bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a novel mechanism of Th2 polarization in mouse CD4+ T cells through nuclear factor erythroid 2-like 2 (Nrf2) regulation
Authors: Kennedy Rebekah C , Liu Sheng , Turley Alexandra E , Freeborn Robert A , Rockwell Cheryl E
Published: January 2023

The nuclear factor erythroid 2-like 2 (Nrf2) transcription factor regulates a battery of detoxification and antioxidant genes following activation by cell stress. The primary role of Nrf2 in immune function was thought to occur as an indirect consequence of oxidative stress. We have previously demonstrated an unknown immunomodulatory role of Nrf2, where activation by the common food preservative, tBHQ, induced Nrf2-dependent skewing of CD4+ T cells towards a Th2 phenotype. Polarization resulted in increased production of Th2 cytokines and DNA binding of the master regulator of Th2 differentiation, GATA3. To date, the molecular mechanism(s) responsible for increased GATA3 activity and subsequent Th2 cytokine protein production are unknown and elucidation of this mechanism(s) is the study objective. Primary mouse CD4+ T cells (WT and Nrf2-null) were treated with tBHQ and GATA3 activity was analyzed. Additionally, Nrf2 binding to putative AREs within an intron and the promoter of the GATA3 gene was investigated in the Th2 clone, D10 cells. Results from these studies show GATA3 gene expression and protein production increased after tBHQ treatment in WT, but not Nrf2-null, mice. In addition, Nrf2 binding to putative AREs in the GATA3 gene was demonstrated in D10 cells by ChIP analysis. The functionality of these putative AREs was established by luciferase assay. Together, results of these experiments suggest that GATA3 is upregulated through a direct mechanism involving Nrf2 at the transcriptional level. Overall, these studies provide strong evidence for the role of Nrf2 in a novel mechanism of Th2 polarization of CD4+ T cells, which could have potential implications in Th2 mediated allergic disorders.
Immune-markers for assessment of wheat allergenicity in mice: comparison of adjuvant-free vs. alum-adjuvant based models
Authors: Gangur Venugopal , Jin Yining , Gao Haoran , Jorgensen Rick , Ng Perry KW
Published: January 2023

Immediate hypersensitivity reaction to wheat proteins is a major type of food allergy in many countries including the USA. Mechanisms of wheat allergenicity are incompletely understood at present. Reliable and validated in vivo immune-markers for assessment of allergenicity potential of different wheat products are unavailable at present. Published mouse models of wheat allergy have mostly used adjuvants to induce wheat allergenicity. However, it is not clear whether such models are useful to develop reliable immune-markers to assess allergenicity of different wheat products. Here we compared the immune-markers of allergenicity between an adjuvant-free transdermal exposure (TDE) model and an alum-adjuvant (AA) mouse model of anaphylaxis induced by salt-soluble wheat protein (SSWP) extract. In the TDE model, Balb/cJ mice were sensitized with SSWP by six weekly exposures. In the AA model, mice were sensitized by intraperitoneal injection of SSWP with alum. In both models, significant IgE as well as MMCP-1 responses were elicited. Analysis of spleen immune-markers using a quantibody multiplex assay, identified IL-4, -5, -17B/E, -20, CCL4,5,11, CXCL4, and CD40L as useful markers in the TDE model. In the AA model, with the exception of IL-5 and -13, none of the immune-markers tested were found useful. Our data demonstrate that the TDE model is better than the AA model for development of in vivo immune-markers for assessment of wheat protein allergenicity. Funding: USDA, National Institute of Food and Agriculture, Hatch project MICL02486 (Accession Number: 1012322) and Agricultural and Food Research Initiative Competitive Program#2018-67017-27876; Michigan State University/FSHN.
Differential modulation of T cell activation
Authors: Swain Pamela , Kam Yoonseok , Romero Natalia , Dranka Brian P. , Dranka Brian P.
Published: January 2023

Dysregulated T cell responses cause inflammation and autoimmune diseases. These conditions are managed with agents to limit and down-modulate the diverse cellular processes that control T cell activation. Cellular metabolism is a primary regulator of immune cell fate and function. T cell activation is correlated with metabolic shifts, especially in glycolysis, providing increased metabolic flux to support high rates of cellular proliferation. In this study we evaluated the impact of immunosuppressive compounds on the magnitude and kinetics of early anti-CD3/CD28 mediated T cell activation from a metabolic perspective. We employed compounds with divergent inhibitory mechanisms in order to understand the relationship of specific inhibitory pathways and metabolic function. Using an Agilent Seahorse XF Analyzer, T cells were activated in situ and activation was quantified in real time using proton efflux rate (PER) as a measure of glycolysis. Here, we report that anti-CD3/CD28-mediated T cell activation was suppressed when cells were pretreated with 20 μM prednisone. In contrast, activation was increased in the presence of 20 μM cyclosporin A. In non-activated cells, neither compound had a measurable effect on metabolism within 30 min of treatment. These data show a correlation in timing of suppression with the context of the drugs action. Future studies will utilize additional inhibitory pathway modulators to further examine the mechanisms of T cell activation in the context of immunosuppression.
Lung-migratory Dendritic Cells traffic into the spleen after influenza virus infection
Authors: Jenkins Meagan M , Bachus Holly , Leon-Ruiz Beatriz , Ballesteros-Tato Andre
Published: January 2023

Initiation of CD8+ T cell responses to influenza virus requires the trafficking of lung-migratory dendritic cells (mDCs) from the lung into the lung-draining LN (mLN). As such, when mDCs are absent or are unable to migrate into the mLN, T cell responses are severely compromised. Importantly, it is generally considered that mDCs die shortly after reaching the mLN. Thus, the current paradigm suggests that priming of influenza-specific T cell responses by lung-mDCs takes place entirely in the mLN. We show here, however, that a fraction of the lung mDCs egress the mLN, enter the blood circulation, and subsequently traffic to the spleen, where they prime influenza-specific CD8+ T cell responses. Mechanistically, egress of mDCs from the mLN is controlled by an S1PR dependent mechanism. As a consequence, blockade of S1P/S1PR interactions prevents the egress of mDCs from mLN, thereby inhibiting the priming of T cell responses in the spleen. Collectively, our results demonstrate a novel DC trafficking pathway and support a new paradigm for how T cell responses to influenza are initiated.
Rapid activation of brain resident memory T cells following neurological insults
Authors: Ayasoufi Katayoun , Namen Shelby , Goddery Emma , Tritz Zachariah , Fain Cori E. , Yokanovich Lila , Jin Fang , Johnson Aaron J.
Published: January 2023

Brain tissue resident memory T cells (TRM) are an emerging lymphocyte of interest. While the phenotype and gene expression pattern of brain TRMs have recently been discussed, their function and response to neurologic insults is not yet understood. Using the described phenotype of TRMs (TCRB+, CD69+, CD4 or CD8+, CD103−, CD44+), we analyzed the reaction of this lymphocyte in the brain to various neurological insults. We found that physical insult induced by an intracranial(ic) injection of PBS resulted in increased TRM numbers within 24 hours post insult. This TRM population later decreased yet remained above baseline by 50 days post insult. Interestingly, TRM populations also increased in the brains of animals as a function of age, suggesting natural insults experienced by mice leads to an ever increasing TRM population in the brain. We then assessed the reaction of TRMs to virus in the brain. Mice infected ic with Theiler’s Murine Encephalomyelitis virus (TMEV) had a marked increase in brain TRMs one day later, which is 4 days prior to detection of traditional TMEV antigenspecific CD8 T cells. This implies TRMs respond to CNS viral infections prior to generation of virus antigen specific responses. Finally, we investigated the role of dendritic cell (DC) antigen presentation in generation of TRMs using our novel MHC class I conditional knockout mice. Conditional ablation of H-2Kb and H-2Db did not disrupt TRM populations in the brain. Our data is the first study to show: 1. Activation of TRMs in the brain preceded antigen specific cell infiltration and 2. MHC class I molecules on DCs are not required to generate brain resident TRMS. Understanding brain TRMs is crucial in elucidating their role in neurodegeneration and/or targeting them in CNS cancers.
CD160 serves as a negative regulator of NKT cells in acute hepatic injury
Authors: Lee Kyung-Mi , Park Gayoung , Kim Tae-Jin , Kim Jeongmin , Lim Seon-Ah , Fu Yang-Xin
Published: January 2023

CD160 and BTLA binds to herpes virus entry mediator (HVEM). Although the function of BTLA as a negative regulator has been well documented, the role of CD160 in NKT cells remains unclear. Upon analysis of CD160−/−mice and their mixed bone marrow chimeras, we found no apparent developmental defects of NKT cells. However, CD160−/−mice demonstrate severe liver injury after in vivo challenges with α-galactosylceramide (α-GalCer) and a large proportion of CD160−/− mice died following Con A challenges with elevated serum AST and ALT levels, due to hyperactivation of NKT cells with significantly elevated levels of IFN-γ, TNF-α, and IL-4. Importantly, inhibition of BTLA by anti-BTLA mAbs aggravated α-GalCer-induced hepatic injury, highlighting that both CD160 and BTLA serve as non-overlapping negative regulators of NKT cells. Our data presents CD160 as an important co-inhibitory receptor that delivers antigen-dependent signals in NKT cells that dampen cytokine production during early innate immune activation processes.
CD4+ T-helper 17 (Th17) signature cytokine, IL-17, mediates CD4 resistance to immune suppression
Authors: Crawford Michael Patrick , Sinha Sushmita , Renavikar Pranav S. , Karandikar Nitin J.
Published: January 2023

CD4+ T-helper (Th) cells are an important component of the immune system that come in several flavors, largely defined by the cytokines they produce. Important immune regulatory mechanisms mediated by CD4+ and CD8+ T-cells keep untoward effector T-cell responses in check. T-helper 17 (Th17) cells, characterized by IL-17 production, play critical roles in the body’s response to infections and cancer and in the pathogenesis of autoimmune diseases such as multiple sclerosis, psoriasis, arthritis, IBD, among others. We hypothesized that different lineages of effector Th cells would exert differential resistance to suppression. To test this, we differentiated purified naïve human CD4+ T-cells along different lineages (Th0, Th1, Th2, Th17) and tested the ability of CD8+ T-cells to suppress them. We observed that Th17 cells were highly resistant to immune suppression by CD8+ T-cells, compared to control Th0 cells. In contrast, Th1 cells were significantly more sensitive to suppression. Th17 resistance was mediated through the action of IL-17A, IL-17F and IL-17AF heterodimer through their receptors (IL-17RA and IL-17RC) on CD4+ T-cells themselves, but not through their action on CD8+ T-cells or APC. Interestingly, Th17 cells derived in different cytokine milieus showed distinct suppressive resistance, with the typical “non-pathogenic” conditions resulting in Th17 cells that were as sensitive to suppression as control Th0 cells. These studies reveal a novel function for IL-17 cytokines in a CD4-intrinsic mechanism of immune resistance that may serve as a critical target for intensive investigation and therapeutic intervention.
RNA-binding protein PCBP1 shapes tolerance and immune response by constraining the formation of regulatory T cells
Authors: Ansa-Addo Ephraim Abrokwa , Huang Huai-Cheng , Iamsawat Supinya , Yu Xue-Zhong , Howe Philip H , Li Zihai
Published: January 2023

Maintaining adequate regulatory T cell (Treg) and effector T cell balance is critical for efficient regulation of tolerance and immune response, respectively. There is increasing evidence that in addition to regulating RNA fate, RNA-binding proteins (RBPs) may also control gene expression and determine the fate of immune cells. Here by studying mice in which the RBP Pcbp1 gene was disrupted in T cells, we define PCBP1 as a global regulator of immune activity that stabilizes immune responses, while repressing the signature programs of Tregs. PCBP1 expression was upregulated in response to T cell activation, but functionally inhibited by TGF-β-mediated phosphorylation. Loss of PCBP1 expression in T cells was associated with dysregulated peripheral homeostasis and impaired effector functions in knockout mice. Indeed, PCBP1-deficient T cells produced less IFN-γ and TNF-α upon stimulation with PMA, plus ionomycin, compared to wild-type littermates. Strikingly, lack of PCBP1 constrained the pathogenicity of effector T cells by conferring resistance to acute graft-vs-host disease in mice. Mechanistically, assessment of the genome-wide function of PCBP1 revealed that it limits the expression of genes associated with commitment to the Treg lineage. Consequently, its absence resulted in a marked increase in the frequency of Foxp3+Treg cells. Together, our findings identify PCBP1 as a distinct regulator of effector T cell functions and the formation of Treg cells in order to control the balance between tolerance and immunity.
A wave of Foxp3+ regulatory T cell accumulation in neonatal liver plays unique roles in maintaining self-tolerance
Authors: Ge Qing , Li Mingyang
Published: January 2023

Newborn animals require a tightly-regulated local and systemic immune environment to govern the development and maturation of multiple organs/tissues even when the immune system itself is far from mature during neonatal period. The regulatory T cells (Tregs) are essential in maintaining immune tolerance/homeostasis and modulating inflammatory responses. The features of Tregs in neonatal liver under steady state are not well understood. To investigate the phenotype, functions, and significance of neonatal Tregs in the liver, we compared the frequency, surface markers, functions, and transcriptome of liver and splenic Tregs in neonatal mice. We found a wave of thymus-derived Treg influx into the liver during the first 1–2-week age window. Depletion of these Tregs between days 7 and 11 after birth rapidly resulted in Th1-type autoimmune hepatitis and metabolic disorder. Compared to splenic Tregs, more Tregs in neonatal liver underwent MHC II-dependent activation and proliferation and acquired higher suppressive functions. The transcriptome profile of these neonatal liver Tregs showed elevated expression of Tbx21, Pparg, and features of Tregs capable of regulating Th1 responses and metabolic homeostasis. These results suggest that neonates have a transient accumulation of Tregs with unique features in the liver. This Treg influx is critical to ensure self-tolerance and liver maturation.
Phenotypic and functional characterisation of expanded antigen-specific regulatory T cells (Ag-sp Treg), towards clinical translation
Authors: Bianchi Joana , Azevedo Rita I. , Vieira Ana I.S. , Ligeiro Dário , da Silva Cláudia L. , de Lacerda João F.
Published: January 2023

Treg infusion for graft-versus-host-disease treatment has been increasingly investigated. Yet, Treg of unknown specificity may suppress graft-versus-leukemia. To ascertain the factors affecting Ag-sp Treg potency and specificity of suppression, we assessed Ag-sp Treg phenotype and function within different expansion conditions and suppression assay (SA) milieus. Ex vivo Treg were co-cultured with monocyte-derived dendritic cells (DC) presenting allogeneic Ag directly (allo-DC) or indirectly (self-DC loaded with allo-lysate). Suppression of effector proliferation and cytokine secretion was assessed in SA with the same DC as in expansion (original) or 3rd party DC (3P) from various donors.

Ag-sp Treg significantly suppressed Tcon and CD8+ T cell responses to original DC. Directly expanded cells suppressed responses to Ag presented direct and indirectly; however, indirectly expanded cells were less suppressive of responses to allo-DC. More potent Treg were less specific in their suppression of Tcon. Still, responses to 3P donors with MHC matches to original DC donor were significantly more suppressed than responses to unmatched 3P donors. Potency of suppression was also negatively correlated to expression of CD86 and CD83 in DC. Finally, FlowSOM analysis showed minimal overlap between Ag-sp Treg and activated Tcon phenotypes, suggesting Ag-sp Treg were pure. K-means clustering of Ag-sp Treg expression of CD137, CD154, CTLA-4, PD-1, CTLA-4 and HLA-DR identified clusters that may be correlated to Treg function.

We propose the specificity of Ag-sp Treg function in the inflammatory milieu is a result of Treg phenotype, type of responders, DC co-stimulation and degree of MHC match between expansion DC and DC present in the milieu.
IL-10 and TGF- β differentially regulate gap junction formation and membrane transfer in macrophages and macrophage-like cells
Authors: Eill Elizabeth , Taffet Steven M
Published: January 2023

In macrophages, the expression and regulation of the gap junction protein connexin43 (Cx43) is poorly understood. Here we demonstrate that inflammatory and anti-inflammatory mediators differentially regulate gap junction plaque formation in primary bone marrow-derived macrophages (BMDMs) and the macrophage-like cell line RAW264.7. RAW264.7 macrophage-like cells were transfected with GFP-tagged Cx43 to visualize Cx43 trafficking. LPS, an inflammatory mediator, is a potent inducer of Cx43 in BMDMs and RAW264-Cx43-GFP but demonstrates limited trafficking into gap junction plaques. Interestingly, the further addition of IL-10, an anti-inflammatory cytokine, resulted in no difference in protein expression but significantly increased gap junction plaque formation. In contrast to IL-10, the addition of TGF-β (beta) decreased Cx43 protein levels and prevented gap junction plaque formation. We utilized a dye transfer assay to assess gap junction intracellular communication. We could not demonstrate any channel-associated transfer of an intracellular dye, calcein. Uniquely, macrophages were observed transferring endosome-like particles containing membrane between cells as visualized with the membrane-bound dye DiI. This appeared to be through long tube-like structures, possibly tunneling nanotubes. This effect was enhanced with IL-10 treatment and decreased with TGF-β. These results suggest that IL-10 and TGF-β differentially regulate Cx43 plaque formation and that this corresponds to changes in the transfer of cellular components in a gap junction channel-independent way. Overall these results suggest a complex and novel regulation of Cx43 gap junction formation in macrophages and macrophage-like cells.
Creatine shapes macrophage polarization by reprogramming L-arginine metabolism
Authors: Ji Liangliang , Zhao Xinbin , Zhang Bin , Kang Lan , Song Wenxin , Zhao Baohong , Xie Wei , Chen Ligong , Hu Xiaoyu
Published: January 2023

Macrophage polarization is accompanied by drastic changes of L-arginine metabolism. Two L-arginine catalytic enzymes, iNOS and arginase 1, are well-characterized hallmark molecules of M(IFN-γ) and M(IL-4) polarization, respectively. The third metabolic fate of L-arginine is generation of creatine that acts as a key source of cellular energy reserve, whereas little is known about regulation and function of creatine metabolism in the immune system. Here, combination of genetic, genomic, metabolic, and immunological analyses revealed that creatine fundamentally reprogrammed macrophage polarization by suppressing M(IFN-γ) yet promoting M(IL-4) effector functions. Mechanistically, creatine inhibited IFN-γ-JAK-STAT1-mediated induction of immune effector molecules including iNOS while supported IL-4-triggered STAT6-dependent upregulation of arginase 1. Depletion of intracellular creatine by ablation of creatine transporter Slc6a8 significantly altered macrophage-mediated immune responses in vivo. These results depicted a previously uncharacterized regulatory loop whereby creatine, a single cellular metabolite, shaped macrophage polarization by regulating L-arginine metabolic enzymes.
The role of the HO-1/Heme axis on macrophage cytokine production
Authors: Choa Ruth , Kambayashi Taku
Published: January 2023

Transfusion Related Immunomodulation (TRIM) is a phenomenon that was first described over 30 years ago when it was reported that patients receiving blood transfusions prior to renal transplants had improved graft survival rates. The prevailing hypothesis for these observations at the time was that donor leukocytes were responsible for the effects; however, transfusion of leukoreduced and irradiated blood products still resulted in TRIM, suggesting that another component of the blood products, such as red blood cells (RBCs), might mediate the immunosuppressive effects.

RBCs have been shown to affect the activation of immune cells and immune responses. For example, monocytes co-cultured with RBCs and stimulated with LPS produce lower levels of TNF-a than those that are not co-cultured. This suggests that RBCs can shape the immune response that develops during antigen presentation.

Splenic red pulp macrophages are critical for clearance of both RBCs and heme, and these processes are essential for maintaining RBC homeostasis and preventing heme-mediated toxicity. Inside macrophages, heme is degraded by the enzyme Heme Oxygenase-1 (HO-1) and HO-1 activity leads to increased IL-10 and decreased TNF-a secretion. We hypothesized that erythrophagocytosis by macrophages during RBC transfusion may result in the activation of a heme-mediated HO-1 dependent signaling pathway that causes immunosuppressive effects. We used the robust TLR stimulation models of LPS and CpG challenge, and tested whether RBC transfusion or heme signaling could modulate the resulting macrophage cytokine response. We found that CORM-2 (a synthetic CO releasing molecule) downregulates the macrophage pro-inflammatory cytokine profile.
TRAF3 is a critical regulator of the expansion of myeloid-derived suppressor cells in mice
Authors: Zhu Sining , Lalani Almin , Sant’Angelo Derek , Covey Lori R , Xie Ping
Published: January 2023

TRAF3 is an adaptor protein and E3 ligase that regulates the signal transduction pathways of the TNFR superfamily, pattern recognition receptors, and cytokine receptors. We previously showed that specific deletion of TRAF3 in myeloid cells leads to spontaneous inflammation and tumor development in aging M-TRAF3−/− mice, which is consistently associated with a striking expansion of CD11b+Gr1+ myeloid cells. Here we report that these CD11b+Gr1+ cells were myeloid-derived suppressor cells (MDSCs), as they exhibited a strong suppressive activity on CD8 T cell proliferation and also displayed a nuclear morphology of immature myeloid cells. Using a chronic inflammation model induced by repeated injections of heat-killed Bacillus Calmette-Guérin (BCG), we showed that young adult M-TRAF3−/− mice also exhibited hyperexpansion of MDSCs after BCG injections. Our in vivo BrdU labeling experiments revealed that MDSC hyperexpansion in M-TRAF3−/− mice was mainly due to the increased proliferation of MDSCs or progenitor cells in the spleen but not in the bone marrow (BM). Interestingly, TRAF3 does not appear to be an intrinsic regulator of the development, survival or proliferation of MDSCs, as suggested by the results of cultured BM progenitor cells and purified splenic MDSCs, in the absence or presence of CSFs or agonists of TLRs or CD40. Furthermore, we identified environmental factors that induced the aberrant expansion of MDSCs in M-TRAF3−/− mice using cytokine protein array analyses, which were further verified by macrophage cultures and in vivo neutralization studies. Taken together, our findings identify TRAF3 as a new regulator of MDSC expansion, and therefore also a critical regulator of chronic inflammation and immunosuppression.
Distinct heat shock protein-mediated immune responses are induced by macrophages and dendritic cells
Authors: Sedlacek Abigail L , Kinner-Bibeau Lauren B , Wang Yifei , Mizes Alicia P , Binder Robert J
Published: January 2023

Heat shock proteins (HSPs) induce potent immune responses against their chaperoned antigens and thus have potential as cancer immunotherapy. APCs, T cells, and NK cells are all required in HSP-mediated immune responses. NK cells are activated indirectly via APCs, but the mechanism of this activation is poorly understood. Interestingly, MΦ are capable of inducing IFNγ production by NK cells, however, DC are not. This is observed in response to multiple HSPs including gp96, calreticulin and HSP90. Thus, APC production of potential mediators of NK cell activation were investigated. Gp96 induces CXCL10 production in MΦ but not DC and this chemokine is necessary for NK cell activation. Since CXCL10 is STAT1 induced, STAT1 phosphorylation (STAT1p) was examined. A unique pattern of STAT1p is observed in MΦ where S727 is phosphorylated early, followed by delayed phosphorylation of Y701. In contrast, DC exhibit delayed phosphorylation of S727, and phosphorylation of Y701 is not observed. Upstream, p38 MAPK is phosphorylated to comparable levels in both APCs. However, basal expression of the phosphatase SHP2 is higher in DC. Blocking SHP2 in DC leads to earlier phosphorylation of S727 but no recovery of Y701 phosphorylation or subsequent induction of IFNγ production by NK cells. Thus, additional phosphatases and inhibitors are likely contributing to these signaling events. Collectively, these data demonstrate that targeting both MΦ and DC in vivo is critical for optimal HSP-mediated immune responses.
TRIM14 promotes endothelial activation via NF-κB signaling pathway
Authors: Li Yong , Fan Daping , Fu Mingui
Published: January 2023

Endothelial activation induced by proinflammatory cytokines is closely associated to the pathogenesis of atherosclerosis and other vascular diseases, however, the molecular mechanisms controlling endothelial activation are not fully understood. Here we report that TRIM14 promotes endothelial activation by enhancing cytokine-induced NF-κB activation. TRIM14 is highly expressed in human vascular endothelial cells, and markedly induced by TNF-α, IL-1β and LPS in both HUVECs and aortas of LPS-injected mice. Overexpression of TRIM14 significantly increased TNF-induced expression of adhesion molecules such as VCAM-1, ICAM-1, E-Selectin and cytokines in human endothelial cells, and by which it facilitated monocyte adhesion to the activated endothelial cells. Conversely, its silencing has opposite effect on endothelial activation. We further found that TRIM14 interacted with TAK1 and NEMO to facilitate the IKK complex formation and enhanced cytokine-induced phosphorylation of IκBα and NF-κB nuclear translocation. Moreover, we found that TRIM14 underwent K63-linked ubiquitination during TNF-induced endothelial activation, which is required for its positive role in the regulation of NF-κB signaling and endothelial inflammation. Meanwhile, NF-κB can directly bind to the promoter regions of TRIM14 gene and control its mRNA transcription. Finally, TRIM14 protein levels were significantly up-regulated in mouse and human atheroma compared to normal arteries. Taken together, the current study demonstrates the new role of TRIM14 in the regulation of endothelial activation and suggests that TRIM14 may be a potential therapeutic target for vascular inflammatory diseases such as atherosclerosis.
CFTR, which not only serves as a TCR signaling molecule but also function as an anion channel, dual-negatively regulates IFN-γ production and tumor immunity in γδ T cells
Authors: Duan Yuanyuan , Li Guangqiang , Xu Miaomiao , Yin Zhinan
Published: January 2023

γδ T cells have been demonstrated to have the MHC independent-broad anti-tumor activities with high IFN-γ production in the infiltration of the tumor tissue, therefore, they become the strongest interest for cancer immunotherapy. Ion channels play critical roles in the cell functions, especially in the regulation of immune cells functions. Cystic fibrosis transmembrane conductance regulator (CFTR) is an important chloride channel and regulator in the apical membrane of epithelial cells. Recently, CFTR was reported to participate in the immune regulation and likely account for the risk of developing cancer. However, little is known about the effect of CFTR on γδ T cells tumor immunity. In this study, we would like to elucidate the regulatory mechanisms and indispensable functions of CFTR on γδ T cells in tumor immunity. We found that genetic deletion of CFTR displayed the increased TCR signaling and elevates IFN-γ release in peripheral γδ T cells, but not in CD4+ T cells. The absence of CFTR in γδ T cells strengthened the TCR signaling. The underlying mechanism of higher TCR signaling was likely that CFTR may participate in TCR signaling via interacting with the PLCγ-1-LCK-ZAP70 signaling complex in γδ T cells. Consistent with the data in mice, CFTR inhibition, which increased intracellular Ca2+ concentration, significantly increased the IFN-γ production in γδ T cells. Finally, CFTR−/−γδ T cells displayed higher cytolytic ability against the B16 melanoma and protected against B16 tumor growth. This study systematically defined the roles of CFTR in the activation of γδ T cells and its potential contribution to tumor immunity, which will enrich the γδ T cells biology and expand the sight for the application of CFTR in γδ T cells cancer immunotherapy.
PD-1 is negatively regulated by LSD1 through interactions with Blimp-1 during acute viral infection
Authors: Neeld Dennis Kyle , Bally Alexander , Lu Peiyuan , Majumder Parimal , Barwick Benjamin , Boss Jeremy M
Published: January 2023

Prolonged exposure to antigen, such as during chronic viral infections and cancer, leads to sustained TCR signaling and ultimately T cell exhaustion, which is in part mediated by up regulation of Programmed cell death 1 (PD-1). Antibody blockade of PD-1 does not work in all instances and can lead to autoimmunity, thus identifying novel tools to manipulate PD-1 are needed. Therefore, we sought to understand the genetic and epigenetic mechanisms that regulate PD-1 during acute and chronic antigen conditions in order to identify additional therapeutic targets to modulate PD-1. We determined the chromatin status at the PD-1 locus and found that activating H3K4me1 and H3K4me2 epigenetic modifications were still present at day 8 during a chronic but not acute LCMV infection. This suggested a failure to remove the active histone marks in chronic infections. Indeed, CD8 T cells lacking lysine-specific histone demethylase 1A (LSD1), a H3K4 demethylase, display significantly higher levels of PD-1 mRNA and protein during acute but not chronic infection. Molecular analysis revealed that Blimp-1 recruits LSD1 to the PD-1 locus to induce silencing during acute infection. Despite Blimp-1 binding to the PD-1 locus during both acute and chronic LCMV infection, it fails to recruit LSD1 during the chronic setting. Additionally, failure to remove H3K4 methylation resulted in less DNA methylation at known PD-1 control regions compared to wild-type cells, consistent with the differential expression of PD-1. Taken together, these results demonstrate that LSD1 downregulates PD-1 by interacting with Blimp-1 during acute, but not chronic LCMV infection, facilitating full epigenetic repression of the locus.
Simultaneous analysis of mRNA and proteins in immune cells using the BD&[trade] Single-Cell Multiplexing Kit and BD&[trade] AbSeq reagents on the BD Rhapsody&[trade] system for high-resolution interrogation of differential immune regulation
Authors: Song Hye-Won , Baracho Gisele V , Bansal Nidhanjali , Taylor Ian , Shum Eleen , Widmann Stephanie , Mortimer Stefanie
Published: January 2023

Obesity can weaken the body’s immune system and trigger chronic inflammation. To understand the root cause of this problem, we used single cell sequencing to examine thousands of immune cells isolated from primary and secondary lymphoid organs as well as adipose tissue and compared a diet-induced obesity mouse model with control mice. Cells were stained with 30 DNA-barcoded antibodies from BD™ AbSeq reagents to enable multiomic analysis, i.e. examining protein alongside mRNA expression in tandem. We also utilized DNA-barcoded universal antibodies from the BD™ Single-Cell Multiplexing Kit, which allowed us to combine 8 samples from different mice and tissue types into a single pooled sample, significantly reducing experimental scale and cost while eliminating potential batch effects. The pooled samples were loaded on the BD Rhapsody™ system to perform cell lysis and individual mRNA and cell barcoding, allowing measurement of ~400 immune-related mRNAs and 30 proteins at the single cell level. We were able to de-multiplex the pooled samples with high specificity after sequencing. The targeted mRNA and AbSeq panel provided robust clustering of immune cell types and showed that genes related to critical immune responses, including inflammation and lymphocyte activation, are differentially regulated in specific immune-cell subsets in obese mouse. Using this multiomic analysis of genes differentially regulated in immune cells from different tissues, we propose a model to explain the immuno-phenotype we observed in obese mouse.

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Single cell transcriptomics resolves activation dynamics and cellular states of human blood and tissue T cells
Authors: Szabo Peter Anthony , Levitin Hanna M , Miron Michelle , Snyder Mark E , Senda Takashi , Yuan Jinzhou , Chen Yim Ling , Bush Erin C , Dogra Pranay , Thapa Puspa , Sims Peter A , Farber Donna L
Published: January 2023

T cells persist as heterogeneous subsets throughout the body and are essential in mounting protective immune responses. In healthy humans, most of our knowledge of T cell activation derives from sampling the peripheral blood and therefore the transcriptional states of tissue T cells, their functional responses to stimulation, and how they relate to T cells in blood have been poorly defined. Here, we profile the activation dynamics of T cells isolated from human lungs (LG), lymph nodes (LN), bone marrow (BM) and blood following TCR-stimulation by single cell RNA-sequencing (scRNA-seq). Analysis of >50,000 individual resting and activated T cells using clustering and new factorization methods reveals lineage-specific gene expression signatures and discrete activation trajectories in all tissues. Between sites, T cells from LG and LN are most distinct, while blood T cells are most similar to those in BM but persist in a more activated basal state. We identify a common transcriptional profile of tissue T cells and detect trace numbers of these cells in the blood. We also define cellular states for resting and activated T cells across tissues, including an interferon-induced state in CD4+ T cells and distinct effector states specific to CD8+ T cells, and uncover new markers of T cell activation that may be central to T cell function. Importantly, we demonstrate that the T cell activation states resolved here serve as a new baseline for defining T cell dysfunction in disease, revealing novel insights into T cell states among tumor-infiltrating lymphocytes from previous studies. Our investigation couples scRNA-seq with new analysis methods to define the activation dynamics of human T cells from disparate anatomical sites on a single cell level.
Engaging with Pacific Communities to support effective dialogue, appropriate outreach and scientific knowledge translation activity
Authors: Sika-Paotonu Dianne , Anitelea Toni , Uiese Tina , Cockburn Alana
Published: January 2023

Indigenous Māori and Pacific population groups remain disproportionately affected by Acute Rheumatic Fever (ARF) and Rheumatic Heart Disease (RHD). ARF is an autoimmune response to untreated throat (and skin) infection by Group A Streptococcal bacteria. Untreated and repeated episodes of ARF or a severe attack, can lead to permanent cardiac damage known as RHD. High rates of ARF and RHD persist in New Zealand and the Pacific Region. To help raise awareness and support knowledge translation efforts that facilitate dissemination of research work and findings of relevance to Pacific populations, appropriate community engagement is needed. The purpose of this work was to host a Pacific community event to support appropriate engagement and dialogue between researchers and Pacific communities in the southern Wellington Region that would facilitate communication and discussion of ARF/RHD related Penicillin research works and findings. A Pacific community event was held on 30th July, 2018 in Porirua that involved Pacific researchers, University Pacific staff a Medical student and various Pacific groups in the area. Scientific information and research focused on Immunological components related to ARF and RHD were presented by a Pacific Biomedical Scientist with the audience participating in a lengthy discussion session that followed. Feedback demonstrated those involved had engaged appropriately and successfully with the Pacific community groups represented with requests made for similar future events to be held. The response to this Pacific community event highlights the importance of cultural awareness being combined with appropriate scientific and knowledge translation communication activity to support effective dialogue.
Development and evaluation of an interactive simulation to teach undergraduate immunology students all stages of monoclonal antibody (mAb) production
Authors: Costabile Maurizio
Published: January 2023

Monoclonal antibodies (mAbs) have revolutionised science since the method was first reported in 1975. The resultant impact of this technique led to Köhler and Milstein being jointly awarded the Nobel prize in 1984. Given the wide use of mAbs in science, it is important that undergraduate immunology students understand how they are produced including the principles of the selection process. Past experience has demonstrated that some undergraduate Immunology students at the University of South Australia (UniSA) experienced difficulties in learning these principles. While there are numerous resources available to students on the internet, they have their shortcomings, such as the focus being on the selection process alone, omitting the prior steps involved and all these resources are completely passive ways of learning. In an attempt to enhance undergraduate student learning of all aspects of mAb production, an interactive simulation was developed. The impact of the simulation was evaluated in 2017 and 2018 with significant improvements in student performance in a quiz and end of semester examination seen for those students that made use of the simulation. Anonymous student feedback was overwhelmingly positive, with students enjoying the new teaching approach. I suggest that interactive simulations can enhance the teaching of complex immunology topics to undergraduate students. The data clearly shows that this approach has significantly enhanced student learning and now forms a core part of teaching mAb production at UniSA. The simulation is freely available to any teaching staff that wish to include it as part of their teaching approach.
Potassium efflux and serum amyloid A3 are involved in IL-1β production by macrophages infected with Mycobacterium tuberculosis
Authors: Jung Bock-Gie , Samten Buka
Published: January 2023

While interleukin (IL)-1β is essential for the protective immunity against tuberculosis infection, it also exacerbates the pathology of tuberculosis. Although previous studies suggested that early secreted antigenic target of 6-kDa (ESAT-6) of Mycobacterium tuberculosis (Mtb) is a potent activator of NLRP3 inflammasome for IL-1β maturation, the mechanisms remain unclear. Therefore, we studied IL-1β production by C57BL/6 mouse bone marrow-derived macrophages (BMDM) infected with Mtb H37Rv, its esat-6 deletion mutant or esat-6 complemented strain. Our data clearly showed that BMDM infected with esat-6 deletion mutant produced significantly reduced IL-1β compared with the cells infected with H37Rv and esat-6 complemented strain despite having comparable levels of pro-IL-1β, implying that ESAT-6 is critical for the maturation but not synthesis of pro-IL-1β. IL-1β production by Mtb-infected BMDM is inhibited by MCC950, a selective NLRP3 inhibitor. Interestingly blocking K+ efflux by high concentrations of extracellular K+ or quinine, an inhibitor of TWIK2 K+ efflux channel, inhibited IL-1β production, but increased bacilli growth in BMDM. Infection of mice with esat-6 deletion mutant showed defects in lung IL-1β production compared with H37Rv. Recombinant ESAT-6 induced IL-1β production in mouse lung. RNA-seq analysis identified serum amyloid A (Saa) 3 as one of the most highly expressed genes in ESAT-6 inoculated mouse lung. Silencing Saa3 in BMDM led to reduced IL-1β production by H37Rv or ESAT-6. These data together imply that Mtb induces secretion of IL-1β in an ESAT-6 dependent manner with the involvement of NLRP3, Saa3 and K+ efflux channel which is also required for Mtb growth control in macrophages.
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

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

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

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.