brain parenchyma. These studies directly address the critical role of microglial MHC class I on antigen specific CD8 T cell responses to an in vivo neuroinflammatory insult.
The host E3-ubiquitin ligase TRIM6 plays an important role in balancing protective innate antiviral immunity and damaging inflammation
Authors: Rajsbaum Ricardo , Aguilera-Aguirre Leopoldo , Hage Adam
Published: January 2023

The host antiviral innate immune response involves activation of multiple signaling pathways that culminate in the production of type I interferons (IFN-I) and inflammatory cytokines, which together control virus infections. However, excessive cytokine production can result in exacerbated disease. To strike the right balance, these signaling pathways are regulated by different molecular processes, including the ubiquitin (Ub) system. We recently reported that the E3-Ub ligase TRIM6 catalyzes the synthesis of unanchored K48-linked poly-Ub chains, which are not covalently attached to any protein, and promote antiviral IFN-I responses by activating the IKKɛ kinase. Two different highly pathogenic viruses, Nipah (NiV) and Ebola (EBOV), hijack TRIM6 to promote virus replication through mechanisms that involve both covalent and unanchored Ub. This indicates that host antiviral factors can have direct pro-viral functions. Consistent with this notion, EBOV replication is decreased in the lungs of TRIM6 knockout mice (Trim6−/−) upon intranasal administration. In contrast, TRIM6 plays a protective role against Influenza (IAV), via the IFN-I pathway, but can exacerbate disease via induction of chemokines and infiltration of inflammatory cells. Although Trim6−/− mice have increased IAV titers, they also show reduced signs of pathology and reduced neutrophil infiltration in the lungs. Phosphorylation of AKT is impaired in Trim6−/− cells resulting in decreased CXCL1 expression, a well-known neutrophil chemo-attractant. These results suggest that TRIM6 regulates multiple innate immune pathways to balance protective and excessive inflammatory responses, and viruses have adapted to hijack TRIM6 to enhance their replication
Early host responses to cattle gastro-intestinal parasite Ostertagia ostertagi
Authors: Bakshi Mariam , Tuo Wenbin , Zarlenga Dante
Published: January 2023

Ostertagia ostertagi is a gastrointestinal parasite of cattle that has a huge economic impact on the US cattle industry. With the rise in drug resistance among nematodes, vaccination is advanced as the most cost-effective alternative; however, nematode vaccines have met with lackluster results thus far. Thus, understanding host-parasite interactions is a critical step for controlling parasite transmission. Reports show that a primary O. ostertagi infection in cattle does not elicit the classic Th2 (anti-inflammatory) paradigm suggesting that an overall mixed response (mixture of Th1, Th2 and other types of Th) may dominate in the absence of a protective response. Little is known about the host innate immune pathways to which O. ostertagi is subjected. This research investigates the early onset of host immune response during the infection by determining the; 1) kinetic responses of immune mediated genes within peripheral blood mononuclear cells using quantitative (q) real time (RT) PCR; and 2) influence of O. ostertagi excretory secretory molecules on host innate immune cells i.e., macrophages, by in-vitro cell activation assays followed by qRT-PCR and immune-cytochemistry. Results show that upon progression of the disease, gene expression of a subset of Toll-like receptor genes increased, indicating active involvement of different cellular immune responses. Additionally, morphological changes in macrophages coupled with transcriptional changes in TNFα and TGFβ supported classical signs of cell activation. These results demonstrate early initiation of host immune responses during a parasitic infection and help further elucidate the complex and intimate relationship between O. ostertagi and its bovine host.