bluuebunny/crossref_metadata_2025 huggingface dataset:
Protective effect of recombinant thrombomodulin on chemotherapy-induced intestinal mucosal injury in mice
Authors: Gaowa Arong , Yamaguchi Takanori , Shimaoka Motomu
Published: November 2024

Intestinal mucositis is a common side effect of chemotherapy. Cisplatin is an effective chemotherapeutic agent for various types of cancers. However, cisplatin-induced intestinal mucositis limits the dose of cisplatin and reduces its therapeutic efficacy. In this study, we investigated the protective effects of recombinant thrombomodulin (rTM), which is a potent anti-inflammatory agent, against cisplatin-induced intestinal mucositis and its possible molecular mechanisms. We first evaluated the effects of rTM on intestinal mucositis caused by cisplatin in mice. Disease progression was monitored by analyzing loss of body weight and histological changes in intestinal tissue. We then investigated the effects of rTM on mouse intestinal organoid formation and growth. Gene expression levels were analyzed by quantitative real-time polymerase chain reaction, Western blotting, and flow cytometry analysis. We found that rTM treatment significantly attenuated the loss of body weight, histological damage, and gene expression levels of pro-inflammatory cytokines such as interleukin-6, tumor necrosis factor-a and high-mobility group box-1 in a cisplatin-treated mouse model in vivo. Furthermore, rTM alleviated the inflammatory cytokine production and apoptosis in a cisplatin-treated intestinal epithelial organoid model in vitro. Taken together, rTM suppresses cisplatin-induced intestinal epithelial cell-derived cytokine production and alleviates intestinal mucositis.
Medium-chain inulin enhances systemic immunity and intestinal health by modulating the gut microbiota and metabolome in C57BL/6 mice
Authors: Zhang Guiguo , Lee Yunkyoung , Kim Eunnyoung , Wang Yunpeng , Cui Jiamei , Wang Xiagojun
Published: November 2024

This study investigated the impact of medium-chain inulin on systemic immunity and its mechanisms. Sixty healthy male C57BL/6 mice, three weeks old, were divided into five groups of twelve, housed in three cages each. They were fed a normal diet, with variations including 0%, 1%, 3%, 5%, or 7% medium-chain inulin. The study found that inulin supplementation at 3% or higher reduced serum cholesterol, triglycerides, and LDL levels, while increasing IgG and IgA levels. Inulin also enhanced intestinal health, as indicated by a reduction in crypt depth, and increases in villus height and villus-to-crypt ratio. Moreover, inulin, particularly at 3% or higher, boosted populations of specific gut bacteria like Candidatus_Saccharimonas, Prevotellaceae_UCG-001, Bifidobacterium, Bacteroides, and Dubosiella. It also raised levels of certain metabolites such as stearamide, fatty acid-hydroxy fatty acids (4:0/18:0), L-malate, orotic acid, and bilirubin. Notably, these inulin-induced changes in microbes and metabolites were positively associated with improved immune and intestinal health. Overall, the study concluded that dietary inulin supplementation above 3% enhances systemic immunity and intestinal health, primarily by promoting beneficial gut bacteria and metabolites.
Unveiling the immune dynamics of Neisseria persistent oral colonization: a roadmap for innovative vaccine strategies
Authors: Alles Mario , Gunasena Manuja , Zia Tauqir , D'Mello Adonis , Bhattarai Saroj , Mulhern Will , Terry Luke , Scherger Trenton , Wijeratne Saranga , Singh Sachleen , Wijeratne Asela , Kasturiratna Dhanuja , Tettelin Hervé , Weyand Nathan , Liyanage Namal
Published: November 2024

Commensals shape host physiology and immunity. Neisseria gonorrhoeae (NG), a human pathogen, causes asymptomatic colonization and animal studies on NG for vaccine-design have been unsuccessful. N. musculi (NM), a mouse commensal, shares host-interaction genes with NG and is a surrogate to study NG colonization.

Eight A/J mice were orally inoculated with NM; uninoculated mice served as controls (n=8). Four from each group were euthanized at day 5 and the rest at day 33. Immune signatures and palate transcriptome were compared between inoculated and control mice using Wilcoxon rank-sum test.

Transcriptomics revealed enriched mucosal immunity and inflammation in colonized tissue. Day 5 data suggested reduced colonization resistance (reduced monocytes), while data at day 33 showed enriched adaptive (memory B cells) and immunoregulatory cells (Tregs) in colonized tissue.

At day 5 of colonization, systemic immune response was pro-inflammatory. Splenic NK cells showed higher IL22, suggesting reinforcement of mucosal immunity during colonization. Systemic immune response displayed reduced enrichment of immune signatures at day 33 of colonization suggesting homeostasis. Positive correlations between NM palate colony counts and enriched immune subsets suggest impact of mucosal colonization on immunophenotypes (p<0.05 for all).

This data is crucial for defining transcriptional and immunological endpoints of NM mucosal colonization, aiding in developing vaccine candidates against NG.
Maternal Protection from Bacterial Translocation in a Murine Model of Late-Onset Neonatal Sepsis (LOS)
Authors: Tamar Christian , Greenfield Kara , Dziekan Evelyn , Cassidy Mercedes , Knoop Kathryn
Published: November 2024

Late-onset neonatal sepsis (LOS), resulting from a bloodstream infection at least 72 hours after birth, is a major contributor to neonatal mortality. In a substantial portion of LOS cases, the causative pathogen is present as a member of the intestinal microbial community before dissemination, implicating intestinal translocation as a source of bloodstream infections. One potential pathway of bacterial translocation is through goblet cell-associated antigen passages, or GAPs, in colonic goblet cells. The presence of luminal EGF, obtained from breastmilk, and subsequent EGF receptor (EGFR) signaling in the intestine inhibits the formation of GAPs. We have established that disruption of EGFR signaling in pups leads to dissemination of the enteric pathogen Escherichia coli to the mesenteric lymph node (MLN). As pathogen dissemination was quickly followed by mortality, we use this as a preclinical model of LOS. Further work with this model focuses on the role of milk-derived immunoglobulins in reducing pathogen colonization and protecting from disease resulting from intestinal translocation. Through cross-fostering litters between dams immunized against E. coli and unimmunized dams, along with a comparison in disease between litters from immunized wild-type mice and IgA-/- mice, we aim to describe the protective role of mammary IgA. These experiments will also provide further implications for the role of human milk and human milk factors in protection from LOS.
The Immunomodulatory Effects and Mechanism of Wogonin for the Treatment of Multiple Sclerosis in a Murine Experimental Autoimmune Encephalomyelitis Model
Authors: Song Ying-Chyi , Yen Hung-Rong
Published: November 2024

Multiple sclerosis (MS) stands out as the most prevalent inflammatory autoimmune disease affecting the central nervous system (CNS). The primary driver of MS involves myelin-specific autoreactive T cells that infiltrate the CNS, initiating attacks on myelinated axons and triggering neuroinflammatory responses. Wogonin, a flavonoid, exhibits pharmacologic properties, including neuroprotective, anticancer, antiviral, anti-inflammatory, and anti-oxyradical effects. Our study demonstrates that wogonin significantly suppresses TLR-dependent DC activation, migration, and the expression of adhesion molecules in LPS-stimulated bone marrow-derived dendritic cells (BMDCs). In the experimental autoimmune encephalomyelitis (EAE) model, preventive treatment with wogonin led to reduced lymphocyte infiltration, prevented demyelination in the CNS, and consequently mitigated disease symptoms. Moreover, wogonin suppressed the release of neuroinflammatory cytokines by activated microglial cells, Th1 cells, Th2 cells, and Th17 cells in vitro and exerted therapeutic effects on attenuating the disease severity in the early disease onset stage and late disease progression stage. Our study suggests that wogonin treatment may not only prevent the onset of MS by inhibiting DC activation and migration but also potentially ameliorate the progression of MS by reducing neuroinflammation. This opens avenues for the development of new approaches in the therapy of autoimmune diseases.
Rapamycin ameliorates renal pathology in anti-Fx1A induced passive Heymann nephritis
Authors: Chunhui He
Published: November 2024

Our study focused on understanding the pathogenesis and treatment of membranous nephropathy (MN), a common cause of nephrotic syndrome (NS) characterized by glomerular basement membrane (GBM) thickening and immune complex deposition.

Through an Fx1A antibody-induced passive Heymann nephritis rat model, we observed a significant increase in kidney size, weight, and kidney-to-body weight ratio in diseased rats. However, oral administration of rapamycin mitigated renal pathology. Rats treated with rapamycin displayed kidney weights comparable to healthy rats, along with improvements in renal health indicators like proteinuria, blood urea nitrogen (BUN), and creatinine levels.

Furthermore, rapamycin-treated rats exhibited reduced inflammatory cytokine expressions (IL1b, IL6, IL10, IFNgama) comparable to healthy rats, in contrast to elevated expression levels in diseased rats. Histopathological analysis revealed diminished complement C3 and C5b-9 deposits, decreased renal tubular dilatation, and mitigated glomerular collapse upon rapamycin treatment.

Our findings indicate that rapamycin effectively improved renal pathology in anti-FX1A-induced passive Heymann nephritis, potentially suggesting a role for autophagy in modulating this condition.
Role of Immuno-Responsive Gene-1 (IRG1) in regulating Efferocytosis in Mouse Model of EAE
Authors: Nematullah Mohammad , FATMA MENA , RASHID FARAZ , Ayasolla Kameshwar , Ahmed MOHAMMAD EJAZ , WATER JEFFERY , MIR SAJAD , ZAHOOR INSHA , RATTAN RAMANDEEP , GIRI SHAILENDRA
Published: November 2024

Multiple sclerosis (MS) is a condition characterized by inflammation and demyelination, primarily affecting individuals in their youth. Numerous studies have documented the buildup of dying cells and myelin debris in the CNS, leading to inflammation and disability. This suggests that enhancing the removal of cellular debris could be a promising approach to address disability and promote the restoration of myelin. Efferocytosis is a process in which phagocytes take in apoptotic cells and help maintain tissue balance by releasing anti-inflammatory cytokines. However, the relationship between efferocytosis and EAE pathogenesis is not well understood. Loss of Irg1 is expected to interfere with the efferocytosis process, causing a strong inflammatory environment in the CNS, which in turn impairs disease resolution. We observed that Irg1 knockout displayed more severe experimental autoimmune encephalomyelitis (EAE) disease compared to wild type. Further, macrophages lacking Irg1 showed upregulation in the levels of NLRP3, GSDMD,and IL1β, along with a decrease in the phagocytic activity. Also, it showed reduced expression of key efferocytosis genes such MerTk, CD206, and arginase 1, suggesting that a lack of Irg1 leads to a highly inflammatory environment, resulting in impaired efferocytosis. Our results indicate that directing interventions towards the Irg1 gene could surpass the constraints of existing drugs and open possibilities for novel techniques in the treatment of MS.
Non redundant roles of group 2 innate lymphoid cells and T-helper 2 cells on anti-helminth immunity
Authors: Femoe Ulrich , Musaigwa Fungai , Li-Yin Hung , Inclan-Rico Juan Manuel , Nicolis Imani , Herbert De'Broski
Published: November 2024

The lung microenvironment is a critical target organ for trapping and killing migratory larvae of the murine hookworm Nippostrongylus brasiliensis during memory type 2 immunity. However, the relative contribution(s) of group 2 innate lymphoid (ILC2s) vs. T-helper 2 (TH2) populations in host protection and subsequent tissue repair are currently unknown. Data show that mice lacking locus control region 1 (LCR1-/-) have a selective defect in ILC2s resulting in impaired larval killing, pulmonary hemorrhage, and increased numbers of IL-17-expressing gdT cells. Adoptive transfer of excess numbers of CD4+ T cells into LCR1-/- mice restored larval killing during the secondary challenge but did not promote lung repair or suppress gdT cell-driven neutrophilic inflammation. Moreover, CD4 T cell depletion experiments demonstrate that while CD4 cells were necessary for the initial priming of ILC2s, CD4 cells were not essential for host-protective Type 2 immunity or tissue repair upon secondary challenge. This work suggests a division of labor between TH2 cells and ILC2 during primary and secondary responses, but that ILC2s serve a non-redundant role in limiting tissue injury through suppression of gd T cell dominant inflammation. Ongoing studies are designed to uncover the immunoregulatory/ tissue reparative molecules used by ILC2 during memory type 2 immunity against helminths.
Enteric pathogen targeting of the inflammasome pathway
Authors: Ricci de Azevedo Rafael , Kailasan Vanaja Sivapriya
Published: November 2024

Pattern recognition receptors-based sensing of microbial products and activities to mount an effective antibacterial defense is an evolutionarily conserved immune strategy. Cytosolic LPS sensing by caspase-11 or -4 and the ensuing noncanonical inflammasome-mediated IL-1 and cell death responses play a central role in the clearance of bacterial pathogens. In response to this selection pressure from the host it is natural that pathogens have developed strategies to actively antagonize or evade inflammasome responses. However, the mechanisms by which bacterial pathogens subvert the noncanonical inflammasome pathway is only beginning to be understood. In this context, this study investigates bacterial modulation of noncanonical inflammasome by utilizing Salmonella entericaserovar Typhimurium as a model enteric pathogen. By screening a library of defined single gene mutants of S. Typhimurium, we found that a mutant that lacks a Salmonella Pathogenicity Island 1 (SPI-1) effector triggers a markedly higher cell death and IL-1 responses in epithelial and professional innate immune cells indicating that this effector protein is a suppressor of inflammasome responses. Further characterization indicated that this effector protein-mediated inhibition of inflammasome responses occurs at the level of gasdermin D, the pyroptotic executioner. Together, this study attributes a novel inflammasome inhibitory function to a Salmonella SPI-1 effector.
Comparison of FOXP3 methylation status using three different methods
Authors: Hudzik Collin , Poulin Matthew
Published: November 2024

The FOXP3 gene is an X chromosome encoded transcription factor that is critical for the function of regulatory T (Treg) cells that maintain immunological self-tolerance and homeostasis. FOXP3 expression is primarily controlled by DNA methylation and the necessary development of Treg cells requires the establishment of Treg-specific DNA methylation patterns. FOXP3 and its epigenetic control genes have been associated with several immune-related conditions such as auto-immunity, asthma, food allergies and cancer. Here, we have conducted a study comparing the sensitivity to methylation status within our assays that cover FOXP3 using pyrosequencing, targeted bisulfite NGS (tbNGS), and methylation-sensitive high-resolution melting (MS-HRM) using a variety of different starting materials in both human and mice. As we transition away from pyrosequencing, we ensure that both tbNGS and MS-HRM result in methylation profiles which do not exhibit significant differences in sensitivity and accuracy to pyrosequencing, while also reducing the cost to the end user.
Anti-TIGIT antibody tiragolumab improves PD-L1 blockade via myeloid and Treg cells
Authors: Guan Xiangnan , Hu Ruozhen , Choi Yoonha , Srivats Shyam , Nabet Barzin , Silva John , McGinnis Lisa , Hendricks Robert , Nutsch Katherine , Banta Karl , Duong Ellen , Dunkle Alexis , Chang Patrick , Han Joy , Mittman Stephanie , Molden Nandini , Daggumati Pallavi
Published: November 2024

Tiragolumab, an anti-TIGIT antibody with an active IgG1/kappa Fc, demonstrated improved outcomes in the phase 2 CITYSCAPE trial when combined with atezolizumab (anti-PD-L1) versus atezolizumab alone. To understand the mechanism(s) of response with this combination, we leveraged tumor pretreatment samples, and peripheral blood mononuclear cells (PBMC) and serum samples (baseline and on-treatment) collected from patients enrolled in the clinical trials, as well as the syngeneic CT26 mouse tumor model by using mouse tiragolumab surrogate antibodies. Bulk RNA-seq, proteomics, scSeq, and flow cytometry were performed. We found that high baseline intratumoral macrophages and Tregs were associated with better outcomes in patients treated with atezolizumab plus tiragolumab but not with atezolizumab alone. Comparing the on-treatment versus baseline serum samples revealed that macrophage activation was associated with clinical benefit in patients receiving the combination treatment. In mouse tumor models, tiragolumab surrogate antibodies inflamed tumor-associated macrophages, monocytes, and dendritic cells through Fc gamma receptors (FcɣR), in turn driving anti-tumor CD8+ T cells from an exhausted effector-like state to a more memory-like one. These results uncover a mechanism of action by which TIGIT checkpoint inhibitors can remodel immunosuppressive tumor microenvironments, and suggest that FcɣR engagement is an important consideration in anti-TIGIT antibody development.
Unraveling the immune dynamics of lung tissue infected with Francisella tularensis in a spatial context
Authors: Arakkal Leanne , Roberts Lydia , Schwarz Benjamin , Bohrnsen Eric , Wehrly Tara , Germain Ronald , Bosio Catharine , Speranza Emily
Published: November 2024

Francisella tularensis (Ftt) is a highly virulent bacterial pathogen whose vaccine offers only short-lived immunity. While CD4+ T cells are required for protection, the mechanism behind waning immunity remains unclear. One potential reason is that the local environment induced by the bacteria inhibits T cell function. We therefore carried out a multi-omics study of the infection microenvironment following vaccination and secondary challenge in mice. Flow cytometry and single cell sequencing analysis revealed that during Ftt secondary challenge, the CD4+ T cells in vaccinated animals show a delayed proliferative response and produced high levels of IFN-γ which correlated with an up-regulation of indoleamine 2,3-dioxygenase (IDO1) by endothelial cells. Importantly, increased IDO1 expression corresponded with decreased levels of tryptophan in the lung as measured by LC/MS. Given the requirement for tryptophan in optimal T cell proliferation, we investigated if IDO1 expression was spatially associated with sites of bacterial replication. We performed multiplex immunohistochemistry targeting bacterial antigen, immune cells, and nutrient depleting enzymes and quantified the proximity of CD4+ T cells to IDO1-expressing cells which arose just outside the points of bacterial replication. Overall, this research uncovered a potential systems level of regulation that is inhibiting T cell function.

All studies were approved by the IACUC and was supported by the IRP of the NIH/NIAID
IL-18 promotes pro-inflammatory cell death in cytotoxic T-cell responses
Authors: Frank-Kamenetskii Anastasia , Varghese Jemy , Klinghoffer Hannah , Carrafiell Olivia , Morrisseette Jeremy , Canna Scott
Published: November 2024

Hyperinflammation is a life-threatening systemic inflammatory state that complicates 11% of all SIRS cases. Clinical observations have identified several genetic and/or acquired suseptibility factors. Though Perforin-deficiency is the best-studied mechanism of hyperinflammatory susceptibility, several genetic and biomarker discoveries support excess IL-18 is another. However, it remains unclear how IL-18 drives hyperinflammation. We hypothesized that excess IL-18 leads to excess IFN-y that operates, at least in part, at the critical point of T-cell activation known as the immunological synapse (IS).

Methods. We observed the effects of IL-18 and IFN-y on three IS parameters: IS duration, cytokine production, and the mode of target cell death. We measured IS duration by live-cell microscopy, and measured cytokine levels in supernatants. We performed experiments in the presence of several cell death inhibitors. In vitro findings were further analyzed in Il18tg and Prf1-/- mice infected with LCMV.

Results . Prf deficiency has been shown to increase IS duration and IFN-y production, however, excess IL-18 shortened IS duration but increased IFN-y production. Moreover, excess IL-18 or the use of BMDCs that were pre-exposed to high levels of IFN-y induced RIPK-1 dependent cell death. These data suggest that T-cell IFN-y, amplified by IL-18, acts on target BMDC to promote necroptosis. Correspondingly, in vivo Nec-1 (RIPK-1 inhibitor) dampened inflammation during LCMV infection.
Lipid activated T cell Inflammation in Type 2 Diabetes
Authors: Ayala Angulo Julio , Lopez Miranda , Wiggins Kiara
Published: November 2024

Current mouse models of Type 2 Diabetes (T2D) do not recapitulate the complexity of the human disease. Chronic inflammation in T2D patients has been described in the literature extensively. However, there are no clear answers describing what molecules are causing this inflammation and how it contributes to disease progression. Classically, T cells are known to mediate immune responses by recognizing protein that is presented to them. Recently, T cells have been shown to respond to lipids that are presented on a molecule called CD1a. This molecule is expressed by humans and not by mice. Using mice expressing human CD1a with comparable CD1a expression to human cells, we show these humanized mice on a high fat diet have human-like T2D including liver and adipose inflammation as well as higher glucose intolerance without insulin intolerance when compared to wildtype mice. We also see elevated immune infiltration into adipose and liver tissue in high fat CD1a mice. In addition, CD1a are more likely to have smaller islet on a high fat diet relative to wildtype as well as sign of pancreatic atrophy, a hallmark of T2D progression.These data validate T cell responses to lipids via CD1a as a potential contributor to T2D and will establish a more physiologically relevant mouse model of T2D through an immunological mechanism.
A Case for idiopathic CD8 T cell lymphocytopenia as a medical condition
Authors: Sung Alexander , Hudig Dorothy , Cruz-Amaya Judith , Dooley Staci , Hess John
Published: November 2024

Idiopathic CD4 Lymphocytopenia (ICL) is a clinical condition where patients (pts) have CD4 T cell counts less than 300/ul of blood, detected twice at least 6 weeks apart, are HIV-negative, and lack familial inheritance or therapy-caused lymphodepletion (A. Lisco et al., NEJM 2023). Many of these pts have papillomavirus-related diseases and cryptococcosis. We propose another low T cell condition, “Idiopathic CD8 Lymphocytopenia (IC8L)”, in which pts would have low CD8 T counts (<165 cells/ul blood, tested twice) with CD4 T cells > 300/ul. I C8L exclusion is based on HIV positivity or immunosuppressive therapies. The IC8L concept started with JH’s concern for pts with recurrent infections and notably low absolute lymphocyte counts that decreased after SARS-CoV-2 infection. Five pts were identified with CD8 counts ranging from 104-155/ul and CD4:CD8 T cell ratios as high as 5.3. For one pt, low CD8 T cells were transient. Three pts had stable CD8 T cell counts and another pt awaits retesting. The three pts (of a cohort of ~580 pts also >65 years of age in the practices of JH&SD) who met the proposed criteria had serious and recurrent infections. We conclude: (1) cause-and-effect relationships between SARS-CoV-2 infections, low CD8 T cells, and infectious diseases are impossible to assess for the individual pts and (2) it is essential to examine a large database to determine occurrence rates of “Idiopathic CD8 Lymphocytopenia” and its risks for infectious diseases.
Evidence of immune activation in pathogenesis of the HLA class II associated disease, podoconiosis
Authors: Gemeda Mikias , Newport Melanie , Howe Rawleig , Chanyalew Menberework , Teferi Tigist , Alcantra Diana , Towler Ben , Altmann Danniel , Boyton Rosemary
Published: November 2024

Podoconiosis is a form of lymphedema that causes bilateral swelling of the lower legs, resulting from exposure of bare feet to volcanic clay soils. There is an association between genetic variation in class II human leukocyte antigen genes and development of podoconiosis, raising the hypothesis that it is caused by an inflammatory immune response to an unknown soil component. Therefore, we aimed to characterise the immune profile and differential gene expression of podoconiosis patients relative to healthy controls. Here we report that, peripheral blood immunophenotyping of T cells indicated podoconiosis patients had significantly higher surface expression of HLA-DR molecules on both CD4 and CD8 T lymphocytes compared to healthy controls, while CD62L expression was significantly lower. The levels of the activation markers CD40 and CD86 were significantly higher on monocytes and dendritic cell subsets in patients compared to the controls. RNA sequencing gene expression data indicated higher levels of transcripts for activation markers, scavenger receptors and inflammation while lower levels of histones, T cell receptors, and immunoglobulin chain expression were found in podoconiosis patients. Our finding provides evidence that podoconiosis is associated with high levels of immune activation and inflammation compared to control subjects with over-expression of genes within the pro-inflammatory axis which could play a role in the pathology and lymphedema in the patients.
Metabolic dysfunction is associated with alterations in gut microbiota in obese adolescents
Authors: Xie Quin , Granato Alessandra , Paun Alexandra , Wong Anthony , Yau Christopher , Chu Lisa , Steele Shawna , Hamilton Jill , Danska Jayne
Published: November 2024

The gut microbiota (MB) has been implicated in adult metabolic dysfunction given evidence of dysbiosis in obesity and type 2 diabetes. However, few studies have investigated the relationship between metabolic disease, inflammation and the gut microbiota in children. We characterized the gut MB and immunometabolic profile of a cohort of obese, non-diabetic adolescents (MI4D) using high throughput fecal DNA sequencing with absolute bacterial quantitation. In the absence of antibiotics, we observed multi-log differences in fecal biomass correlated with stool water content and MB composition. Moreover, fecal bacterial biomass was associated with metabolic measures including serum triglycerides and insulin resistance in a sex-dependent manner. Mass cytometry demonstrated no significant associations of major immune cell subsets to fecal biomass or metabolic markers. In contrast, there were male-specific associations between serum markers of systemic inflammation and neutrophil activity with fecal biomass and waist circumference. Given the infiltration of visceral adipose tissue by myeloid cells in metabolic inflammation, our findings suggest that lower fecal biomass is associated with elevated systemic inflammation, especially in obese adolescent males. This study expands the understanding of sex-specific mechanism driving metabolic dysfunction in obese youth and suggests that gut MB alterations contribute to their metabolic and inflammatory dysregulation before diabetes diagnosis.
Bimodal single cell RNA sequencing reveals changes in CD4+ T cell transcriptomes with age
Authors: Khan Amir , Oliaeimotlagh Mohammad , Kumar Sunil , Hedrick Catherine , Ley Klaus
Published: November 2024

Aging affects the human immune system, resulting in modified autoimmunity, responses to infection and cancer. Here, we studied changes in CD4 T cells in subjects with and without coronary artery disease (CAD). PBMCs of 61 men and women who underwent cardiac catheterization were interrogated by single cell RNA sequencing combined with 49 protein surface markers via CITE-Seq. After quality, we identified 139,343 PBMCs from these patients, 40,816 were CD4 T cells, identified as CD19- CD3+ CD4+ CD8-. Linear regression was used to assess the impact of age on gene expression in these cells, adjusted for disease severity, sex, statin status, diabetes, and smoking status. As expected, the surface markers CD25 and CD45RO were positively and CD45RA negatively correlated with age. Across all subjects, 24 genes: GLUL, F5, CXCL10, CTSA, IL32, IFITM1, LAT2, TNFRSF8, RPN2, HLA.A, RGS1, TRAT1, TIGIT, STAT1, BAX, MKI67, CLC, LGALS1, F13A1, SPON2, CD163, IL3RA, VSIG4, CD200 were significantly correlated with age. Other genes were correlated in cases, but not controls: IL2RA, THBS1, VPREB3, PIK3AP1, TNFSF8, PAX5, IL2, LIF, CLEC4E, MMRN1, KLRC4, ADGRG3, PTPRC, ZNF683, IL22, TNFRSF25, CAVIN2, SYNE2, CD72, CD300A. Significant transcriptomic changes with age were observed in naïve, Th1, Tregs and exTregs CD4+ T cells. We conclude that age significantly changes the proportions and transcriptomes in CD4 + T cells. Also, sex, diabetes and use of statin confound these changes with age.
Prostaglandin I2 signaling in Treg restrains ST2 expression and associated pathogenicity
Authors: Norlander Allison , Abney Masako , Peebles Jr. R Stokes
Published: November 2024

We reported Prostaglandin I2 (PGI2) signaling within Treg is critical for Treg suppressive function in allergic airway inflammation (AAI). Interestingly, pathogenic Treg that have impaired suppressive capacity and promote AAI have greater ST2 expression. We hypothesized that Treg PGI2 signaling limits ST2 expression via restraint of β-Catenin signaling in AAI to prevent Treg pathogenicity. The percentage of ST2+ Tregs was elevated at baseline in IP deficient (IP KO) Treg, which lack the receptor for PGI2, compared to WT Treg. To further test our hypothesis, we utilized an Alternaria alternata (Alt) extract sensitization and challenge protocol. Alt-challenged mice that lacked IP signaling only on Treg (Foxp3YFPcrexIPflox) had elevated BAL IL-13, BAL eosinophils, lung vascular remodeling, and lung ST2+ Treg percentages compared to Foxp3YFPcre controls. Moreover, there were elevated total lung ST2+IL13+ Treg and ex-Treg in Alt-challenged fate-mapping IP KO mice compared to fate-mapping controls. Interestingly, BAL IL-13, BAL eosinophils, lung ST2+ Treg percentages were significantly decreased in Alt-challenged mice that had Treg specific deficiency for both IP and β-Catenin (Foxp3YFPcrexIPfloxxCtnnb1flox) compared to Foxp3YFPcrexIPflox mice. In summary, we demonstrate an association of Treg ST2 expression with enhanced Treg pathogenicity in a mouse model of AAI in IP deficient Treg mediated by β-Catenin. These data support PGI2 as a therapeutic for asthma.
BLOC1S1 deficiency promotes differentiation of Inflammatory Th2 cells and exacerbates atopic dermatitis and allergic airway inflammation in mice
Authors: Sharma Rahul , Wu Kaiyuan , Han Kim , Sack Michael
Published: November 2024

The levels of biogenesis of lysosome organelles complex 1 subunit 1 (BLOC1S1) control mitochondrial and endolysosome organelle homeostasis and function. Reduced fidelity of these vacuolar organelles is increasingly being recognized as influential in instigating cell-autonomous immune cell activation. We reasoned that exploring the role of BLOC1S1 in CD4+ T cells may further advance our understanding of regulatory events linked to mitochondrial and endolysosomal function in adaptive immunity.

We report that BLOC1S1-/- CD4+ T cells from mice and humans show enhanced Th2 activation. Transcript levels of the canonical transcription factors driving CD4+ T cell showed that the Th2 regulator GATA3 was exclusively induced in BLOC1S1-/- CD4+ cells. In parallel, in response to both T cell receptor activation and to Th2 polarization, the levels of IL-4, IL-5 and IL-13 were markedly induced in the absence of BLOC1S1. At a functional level, BLOC1S1-/- mice displayed increased susceptibility to allergic conditions, including atopic dermatitis and asthma. At the organelle level, mitochondrial DNA leakage is evident with STING accumulation on lysosomes and activation of the cGAS-STING NF-kB pathway to drive Th2 polarization. The genetic knockdown of STING in cultured CD4+ T cells ameliorates this immune regulatory cascade. In conclusion, we provide the first evidence that BLOC1S1 is essential in regulating Th2 cell expansion and developing type 2 allergic responses.
A Novel CD28-Targeting Molecule with Potent Therapeutic Impact on Asthma
Authors: Kuo Chieh-Ying , Chen Li-Chen , Kuo Ming-Ling
Published: November 2024

It has been well established that cytokines produced by T helper 2 (Th2) cells are responsible for asthmatic pathogenesis. Thus, the blockade of co-stimulatory molecules may control Th2-cell activation and attenuate asthmatic responses. We firstly identified Cyn-1324, a small compound, with high binding affinity to CD28 and can disrupt the interaction between CD28/CD80. This study investigated whether Cyn-1324 serves as an immunomodulator via blocking CD28/CD80 signaling, leading to the interference of CD4+ T cell activations and the relief of allergic asthma. Among house dust mite (HDM)-induced asthmatic mice model, the intranasal administration of Cyn-1324 significantly reduced airway resistance, eosinophilia in lung, and Th2-associated cytokines production in antigen-stimulated lymphocytes culture supernatants. Furthermore, we further studied the immunosuppression effect of Cyn-1324 in vitro. Cyn-1324 decreased not only murine but also human T-cell proliferation and IL-2 secretion, through the induction of T-cell anergy. This is the first identified compound that directly binds to CD28 and leads to T-cell anergy, but not the induction of Tregs. In addition, these findings suggest the potential of Cyn-1324 as an immunosuppressive agent capable of attenuating allergic responses through the blockade of the CD28/CD80 interaction. Cyn-1324 emerges as a promising candidate for further exploration in developing immunosuppressive therapies for allergic or autoimmune diseases.
GluK2 agonism suppresses dermal mast cell activation and proliferation
Authors: Zhang Youran , Keshari Swapnil , Das Jishnu , Yang Yufan , Zhang Jiying , Sumpter Tina , Kaplan Daniel
Published: November 2024

It has been recently appreciated that mast cells respond to glutamate in the context of neural transimtter release and tissue injuries. However, the precise subtype of glutamate receptor expressed on dermal mast cells at steady states and its function remain to be elucidated.

Herein, we identified the kainate receptor GluK2/KA2 as the predominant glutamate receptor expressed on dermal MCs at the mRNA and protein level. GluK2 agonists such as glutamic acid and kainic acid suppressed the activation of cultured peritoneal mast cells (PMCs) in response to MrgprB2 ligand compound 48/80, corroborating a previously demonsrated neuroimmune pathway.

The GluK2 selective agonist SYM2081 downregulated MrgprB2 expression and inhibited compound 48/80-mediated PMC activation. SYM2081 suppresed various mast cell-dependent inflammation models in vivo, such croton oil-induced irritant dermatitis, DNFB contact hypersensitivity, and LL-37 induced rosacea. The suppressive effect of SYM2081 on mast cells holds true in a model of human skin explants as well.

Transcriptomic analysis revealed that GluK2 agonism suppresses proliferation pathways in mast cells and downregulates granule content synthesis, such as mast cell chymase 1. Together, this study identified a previously unappreciated glutamate receptor on dermal mast cells and raised the interesting posibility of using selective GluK2 agonists to as a therapeutic agent for inflammatory conditions where MCs play a key role in the pathogenesis.
A model for cross-disciplinary and cross-institutional student collaboration for undergraduate immunologists
Authors: Singh Jastaranpreet
Published: November 2024

Immunology is a cross-disciplinary field, existing at the interface of biology, engineering and medicine. Therefore, a crucial skill for immunologists to develop is their ability to collaborate with others who have vastly different expertise and skillsets, such as biomedical engineers and statisticians. Undergraduate students at the University of Toronto (UofT) have few formal opportunities to engage in these types of collaborations. Most team-based projects allow students to work with fellow immunology peers, or students from other biology disciplines; however, the opportunity to do so with students in other fields is rare. Even fewer opportunities exist to collaborate with students from other academic institutions. To address this gap, two undergraduate courses, IMM360 (Scientific Methods and Research in Immunology, UofT) and BMEG372 (Biomedical Materials and Drug Delivery, University of British Columbia), were knit together in September-December 2022 and 2023. Here, we present our scaffolded approach for promoting collaboration on a cross-disciplinary, cross-institutional project. Furthermore, we will report student attitudes towards cross-disciplinary collaboration, which were assessed via surveys both at the start and end of the collaborative project. We hope that learnings from this work will inform an effort to weave cross-disciplinary collaboration throughout all years of the Immunology undergraduate program at the University of Toronto and other institutions.
Oxidized phospholipids regulate tenocyte function via the induction of amphiregulin in dendritic cells
Authors: Pinnaro' Veronica , Kirchberger Stefanie , Künig Sarojinidevi , Gil Cantero Sara , Della Porta Giovanna , Stöckl Johannes
Published: November 2024

Inflammation is a driving force of tendinopathy. Oxidation of phospholipids by free radicals is a consequence of inflammatory reactions. Here we have studied the impact of oxidized phospholipids (OXPAPC) on the function of human tenocytes. We observed that treatment with OXPAPC did not alter the morphology, growth, and capacity to produce collagen in healthy or diseased tenocytes. Since OXPAPC is a known modulator of the function of immune cells, we analyzed whether OXPAPC-treated immune cells might influence the fate of tenocytes. Treatment of immature, monocyte-derived dendritic cells with OXPAPC (Ox-DCs) was found to enhance the proliferation of tenocytes, in particular tenocytes from diseased tendons.

In contrast, the production of collagen by tenocytes was not altered upon co-culture with Ox-DCs. Since the supernatant of Ox-DCs was also able to stimulate the growth of tenocytes, we searched for a growth factor in the secretome of Ox-DCs and could identify amphiregulin (AREG), a ligand for EGFR. The addition of recombinant AREG to tenocytes was indeed a stimulus of proliferation. Diseased tenocytes were found to express higher levels of EGFR compared to tenocytes isolated from healthy donors and show a stronger proliferative response. In summary, inflammatory reactions in the tendon might lead to the oxidation of phospholipids which can trigger the production of AREG in resident DCs and thereby stimulate the proliferation of tenocytes.
Cx3cr1 deficiency exacerbates glomerulonephritis in MRL/lpr mice through modulating renal infiltration of IL-17 producing double negative T cells
Authors: Estaleen Rana , Oakland David , Montano Hilary , Alajoleen Razan , Lu Ran , Amin Pavly , Shakeri Aida , Reilly Christopher , Luo Xin
Published: November 2024

The CX3CR1 chemokine receptor initiates intracellular signaling cascades responsible for cellular activity, proliferation, and survival. CX3CR1 loss-of-function leads to exacerbation of chronic kidney disease in patients. Renal disease is a severe complication that often occurs in patients with systemic lupus erythematosus (SLE). We had previously reported that Cx3cr1 deficiency exacerbated glomerulonephritis in lupus-prone MRL/lpr mice. In our current studies, we sought to investigate the role of Cx3cr1-deficiency at different stages of SLE disease. We investigated glomerulonephritis development and renal infiltrating immune cells in MRL/lpr mice at 3, 7, 11, and 15 weeks of age. Inflammation in lupus-like disease was initiated at an earlier time-point in the Cx3cr1-deficient (Cx3cr1-/-) mice when compared to Cx3cr1-wildtype (Cx3cr1+/+) mice, where Cx3cr1-/- mice had significantly higher proteinuria at all time points. Double negative (DN) T cells which are pathogenic in lupus were found to be significantly expanded in the kidney of Cx3cr1-/- MRL/lpr mice at 3, 7, and 11 weeks of age with alterations in the production of two IL-17 isoforms, IL-17A and IL-17F. Our data suggest that CX3CR1 plays a role in a balanced production of IL-17A and IL-17F by DN T cells and that Cx3cr1 deficiency may alter disease pathogenesis through modulation of IL-17 isoforms.
Role of type Ⅰ interferon signaling in kainic acid-induced seizure model
Authors: Ma Jeong-Hwa , Hwang Inhwa , Yu Je-Wook
Published: November 2024

Type Ⅰ interferon (IFN) signaling has emerged as a significant factor in brain development and homeostasis. Also, its dysregulation has been associated with various central nervous system (CNS) diseases, including Alzheimer's disease. According to a recent study, microglia in kainic acid-induced seizures show an increase in interferon beta responsiveness. However, the specific role of type Ⅰ IFN signaling in seizure-related processes remains elusive. Therefore, in this study, we aimed to investigate the function of type Ⅰ IFN signaling in seizures. Here, we found the upregulation of interferon beta and interferon-stimulated genes in the hippocampus of mice subjected to kainic acid, which indicates the involvement of type Ⅰ IFN signaling in seizure events. To examine the specific roles of type Ⅰ IFN signaling in seizures, we induced seizures in C57BL/6 wild-type mice and IFNAR1 knockout mice and compared the severity of seizures and subsequent events. Interestingly, IFNAR1 knockout mice showed rapid rises in seizure severity in the early stages, but later exhibited a decrease in severity compared to the wild-type. On the other hand, interferon signaling did not have a significant impact on late-phase gliosis after seizures. These data provide novel insights into the crucial role of the interferon pathway in seizure progression, suggesting its potential as a therapeutic target for the treatment of seizures and epilepsy.
Maternal Infection and altered fetal immune programming.
Authors: Coronado J
Published: November 2024

The maternal-fetal interface involves complex and balanced interactions between mother and fetus, which support the development of the fetal immune system. While we know maternal helminth infection during pregnancy is known to disrupt this relationship and affect neonatal immune development, the fundamental mechanisms that drive fetal immune programming is not completely understood. To further examine how maternal parasitic infection influences offspring immunity, we established a model in which female mice prior to mating are infected with the persistent mouse intestinal helminth, Heligmosomoides polygyrus (HP). One week after infection, female mice are mated, and the progeny are aged to adulthood. The offspring are then challenged with influenza virus. Intriguingly, we found that offspring born from mothers with HP infections were less susceptible to influenza challenge than offspring born from uninfected mothers. This data implies that the effects of maternal helminth infection are long lasting, and potentially involve fetal programming during the early development of the immune system.
Investigating Neutrophil Subpopulations involved in Acute Lung Injury in Pediatric Patients with Congenital Heart Disease using a Single Cell Approach
Authors: Gianoli Stefano
Published: November 2024

Congenital Heart Disease (CHD) is a significant health concern (4-10/1,000 live births). Morbidity in CHD includes a younger age, thrombotic complications, and organ dysfunction. Acute lung injury (ALI) occurs intra/postoperatively in 2–3% of pediatric patients, particularly neonates and infants. Neutrophil recruitment to the lungs during CPB-induced ALI is noted. Recognizing the dual role of neutrophils in both defense and tissue damage, a nuanced strategy is essential.

Our preliminary single-cell RNA sequencing (scRNAseq) analysis of blood from pediatric patients undergoing longer (>=3 hours) versus shorter (<3 hours) CPB surgery reveals distinct neutrophil subpopulations to be enriched post-CPB and associated with ALI. Specifically, in patients with a longer CPB, the IFTIM2 gene, associated with type I interferon responses, is enriched. Pathway analysis confirms associations with interferon responses, chemotaxis, cell migration, reactive oxygen species, and cell death. Preliminary data shows that upregulated β2 integrin levels post-longer CPB suggest a predisposition to ALI.

This insight aims to guide the design of targeted therapies for specific neutrophil subpopulations while preserving their critical functions. This study will improve the treatment of these patients, addressing a crucial and critical gap in pediatric cardiac intensive care.
CD8+ T cells recognize de novo epitopes induced by TDP-43 dysfunction.
Authors: Chizari Shahab , Saigal Vidur , Gu Albert , Malaspina Andrea , Fratta Pietro , Jiang Jenny
Published: November 2024

Cytoplasmic aggregation and dysfunction of the DNA-binding protein, TDP-43, is an underlying pathology in disorders including amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), and inclusion body myositis (IBM). The functional loss of TDP-43 results in aberrant splicing and the inclusion of cryptic exons in hundreds of mRNA transcripts which ultimately lead to the expression of de novo proteins. Whether these de novo proteins can stimulate and activate T cells is unknown but would be critical in understanding the pathogenesis of these disorders. Cryptic peptides are strong candidates for T cell responses as they are likely not presented during T cell selection. Additionally, it has been demonstrated that there are highly differentiated and clonal populations of CD8+ T cells circulating the blood of patients with ALS-4. Therefore, we hypothesize that cryptic peptides induced by TDP-43 aggregation could be recognized by clonally expanded CD8+ T cells that exhibit an activated phenotype. In exploration of our hypothesis, PBMCs from ALS and IBM donors were collected and HLA-genotyped. Cryptic epitopes were bioinformatically predicted using a sliding window approach. From this epitope library, we have generated barcoded and fluorescently tagged peptide-MHC tetramers to capture and single cell sequence cryptic peptide-specific T cells. Preliminary results highlight several populations of circulating CD8+ T cells capable of recognizing cryptic peptides in ALS and IBM.