bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-10 enhances mast cell activation and is essential for the development of IgE-mediated experimental food allergy
Authors: Mathias Clinton B , Rovatti Jeffrey , Polukort Stephanie , Carlson Logan , Thompson Chelsea , Ser-Dolansky Jennifer , Kinney Shannon , Schneider Sallie S
Published: January 2023

IL-10 is a key pleiotropic cytokine that can both promote and curb Th2-dependent allergic responses. Herein we demonstrate a novel role for IL-10 in the development of IgE-mediated food allergy. Oral ovalbumin challenge in sensitized BALB/c mice resulted in a robust intestinal mast cell response accompanied by allergic diarrhea, mast cell activation and a predominance of Th2 cytokines, including enhanced IL-10 expression. In contrast, the development of intestinal anaphylaxis including diarrhea, mast cell activation, and Th2 cytokine production was significantly attenuated in IL-10−/− mice compared to WT controls. Furthermore, IL-10 directly promoted the activation of mast cells. Administration of IgE and antigen induced the development of passive anaphylaxis in WT mice, which was attenuated in IL-10−/− animals. However, treatment of IL-10−/− mice with recombinant IL-10 prior to IgE administration restored the development of passive anaphylaxis in these animals. Finally, adoptive transfer of WT mast cells restored allergic symptoms in IL-10−/− mice. Our observations demonstrate a critical role for IL-10 in driving mucosal mast cell activation, suggesting that in its absence, mast cell function is impaired, leading to attenuated food allergy symptoms.
Saturated fatty acids promote allergic (Th2) cytokine responses by activation of unfolded protein response (UPR) and ER stress
Authors: Samavedam Unni Krishna SRL , Khodoun Marat , Wu David , Hogan Simon P , Finkelman Fred D
Published: January 2023

Endoplasmic reticulum (ER) stress and an unfolded protein response (UPR) are stimulated by saturated, but not unsaturated fatty acids. We previously showed that saturated medium chain triglycerides (MCT) promote food allergy induction and increase epithelial cell expression of 3 pro-Th2 cytokines, TSLP, IL-25 and IL-33, which stimulate a Th2 cytokine response. These observations led us to hypothesize that the high concentrations of saturated triglycerides in two common food allergens, cow’s milk and chicken eggs, promote food allergy by inducing an epithelial cell UPR response, which in turn, causes these cells to express pro-Th2 cytokines. Indeed, studies with transformed human intestinal epithelial CACO2 cells demonstrated that MCT, egg yolk plasma (the liquid, triglyceride fraction of eggs) and cow’s milk cream each increase expression of the UPR-associated PERK, BiP, CHOP, XBP1 and/or XBP1s genes after 6 hours, followed by increased expression of all 3 pro-Th2 cytokine genes after 24 hours. Each of these stimuli, but not but not protein-rich, lipid-poor egg white, also increase epithelial permeability. Further, the increases in pro-Th2 cytokine expression are blocked by each of 3 UPR inhibitors: metformin, 4PBA and TUCDA, and the egg yolk plasma-induced increase in epithelial permeability is blocked by the only UPR inhibitor tested (metformin). Additionally, monoclonal antibodies (mAbs) to IL-25, IL-33 and TSLP each individually block the induction of egg white food allergy by a combination of egg white and MCTs, and a combination of these mAbs strongly suppress established egg white food allergy. These observations support our hypothesis and provide an explanation for the strong allergenicity of cow’s milk and chicken eggs.
Reduced Suppressor of Cytokine Signaling-1 levels in SLE patients correlates to enhanced STAT1 activation
Authors: Sukka-Ganesh BhagyaLaxmi , Li Yi , Reeves Westley H , Larkin Joseph
Published: January 2023

Systemic Lupus erythematosus (SLE) is a chronic inflammatory autoimmune disorder with unknown etiology. Although the specific events dictating SLE pathology remain unclear, abundant evidence indicates a critical role for dysregulated cytokine signaling in disease progression. Notably, the suppressor of cytokine signaling (SOCS) family of intracellular proteins, in particular the kinase inhibitory region (KIR) bearing SOCS1 and SOCS3, play a critical role in regulating cytokine signaling. Here in this foundational study, we test the hypothesis that regulation of cytokine signaling in SLE patients may be perturbed by a lack of KIR bearing SOCS1 and SOCS3. We analyzed 34 SLE patients, segregated by disease activity (SLEDAI) and prednisone treatment, in comparison with 11 healthy controls. Real time RT-PCR and Western blot analysis showed significant reductions in SOCS1 and SOCS3 in PMBC’s of SLE patients by both mRNA and protein expression when compared to controls. Notably, decreased SOCS1, but not SOCS3 protein levels in the SLE patients were inversely correlated to activation of STAT1, but not Erk 1/2 or Akt. Notably, the inverted SOCS1/pSTAT1 ratio correlated to significantly enhanced MHC class II levels amongst SLE patients. These studies represent a critical first step in implicating a role of SOCS1 and SOCS3 deficiencies in the progression of human SLE, providing impetus for the performance for a larger multi-centered examination. Finally, these studies point to the possibility that peptides, previously shown to mimic SOCS signaling and inhibit rodent autoimmune disease, may have efficacy in the treatment of human SLE.
Identification of Ezrin-Radixin-Moesin proteins as novel regulators of pathogenic B-cell receptor signaling and tumor growth in diffuse large B cell lymphoma
Authors: Gupta Neetu , Pore Debasis , Danda Avinash , Yan Di , Phillips James , Lindner Daniel , Hill Brian , Smith MItchell , Hsi Eric
Published: January 2023

Diffuse large B cell lymphoma (DLBCL) is a hematological cancer associated with an aggressive clinical course. The predominant subtypes of DLBCL display features of chronic or tonic B cell antigen receptor (BCR) signaling. However, it is not known if the spatial organization of the BCR contributes to regulation of pro-survival signaling pathways and cell growth. Here, we show that primary DLBCL tumors and patient-derived DLBCL cell lines contain high levels of phosphorylated Ezrin-Radixin-Moesin (ERM) proteins. The surface BCRs in both activated B cell and germinal B cell subtype DLBCL cells co-segregate with phosphoERM suggesting that the cytoskeletal network may support localized BCR signaling and contribute to pathogenesis. Indeed, ablation of membrane-cytoskeletal linkages by dominant negative mutants, pharmacological inhibition and knockdown of ERM proteins disrupted cell surface BCR organization, inhibited proximal and distal BCR signaling, and reduced the growth of DLBCL cell lines. In vivo administration of the ezrin inhibitor retarded the growth of DLBCL tumor xenografts, concomitant with reduction in intratumor phosphoERM levels, dampened pro-survival signaling and induction of apoptosis. Our results reveal a novel ERM-based spatial mechanism that is coopted by DLBCL cells to sustain tumor cell growth and survival.
Monocytes and alveolar macrophages skewing in a non-human primate model of HIV-associated pulmonary arterial hypertension (PAH)
Authors: Schweitzer Finja , Kling Heather M , Tarantelli Rebecca , Stehle Alexandria , Simon Mark A. , Norris Karen A
Published: January 2023

Background

PAH is a progressive disease of the pulmonary vasculature and right heart, with an increased prevalence in HIV-infected individuals. The causes of HIV-PAH are not known, though evidence suggests immune-mediated pathogenesis. We hypothesize that chronic inflammation contributes to increased pro-inflammatory monocytes and fibrotic alveolar macrophages, which may promote the vascular remodeling characteristic of PAH. We have developed a primate model of simian immunodeficiency virus (SIV)-PAH. This model was used to address the relationship between phenotypic changes in monocyte/macrophage and the development of SIV-PAH.

Methods

Macaques (n=22) were infected with SIV and serial blood and bronchoalveolar lavage fluid cells were analyzed by flow cytometry. Serial right heart catheterizations were performed to assess mean pulmonary arterial pressure (mPAP).

Results

Nine macaques showed no change in mPAP over the course of infection, whereas 13 developed PH (mPAP >25mm Hg). Higher frequencies of pro-inflammatory, non-classical monocytes were observed in PH+ animals (p=0.0001), correlating with mPAP (p=0.03). PH- animals had a higher frequency of phagocytic, classical monocytes (p=0.046), negatively correlating with mPAP (p=0.02). PH+ animals had a higher frequency of fibrotic M2a macrophages (p=0.02), correlating with mPAP (p=0.035). PH- animals had a higher frequency of regulatory, M2c macrophages (p=0.0003), negatively correlating with mPAP (p=0.005).

Conclusion

These results support the hypothesis that HIV-associated PAH is associated with skewing of monocytes and macrophages toward a pro-inflammatory and fibrotic phenotype that contributes to vascular remodeling associated with HIV-PAH.
Innate correlates of influenza clinical outcomes in a multicenter, longitudinal study of critically ill children
Authors: Fiore-Gartland Andrew , Panoskaltsis-Mortari Angela , Agan Anna A , Thomas Paul Glyndwr , Hertz Tomer , Randolph Adrienne
Published: January 2023

Paradoxically, influenza infection can trigger pro- and anti-inflammatory responses, both of which can lead to significant morbidity and mortality. Inflammation can lead to septic shock and acute lung injury (ALI), while suppression can slow viral clearance and increase susceptibility to bacterial coinfection (BCo). We collected and analyzed lung and peripheral blood samples from a multi-center cohort of critically ill children with influenza infection (N = 171) to better understand innate signatures of disease. Samples were collected at admission to the PICU and assayed for levels of 42 cytokines and chemokines. We found intra-compartment analyte correlations were high, while those between compartments were comparatively weak. To increase statistical power and identify groups of co-signaling cytokines, we pre-specified an unsupervised hierarchical clustering analysis to form influenza-specific modules. Some modules were consistent with the literature; e.g. IL4, IL5, IL9 and IL13 (Th2-like) clustered within both compartments. We hypothesized that modules would associate with shock, ALI and death or near death (ECMO). A pro-inflammatory PB module including IL6, IL8, IL10, IP10, GCSF and MCP1 was associated with shock (FWER p<1e-4; OR 3.4) and ALI (FWER p=0.003; OR 2.2), validating previous studies. Inverse associations were also identified with lung and serum modules, only after adjusting for patients’ mean analyte levels, suggesting that relative levels are important. An integrative analysis of lung and PB analytes showed that BCo has a distinct signature from shock that can be used as a biomarker to distinguish the two. Significant modules also generated hypotheses about analytes that could be targeted by novel therapies.
The lectin complement pathway and Ficolin-2 in patients with necrotizing soft tissue infection
Authors: Hansen Marco Bo , Rasmussen Lars Simon , Pilely Katrine , Hellemann Dorthe , Hein Estrid , Madsen Martin Bruun , Hyldegaard Ole , Garred Peter
Published: January 2023

Background

Mannose-binding lectin (MBL) and ficolins are pattern recognition molecules (PRMs) that play an important role during infection through activation of the lectin complement pathway. We assessed whether blood concentrations of the PRMs were associated with mortality in patients with necrotizing soft tissue infection (NSTI).

Methods

We conducted a prospective, observational study over 25 months involving 135 NSTI patients with a maximum follow-up time of 2.7 years. Plasma PRM level were measured upon admission. Non-infected surgical patients served as controls.

Results

PRMs levels of the lectin complement pathway were significantly lower compared to non-infected patients. Ficolin-2 level below median was associated with mortality at the end of follow-up (p = 0.007, log-rank), whereas no significant association was found for MBL, Ficolin-1 and Ficolin-3. Ficolin-2 level below median had a positive predictive value of 0.27 and a negative predictive value of 0.94 for 28-day mortality. When optimal cut-off was used, the multivariate cox-regression analysis revealed that low Ficolin-2 level was independently associated with 28-day mortality when adjusted for age, gender and chronicity (HR 6.27 [95% CI, 2.28–17.21], p < 0.0001) and also when SAPS II was included (HR 3.16 [95% CI, 1.03–9.73], p = 0.045).

Conclusions

All PRMs of the lectin complement pathway were significantly lower in patients with NSTI than in non-infected controls upon admission. Only plasma Ficolin-2 was significantly associated with both short-term and long-term mortality. Additionally, a high Ficolin-2 level above the median indicated a 94% chance of surviving the first 28 days after admission.

ClinicalTrials.gov Identifier: NCT2180906.
Beta-arrestin 2 plays a critical role in sepsis-induced cardiac dysfunction
Authors: Yin Deling , Yan Hui , Li Hui , Daniels Christopher , Singh Krishna , Chua Balvin , Stuart Charles , Caudle Yi , LeSage Gene
Published: January 2023

Sepsis, a major clinical problem with high morbidity and mortality, is caused by overwhelming systemic host-inflammatory response. β-arrestin 2, a key regulator and scaffolds of various signaling, modulates cell survival and cell death in various systems. However, the effect of β-arrestin 2 on sepsis-induced cardiac dysfunction is not yet known. Here, we show that β-arrestin 2 overexpression significantly enhances animal survival following cecal ligation and puncture (CLP)-induced sepsis. Importantly, overexpression of β-arrestin 2 in mice prevents CLP-induced cardiac dysfunction. In addition, β-arrestin 2 overexpression dramatically attenuates CLP-induced myocardial gp130 and p38 mitogen-activated protein kinase (MAPK) phosphorylation levels following CLP. Therefore, β-arrestin 2 prevents CLP-induced cardiac dysfunction through gp130 and p38. These results suggest that modulation of β-arrestin 2 might provide a novel therapeutic approach to prevent cardiac dysfunction in patients with sepsis.
Strain differences in lung inflammation and injury following progressive exposure to tobacco smoke in rats
Authors: Pham Alexa , Uyeminami Dale , Pinkerton Kent
Published: January 2023

Rationale

Chronic obstructive pulmonary disease (COPD) is currently the third leading cause of death worldwide and has been strongly associated with lung inflammation. To determine whether susceptibility to lung inflammation can lead to an enhancement of COPD-like features, we chose to examine normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rats following progressive exposure to tobacco smoke (TS) to determine the relative degree of lung injury and inflammation in each strain of rat.

Methods

WKY and SH rats were exposed to either filtered air or tobacco smoke at a particulate concentration reaching 80mg/m3 for 4, 8, or 12 weeks. Necropsy was performed 24 hours after the last exposure, followed by bronchoalveolar lavage (BAL) fluid to determine total and differential cell count. Histological scoring and immunohistochemical staining for M1 (classically activated) and M2 (alternatively activated) macrophages were performed on paraffin embedded and sectioned lung tissues.

Results

Following progressive smoke exposure, TS-exposed SH rats demonstrated significant increases in BAL total cell number, including elevated numbers of macrophages and neutrophils and higher inflammation scores in lung tissues compared to their FA control and TS-exposed WKY rats. Moreover, TS-exposed SH rats also had increased expression of the M1 but not the M2 markers in alveolar macrophages compared to their FA control and TS-exposed WKY rats.

Conclusion

These findings suggest that progressive tobacco smoke exposure contributes to persistent lung inflammation that is enhanced by rat strain susceptibility in the genesis of COPD.
Cigarette smoke extracts enhance neutrophil elastase-induced IL-8 production via up-regulation of proteinase-activated receptor-2 in human bronchial epithelial cells
Authors: Lee Kyoung-Hee , Jang An-Hee , Jeong Jiyeong , Lee Chang-Hoon , Yoo Chul-Gyu
Published: January 2023

Inflammation, oxidative stress, and protease-antiprotease imbalance have been suggested as a pathogenic triad in chronic obstructive pulmonary disease (COPD). However, little is known about how they interact. The objectives of this study were to elucidate the effect of cigarette smoke extract (CSE) on the neutrophil elastase (NE)-induced inflammatory response and its molecular mechanism in bronchial epithelial cells. NE activated extracellular signal-regulated kinase (ERK) and induced IL-8 production. Blocking ERK activation by MEK inhibitor (U0126) suppressed NE-induced IL-8 secretion and pre-incubation with proteinase-activated receptor 2 blocking peptide (PAR2BP) inhibited both NE-induced ERK activation and subsequent IL-8 release, suggesting that NE-induced IL-8 production is dependent on PAR2-mediated ERK activation. Interestingly, pre-exposure with CSE markedly enhanced NE-induced IL-8 production. Knock-down of PAR2 expression by transfection with siRNAs blocked the synergistic effect of CSE on NE-induced IL-8 production. Therefore, we next investigated the effect of CSE on PAR2 expression. CSE, but not NE, increased the expression of PAR2 mRNA and surface membrane protein. Inhibition of p38 MAP kinase reduced PAR2 expression by CSE, while inhibition of ERK and Akt pathway was without effect. Moreover, p38 inhibition completely abrogated CSE-induced enhancement of IL-8 production in NE-treated cells. Taken together, these results suggest that CSE up-regulates PAR2 in normal human bronchial epithelial cells, thereby enhancing the inflammatory response to NE.
Adipose Fatty Acid Binding Protein Promotes Saturated Fatty Acid-induced Cell Death through Ceramide Production in Macrophages
Authors: Zhang Yuwen , Rao Enyu , Zeng Jun , Hao Jiaqing , Sun Yanwen , Sauter Edward , Cleary Margot , Li Bing
Published: January 2023

Macrophages play a critical role in obesity-associated chronic inflammation and disorders. However, the molecular mechanisms of how macrophages respond to elevated fatty acids (FAs) and contribute to sterile inflammation in obesity remain to be fully elucidated. Here, we report a new mechanism by which dietary FAs, in particular saturated FAs, were able to directly trigger macrophage cell death. We demonstrated that excess saturated FAs, but not unsaturated FAs, induced the production of cytotoxic ceramides in macrophages. Most importantly, expression of adipose fatty acid binding protein (A-FABP) in macrophages facilitated metabolism of excess saturated FAs for ceramide synthesis. Inhibition or deficiency of A-FABP in macrophages decreased saturated FA-induced ceramide production, thereby resulting in reduced cell death. Furthermore, A-FABP-deficient obese mice exhibited increased percentage of CD36+ macrophages in peripheral as compared to WT control mice. Altogether, our data suggest that excess saturated FAs in diets serve as new triggers in inducing ceramide production and macrophage cell death through elevated expression of A-FABP, thus contributing to sterile inflammation in obesity.
The type 2 diabetes-associated Th17 cytokines IL-21 and IL-22 promote breast cancer cell survival.
Authors: Nicholas Dequina , Strissel Katherine J , Andrieu Guillaume , Tran Anna H , Nikolajczyk Barbara S , Denis Gerald V
Published: January 2023

Obesity-induced inflammation has taken center stage as a driving force in metabolic disease and type 2 diabetes (T2D). As the epidemic of obesity deepens, it has become imperative to dissect mechanisms by which obesity-induced inflammation contributes to increased cancer morbidity. Recent studies have shown that increased breast cancer mortality rates are associated with obesity-related T2D. Our goal is to understand the immunological mechanisms in T2D that contribute to increased breast cancer risk. We previously showed that PBMCs treated with T cell-targeted stimuli produce a pro-inflammatory Th17 cytokine profile that differentiates T2D from obese non-T2D subjects. We therefore tested the hypothesis that Th17 cytokines support breast cancer cells. Proliferation assays showed that IL-21 and IL-22, but not IL-17A/F, or the Th1 cytokine IL-6, maintained the proliferation of MCF7 cells, an estrogen dependent breast cancer cell line, in the absence of estrogen. Building on this result, we tested the effect of IL-21/22 on MCF7 survival. Immunoblots and mRNA arrays demonstrated that IL-21 but not IL-22 upregulated steady-state expression of BCL-2 family survival mRNA/protein. Both IL-21 and IL-22 survival was blocked by JQ1, an inhibitor of the Bromodomain and ExtraTerminal (BET) family of chromatin regulating proteins. The inferred cross-talk between BET- and Th17 cytokine-triggered pathways implicate the Th17 cytokine signature from PBMCs of patients with T2D in the higher recurrence of post-menopausal breast cancer after tamoxifen treatment. The public health impact of these data is that monitoring Th17 cytokine production by PBMCs may inform risk assessment for relapse or recurrence in breast cancer patients with T2D.
Human adenosine deaminases control the immune cell responses to activation signals by reducing extracellular adenosine concentration
Authors: LIU Chengqian , Mukienko Yulia , Wu Chengxiang , Zavialov Andrey
Published: January 2023

Adenosine rapidly accumulates in the sites of inflammation and tumor growth. It binds to adenosine receptors expressed on the cell surface of immune cells and induces either suppression or activation of inflammatory responses to pathogens. In humans the level of extracellular adenosine is regulated by two adenosine deaminases ADA1 and ADA2. Decrease in ADAs concentration due to genetic defects in the ADA genes leads to serious perturbation in the immune system function while increase in ADA activity associates with numerous immune diseases and cancers. The immune responses to extracellular adenosine have largely been studied using pharmacological approach where non-hydrolysable adenosine receptors agonists substitute adenosine to form the activated state of adenosine receptors. On contrary, adenosine receptors bound to adenosine receptor antagonists mimic inactivated state of adenosine receptors.

Here, the effect of adenosine receptor agonists and antagonists on the monocytes function as well as and T helper cell proliferation and differentiation was compared with the effect of adenosine and adenosine deaminases. It was demonstrated that adenosine deaminases control the immune cells responses to activation signals by reducing the concentration of extracellular adenosine and that the cells sensitivity to adenosine greatly depends on the type of the cell activation. Therefore, our data suggests that ADAs could be considered as new drug candidates for the treatment of immune disorders and cancers.
ImmuneSpace: Enabling integrative modeling of human immunological data
Authors: Sauteraud Renan , Dashevskiy Lev , Finak Greg , Gottardo Raphael
Published: January 2023

Recent technical advances have transformed the field of immunology. We are now capable of measuring features of immune responses, including B- and T-cell specificity and repertoire, serum and intracellular cytokines, and more, on a scale never imagined before. As a consequence, the generation of big data sets has become routine and there is an urgent need for an analysis platform to facilitate data exploration and integration across assays and studies. Here we present ImmuneSpace, the data repository and analysis platform of the Human Immunology Project Consortium (HIPC). The HIPC program, funded by the NIH, is a multi-center collaborative effort to characterize the status of the immune system in different populations under diverse stimulations and disease states. This ongoing effort has generated large amounts of varied high-throughput, high-dimensional biological data (flow cytometry, CyTOF, RNA-Seq, Luminex, among others). All data generated to date by HIPC, along with other selected datasets generated by other NIAID funded projects, have been made publicly available through ImmuneSpace and are ready to be explored using visualization and analysis tools built in ImmuneSpace. To this end, we hope that ImmuneSpace will act as a central immunological hub, allowing experimentalists, statisticians, and bioinformaticians to freely retrieve, explore and compare data across assays and across studies generated within and outside of HIPC.
Complement component C1q limits osteoarthritis pathology by regulating macrophage activation
Authors: Raghu Harini , Lingampalli Nithya , Wang Qian , Wong Heidi , Lepus Christin , Mao Rong , Robinson William H
Published: January 2023

Osteoarthritis (OA), the most common form of arthritis in the world, is characterized by articular cartilage breakdown in synovial joints. There is evidence of low-grade inflammatory responses in OA, but their contribution to OA pathogenesis is unclear. Studies have demonstrated that dysregulated complement activity is involved in OA pathogenesis. The complement component, C1q, is known to regulate inflammation via mechanisms involving apoptotic cell clearance and macrophage activation. Thus, we hypothesized that C1q is a key negative regulator of inflammatory mechanisms in OA. Using a combination of NanoString-based mRNA quantification and Luminex-based analyses for cytokine profiling, we found that differentiation of human monocyte-derived macrophages in C1q-depleted serum followed by stimulation with cartilage debris resulted in enhanced pro-inflammatory (e.g., IL1β) and reduced anti-inflammatory (e.g,. IL10) cytokine expression and secretion compared to cells grown in normal human serum. To definitively establish a regulatory role for C1q in OA, we used a well-established destabilization of the medial meniscus (DMM) model of knee OA in genetically-deficient mice. We found that knees of C1q-deficient mice subjected to DMM developed worse OA pathologies including exacerbated cartilage damage, osteophyte formation and synovitis relative to C1q-sufficient mice. Our data suggest that C1q deficiency exacerbates inflammation and cartilage damage through mechanisms involving dysregulated macrophage activation. Understanding the precise mechanisms underlying C1q-mediated regulation of inflammation may lead to the identification of novel targets for the treatment of inflammatory diseases including OA.
Integrin activation controls regulatory T cell identity and stability
Authors: Klann Jane Elizabeth , Kim Stephanie H , Remedios Kelly A , Metz Patrick J , Snider Adam K , Lopez Justine , Cantor Joseph M , Ablack Jai N , Mack Lauren A , Zheng Ye , Lu Li-Fan , Bui Jack D , Ginsberg Mark H , Petrich Brian G , Chang John T
Published: January 2023

Maintenance of the regulatory T (Treg) cell pool is essential for peripheral tolerance and prevention of autoimmunity. Integrins, heterodimeric transmembrane proteins consisting of α and β subunits that mediate cell-cell and cell-extracellular matrix interactions, have been shown to play an important role in facilitating cell contact-mediated suppression by Treg cells. Here we show that integrin activation plays an essential, previously unappreciated role in maintaining the stability of the Treg cell pool. Treg cell-specific loss of talin1, a β integrin-binding protein, or expression of talin1(L325R), a mutant that selectively abrogates integrin activation, resulted in dysregulation of Treg cell identity and lethal systemic autoimmunity. Moreover, the absence of sustained interactions between the integrin LFA-1 on Treg cells and its ligand ICAM-1 on dendritic cells reduced the expression of Foxp3 and caused Treg cells to adopt an effector CD4+ T cell-like phenotype. Taken together, these results reveal a critical role for tonic, integrin-mediated signals in controlling peripheral tolerance by virtue of maintaining Treg cell identity and stability.
Factor VIII-specific CAR regulatory T cells modulate murine anti-factor VIII immune responses
Authors: Fu Richard , Lyle Meghan J. , Wang Xuefeng , Miao Carol H.
Published: January 2023

The immune response to factor VIII protein (FVIII; F8 in constructs) limits the effectiveness of treatments for hemophilia A (HemA) patients. Previously we demonstrated that regulatory T cells (Tregs) play a pivotal role in modulating anti-FVIII immune responses. For application of adoptive Treg therapy, we successfully expanded highly suppressive murine polyclonal Tregs antigen specifically in vitro. However, the FVIII-specific Tregs in the polyclonal population are still in very small numbers. Thus, we explored the strategy to generate FVIII-specific Tregs using the chimeric antigen receptor (CAR) approach. Lentiviral vector (LV) incorporating a high-binding anti-FVIII scFv linked to the CAR signaling domains and fused with a Foxp3 cDNA (F8CAR-Foxp3-LV) was prepared and used to transduce murine CD4+T cells. Flow cytometry analysis confirmed extracellular scFv and intracellular Foxp3 expression in transduced cells (F8CAR-Tregs). In vitro FVIII-specific suppressive assay showed that transduced cells had significantly higher suppressive activity than untransduced cells towards murine effector T cells. In addition, 1×106 transduced cells and untransduced cells were adoptively transferred into HemA mice and the treated mice were subsequently challenged with FVIII plasmid injected hydrodynamically. The anti-FVIII antibody titers are evaluated overtime. It is expected that F8CAR-Foxp3-LV transduced cells will prevent or decrease the production of anti-FVIII antibodies in HemA mice. We anticipate that compared with nonspecific or polyclonally expanded Tregs, FVIII-specific CAR Tregs will exert superior suppressive activity towards anti-FVIII immune responses without triggering systemic immune suppression.
Identify the differential response of Kupffer cell subsets during liver inflammation
Authors: Soysa Dilini Radika , Crispe Ian N
Published: January 2023

Kupffer cells (KC) are the resident macrophages that reside within the lumen of the liver sinusoid. Based on the origin there are two different sub sets of liver kupffer cells. The long-lived, radio-resistant subset also known as the sessile KC, derives from the progenitor cells generated during development in the yolk sac and maintains through self-renewal. The short-lived, radio-sensitive subset originates from hematopoietic stem cells. KCs play a major role in liver homeostasis, hepatic injury and induction of fibro-genesis as well as resolution of inflammation and fibrosis. Determination of the exact role of those ontogenetically different KC subpopulations in those critical liver functions is eluded by the inability to distinguish between the two subsets. Sessile KCs cannot be isolated and hence need an in situ method to study their gene expression. We propose to use the Ribo tag approach combined with Cre recombinase driven by a macrophage specific promoter (Emr1) to isolate actively translating polyribosomes using a ribo immunoprecipitation technique. The isolated KC subset-specific RNA will be used to analyze the transcriptome during both steady state conditions and liver inflammation.
Epithelial and bone marrow derived sources of Cd1d contribute to wound repair
Authors: Borkowski Andrew William , Havran Wendy L
Published: January 2023

Wound healing is a multifaceted process that depends on both epithelial and bone marrow derived cells to promote proper healing. Recent publications have shown that a subset of T cells in the skin are activated by certain lipids bound to CD1 molecules. Cd1d, the isoform of CD1 expressed in mice, is an MHC-like molecule that binds lipid antigens and is expressed by Langerhans cells (LCs), macrophages, B cells, and wounded keratinocytes. As our lab has previously shown that T cell activation in the skin promotes wound healing, we hypothesize that Cd1d is an important component of normal wound healing. To investigate this hypothesis, we administered full thickness wounds to the dorsal skin of WT and Cd1d−/− mice and analyzed wound closure. Cd1d−/− mice had a significant delay in wound healing between days 1 and 5 compared to WT controls. Analysis of ex vivo wound healing failed to show any difference in wound closure rates between WT and Cd1d−/− skin. This suggested that an infiltrating cell type is at least partially responsible for the observed difference in in vivo wound healing. Transfer of WT bone marrow cells into lethally irradiated Cd1d−/− mice did not rescue the wound healing defect observed in Cd1d−/− mice. Interestingly, transfer of Cd1d−/− bone marrow into WT mice caused a delay in wound healing. Analysis of wounded skin demonstrated that significantly fewer macrophages and LCs were present in Cd1d−/− epidermis. We conclude that the Cd1d-mediated wound healing defect is dependent on both resident LCs and infiltrating macrophages, and continue to investigate how Cd1d expressed by LCs promotes wound repair as studies have shown that LCs have an immunosuppressive effect in models of contact hypersensitivity and UVB-induced inflammation.
Ceramide mediates FasL-induced caspase 8 activation in colon carcinoma cells to enhance FasL-induced cytotoxicity by tumor-specific cytotoxic T lymphocytes
Authors: Coe Genevieve , Redd Priscilla , Paschall Amy V , Lu Chunwan , Albers Thomas , Lebedyeva Iryna , Liu Kebin
Published: January 2023

Cytotoxic T Lymphocytes (CTLs) are the major effectors of the host cancer immune surveillance. FasL-induced cytotoxicity is one of the two effector mechanisms that CTLs use to kill tumor cells. Fas is the physiological ligand of FasL and its expression and function is often deregulated in cancer cells. Ceramide is a sphingolipid metabolite that mediates Fas function. We aimed at testing the hypothesis that ceramide analogs are effective in modulating Fas function to sensitize colon carcinoma cells to FasL-induced apoptosis by tumor-specific CTLs. We show that Fas is expressed in human colon carcinoma cells. However, human colon carcinoma cells are not sensitive to FasL-induced apoptosis. Based on structures of existing ceramide analogs and ceramidase inhibitors, we used rationale design and synthesized twenty ceramide analogs as Fas function modulators. Six of these twenty ceramide analogs, IG4, IG7, IG8, IG14, IG17 and IG19, exhibit potent activity in sensitization of human colon carcinoma cells to FasL-induced apoptosis. At the molecular level, we observed that these six ceramide analogs dramatically increased FasL-induced activation of caspase 8, an essential initiator caspase of the Fas receptor-death-inducing signaling complex. Finally, a sublethal dose of ceramide analog significantly increased CTL-mediated and FasL-induced apoptosis of colon carcinoma cells. We have therefore developed six novel ceramide analogs that act at sublethal doses to enhance the efficacy of tumor-specific CTLs and hold great promise for further development as adjunct agents in CTL-based colon cancer immunotherapy.
A dynamic (epi)genomic landscape of CD8+ T cell differentiation drives cell-fate decisions
Authors: Amezquita Robert A , Gray Simon M , Guan Tianxia , Kaech Susan M , Kleinstein Steven
Published: January 2023

Long-lived memory CD8+ cytotoxic T lymphocytes (CTLs) are a critical component of long-term protective immunity against intracellular pathogens following infection or vaccination. During many acute viral infections, memory CD8 T cells arise from a heterogeneous pool of CTLs that contains terminal effector (TE) cells, which are highly-functional terminally differentiated CTLs that subsequently undergo programmed contraction, and memory precursor (MP) cells, which are less differentiated effector cells that have the greatest potential to persist and seed the memory T cell pool. How these effector CTLs adopt these distinct cell fates is an important question. Several key transcription factors (TFs) have been discovered that regulate the development of these two effector CTL subsets and promote the acquisition of their unique gene signatures, but it is unclear how the epigenome contributes to the development of terminally differentiated TE and mulitpotent MP cells. To understand this question, we are profiling RNA expression, histone modifications, and DNA accessibility to dissect the (epi)genomic network throughout the early CTL response to characterize the genetic networks controlling TE and MP cell differentiation during LCMV infection. Specifically, we are focusing on how changes in enhancer utilization and chromatin remodeling are regulated by key TFs, such as T-bet and ZEB2, in controlling the expression of target genes that regulate effector cell fates. This work will provide greater insight into how T cell memory forms after infection and vaccination, which could inform future T cell based vaccine strategies to improve immunity against foreign pathogens.
High School students conduct research on the neonatal immune response as part of the Students and Teachers As Research Scientists (STARS) program
Authors: Shornick Laurie P , Mares Kenneth R
Published: January 2023

The Students and Teachers As Research Scientists (STARS) program offers academically-talented high school students in the Saint Louis metropolitan area an opportunity to do research in a variety of scientific fields. Fourteen students have been mentored in my immunology laboratory during the last 9 summers as a part of the STARS program. Students take on small-scale, hypothesis-driven independent projects related to the neonatal immune response of respiratory viral infection using a mouse model. The high school students take basic safety training, blood borne pathogen training and biohazard animal training within the first few days of the program. Each student learns the basic mouse model and then specific techniques (immunostaining, western blot analysis, qPCR and flow cytometry) needed to test to their hypothesis. Students learn how to keep laboratory notebooks, how to find and read primary journal articles, and basic laboratory skills. While the main goal of the program is to teach the scientific process, other structured activities include lectures by nationally-known scientists, information concerning the higher education admissions process, and social activities. The program culminates with a written research paper and a short powerpoint presentation. The papers are written in the style of a peer-reviewed journal article in the field of immunology; each student receives assistance in writing from the primary mentor and from a STARS advisor. Since its inception in 1987, the STARS program has sent on more than 2,000 students with research experience to top universities.
An Immunology Course-Based Research Experience for Undergraduate Students that Introduces Computational and Modeling Skills
Authors: Kurt Robert A , Liew Chun Wai
Published: January 2023

Previously, we developed a computational modeling lab for undergraduate immunology students that introduced them to the SIR model and the Vensim modeling program. In short, students modeled an epidemic of strep throat and were required to model the Susceptible (S), Infected (I) and Recovered (R) in order to find the best approach to decreasing the length of the epidemic and the number of people infected. While this lab experience was valuable in teaching some basic immunology and modeling skills, the short time frame for the lab provided limited exposure to the importance and potential of modeling skills in immunology. Here we developed a semester-long course-based research experience (CRE) where students develop their own hypothesis, design the experiments, conduct the studies, and analyze their findings. Moreover, in addition to the wet lab component that exposes students to immunology techniques such as flow cytometry, qRT-PCR, cell separation and tissue culture, the students model a signaling pathway providing them with experience in computational modeling. We developed a software package that generates a computational model in Netlogo from the specifications provided by the students. The computational approach allows students to model any type of cell signaling cascade providing a large number of possible hypotheses that can be tested. We piloted this approach with four first-year students with no immunology or modeling/programming experience and the students successfully generated and tested their own hypotheses, and using Netlogo they modeled several different G Protein Coupled Receptor signaling cascades in CD4 and CD8+ T cells. We are in the process of running this CRE with students enrolled in a 200 level immunology class.
Kinesthetic Learning Modalities’ Approach in Understanding Concepts of Hypersensitivities Immunological Reactions.
Authors: Lana Valentina , Royan Ayla , Fazal Nadeem
Published: January 2023

It is now generally accepted that there are four learning modalities. Auditory learners learn best by listening so lecturing would be the way they would prefer to learn. Visual learners are those who prefer the printed word. They would learn best by reading or responding to the use of an overhead projector type of teaching. Tactile learners learn best by manipulating objects so they would prefer laboratory exercises. Kinesthetic (also known as whole body learners) prefer to learn through physical activities. This is the group of learners who would benefit most by this type of role-playing workshop. There are three basic modalities to process information to memory: visual (learning by seeing), auditory (learning by hearing), and kinesthetic (learning by doing). We tested role-playing modality at College of Pharmacy Chicago State University. This was done during spring semester where pharmacy year 1 students were taking Immunology as a didactic course. The students were divided into different small learning groups of 7–10 students in a class of 90 students and were made to role-play different types of hypersensitivity reactions. The role-playing acts were acted up in front of the whole class and also recorded. Asking Likert-type ordinary scale data analysis questions assessed the effectiveness of this modality and student learning. A statistically significant number of students found this immunology role-playing exercise as a fun and very beneficial activity. Students strongly agreed that this group-base role-playing approach was very powerful in conceptual understanding of hypersensitivity reactions, which is a clinically important and significant immunology topic.
Mechanisms of Non-canonical Inflammasome Activation by Rickettsia
Authors: Smalley Claire A , Bechelli Jeremy , Walker David , Fang Rong
Published: January 2023

Rickettsiae are Gram-negative, obligately intracellular bacteria that infect macrophages, however, the molecular mechanisms involved in the interactions of rickettsiae with macrophages remain poorly understood. We have found that R. australis activates the inflammasome in mouse primary macrophages as evidenced by caspase-1/11-dependent secretion of IL-1β as well as the cleavage of caspase-1 in both cell lysates and supernatant. In the present study, we show the cleavage of caspase-11 and the induction of pyroptosis in mouse macrophages upon infection with R. australis. Furthermore, inhibitors of caspase-4 significantly reduced the levels of IL-1β secreted by infected human macrophages. Our results suggest that rickettsiae also activate caspase-11/4-dependent noncanonical inflammasome in macrophages. We are currently investigating the role of cytosolic rickettsial lipopolysaccharide (LPS) in activating noncanonical inflammasome in mouse and human macrophages and the molecular mechanisms involved. Considering the distinct features of rickettsial LPS from other obligately intracellular Gram-negative bacteria, our studies will not only provide novel activating mechanisms of noncanonical inflammasome by employing rickettsiae as an infection model, but also help us to better understand the immune pathogenesis of fatal rickettsioses.
Microbiota regulated IL-25 protects from Clostridium difficile infection via eosinophils
Authors: Buonomo Erica L , Cowardin Carrie , Wilson Madeline , Saleh Mahmoud , Petri William
Published: January 2023

Clostridium difficile infection (CDI) occurs when the microbiota has been disrupted, stressing the beneficial role of commensal bacteria. The ability of the microbiota to contribute to protection has historically been linked to the induction of host factors such as bile acids and out competing the pathogen for nutrients and space. The influence of the microbiota to modulate the host immune response to dictate CDI outcome remains unexplored. Here, we show that IL-25, a cytokine regulated by the microbiota, is suppressed in the intestine during CDI in humans and mice. Administration of IL-25 protected mice from CDI-associated mortality and enhanced the integrity of the epithelial tissue in spite of equivalent quantities of C. difficile bacteria and toxin in the gut. Surprisingly, we identified eosinophils, a cell previously unexamined during CDI, as the cellular mechanism by which IL-25 protects. Using two distinct models, IL-25 was unable to reduce mortality or prevent gut barrier disruption in the absence of eosinophils. Thus, our study identifies IL-25 as the first immune mediator that is both regulated by the microbiota and enhances survival during CDI. This work provides a novel mechanism by which induction of innate immune responses, specifically eosinophils, can reduce the severity of CDI by protecting host tissue.
Proteasome degradative activity regulates CD8+ T lymphocyte metabolism and fate specification
Authors: Widjaja Christella E , Metz Patrick J , de Bruin Gerjan , Leestemaker Yves , Savas Jeffrey N , Phan Anthony T , Goldrath Ananda W , Yates John R , Florea Bogdan I , Overkleeft Hermen S , Ovaa Huib , Chang John T
Published: January 2023

During an immune response to microbial infection, CD8+ T lymphocytes can undergo asymmetric division, giving rise to daughter cells that exhibit distinct tendencies to adopt the terminal effector and memory fates. Here we show that ‘pre-effector’ and ‘pre-memory’ cells arising from the first CD8+ T cell division in vivo exhibit low and high rates of proteasome degradative activity, respectively. Pharmacologic inhibition of proteasome activity in activated CD8+ T cells resulted in increased inflammatory cytokine production and effector cell-associated molecule expression in vitro, as well as a loss of survival capacity and an impaired recall response to secondary infection in vivo. By contrast, increasing proteasome activity using a novel small molecule proteasome activator resulted in augmented memory cell-associated characteristics in vitro and an increased proportion of central memory cells with enhanced recall capabilities in vivo. Transcriptomic and proteomic analyses revealed that modulating proteasome activity in activated CD8+ T cells affected processes associated with cellular metabolism. These proteasome-induced metabolic consequences were mediated, in part, through asymmetric segregation of Myc, a transcription factor that has been shown to control glycolysis and proliferation, during cell division. Taken together, these results suggest proteasome degradative activity as a key regulator of CD8+ T lymphocyte fate specification by virtue of effects on cellular metabolism.