bluuebunny/crossref_metadata_2025 huggingface dataset:
ICOSL-mediated signaling is essential for the survival and functional maturation of germinal center B cells through the classical NF-κB pathway (IRM10P.611)
Authors: Zheng Biao , Xu Guiliang , Chen Xi , Marinova Ekaterina , Han Shuhua
Published: January 2023

The molecular mechanisms by which helper T-cells support B-cell activation, survival and functional differentiation in the germinal center (GC) are not well-defined. To date, signaling through CD40 by CD40L is the only established receptor-ligand signaling pathway initiated by helper T-cells. Here we show that helper T-cells provide helper signal to B-cells through the inducible T-cell co-stimulator (ICOS)-ICOS ligand (ICOSL) interaction. We found that reverse ICOSL signaling on GC B-cells is essential and sufficient to promote GC B-cell survival and functional maturation; whereas CD40-mediated signaling plays a minor role during GC reaction, although critical in initiating the GC response. In vivo ICOSL engagement by ICOS-Ig fusion protein can rescue impaired GC formation and antibody production in ICOS-deficient mice. Various lines of ICOSL mutant mice, including pan-B cell-specific ICOSL-deficient mice, GC B cell-specific ICOSL-deficient mice, and ICOSL intra-cellular domain mutant mice, all show defective humoral response and impaired GC reaction. ICOSL reverse signaling in GC B cells is dependent on activation of the classical NF-kB signal pathway, resulting in increased BCL2, c-MYC and FOXO1 expression that supports GC B cell survival and differentiation. Thus, CD40-CD40L interaction mainly supports the initiation of GC response, whereas ICOS-ICOSL collaboration provides bi-directional signals that support the maintenance and functional maturation of GC B cells.
Boosting antitumor immunity through TIM1 (IRM11P.631)
Authors: chen xiaojuan , Wang Xuefeng , Lu Binfeng
Published: January 2023

The lack of co-stimulation, as well as the dominance of co-inhibition, of type 1 lymphocytes, such as CD8+ T cells and NK cells, is a principal immunosuppressive mechanism in the tumor microenvironment. Conversion of the tumor immunosuppressive microenvironment to one that favors cancer-eradicating immune responses is therefore a goal for the development of more effective cancer immunotherapy. Targeting co-stimulatory pathways represents one attractive therapeutic strategy. T cell Ig mucin (TIM) proteins are structurally related to type I membrane glycoproteins. Previous studies have shown that TIM-1 is highly expressed on activated CD4+ T cells, facilitates the activation of Th1 cells, Th17 cells, and Th2 cells, and is implicated in the pathogenesis of human allergy and autoimmune diseases. These data support an important role for TIM-1 in regulating CD4+ T cell function. However, the role for TIM-1 in CD8+ T cell- or NK cell-mediated immune responses, including antitumor immune responses, is unknown. Here we showed that tumoral expression of antitumor cytokines such as IL-12 and IL-33 upregulated TIM-1 on tumor-infiltrating CD8+ T cells and NK cells. In addition, we demonstrated that TIM1 mAbs greatly inhibited tumor growth, resulting in greatly elevated cellular immune responses against tumor. The in-depth mechanistic insights and implication in tumor immunity are gained by our studies. Our study establishes TIM-1 as a potential target for enhancing antitumor immunity.
Sodium arsenic and ultraviolet-radiation synergistically induce cell injury in lens and retinal epithelial cells occurring migration of HMGB1 (IRM11P.639)
Authors: Park SuJung , Shin Jeon-Soo
Published: January 2023

Although sodium arsenic (As) and ultraviolet radiation (UVR) can induce cell injury through either DNA damage or reactive oxidative species (ROS) in various epithelial cells, whether damaged-associated molecular pattern (DAMP) involved in cell injury is still unclear. To determine the association of HMGB1, one of DAMP molecules that can be secreted by oxidative stress, we investigated cell injury in human lens epithelial cell (HLE B3) and human retinal epithelial cell (ARPE-19) using cell viability assay, western blot, confocal microscopy and flow cytometry (FACS analysis). In both HLE B3 and ARPE-19 cells, DNA damage marker, cleaved PARP and cell viability assay showed that UVR synergistically increased As-caused cell death(injury). During UVR and As-caused cell injury, HMGB1 was translocated from nucleus to cytosol and then secreted to extracellular region. ROS scavenger N-acetylcystein (NAC) and mito-tempo inhibited both translocation and secretion of HMGB1 in dose-dependent manner, implicating that migration of HMGB1 is closely associated with ROS generation in mitochondria. Taken together, we concluded that HMGB1 is associated in cell injury caused by UVR and As. Nevertheless how migrated HMGB1 plays a role in UVR-As caused cell injury of HLE B3 and arpe-19 cells is still unclear.
TLR9 plays a critical role in sepsis-induced inflammatory response (IRM11P.623)
Authors: Yin Deling , Denney James , Yan Hui , Hu Dan , Caudle Yi , Li Hui
Published: January 2023

Sepsis, a major clinical problem with high morbidity and mortality, is caused by overwhelming systemic host-inflammatory response. Toll-like receptors (TLRs) play a fundamental role in induction of hyperinflammation and tissue damage in sepsis. In this study, we demonstrate a protective role of TLR9 inhibition against the dysregulated inflammatory response and tissue injury in sepsis. TLR9 deficiency decreased the mortality of mice following cecal ligation and puncture (CLP) -induced sepsis. TLR9 knockout mice showed dampened p38 activation and augmented Akt phosphorylation in the spleen, lung and liver. In addition, TLR9 deficiency decreased the levels of inflammatory cytokines and attenuated splenic apoptosis after CLP. These results indicate that TLR9 inhibition might offer a novel therapeutic strategy for the management of sepsis.
Exosomal miRNAs regulate inflammatory responses (IRM11P.624)
Authors: Alexander Margaret , O'Connell Ryan
Published: January 2023

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression. Typically, miRNAs are thought to function within the cells where they are transcribed. However, recent data shows that miRNAs are transferred between cells and mediate target gene repression. miR-155 and miR-146a are critical miRNAs that regulate inflammation. We found that miR-155 and miR-146a were released from dendritic cells (DCs) within exosomes and transferred to recipient DCs. These miRNAs were associated with RISC and inhibited their mRNA targets in recipient cells. Following uptake, miRNAs reprogramed the cellular response to endotoxin, where exosomal miR-155 enhanced and miR-146a reduced inflammatory gene expression. miR-155 and miR-146a were also found in exosomes and passed between immune cells in vivo. Additionally, injection of miR-146a containing exosomes delivered miR-146a to various tissues and decreased inflammation in response to endotoxin, while miR-155 containing exosomes had the converse effect. This suggests that exosomal miRNAs could be used as therapy for inflammatory diseases. Further characterization of the loading and functional transfer of miRNAs should make therapeutic approaches more feasible. Additionally, we aim to explore the importance of endogenously transferred miRNAs during immune responses in vivo. Our research will help unravel both the therapeutic potential of exosomal miRNAs and determine the biological relevance of transferred miRNAs in immune responses.
Down regulation of genes related to selenium signaling pathway in in-vivo transmigrated neutrophils from patients with Chronic Kidney Disease (IRM11P.627)
Authors: MANSOURI LADAN , Moshfegh Ali , Hylander Britta , Jacobson Stefan , Lundahl Joachim
Published: January 2023

The most common causes of morbidity in patients with Chronic Kidney Disease (CKD), emerge from the dysregulation of immune system. Granulocytes are less functional, in terms of transmigration to the inflammatory sites. We analyzed the gene expression of neutrophils from peripheral blood, and in-vivo transmigrated neutrophils, collected by skin chamber technique, in CKD patients as well as healthy controls. We found that, transmigration induced a significant change in the gene expression, and kidney failure had different impacts on the gene expression of neutrophils in peripheral blood, and upon in vivo transmigration, comparing to healthy subjects. After Cluster analysis of the significant genes, and subjecting them to signaling pathways, we found selenium signaling pathway as one of those, which were significantly impaired in transmigrated neutrophils from CKD patients. This comprised down regulation of genes such as; Glutathione peroxidase-1 (GPx-1), Superoxide dismutase-2 (SOD2), Peroxiredoxin-1(PRDX1), and Selenoprotein W (SelW) gene. Selenoproteins play a major role in antioxidant defense, thus our results may indicate that the CKD patients have a deficiency in these components, leading to the oxidative stress in neutrophils, and their impairment. These findings, however, warrant further investigation, and we are currently in the process to setup an in-vitro system to analyze the effect of selenium compounds on different functions of neutrophils from CKD patients.
Human tonsil naive B cells and germinal center B cells show distinct behaviors upon contact of antigen in lipid bilayers
Authors: Kwak Kihyuck , Sohn Haewon , Brzostowski Joseph , Manzella-Lapeira Javier , Pierce Susan K
Published: January 2023

During antibody responses to T-dependent antigens B cells undergo clonal expansion and somatic hyper-mutation in the dark zones of germinal centers (GCs) and subsequently undergo isotype switching and affinity based selection in the GC light zones. Only a small portion of B cells survive from GC reactions and differentiate into either plasma cells or memory B cells. However, the mechanism underlying selection within the GC is not fully understood. We compared the behavior of human tonsil naïve (IgD+, CD10−) and GC (IgD−, CD10+) B cells as they engaged antigen (using Light chain specific antibodies as a surrogate antigen) incorporated into planar lipid bilayers using a combination of TIRF and confocal microscopy. We observed marked differences in the cellular dynamics of naïve and GC B cells. Upon touching the antigen-containing bilayer naïve B cells rapidly form B cell receptor (BCR) micro-clusters that grow rapidly with time and signal for Ca++ fluxes. Within five minutes naïve B cells formed stable areas of contact with the bilayers. In contrast, GC B cells only slowly form BCR micro-clusters that grow slowly with time and signal for delayed Ca++ fluxes. Notably, the behavior of GC B cells over the same time period is far more dynamic with only transient interactions with the bilayer and active ruffling in the cells periphery suggesting that the GC B cells in contrast to naïve B cells continue to sample antigen for longer periods of time. Experiments in progress are aimed at relating the observed behaviors to the affinity of naïve and GC B cells for antigen.
Retinoic acid regulates immune responses by promoting IL-22 production and modulating S100 protein in viral hepatitis
Authors: Liang Yuejin , Jie Zuliang , Yi Panpan , Wan Xianxiu , Wang Wei , Tang Hui , Kwota Zakari , Soong Lynn , Cong Yingzi , Zhang Kangling , Sun Jiaren
Published: January 2023

The effector T cell responses promote viral clearance and disease resolution in viral hepatitis, but can also mediate tissue damage; however, much less is known as to how the liver protects itself against the injury. In this study, we have revealed a retinoic acid (RA)-mediated, hepatoprotective mechanism in adenovirus-induced hepatitis in mice. We found that RA treatment promoted hepatoprotective cytokine IL-22, but inhibited the pro-inflammatory factor IL-17, from gd T and double-negative T cells, and that the hepatic IL-17 and IL-22 production were regulated via the mTOR/PI3K signaling pathway. Moreover, we found that RA modulated the magnitude of antigen-specific T cell responses via down-regulating dendritic cell (DC) co-stimulatory molecule expression and its migratory capacity. Mechanistically, RA treatment inhibited Calcium-binding S100 family proteins (S100A4/6/10) and the NF-κB/ERK signaling pathways on DCs. The inhibition of novel S100A4 resulted in impaired DC migration from inflamed tissues to lymph nodes for T cell priming. Collectively, our study has revealed a previously unappreciated role and molecular mechanism of RA in modulating immune responses and protecting the liver from viral hepatitis.
IL-6 influences the balance between M1 and M2 macrophages in a mouse model of irritant contact dermatitis
Authors: Luckett-Chastain Lerin , Calhoun Kaitlin , Schartz Tayler , Gallucci Randle M
Published: January 2023

Irritant contact dermatitis (ICD) is an acute inflammatory response that is ranked the 2nd most prevalent occupational injury associated with workman’s compensation. Interleukin-6 (IL-6), a pro-inflammatory cytokine produced in the skin, is closely associated with healing and decreased levels are associated with more severe ICD. The phenotypic nature of the macrophage infiltrate during skin inflammation can greatly influence damage and healing during ICD. The pro-inflammatory, classically activated (M1) macrophage produce cytokines such as IL-6, IL-1, and TNFα, while alternatively activated (M2) macrophages produce IL-10 and TGFβ, and are thought to be associated with tissue repair. IL-6 may play a regulatory role in mediating the M1 versus M2 phenotypic change in skin. To examine the role of IL-6 in ICD, IL-6KO and C57 mice were exposed to benzoalkonium chloride (BKC) for 7 days. IL-6KO mice displayed decreased expression of CD206 (M2) as compared to CD86 (M1) and the ration of CD86:CD206 was significantly increased in IL-6KO mice compared to control. Multiplex protein analysis showed expression of multiple M1-related cytokines such IFN-g, CCL3, CCL4, CCL5, and CCL7 significantly modulated in IL6-KO BKC treated mice compared to C57. Additionally, M2 related cytokine/chemokine receptors IL-4R and CCR1 were significantly dysregulated in IL-6KO BKC treated mice. IL-6 treatment of RAW cells enhanced the expression of CD206 in manner similar to IL-4. Overall, these results further confirm a role for IL-6 in macrophage polarization and could eventually result in the development of therapies for dermatitis based on this cytokines activity.
Novel IL12Rβ1 mutation impairs Th1 response in heterozygote patient.
Authors: Sonder Soren Ulrik , Plassmeyer Matthew , Alpan Oral
Published: January 2023

The interleukin-12 Receptor beta-1 chain (12Rβ1) is a subunit in the functional IL12 and IL23 receptor that binds the IL12 p40 subunit of the IL12 and IL23 heterodimers. Patients that are homozygote for “lack of function” mutations in IL12Rβ1 are mainly characterized by defects in the Th1 response resulting in recurrent and severe disease caused by infections with poorly pathogenic mycobacteria and salmonellae. Family members that are heterozygote for these mutations are clinically unaffected.

Here we describe a case of a 62 year old female patient that is heterozygote for the novel 12Rβ1 mutation Q238E located in the cytokine binding region. The patient has recurrent infections, poor wound healing, osteomyelitis and persistently positive PPD. Lymphocyte count in the lower end of the normal range. Using flow cytometry analysis of whole blood we showed above normal percentages of NK cells and below normal percentages of T-cells. The patients Th1 response was studied by stimulating PBMC from the patient and healthy controls with IL12 and measuring IFNγ in multiple cell types using flow cytometry. The surface expression of IL12Rβ1 does not differ between the patient and controls; however the results demonstrated a reduction in IFNγ producing CD4+ and CD4+/CD45RO+ cells in the patient compared to the controls.

Functional screening for IL12 response may provide a powerful tool in evaluating other patients with recurring infections resulting in more accurate diagnose and efficient treatment.
IL-4R signaling in B cells mediates control of IL-17A dependent emphysema triggered by N. brasiliensis infection.
Authors: Chen Fei , Wu Wenhui , Millman Ariel , Palma Mark J , Gause William C
Published: January 2023

The intestinal nematode parasite, Nippostrongylus brasiliensis (Nb), transiently resides in the lung and causes lung damage, which is then rapidly repaired. However, the repaired lung eventually develops emphysema and the mechanisms contributing to this chronic lung pathology remain poorly defined. Here we show that emphysema, as measured by mean linear intercept measurements (Lm) of alveolar spaces, develops as early as 7 days after Nb inoculation and is associated with increased gene expression of Il17A, neutrophil elastase (ELA), and macrophage matrix metalloproteinase 12 (Mmp12) in the lung. Transfer of Nb-primed neutrophils or macrophages to naïve recipients results in emphysema, and these cells expressed high level of ELA and Mmp12, respectively. Neutralization of IL-17A using anti-IL-17A antibody curbed development of emphysema, and decreased influx of neutrophils in the lung and gene expression of Il17A. Parasite infection in B cell deficient Jh−/− mice results in a more severe emphysema when compared to wild-type mice or FcRγ−/− mice, and is associated with higher numbers of neutrophils and macrophages, as well as increased expression of Il17a, ELA2 and Mmp12 in the lung. Transfer of B lymphocytes from wild-type mice or IL-10−/− mice, but not from IL4Rα−/− mice, to recipient Jh−/− mice can restore control of emphysema and downregulate IL-17A. B cells from lung tissue of Nb-primed wild type but not IL4Rα−/− mice expressed high level of Relmα. Taken together, these data suggest that IL-17A triggers infiltration of neutrophils and macrophages that contribute to chronic emphysema, which is controlled by IL-4R dependent B regulatory cells expressing high levels of Relmα.
Characterization of Th17-like Tregs during late stages of infection with B. pertussis in mice. Possible immunomodulation by type I interferon.
Authors: Smith Andrew A , Jakoush Tarek A , Silva Elvia , You Hana , Wu Victoria , Segal-Abramson Tzvia
Published: January 2023

Whooping cough is a highly contagious respiratory disease caused by Bordetella pertussis (Bp). Recently, the prolonged nature of whooping cough has been associated with a delay in Th17 responses, which are essential to resolve the infection. While investigating the IFNα-driven delay in Th17 responses during Bp infection, we noted a parallel rise of Th17-like Tregs secreting IL-17. Similar T cell hybrids have demonstrated either inflammatory or suppressor function.

In this study we phenotypically and functionally characterized Th17-like Tregs in mice infected with Bp to test the hypothesis that these cells are derived from Tregs and display a suppressor role to specifically control the robust rise in Th17 inflammation during late stages of infection.

Phenotypic analysis of the Th17-like Tregs revealed high frequencies expressing cytokines and trafficking markers similar to that of Th17. However, the abundance of Th17-like Treg expressing regulatory markers such as CTLA-4, OX-40 and PD-1, resemble a Treg profile. Furthermore, Th17-like Tregs express the thymic-derived nTreg marker, Helios, and were detected in the lungs during late infection but not in the blood or thymus, which suggests their derivation from lung nTregs. When IFNα was spiked into a Th17-polarized allogeneic assay, T-cell differentiation skewed towards Th17-like Tregs. In vitro functional analyses of these cells confirmed their suppressive nature. These data imply that there is a balance between IFNα Treg-favoring conditions and a Th17-polarizing environment that push Tregs to a Th17-like state during Bp infection.

This study contributes valuable information about adaptive IL-17 responses that are crucial to the resolution of whooping cough.
Sirt1 decreases TLR2-mediated Mac-1 expression in atherosclerosis
Authors: LEE SEUNG-JIN , KIM CHI DAE
Published: January 2023

Sirtuin (Sirt) 1 has been demonstrated to exerts protective effects in the progression of atherosclerosis, however, the underlying mechanisms are unclear. Thus, this study investigated the the role of Sirt1 in TLR2-mediated monocyte adhesion to vascular endothelium, which is a key early event in macrophage foam cell formation. The atherosclerotic lesion formation in aortic sinus was significantly increased in Pam3CSK4-treated wild type (WT) mice, which was significantly inhibited in Sirt1 Tg mice group. An en face immunohistochemistry of endothelial surfaces revealed a marked increase in monocyte adhesion to aortic endothelium in WT mice treated with Pam3CSK4, which was significantly attenuated in Sirt1 Tg mice. Likewise, the adhesion capacity of primary monocytes isolated from Pam3CSK4-treated WT mice was higher than those of monocytes from Sirt1 Tg mice. In in vitro study, Pam3CSK4 increased monocyte adhesion to endothelial cells with an enhanced Mac-1 expression. These were attenuated by a recombinant Sirt1, as well as by molecular overexpression of Sirt1 in monocytes. In addition, we found that Pam3CSK4-reduced Sirt1 expression in monocytes was regulated by the transcription factors, NF-kB and CREB. The role of NF-kB and CREB in Pam3CSK4-reduced Sirt expression was confirmed by siRNA knockdown of Sp1 and NF-kB. Sirt1 supresses atherosclerosis progression by TLR2-mediated monocyte adhesion to endothelium through Mac-1 expression, which is mediated by NF-kB and CREB pathways in monocytes, consequently leading to the development of therapeutic interventions for regulating Sirt1 expression in atherosclerosis.
Role of oxidative stress in the suppression of immune responses in peripheral blood mononuclear cells exposed to combustible tobacco product preparations
Authors: Arimilli Subhashini , Damratoski Brad , Prasad G.L.
Published: January 2023

Cigarette smoking is a major risk factor for several human diseases. Chronic inflammation, resulting from increased oxidative stress, has been suggested as a mechanism that contributes to the increased susceptibility of smokers to cancer and microbial infections. We have previously shown that whole smoke-conditioned medium (WS-CM) and total particulate matter (TPM) prepared from Kentucky 3R4F reference cigarettes potently suppressed agonist-stimulated cytokine secretion and target cell killing in peripheral blood mononuclear cells (PBMCs) under ex vivo conditions. Here, we sought to investigate the role of oxidative stress from cigarette smoke exposure in the compromised immunity observed in smokers. Particularly, we investigated the mechanisms of WS-CM and TPM induced suppression of select cytokine secretions in TLR agonist-stimulated cells, and target cell killing by effector cells in PBMCs. Pre-treatment with N-acetyl cysteine (NAC), a precursor of reduced glutathione and an established antioxidant, protected against DNA damage and cytotoxicity caused by exposure to WS-CM and TPM. Similarly, secretion of IFN-γ, TNF, IL-6 and IL-8 in response to TLR-4 stimulation was restored by NAC. Target cell killing, which is used as a functional measure of cytolytic cells in PBMCs, is suppressed by WS-CM. Pretreatment with NAC restored the target cell killing in WS-CM treated PBMCs. This was accompanied by higher perforin levels in the effector cell populations. Collectively, these data suggest that reducing oxidative stress caused by cigarette smoke components restores select immune responses in this ex vivo model.
Myeloid cell regulation by CD200 signalling in atherosclerosis
Authors: Kassiteridi Christina , Cole Jennifer , Goddard Michael , Green Patricia , Park Inhye , Danso-Abeam Dina , Monaco Claudia
Published: January 2023

Background

Atherosclerosis, the major risk factor for cardiovascular disease and the leading cause of death worldwide, is a multifactorial chronic inflammatory disease. Deletion of CD200 in vivo increases myeloid cell numbers and activation resulting in enhanced susceptibility to autoimmune diseases and infection. However, the importance of CD200 in atherosclerosis development is still unknown.

Methods and Results

To understand the role of CD200 signalling, both the effect of CD200 deletion and provision were assessed in a murine model of atherosclerosis. Firstly, CD200 deficiency in hypercholestorelemic apolipoprotein E deficient (ApoE−/−) mice accelerates advanced atherosclerotic lesion formation in the aortic roots. Moreover, APOE−/− CD200−/− mice exhibit significant increase in specific myeloid cell populations in spleen, blood and aorta. Secondly, the role of CD200R ligation has been examined in a murine model of carotid injury. ApoE−/− mice underwent surgery for placement of a perivascular collar and were treated with 10mg/kg of a CD200-Fc fusion protein. CD200-Fc fusion protein treatment attenuates neointima development and, interestingly, affects macrophage accumulation and polarization.

Conclusions

Our data indicate that CD200 is an important modulator of myeloid cell function and phenotype in atherosclerosis and suggest that targeting the CD200-CD200R pathway holds promise as a potential therapeutic strategy in atherosclerosis.
Prenatal methyl-donor supplemented diet increases endoplasmic reticulum stress and inflammation in adipose tissue CD11b+ cells in F1 mice
Authors: Mau Theresa , Ghosh Amiya , O’Brien Martin , Yung Raymond
Published: January 2023

The risk for development of metabolic and cardiovascular diseases in later life is increasing, as the population is becoming overweight and obese at earlier ages. Early-life nutritional programming influences late-life disease susceptibility across generations, in part through epigenetic mechanisms including DNA methylation. Using mouse models, it has been shown that prenatal diet rich in methyl donors (L-methionine, vitamin B-12, folic acid, choline, betaine, zinc) can increase airway reactivity but improves cardiovascular disease outcomes in F1 mice. Here, we used a prenatal diet enriched with methyl-donors to study the impact of the prenatal diet in the context of diet-induced obese F1 C57Bl/6 mice fed a 40% high-fat diet (HFD) for 16 weeks. F1 mice exposed to the prenatal methyl donor supplemented diet have increased number of adipose tissue ‘crown-like structures’ consisting of primarily CD11b+ cells. The prenatal diet was found to increase the expression of adipose tissue endoplasmic reticulum (ER) stress response genes Chop, Bip, and Atf4 in HFD F1 mice. Obesity-induced ER stress has been linked to adipose tissue chronic inflammation. We have previously established that ER stress contributes to aging adipose tissue macrophage inflammation. Interestingly, Mcp-1 and Tnfα gene expression were found to be increased in the CD11b+ cells of F1 mice adipose tissue. The results strongly support an important role of prenatal methyl-donors exposure in modulating diet-induced obesity ER stress response and inflammation in offspring.
Stress-induced mast cell activation contributes to atherosclerotic plaque destabilization
Authors: Lagraauw Max , Wezel Anouk , van der Velden Daniel , Kuiper Johan , Bot Ilze
Published: January 2023

Mast cells accumulate in the perivascular tissue during plaque progression and upon activation release pro-inflammatory cytokines and extracellular matrix degrading enzymes which fuel the inflammatory process and decrease lesion stability. Interestingly, mast cells have been shown to co-localize with peripheral neurons and contain receptors for various hormones and neuropeptides which are released upon exposure to stress. Therefore, we hypothesize that stress-induced neuronal activation of mast cells contributes to plaque destabilization.

In apoE−/− mice the acute stress induced by 120′ restraint caused a significant increase in mast cell activation in the heart (37.3 ± 1.8% vs. 50.1 ± 4.9%). In parallel with a rise in serum corticosterone levels we observed a transient increase mast cell-specific β-hexosaminidase levels and the pro-inflammatory cytokine, interleukin-6 in the stressed mice compared to controls (55.9 ± 11.0 pg/ml vs. 29.6 ± 5.49 pg/ml). Subsequent characterization of atherosclerotic lesions in the aortic root revealed a significant reduction in lesion collagen content and a higher incidence of intraplaque hemorrhages, a hallmark of the vulnerable plaque (38.5% vs. 7.7%).

Importantly, both these effects were reduced in mice treated with the mast cell stabilizer cromolyn and completely abolished in mast cell deficient (apoE−/−/Kit(W-sh/W-sh)) mice, thus strongly indicating the involvement of a mast cell-dependent response to stress in atherosclerotic plaque destabilization.

We demonstrate that acute stress activates mast cells near atherosclerotic lesions and contributes to plaque destabilization, identifying acute stress as a risk factor for acute cardiovascular syndromes.
Regulatory T cells and IL10 suppress pulmonary host defense during early-life exposure to combustion generated radical containing ultrafine particulate matter.
Authors: Jaligama Sridhar , Yadav Nikki , Minso Jagila , Saravia Jordy , Lee Greg , Cormier Stephania
Published: January 2023

Exposure to elevated levels of particulate matter (PM) is associated with adverse respiratory health and increased risk of morbidity and mortality due to respiratory tract viral infections in infants. Our previous studies demonstrated combustion derived PM (CDPM) induced immunosuppression allowing for enhanced influenza disease severity and mortality in neonatal mice. Current studies are aimed at understanding the underlying mechanisms. Neonatal mice (3 day old) were acutely exposed to Air or DCB230 (CDPM), and infected with influenza virus (Flu), Air/Flu and DCB/Flu respectively. Pulmonary T cell profile and immunosuppressive cytokines were analyzed. At 10 days-post exposure, a significant increase in pulmonary Tregs and IL10 coinciding with decrease in protective T helper 1 (Th1) and cytotoxic T cell (Tc1) responses was observed in DCB/Flu mice compared to Air/Flu mice. Further, depletion of Tregs (using anti-CD25 antibody) in DCB mice prior to flu infection increased both Th1 and Tc1 cells, whereas adoptive transfer of DCB-induced Tregs into Air mice and later infection with flu suppressed these responses. To elucidate the role of IL10 in DCB/Flu mice, Air exposed neonatal mice were treated with recombinant IL10 (rIL10) and subsequently infected with flu (rIL10/Flu). rIL10/Air/Flu mice had increased morbidity and pulmonary viral load compared to Air/Flu mice, whereas, IL10 deficient DCB/Flu mice exhibited reduction in pulmonary viral load and morbidity compared to wild-type mice indicative of enhanced protection against flu. Together, these studies demonstrate that PM- induced Tregs and their effector cytokine, IL10, suppress adaptive T cell responses leading to increased influenza severity in infected neonatal mice.
Alveolar macrophages regulate granulomatous inflammation in a mouse model of chronic beryllium disease.
Authors: McKee Amy , Mack Douglas , Fontenot Andrew
Published: January 2023

Chronic beryllium disease is a granulomatous lung disease orchestrated by beryllium-specific CD4+ Th1 cells. In addition to its role as a pathogenic antigen, beryllium has potent effects on innate immune cells. In mice, beryllium exposure causes cellular death and release of alarmins, including IL-1α and DNA. This is associated with accelerated migration of pulmonary dendritic cells from the lung to the draining lymph nodes, upregulation of costimulatory molecules on their surfaces and enhanced priming of effector Th1 cells. These effects are MyD88-dependent but independent of the inflammasome. Our previous characterization of a mouse model of berylliosis showed that in contrast to WT mice, HLA-DP2 transgenic mice exposed to beryllium develop beryllium-specific Th1 responses and peribronchovascular infiltrates in the lung. We wanted to investigate the role of alveolar macrophages in this model using depletion with clodronate liposomes. Intratracheal instillation of mice with clodronate liposomes depleted alveolar macrophages but dendritic cell migration to the draining lymph nodes remained intact. HLA-DP2 transgenic mice depleted of alveolar macrophages generated beryllium-specific Th1 responses in both lung and spleen that were similar to those in mice treated with control PBS-containing liposomes. Surprisingly, however, the depletion of alveolar macrophages severely exacerbated the development of granulomas in the lung. These results implicate alveolar macrophages in regulating granulomatous inflammation in the lung.
Sirt1 modulates AID expression, class switch DNA recombination and somatic hypermutation by repressing NF-kB activity
Authors: shen Tian , Sanchez Helia Nasrollahi , Zan Hong , Casali Paolo
Published: January 2023

As we have suggested, epigenetic factors, such as histone deacetylases (HDACs), play critical roles in modulating the maturation of the antibody response. This entails class switch DNA recombination (CSR) and somatic hypermutation (SHM). CSR and SHM require AID and diversify the specificity and effector functions of antibodies. By showing that HDAC inhibitors (HDIs) that selectively block Classes I/II HDACs (the “classical” Zn-dependent HDACs together with Class IV HDAC), impaired AID and Blimp1 expression, CSR, SHM and plasma cell differentiation, we have previously outlined an important role of these Zn-dependent HADCs in the modulation of antibody response. We found that Sirt1, one of the NAD+-dependent Class III HDACs or sirtuins (Sirts), played an important role in the regulation of CSR and SHM. Sirt1 deacetylated p65, an element of the canonical NF-kB pathway, which mediates AID induction, thereby repressing NF-kB activity. Sirt1 deficiency in B cells led to NF-kB hyperacetylation and activation, which resulted in enhanced AID expression and increased CSR to IgG, IgA and IgE. Conversely, Sirt1 deletion in AID expressing B cells of AicdacreSirt1fl/fl mice, whose B cells contain the normal complement of Aicda genes together with a Cre gene under control of an extra Aicda promoter, resulted in upregulated AID expression, increased CSR and SHM, and enhanced T-dependent and T-independent antibody response. These findings outline an important B cell-intrinsic role of Sirt1 in the modulation of the antibody response. Such a role is independent from and may be in addition to the histone deacetylation activity of this Class III HDAC.

Supported by the NIH grants AI 105813, AI 079705 and ALR TIL grant ALR 295955.
Ezrin regulates inflammation by limiting B cell interleukin-10 production
Authors: Gupta Neetu , Pore Debasis , Matsui Ken , Parameswaran Neetha
Published: January 2023

In addition to their essential role in antibody production, B cells regulate immune responses by secreting effector cytokines. Interleukin 10 (IL-10) produced by B cells is important for controlling inflammation and autoimmunity, underscoring the importance of identifying mechanisms regulating B cell IL-10 production. We have previously demonstrated that the membrane-cytoskeleton linker protein ezrin regulates B cell antigen receptor organization and signaling. Further, we have shown that ezrin undergoes rapid dephosphorylation in naïve B cells treated with LPS, suggesting that it may regulate TLR4-mediated IL-10 production. In the present study, we demonstrate that conditional deletion of ezrin in B cells increases IL-10 production induced by TLR4, but not TLR2 or TLR9, ligation. The MyD88-independent TRIF-TBK1-IRF3 pathway is required for ezrin-deficient B cells to produce higher IL-10 upon LPS stimulation. Treatment of B cells with a novel small molecule inhibitor of ezrin induces its dephosphorylation, and increases LPS-induced NF-κB and IRF3 activation and IL-10 secretion, indicating a role for Thr567 phosphorylation of ezrin in limiting IL-10. Loss of ezrin in B cells results in dampened pro-inflammatory response to a sub-lethal dose of LPS in vivo, which is dependent on increased IL-10 production. Taken together, our data yield new insights into molecular and membrane-cytoskeletal regulation of B cell IL-10 production, and reveal ezrin as a potential therapeutic target in inflammatory diseases.
Hotspots for hormone response elements in Sα switch regions of immunoglobulin heavy chain loci; how estrogen may influence class switch recombination (CSR) and IgA/IgG isotype expression by activated B cells
Authors: Hurwitz Julia L , Jones Bart G. , Sealy Robert E , Xu Beisi , Fan Yiping , Partridge Janet F. , Maul Robert W. , Gearhart Patricia J , Penkert Rhiannon
Published: January 2023

B cell immune responses differ between genders. To investigate mechanisms responsible for these differences, we activated murine B cells with mitogens in vitro in the presence or absence of hormones. We found that additions of estradiol and thyroid hormones to mitogen-activated B cell cultures altered IgA/IgG expression patterns. Chromatin immunoprecipitation (ChIP) analyses then showed that estrogen receptor (ER) associated with the anti-apoptotic bcl-2 gene in LPS-stimulated murine B cells, but the interaction was inhibited by estrogen, possibly explaining variant isotype expression patterns. In search of additional mechanisms, we queried mouse and human immunoglobulin heavy chain switch (S) regions for potential hormone response elements (HRE). In both human and mouse loci, the DNA sequences of Sμ and Sα encompassed an extraordinary number of HRE. For example, there were more than 70 potential estrogen response elements (ERE, G/AG/AT/CT/CG/AnnnTGAC/GC) in Sα regions of both human and mouse loci, yet not a single ERE in Sγ2. Results suggest that hormones impact antibody isotype expression by both indirect and direct mechanisms; they might alter B cell growth, but might also up- and down-regulate class switch recombination (CSR) by direct receptor interactions with Sμ and Sα.
CD277 reduces B cell Activation by HIV Env Immunogens
Authors: Coleman Michelle , Corey Daniel , Ring Aaron R , Corey Lawrence , Weissman Irving L , Stamatatos Leonidas
Published: January 2023

CD277 is a human B7-like molecule. involved in activation of Vγ9Vδ2 T cells. Its counter-receptor is unknown and its role in B cell activation has not been described. B cell lines were stably transduced with the IgG isotype of the inferred germline B cell receptor (BCR) form of the broad and potent HIV-1 neutralizing antibody, VRC01, and stimulated with recombinant HIV Env in Ca2+-dependent activation assays. CD277 ectodomain proteins partially inhibited Ca2+ flux in response to heptameric and trimeric HIV Env. Similar observations were made with B cells expressing an anti-HA BCR, when stimulated with recombinant HA. We conclude that the reduction in B cell activation recorded in the presence of CD277 was not specific for the HIV Env. In contrast, CD277 completely abolished B cell activation that was induced by cross-linking the same BCRs with anti-Fab antibodies. To identify molecules that may serve as counter-receptors for CD277 binding, a fluorescently coupled CD277 ectodomain was utilized to conduct a FACs-based negative selection screen from a pooled lentiCRISPR-Cas9 knockout library (GeCKO). This strategy resulted in a significant reduction in library diversity with the overall sgRNA pool by the end of the screen restricted to a few, highly abundant sgRNAs. Specific binding assays among putative counter-receptors are ongoing. Once identified, CD277 counter-receptors could be targeted for inhibition in vaccination studies, to enhance B cell responses to antigen such as HIV Env. These data indicate an important physiological role for CD277 in inhibiting B cell activation and its role in influencing the B cell repertoire should be pursued.
Single molecular force sensitivity and threshold for the activation of B cell receptors
Authors: Liu Wanli , Wan Zhengpeng
Published: January 2023

B lymphocytes show remarkable mechanosensing capability by utilizing the surface expressed B cell receptors (BCRs) to sense antigens. However, the sensitivity and threshold for the activation of BCRs resulting from the stimulation by mechanical forces are unknown. Here we systemically addressed this question using a ds-DNA based tension gauge tether (TGT) system serving as a predefined single molecular force gauge ranging from 12 to 56 pN to restrict BCR and antigen tension. Utilizing total internal reflection fluorescence microscopy, we observed that IgM-BCR activation is sensitive to single molecular forces. We observed an obvious multi-threshold effect with a low-end 12–16 pN force which triggered a weak activation, whereas a 23–43 pN force initiated a medium-level activation, and a high-end 50–56 pN force produced a strong activation. Such patterned dependence on mechanical forces with multi-threshold effects did not strictly rely on myosin IIA, dynein and integrin. However, the break-through of the high-end but not the medium-level and low-end mechanical force threshold in IgM-BCR activation required myosin IIA and the inside-out activation of integrin. Surprisingly, we found that the activation of the isotype-switched IgG-BCR or IgE-BCR only requires an extremely low threshold of less than 12 pN, providing an explanation for their rapid activation in response to antigen stimulation. Mechanistically, we found that the cytoplasmic tail of the IgG-BCR heavy chain is both required and sufficient to account for the low mechanical force threshold to activate the IgG-BCR. These results defined the single molecular force sensitivity and threshold that are required to activate different isotyped BCRs.
mTOR Complex Signaling through the SEMA4A–Plexin B2 Axis Is Required for Optimal Activation and Differentiation of CD8+ T Cells
Authors: Ito Daisuke , Kumanogoh Atsushi
Published: January 2023

Mammalian target of rapamycin (mTOR) plays crucial roles in activation and differentiation of diverse types of immune cells. Although several lines of evidence have demonstrated the importance of mTOR-mediated signals in CD4+ T cell responses, the involvement of mTOR in CD8+ T cell responses is not fully understood. In this study, we show that a class IV semaphorin, SEMA4A, regulates CD8+ T cell activation and differentiation through activation of mTOR complex (mTORC) 1. SEMA4A−/− CD8+ T cells exhibited impairments in production of IFN-γ and TNF-α and induction of the effector molecules granzyme B, perforin, and FAS-L. Upon infection with OVA-expressing Listeria monocytogenes, pathogen-specific effector CD8+ T cell responses were significantly impaired in SEMA4A−/− mice. Furthermore, SEMA4A−/− CD8+ T cells exhibited reduced mTORC1 activity and elevated mTORC2 activity, suggesting that SEMA4A is required for optimal activation of mTORC1 in CD8+ T cells. IFN-γ production and mTORC1 activity in SEMA4A−/− CD8+ T cells were restored by administration of recombinant Sema4A protein. In addition, we show that plexin B2 is a functional receptor of SEMA4A in CD8+ T cells. Collectively, these results not only demonstrate the role of SEMA4A in CD8+ T cells, but also reveal a novel link between a semaphorin and mTOR signaling.
αv integrins engage autophagy components to regulate B cell immune response
Authors: Acharya Mridu , Sokolovksa Anna , Tam Jenny M , Vyas Jatin M , Stuart Lynda , Lacy-Hulbert Adam
Published: January 2023

Signaling through Toll-like receptors (TLR) such as TLR7 and TLR9 has been implicated in B cell activation by self-antigens and development of autoimmunity. Our recent work based on murine models has identified a novel mechanism by which a family of adhesion molecules called αv integrins and the autophagy pathway limit excessive B cell responses to antigens containing TLR ligands and self-derived antigens.

Specifically, we have found that αvβ3 integrin regulates B cell TLR signaling by directing trafficking and maturation of TLR containing endosomes and engaging components of the autophagy pathway. When B cells lack either αv integrins or autophagy components LC3 and Atg5 endosomal trafficking of the TLRs is disrupted and leads to enhanced TLR signaling and increased B cell responses. We have used av conditional knockout mice to determine the consequences of loss of this αvβ3-mediated immune regulation. Mice lacking B cell av show increased antibody responses to antigens containing TLR-ligands and develop increased levels of autoantibodies.

We therefore propose that αv-mediated TLR regulation serves to maintain the balance between protective immunity to microbes and potential pathological autoimmune responses. We are currently studying the role of αv signaling and autophagy components in development of class switched, high affinity antibodies in response to antigens in the context of infection with a pathogen or in response to self-antigens.
Aberrant localized LTB4 production exacerbates methicillin-resistant Staphylococcus aureus skin infection in diabetic mice.
Authors: Brandt Stephanie L , Wang Soujuan , Carrasco Sebastian , Dejani Naiara , Glosson-Byers Nicole , Serezani Carlos Henrique
Published: January 2023

A major cause of severe skin infections is methicillin-resistant Staphylococcus aureus (MRSA). Immunotherapeutic strategies are needed to treat infections. Neutrophils and macrophages control MRSA skin infection but undue inflammation causes injury. The bioactive eicosanoid leukotriene B4 is a potent chemoattractant and enhances phagocyte effector functions in naïve mice. However we have shown that diabetic mice produce chronic LTB4 which promotes susceptibility to sepsis but the role of LTB4 in localized infections is unknown. We hypothesize that aberrant LTB4 drive excessive inflammation during MRSA skin infection in diabetics.

Diabetic mice at day 1 post infection had larger infected areas, bacterial and apoptotic cell burdens than nondiabetic controls. By intravital microscopy we observed more neutrophil recruitment in diabetic mice. These effects correlated with high levels of LTB4 and its receptor BLT1. At day 9 post infection nondiabetic mice were nearly healed but diabetic mice still had cell infiltration, high bacterial and apoptotic burdens, which associated with high levels of inflammatory mediators.

To test whether chronic LTB4 causes uncontrolled infection, diabetic mice were treated daily with a topical ointment containing BLT1 antagonist. The treatment reduced infection area, bacterial burden and neutrophil recruitment. We also detected fewer apoptotic cells and lower expression of inflammatory mediators. These findings reveal that LTB4 is detrimental for diabetics by chronically inducing a damaging inflammatory response. We observe that topical BLT1 antagonist treatment improves host defense in diabetic mice and promises to be an effective therapeutic to treat MRSA infections in diabetic patients.