bluuebunny/crossref_metadata_2025 huggingface dataset:
P2X3 Potentiates IgE Mediated Mast Cell Function
Authors: Burchett Jason , Ryan John J
Published: August 2023

The rising incidence of allergic disease is a public health challenge needing novel interventions. Mast cell activation by IgE is an established feature of allergy, but fundamental aspects of IgE-induced signaling remain unclear. Prior data from our lab testing the effects of fluoxetine on IgE-driven mast cell function indicated a role for the purinergic receptor P2X3. The P2X3 receptor is an ATP-activated cationic channel most often associated with pain that is expressed on the surface of mast cells. IgE cross-linkage elicits a rapid release of ATP from mast cells, suggesting ATP-P2X3 receptor signaling may potentiate IgE-driven responses. We show that diminishing P2X3 function pharmacologically and genetically reduces IgE-mediated cytokine production. P2X3 knockout bone marrow-derived mast cells showed a diminished capability to produce cytokine in vitro when stimulated by IgE cross-linkage. The P2X3 inhibitors BLU-5937 and Gefapixant administered intraperitoneally suppressed cytokine levels measured in the plasma of mice subjected to the mast cell-dependent passive systemic anaphylaxis model (PSA). Furthermore, P2X3 knock-out mice showed reduced IL-6 compared to wild-type mice in the PSA model. Interestingly, inhibition or deletion of the P2X3 receptor showed no effect on mast cell degranulation in vitro and did not reduce hypothermia during PSA. We propose that an ATP-P2X3 autocrine or paracrine loop augments mast cell-mediated inflammation, thus offering a potential new target for the treatment of allergic disease.

RO1 AI164710-01, TL1 DK 132771-1
Are commercial soy-sauce products naturally gluten-free? Preliminary evaluation of selected products sold in the USA
Authors: Shivaramaiah Shambhavi Devarahalli , Shah Yash , Jorgensen Rick Dean , Gangur Venugopal
Published: August 2023

Gluten present in wheat, rye, and barley has been linked to 3 types of immune mediated disorders: Celiac disease, non-celiac gluten sensitivity and wheat hypersensitivity (or wheat allergy). Consequently, detection of gluten in food products is routinely used to validate gluten-free products meant for gluten-sensitive subjects. Fermentation processing has been shown to be effective in degrading food allergens including gluten. Most commercial soy sauce products (SSPs) use wheat in addition to soybean as a major ingredient in their production, thus presence of wheat gluten is expected in such products. Here we tested the hypothesis that commercial SSPs sold in the retail market in the USA will test positive for gluten independent of the country of origin. Commercial SSPs were tested for gluten presence using gluten test kit (LOD, 5 ppm). The test kit was validated for positivity using purified gluten extracted from durum wheat flour and for negativity by screening 2 commercial gluten-free SSPs. The following products have been tested: 4 products from China, 3 from USA, 1 from Canada and Taiwan respectively. Commercial SSPs when used without dilution in the food extract buffer rendered the test kit system invalid for testing. Therefore, a dilution of 1/10 was used in testing. Despite identifying wheat as an ingredient on the product label, all SSPs from USA, Canada, and Taiwan tested negative for gluten. Among these products, only 1 from china tested slightly positive for gluten. These results demonstrate that most commercial SSPs sold in the united states, independent of country of origin, used in this study test negative for the gluten content despite identifying wheat as a major ingredient on the labels. Further product screening is in progress.

USDA/NIFA
The ESCRT protein CHMP5 controls skeletal muscle homeostasis and coordinates myeloid cell-mediated tissue repair
Authors: Trichka Josephine , Adoro Stanley
Published: August 2023

Macrophages promote skeletal muscle homeostasis by participating in crosstalk with satellite cells, fibroadipogenic precursors, and myofibers that drives regeneration after injury. Dysregulation of this crosstalk contributes to conditions like myopathy, inefficient wound healing, and sarcopenia. Despite their central role in muscle homeostasis and regeneration, the tissue-intrinsic mechanisms that coordinate the recruitment and activity of these macrophages are poorly understood. In this study, we investigated how skeletal muscle homeostasis is regulated by the ESCRT protein CHMP5. CHMP5 was initially characterized as a member of the ESCRT family of proteins that coordinate membrane scission events in eukaryotic cells. However, recent studies by us and others have revealed non-canonical roles for CHMP5 wherein CHMP5 promotes the stability of client proteins required for cellular differentiation and cell fate decisions in hematopoietic and non-hematopoietic tissues. Using an in vitro model of myogenesis, we found that CHMP5 knockdown impaired the differentiation of C2C12 myoblasts into myotubes. Furthermore, when wild-type BMDMs were cocultured with CHMP5-knockdown C2C12 myoblasts, they failed to polarize into M2-like macrophages. In vivo, mice with muscle-specific CHMP5 heterozygous deletion (CHMP5hetmice) displayed diminished myeloid cell presence in muscle following cardiotoxin-induced injury. 12 days post-injury, histologic and flow cytometric analyses of muscles from CHMP5hetmice revealed less regenerative progress than littermate controls. These data suggest a critical function for CHMP5 in promoting muscle homeostasis both in the steady state and in the context of injury and inflammation.

Supported in part by intramural funding from the National Cancer Institute, T32 GM007250, T32 NS077888-08, and NIH R01AI143992-03.
NF kappa B regulator Bcl3 controls development and function of classical dendritic cells required for resistance to Toxoplasma gondii
Authors: Guha June
Published: August 2023

The atypical IκB family member Bcl3 associates with p50/NF-κB1 or p52/NF-κB2 homodimers in the nucleus, and positively or negatively modulates transcription in a context-dependent manner. Since Bcl3 expression in dendritic cells (DC) is pivotal for antigen presentation and since classical DCs (cDC) are major antigen presenting cells, we investigated the role of Bcl3 specifically in cDCs in vivo by crossing Zbtb46 cre mice with Bcl3flx/flx mice. Bcl3flx/flx Zbtb46 cre mice were as susceptible to lethal T. gondii infection as total Bcl3−/− mice and generated poor Th1 immune responses. Consistent with this, compared to wildtype controls, splenic Xcr1+ Bcl3-deficient cDC1 cells were defective in presenting Ova antigen to OT-I cells both for Ova257–264 peptide and after infection with Ovalbumin-expressing T. gondii. Moreover, splenic CD4+ and CD8+ T cells from infected Bcl3flx/flx Zbtb46 cre mice exhibited decreased T. gondii-specific priming as revealed by both reduced cytokine production and reduced T. gondii-specific tetramer staining. Consistent with this, splenocyte single cell RNA seq (scRNAseq) in infected mice revealed Bcl3-dependent expression of genes involved in antigen processing in cDCs. We also identified by scRNAseq, a unique Bcl3-dependent hybrid subpopulation of Zbtb46+ DCs co-expressing the monocyte/macrophage transcription factor Lysozyme M. Likewise, by flow cytometry we identified two T. gondii-induced hybrid subpopulations of Bcl3-dependent cDC1 and cDC2 cells both expressing monocyte/macrophage markers, designated as icDC1 and icDC2. Together, our results indicate that Bcl3 in classical DCs is a major determinant of protective T cell responses and survival in T. gondii-infection.
Complement Turning Violent in Ultra-Rare Genetic Disorder
Authors: Pandey Manoj K , Trivedi Vyoma Snehal , Magnusen Albert Frank , Rani Reena , Woods Christopher K , DiPasquale Betsy Ann
Published: August 2023

Sandhoff disease (SD) is an ultra-rare lysosomal storage disorder (LSD), which affects ~ 1/1000, 000 live birth. SD is caused by genetic deficiency of beta (β) Hexosaminidase and resulting excess central nervous system (CNS) synthesis of GM2 ganglioside (GM2) and its impact on neuron death. The exact mechanisms underlying such GM2-driven neuron death are unknown in SD. Glucosylceramide (GC) induced complement 5a (C5a) and its C5aR1 activation causes tissue inflammation in experimental and clinical Gaucher disease. Additionally, C5a-C5aR1 axis was found to sparks CNS inflammation and neurodegeneration in several brain diseases including, intracerebral hemorrhage, traumatic brain injury, myasthenia gravis, amyotrophic lateral sclerosis, neuromyelitis optica spectrum, Alzheimer, and Huntington’s diseases. Here, we determined the increased brain levels of C5a and C5aR1 in a beta (β) hexosaminidase inhibitor (HABI)-induced experimental mouse model of SD, when compared to vehicle (PBS) injected WT control mice. Also, HABI-induced experimental mouse model of SD showed increased microglial cells activation, massive brain generation of pro-inflammatory cytokines and loss of neurons, when compared to vehicle injected WT mice. To assess the relevance of C5a-C5aR1 axis activation for brain inflammation in SD, we targeted β hexosaminidase with HAB-I in WT and C5aR1−/− mice. Strikingly, as compared to HABI-injected WT mice, HABI-injected C5aR1−/− mice caused marked reduction in microglial cell activation, brain production of pro-inflammatory cytokines, and the loss of neurons. These data suggest that the C5a-C5aR1 axis is a critical driver of neuroinflammation and neurodegeneration in SD.

No Funding
The role of complement C3 in the outcome of regional myocardial infarction
Authors: Fang Zhou
Published: August 2023

Coronary heart disease leading to myocardial ischemia is a major cause of heart failure. A hallmark of heart failure is myocardial fibrosis. Using a murine model of myocardial ischemia/reperfusion injury (IRI), we showed that, following IRI, in mice genetically deficient in the central factor of complement system, C3, myocardial necrosis was reduced compared with WT mice. Four weeks after the ischemic period, the C3−/−mice had significantly less cardiac fibrosis and better cardiac function than the WT controls. Overall, our results suggest that innate immune response through complement C3 plays an important role in necrotic cell death, which contributes to the cardiac fibrosis that underlies post-infarction heart failure.
CRISPR screening for cytotoxic granule degranulation pathway in CD8 T cell
Authors: Shibata Hirofumi , Golec Dominic P , Phelan James , Johansen Kristoffer , Huang Bonnie , Schwartzberg Pamela L
Published: August 2023

Degranulation of perforin-containing cytotoxic granules is important for NK cells and CD8 cytotoxic T lymphocytes (CTLs) to kill infected cells or tumors. Defects in this pathway cause the primary form of hemophagocytic lymphohistiocytosis (HLH), which is known as familial HLH (FHL) syndrome. Most of reported FHL causative genes encode molecules that are involved in the intracellular trafficking of cytotoxic granules and their fusion to the plasma membrane, followed by the delivery of their contents into the target cell through immunological synapses. However, other components essential for cytotoxic granule degranulation for CTLs are not well understood.

To identify indispensable molecules for cytotoxic granule degranulation, we established an efficient in vitro CD8 amplification and degranulation system to perform a CRISPR-screening. To test this system, we transduced OT-I CD8 cells with a gRNA library targeting PIP3 binding proteins, which are important for cell adhesion, subcellular trafficking and T cell activation. Cells were harvested at short (Day 8) and medium-timepoints (Day 15) to evaluate the frequency of gRNAs in the degranulated (CD107+) and non-degranulated fractions.

Of the top 10 hits of genes whose deletion suppressed degranulation, five were already listed in the 2022 IUIS classification of inborn error of immunity. We will use individual knockouts to evaluate how these genes affect degranulation. To expand to genome-wide screening, we have also subcloned a full library of sgRNAs into our high efficiency retroviral vector. Thus, we established an efficient assay system to perform a CRISPR-screening for CD8 T cells and can detect genes affected by primary immunodeficiencies.

This work was supported by the Intramural Research Program of NIAID, NIH.
A20 is a negative regulator of both NF-κB and JNK signaling following T cell receptor stimulation in humans
Authors: Dabbah-Krancher Gina L , Ruchinskas Allison , Snow Andrew L
Published: August 2023

A20 is an NF-κB-induced, dual function ubiquitin editing enzyme that negatively regulates NF-κB signaling induced by TNF and other innate immune receptors, constraining inflammation. Indeed, A20 haploinsufficiency drives a severe autoinflammatory disease in humans. Although A20 serves a similar negative feedback function for T cell receptor (TCR) signaling, the molecular mechanisms utilized and their ultimate impact on human T cell function remain unclear. TCR engagement triggers the assembly of the CARD11-BCL10-MALT1 (CBM) protein complex, a signaling platform that governs the activation of downstream transcription factors including NF-κB and c-Jun. We employed an extensive set of mutant expression constructs to better elucidate how A20 regulates TCR signaling output in Jurkat T cells. Intriguingly, A20 knockout cells showed enhanced, sustained NF-κB and JNK signaling after stimulation, which was reversed upon wild-type (WT) A20 complementation. A20 deubiquitinase function was entirely dispensable for NF-κB and JNK regulation, whereas zinc finger domains that confer E3 ligase activity (ZnF4) and linear ubiquitin-binding capacity (ZnF7) were required. Abolishing MALT1-dependent proteolytic cleavage of A20 did not affect suppressive function, although neither N- nor C-terminal A20 cleavage fragments retained regulatory capacity. We also found that CRISPR-mediated deletion of A20 adversely affects the survival and expansion of primary human T cells. Ongoing studies will determine how A20 deletion affects human T cell effector responses after stimulation, and decipher how dysregulated NF-κB and/or JNK signaling contributes to T cell-intrinsic abnormalities that perturb adaptive immune homeostasis.
An old dog with ‘new’ tricks: the MDR1 transporter in T cell-mediated immunity
Authors: Balasubramanian Akshaya , Chen Mei Lan , Hegner Courtney Lynn , Sundrud Mark S
Published: August 2023

Multidrug Resistance Protein 1 (MDR1) is an orphan ATP-binding cassette transporter, originally discovered for removing cytostatic drugs from resistant tumor cells. However, MDR1 is also functionally expressed in many normal cell types. Work from our group has shown that MDR1 is constitutively expressed in cytotoxic T cells (CTLs), where it promotes CTL-mediated immunity to infection. Adoptive transfer of Lymphocytic choriomeningitis virus (LCMV)-specific MDR1 (Abcb1a/b)-deficient naïve CTLs into wild-type recipients, followed by LCMV infection, has revealed that MDR1-deficient CTLs fail to accumulate to wild-type levels in response to either acute (Armstrong) or chronic (Clone-13) LCMV infection. This defective accumulation is observed as early as 5-days post-infection, suggesting that endogenous MDR1 transport activity may preferentially regulate early T cell receptor (TCR)-elicited CTL functions. New single cell (sc)RNA-seq experiments, coupled with RNA velocity and lineage trajectory analyses, suggest that MDR1 expression is induced rapidly during TCR activation, coinciding with the induction of other genes involved in mitochondrial and redox metabolism. In vitro-activated CTLs lacking MDR1 also experience oxidative stress, mitochondrial dysfunction and display mitochondrial localization of MDR1. Together, these data support a new model of MDR1 function, in which MDR1-dependent transport of endogenous substrates suppresses oxidative stress and supports redox homeostasis during periods of metabolic activation. In turn, these concepts provide a novel framework for improving the efficacy and durability of vaccines, and for elucidating metabolic vulnerabilities of multidrug-resistant cancers.
An inter-species amino acid variation modulates signaling strength and function of CD28 in vitro and in vivo
Authors: Brady Alexander , McGeachy Mandy
Published: August 2023

Animal models do not always reflect the human condition. One such example was a phase one clinical trial of a CD28 super agonist antibody (CD28SA). In mice, CD28SA treatment expanded Tregs and reduced autoimmune disease; yet when human volunteers received CD28SA, they experienced life-threatening cytokine storm and multi-organ failure. CD28 is constitutively expressed on T cells and provides the canonical co-stimulatory “signal 2” needed for T cell activation. The proline residues in the CD28 PYAP domain are required for downstream signaling and this motif is conserved across species. However, the human PYAP domain is adjacent to an additional proline, while murine PYAP is followed by an inactive alanine. Previous in vitro studies have suggested this single amino acid variation modulates downstream signaling. We have developed a “humanized” mouse with a CD28 A to P substitution at residue 210 adjacent to the PYAP domain (CD28A210P). In vitro, CD28A210P T cells had increased p-Akt, JunB abundance, and produced more IL-2 compared to WT T cells when stimulated in vitro. In vivo, immunization with antigen/CFA emulsion, CD28A210P mice display increased antibody production, suggestive of enhanced CD28-mediated Tfh activity. In addition, we assessed the response of the “humanized” CD28 mice to the CD28 super agonist antibody. After injection with CD28 super agonist, CD28A210P mice had significantly increased weight loss, elevated and sustained pro-inflammatory cytokine production, and less Treg expansion compared to WT mice. This study suggests interspecies variation in CD28 signaling due to differences outside of the well-studied signaling domains can alter outcomes of CD28 ligation and promote divergent outcomes when targeted in vivo.
Single-Cell RNA Sequencing Reveals Disease Stage-Dependent Transcriptomic Profiles of Regulatory B Cells in Systemic Lupus Erythematosus
Authors: Alajoleen Razan M , Daamen Andrea , Timilesena Prakash R , Li Song , Lipsky Peter E , Luo Xin
Published: August 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease associated with abnormal activation of immune cells. Regulatory B (Breg) cells are a B cell subset that negatively regulate immune responses via secretion of immunoregulatory cytokines interleukin (IL)-10, IL-35, and transforming growth factor (TGF)-β. We had previously shown that in lupus-prone MRL/lpr mice, pre-disease Breg cells were more potent in suppressing autoimmunity than active-disease Breg cells. In this study, using single-cell RNA (scRNA) sequencing, we profiled ~10,000 Breg cells from female MRL/lpr mice at the pre-disease (6–8 weeks of age) vs. active-disease (10–12 weeks of age) stages. Based on known markers of Breg subsets, scRNA analysis identified respective clusters as transtional-2 marginal zone precursor B cells (T2-MZP), marginal zone B cells (MZB), transitional 1 B cells (T1), germinal center B cells (GC), and B1 B cells. Our data showed that the pre-disease Breg cells were predominantly T2-MZP and MZB cells. Follicular B cells outside the marginal zone, on the other hand, are significantly increased in the active-disease stage. Two long noncoding RNAs (lncRNAs), Malat1 and Xist, were significantly increased in active-disease Breg cells, where the expression of Lglas3, Slpi, and Spp1 was also increased. Notably, active-disease Breg cells exhibited an exhausted phenotype with increased expression of T-bet (Tbx21). Collectively, our findings suggest that the SLE disease stage-dependent functions of Breg cells are regulated at the transcriptional level. In future experiments, we will determine whether the immunosuppressive functions of active-disease Breg cells can be restored by knocking down the differentially overexpressed genes.
CSF1R blockade improves clinical parameters in prediabetic rhesus macaques
Authors: Stahr Natalie Ann , Phou Amy , Hattler Julian , McGuire David , Kim Woong-Ki
Published: August 2023

It is unknown whether dysregulated CSF1Rhi monocytes/macrophages drive the progression to diabetes and if their removal and endogenous replacement with fresh macrophages resolves disease. Therefore, we hypothesize that depletion of activated CSF1R+ tissue macrophages using a monoclonal antibody against CSF1R and replacement with freshly differentiated macrophages will alleviate inflammation in tissues and ameliorate diabetes-associated disease in prediabetic rhesus macaques.

We identified 6 aged Indian rhesus macaques with heightened fasting blood glucose as well as higher area-under-the-curve in an intravenous glucose tolerance test (IVGTT), indicating prediabetes. Three animals were treated with a rhesus IgG1 recombinantanti-CSF1R antibody administered i.v. (15 mg/kg) once every two weeks for 6 weeks. An IgG1 control was administered to the remaining 3 animals. Clinical parameters such as blood pressure, plasma levels of total cholesterol, glucose, insulin, adiponectin, colony stimulating factor 1 (CSF1), IL1β, monocyte chemoattractant protein 1 (MCP-1) were monitored throughout treatment and up to 8 weeks after the 3rd injection. A 27-color flow cytometry panel were used to monitor changes in immune cell populations throughout the study.

Treatment with anti-CSF1R resulted in decreased total cholesterol, increased adiponectin, increased CSF1, and increased IL1β levels in the blood throughout the study. Flow cytometry experiments confirmed the depletion of CD16+ monocytes (known to be CSF1R+) in treated animals.

Treatment with anti-CSF1R resulted in improvement in multiple clinical parameters associated with the progression to type-2 diabetes mellitus.
Complete tolerogenic response adjuvant stimulates Treg response to immunization
Authors: Janjam Sri Sushma Santhi , Geng Yajun , Yan Zhaoqi , Shin Soo Min , Joshi Kanak , Panicker Anjali Jacob , Shankar Archana , Elangovan Ramya , Koehler Jason , Gnanasekar Varun , Williams Amanda , Feng Addysen , Ellis Christian , Vattikota Anirudh , Chen Aoshuang , Zheng Guoxing
Published: August 2023

We identified the lowest composition necessary to generate a vaccination adjuvant that promotes a Treg response following immunization in mice and named it “complete tolerogenic adjuvant.” This novel adjuvant may allow us to utilize the well-established “antigen plus adjuvant” method of vaccination to induce Treg cell-mediated antigen-specific immunosuppression. The minimal composition is dexamethasone, rapamycin, and monophosphoryl lipid A with the following mass ratios: 8:20:3. We have demonstrated why immunosuppressive and immunogenic substances are both required for the formation of genuine adjuvants for Treg cells by dissecting the roles of each of these components during vaccination. Currently, research is ongoing in our group to begin to assess the usefulness of this adjuvant for antigen immunization in murine models of human autoimmune/inflammatory diseases. Specifically, we are evaluating the tolerogenic efficacy of this adjuvant for vaccination regimen targeting the apolipoprotein B antigen, using the ApoE−/− mouse model of atherosclerosis. These studies are expected to help drive the development of further, perhaps more effective, complete tolerogenic adjuvants that could be used to create a plethora of new, novel vaccines for treating immunological illnesses.

Master program and NIH grant
Restoring T cell function to promote resolution of chronic HBV infection
Authors: Rexhouse Catherine , Bailey Jacob T , Moshkani Safiehkhatoon , Cimino Jesse L , Robek Michael D
Published: August 2023

An estimated 296 million people worldwide suffer from chronic hepatitis B virus (HBV) infection, with 1.5 million new infections occurring each year despite the availability of an effective vaccine. Chronic HBV infection persists in a dysfunctional immune environment, manifested by an ineffective T cell response to the virus. Patient HBV-specific CD8+ T cells have diminished proliferative capacity and cytokine production, and they exhibit increased and sustained expression of inhibitory immune checkpoint receptors. In mice with persistent HBV replication, reduction of viral antigens can be achieved by therapeutic immunization with alphavirus- and vesiculovirus-based vaccine platforms expressing HBV antigens. However, blocking PD-1 and CTLA-4 signaling may further enhance antiviral effector T cell function and therapeutic efficacy. Using a mouse model of HBV replication in which mice are transduced with adeno-associated virus encoding the HBV genome (AAV-HBV), we investigated the role of these inhibitory receptors in the T cell response to HBV. Antibody-mediated blockade of either PD-1 or CTLA-4 independently in AAV-HBV mice did not promote viral clearance. Likewise, PD-1 and CTLA-4 inhibition combined with therapeutic immunization with VSV expressing an HBV antigen did not improve anti-HBV T cell responses or virus clearance in high-antigen mice with persistent viral replication. However, in mice with lower initial antigen loads, blockade of these receptors along with VSV therapeutic immunization resulted in an enhanced decrease of circulating HBV antigen. These results suggest new methodologies for improving HBV-specific immune responses to promote a functional cure of the infection.

Supported by the National Institute of Allergy and Infectious Diseases and the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under award numbers R01AI148354 and R44DK113858. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Developing a Robust and High Throughput Assay for Screening Innate Immune Training Molecules
Authors: Forbes Greta E , James Heera , Henderson Mark , John Sinu P , Fraser Iain D C
Published: August 2023

Innate immune training molecules have several potential applications in the biomedical field. We developed an assay to screen for new training molecules by generating HiBiT tagged TNF cells in the THP1 monocytic cell line through CRISPR knock-in. The HiBiT tag, an 11 amino acid long peptide, was fused to the TNF C-terminus to produce luminescence upon addition of the LgBiT substrate. To optimize the assay, we first titrated different concentrations of multiple TLR ligands including pIC, p(dAdT), PGN, FLGN, LPS, R848, and P3C. LPS, PGN, and P3C induced the strongest signal to noise (S/N) ratio in the pool of TNF-HiBiT cells. From the TNF-HiBiT pool, we then single cell cloned and characterized them for optimum TNF response. The clones D3 and D6 had significantly higher S/N ratio compared to the pools, especially in response to P3C and PGN. To optimize the TNF-HiBiT cells for a training screen, we used previously known training molecules such as Syk kinase inhibitor, beta-Glucan and Rutaecarpine, to train multiple TNF-HiBiT cell clones. We found that the clones that induced a lower TNF signal upon acute TLR activation exhibited a more robust training phenotype compared to the clones that induced a more robust acute TNF signal. We found therefore that the optimal protocol to assess trained immunity is to use lower concentrations of activating TLR ligands (0.1, 0.1, and 0.0001 ng/mL of P3C, LPS and PGN) in the trained cells. This may be due to saturation of the TNF-HiBit reporter signal when cells are challenged with higher TLR ligand concentrations (10, 100, LPS, and 10 ng/mL of P3C, LPS and PGN). In brief, we have developed and optimized a robust assay for screening novel training molecules.

This work was supported by the Intramural Research Program of NIAID, NIH

Supported by grants from Intramural Research Program of NIAID, NIH and the Office of AIDS Research at NIH.
Tpl2−/− macrophages exhibit impaired mitochondrial respiration and function in response to LPS stimulation
Authors: Fahey Denise Lyn , Watford Wendy Tharpe
Published: August 2023

Macrophages are instrumental in maintaining immune homeostasis, and their dysfunction or depletion is tied to many human diseases, including autoimmunity, obesity, and cancer. Activated macrophages modify their cellular metabolic pathways, such as glycolysis and oxidative phosphorylation, to drive their diverse functions. Many metabolic processes occur in the mitochondria, including oxidative phosphorylation, metabolite production, and reactive oxygen species (ROS) generation. Previous research demonstrated that tumor progression locus 2 (Tpl2, MAP3K8, or COT) is required for the induction of oxidative phosphorylation and the alternatively activated phenotype in macrophages during Schistosome infection; however, Tpl2 regulation of cellular metabolism in classically activated macrophages remains unexplored. We hypothesize that Tpl2 normally promotes oxidative phosphorylation, and its absence causes mitochondrial dysfunction. LPS-stimulated Tpl2−/− bone marrow-derived macrophages (BMDMs) had decreased oxygen consumption, basal respiration, ATP production, and maximal respiration compared to LPS-stimulated wild type BMDMs. Additionally, Tpl2−/− BMDMs exhibited higher basal and LPS-stimulated ROS production than wild type BMDMs. These data suggest that Tpl2−/− mitochondria are functionally defective, and the absence of TPL2 increases mitochondrial oxidative stress. Together, this may reveal the underlying mechanism for defective Tpl2−/− macrophage behavior. Understanding the role of Tpl2 in classically activated macrophages will provide a foundation for developing targeted therapies to treat cancer and autoimmune diseases.

Supported by grant from NIH (R21 AI147003).
ZIP8 impacts protease activity in macrophages in response to Streptococcus pneumoniae
Authors: Haq Sabah , Smith Deandra R , von Pein Jessica B , Sweet Matthew J , Knoell Daren L
Published: August 2023

Zinc (Zn) is essential for immune function and Zn deficiency increases the incidence of community acquired pneumonia. The Zn transporter ZIP8 is required by myeloid cells to defend against pathogens. Previously, we reported that S. pneumoniae infected ZIP8 knockout mice (Zip8KO) have increased bacterial burden and mortality and that Zip8KO bone marrow derived macrophages (BMDMs) have reduced phagocytic activity. Others have shown that Zn induces cathepsin expression via the TFEB pathway. However, it is unknown whether ZIP8 impacts TFEB-dependent phagolysosomal processing in macrophages. Based on this, we hypothesized that ZIP8 facilitates phagolysosomal processing and eradication of bacteria via TFEB.

BMDMs were derived from wild type and Zip8KO mice and then exposed to S. pneumoniae (MOI=10) for 1 hour. At 6–18 hours post challenge we assessed cathepsin B and L mRNA and protein levels as well as total protease, cathepsin B and L activity. In conjunction, we measured p-TFEB and total TFEB proteins levels.

Active TFEB and cathepsin B protein levels were significantly lower in Zip8KO BMDMs with no difference in cathepsin L. Strikingly, Zip8KO BMDMs had significantly reduced total protease, cathepsin B and cathepsin L activity which also corresponded with decreased mRNA levels.

Based on these findings we predict that ZIP8 loss in macrophages impairs phagolysosomal processing and bacterial eradication in a TFEB-dependent manner. Overall, this warrants future studies given the high incidence of Zn deficiency and the rs13107325 defective ZIP8 allele in humans.

National Heart, Lung and Blood Institute #1R01HL156952-01-A1 (DLK)
Tryptophan diet promotes myeloid-derived suppressor cell response via aryl hydrocarbon receptor in Sjogren’s syndrome
Authors: Lin Xiang
Published: August 2023

Myeloid-derived suppressor cells (MDSCs) are heterogeneous myeloid cell populations with immunosuppressive capacity, which contribute to immune regulation and tolerance induction. We previously reported impaired MDSC function in patients with primary Sjögren’s syndrome (pSS) and mice with experimental SS (ESS). In this study, we first found that aryl hydrocarbon receptor (AhR) was highly expressed by human and murine polymorphonuclear MDSCs (PMN-MDSCs). Indole-3-propionic acid (IPA), a natural AhR ligand produced from dietary tryptophan, significantly promoted PMN-MDSC differentiation and their suppressive function on CD4+ T cells. In contrast, feeding tryptophan-free diet resulted in decreased PMN-MDSC response, a phenotype that could be reversed by IPA supplementation. Notably, AhR expression was reduced in PMN-MDSCs during ESS development, while AhR antagonism resulted in exacerbated ESS pathology and dysregulated T effector cells, which could be phenocopied by tryptophan-free diet. Interferon regulatory factor 4 (IRF4), a repressive transcription factor, was upregulated in PMN-MDSCs during ESS progression. ChIP analysis revealed the binding capacity of IRF4 to the promoter region of AhR, while IRF4 deficiency markedly enhanced AhR-mediated PMN-MDSC responses. Furthermore, tryptophan-enriched diet or dietary supplementation of IPA markedly ameliorated salivary glandular pathology in ESS mice with restored MDSC immunosuppressive function. Together, our results identify a novel function of AhR in modulating PMN-MDSC response and demonstrate the therapeutic potential of targeting AhR for the treatment of pSS.
CD4+ Treg expansion arrests disease progression in a pre-clinical model of alopecia areata
Authors: Lensing Maddison , Connell Samuel J , Kahl Payton , Zhu Zhaowen , Jabbari Ali
Published: August 2023

Alopecia Areata (AA) is a prevalent autoimmune disease that results in nonscarring hair loss. Disease is associated with the collapse of an immune privileged state of the hair follicle, characterized by increased MHC expression, immune cell infiltration and the presence of pro-inflammatory cytokines in the microenvironment. CD8 T cells have been identified as the main contributors to disease and the activating receptor NKG2D can identify this pathogenic population. There is currently one FDA-approved drug for AA, a JAK1/2 inhibitor, which is thought to act by dampening inflammatory mediators during disease. However, the potential of adverse effects and refractory patients necessitates the development of alternative therapeutics. Emerging data from our lab suggests that the observed expansion of CD4 T regulatory cells (Tregs), relative to effector CD8 T cells during AA, is inadequate to prevent disease onset. We hypothesized that pharmacologic enhancement of Treg numbers may restrain development of AA. To address this, we investigated the protective potential of Treg expansion using a murine model of AA. In vivo expansion of CD4+ CD25+ FoxP3+ Tregs was achieved using an IL-2 cytokine/antibody complex (IL-2C), which is known to selectively expand and enhance Tregs. We found that 6 weeks of treatment with IL-2C halted the progression of hair loss, while control mice exhibited robust hair loss. Additionally, IL-2C treatment led to a reduction in NKG2D+ CD8 T cells and IFNγ-producing CD8 T cells found infiltrating the skin, suggesting that increasing Tregs in the system is an effective strategy for restraining disease. Our findings provide translational insight for the use of Treg enrichment as a therapeutic strategy for patients with AA.

Supported by grants from NIH (R01 AR077194) and Dept. of VA (I01 BX004907)
Characterization of the immunosuppressant activity of the novel biogenic amine 3HKA and its analog derivatives
Authors: Martinez Aitziber Buque , Clement Cristina , Shetty Madhur , Santambrogio Laura
Published: August 2023

Tryptophan (Trp) is an essential amino acid which in mammalian cells 90% is processed through the kynurenine pathway. Kynurenine metabolites are among the most potent regulators of immune responses. Herein we report the characterization of a novel immunosuppressant biogenic amine, 3-hydroxy-L-kynurenamine (3HKA), generated through an uncharacterized lateral pathway of Trp catabolism, involving its decarboxylation by the aromatic amino acid decarboxylase. 3HKA is generated by both professional (Dendritic Cells, DC) and non-professional (Lymphatic Endothelial Cells) APC. Quantitative phosphoproteomic analysis of human-monocytes (CD14+) derived DCs, identified key signaling transduction receptors, IFNR1, and signaling molecules from the JAK/STAT and cAMP mediated PKA pathways, regulated by 3HKA. Cell-based assays coupled with ELISA platforms showed that 3-HKA inhibited the IFN-g-mediated STAT1/NF-kB pathway in human DCs, and consequent decreased the release of pro-inflammatory chemokines and cytokines IL-1, TNFa, IL-6, and IL12p70. In vivo, 3-HKA reduced the intestinal inflammation in a model of dextran sodium sulfate-induced colitis. Furthermore, flow cytometry analysis revealed that 3-HKA increased the number of T-cells expressing 2, 3 or 4 exhaustion markers (PD-1, CTLA4, LAG3 or TIM3), exclusively in CD8+CD4− cells. We used, in silico approaches for ligand and pharmacophore-based drug discovery and developed 30 novel 3-HKA analogues with ADMET predicted drug-like properties. Structure-activity relationships studies, leaded to identify compounds with improved anti-inflammatory profiles. The novel anti-inflammatory 3-HKA analogues showed promising activity for the treatment of autoimmune diseases
Targeting of GPR65 with Small Molecules alters Th17 cell differentiation
Authors: Hernandez Jeniffer B , Cave Si , Patel Dhruv , Liang Yuqiong , Yang Qiyuan , Wiater Ezra , Zheng Ye , Montminy Marc
Published: August 2023

GPR65 has been shown to be a critical regulator of Th17 pathogenicity. Loss of GPR65 in mice results in a decrease in Th17 cells and reduced susceptibility to experimental autoimmune encephalomyelitis. The CREB/CRTC2 pathway has emerged as an important regulator of immune function. We have previously shown that the CREB/CRTC2 pathway modulates autoimmune disease by promoting differentiation of Th17 cells. In this study we performed RNA-seq to identify Th17 genes regulated by the CREB/CRTC2 pathway. Our RNA-seq analysis led us to uncover the first mechanism of regulation of the orphan receptor GPR65 by the CREB/CRTC2 pathway. We show that GPR65 is a target of the CREB/CRTC2 pathway through expression studies and chromatin immunoprecipitation. In addition, we show that targeting of GPR65 with small molecules alters Th17 cell differentiation. Understanding the regulation of GPR65 will be crucial in developing small molecules to treat patients with Th17-mediated diseases.
B Cell Targeting mAb–siRNA Conjugates Reduced Autoantibody in Experimental Myasthenia
Authors: Huda Ruksana
Published: August 2023

Conjugates of monoclonal antibodies (mAbs) and siRNA have been used for target cell-specific gene repression. B cells are integrally involved in the anti-Acetylcholine receptor autoantibody-mediated pathogenic development of autoimmune myasthenia gravis (MG). Here we show that simultaneous targeting of the critical receptors expressed on pathogenic B-cell subsets with the optimized dose of mAb-siRNA conjugates markedly depleted target B cells and substantially reduced pathogenic autoantibody in a mouse model of MG. The results show the potential of mAb-conjugates in the treatment of myasthenia gravis.

Supported by grants from AFM-Telethon, France
14-3-3ζ Pseudogenes: Physiologically and powerful evolutionary forces in the genome
Authors: Lughmani Haroon Yahya , Chakravarti Ritu , Chattopadhyay Srestha , Patel Hrushil
Published: August 2023

Contrary to the rigid dogma ruling out the phenotypic relevance of pseudogenes, recent RNA-seq and meta-analyses indicate that 10% of all pseudogenes are transcribed, and 40% of those pseudogenes are translated into protein. This investigation unpackages the 14-3-3ζ (YWHAZ) gene, which has been shown to regulate cytokine signaling, induce antibody response, and reduce inflammation in rats with rheumatoid arthritis. The original YWHAZ gene is located on the 8th human chromosome. Our detailed investigation in the human genome shows that there are ten pseudogenes of YWHAZ. Located across the human genome, these pseudogenes display unique properties, such as having allelic SNPs for Body Mass Index, Prostatic Neoplasms, and Macular Degeneration. Diverse mRNA expression profiles skewed towards reproductive organs and an unusually high transcription rate (9 of 10 being actively transcribed) suggest a strong physiological relevance. Additionally, contrary to the prevailing pseudogene formation theory where all exons are transcribed, YWHAZ pseudogenes have omitted the sixth exon’s UTR region, resulting in a more compact, equally functional gene structure. The evolution of the YWHAZ pseudogene from the parent gene’s framework, combined with some pseudogenes having allelic properties, demonstrates how pseudogenes may be the body’s way to repackage and repurpose parent genes for a more diverse genome and proteome.
The CD16 and CD32b Fc-gamma receptors regulate antibody-mediated responses in mouse natural killer cells
Authors: Aguilar Oscar A , Gonzalez-Hinojosa Maria D R , Arakawa-Hoyt Janice S , Millan Alberto J , Gotthardt Dagmar , Nabekura Tsukasa , Lanier Lewis L
Published: August 2023

Natural killer (NK) cells are innate lymphocytes capable of mediating immune responses without prior sensitization. NK cells express Fc-gamma receptors (FcγR) that engage the Fc region of IgG. Studies investigating the role of FcγR on mouse NK cells have been limited due to lack specific reagents. In this study, we characterize the expression and biological consequences of activating mouse NK cells through their FcγR. We demonstrate that most NK cells express the activating CD16 receptor, and a subset of NK cells also express the inhibitory CD32b receptor. Critically, these FcγRs are functional on mouse NK cells and can modulate antibody-mediated responses. We also characterized mice with conditional knock-out alleles of Fcgr3 (CD16) or Fcgr2b (CD32b) in the NK and innate lymphoid cell (ILC) lineage. NK cells in these mice did not reveal any developmental defects and were responsive to cross-linking activating NK receptors, cytokine stimulation, and killing of YAC-1 targets. Importantly, CD16-deficient NK cells failed to induce antibody-directed cellular cytotoxicity (ADCC) of antibody-coated B cell lymphomas in in vitro assays. In addition, we demonstrate the important role of CD16 on NK cells using an in vivo model of cancer immunotherapy using anti-CD20 antibody treatment of B cell lymphomas.

This project was funded by fellowships from Burroughs Wellcome Fund (BWF) and the Cancer Research Institute to O.A.A., and grants from the Parker Institute for Cancer Immunotherapy (PICI), and the NIH (AI068129 and AI146581) to L.L.L.
BPA modulates the neutrophil response and cytokine environment in a model of tail fin regeneration
Authors: Walser-Kuntz Debby , Wang Hongshen , Harmon Bailey
Published: August 2023

The ER-α and β ligand bisphenol A (BPA) is used in the production of polycarbonate plastic and epoxy resins. BPA presents a potential environmental challenge to both human and wildlife immunity due to its widespread use and its resulting presence in surface water and wastewater runoff. We observed that BPA exposure downregulates the expression of a neutrophil-recruiting chemokine in both mice (cxcl1) and zebrafish (cxcl8) and that BPA exposure decreases neutrophil recruitment to a tail injury in zebrafish. Neutrophils are essential for the removal of damaged tissue and invading microbes, thus facilitating tissue regeneration of the tail fin. To investigate BPA’s effects on larval zebrafish tail regeneration, we induced a tail injury at day 3 post fertilization (dpf) and found that fish exposed to 100 ng/ml BPA regenerate a smaller tail fin area at 3 dpi (p-value < 0.01). We hypothesized that neutrophil recruitment could be tied to regeneration by altering the expression of inflammation- and regeneration-related genes. 3 dpf embryos exposed to BPA show modulated levels of background wnt10a and il-10 expression compared to controls. BPA exposure reduces the 3 hour post-injury inflammatory response by decreasing both il-1 and il-6 expression. By modifying the cytokine environment both prior to and following infection and injury, BPA impacts both the inflammatory response and tissue regeneration. Multiple cytokine and chemokine genes are regulated by the transcription factor NF-kB and Wnt plays a key role in tissue regeneration; these two proteins also cross regulate one another. The impact of BPA on wnt10a and nfkb expression following tissue injury and LPS exposure will be discussed.

Supported by Carleton College.
Regulation of CCR6 Expression on Epidermal γδ T Cells in Response to Damage or Stress in the Skin
Authors: Castellanos Tanya , Lawler William Keele
Published: August 2023

Chronic conditions, including type 2 diabetes and psoriasis, interfere with the timing and location of necessary inflammatory cellular functions. Inflammation in the skin is at least partly mediated by epidermal γδ T cells in humans and mice, which secrete growth factors, cytokines and chemokines. However, less is known about which chemokines regulate epidermal γδ T cells and whether functional epidermal T cell subsets exist. One signaling pathway of interest is the C-C Motif Chemokine Receptor 6 (CCR6)/C-C Motif Chemokine ligand 20 (CCL20) axis, which recruits T cells to the epidermis for wound repair. To investigate how this signaling pathway initiates and regulates epidermal γδ T cell functions, we utilized flow cytometry, publicly available scRNA-seq data. Luminex and immunofluorescence microscopy were utilized to evaluate the location and timing of CCR6 expression by epidermal γδ T cells. Initial findings indicate that CCR6 expression is upregulated in a subset of activated epidermal γδ T cells and that CCL20 induces pro-inflammatory IL-17-biased gene expression. Analysis of publicly available single cell RNA sequencing data from mice with psoriasis show that predominantly proinflammatory genes are upregulated in the CCR6+ epidermal γδ T cell subset. Some proinflammatory genes that are expressed include IL-22, IL-17F and IL-17A. Further, upregulation of CCR6 expression by a subset of epidermal γδ T cells begins within the first hours post wounding and remains elevated until a decline is observed 72 hours post wounding.Further understanding of the role of CCR6 in inflammation can lead to the development of novel treatments for conditions such as chronic non healing wounds or psoriasis.
Distinct hypoxic molecular mechanisms differentially regulate human vs. mouse Interleukin 1β gene expression in monocytes/macrophages
Authors: Macar David A , Pulugulla Sree H , Adamik Juraj , Mitta Priyanka , Galson Deborah L , Auron Philip E
Published: August 2023

Interleukin 1β (IL-1β) is an important cytokine, whose regulation is distinct from that of Tumor Necrosis Factor (TNF). We previously reported that LPS/TLR4 initiates hypoxia in mice, with HIF-1α binding the murine Il1b gene, suggesting that TLR4 directly activates Trained Innate Immune Memory (TIIM). To address whether human monocyte IL1B gene regulation parallels that of mouse, mRNA and chromatin immunoprecipitation (ChIP) kinetics, with small molecule inhibitors under either normoxia or hypoxia, were evaluated. IL1B transcription kinetics in Lipopolysaccharide (LPS)-treated human THP-1 monocytes revealed an Immediate-Early (IE) phase of IL1B gene induction, requiring Spi1/PU.1, NF-κB and C/EBPβ transcription factors (TF), followed by a late-phase of lower-level continuous non-tolerized TIIM expression that depends upon hypoxia-inducible TFs. We observed that Spi1/PU.1 is required for IL1B gene transcription, performing a critical role in inducing the promoter for IE expression via a TAF1-independent mechanism. Late HIF-2α-dependent IL1B expression is due to LPS pseudo-hypoxic stabilization of the labile HIF factor. In contrast to IE activation, late-phase transcription revealed minimal dependence on NF-κB due to human-specific competition for an overlapping NF-κB/HIF enhancer element. Our studies reveal that IL1B in naïve THP-1 human monocytes binds HIF-2α, not HIF-1α, while PMA treatment morphologically polarizes THP-1 macrophages to bind both HIF isoforms at IL1B. Additionally, bioinformatic screening revealed other immunologically important genes regulated by similar mechanisms, providing insight for novel therapeutic targeting of transcription factors other than NF-κB.

Interleukin Foundation, Denver Colorado