bluuebunny/crossref_metadata_2025 huggingface dataset:
Follicular Regulatory T cells Positively Regulate Follicular Helper T Cells, Germinal Center B Cells and IgE Response in Peanut Allergic Mice
Authors: Xie Markus Ming , Liu Hong , Dent Alexander
Published: January 2023

Food allergy remains a major health problem in developed countries such as United States. However, the immunological mechanisms that control peanut allergy are largely unknown. In an oral sensitization food allergy model using peanut protein and cholera toxin (PCT), we found mice (Bcl6FC: FOXP3cre Bcl6 fl/fl) deficient in follicular T regulatory (Tfr) cells had lower follicular helper T (Tfh) cells and germinal center B (GCB) cells after two challenges. The decrease in Tfh/GCB cells was seen in both mesenteric Lymph Node and spleen responses. In this two-challenge model, peanut specific IgE, IgG1 and IgA antibody (Ab) responses were sharply decreased in Bcl6FC mice compared with wild type (WT) ones. However, after eight PCT challenges, Tfh and GCB cells were similar in Bcl6FC mice compared with WT, and peanut specific IgE was elevated in the absence of Tfr cells. We also show that Tfh/GCB cells are required for IgE production in this food allergy model. In mice with Blimp1-deficiency in T regulatory (Treg) cells (Blimp1FC: FOXP3cre Blimp1 fl/fl), Tfr, Tfh and GCB cells were significantly higher compared with WT or Bcl6FC mice while Treg derived IL-10 was much lower. Peanut specific Ab responses trended lower in Blimp1FC mice after two PCT challenges. Our data presented here is the novel evidence showing a positive regulation of Tfh/GCB cells and IgE response by Tfr cells, and suggest that a key regulatory pathway of Tfh/GCB cells and IgE response involves IL-10 production by Tfr cells. Supported by The AAI Careers in Immunology Fellowship Program.
Pam3CSK4 Induces B Cells to Release FasL+ Killer Exosomes That Suppress Allergic Asthma
Authors: Lundy Steven K. , Yang Jennifer , Taitano Sophina , van der Vlugt Lucien
Published: January 2023

We have previously shown that mouse B lymphocytes can express Fas ligand (FasL) on their cell surfaces and can kill peptide antigen-specific T helper cells. In humans, FasL protein produced by human B cells was exclusively found intracellularly on the surface of exosomes. In the current study we determined that mouse B cells are a major source of FasL+ exosomes, as well as contributing significantly to the Granzme B+ exosome pool. The major B cell subsets in the spleen and lungs that produced FasL and Granzyme B were marked by CD9 and/or CD107a expression. We tested a panel of toll-like receptor ligands in combination with anti-IgM and CD40 ligand for their ability to activate killer function in purified mouse B cells. The TLR2 ligand Pam3CSK4 was a potent inducer of surface FasL expression and FasL+ exosome release from B cells. Using a mouse model of OVA-specific allergic airways disease, we showed that transfer of Pam3CSK4-induced B cell-derived exosomes diminished total numbers of eosinophils and CD4+ T cells in the bronchoalveolar lavage fluid (BAL). Lung TH cell apoptosis was higher in the mice that received exosome transfer. Lung cells produced less IL-4 and IL-5 upon restimulation with OVA in the exosome-treated mice. Effects of Pam3CSK4-induced exosomes on BAL cell infiltration, TH cell apoptosis and cytokine production were all diminished when exosomes were derived from mice with a functional mutation of the FasL gene. These data indicate a potential role for B cell-derived killer exosomes in control of allergic airways disease.
Syndecan-1 (CD138) Regulates Competition Between Antibody Secreting Cells for Survival
Authors: Fooksman David R. , McCarron Mark
Published: January 2023

Long-lived antibody secreting cells (LLASCs) are critical immune effector cells constitutively secreting high affinity antibody providing prophylaxis against infections. In contrast to live attenuated viruses which can provide lifelong immunity, protein-based vaccines are known to poorly induce LLASCs, often requiring multiple boosting to generate sufficient antibody titer. The factors that control LLASC production are poorly understood, but are critically important for all humoral-based vaccines. Survival of LLASCs is chiefly mediated by soluble cytokines, such as APRIL in the BM, and IL-6 in secondary lymphoid tissues. After immunization, antibody secreting cells (ASCs) express variable levels of CD138, and its function was unclear, since it can bind 100+ proteins, and CD138−/− mice have no overt phenotype. However, we have discovered a direct role for CD138 for potent antibody responses, by controlling ASC maturation and survival after immunization via cytokine signaling. LLASCs express the highest level of CD138 among all cells, in comparison to newly minted ASCs, which express intermediate levels of CD138. We propose that high expression of CD138 provides LLASCs with higher binding and accumulation of IL-6 and APRIL, which are limiting, leading to a competitive advantage over new ASCs for survival.
Role of Astrocyte Activation in Experimental Cerebral Malaria
Authors: Tunon-Ortiz Arnulfo B.N. , King Thayer P. , Mimche Patrice N. , Lamb Tracey J.
Published: January 2023

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum (Pf) with a high lethality rate. Key features at the cellular and molecular level are endothelial cell (EC) activation, blood-brain barrier (BBB) dysfunction, pro-inflammatory cytokine signaling, and sequestration of infected red blood cells (iRBCs). While research on malaria as a peripheral infection is extensive, there is still much left to investigate in terms of its neuropathological progression. Astrocytes are cells that maintain the stability of the BBB, yet nothing is known about their role in maintaining the BBB during cerebral malaria. In the mouse model of experimental cerebral malaria (ECM), we have used flow cytometry to determine that astrocytes in the cerebellum and frontal cortex of mice infected with Plasmodium berghei ANKA (PbA) are both activated at 6 days PI, a time-point correlated with breakdown of the BBB. Furthermore, EphA2, a receptor tyrosine kinase, is upregulated in malaria and plays a critical role in BBB disruption. We show increases in EphA2 densities on astrocytes in ECM. The implications of these data for BBB disruption in cerebral malaria will be discussed.
Endogenous cannabinoid, anandamide mitigates acute lung injury by altering the microbiome and metabolome profile in the gut-lung axis with the induction of an anti-inflammatory response
Authors: Sultan Muthanna Ali , Nagarkatti Prakash , Ngarkatti Mitzi
Published: January 2023

Staphylococcus Enterotoxin B (SEB) is a superantigen that can activate ~30% of T cells. Inhalation of SEB can lead to toxic shock syndrome and death. We have shown that anandamide (AEA) is effective in ameliorating SEB-induced ALI. Cross-talk between microbiome through the gut-lung axis (GLA) can influence the immune and respiratory functions. In the current study, we found that AEA treatment alleviated SEB-induced ALI in mice by alteration in the microbiome and the metabolome profile especially short-chain fatty acid (SCFA) production in the GLA. Sequencing of the V3–V4 region of 16S rRNA of colon, cecal flushes and lungs was performed and analysis showed that AEA treatment led to a significant increase, starting from the phylum level in Bacteroidetes until genus Aldercreutzia. Mass Spectrometric (MS) analysis revealed that there was a significant increase in the SCFAs in AEA treatment group, especially iso-butyrate, which may be responsible for induction of T regulatory cells (T regs). Flow cytometry data showed that AEA increased CD4+FOXP3+ in the lung compared to vehicle-treated SEB-induced disease group. Our study also showed that AEA treatment significantly decreased the infiltration of inflammatory cells in the lungs. Furthermore, AEA treatment led to a significant decrease in gut and lung leakage, and pro-inflammatory cytokines (IL-2 and TNFα) while increasing anti-inflammatory cytokines (IL-10). Collectively, these data suggest that AEA ameliorates ALI and prevents leakage through modulation of microbiome profile and the metabolome in the GLA while promoting an anti-inflammatory response to include induction of Tregs and decrease in pro-inflammatory cytokines.
Microbiota Regulation of Pro-atherogenic Phagocytes − Separating Metabolic and Inflammatory Mechanisms.
Authors: Eshghjoo Sahar , Ding Yufang , Hunt Carrie , Jayaraman Arul , Alaniz Robert Christopher
Published: January 2023

A significant hallmark of the chronic inflammatory disease, atherosclerosis, is the accumulation of pro-inflammatory M1-like macrophages (macs) and dendritic cells (DCs) in coronary arteries that respond to pro-atherogenic stimuli and fail to digest lipids that contribute to the formation of foam cells in atherosclerotic plaques. Mechanisms that reduce Mac/DC inflammation, increase lipid degradation, and prevent foam cell formation are likely to decrease progression of atherosclerosis. Mechanisms that regulate autophagy in macs and DCs will likely reveal targets for the prevention of atherosclerosis.

Mouse models of high-fat diets demonstrate a link between microbiota composition and the development of obesity, diabetes, and atherosclerosis through several mechanisms best demonstrated by the microbiota-dependent production of TMAO that promotes atherosclerosis.

We have identified a tryptophan-derived microbiota metabolite, indole, that has novel immunomodulatory properties. This strong association between obesity, inflammation, and the microbiota has led us to the hypothesis that microbiota-derived metabolites in the GI tract might regulate dietary saturated fat-induced inflammation. To test this hypothesis, we generated bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) and tested the effects of indole on these cells in the context of saturated fatty acid-induced inflammation. We investigated pro-inflammatory cytokine production, M1 macrophage development, and metabolic flux. Here, we demonstrate that indole reduces saturated fatty acid-derived inflammation in BMDCs and BMDMs and regulates metabolic programming of phagocytes by inducing autophagy on a level similar to rapamycin.
VCAM-1 might be a potential angiogenic biomarker for Metal-on-Metal prosthetic device and FSH-induced inflammation
Authors: Wood Steven C. , Jamison Joshua
Published: January 2023

Follicle stimulating hormone (FSH) rises in peri-menopausal women and remains elevated during menopause. Bone loss, previously attributed to estrogen, may be due to direct effect of FSH. On this background of decorticated bone, hips are replaced due to fractures or osteoarthritis. Metal-on-metal (MoM) hips replacements were developed as they were thought to be longer lasting and robust implants. However, in women, they are failing at a significantly higher rate when compared to men. Failure modes include pain, device loosening due to osteolysis and exuberant blood vessel formation resulting in pseudotumor formation. Recent work from our lab has demonstrated the molecular interaction of the wear particles from MoM with immune cells and the release of inflammatory and pro-angiographic factors such as IL-1b, TNF-a and IL-8. Therefore we hypothesize that binding of monocytes to the endothelium induced via FSH and MoM wear particles should stimulate angiogenesis that would increase detectable inflammatory biomarkers. Vascular inflammatory markers were investigated with FSH and MoM particle stimulation of endothelial cells and bound monocytes. From these data, soluble VCAM-1 protein levels increased during monocyte-endothelial co-culture induced by FSH and MoM wear particle co-stimulation. FSH stimulation was also associated with increased monocyte binding and endothelial tube thickness. These data indicate that FSH promotes vascular endothelial cell cohesion and monocyte recruitment, which may lead to increased VCAM-1 expression towards combined MoM wear particle and FSH stimulation. Our findings suggest that sVCAM-1 may be a biomarker of pseudotumor formation elicited by MoM wear particles in women with elevated FSH.
PD-1H facilitates phagocytic clearance of HIV infected T cells by monocyte derived macrophages and dendritic cells
Authors: Petersen Sean , Xu Xuequn , Camarillo Cynthia , Yi Guohua
Published: January 2023

Phagocytosis is a critical component of the immune response to viral infection, resulting in the clearance of infected cells, while minimizing exposure of uninfected cells. HIV is able to evade this response through suppression of key regulators of phagocytosis. Programmed death 1- homologue (PD-1H) has been identified as a co-inhibitory molecule that suppresses activation of T cells during infection and cancer immunogenesis. However, our evidence indicates that PD-1H may also play a significant role during immune response to HIV infection via upregulation and subsequent phagocytosis of infected T cells. HIV-induced apoptosis and monocytic cell engulfment was tested utilizing CEMSS T cells as target cells and monocyte derived macrophages (THP-1), as phagocytic cells. Cells were infected with a GFP-labeled HIV strain, NL4-3. HIV-infected T cells displayed greater apoptotic activity (approximately 9.0%) relative to mock-infected controls. Concurrently, phagocytosis of HIV-infected T cells increased approximately 4-fold. Expression of PD-1H on infected T-cells was detected on 16.7% of cells, which correlated with increased phagocytosis. When an antagonistic antibody against PD-1H was used, the number of phagocytic cells was reduced by a factor 2, which was replicated utilizing human stem cell derived dendritic cells. Phagocytosis was also confirmed by the upregulation of IL-1β, which was 5 fold higher relative to controls. Here we demonstrate that PD-1H facilitates monocytes-derived macrophages or DCs to phagocytize HIV-infected T cells in cell based assays. Based on these results we believe that further study in humanized mouse models of HIV infection is warranted.
Epithelial Cell Control of Adaptive Immune Plasticity in Periodontal Disease
Authors: Wallet Shannon , Delitto Andrea , Rocha Fernanda , Amador Byron , Sorenson Heather
Published: January 2023

In periodontal disease (PD), subgingival bacteria initiate and sustain a non-resolving inflammation that is ineffective at controlling the infection. T cells are critical for proper control of infection, but also homeostasis at mucosal surfaces including that of the periodontium. We have previously demonstrated that PD is associated with the local expression of cytokines associated with induction of Th(c)1/Th(c)17 plasticity, whereby loss of oral epithelial cell (OEC) MyD88-dependent-signaling results in exacerbation of this response. The objective of this study was to determine in which T-cell compartment plasticity occurs during PD and whether MyD88-dependent OEC signaling regulates this plasticity. Mice with MyD88-sufficient (B6) or MyD88-deficient (B6OEC.MyD88) OEC were subjected to oral polymicrobial infection for six weeks, after which the severity of disease as measured by alveolar bone loss and immunophenotype of the cellular infiltrate were evaluated. A temporal reduction in the frequency of Th(c)17 cells along with an increased frequency of Th(c)1 and Th(c)1/Th(c)7 cells was observed in B6 mice as PD progressed. This conversion was concomitant with a decrease in the frequency of regulatory T-cells and soft tissue IL10 and TGFβ expression. This immunophenotype was exacerbated in time of onset and magnitude in B6OEC.MyD88mice and was associated with an increase in alveolar bone loss. Together these data demonstrate that T-cell plasticity plays a role in PD whereby OEC-MyD88-signaling at least in part controls this plasticity. These data suggest that OECs can be a target for local modulation of T-cell plasticity as an intervention for the treatment of periodontal disease.
Characterizing the function of the CD300e immunoregulatory molecule.
Authors: Chen Kate , Silveira Pablo , Lo Tsun Ho , Hart Derek , Clark Georgina
Published: January 2023

The CD300 molecules are a family of immunoglobulin-like molecules that have immune regulatory functions. It includes both inhibitory and triggering members, which are highly conserved within the family as well as across species. CD300e, the most restricted member of the family, is expressed in low amounts in most of tissue, whereas its expression is upregulated in different cancers, such as, colorectal, stomach, liver, ovarian and pancreatic cancer. CD300e is highly expressed on CD14+ monocytes. Differentiation of monocytes into dendritic cells (DCs) and macrophages down regulated their CD300e expression. As no monoclonal antibodies are available yet to mouse CD300e, we generated a CD300e knock-out (KO) mouse as an alternative approach to study its biology. We have characterized the changes in immune cells populations resulting from CD300e deletion. The CD300eKO mice are in general fit and reproductively healthy. Mice with CD300e deficiency has a significantly reduction in their circulating leukocytes compared to wild type mice. Hematopoietic stem cells from CD300eKO mice were capable of differentiating in vitro into DC or macrophages with normal antigen uptake and T cell stimulatory capacity. We are using CD300eKO mice to elucidate CD300e function by further testing a variety of functions in monocytic cells.
Detection of chicken interleukin-23, a heterodimeric cytokine with monoclonal antibodies reacting p19 and p40 subunits
Authors: Kim Woohyun , Lim Yeaseul , Lillehoj Hyun S.
Published: January 2023

Interleukin-23 (IL-23) is a heterodimeric cytokine consisting of two covalently linked subunits, p19 (IL-23α) and p40 (IL-12/IL-23β) which is shared with IL-12. IL-23 plays an important bridging role between the innate and adaptive immune responses. Recently, chicken IL-23 (chIL-23) has characterized and shown functional similarity to mammalian IL-23 in production of IFN-γ from T cells and macrophages. In the present study, thus, we developed mouse monoclonal antibodies (mAbs) against two chIL-23 subunits, p19 and p40 and characterized their specificities in various immunoassays. To differentially detect IL-12 and IL-23 which share p40 subunit, both were expressed functionally by cotransfection with genes encoding IL-12α (also known as p35 subunit) and IL-12/IL-23β, or IL-23α and IL-12/IL23β into mammalian system. By several immunoassays including Western blot, and immunocytochemistry, several IL-23α mAbs react with IL-23 were found out. We also found some IL-12/23β mAbs react with IL-12 and IL-23 and some react with IL-23 only, but not with IL-12. Using these mAbs, we developed capture ELISA assay to quantify native IL-23 from various samples. One of the mAbs also exhibited its activity to neutralize the IL-23 in production of IFN-γ in chicken lymphocytes. We believe that these mAbs will be important immune reagents for basic and applied research of IL-23 in poultry.
Lipocalin2 mediates anti-inflammatory functions through the inhibition of STAT3 and activation of STAT5 signaling in bone marrow derived macrophages
Authors: samuels janaiya , Rayalam Srujana , Shashidharamurthy Rangaiah
Published: January 2023

Lipocalin2 (Lcn2), an innate immune protein, is known to be significantly up-regulated during inflammatory disorders including arthritis, however, its molecular mechanisms remain to be elucidated. We have shown that Lcn2-knockout (Lcn2KO) mice developed more severe serum-induced arthritis (STA) compared to wild-type (WT) mice with significantly reduced neutrophil infiltration but considerably more macrophage migration. Therefore, we have investigated the possible role of Lcn2 in regulating macrophages. Bone marrow cells were isolated from Lcn2KO and WT mice and polarized to classical or inflammatory (M1) and alternate or anti-inflammatory (M2) macrophages. The polarization of M1 (iNOS) and M2 (arginase-1) phenotypes was confirmed by Western blot analysis. We did not observe a difference in polarization of Lcn2KO and WT macrophages suggesting that Lcn2 may not alter the polarization of macrophages. However, anti-inflammatory cytokines such as TGF-β1 and IL-10 were significantly reduced in either LPS or IC-stimulated M2 phenotypes from Lcn2KO mice compared to WT mice. In agreement, we observed systemic elevated levels of pro-inflammatory cytokines in Lcn2KO compared to WT mice during STA. Further, we’ve demonstrated that Lcn2 deficient M1 macrophages displayed increased STAT3 activation compared to WT cells. In addition, WT M2 phenotype exhibited elevated STAT5 activation. Taken together, these results suggest that Lcn2 may promote the down-regulation of pro-inflammatory cytokines and up-regulate anti-inflammatory cytokines possibly through STAT3 and STAT5 signaling pathways as a potential negative feedback loop in order to limit the extent of inflammation during autoimmune arthritis conditions.
Intragenic transcriptional interference regulates the human immune ligand MICA.
Authors: O’Callaghan Chris , Lin Da , Hiron Thomas K.
Published: January 2023

Regulation of MICA expression is incompletely understood, but human MICA can be upregulated in cancer cells, virus-infected cells and rapidly proliferating cells. Binding of MICA to the activating NKG2D receptor on cytotoxic immune cells promotes elimination of the cell expressing MICA. We noted that MICA has tandem promoters that drive overlapping forward transcription. We show that the MICA gene contains a conserved upstream promoter that expresses a non coding transcript. Transcription from the upstream promoter represses transcription from the standard downstream MICA promoter in cis through transcriptional interference. The effect of transcriptional interference depends on the strength of transcription from the upstream promoter and quantitative studies show that it is described by a simple reciprocal repressor function. The time course of transcriptional interference coincides with recruitment at the standard downstream promoter of factors involved in nucleosomal remodeling during transcription. Transcriptional interference is demonstrated in the regulation of MICA expression by the physiological inputs interferon-γ and interleukin-4, that both act through regulatory DNA elements in the upstream promoter. These findings have significant implications for the understanding of MICA expression. Transcription factors activating the downstream promoter will upregulate MICA expression, whereas transcription factors activating the upstream promoter will downregulated MICA expression. A genome-wide analysis indicates that transcriptional interference between tandem intragenic promoters may be involved in regulating the expression of multiple other human genes.
Critical role of miR-23b in late sepsis-induced immunosuppression.
Authors: Yin Deling , Caudle Yi , Zhang Haiju
Published: January 2023

Sepsis is characterized with immunosuppression in late phase. microRNA-23b (miR-23b) is a multiple functional miRNA, which has been reported to prevent multiple autoimmune diseases through the regulation of inflammatory cytokine pathways. We hypothesize that miR-23b plays a role in the pathogenesis of sepsis. Our study verified the effect of miR-23b on sepsis induced immunosuppression. We found miR-23b expression was upregulated in early sepsis and sustained in late sepsis. The activation of TLR4/9/p38/STAT3 signal pathway contributes to the production of miR-23b in CLP-induced sepsis. Simultaneous in vivo blockade of miR-23b with inhibitor injection after sepsis initiation decreased TUNEL positive cells in spleen, improved survival via reducing the mortality by 42%. MiR-23b inhibitors restored the T cell immunoreactivity by alleviating the development of T cell exhaustion and producing smaller amounts of immunosuppressive interleukin 10 (IL-10) and IL-4 in late sepsis after LPS stimulation. We also demonstrated that miR-23b triggered apoptosis and T cell exhaustion to mediate immunosuppression in late sepsis by inhibiting the activation of non-canonical NF-κB signal and promoting pro-apoptotic signal pathway via targeting NIK, TRAF1 and XIAP. Circulating miR-23b serum levels might be a potential useful marker to predict survival in patients with critical sepsis. Blocking the expression of miR-23b may be an effective approach for prevention and treatment of sepsis-induced immunosuppression.
BCAP, a novel TLR-IL1R signaling adapter, regulates the differentiation of pathogenic Th17 cells
Authors: Deason Krystin L. , Troutman Ty Dale , Jain Aakanksha , Pasare Chandrashekhar
Published: January 2023

Toll-IL-1R homology (TIR) domains are found within adaptor proteins involved in the signaling of Toll like receptors (TLRs) and Interleukin 1 receptor (IL1R) families. Previous work by our lab identified a TIR domain in the protein B cell adaptor for phosphoinositide 3-kinase (BCAP) and determined a role for BCAP in TLR signaling in myeloid cells. Due to the shared use of TIR domains by TLR and IL1R signaling pathways, we hypothesized that BCAP would also be involved in signaling downstream of the IL1 family of receptors. The IL1 cytokine family has been shown to play a major role in T cell activation, survival, and differentiation; IL1b, in particular, plays a critical role in Th17 lineage cells. We have discovered that BCAP functions downstream of IL1R in CD4 T cells and regulates Th17 differentiation and function. IL1b-induced PI3K-Akt signaling was compromised in BCAP deficient T cells, which contributes to decreased mTOR activation, decreased glycolysis, and defective Th17 differentiation. Transcriptional analysis of BCAP deficient CD4 T cells led to the finding that BCAP is critical for the expression of genes associated with pathogenic Th17 lineage cells. Mice specifically lacking BCAP in T cells have normal development of steady state Th17 cells in vivo yet have decreased experimental autoimmune encephalomyelitis (EAE) disease. This study establishes BCAP as a critical link between IL1R and the metabolic status of activated Th17 cells and further demonstrates that BCAP is an important adapter molecule that functions downstream of IL1R and is critical for the generation of pathogenic Th17 cells in vivo.
Inhibition of Inositol-Requiring Enzyme 1 α in Established TH2 Cells Decreases the Secretion of Cytokines IL-5 and IL-13, but Not IL-4
Authors: Kemp Kyeorda L. , Poe Cody , Youngblood CheyAnne
Published: January 2023

TH2 cells are crucial in immune responses against extracellular pathogens; however, they can play a predominant role in a number of diseases due to inappropriate IL-4, IL-5, and IL-13 cytokine expression. Therefore, understanding how these cells develop and regulate their effector functions is of great importance. In response to an excess of unfolded proteins, serine-threonine kinase Inositol-Requiring Enzyme 1 α or IRE1α activates the unfolded protein response, UPR, which promotes increased protein folding capacity. T cell activation induces ER stress, and inhibition of IRE1α RNase activity in activated primary cells, via treatment of the cells with the commercially available drug 4μ8c, results in the reduction of IL-5, IL-4, and IL-13. Prior to this work, it was unknown if 4μ8c could inhibit TH2 cytokines in established TH2 cells. Treatment of a mouse TH2 cell line and differentiated human TH2 cells with 4μ8c results in inhibition of IL-5 and IL-13, but not IL-4 as measured by ELISA. The reduced cytokine expression is not due to differences in mRNA stability or mRNA levels; it appears to be due to a defect in secretion, as the cells appear to produce cytokines IL-5 and IL-13 as measured by flow cytometry. This suggests that the inhibition of these cytokines is due to post-translational processes. IL-5 and IL-13 produced by TH2 cells are critical for promoting chronic, inflammatory asthma, and 4μ8c blocks their expression in T cells in vitro. Future studies will determine if 4μ8c treatment can ameliorate the effects of these cytokines in a disease model. This work is supported by the NSU Faculty Research Council grant and OK-INBRE NIH P20GM103447.
Functional dissection of a novel IL-22 super-enhancer
Authors: Saini Ankita , Li Shasha , Bando Jennifer K. , Collins Patrick , Cella Marina , Colonna Marco , Oltz Eugene M.
Published: January 2023

Innate lymphoid cells (ILCs) are an emerging family of immune cells that serve essential roles in initiation, regulation and resolution of different immune responses. ILCs respond promptly to various signals present in their microenvironment by producing an array of cytokines and thereby directing the immune responses. These cells have been categorized as ILC1, ILC2 and ILC3 depending upon the effector cytokines secreted and transcription factors expressed by them. The signature effector cytokines produced by ILC3s are IL-22 and IL-17, providing protective immunity against extracellular pathogens. Another crucial facet of IL-22 function is its maintenance of mucosal homeostasis by modulating processes like tissue repair. However, there is a void of knowledge about genomic regions regulating IL-22 in different immune cells. In this regard, recent studies from our lab revealed a novel super-enhancer (SE), regulatory region composed of a large cluster of conventional enhancers, which likely regulates IL-22 and, perhaps, the neighboring IFNG gene. To dissect ILC3-specific functions of enhancers within the SE regulatory region, we are exploiting a murine cell line, MNK3, which mimics ILC3 functions in vitro and in vivo. We found that CRISPR-Cas9 mediated deletion of 5′ and 3′ end of this novel SE abrogated IL-22 expression in MNK3 cells. Furthermore, to identify cell and/or agonist specific elements of SE we set up a CRISPRi screen for which we generated a stable line of MNK3 expressing Dox inducible KRAB-dCas9. Together, our approach will define the cis-elements that regulate IL-22 in distinct immune cells and hence opening the field for novel therapeutic interventions for inflammatory diseases including autoimmune disorders.
Comparison of the open chromatin landscapes of effector and anergic T helper cells
Authors: Torres Annabel , Johnson Matthew , Wells Andrew D.
Published: January 2023

Costimulation through the TCR and CD28 drives T cell activation and differentiation. TCR engagement without CD28 results in anergy, a dominant state refractory to further stimulation. To understand how signals from CD28 and/or TCR imprint anergic and effector cell fates, we analyzed the genome-wide patterns of open chromatin in TCR or TCR/CD28-costimulated CD4+ T cells using ATAC-seq. Remarkably, although anergy represents a loss of T cell function, anergic cells exhibited 40% more open chromatin regions (OCR) than effector cells. Over 85% of the accessible effector genome was shared with anergic cells, and only 6,400 OCR were effector-specific. Conversely, only 60% of the accessible anergic genome overlapped with effectors, while 24,000 OCR were anergy-specific. Nearly one-fifth of effector-specific OCR were located in promoters, and were enriched for ETS, BCL6, and KLF consensus binding motifs. The remaining 5300 OCR, putatively representing the effector-specific enhancer landscape, were enriched for CTCF, ETS, BATF, IRF, and E2F motifs. Conversely, only 5% of anergy-specific OCR were in promoters, and these regions were enriched for motifs for FOS, ETS, IRF, SMAD3, and EGR1. The remaining 23,000 OCR that putatively constitute the anergy-specific enhancer landscape were specifically enriched for NFkB, TBET, NFAT, and Nur77 motifs. These results suggest that while CD28 cooperates with the TCR to activate a minor, effector-specific component of the T cell gene regulatory architecture, the major epigenomic role of costimulation is to actively oppose remodeling of the anergy-associated gene ‘regulome’ imprinted by default by the TCR.
T cell-independent mechanisms for protection against Mycobacterium tuberculosis
Authors: Izzo Angelo A. , Creissen Elizabeth , Izzo Linda , Troudt JoLynn , Bickett Thomas
Published: January 2023

Mycobacterium tuberculosis is responsible for millions of deaths each year and with the rise of drug resistance, the need for an effective vaccine is paramount. Currently, the Mycobacterium bovis strain, BCG is the only vaccine available, which provides inconsistent efficacy against pulmonary tuberculosis. In the mouse model, BCG induces adequate protection against M. tuberculosis infection, even in the absence of T cells. In our preliminary studies, subcutaneous BCG vaccination sufficiently protected mice when given 7 days prior to pulmonary infection with M. tuberculosis, reinforcing the T cell-independent nature of immunity and leading us to hypothesize that innate immunity was responsible. Various studies have defined a significant role for innate immunity during BCG vaccination in what has been termed “trained innate immunity”. However, our studies suggest that whereas “trained innate immunity” may play a role in BCG induced protection against non-mycobacterial diseases, this may not be the case for M. tuberculosis infection. We have found that the ability of BCG to replicate is key and that this may be due to its residence within phagocytic cells, as not all cells of the innate immune system are required for this outcome, based on selective cell depletion studies. As the effect of BCG on innate immunity is beneficial for other diseases, this study focuses on the early immune response shortly after BCG vaccination in the mouse model to help elucidate the mechanisms through which BCG stimulates protection and induces killing of M. tuberculosis. Our studies uncover novel mechanisms of BCG induced immunity that may be important to understanding new vaccination strategies.
CARDS toxin is critical for mediating the early inflammatory response during Mycoplasma pneumoniae infection
Authors: Segovia Jesus A. , Somorajan Sudha R. , Cagle Marianna P. , Baseman Joel B. , Kannan Thirumalai R.
Published: January 2023

Mycoplasma pneumoniae is a leading cause of bacterial community-acquired pneumonia and is implicated in initiation and exacerbation of asthma. One of the characteristic signs of M. pneumoniae infection in humans is an imbalanced proinflammatory immune response. A recently identified M. pneumoniae virulence factor is the Community-Acquired Respiratory Distress Syndrome (CARDS) toxin, an ADP-ribosylating and vacuolating protein produced abundantly during infection that is capable of displaying inflammation and pathology similar to infection with live M. pneumoniae. Our prior studies have focused heavily on the potent inflammatory effects of CARDS toxin in vitro, however its exact role during infection is unclear. We hypothesized that an M. pneumoniae strain lacking CARDS toxin expression would be unable to trigger the robust inflammatory response in vivo that is necessary for M. pneumoniae pathogenesis.

We genetically modified the clinical isolate M. pneumoniae S1 strain to generate a CARDS-deficient mutant we designated M7. M7 and S1 were used in vivo to identify changes in the proinflammatory response. Interestingly, we observed a drastic difference in the dynamics of M. pneumoniae infection during the early course of infection. Secretion of the cytokines TNF-α and CXCL1 were both significantly altered early during infection in M7-infected mice. The effects of this response led to a significant impact on Mycoplasma survival. By utilizing a novel CARDS toxin-deficient mutant of M. pneumoniae, our findings provide valuable insight into our understanding of the innate immune response against M. pneumoniae and implicate CARDS toxin as a potentially important target for therapeutic intervention during M. pneumoniae infection.
Innate lymphoid cell composition shift upon Campylobacter jejuni induced colitis, a process inhibited by targeting mTOR signaling in Il10−/− mice
Authors: Tang Ying , He Zhen , Shirley James F. , Ohland Christina L. , Pope Jillian L. , Jobin Christian , Glover Sarah
Published: January 2023

Innate lymphoid cells (ILCs) are important innate effector cells involved in host mucosal immunity. The role of ILCs in regulating homeostasis following commensal bacterial colonization and enteropathogenic bacterial infection is unclear. Herein, we profiled ILC repertoire following Campylobacter jejuni (C. jejuni)-induced colitis as well as responses following anti-inflammatory treatment. Germ-free (GF) Il10−/− mice were colonized with SPF biota or human clinical isolate C. jejuni 81-176. Rapamycin (1.5mg/kg, daily), an inhibitor of mTOR signaling, was used to inhibit C. jejuni-induced colitis. ILC profile was assayed by Flow Cytometry analysis using specific markers (Lineage marker, CD90.2, CD127, CD117, ST2, and NKp46). Inflammatory cytokine production was detected using RNAscope. Compared to WT-C57Bl6 mice, ILC distribution was similar in absence of intestinal microbiome (GF mice), whereas presence of microbiota leaded to elevated NCR−ILC3 (30% vs 5%, P< 0.01) and lower ILC2 (1% vs 21%, P< 0.05) in the colon of Il10−/− mice. C. jejuni-induced colitis caused an increase of ILC1 (50% vs 18%, P< 0.0001) with decrease of NCR−ILC3 (13% vs 43%, P< 0.0001) in the colonic lamina propria, compared to uninfected mice. RNAscope revealed an augmented inflammatory cytokines, such as IFN-γ, IL-17A, TNF-α, and IL-22, in mucosal immune cells, and elevated IL-33 expression in epithelial cells. Rapamycin treatment inhibited C. jejuni-induced colitis (histology score 0.07 vs 4.0, P< 0.0001), which associated with re-establishment of ILC profile and decreased cytokine production. In conclusion, ILC trans differentiation correlates with the development of C. jejuni-induced colitis, a process inhibited by blocking mTOR signaling.
LIGHT-HVEM Signaling in Group3 Innate Lymphoid Cells Protects Against Enteric Bacterial Infection
Authors: Seo Goo-Young , Shui Jr-Wen , Takahashi Daisuke , Song Christina , Wang Qingyang , Kim Kenneth , Mikulski Zbigniew , Chandra Shilpi , Giles Daniel A. , Zahner Sonja , Kim Pyeung-Hyeun , Cheroutre Hilde , Colonna Marco , Kronenberg Mitchell
Published: January 2023

Innate lymphoid cells (ILC) are important regulators of early infection at mucosal barriers. They are known to be activated by cytokines and neuropeptides, but it remains to be determined if they integrate other signals in providing protection. The herpes virus entry mediator (HVEM), a member of the TNF receptor superfamily, is expressed by all intestinal ILC subsets. Here, we show that HVEM signaling of ILC3 is important for host defense against oral infection with the enteric bacterial pathogen Yersinia enterocolitica (Y. enterocolitica). Surprisingly, IFNγ production by CCR6 negative ILC3 was strongly implicated in protection, likely because these cells were more numerous than other innate lymphocytes that produced IFNγ early after infection. We identified the TNF superfamily member LIGHT, as the ligand inducing HVEM signals in ILC. Therefore, our results demonstrate a novel role for LIGHT-HVEM signaling in regulating ILC3 IFNγ production and protection following infection.
Interplay between type I interferon signaling and AIM2 inflammasome in Francisella novicida infection
Authors: Zhu Qifan , Kanneganti Thirumala-Devi
Published: January 2023

Inflammasomes are crucial for mediating protection against microbial infection. Type I interferon (IFN) signaling is required for driving activation of the AIM2 inflammasome in macrophages in response to infection by Francisella tularensis subspecies novicida (F. novicida). Here, we showed that mice lacking type I IFN signaling had intact inflammasome responses after F. novicida infection. Mice lacking the type I IFN receptor subunit, IFNAR1 or IFNAR2, and core components of the type I IFN signaling pathway were resistant to infection by F. novicida compared with wild-type mice, whereas mice lacking AIM2 were more susceptible. Importantly, we found that mice lacking both AIM2 and IFNAR2 were more resistant to the infection with an impaired ability to induce activation of apoptotic caspases than infected wild-type mice. These results suggested that while type I IFN signaling and AIM2 were essential for activation of the Francisella-induced inflammasome in macrophages, they have strikingly opposing effects in vivo. Furthermore, type I IFN signaling is a dominant regulator of anti-microbial activity during F. novicida infection in vivo even in the absence of AIM2 responses.
Gasdermin-D controls cytokine responses in Francisella infection.
Authors: Banerjee Ishita , Behl Bharat , Shrivastava Gaurav , Russo Ashley J. , Vanaja Sivapriya Kailasan , Rathinam Vijay A.
Published: January 2023

The innate immune system is essential for detection and elimination of pathogenic microbes. Francisella is an intracellular pathogen that replicates in the cytosol of macrophages. In the cytosol, it can be recognized by host defense sensors. Francisella is sensed by cyclic GMP-AMP synthase (c-GAS) leading to the production of type I Interferons (IFN). It can also be recognized by the PYHIN DNA sensor absent in melanoma (AIM2) resulting in the activation of inflammasome responses i.e. production of pro-inflammatory cytokines IL-1b and IL-18 as well as pyroptosis. While mice deficient in inflammasome components are more susceptible to Francisella infection, type I interferon signaling deficient mice are resistant to Francisella bacterial infection. Inflammasome activation results in gasdermin-D cleavage leading to pyroptotic cell death. Our results show that gasdermin-D activation plays an important role in the nature of cytokine responses to Francisella infection, therefore influencing the outcome of the infection. These findings have major implications in understanding the balance of cytokine production in response to cytosolic immune surveillance.
Anti-Flagellin antibodies dampen TLR5 and NLRC4 innate immune responses to flagellin
Authors: Yeoh Beng San , Gewirtz Andrew T. , Vijay-Kumar Matam
Published: January 2023

The host immune system is constantly exposed to diverse microbial ligands, including flagellin (FliC; a ligand for TLR5 and NLRC4) and lipopolysaccharide (LPS; a ligand for TLR4), whose ability to induce immune tolerance to subsequent exposure is well-documented in vitro. In this study, we investigated the extent to which FliC and LPS could induce immune cross-tolerance to one another in vivo, as well as the interaction between the innate and adaptive immune response to FliC. Mice pre-treated with LPS displayed attenuated serum keratinocyte-derived chemokine [KC; alias interleukin (IL)-8] and IL-6 response to FliC; however, the administration of FliC followed by LPS instead resulted in an additive response. LPS also augmented the serum IL-18 response to FliC; the increment was more pronounced when LPS was administered before, than after, FliC. Intriguingly, the generation of anti-FliC antibodies was notably impaired in mice sequentially challenged with LPS and FliC, compared to mice given only FliC. A strong negative correlation between high anti-FliC titer and reduced KC, IL-6 and IL-18 responses to FliC re-challenge was observed in these mice, suggesting that adaptive immunity could tolerize TLR5 and NLRC4. Indeed, the serum from WT mice with high anti-FliC titer, but not Rag1KO mice, was able to dampen FliC-induced secretion of IL-8 from the human epithelial cell line HT-29. These observations may be relevant in the context of bacterial infection, as we demonstrated that anti-FliC response are protective against systemic infection by Salmonella typhimurium. Taken together, our study delineates a distinct co-operative and reciprocal interaction between the innate and adaptive arms of immunity in modulating their responses to FliC.
Pyrin Inflammasome Regulates Tight Junction Integrity to Restrict Colitis and Tumorigenesis
Authors: Sharma Deepika , Malik Ankit , Guy Cliff , Vogel Peter , Kanneganti Thirumala-Devi
Published: January 2023

Mutations in the Mediterranean fever gene (MEFV or pyrin) are associated with hereditary autoinflammatory disease and severe IBD. Inflammatory bowel diseases (IBD) increase the risk for colorectal cancer. Expression of MEFV, a sensor protein that the initiates assembly of the inflammasome complex, is increased in colon biopsies from patients with colorectal cancer. We investigated the role of pyrin in intestinal homeostasis in mice. Mefv−/− and C57/BL6 mice were subjected to azoxymethane - dextran sodium sulfate (AOM-DSS) model of colitis and tumorigenesis. Pyrin was expressed in colons of control mice and expression increased during chronic and acute inflammation. Mefv−/− mice developed more severe colitis than control mice, with a greater extent of epithelial hyperplasia and a larger tumor burden. Mefv−/− mice had increased epithelial permeability early during DSS administration and increased levels of inflammatory cytokines and chemokines during acute colitis. On the other hand, IL18, which depends on the inflammasome for maturation and release, was significantly lower in colons of Mefv−/− mice. Further, the inflammatory milieu in Mefv−/− mice was pro-tumorigenic with increased STAT3 activation, and stem cell activity in the colon and reduced cytotoxic T cell activation in draining lymph nodes and spleen. Administration of rIL18 to Mefv−/− mice reduced epithelial permeability, intestinal inflammation, the severity of colitis, and colon tumorigenesis. In conclusion, we found that pyrin (MEFV) is required for inflammasome activation and IL18 maturation, which promote intestinal barrier integrity and prevent colon inflammation and tumorigenesis.
Distinct lymph node entry efficiencies for CD8+ and CD4+ T cells are eliminated during malnourishment.
Authors: Gubbels Bupp Melanie R. , Gibson David , Goulmamine Syreen , Little Amie , Murphy Sarah , Patrick Kristin , Thoner Timothy
Published: January 2023

Previous work has suggested that glucocorticoids upregulate expression of the IL-7 receptor component, CD127 on peripheral T cells, even though the total number of peripheral T cells declines dramatically during malnourishment. We have proposed that CD127 up-regulation contributes to peripheral T cell diminishment by increasing the scavenge rate of IL-7 and thereby providing a mechanism to rapidly adjust the total number of T cells during malnutrition. As such, each malnourished T cell would receive a higher dose of IL-7 than control T cells. We next wondered if increased exposure to IL-7 during malnourishment might confer additional energy-saving behaviors. Thus, we compared lymph node entry rates of adoptively-transferred malnourished and control T cells in malnourished and control recipients. As expected, control CD4+ T cells were more efficient than control CD8+ T cells at entering the lymph nodes. Interestingly, regardless of recipient diet, malnourished CD4+ and CD8+T cells entered the lymph nodes at equivalent rates. The molecular events that enable faster CD4+ T cell entry into lymph nodes in control mice is currently unknown. An improved understanding of T cell-intrinsic changes that occur during malnourishment should enhance our knowledge of CD4+ and CD8+ T cell migration, as well as uncover strategies to improve vaccination responses in malnourished children.
Memory CD8+ T Cell Development is Controlled by the Sympathetic Nervous System
Authors: Farrar John David , Estrada Leonardo , Agac Didem , Wise Aaron , Maples Robert , Chen Beibei , Cobanoglu Murat Can
Published: January 2023

Immune memory is critical for protection from repeated infections by pathogenic organisms and is the basis for the success of vaccines. Memory CD8+ T cell development is guided by important signals that alter their function and transcriptional program, enabling them to persist as pools of primed memory cells capable of rapid response. Lymphoid organs are highly innervated by sympathetic neurons that secrete norepinephrine as the primary neurotransmitter, which places the nervous and immune systems in direct communication. CD8+ T cells express the β2-adrenergic receptor (ADRB2), and we found that it was required for the development of long-lived memory cells in response to virus infection. The failure of Adrb2−/− T cells to form memory populations correlated with profound differences in gene expression and predicted upstream regulators during the early stages of T cell priming. Thus, the sympathetic nervous system controls memory CD8+ T cell development through ADRB2 signaling.