bluuebunny/crossref_metadata_2025 huggingface dataset:
Inflammasome-mediated sensing of multi-component pore-forming toxins
Authors: Man Si Ming
Published: January 2023

Host recognition of microbial components is essential in mediating activation of an effective immune response that drives clearance of the pathogen. Cytosolic innate immune sensors and the inflammasome can recognize both intracellular and extracellular pathogens. Here, we identified a bacterial factor secreted by the extracellular bacterial pathogen Bacillus cereus which engages activation of the inflammasome without entering the host cytoplasm. The identity of the secreted factor is a multi-component enterotoxin called hemolysin BL (HBL) that is highly conserved amongst B. cereus strains. The three subunits of HBL bind to the cell membrane in a single and linear order, forming a lytic pore inducing efflux of potassium, activation of caspase-1, secretion of IL-1β and IL-18, and cell death. Further, HBL-producing B. cereus induces rapid inflammasome-mediated mortality in the host within 24 hr. Pharmacological inhibition of the NLRP3 inflammasome using MCC950 efficiently prevented B. cereus-induced lethality. Overall, our results reveal that cytosolic sensing of a membrane-bound multi-subunit toxin via the inflammasome is central to the innate immune recognition of B. cereus infection. Therapeutic modulation of innate immune sensing pathways can lead to enhanced host protection against deadly bacterial infections.
Conditional disruption of Hematopoietic protein-1 in mice reveals an essential role for Hem-1 in myeloid cell functions
Authors: Suwankitwat Nutthakarn , Park Heon , Iwata Terri , Libby Stephen , Liggitt Denny , Iritani Brian M
Published: January 2023

Hematopoietic protein1 (Hem-1) is a hematopoietic cell-specific subunit of WAVE (WASP-family verprolin homologous protein) complex, which acts downstream immune receptors (including BCR, TCR, TLR, and cytokine receptors) to stimulate filamentous actin (F-actin) polymerization. Inactivating mutations in NCKAP1L, the gene encoding Hem-1, have been recently associated with Primary Immunodeficiency Disease in humans (unpublished), and high NCKAP1L expression has been associated with a poor prognosis in Chronic Lymphocytic Leukemia. Using constitutive Hem-1 null mice, we previously published that Hem-1 is critical for normal lymphopoiesis and innate immunity. However, the cell autonomous functions of Hem-1 in individual immune cell types remain an enigma. The objective of this study was to define the cell autonomous roles of Hem-1 in myeloid cells. We created conditional Nckap1lfl/flmice, which were bred to LyzMCre mice to delete Hem-1 specifically in myeloid cells. We found that neutrophils from myeloid cell specific Hem-1 null mice exhibited defective F-actin polymerization and impaired migration in response to the chemoattractant fMLP. Hem-1 deficient macrophages were unable to efficiently phagocytose live bacteria. Myeloid specific Hem-1 null mice were much more susceptible to influenza virus (H1N1/PR8) infection based on significantly increased body weight loss, increased peribronchial inflammation, reduced alveolar macrophage number, and increased pro-inflammatory cytokines in bronchiolar lavage fluid. Collectively, our results reveal previously uncharacterized cell autonomous roles for Hem-1 in primary myeloid cells, and suggest that Hem-1 is critically important for effective anti-viral immunity.
Syk-dependent Glycolytic Reprogramming in Dendritic Cells Regulates IL-1-beta Production to Fungal-assoicated Ligands in a TLR-independent Manner
Authors: Amiel Eyal , Thwe Phyu M. , Fritz Daniel , Snyder Julia
Published: January 2023

Dendritic cells (DCs) activated via TLR ligation experience metabolic reprogramming, in which the cells are heavily dependent on glucose and glycolysis for the synthesis of molecular building blocks essential for maturation, cytokine production, and the ability to stimulate T cells. Although the TLR-driven metabolic reprogramming events are well documented, fungal-mediated metabolic regulation via C-type Lectin Receptors such as Dectin-1 is not clearly understood. Here, we show that activation of DCs with fungal-associated ligands induces acute glycolytic reprogramming that supports the production of IL-1b via inflammasome activation. This acute glycolytic induction to fungal-associated ligands is depending on Syk signaling in a TLR-independent manner, suggesting now that different classes of innate immune receptors functionally induce conserved metabolic responses to support immune cell activation. These studies provide new insight into the complexities of metabolic regulation of DCs immune effector function regarding cellular activation associated with protection against fungal microbes.
Role of NLRC5 and IRF1 in the induction of MHC class I
Authors: Vijayan Saptha , Meissner Torsten B. , Lee Kyoung-Hee , Liu Yuen-Joyce , Downs Isaac , Sidiq Tabasum , Zhang Chi , van den Elsen Peter J. , Kobayashi Koichi S.
Published: January 2023

MHC class I and MHC class II plays a major role in adaptive immune responses through activation of CD8+ T cells or CD4+ T cells respectively, by presenting intracellular or extracellular antigens. The MHC class I transactivator (CITA), NLRC5 was recently identified as a key transcriptional regulator of MHC class I and related genes. Although promoters of both MHC class I and class II genes share similar cis-regulatory elements, the specificity of NLRC5 to MHC class I gene transactivation remains unclear. To delineate the specificity of NLRC5 in NLRC5-dependent MHC classs I transactivation, we performed co-immunoprecipitation with different deletion mutants of IRF1. The results were further confirmed in mice lacking Irf1 and Nlrc5. We found that the transcription factor IRF1 associates with NLRC5, enabling efficient transactivation of MHC class I and related genes. Double deficiency of Irf1 and Nlrc5 resulted in the severe reduction of MHC class I and related gene expression in vitro and in vivo. Therefore, IRF1 is a key component that determine the specificity of NLRC5-dependent MHC class I gene transactivation, and their involvement in the CITA enhanceosome is critical for MHC class I-dependent immune responses. Further studies on the interaction of IRF1 and NLRC5 would shed light on the potential therapeutic intervention on the MHC class I related diseases such as auto-immune disorders, cancer immunity and in organ transplantation
TLR-2, TLR-3 and TLR-4 agonists induce synthesis of IFN-γ in human M1 but not in M2a, M2b and M2c macrophages through the activation of S6K1-S6 pathwayγ
Authors: Kumar Ashok , Iqbal Salma , Mukerjee Ananda , Gajnayaka Niranjala , Ahmad Faria , Crawley Angela
Published: January 2023

IFN-γ, a pro-inflammatory cytokine produced primarily by T cells and NK cells, activates macrophages and engage mechanisms to control pathogens. Although murine macrophages have been shown to produce IFN-γ, very little is known about IFN-γ production by normal human macrophages and their subsets. Herein, we show that priming of human monocyte-derived macrophages (MDM-M0) with IL-12 and IL-18 or IFN-γ alone leads to IFN-γ production in M1 macrophages that is significantly enhanced transcriptionally as well as at the protein level following stimulation with Toll-like receptors (TLR)-2, TLR-3 or TLR-4 agonists in contrast to the M0, M2a, M2b and M2c macrophages. The macrophage derived IFN-γ is biologically active and can activate macrophages in an autocrine manner. To determine the signalling pathway responsible for TLR-4-induced IFN-γ production in M1 macrophages, our results show that TLR-4-induced IFN-γ production is regulated by mTORC-1/2, PI3K, p38 and JNK MAPKs and S6Kinase-S6 pathway. The S6K1-S6 pathway plays a crucial role in TLR-induced IFN-γ production in M1 macrophages as IFN-γ-induced S6 phsophorylation (Ser 235/236) was inhibited by the inhibitors specific for PI3K, p38-MAPK and JNK MAPK, and mTORC-1/2 suggesting that S6K1-S6 pathway plays a critical role in TLR-4 induced IFN-γ production in human M1 macrophages.
Ikaros zinc finger transcription factors as novel regulators of STAT factor activity
Authors: Read Kaitlin , Sreekumar Bharath , Powell Michael Duane , Jones Devin , Oestreich Kenneth J
Published: January 2023

Cytokine signaling is important for the differentiation and function of a number of cell types, including lymphocyte populations. One mechanism by which cytokine signals are propagated is via phosphorylation-mediated activation of Signal Transducer and Activator of Transcription (STAT) factors, which directly regulate cell type-specific gene programs. We recently identified novel regulatory modules composed of STAT and Ikaros Zinc Finger (IkZF) transcription factors in CD4+ T cell populations. Specifically, we found that IkZF/STAT modules composed of Aiolos/STAT3 and Eos/STAT5 directly activate genes associated with T follicular helper (TFH) and T helper 1 (TH1) gene programs, respectively. Here, we extend these findings and show that IkZF factor expression correlates with STAT activation, suggesting that IkZF proteins may regulate STAT factor activity in CD4+ T cells. We find that overexpression of Aiolos, but not the IkZF factors Ikaros and Eos, results in increased STAT3 activation. Conversely, overexpression of Eos, but not Ikaros or Aiolos, results in increased activation of STAT5. Importantly, when we performed these studies using IkZF protein mutants incapable of interacting with STAT factors, the observed increase in STAT factor phosphorylation was lost. We also find that in CD4+ T cells, Eos deficiency correlates with reduced STAT5 activation, while Aiolos-deficient cells display reduced STAT3 activation. Finally, we observe differential IkZF factor expression across CD4+ T cell subsets. When coupled with known differences in STAT factor activation, these findings suggest that IkZF/STAT regulatory modules may broadly regulate T helper cell differentiation.
Single cell profiling reveals unique stages of NK cell development
Authors: Beaulieu Aimee M
Published: January 2023

Natural killer (NK) cells are potent cytotoxic lymphocytes that provide critical host defense against pathogens and malignancy. Current understanding of the molecular and cellular events that define the stage-wise progression of NK cell development is limited. Here, we use single-cell RNA sequencing (scRNA-seq) to identify unique stages of NK cell development. Our data reveal previously unappreciated heterogeneity within the progenitor and immature NK cell compartments. Notably, we find that these compartments are comprised of several unique subpopulations that express distinct transcriptional and epigenetic factors, intracellular signaling molecules, metabolic enzymes, and cell cycle regulators. Collectively, our data provide important insights into the molecular events that underlie NK cell development, and have the potential to inform NK cell-based therapies aimed at treating cancer and infectious disease in humans.
Predicting Natural Killer Cell Behavior with Mathematical Models
Authors: Millan Alberto Jovani , Sindi Suzanne , Manilay Jennifer
Published: January 2023

Natural killer (NK) cells are specialized lymphocytes with an innate ability to eliminate virally infected and cancerous cells, but the mechanisms that control NK cell development and cytotoxicity are incompletely understood. We hypothesize mathematical models can be used to predict how to direct NK cell development toward mature NK cells. We observed Sostdc1-knockout (KO) mice to display a partial NK cell developmental block. Building from these studies, a best fit model was simulated in which rates of transition and differentiation and cell death was determined by ordinary differential equations of deterministic compartmental models. This approach indicated that NK cell proliferation rates are not necessary to predict WT and KO population outcomes. We further hypothesize that clusters of highly cytotoxic NK cells can be identified computationally, using a combination of Ly49 receptor expression, in vitro and in vivo cytotoxicity assays using β2m−/− targets, and viSNE analysis of flow cytometry data. Sostdc1-KO NK cells also display defective cytotoxicity. We identified that KO splenic CD27+CD11b+ transitional NK cells express lower frequencies of inhibitory Ly49G2, but higher frequencies of activating Ly49H+ and D+ cells. Our viSNE results identified 3 NK cell clusters displaying a “superactive” phenotype that were virtually absent in KO mice; consistent with our observations of their hyporesponsiveness. Taken together, these data support a role for Sostdc1 in the regulation of NK cells and could provide insights into novel biological parameters to expand active NK cell numbers with high killing efficiency for immunotherapies.
Heterogeneity in differentiation capacity among ILC precursors in human peripheral blood and tissues
Authors: Kokkinou Efthymia , Gutierrez-Perez Irene , Pandey Ram Vinay , Rao Anna , Mazzurana Luca , Almer Sven , Höög Charlotte , Lindforss Ulrik , Bryceson Yenan , Mjösberg Jenny
Published: January 2023

Background

The origin of human Innate Lymphoid Cells (ILCs) remains less well characterised compared to mice. Single-cell RNA-sequencing recently revealed a population of ILC precursors (ILCP) expressing CD62L and CD45RA, which shows diversity in frequencies among blood, tonsil and intestine.

Objective

We set out to determine the differentiation capacity and epigenetic landscape of blood, tonsil and intestinal ILCP.

Methods

Blood, tonsil and intestinal ILCP were sort-purified on the basis of CD62L and CD45RA (CD62L+/−CD45RA+/−) and cultured on OP9-DL1 stromal cells under specific cytokine conditions. Additionally, genome-wide chromatin accessibility was measured using ATAC-sequencing.

Results

We show that all ILCP subsets in blood and tonsil were able to differentiate to IFN-γ-producing ILC1/NK cells. Whereas all blood ILCP subsets readily differentiated to IL-13-producing ILC2, no or very few ILC2 were generated from tonsil ILCP. In contrast, tonsil ILCP subsets showed higher ability to generate IL-22-producing ILC3, compared to blood ILCP. These data indicate that ILCP in blood are poised towards ILC1/ILC2 while ILCP in tonsil favours ILC1/ILC3 differentiation. To further dissect their development, we are currently analysing global epigenetic information of tonsil ILCP, aiming to delineate their chromatin landscape. Finally, we investigated the ILCP in intestinal samples and detected accumulation of CD62L−CD45RA+ ILCP in the inflamed mucosa of patients with inflammatory bowel disease (IBD) as compared to the non-inflamed tissue. We are currently analysing the potency of those cells.

Conclusion

Heterogeneity in ILC differentiation in blood and tissues could further reveal novel targets for IBD treatment.
M. tuberculosis antigen-specific T-cell function in breast milk of HIV-infected mothers
Authors: Warr Alex Jay , Shah Javeed , LaCourse Sylvia , Kinuthia Jon , Maleche-Obimbo Elizabeth , Cranmer Lisa , Nguyen Felicia , Matemo Daniel , John-Stewart Grace , Hawn Thomas
Published: January 2023

Objective

Breast milk (BM) is a mucosal compartment containing T cells, however, little is known regarding BM T-cell functional capacity or role in infant immunity. We hypothesized that BM T cells have M. tuberculosis (Mtb)-specific Th1 responses that are compartment-specific and differ from peripheral blood (PB) responses.

Methods

HIV-infected mothers and their infants were enrolled in a randomized clinical trial of isoniazid to prevent Mtb infection in Kisumu, Kenya. Maternal BM and PB were collected at 6–10 weeks postpartum. Cells were stimulated with Mtb whole cell lysate and controls. We measured frequencies of T cells expressing CD4 and CD8 and intracellular cytokines IFN-γ, IL-2, and TNF-α.

Results

Among 12 mothers with >1000 CD3+ cells evaluable, 33% had Mtb-specific IFNγ responses, 66% had Mtb-specific IL2 responses, 66% had Mtb-specific TNF responses, and 25% had combined IFNγ, IL2, and TNF responses. The most common Mtb-specific CD4 cytokine profile was IFNγ-IL2+TNF+ (66%) and the most common CD8 profile was IFNγ+IL2-TNF+ (58%). When compared to PB profiles (N=25), BM had higher polyfunctional CD4 cells expressing IFNγ-IL2+TNF+ (median 0.11 BM vs 0.0 PB, p=.005) and lower frequencies of IFNγ+IL2-TNF− CD4 cells (median 0.0 BM vs 0.03 PB, p=.002). CD8 cells in BM had higher frequencies of IFNγ+IL2-TNF+ cells compared to PB (median 0.05 BM vs 0.0 PB, p=.0002).

Conclusions

Our data demonstrate the presence of Mtb antigen-specific Th1 cells in BM with cytokine profiles distinct from PB responses. Defining BM Mtb-specific immune responses may inform novel vaccine strategies. Studies are ongoing to examine correlations between maternal BM and PB Mtb-specific T-cell responses and infant responses to BCG vaccination.
Differential phenotypes and localization of CD8 T cell responses to acute and chronic viral infection
Authors: Hardman Blair K , Osborne Lisa
Published: January 2023

Norovirus represents the major viral source of gastroenteritis worldwide and the leading cause of foodborne illness in North America. Most infections are acute, symptomatic, and rapidly cleared but some cases persist asymptomatically or induce post-infectious irritable bowel syndrome. Despite the global economic burden of these infections no vaccine to prevent disease exists. Using immune correlates of acute vs chronic infection in mouse models following infection with related strains of murine norovirus, MNV-CW3 and MNV-CR6 respectively, this research interrogates the mechanisms underlying strain-specific differential antiviral CD8 T cell responses. The phenotype and quantity of MNV specific CD8 T cells are analyzed by flow cytometry. Since MNV-CW3 and -CR6 replication is higher in the small and large intestine, respectively, immunofluorescent microscopy is used to determine whether CD8 T cells are preferentially recruited to either tissue. Combining these complementary techniques provides novel insight into mechanisms governing intestinal antiviral T cell responses. Preliminary data shows activated MNV-specific CD8 T cells first accumulate in the MLN, suggesting this is the site of immune activation following oral infection with either virus. However, the magnitude and kinetics of T cell activation are reduced in CR6-infected mice compared to CW3-infected mice. This data suggests that these highly related viral strains induce activation of distinct populations of, or pathways in, APC populations with long-lasting effects on the outcome of infection. These results may have broad impacts on our understanding of how non-latent, chronic viral infections persist within a host.
Mucosal immune mediated protection of the CNS following olfactory viral infection
Authors: Moseman E. Ashley
Published: January 2023

Barrier immunity, particularly at mucosal surfaces, provides a first line of defense against invading pathogens. Unlike classical mucosal barrier surfaces in the gut and respiratory tract, the olfactory epithelium within the nose is a specialized barrier structure containing layers of olfactory sensory neurons (OSNs) dedicated to our sense of smell. Within nasal airways, OSNs are vulnerable to infection but also directly connected to the brain, thus anatomically, the olfactory epithelium functions as a mucosal barrier to the central nervous system (CNS). Upper respiratory viral infections can damage the olfactory neuroepithelium and are associated with loss of olfaction. Neurotropic viruses, such as vesicular stomatitis virus (VSV), readily infect OSNs, yet the olfactory epithelial response to infection is unknown. VSV infection drove a massive influx of hematopoietic cells including antigen presenting cells and antigen specific CD4+ and CD8+ T cells that established residence within the olfactory epithelium after viral clearance. Upon rechallenge, neutralizing antibody completely protected the neuronal tissues of the olfactory mucosa and the CNS, however this protection appeared to be secretory IgA independent. Resident memory T cells within the olfactory mucosa rapidly produced effector cytokines and prevented heterotypic viral challenge from accessing the CNS. Olfactory VSV infection also resulted in widespread viral and immune mediated tissue damage that contributed to permanent changes in airway turbinate structure. Collectively, these studies offer insight into how the olfactory barrier remodels its neural-immune architecture to provide enhanced protection following a viral challenge.
Host immunity modulates efficacy of fecal microbiota transplantation treatment of Clostridium difficile infection
Authors: Abt Michael C , Pamer Eric G. , Littmann Eric R. , Zarin Isma , Carter Rebecca A.
Published: January 2023

Clostridium difficile is an opportunistic pathogen that infects the large intestine following perturbation of the intestinal microbiota causing epithelial damage and debilitating, potentially fatal, colitis. Current antibiotic treatment options result in high recurrence rates, highlighting the need to identify alternative approaches to control this disease. Fecal microbiota transplantation (FMT) is clinically proven to efficiently clear recurrent C. difficile infection. Despite remarkable efficacy, implementation of FMT therapy is limited due to inadequate understanding of FMT mechanism of action. We use a murine model of persistent C. difficile infection to demonstrate a critical role for the host’s immune system in supporting efficacy of FMT. Immunodeficient Rag1−/− and littermate Rag1HET mice infected with C. difficile exhibit comparable establishment of persistent infection with no difference in bacterial burden or toxin levels in the cecum. Following FMT, Rag1HET mice resolve C. difficile infection while cohoused Rag1−/− mice fail to clear the infection. Analysis of intestinal microbial communities following FMT demonstrates that the microbiota of Rag1HET mice assimilate toward the composition of the FMT donor, while Rag1−/− mice exhibit impaired engraftment of bacterial species associated with 2° bile acid biosynthesis. Conversion of 1° bile acids to 2° bile acids in the cecum drives resolution of C. difficile. Metabolomic analysis of cecal content from Rag1HET mice following FMT reveals restoration of 2° bile acids while the cecal content from FMT-treated Rag1−/−mice remain comprised of 1° bile acids. These data support a role for host immunity in determining efficacy of microbiota-based therapeutics.
Protective effect of resveratrol on the integrity of alveolar and intestinal epithelial barrier in SEB-induced acute lung injury
Authors: Alghetaa Hasan F , Mohammed Amira , Nagarkatti Mitzi , Nagarkatti Prakash S
Published: January 2023

Acute lung injury (ALI) is a state of inflammation that breaks down the lung endothelial and epithelial cell barriers. In the current study, we investigated the role of resveratrol (RES) in regulating the expression and functions of tight junction proteins (TJP) in epithelial cell responses following exposure to this superantigen. To this end, C3H mice were given resveratrol orally twice prior to intranasal challenge with lethal SEB doses. 16S rRNA results showed that there were microbes transported in the blood in addition to the lung and colonic tissues. For this purpose, we used a reporter E. coli-GFP labeled bacterium to monitor and examine the viability of this bacterium in case of leak from lung into blood and other body compartments during the peak of ALI. The flow cytometry results showed significant reduction in copy numbers of this reporter in the blood in RES-treated mice in comparison to vehicle-treated mice. However, bacteriological examination for viability by culture of the blood showed all were negative for all experimental groups. Mouse transcriptome array showed increase in TJP gene expression in colonic and alveolar epithelial cells of RES-treated mice. Some of these genes were validated with qPCR. We found that there was an increase in the expression of some TJP genes following RES treatment. Commensals and probiotics are also known to decrease intestinal barrier dysfunction caused by inflammatory cytokines. Therefore, mass spectrometry (MS) analysis showed increase in the metabolome in colons of RES-treated group that help in strengthening of intestinal barrier. In conclusion, RES treatment increased directly or indirectly the rigidity of cell boundaries resulting in amelioration of ALI-dependent complications.
Skin-recirculating NK cells express α4β1 integrin, E-selectin ligand, and produce IFNγ
Authors: Riley John T , Hunka Julia , Debes Gudrun F.
Published: January 2023

NK cells are increasingly recognized as important players of skin immunity in health and during skin diseases, including skin cancers and autoimmunity. Despite their importance, little is known about the functional and migratory properties of skin-associated NK cells. Here, we employed afferent lymph vessel cannulation in sheep to assess NK cells re-entering the lymphatic system from the skin. NK cells (CD3−Nkp46+) consistently comprised 2–5% of total lymphocytes traveling in afferent lymph. The majority of skin-recirculating NK cells expressed perforin and readily produced IFNγ upon stimulation, indicating continuous recirculation of pro-inflammatory NK cells through unperturbed skin. Skin lymph NK cells expressed high levels of α4β1 integrin, similar to other circulating NK cells including those in blood and mesenteric lymph. Furthermore, a subset of lymph-borne NK cells expressed E-selectin ligand (CLA), suggesting a preference to return to skin in a manner similar to skin-homing T cells. Consistent with organ-selective as opposed to ubiquitous homing we observed that skin-recirculating NK cells lacked the gut-homing molecule, α4β7 integrin. In chemotaxis assays, skin-draining NK cells responded strongly to CCL21, a ligand for ‘tissue exit receptor’ CCR7, suggesting that NK cells use CCR7 to egress from skin via lymph. Our results support a model in which skin-specific NK cells with proinflammatory properties continuously survey the skin and are readily available for potential NK memory responses. Additional studies are underway to further assess the function and tissue tropism of skin-draining NK cells.
Aryl hydrocarbon receptor activation does not impact the number of myeloid-derived suppressor cells in the spleens of mice immunized with cholera toxin
Authors: King Aspen E , Schoenherr Elleanour M , Metten Madeline R , DeKrey Gregory K
Published: January 2023

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates immune responses. Previous studies in this laboratory have found that AhR activation with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice suppresses the anti-cholera toxin (CT) IgA response, but fecal antibody levels recover at least two weeks earlier than serum levels after repeated immunization. Studies by others have shown that TCDD exposure increases splenic myeloid-derived suppressor cell (MDSC) numbers in mice, and MDSC have been associated with enhanced IgA responses. In this study, we hypothesized that TCDD exposure would increase MDSC numbers in CT-immunized mice. Female C57Bl/6 mice were administered peanut oil (vehicle) or TCDD (40 μg/Kg) orally on day 0, and each animal was orally immunized with cholera toxin (CT, 10 μg/Kg) on days 1 and 8. On day 14, CT-specific serum IgA levels (ELISA) were significantly suppressed by TCDD exposure to 21.6% of control (P < 0.05), but fecal IgA levels were not significantly different. Total spleen cell numbers were significantly reduced by TCDD exposure to 72.9% of control (P < 0.05), but neither the percent nor number of MDSC (CD11b+ Gr1+, by flow cytometry) per spleen was significantly altered: 2.21% ± 0.48% and 1.91% ± 0.23% (450,000 ± 98,000 and 290,000 ± 34,000) for control and TCDD-treated, respectively (mean ± SEM). These results suggest that AhR activation differentially impacts antigen-specific IgA responses between serum and feces in cholera toxin-immunized mice, but not the number of MDSCs.
Formation of Human T Cell Mucosal Resident Memory Starts in Early Life with the Intestines
Authors: Connors Thomas , Thapa Puspa , Yopes Margot C , Guyer Rebecca S , Li Mei Mei , Snyder Mark E , Farber Donna L
Published: January 2023

Memory T cells are essential for optimal protection from previously encountered pathogens. Immune responses in infants lack the benefit of memory and are intrinsically distinct from those in adults. Early life adaptive immune responses have been characterized by increased generation of terminal effector subsets with decreased production of long lived memory. Here we investigate the origin of memory T cells utilizing flow cytometry and immunohistochemistry to characterize proportions and localization of T cell subsets in mucosal (intestine/lung) tissue from infant organ donors (n=12). At birth lung T cells are predominantly naïve and localized to the interstitium of the airways with memory accumulating over the first year of life. In contrast, significant proportions of memory phenotype T cells can be found in the lamina propria and intraepithelial layer of the intestine shortly after birth along with naïve cells. Effector memory subsets could be detected in robust proportions, highest in the jejunum, from 3 organ donors all less than 8 days of age. Additionally these memory T cells displayed high levels of canonical resident memory markers (CD69 and CD103). We conclude that mucosal tissues are host to naïve cells which cannot be attributable to circulating populations and that memory subsets are generated within these mucosal sites early in life. Importantly T cells in the youngest donors (ages 0–8 days) displayed resident memory phenotypes raising the possibility of prenatal development of adaptive memory responses populating the intestine. These results warrant additional investigation to determine the persistence and functionality of these subsets and emphasize the importance of the intestine to early life adaptive immunity.
Effect of Muscarine and Atropine on the production of myeloperoxidase and proinflammatory cytokines in small intestine
Authors: RESENDIZ-ALBOR ALDO ARTURO , Arciniega-Martinez Ivonne Maciel , Santamaria-Chavez Monica Miguel , Rebollar-Ruiz Xochitl Abril , Campos-Rodriguez Rafael
Published: January 2023

The cholinergic system it’s known as a modulator of the intestinal immune system. The aim of this work was to analyze the impact of muscarine and atropine on the production of myeloperoxidase (MPO) on lamina propia of small intestine as well as other local effects, like stimulation of goblet cells and mucus secretion. Three groups of male BALB/c mice were administred by i.p. route: a) control (SS), b) atropine (4 mg/kg) and c) muscarine (50 ng/kg). At 7 day of treatment, histological samples were obtained for Naphtol AS-D method to MPO activity analysis, immunofluorescence assay to localize MPO secreting cells and Flow Cytometry for intracellular TNF-α, IFN-γ, IL-2 and IL-6 measures. In the analysis of the histological samples it was observed that in the group treated with muscarine the number and size of goblet cells and mucus were augmented in the lumen. The number of neutrophils in the villus was also augmented in the muscarine group in comparison of treated with atropine. On the other hand, the immunofluorescence had an increased number of positive cells and activity in tissue to MPO in the muscarine group. In the Flow Cytometry analysis for lamina propia and Peyer’s patches, IFN-γ and TNF-α were augmented in the muscarine group, while IL-2 and IL-6 decreased. In this study, the MPO can be regulates immune functions such as increasing cytokines in macrophages and endothelial cells, also suppressing DC’s function of antigen presentation and switch to adaptive immunity which can lead to more innate response, and increased production of proinflamatory cytokines by lymphocytes.
The E3 Ligase VHL Controls Lung Inflammation by Regulating Epithelium-ILC2 Cross-talk
Authors: Han Jihye , Wan Qingqing , el Baghdady Sarah , Lee Jeeho , Liu Yun-Cai
Published: January 2023

Group 2 innate lymphoid cells (ILC2) are a specialized subset of lymphoid effector cells that play an important role in allergic inflammation in response to host-derived cytokines and alarmins. Pulmonary epithelium play a critical role in the initiation of type 2 immunity by production of cytokines, such as TSLP, IL-25 and IL-33 after exposure to allergens. However, the epithelium-derived signals and their regulatory mechanisms that directly regulate ILC2 remain unclear. Here we report that conditional deletion of the E3 ubiquitin ligase VHL in lung epithelial secretory club cells caused a selective increase of ILC2 under steady state and during pulmonary inflammation, resulting in exacerbated type 2 immune responses accompanied by increased eosinophil infiltration. VHL deficiency in lung epithelial cells upregulated the number and activity of ILC2, which was rescued by hypoxia-inducible factor 1α(HIF1α) ablation. Our findings indicate that VHL-HIF pathways control allergic inflammation in the lung epithelium by regulating ILC2, but the mechanisms of their regulation need to be identified.
TLR4 signaling regulates an inflammatory response to food allergens
Authors: Campbell Evelyn , Nagler Cathryn
Published: January 2023

Allergies have become increasingly more prevalent over recent years, and this increase has been found to correlate with dysbiosis of the gut microbiota. Bacterial signaling through TLR4 is known to be play a role in allergic sensitization, but the specific host-bacterial interactions and cellular immunological networks have not been elucidated. Preliminary data from our lab has shown that colonization of germ-free mice with feces from a subset of cow’s milk allergic (CMA) infant induces Serum-Amyloid A1 (Saa1) expression in the small intestine epithelium. Saa1 is an acute-phase protein that is part of a transcriptome signature associated with a Th17 response induced by adherent bacteria in the small intestine. Moreover, we show that in mice with a global ablation of TLR4, upregulation of Saa1 in abolished. Because dendritic cells (DCs) are known to be important for mediating intestinal immune responses, we sought to determine the roll of CD11c+ cells in mediating Saa1 upregulation. Antibiotic-treated CD11ccre TLR4fl/fl SPF mice were colonized with CMA feces and intestinal epithelial cells from the ileum were isolated to determine relative expression of Saa1 compared to antibiotic-treated SPF mice. Results from these experiments show that ablation of TLR4 signaling in CD11c+ cells reduces Saa1 upregulation in the ileal epithelium. Future directions of this work include examining immune cell populations in the small intestine draining lymph nodes and lamina propria to determine if this phenotype increases type 3 inflammation to exacerbate an allergic response.
Lymphotoxin receptor beta expression by neutrophils prevents metabolic activation and colitis pathogenesis
Authors: Giles Daniel A , Riffelmacher Thomas , Zahner Sonja , Andreyev Alexander Y. , McArdle Sara , Perez Tamara , Van der Gracht Esme , Morris Venetia , Tumanov Alexei , Kronenberg Mitchell
Published: January 2023

Inflammatory bowel disease, while multifactorial, is largely characterized by an exacerbated intestinal immune activation. However, the critical mechanisms regulating immune activation remain to be fully defined. We have previously reported that a member of the TNF superfamily, TNFSF14 (LIGHT), and its interaction with the lymphotoxin beta receptor (LTβR), is required for preventing severe disease in a mouse model of dextran sulfate sodium (DSS) induced colitis. Thus, we aimed to determine the cell type and mechanism critical to LTβR mediated protection from DSS-induced colitis. Employing the use of LTβRfl/fl mice and tissue-specific Cre-mediated deletion, we found that neutrophils were a critical LTβR expressing cell type. Mrp8-Cre driven deletion of neutrophil LTβR expression resulted in exacerbated DSS-induced colitis and drastically increased colonic neutrophil accumulation. Mechanistically, RNASeq analysis of neutrophils revealed transcriptional alterations in genes associated with cellular metabolism, and mitochondrial function in particular. Ex vivo studies of bone marrow neutrophils from LTβRfl/fl x Mrp8-Cre mice, compared to Cre-controls, revealed increased mitochondrial mass and number, as well as elevated mitochondrial ROS production. Subsequent metabolic studies demonstrated that LTβR deficient neutrophils exhibited elevated mitochondrial respiration and glycolysis. Interestingly, these increased levels of mitochondrial mass, ROS production and glycolytic activity persist in LTβR deficient colonic neutrophils during DSS-induced colitis. In sum, our results demonstrate that neutrophil LTβR signaling plays a critical role in the immune activation associated with DSS-induced colitis.
IFN-β induces IL-6 production by B cells to promote TH17-mediated neuroinflammation
Authors: Agasing Agnieshka M , Gawde Saurabh , Siebert Nadja , Ruprecht Klemens , Paul Friedemann , Axtell Robert C.
Published: January 2023

Both type I interferon (IFN-I) and T helper 17 (TH17) drive pathology in neuromyelitis optica spectrum disorder (NMOSD) and TH17-induced experimental autoimmune encephalomyelitis (TH17-EAE). This is paradoxical because the prevalent theory is that IFN-I inhibits TH17 function. Here, we identify that IFN-I promotes IL-6 production from B-cells to exacerbate TH17-mediated disease in NMOSD and TH17-EAE. We identified that high levels of IL-17, IFN-I chemokines and IL-6 correlate with severe disability in NMOSD. Furthermore, IL-6 levels were significantly lower in patients treated with anti-CD20 antibody therapy. In mice, IFN-I treatment elevated IL-6 and exacerbated TH17-EAE. Strikingly, therapeutic blockade of IL-6 attenuated disease in IFN-I treated mice. We further demonstrate that IFN-I stimulates B cells to produce IL-6 which in turn drives pathogenic TH17 differentiation. These findings identify potential prognostic biomarkers for NMOSD, define molecular processes behind NMOSD pathogenesis and clarify the paradox surrounding IFN-I and TH17-induced disease.
Synergistic effect of IL-27 and retinoic acid on the expression of CD39 on mouse Treg cells
Authors: Maslanka Tomasz , Gregorczyk Izabela , Jasiecka-Mikołajczyk Agnieszka , Jaroszewski Jerzy
Published: January 2023

CD39, NTPDase 1, confers a stronger suppressive activity on Foxp3+CD25+CD4+ regulatory T (Treg) cells. Therefore, the induction of CD39 expression on Treg cells may be a promising drug target for autoimmune disorders. This study investigates the effect of IL-27 and all-trans-retinoic acid (ATRA) on CD39 expression by murine Treg cells in the course of in vitro activation. The mean percentage of CD39+ cells within Treg cell subset in untreated, IL-27-treated, or ATRA-treated cultures was 9.57, 13.29 and 14.31, respectively. Thus, the mean percentage of CD39-expressing Treg cells induced by exposure to IL-27 or ATRA alone (i.e. after deduction of control values) was 3.72 and 4.73. Co-exposure to IL-27 and ATRA induced a considerably greater increase in the percentage of CD39-expressing Treg cells compared to that produced by either IL-27 or ATRA alone. This effect was spectacular, since the mean percentage of CD39-expressing Treg cells was found to be 31.45. Thus, the combined exposure to IL-27 and ATRA induced CD39 expression on 21.88% Treg cells. This value was significantly greater than the mean percentage of CD39+ cells within the Treg cell subset calculated by summing up the values of this parameter obtained in the cultures treated with IL-27 or ATRA alone (i.e. 8.45%), which proves a strong superadditive effect between both agents. In conclusion, both IL-27 or ATRA have potential to induce CD39 expression on Treg cells. However, this effect seems to be relatively weakly expressed, and therefore its clinical importance is uncertain. The combined exposure to IL-27 and ATRA leads to a strong synergistic effect on CD39 expression by Treg cells, what may have important clinical implications in future.
Restoring the balance in autoimmunity: the promise of IL-2 mutein
Authors: Khoryati Liliane , Pham Minh Nguyet , Sherve McKenna , Kumari Swarnima , Bogdani Marika , Campbell Daniel J. , Gavin Marc
Published: January 2023

The development and function of regulatory T cells (Treg) relies on interleukin-2 (IL-2), and Treg efficiently compete with other cells for IL-2 by expressing the highest levels of the IL-2 receptor alpha-chain (CD25). Accordingly, defects in Treg and the IL-2 pathway have been reported in several autoimmune diseases including type 1 diabetes (T1D). Low-dose IL-2 therapy has shown promising results in autoimmunity; however, autoreactive immune cells can also be activated by IL-2, and the pleiotropic effects of IL-2 raise concerns regarding its widespread utility. Hence, there is a need to develop novel IL-2 based therapeutics with enhanced Treg-specificity. Hypothesizing that reduced IL-2 affinity for IL-2Rβ should increase dependency on high CD25 expression and enhance Treg-selectivity, we generated and screened twenty-eight Fc-fused murine IL-2 muteins (Fc.IL-2 mutein), and identified an optimized mutein that showed reduced signaling but prolonged association with CD25. In vivo, this optimal mutein led to greater enrichment and Ki-67 induction in Treg compared to wild-type (WT) IL-2, and reduced agonism of effector cells, resulting in an improved Treg:effector ratio. In the non-obese diabetic mouse model of T1D, we observed significant protection from disease in mice treated with Fc.IL-2 mutein compared to WT IL-2. The enhanced efficacy of the mutein was most pronounced at high doses, revealing preferential Treg stimulation across a wide dose range. Thus, Fc.IL-2 mutein treatment represents a safer and more efficient alternative for the WT IL-2 therapy, allowing for selective Treg enrichment and a targeted control of the autoimmune response.
Investigating heme regulation of REV-ERBα activity in Th17 cells
Authors: Mosure Sarah A , Kojetin Douglas , Solt Laura A
Published: January 2023

T helper 17 (Th17) cell dysregulation is implicated in the pathogenesis of autoimmune and chronic inflammatory diseases. Because current therapeutics exhibit negative side effects or are only successful in small patient subsets, there is a need to identify better Th17 cell-specific drug targets. We recently showed that the nuclear receptor (NR) REV-ERBα represses Th17 cell development and pathogenesis. As ligand-regulated transcription factors, NRs like REV-ERBα are amenable to pharmacological modulation, making REV-ERBα a viable target for the treatment of Th17 cell-mediated diseases. However, the mechanisms underlying ligand regulation of REV-ERBα in Th17 cells remain unclear.

REV-ERBα-mediated repression of target genes is enhanced by its endogenous ligand, heme, in other cell types, but heme’s role in Th17 cells is largely unexplored. To investigate heme’s role in Th17 cells, we differentiated naïve CD4+ T cells under Th17-polarizing conditions in vitro and found heme treatment repressed differentiation. To assess whether the effect was due to activation of REV-ERBα, we heme-treated cells lacking REV-ERBα and found the repression was partially ablated. We also found that overexpression of a REV-ERBα mutant incapable of binding heme relieved repression of Th17 cell differentiation relative to overexpression of wild type REV-ERBα. Our findings suggest that heme negatively regulates Th17 cell development, in part by activating REV-ERBα, which may require heme binding to repress target genes. Characterizing REV-ERBα regulation by heme may offer insight into mechanisms of Th17 cell pathogenesis, as well as inform the design of therapeutics for autoimmune and chronic inflammatory diseases.
Inhibition of the Cullin RING E3 ubiquitin ligase by MLN4924 targets DEPTOR degradation in Tregs to enhance their stability and functional activity
Authors: Wedel Johannes , Liu Kaifeng , Briscoe David M
Published: January 2023

DEPTOR is an intracellular mTOR_binding protein in CD4+ Tregs that functions to enhance lineage stability and suppressive activity. Following activation, DEPTOR is targeted for degradation by the Cullin RING E3 ubiquitin ligase, thus decreasing its expression which we postulate results in Treg instability. In this study, we wish to test if small molecule inhibitors that sustain DEPTOR levels can promote Treg function. Human CD4+ T cells were stimulated with anti-CD3/CD28 with/without MLN4924 (1–100 nM) for 6 hours. By Western blot analysis, activation was associated with a time-dependent reduction in DEPTOR protein levels, but MLN4924 sustained its expression. Using in vitro Treg stability assays, we find that human CD4+CD25hiCD127lo Tregs loose FOXP3 expression over 7 days in culture, but retain expression when cultured in the presence of MLN4924 (100 nM) (P<0.01; 23% vs. 4% FOXP3+). In vivo, the treatment of C57BL/6 recipients of BALB/c cardiac allografts with MLN4924 (60 mg/kg) every 12 hours for the first five days post-transplant significantly (P<0.01) prolonged allograft survival (mean survival 18 days vs. 7 days, n=6/group). Furthermore, MLN4924 was not efficient to prolong graft survival when CD4-specific DEPTOR knockout mice (C57BL/6) were used as recipients (P<0.01). Collectively, these data indicate that inhibitors of the Cullin RING E3 ubiquitin ligase have potent immunosuppressive effects in part through targeting DEPTOR degradation in CD4+ T cells and by increasing Treg stability and immunoregulation in vivo. These findings have broad application following transplantation to augment tolerance, and as novel therapeutics to treat autoimmune disease.
Decreased CD4+-Th/CD8+-Tc ratio in Bone marrow at diagnosis of acute myeloid leukemia Predicts Increased Risk of Graft-Versus-Host-Disease in Transplant Patients
Authors: Wang Weiwei , Li Haibo , Fan Guang , Shen Lisong
Published: January 2023

Introduction

Acute myeloid leukemia (AML) is an aggressive disease with high morbidity and mortality. Transplant is one of the most important treatments for AML. However, graft-versus-host-disease (GVHD) remains one of major problems in transplant patients. Since immune repertoire plays an important role in the GVHD occurrence, we investigate the relationship between lymphocyte subsets and the risk of GVHD.

Methods

60 initial diagnostic flow data of AML transplant patients were reviewed (Oregon Health and Science University 2010 to 2016, 30 females and 30 males, with a median age of 51.417 years, range 13–72). 36 patients with GVHD including acute and chronic GVHD while 24 patients without GVHD. The lymphocyte subsets were analyzed including CD3+T-cells, CD3+CD4+Th, CD3+CD8+Tc, CD4/CD8 ratio, CD19+CD20+B, CD19+CD5+B1, CD3+CD56+NKT, CD3−CD56+NK, and three NK subsets (CD56brightNK, CD56normalNK, CD56dimNK).

Results

As to the overall survival (OS), there’s no statistic difference between age<60 years old or age ≥60 years. Compared with non-GVHD group, decreased percentage (%) of Th, increased % of Tc & low Th/Tc ratio were observed in GVHD group (P=0.0281, P=0.0394, P=0.0340 accordingly). Receiver operating characteristic (ROC) analysis indicates that low Th/Tc ratio predicts increased risk of GVHD (P=0.0231, AUC=0.663, 95% Confidence Interval is 0.530 to 0.780). There’re no statistic differences between GVHD and non-GVHD for all other lymphocyte subsets percentage (P>0.05).

Conclusions

AML patients with GVHD post transplant have a lower Th cell percentage and higher Tc cell percentage at diagnosis. This is to say, decreased Th/Tc ratio in BM of initial diagnosed AML patients may predict a high risk of GVHD post transplant.