bluuebunny/crossref_metadata_2025 huggingface dataset:
Ketone body beta-hydroxy butyrate deactivates NLRP3 inflammasome in myeloid cells (CAM1P.153)
Authors: Goldberg Emily , Youm Yun-Hee , Nguyen Kim , Alnemri Emad , Dixit Vishwa
Published: January 2023

The NLRP3 inflammasome is a major regulator of sterile inflammation. It is now known that many age-related diseases are NLRP3-dependent, including metabolic disease, cognitive decline, bone loss, and thymic demise. Therefore, decreasing inflammasome activation during aging may prevent many chronic diseases known to limit healthspan. Calorie restriction and prolonged fasting reduce inflammation. During fasting, the body uses fat for energy, leading to an increase in ketone body production as a byproduct of fatty acid oxidation. We tested the hypothesis that switching the substrates from glucose to ketones could inhibit inflammasome activation in myeloid lineage cells, macrophages and neutrophils. Here we present novel data that ketone body beta-hydroxy butyrate (BHB) inhibits NLRP3-dependent IL-1β secretion. Notably, related short chain fatty acid, butyrate does not impact the inflammasome and oxidation of ketones for energetic utilization was not required for inflammasome deactivation in myeloid cells. Instead, ketones directly inhibit inflammasome complex assembly by blocking ASC oligomerization and prionization. BHB also inhibits NLRP3 dependent inflammasome activation in vivo and in neutrophils of aged mice. Therefore, ketones may represent an anti-inflammatory intervention to prevent chronic inflammation during aging that is normally associated with functional decline and death in the elderly.
A small animal model of immune reconstitution inflammatory syndrome following antiretroviral therapy (CAM1P.159)
Authors: Huante Matthew , Nusbaum Rebecca , Vijayakumar Sudhamathi , Sutjita Putri , Cirillo Jeffrey , Ferguson Monique , Gelman Benjamin , Valbuena Gustavo , Endsley Janice
Published: January 2023

In HIV-infected patients with latent or subclinical tuberculosis (TB), the development of immune reconstitution inflammatory syndrome (IRIS) following restoration of T cell populations by anti-retroviral therapy (ART) can produce a life threatening inflammatory reaction. The objective of this study was to use our humanized mouse model of Mycobacterium tuberculosis (Mtb) and HIV co-infection to develop a small animal model of IRIS. Humanized BLT mice (HuMice) were infected i.n. with Mtb H37Rv, and following development of TB disease were given TB chemotherapy (rifampicin and isoniazid) by oral gavage. Bacteria were not detected in the lung following TB chemotherapy, but low numbers of Mtb were detected two months after stopping drug treatment, which mimics subclinical TB. When Mtb-infected and TB drug-treated HuMice were infected i.v. with HIV-1 Ada, the animals developed HIV-1 co-infection as determined by the presence of the HIV p24 capsid protein in peripheral blood by ELISA. ART was performed to reduce viral load in blood plasma and restore T lymphocyte function in animals with subclinical TB. In ongoing studies, the development of inflammatory responses, Th1 cell activation and lung pathology are being determined to understand how immune recovery, due to ART, drives inflammation in the setting of subclinical TB. This model will facilitate investigations to understand the mechanistic basis for IRIS and identify targets for host-directed therapies.
The role of HMGB1's critical cysteines are explored in human monocytes (CAM1P.151)
Authors: Bauer Eileen , Deng Peng , Gollapudi Venkata Sashi , Shapiro Richard , Li Tunliang , Billiar Timothy
Published: January 2023

High Mobility Group Box 1 protein, HMGB1 is a prototypical DAMPs. While HMGB1 is present in the nucleus of every cell under homeostatic conditions, cellular stress leads to its release into the extracellular milieu, where it activates a variety of pattern recognition receptors. HMGB1 has multiple functions. Under certain conditions HMGB1 acts as a cytokine, causing the release of pro-inflammatory cytokines including TNFα. In addition, HMGB1 may act as a chemokine. We now know that HMGB1 contains three critical redox-sensitive cysteines (C23, C45, C106). Modifying the redox state of these cysteines determines the action of HMGB1. Here we created 3 HMGB1 mutants replacing the cysteines with serines and systematically evaluated their role in human monocytes based on cytokine expression. Human monocytes are classically used to evaluate HMGB1’s ability to produce TNFα thus creating a comparison to other published work on HGMB1. While it is known that a disulfide bond between C23 and C45 is necessary for the cytokine like effect of HMGB1 our data show that the presence of all three cysteines is necessary for this action. Also, mutation of C23 or C106 to serine led to suppression of basal TNF release, changes in autophagy induction and signaling events. This suggests that terminal oxidation of either of these cysteines acts to suppress inflammation. Understanding the role of these cysteines hopefully will allow us to create better therapeutics targeting diseases driven by HMGB1.
IQGAP1 critically promotes CXCR4 chemokine receptor expression and signaling by regulating CXCR4 intracellular trafficking through EEA-1 endosomes (CCR3P.212)
Authors: Hedin Karen , Bamidele Adebowale , Kremer Kimberly , Hirsova Petra , Gores Gregory , Billadeau Daniel
Published: January 2023

IQGAP1 is a ubiquitous, cytoskeletal-interacting scaffold protein frequently overexpressed in cancer. CXCR4 is a chemokine receptor that signals upon binding stromal cell-derived factor 1 (SDF-1, CXCL12). CXCR4 regulates lymphocyte migration and development and cancer cell proliferation and metastasis. Here, we show that IQGAP1 potently enhances CXCR4’s expression, trafficking, and function in the Jurkat T cell line and epithelial cancer cells. The SDF-1-induced cell migration and activation of ERK MAPK were impaired in IQGAP1-deficient cells, even after forced CXCR4 overexpression. Exploring the mechanism, we found that SDF-1 treatment induced CXCR4 endocytosis and caused IQGAP1 to bind a-tubulin and localize to EEA-1 endosomes containing endocytosed CXCR4. In IQGAP1-deficient cells, CXCR4-containing EEA-1 endosomes accumulated at the distal ends of microtubules instead of moving towards the centrosome. This inhibited CXCR4’s signaling and cell-surface expression by preventing CXCR4 from trafficking into the Rab11 endosomes previously shown to be a critical site of CXCR4 signaling and recycling to the cell-surface. In contrast, IQGAP1 depletion had no effect on TCR trafficking, which does not require EEA-1 endosomes. Together, these results characterize a novel function of IQGAP1 as a regulator of EEA-1 endosomes, and in addition identify IQGAP1 as a critical enhancer of CXCR4 cell-surface expression and signaling in lymphocytes and epithelial cancer cells.
A novel small molecule inhibitor targeting gp130 (CCR3P.217)
Authors: Heo Tae-Hwe , Park Kyung-Yeon , Park Yeon-Hwa
Published: January 2023

Interleukin-6 (IL-6) is a major causative factor of inflammatory disease. Although IL-6 and its signaling pathways are promising targets, orally available small molecule drugs specific for IL-6 have not been developed. To discover IL-6 antagonists we screened our in-house chemical library and identified a novel synthetic compound as a candidate IL-6 blocker. The activity, mechanism of action, and direct molecular targets of the compound were investigated. A reporter gene assay showed that the compound suppressed activation of signal transducer and activator of transcription-3 (STAT3) induced by IL-6, but not activation induced by leukemia inhibitory factor. In addition, the compound downregulated IL-6-stimulated phosphorylation of STAT3, gp130, and JAK2 protein in HepG2 cells and substantially inhibited IL-6-dependent TF-1 cell proliferation. The compound antagonized IL-6-induced TNF-α production in vivo, but did not affect IL-2-induced TNF-α production. In pathologic models, oral administration of the compound ameliorated collagen-induced arthritis and acute pancreatitis in mice. We subsequently demonstrated direct and specific interaction between the compound and gp130, which was measured by SPR analysis (KD = 7.4 μM) and was simulated by a docking study. Taken together, our data suggest that the compound is a novel synthetic IL-6 inhibitor that functions through direct binding to gp130.
Identifying novel mechanisms regulating expression of inflammatory chemokine CXCL2 (CCR3P.206)
Authors: Zhang Xian , Lou Ning , Lennard Richard Mara , Yu Jin , Kindy Mark
Published: January 2023

Mammalian cells produce inflammatory mediators in response to the innate immune signal and their expression is tightly regulated. Chemokine (C-X-C motif) ligand 2 (CXCL2), also known as MIP2-alpha, belongs to the CXC chemokine family. CXCL2 is chemotactic for neutrophils. Elevated expression of CXCL2 is associated with many inflammatory and autoimmune diseases. Recently we have found that the Fli-1 transcription factor is a novel regulator in modulating the expression of many inflammatory mediators. including MCP-1,CCL5) and IL-6. Since we found that lupus-prone mice with reduced expression of Fli-1 had significantly decreased neutrophil infiltration into the kidneys, we investigated if Fli-1 regulates the expression of CXCL2. We found that Fli-1 protein expression in endothelial cells transfected with Fli-1 specific siRNA was significantly decreased compared to the expression of Fli-1 in cells transfected with control siRNA after stimulation by Toll-like receptor (TLR) 4 ligands, and tumor necrosis factor (TNF) alpha. The production of CXCL2 in endothelial cells with LPS stimulation is dose-dependent. We found that Fli-1 binds to the CXCL2 promoter by Chromatin immunoprecipitation‎ (ChIP) assay. Transient transfection assays show that Fli-1 drives transcription from the CXCL2 promoter in a dose-dependent manner. These results indicate that Fli-1 is a novel, critical transcription factor in regulating the expression of the inflammatory chemokine CXCL2.
Interleukin-12 elicits various responses of splenocytes from different mouse strains (CCR3P.207)
Authors: Nguyen Khue , Koppolu Bhanu , Smith Sean , Ravindranathan Sruthi , Zaharoff David
Published: January 2023

Interleukin - 12 (IL-12) is a pivotal linkage between innate and adaptive immunity via its pleiotropic functions on immune system. IL-12 has been shown to provide anti-tumor signals in mouse models including the ability to recruit immune cells, induce the production of interferon - γ (IFN-γ), tumor necrosis factor (TNF) and promote anti-angiogenesis. IL-12 bioactivity significantly varies between mouse strains. However, the mechanism resulting in various responses remains unclear. In this research, bioactivity of IL-12 was determined by quantifying IFN- γ production by splenocytes collected by three different mouse strains (C3H/HeJ, C57BL/6J, BALB/cByJ) in response to IL-12. Our data show that C3H/HeJ mouse strain produces increased levels of IFN-γ when compared with other mouse strains. IL-12 receptor (IL-12RB1) of splenocytes was quantified by fluorescence-activated cell sorting (FACS). Our studies show that the expression of IL-12RB1 by several key immune cell types including CD4 T-cells, CD8 T-cells, NK T-cells and B cells was significantly higher in C3H/HeJ splenocytes compared to splenocytes from C57BL/6J or BALB/cByJ. The higher expression of IL-12RB1 by C3H/HeJ mouse splenocytes leads to a significantly stronger response to IL-12 compared to other mouse strains. Our studies provide a valuable insight into variation of IL-12 bioactivity between different individuals. Further studies will be conducted to better understand in vivo bioactivities of IL-12 on mouse models.
The role of miR-204 in early B cell development. (HEM1P.219)
Authors: Medina Kay , Xu Zhiuhu , Gwin Kimberly
Published: January 2023

The generation of B lineage lymphocytes in bone marrow is contingent upon the combinatorial activities of transcription factors, signaling molecules, chromatin modifiers, and microRNAs. One transcription factor important for the generation of B cell precursors is the homeodomain protein HoxA9. Equally important as HoxA9 function in facilitating lymphoid lineage specification is silencing HoxA9 activity upon commitment to the B cell fate as sustained expression impairs later stage B cell differentiation. We previously showed that transcripts for the B cell specification factor EBF1 show an inverse expression pattern to HoxA9. Furthermore, EBF1 does not directly suppress HoxA9 transcription. Hox proteins are regulated by microRNAs in some developmental contexts. MicroRNA profiling of an EBF1-/- cell line stably expressing an inducible EBF1:ER fusion protein revealed a single miR induced by EBF1 activity, miR-204. Importantly, forced expression of miR-204 in EBF1-/- cells repressed transcription and protein levels of the HoxA9 co-factor, Meis1. We hypothesized that if Meis1 is a critical target of miR-204, then sustained expression of miR-204 should phenocopy conditional deletion of Meis1 or HoxA9. Indeed, we determined that forced expression of miR-204 in wildtype multipotent progenitors impaired B cell differentiation, similar to loss of HoxA9. These data suggest a novel role for EBF1 in B cell development, induction of miR-204 to silence a HoxA9-Meis1 transcriptional axis.
c-Myb coordinates survival and expression of key signaling components required for the pre-BCR checkpoint (HEM1P.220)
Authors: Bender Timothy , Daamen Andrea , Fahl Shawn
Published: January 2023

The c-Myb transcription factor is abundantly expressed during B cell development and is down regulated as the cells mature. We targeted Myb in mice with loxP sites (Mybf/f). By crossing these mice to other strains that direct Cre expression to different stages of B cell development (Rag1-cre, Mb1-cre, CD19-cre) we are able to assess subsequent B-lineage maturation. In Mybf/f Mb1-cre mice, c-Myb is required for B cell differentiation beyond the pre-pro-B cell stage and survival of CD19+ pro-B cells. c-Myb controls survival of pro-B cells by suppression of the proapoptotic factors Bmf and Bim. We find that IL-7 signaling represses the expression of Bmf and Bim. c-Myb controls the expression of CD127 but forced expression of CD127 is not sufficient to repress Bmf and Bim or rescue survival of c-Myb deficient pro-B cells. Examination of negative mediators of IL-7 signaling revealed increased SOCS3 mRNA in c-Myb deficient pro-B cells and forced expression of SOCS3 suppressed proliferation and survival of CD19+ pro-B cells in the presence of IL-7. Surprisingly, Mybf/f CD19-cre Bcl2-Tg mice contain severely reduced large and small pre-B cell compartments. Examination of mRNA expression in these cells revealed that c-Myb controls expression of key regulators of the pre-BCR checkpoint, including CD127, lambda-5, cyclin D3 and Cxcr4. c-Myb coordinates survival in pro-B cells with the expression of key components of the signaling pathways that drive the pro-B to pre-B cell transition.
Abnormal thymocyte development in Mbnl1 knockout mice (HEM2P.240)
Authors: Scotti Marina , Shin Jihae , Batra Ron , Finn Dustin , Swanson Maurice
Published: January 2023

Transcriptional regulation has been shown to play a vital role in thymocyte development, however co-/post-transcriptional modifications are also important regulatory mechanisms for T cell development. Here, we demonstrate that loss of Mbnl1, a developmentally regulated alternative RNA processing factor, leads to dysregulation of thymocyte development. Mbnl1-/- mice develop thymic hyperplasia due to accumulation of CD4+CD8+ double positive thymocytes. Mbnl1-/- thymocytes are resistant to steroid-induced apoptosis in vitro and current studies are focused on characterizing Mbnl1-/- thymocyte proliferation defects as well as peripheral T cells in Mbnl1-/- mice. Whole thymic transcriptome comparisons between Mbnl1-/- and wild type mice indicate 476 mis-splicing events in >300 genes, including those involved in T cell receptor assembly and signaling. Additionally, 688 differentially expressed genes identified between wild type and Mbnl1-/- (FDR < 0.01) demonstrate enrichment for immune processes, such as lymphocyte activation and stimulus response. Since Mbnl1 regulates alternative splicing and polyadenylation during the postnatal period, this study suggests that fetal to adult RNA processing switches are critical for normal thymocyte development.
Divergence in the requirements for HEB factors in the generation and function of fetal-derived versus adult-derived γδ T cells (HEM2P.245)
Authors: Anderson Michele , In Tracy
Published: January 2023

Gamma-delta (γδ) T cells are involved in mucosal immunity, wound healing, and tumor immunosurveillance. γδ T cells are programmed in the thymus to produce either IL-17 or IFNγ, but the molecular basis of this programming is not well understood. Here we show that mice lacking the E protein transcription factor HEB are defective in their ability to generate Vγ4 γδ T cells in fetal thymic organ culture. Since the HEB null allele is embryonic lethal, we generated mice lacking HEB only in hematopoietic cells by crossing HEBfl/fl mice with Vav-Cre mice (HEBfl/fl;vav-cre). These mice show a partial block in αβ T cell development, as seen in the fetal thymus of HEB-/- mice. In stark contrast to fetal gd T cells, however, the frequency of Vγ4 γδ T cells in the thymus of the HEBfl/fl;vav-cre mice is the equivalent to that in the WT thymus. Nonetheless, there is a skewing toward the CD27+ γδ T cells and away from CCR6+ γδ T cells, in both Vγ4+ and Vγ4- populations, suggesting a biased programming towards an IFNγ producing fate and away from the IL-17 producing fate. Moreover, there is a selective expansion of CD27+ Vγ4+ γδ T cells in the spleen, at the expense of other γδ T cell subsets, and a loss of CCR6+ Vγ4 γδ T cells in the lungs of HEBfl/fl;vav-cre mice. These results reveal that fetal Vγ4 cells are HEB-dependent, while adult Vγ4 cells appear to not require HEB, and strongly suggests that HEB is required for the generation and/or maintenance of IL-17 producing γδ T cells.
HIV-1 promotes neutrophil infiltration and lung damage in humanized mice co-infected with Mycobacterium tuberculosis (HUM1P.266)
Authors: Nusbaum Rebecca , Huante Matthew , Sutjita Putri , Calderon Veronica , Vijayakumar Sudhamathi , Aronson Judith , Hunter Robert , Actor Jeffrey , Cirillo Jeffrey , Valbuena Gustavo , Endsley Janice
Published: January 2023

Co-infection with human immunodeficiency virus (HIV) and Mycobacterium tuberculosis (Mtb) promotes aggressive disease through mechanisms that are poorly understood. A recent study suggests an association between peripheral blood neutrophil count and Mtb load in the sputum of human subjects with HIV co-infection. We used our humanized BLT mouse (HuMouse) model of HIV/Mtb co-infection as an investigative tool to study the role of neutrophils in co-infection pathobiology. Humanized mice were infected intravenously with HIV-1 for 3 weeks and then co-infected intranasally with Mtb. Compared to HuMice with Mtb or HIV mono-infections, increased neutrophil numbers were observed in the lungs of co-infected animals, as determined by detection of myeloperoxidase using immunohistochemistry. Consistent with the role of neutrophils to promote immune-mediated pathology in TB disease, greater tissue necrosis and increased mycobacterial burden was observed in the lungs of co-infected animals. In support of these findings, an increased pulmonary production of cytokines and chemokines associated with neutrophil recruitment including IL-8, CXCL5, fractalkine, and IL-17 were also observed in co-infected animals. These studies identify mechanisms for immune-mediated pathology due to HIV/Mtb co-infection that can be targeted to restore immune balance in the lung and complement antimycobacterials.
Oxidized low density lipoprotein immune complexes act as a priming signal for the NLRP3 inflammasome (HUM1P.253)
Authors: Rhoads Jillian , Wilhelm Ashley , Moore Jared , Major Amy
Published: January 2023

Antibodies to oxidized LDL (oxLDL) and resulting immune complexes (ICs) are a prominent feature of atherosclerosis and autoimmune diseases associated with increased atherosclerosis. Although levels of oxLDL-ICs correlate with disease severity, it is unclear whether these ICs play an active role in disease pathogenesis. We hypothesize that oxLDL-ICs exacerbate atherosclerosis by signaling through multiple receptors on dendritic cells (DCs) resulting in an inflammatory adaptive immune response. To test this hypothesis, bone marrow derived dendritic cells (BMDC) were incubated with oxLDL or oxLDL-IC and cytokine levels in culture supernatants were measured by ELISA. Results indicated a 10-fold increase in IL-1β production from BMDC treated with oxLDL-IC compared to oxLDL, suggesting a role for the inflammasome in oxLDL-IC activation of DC. Pre-treatment of BMDC with a caspase 1 inhibitor and use of NLRP3-/- BMDC abolished IL-1β secretion in response to oxLDL-IC, further implicating the inflammasome. Additional investigation using Fab fragments and a TLR4 inhibitor uncovered a role for both FcγR and TLR4 in oxLDL-IC mediated IL-1β production. Finally, BMDC pre-treated with oxLDL-IC, but not oxLDL, and incubated with CD4+ OT-II T cells in the presence of OVA peptide skewed T cells towards a Th17 phenotype. Taken together, these data suggest a novel and pathologic role of oxLDL-ICs in activating the NLRP3 inflammasome and promoting an inflammatory T cell phenotype.
Hyper-responsive TLR7 and TLR9 activation in NADPH oxidase-deficient human B cells (HUM1P.272)
Authors: McLetchie Shawna , Gardiner Gail , Blum Janice
Published: January 2023

Chronic Granulomatous Disease (CGD) is an inherited immunodeficiency linked with mutations in the multi-component leukocyte NADPH oxidase. Phagocytic cells deficient in NADPH oxidase fail to produce sufficient levels of the reactive oxygen species (ROS) necessary to clear engulfed pathogens. Though the hyperactive immune response associated with CGD is widely described in phagocytes, the effects of oxidase-deficiency on B cell functions has not been well studied. In B cells, recognition of microbial single-stranded RNA or unmethylated DNA by intracellular toll-like receptors (TLR) 7 and 9, respectively leads to NADPH oxidase derived ROS production. Here, studies demonstrate hyperactive responses to endosomal TLR stimulation in B cell lines derived from patients with mutations in either the p40phox or p47phox subunits of the NADPH oxidase which compromise its function. NADPH oxidase-deficient B cell lines expressed enhanced levels of TLR7 and TLR9 mRNA compared to cells reconstituted to restore oxidase activity. TLR7/9 stimulation of oxidase-deficient B cells resulted in greater cytokine production and p38 MAP kinase activation than oxidase-sufficient B cells. These data are consistent with the autoimmune predisposition associated with CGD and suggest that B cells may also contribute to hyperactive immune responses detected in individuals with CGD.
The role of indoxyl sulfate, a circulating uremic toxin, for inflammatory responses in the patients with end-stage renal disease (HUM1P.304)
Authors: Hwang Yuri , Jang Ji yeon , Chung Yeon-Ho , Kim Bonah , Kim Dong Hyun , Park Sungha , Chung Doo Hyun , Lee Won-Woo
Published: January 2023

In the ESRD patients, a major cause of death is cardiovascular disease (CVD) and its pathologic processes have been suggested to link to uremia-related chronic inflammation. Although concept of ESRD-related immune dysfunction is well-accepted, little is known about how uremic toxins affect cellular immunity involved with pathogenesis of CVD in the patients. Thus, we investigated phenotypic and functional features of CD4 T cells and monocytes in the ESRD patients and their immune responses mediated by indoxyl sulfate (IS), a key uremic toxin in order to explore the pathogenic roles of these cells for vascular endothelial cells (VEC). In ESRD patients, CD4+CD28null T cells and CD16+ monocytes were expanded as compared with HC. To explore how uremic milieu affects immune responses, monocytes were stimulated with IS. These monocytes produced a large amount of TNF-α through aryl hydrocarbon receptor. TNF-α stimulated VEC greatly produced CX3CL1, a ligand of CX3CR1 which is overexpressed by CD4+CD28null T cells and CD16+ monocytes. CD4+CD28null T cells are preferentially recruited by CX3CL1 and moreover, CD4+CD28null T cells have cytotoxic capability allowing for induced apoptosis of VEC in response to TCR stimulation. Our findings suggest that IS-mediated immune dysfunction may play a critical role for development and accelerated progression of CVD through VEC damage in the ESRD patients.
The MTA1-TG2 is involved in self-limitation of monosodium urate crystal-induced inflammation by upregulating the levels of active TGFβ1 leading to inhibition of JAK2 signaling (HUM1P.305)
Authors: Tsay Gregory , Yen Jia-Hau , Lin Ling Chung , Leong Pui Ying , Chen Jiunn-Horng , Szondy Zsuzsa
Published: January 2023

Abstract Introduction: Transglutaminase 2 (TG2), a protein crosslinking enzyme with multiple biochemical functions, has been connected to various inflammatory processes. In this study, the involvement of TG2 in monosodium urate (MSU) crystal-induced inflammation was studied. Conclusions: These findings reveal an inherent regulatory role of the MTA1-TG2 pathway in the self-limitation of MSU crystal-induced inflammation via positively regulating the levels of active TGF-b in macrophages that opposes the MSU crystal- induced JAK2 -dependent pro-inflammatory cytokine formation.
Loss of NLRX1 results in increased intestinal pathology and T cell responses in mice with inflammatory bowel disease (HUM1P.312)
Authors: Eden Kristin , Hontecillas Raquel , Viladomiu Monica , Washington Philipson Casandra , Carbo Adria , Leber Andrew , Philipson Noah , Tattoli Ivan , Girardin Stephen , Allen Irving , Bassaganya-Riera Josep
Published: January 2023

NLRX1 is a mitochondrial-associated NOD-like receptor that modulates antiviral immunity, cellular stress, autophagy, and reactive oxygen species (ROS). The role of NLRX1 in inflammatory bowel disease remains unknown. This study aimed to characterize NLRX1-mediated mechanisms of protection from IBD. We investigated the ability of NLRX1 to modulate gut pathology, inflammation and immunity by using DSS and CD4+CD45RBhigh transfer colitis models. Colons, spleens, and mesenteric lymph nodes were excised for characterizing immune cell subsets, histological analyses, cytokine and autophagy expression, NF-κB activity, and ROS production. The loss of NLRX1 increased severity of disease and colonic histopathology in both models of IBD. Colons of NLRX1-/- mice had significantly increased epithelial ulceration and leukocyte infiltration in the DSS model, while recipients of NLRX1-/- CD4+ T cells had increased leukocytic infiltration, proliferation, fibrosis, and crypt abscessation in both colon and ileum. The loss of NLRX1 increased numbers of Th1, Th17, and Treg in the spleen, increased colonic NF-κB activity, upregulation of IL-17, IFNγ and TNF-α production, and increased ROS production. NLRX1 ameliorates intestinal pathology during IBD by acting as an internal thermostat that modulates the balance of effector versus regulatory CD4+ T cell responses, and suppressing colonic NF-κB activity, inflammatory cytokine expression, ROS production, and autophagy.
VDJServer: a web-accessible analysis portal for immune repertoire sequence data (HUM1P.317)
Authors: Cowell Lindsay , Fonner John , Jordan Christopher , Levin Mikhail , Mock Stephen , Monson Nancy , Rounds William , Salinas Edward , Scarborough Walter , Scheuermann Richard
Published: January 2023

Despite the importance and widespread application of detailed antigen receptor repertoire profiling via high-throughput sequencing, there is currently no suite of software tools for seamless, reproducible analysis of repertoire sequence data. Tools exist for only a subset of analysis tasks. They do not function together and are difficult to reuse without modification that requires bioinformatics expertise. This leaves researchers to perform repetitive and error-prone tasks by hand, to develop internal, idiosyncratic algorithms not easily generalizable or systematically applied, and to expend significant manual labor reformatting primary and derived data for passing between tools. This approach is not only error-prone and time- and labor-intensive, but it comes at the expense of reproducibility, both within and between research groups. We have addressed this problem by developing VDJServer, a suite of repertoire analysis tools accessible via a web interface that allows users to upload a set of antigen receptor repertoire sequences and pass them through a seamless, customizable workflow that executes all steps in the analysis and generates analysis reports complete with data summary tables, statistical analyses, figures, and workflow logs. VDJServer runs on the high-performance computing resources at the Texas Advanced Computing Center, thus enabling users to process extremely large data sets. VDJServer is funded by the NIAID and is freely available.
Mouse models for immunology research available from the Jackson Laboratory Repository (HUM1P.318)
Authors: Valenzuela Alicia , Fancher Karen , Rockwood Stephen , Sasner Michael
Published: January 2023

The Jackson Laboratory Repository serves as a centralized facility for the development, distribution and cryopreservation of mouse models of human biology and disease. Hundreds of new strains are added annually to one of the largest collections of characterized mouse strains available. This poster presents our newest knockout (KO) strains involving genes associated with cell-mediated and humoral immunity. Lag3 (lymphocyte activation 3) KO mice allow further characterization of Treg suppressive function. Pvr (CD155)-null mice carry a lacZ reporter and exhibit inefficient IgA/IgE responses in response to challenge. The loxP-flanked Bcl6 mice carry a conditional allele involved in the differentiation of CD4 T cells into follicular Th cells. Two strains provide reporter/KO mutants of genes involved in the complement pathway, C1qa and Cfb. In addition, the hematopoietic cytokine, Il3, joins our extensive collection of interleukin mutants. New research tools include the following: a strain with EGFP and cre expression from the Zbtb16 (PLZF) promoter, in which expression is directed to the NKT cell lineage; a cre knockin (KI) into the Cx3cr1 promoter region for fate-mapping studies of the monocyte/macrophage compartment; and a ROSA26 KI that features a two-color fluorescent Cre reporter (widespread red fluorescence prior to cre exposure and localized green fluorescence following cre exposure). Researchers are encouraged to deposit mice to the Repository: www.jax.org/donate-a-mouse.
The microbiome regulates the eosinophil requirement for development of allergic lung inflammation (HYP2P.337)
Authors: Stokes Kindra , Wood Amie , August Avery
Published: January 2023

The diversity of gastrointestinal and respiratory microflora is essential in the shaping the host immune responses. In particularly, the propensity for Th2 allergic responses and asthma has recently been linked to the dysbiosis of the gut microbiome. Thus, the early establishment of particular microflora may potentially explain why some individuals are more predisposed to develop asthma and some do not. Eosinophils may also make differential contributions to allergic asthma in different individuals, and we have previously shown that development of allergic asthma is dependent on eosinophils. Here we report that C57Bl/6 ΔdblGATA eosinophil deficient mice housed under “less-restricted specific-pathogen-free” (LR-SPF) conditions are protected against allergic asthma compared to mice house under “restricted specific-pathogen-free” (R-SPF) conditions. 16s rRNA analysis of gut and lung microbiome reveal differences in lung microbiota diversity, suggesting a potential explanation for this phenomenon. Furthermore, consistent with the “microbiota hypothesis”, reducing the microbiota in ΔdblGATA mice by antibiotic treatment resulted in enhanced susceptibility to developing allergic airway inflammation comparable to WT mice. Thus, we conclude that shifts in microbiota can affect the requirement for eosinophil-mediated Th2 responses.
Alteration of human blood type 2 follicular helper T cells and regulatory B cells underlies comorbid association of allergic rhinitis with bronchial asthma (HYP2P.338)
Authors: Kamekura Ryuta , Shigehara Katsunori , Jitsukawa Sumito , Kawata Koji , Himi Tetsuo , Ichimiya Shingo
Published: January 2023

Allergic rhinitis (AR) is the most common allergic inflammatory disorder of the airway and its increasing worldwide prevalence has made it a global health problem. AR frequently precedes bronchial asthma (in 10 to 40% of cases), and the presence of AR is considered to be a risk factor for new onset of asthma. However, little is known about the mechanism by which AR advances to a state of AR with bronchial asthma (AR+Asthma). To determine the pathophysiologic features of AR and AR+Asthma, we examined subsets of follicular helper T (Tfh) cells and regulatory B (Breg) cells in peripheral blood from AR and AR+Asthma patients. The results demonstrated polarization of Tfh2 cells within Tfh cell subsets in both AR and AR+Asthma cases. Interestingly, Breg cells were decreased in AR cases and, more extensively, in AR+Asthma cases. Addtionally, we found significant correlations of fractional exhaled nitric oxide and blood eosinophil levels with the index %Tfh2 cells per %Breg cells. Our findings indicate that depletion of Breg cells under the condition of Tfh2 cell skewing is a putative exaggerating factor of AR to bronchial asthma.
Gene expression profile of allergen-experienced group 2 innate lymphoid cells (HYP2P.323)
Authors: Mathae Laura , Martinez-Gonzalez Itziar , Takei Fumio
Published: January 2023

We previously identified group 2 innate lymphoid cells (ILC2s) in mouse lungs. Upon allergen encounter, they produce type 2 cytokines and mediate T cell-independent allergic lung inflammation and also promote Th2 responses. We have recently found that allergen-treatments of mice result in significant increase in the number of ILC2s in the lung and lymph node even after the resolution of inflammation for several weeks. The allergen-experienced ILC2s are more responsive to unrelated allergens than naive ILC2s and rapidly induce allergic lung inflammation. To characterize the allergen-experienced ILC2s, we have performed microarray analysis of ILC2s purified from naïve mice and allergen-treated mice 4 days, 2 weeks, and 4 months after the treatment. The genes that are differentially expressed at different time points include those encoding chemokines, cytokines and their receptors, cell cycle and proliferation related proteins, adhesion molecules, and transcription factors. Gene Set Enrichment Analyses of the ILC2 populations revealed that allergen-experienced ILC2s are enriched for the gene signature of memory T cells. These results suggest that activated ILC2s undergo cell intrinsic changes and acquire memory-like features that are governed by the same set of genes as those responsible for memory T cell differentiation.
PARP inhibition by olaparib or gene knockout blocks asthma manifestation in mice by modulating CD4+ T cell function (HYP2P.324)
Authors: Ghonim Mohamed , Pyakurel Kusma , Ibba Salomi , Al-Khami Amir , Wang Jeffery , Rodriguez Paulo , Rady Hamada , El-Bahrawy Ali , Lammi Matthew , Ramsay Alistair , Mansy Moselhy , Ochoa Augusto , Naura Amarjit , Boulares Hamid
Published: January 2023

An important portion of asthmatics do not respond to current therapies. Thus, the need for new therapeutic drugs becomes urgent. We have demonstrated a critical role for PARP in experimental asthma. In the current study, we test the efficacy of Olaparib, a PARP inhibitor, recently introduced in clinical trials against cancer in blocking established asthma traits in animal models of the disease and determine the mechanism by which it achieves such protection. We show that administration of olaparib in mice post-challenge conferred a robust reduction in airway eosinophilia, mucus production and hyperresponsiveness even after repeated challenges with ovalbumin. The protective effects of olaparib were linked to a suppression of Th2 cytokines and ova-specific IgE with an increase in the Th1 cytokine IFN-g. These traits were associated with a decrease in splenic CD4+T cells and concomitant increase in T-regulatory cells. Adoptive transfer of Th2-skewed OT-II-WT CD4+T cells reversed both Th2 and Th1 cytokines and ova-specific IgE production in ovalbumin-challenged naïve PARP-1-/- mice suggesting a role for PARP-1 in CD4+T but not B cells. Our ex vivo studies show that olaparib treatment markedly reduced CD3/CD28-stimulated gata-3 and il4 expression in Th2-skewed CD4+T cells while causing an elevation in t-bet and ifn-g expression in Th1-skewed CD4+T cells. Our findings show the potential of PARP inhibition as a viable therapeutic strategy to be tested in human clinical trials
Sex-dependent differences in lung inflammation and M2-macrophage responses in asthma (HYP2P.328)
Authors: Keselman Aleksander , Heller Nicola
Published: January 2023

​Asthma is a chronic airway inflammatory disease that exhibits a sex difference. Alveolar macrophages acquire an M2-phenotype in asthma and in response to IL-4 in vitro. M2 macrophages correlate with poor lung function and pathology in asthmatics. We hypothesized that macrophages from female mice are intrinsically more responsive to IL-4 than male macrophages. Western blot demonstrated greater expression of the M2-marker, Ym-1 (~14 fold), in IL-4-activated bone marrow-derived macrophages (BMM) from female mice compared to male mice. We next assessed alveolar macrophages from male and female OVA-sensitized and -challenged mice for expression of M2-markers by FACS and western blot. We measured enhanced eosinophilia and increased Ym-1 expression in alveolar macrophages from female mice compared to male mice following OVA challenge. Estrogen enhances macrophage activation but the impact on lung macrophages during asthma is poorly understood. We observed a greater percentage of macrophages in the lungs of estrogen-implanted female mice that had undergone ovariectomy, compared to placebo-implanted females, following OVA challenge. These data suggest both intrinsic and estrogen-enhanced M2 macrophage polarization in females compared to males. Understanding intrinsic and estrogen-mediated sex differences in asthma is paramount to identify novel molecular targets and improve therapeutics in women with hard-to-treat asthma.
Repeated low dose injection of cobalt chromium microparticles in a mouse knee model induces robust inflammation with cartilage and bone loss (INC1P.345)
Authors: Palma Mark , Mishra Pankaj , Millman Ariel , Hallab Nadim , Beebe Kathleen , Benevenia Joseph , Gause William
Published: January 2023

Total joint arthroplasty is a well accepted orthopaedic procedure for the treatment of many pathological joint conditions, but the release of metallic wear debris such as cobalt chromium (CoCr) microparticles can lead to pain, osteolysis, and implant failure. Our studies of periprosthetic tissue samples from patients undergoing revision surgery have shown that these particles induce an M2 macrophage phenotype as evidenced through gene expression analysis and CD68 immunostaining. We used an intra-articular murine knee model to further examine the synovial tissue response to CoCr and have shown that a single high dose (10mg) inoculation of CoCr microparticles triggers a robust type 2 immune response characterized by the presence of alternatively activated macrophages, neutrophils, and eosinophils 2 days after injection. We further examined the long term effects of repeated alternate day low dose (0.5mg) injection of CoCr microparticles on synovial tissues over 14 days. Tissues exposed to CoCr microparticles were found to have increased clinical scores for inflammation with loss of cartilage and bone tissue at day 14 when compared to controls. Together these studies indicate that exposure of synovial tissues to CoCr induces an innate type 2 immune response with long term hard tissue damage. These studies may ultimately lead to the identification of targeted therapeutic options to locally treat implant associated pain and potentially increase implant longevity.
DOCK8 regulates protective immunity by controlling the function and survival of RORγt+ ILCs (INC1P.402)
Authors: Singh Akhilesh , Eken Ahmet , Fry Mallory , Bettelli Estelle , Oukka Mohamed
Published: January 2023

Retinoic-acid receptor-related orphan receptor-γt-positive (RORγt+) innate lymphoid cells (ILCs) produce interleukin (IL)-22 and IL-17, which are critical for protective immunity against enteric pathogens. The molecular mechanism underlying the development and survival of RORγt+ ILCs is not thoroughly understood. Here we show that Dedicator of cytokinesis 8 (DOCK8), a scaffolding protein involved in cytoskeletal rearrangement and cell migration, is essential for the protective immunity against Citrobacter rodentium. A comparative RNA sequencing-based analysis reveals an impaired induction of antimicrobial peptides in the colon of DOCK8-deficient mice, which correlates with high susceptibility to infection and a very low number of IL-22-producing RORγt+ ILCs in their GI tract. Furthermore, DOCK8-deficient RORγt+ ILCs are less responsive to IL-7 mediated signaling, more prone to apoptosis and produce less IL-22 due to a defect in IL-23-mediated STAT3 phosphorylation. Our studies reveal an unsuspected role of DOCK8 for the function, generation and survival of RORγt+ ILCs.
Respiratory syncytial virus disease is mediated by age-variable IL-33 and innate lymphoid cells (INC1P.347)
Authors: Saravia Jordy , You Dahui , Shrestha Bishwas , Lee Greg , Jaligama Sridhar , Rovnaghi Cynthia , Bagga Bindiya , DeVincenzo John , Cormier Stephania
Published: January 2023

Respiratory syncytial virus (RSV) is the most common cause of infant hospitalizations and represents an enormous health burden worldwide. Severe RSV infections are a significant risk factor for childhood asthma, but the pathogenic mechanisms responsible for RSV induced airway disease remain elusive. We and others have shown that age at time of initial infection is a crucial determinant of RSV immunopathogenesis (i.e., Th2 inflammation, eosinophilia, airway hyperresponsiveness, mucus). Using an age-appropriate mouse model of RSV that phenotypically recapitulates severe Th2-biased RSV seen in human infants, we show that this age-related disparity in immunopathogenesis is related to enhanced IL-33 production and increased group 2 innate lymphoid cells (ILC2s) in lungs of neonatal mice compared to adult mice. In nasal aspirates from RSV-infected human infants, IL-33 was elevated in the youngest of patients which further indicates age-variable regulation in response to RSV infection. These data provide a previously unknown mechanism involving ILC2s and IL-33 in RSV mediated human asthma, and represent the first evidence of differential ILC2/IL-33 regulation with respect to very early age.
The protective role of γδ T cells in neonatal influenza (INC1P.348)
Authors: GUO XIZHI , Dash Pradyot , Calverley Matthew , Thomas Paul
Published: January 2023

Influenza virus infection is a continuing world-wide health threat, especially in infants and the elderly. Neonates are highly susceptible to influenza infection, which is often a cause of hospitalization. γδ T cells are the first T cells to develop during embryogenesis and dominate among the neonatal immune system components, as conventional αβ T cell responses are functionally less prominent. Hence, γδ T cells are poised to play a critical role in neonatal infections; however, the specific role of γδ T cells in neonatal influenza infection remains to be elucidated. Using a neonatal mouse model of intranasal influenza infection, we observed the accumulation of γδ T cells within two days after infection. By utilizing the Nur77-GFP reporter mice, we demonstrated that the activation of these γδ T cells was via the T cell receptor. IL-17-producing γδ T cells, rather than IFN-γ-producing cells, dominated in the primary response. A comparison between wild-type and TCRδ-deficient neonates identified a protective function of γδ T cells in neonates. Reduced weight loss and an increased survival rate were observed in the wild-type compared to TCRδ-deficient animals. In infected lungs, wild-type neonates also had more robust IFN-β production and increased inflammatory cytokine production. Our findings demonstrate a vital role for γδ T cells in mediating protection during neonatal influenza virus infection, which may provide a new therapeutic target for pediatric influenza treatment.
Microglial CX3CR1 is required for M2 polarization after intracerebral hemorrhage (INC1P.350)
Authors: Taylor Roslyn , Hammond Matthew , Ai Youxi , Sansing Lauren
Published: January 2023

Intracerebral hemorrhage (ICH) occurs when a blood vessel ruptures in the brain parenchyma, activating microglia. Once activated, microglia may polarize into an M1, pro-inflammatory phenotype, or an M2 phenotype associated with wound repair. Microglia express the chemokine receptor CX3CR1, which regulates microglial neurotoxicity. We have previously shown that mice with CX3CR1-null microglia have worse functional outcomes 14 days after ICH. We hypothesize that CX3CR1-null microglia fail to transition to an M2 phenotype and do not aid in tissue repair. C57BL/6 (WT) and CX3CR1GFP/GFP (CX3CR1-null) mice were irradiated and reconstituted with bone marrow from CD45.1 mice to make bone marrow chimeras. ICH was induced by injecting 20ul of WT whole blood into the right striatum. WT mice were sacrificed 1-14 days after ICH to examine changes in gene expression. Microglia were sorted and qRT-PCR was performed. Chimeras were sacrificed 14 days after ICH and perihematomal regions were harvested for intracellular staining (ICS). At 12 hours after ICH, microglia have high IL-6 gene expression (M1). However, M2 markers arginase-1, TGF-β and SOCS3 increase over time, suggesting microglia transition from M1 to M2 over the first 2 weeks after ICH. By ICS, CX3CR1-null microglia had significantly less CD206 (p<0.01) and BDNF (p<0.001) than WT microglia 14 days after ICH. Our data suggest that microglia transition from M1 to M2 after ICH and that CX3CR1 is required for M2 polarization.
Mesenchymal cell effect on the immune response in renovascular hypertension (INC1P.352)
Authors: Grande Joseph , Azimi Camillia , Hartono Stella
Published: January 2023

Atherosclerotic renal artery sclerosis (RAS) is a significant public health concern, affecting 7% of the population over 65 years old and conveying an increased risk for cardiovascular as well as renal disease. Recent reports have failed to show a benefit of renal artery stenting to treat RAS. We tested the hypothesis that mesenchymal stem cells (MSC) slow the progression of renal injury by reducing inflammation. MSCs were isolated and cultured from murine adipocyte tissue and injected via the carotid artery into the animals 4 weeks after inducing RAS, a time at which the stenotic kidney has undergone severe atrophy. Conjugated microsphere labeling and flow cytometry confirmed that the MSCs migrate to the stenotic kidney. Half of the treatment group demonstrated a 90% reduction in atrophy and significantly lower level of regulatory T cell (Treg) infiltration while the other half demonstrated no change when compared to vehicle treated RAS mice. Array analysis showed a significant down-regulation of NOS2 (M1 macrophages), RORc (Th 17 cells), and CCR4 (associated with M1 macrophages) within those mice with low renal atrophy. The results suggest that under certain conditions, MSCs have a possible effect on the inflammatory environment present in RAS mice. Future studies will determine whether intervention prior to development of severe atrophy will increase therapeutic efficacy of MSC.