bluuebunny/crossref_metadata_2025 huggingface dataset:
The development of a gut-specific immune response to an oral antigen is dependent on α4β7 mediated lymphocyte trafficking
Authors: Sang Allison L , Redhu Naresh S , Lee Dooyoung , Dhang Fyonn , Bonesteel Raegan , St. Gelais Sarah , Zhong Cheng , Cui Dan , Harrison Bryce , Troast Dawn , Bursavich Matthew G , Lippa Blaise , Rogers Bruce N , Ray Adrian S , Wong Jamie
Published: January 2023

Objective

Vedolizumab has been shown to reduce the antibody response to an oral cholera, but not intramuscular hepatitis B vaccine in healthy subjects (Wyant, Gut 2015). The generation of an immune response to an oral antigen (Ag) begins at inductive sites such as Peyer’s patches (PP) followed by cell migration to effector sites in the gut. The integrin α4β7 is known to play a major role in cell trafficking to gut-associated lymphoid tissues. This study assessed whether MT-102, a potent and selective small molecule α4β7 inhibitor has similar effects on immunizations to those reported in the clinic for vedolizumab.

Methods

An in vivo immunization model system was established to determine the effects of MT-102, dosed via osmotic minipump, on the development of a gut-specific immune response. Mice were orally immunized with cholera toxin (CTX), and anti-CTX antibodies were quantified in fecal and plasma samples. The impact of MT-102 on lymphocytes within the PP of immunized mice was determined by flow cytometry.

Results

MT-102 delayed the production of anti-CTX IgA and IgG antibodies as measured in fecal samples without perturbing total gut IgA levels. Furthermore, MT-102 also inhibited the levels of Ag-specific antibodies in the plasma following repeated CTX oral immunizations. Blocking α4β7 induced a decrease in the frequency of B cells in the PP of treated mice compared to the control group.

Conclusion

α4β7-specific inhibitor, MT-102, effectively diminished the mucosal antibody response to an orally delivered Ag in mice. These results are consistent with what has been reported clinically in vedolizumab-treated subjects and thus establishes that potent small molecule inhibitors of α4β7 can replicate the pharmacology of an antibody.
Enteric glia regulates the expression of neutrophil inflammatory factors during acute inflammation
Authors: Padilla Rolon Darwing S , Gulbransen Brian D
Published: January 2023

Inflammation is an event in functional gastrointestinal disorders that has detrimental effects in the enteric nervous system. Recent advances in our understanding of the immune and enteric nervous system indicate that enteric glial cells modulate immune cells in the gastrointestinal tract. Previous single-nuclei multiomic analysis has shown that genes that encode for and influence neutrophil function, Neutrophil cytosolic factor 4 and C-X-C motif chemokine ligand 5, are changed in enteric glia during peak colitis. We hypothesize that enteric glia regulate the expression of neutrophil modulating factors during acute inflammation. We tested this using the acute dinitrobenzene sulfonic acid colitis model. Animal body weight and gross macroscopic damage were recorded as indications of overall inflammation and tissue was harvested for immunohistochemical analysis. Enteric glial cells were labeled with antibodies against Neutrophil cytosolic factor 4 and C-X-C motif chemokine ligand 5. Results suggest no significant difference in labeling for immunological markers in enteric glial cells of dinitrobenzene sulfonic acid treated mice. Results also show labeling in some neurons that may or may not be specific. Further experimentation is needed to elucidate the mechanisms of enteric neurons and glia in gut inflammation. These studies will contribute to further understand how immune responses are regulated in the gut and impact the general knowledge of gut disease processes.

This work was supported by the National Institute of Health grants R01DK103723 and R01DK120862 to BDG and DPR was supported by the National Institute of Health Bridge to the Ph.D. in Neuroscience grant 2R25NS090989.
MOSPD2 regulates the activation state of αLβ2 integrin to control monocyte migration
Authors: Mendel Itzhak , Salem Yaniv , Propheta-Meiran Oshrat , Kafri Pinhas , Yacov Niva , Breitbart Eyal
Published: January 2023

Monocytes are innate immune cells that drive the chronicity of various inflammatory diseases, making them an attractive target for therapeutic intervention. Monocyte migration to inflamed tissues involves multiple steps of interaction with the vascular endothelium and the extra-cellular matrix (ECM), a process mainly mediated through conformational transitions in cell surface integrins. We previously described Motile sperm domain-containing protein 2 (MOSPD2) as a surface protein expressed on myeloid cells that is essential for the migration of monocytes and a key regulator of inflammatory disease pathogenesis. Therefore, a possible role for MOSPD2 in regulating adhesion and integrin activation in monocytes was assessed.

Silencing of MOSPD2 expression in THP-1 monocytic cell line significantly increased the adhesion of monocytes to various ECM ligands of integrins. Employing VB-601, a humanized anti-human MOSDP2 monoclonal antibody, on wild-type THP-1 cells or primary human CD14 monocytes similarly increased cell adhesion to ECM ligands as well as adhesion molecules. At the molecular level, silencing of MOSPD2 or blocking MOSPD2 using VB-601 led to a transition in integrin αLβ2 conformation into active high affinity binding-form and to the phosphorylation of adhesion-associated pathways. Co-precipitation experiments show that MOSPD2 binds integrin-β2, but not integrin-β1.

Our results reveal a novel regulatory mechanism on monocyte migration in which MOSPD2 governs the balance between monocyte adhesion and release from adhesion molecules and ECM. Targeting MOSPD2 has the potential to treat inflammatory diseases by stemming monocyte infiltration into inflamed tissues.
Tissue ischemia induces mobilization of pro-angiogenic neutrophils from the spleen
Authors: Leite Catarina , Parv Kristel , Seignez Cédric , Ng Henry , Lindsay Robin S , Christoffersson Gustaf , Phillipson Mia
Published: January 2023

Pro-angiogenic neutrophils (PAN) form a small subset of circulating neutrophils expressing CD49d+ VEGFR1hi CXCR4hi, which play a crucial role in the revascularization of transplanted islets.

To determine the role of PANs in re-establishing tissue perfusion following a large ischemic event we used the mouse model for peripheral artery disease, the hindlimb ischemia (HLI) model. We found that numbers of PANs in the affected gastrocnemius muscle increased ≈4 times 3h following ischemia induction and reached its peak 2 days following ischemia onset. Similarly, in patients undergoing surgery where peripheral perfusion is transiently clamped, numbers of circulating PANs increase ≈3 times 30 min post-HLI.

In healthy mice, PANs are found in high frequency in the spleen (spleen: 9,8% of total neutrophils, blood: 0,78%). Splenectomy prior to induction of HLI reduced recruitment of PANs to the ischemic muscle, which demonstrates that the spleen acts as a reservoir for PANs.

Furthermore, HLI downregulated the CXCR4-ligand CXCL12α in the splenic red pulp. This was prevented by chemically induced sympathectomy, resulting in increased retention of PANs in spleen, reduced PAN accumulation in the ischemic muscle and reduced blood flow recovery. Therefore, we propose that the downregulation of CXCL12α controlled by sympathetic nerve signaling pathways drives retention of PANs in the spleen, thereby regulating their release in response to HLI.

In summary, we identified that the spleen contains a peripheral pool of PANs and emphasize the pivotal role of the neuro-immune axis in the recruitment of PANs to the site of ischemia. These discoveries open new avenues for drug development to limit damage and accelerate healing following ischemic events.

This study was supported by the Swedish Research Council, Knut and Alice Wallenberg foundation, The Swedish Cancer Society, and O. E. and Edla Johansson’s foundation.
Diesel exhaust particles induced inflammatory responses are associated with executive functions deficits in juvenile mice
Authors: Torres Nicole Arianne Pagan , Rosa Hector J , Menendez Daviana L , Casillas Karla M , Mendez Loyda B
Published: January 2023

Epidemiological studies have reported associations between exposure to ambient particulate matter (PM) and neurocognitive impairments. Children are particularly vulnerable to the effects of PM since their CNS is still in development, especially in regions related to executive functions (EFs). Toxicological studies have reported oxidative stress and neuroinflammatory responses in mice exposed to different types of PM. However, most studies have focused on prenatal, perinatal, and adult PM exposures. The goal of this study was to assess if PM exposure impairs the postnatal development of EF. We hypothesized that exposure to diesel exhaust particles (DEP) will impair EFs by inducing inflammatory responses in the CNS and lungs. To test this hypothesis, female and male C57BL/6J mice (n=14/group) were exposed intranasally to either saline or increasing doses of DEP during postnatal days (PND) 25 to 33. General cognitive abilities and executive function were evaluated on PND 36–38 with the Puzzle Box paradigm. Mice were euthanized on PND 39 and the tissue was collected to measure the concentration of proinflammatory cytokines in the CNS and assess inflammatory cell influx into the lungs. In the brain, the levels of IL-6 and GM-CSF, but not TNFα, significantly increased in a dose-dependent manner. In the lungs, a dose-dependent influx of inflammatory cells was also observed. The results showed that mice exposed to DEP had concomitant deficits in cognitive processes related to executive functions and inflammatory responses in the brain and lungs. This suggest that DEP induced pro-inflammatory responses might be associated with deficitis in the postnatal development of EFs. In the future, the role of microglia activation will be explored.

Supported by Puerto Rico IDeA Network of Biomedical Research Excellence 5P20GM103475-18.
An unexpected role for IRF8 during human T cell development.
Authors: Taghon Tom N , Liang Kai Ling , Lavaert Marieke , Roels Juliette , Strubbe Steven , Lambrechts Nina , De Vos Tamara , Boehme Lena , Van Hulle Jolien , Oatman Nicole , Putteman Tom , Velghe Imke
Published: January 2023

Lineage tracing studies or other genetic approaches have allowed investigators to obtain detailed insights into the molecular mechanisms that control T cell development in the mouse. However, our understanding of how this process is regulated in human has remained unclear. Using various genome-wide approaches, such as bulk RNAseq, ATACseq, ChIPseq and scRNAseq, we have recently obtained important novel insights into the molecular mechanisms through which human hematopoietic progenitor cells are converted into T-lineage committed precursors, and further into the TCRαβ and −γδ lineages. Specifically, we identified two distinct populations of thymus seeding precursor cells (TSPs) of which one population, TSP2, expressed IRF8, a transcription factor that is mainly associated with dendritic cell development. Consistently, IRF8-expressing TSPs have both T− and DC-linage potential and are characterised by the coexpression of CD127 and CD123. We discovered that Notch activation, the main initiator of T-lineage specification, induces IRF8 expression in human ETPs, in contrast to in mouse, and that IRF8 is transiently expressed during early human T cell development. As T cell precursors commit to the T-cell lineage, IRF8 is repressed by GATA3, while IRF8 levels rise for ETPs that differentiate into plasmacytoid dendritic cells. Importantly, IRF8 is required for the generation of CD34+ CD127+ CD123+ T/DC precursors and low level IRF8 activity enhances their generation from hematopoietic progenitors. Thus, using these approaches, we have identified an IRF8-dependent T cell precursor subset and revealed unique molecular events that control early T cell development in human.

Supported by grants from the Fund for Scientific Research Flanders (FWO), the Foundation Against Cancer (STK), the Chan Zuckerberg Initiative (CZI) and Ghent University (BOF Ugent).
Ezrin promotes antigen receptor diversity during B cell development by supporting immunoglobulin heavy chain variable gene recombination
Authors: Aysola Varun , Labib Christina , Gupta Neetu
Published: January 2023

Genome-level rearrangements of immunoglobulin genes during B cell development are critical for generation of a diverse repertoire of B cell antigen receptors (BCRs) that bind to a multitude of foreign antigens and some self-antigens. Bone marrow B cell development involves a variety of cell-cell interactions, cell migration and receptor signaling that likely benefit from the activity of membrane-cytoskeletal reorganizing proteins. However, the specific contribution of such proteins towards BCR repertoire diversification is poorly understood. Ezrin is a membrane-cytoskeletal linker protein that regulates mature B cell activation through spatial organization of the BCR. We employed next generation sequencing to investigate if Ezrin plays a role in immunoglobulin heavy chain rearrangements and generation of BCR diversity in developing bone marrow B cells. BCR repertoire development occurred stochastically in B cell progenitors from both control and B cell conditional Ezrin-deficient mice. However, the loss of Ezrin resulted in fewer unique CDR3s in the BCRs and reduced Shannon entropy. Ezrin-deficient pro-, pre- and immature B cells utilized similar number of joining (J) genes but significantly fewer variable (V) genes, thereby decreasing V-J combinatorial diversity. V-J junctional diversity, measured by CDR3 length, nucleotide additions and deletions, was also altered in Ezrin-deficient pro-, pre- and immature B cells. Mechanistically, Ezrin-deficient bone marrow B cells showed a marked decrease in RAG1 gene expression, indicating a less efficient DNA recombination machinery. Overall, our results demonstrate a novel role for Ezrin in shaping the BCR repertoire through combinatorial and junctional diversification.

Supported by grants from NIH (R01 AR067705) to N.G.
Epigenetic Regulation During Maternal Schistosomiasis Results in Aberrant B cell Maturation and Tolerance
Authors: Gibbs Lisa C , Oviedo Juan Marcos , James Kimra , Fairfax Keke C
Published: January 2023

Maternal helminth infections are a global public health concern that correlate with altered infant immune responses to childhood immunizations and infection. A mechanistic understanding of how maternal infection and inflammation alters the immune responses of offspring is lacking but is critical to decrease childhood morbidity and to understand the consequences of specific long-lived immunity defects. Using our model of maternal Schistosoma mansoni infection, we have shown that murine pups born to mothers chronically infected with Schistosoma mansoni have reduced responses to vaccinations, corresponding to what has been reported in humans. To determine the origin of this humoral immunity defect, we began investigating the plasticity and functionality of progenitors of lymphoid cells critical for a protective humoral response. We found an increase in the common lymphoid progenitors (CLPs) in the bone marrow of pups from Schistosome infected mothers, but a marked decrease in immature and transitional B cells in male mice, indicating sex-specific limitations during B cell maturation. Using single cell V(D)J sequencing, we found only male pups from infected mothers have a more restricted repertoire compared to male mice from uninfected mothers after immunization, leading to decreased antigen-specific B cells in the germinal center of the draining lymph node. We hypothesize that epigenetic regulation of key immune genes is the mechanistic root of long-lived defects in humoral immunity to foreign antigens during maternal Schistosomiasis.

Supported by a grant from NIH (R01 AI135045)
Transcription factor TFII-I fine tunes innate properties of B lymphocytes
Authors: Kaila Mary , Singh Amit , De Supriyo , Mazan-Mamczarz Krystyna , Sen Ranjan , Roy Ananda
Published: January 2023

The ubiquitously expressed transcription factor TFII-I is a multifunctional protein with pleiotropic roles in gene regulation and, germline deletion of the Gtf2i gene leads to embryonic lethality. To delineate the physiological role of TFII-I in murine B cells, we carried out conditional deletion of Gtf2i. Here we report a surprising phenotype that suggests a role for TFII-I in homeostasis of innate properties of B lymphocytes. B cell transcriptome and chromatin landscape is skewed towards myeloid like features in the absence of TFII-I. Gtf2i ablated B cells also demonstrate enhanced sensitivity towards LPS induced transcription and proliferative capacities, features associated with innate immune cells. Finally, increased IgG3 switching, which is normally associated with inflammation, was observed in the absence of TFII-I. These results suggest an unexpected role for TFII-I in maintaining immune homeostasis and provide further clues for GTF2I polymorphism associated with B cell dominated autoimmune diseases.

This work was supported in part by the intramural program of the NIH, National Institute on Aging (NIA)
A Snap Shot of Complement Gene Expression and Presence of Complement Proteins in Synovial Biopsies from Early Rheumatoid Arthritis Patients
Authors: Banda Nirmal K , Deane Kevin D , Seifert Jennifer , Strickland Colin , Bemis Elizabeth , Jordan Kimberly , Goldmann Kationa ,   Accelerating Medicines Partnership (AMP) RA/SLE Network, Morgan B Paul , Lewis Myles J , Pitzalis Costantino , Moreland Larry W , Holers V Michael
Published: January 2023

The etiology of rheumatoid arthritis (RA) is unknown. Previous studies of mouse models of RA have strongly implicated the alternative and lectin pathways of the complement system in disease pathogenesis. Here we explored the Pathobiology of Early Arthritis Cohort (PEAC) tissue RNA sequencing (RNA-seq) database and identify correlations among complement gene expression, Fc receptor expression and clinical severity, measured as disease activity score 28 - erythrocyte sendimentation rate (DAS28-ESR), in both blood and synovium. We also evaluated the biodistribution of complement activation pathway proteins and inhibitors using Multispectral ImmunoHistoChemical (MIHC) staining. Ultrasound guided synovial biopsies (n = 23), obtained from Accelerating Medicines Partnership (AMP) studies, were subjected to MIHC for various complement proteins. Our analyses revealed that in the synovium, but not in blood, significant positive correlations existed between complement gene expression and DAS28-ESR for C2, CFB, FCN1, C3AR1, C5AR1, and CR1. Surprisingly, levels of MASP1, Colec12, C5 and C6 RNA inversely correlated with baseline DAS28-ESR. After 6 months therapy, baseline CFHR4 positively correlated with delta DAS28-ESR. In the synovium, there were also significant positive correlations between DAS28-ESR and FcγR1A, FcγR1B, FcγR2A and FcγR3A. In early RA (ERA) synovium, a significantly (p < 0.05) higher levels of cells expressed CFH compared with CFB and CFHR4. We also found regional imbalance between C3 and CFH in ERA synovial biopsies. ERA synovial biopsies implicate the complement system in early disease and reveal intriguing differences among factors in clinical relevance, outcome and localized tissue dysregulation.

Supported by R01AR51749-16
Metabolic Dysfunction Governs Regulatory T Cell Inflammatory Response during Inflammatory Bowel Disease
Authors: Bamidele Adebowale , Sagstetter Mary , Hirsova Petra , Ramos Guilherme Piovezani , Westphal Maria , Faubion William A
Published: January 2023

Background

Significant proportion of inflammatory bowel disease (IBD) patients respond inconsistently to therapies, underscoring disease complexity. Interleukin 21 (IL21) is highly expressed during IBD. In addition to T helper (Th) cells, inflammatory regulatory T cells (Tregs) have been linked to refractory IBD. Thus, we explored the metabolic role of IL21 in inducing Treg dysfunction and IBD.

Methods

Human Tregs and Th cells were subjected to transcriptional profiling and metabolic phenotyping. Colitis was induced in Rag1−/− mice by naïve CD4 T cell adoptive transfer.

Results

IL21 stimulation of human Tregs induced glycolysis and OXPHOS. In agreement, IL21 enhanced the expression of genes associated with glycolysis, anabolism, and OXPHOS, leading to inflammatory cytokine production. Mechanistically, we found disruption to mitochondrial integrity with concomitant activation of glycogen synthase kinase 3 (GSK3) β, a kinase known to prevent mitochondrial pyruvate metabolism. IL21-induced GSK3β activation was accompanied by pyruvate and lactate buildup. Notably, GSK3 inhibition or supplementation with membrane-permeable methyl pyruvate abrogated metabolic wiring of and inflammatory responses by IL21-stimulated Tregs and effector Th cells. These results suggest that impaired mitochondrial pyruvate metabolism is a feature of inflammatory CD4 T cells. Lastly, GSK3 inhibition prevented pathogenic CD4 T cell-induced colitis in mice, as evidenced by reduced Mouse Colon Histology Index and serum inflammatory cytokines.

Conclusions

IL21 engages Tregs in a hypermetabolic state that augments inflammatory cytokine production. Therefore, desensitizing CD4 T cells to IL21 may also augment Treg function during IBD.

Supported by NIDDK award K01DK124358, the Center for Cell Signaling in Gastroenterology (P30DK084567), and the Mayo Clinic Center for Biomedical Discovery.
Lung memory B cells promote local IgE production in the respiratory tract
Authors: Nelson Alexander J , Wu Yee Ling
Published: January 2023

Allergen-specific IgE mediates allergic asthma. IgE responses including class-switch recombination (CSR) to IgE are tightly regulated. However, the regulation of local CSR to IgE at mucosal sites is poorly understood. This study aims to elucidate cellular sources that promote IgE CSR and mechanisms that regulate IgE production in the respiratory tract.

To model allergic airway inflammation, we repeatedly administered intranasal ragweed pollen and OVA to mice. We profiled B cell responses to OVA in the lung, the lung-draining mediastinal lymph nodes (medLN), spleen and bone marrow. We used fluorescent reporter mice to track IgE CSR and identify B cell subsets that promote IgE CSR by flow cytometry and microscopy.

We found that intranasal allergen exposure elicited robust local B cell responses with the formation of OVA-specific B cells in the medLN and OVA-specific IgE in the airway. OVA-specific germinal center B cells and memory B cells (MBCs) accumulated in the lungs. Using fluorescent reporter mice, we showed that IgG1+ MBCs are the predominant IgE-switching cells. Importantly, the number of IgE-switching MBCs are the highest in the lungs, and IgG1+ MBCs exhibit an increased frequency of IgE CSR in the lungs compared to lymphoid tissues. These suggest that the lung environment favors IgE production. RNA-seq analyses comparing lung and circulating MBCs revealed elevated expression of Tnfsf4 (OX40L) and Icosl on lung MBCs, suggesting a higher propensity for lung MBCs to engage T cells.

Together, these data demonstrate that the allergic lung is a major site of IgE CSR with IgG1+ MBCs promoting local IgE CSR. Our ongoing work focuses on delineating the B cell-intrinsic and extrinsic factors that promote IgE production in the respiratory mucosa.

Supported by grants from NIH (1F31 HL156459, 5T32 AI007508, 1R21 AI159456)
VEGFR3-driven pulmonary lymphangiogenesis exacerbates induction of bronchus-associated lymphoid tissue in allergic airway disease
Authors: Gomez Medellin Jorge E , Hollinger Maile Kananiokala , Rosenberg Jillian , Blaine Kelly , Kurtanich Trevin , Ankenbruck Nick , Hrusch Cara Lynn , Sperling Anne I , Swartz Melody A
Published: January 2023

Asthmatic lung samples present with both a higher density of pulmonary lymphatic vessels and a higher incidence of bronchus associated lymphoid tissue (BALT). Here, we asked whether lymphangiogenesis, stimulated by the VEGF-C/VEGFR-3 signaling axis in lymphatic endothelial cells (LECs), plays a role in promoting BALT in mouse models of allergy. First, we determined that chronic intratracheal instillation of house dust mite (HDM), a clinically relevant allergen, recapitulates both lymphangiogenesis and BALT induction. Intratracheal stimulation of VEGFR-3 in LECs exacerbated BALT, while blocking VEGFR-3 signaling reduced BALT. Furthermore, in transgenic mice with an expanded pulmonary lymphatic network (induced prior to allergen challenge), we found an exacerbated BALT response upon chronic HDM inhalation. Recent studies have determined that LEC-derived CXCL13 plays an important role in secondary lymphoid structure organogenesis, and thus we pondered whether LEC-derived CXCL13 could play a role in the development of tertiary lymphoid structures. Indeed, we observed an increase in lung infiltration by CXCR5+ cells when we used VEGF-C to modulate the chronic allergic response. Finally, we found that the VEGF-C exacerbated BALT phenomenon was indeed CXCL13 dependent. Altogether, these results suggest a causative role for pulmonary lymphatics in mediating BALT induction in chronic allergic airway inflammation.
Choline acetyltransferase attenuates inflammation in a murine model of DSS colitis and sepsis
Authors: Tsaava Tea , Tynan Aisling , Hepler Tyler D , Gabalski Arielle H , Hide Daniel M , Li Jian Hua , Chang Eric H , Tracey Kevin J , Chavan Sangeeta S
Published: January 2023

Excessive immune cell activation and cytokine release leading to inflammatory conditions are associated with bidirectional immune system-brain communication and other physiological responses. The vagus nerve conveys sensory information to the brain and brain derived immunoregulatory signals suppressing peripheral cytokine levels and inflammation. Acetylcholine (A mediated cholinergic signaling has been implicated in this regulation. However, the possibility of controlling inflammation by peripheral administration of choline acetyl transferase (ChAT), enzyme that catalyzes biosynthesis of acetylcholine, is unexplored. We studied the administration of ChAT in endotoxemia, sepsis and DSS colitis models. Intraperitoneal administration of ChAT significantly (p=0.002) suppresses serum levels of TNF (ChAT 1062.0pg/mL ± 113.0 vs vehicle 1711.0pg/mL ± 210.2) during murine endotoxemia. In a murine CLP sepsis model, administration of pegylated ChAT (PEG-ChAT) significantly improves survival (p=0.015), with a 55.6% survival in PEG-ChAT treated mice compared to a 22.2% survival in vehicle mice. In a preclinical model of inflammatory bowel disease, ChAT administration significantly improves body weight gain (d14 p=0.01 ChAT mice -5.1%±1.1 vs vehicle mice -15.5%±2.9), disease score (d11 p=0.006 ChAT mice 0.17DAI±0.05, vs vehicle mice 0.66DAI±0.13), and colon length (p=0.006 ChAT mice 7.15cm±0.2 vs vehicle mice 6.2cm±0.2) compared to vehicle. These results indicate that administration of ChAT inhibits TNF levels in acute endotoxemia and attenuates disease severity in murine models of sepsis and DSS-induced colitis, suggesting that further study of ChAT as an experimental anti-inflammatory therapeutic is warranted.

Supported by grant from NIH to KJT and SSC.
PD-L1 plays a B cell-intrinsic role in suppressing the antibody response and anti-tumor immunity.
Authors: Cervantes Christian , Khan Mustafa , Fernandez Maria , Liu Zexuan , Hakeem Zainab , Viswanadhapalli Suryavathi , Gupta Harshita B , Curiel Tyler J , Yan Hui , Vadlamudi Ratna , Xu Zhenming
Published: January 2023

The discovery that PD-L1 inhibits PD-1 on CD8+ T cells has led to new mechanistic insights into the regulation of host immune responses. The contribution of PD-L1 expressed by different immune cell types remains poorly understood, partly due to the lack of cell type-specific Cd274 (encoding PD-L1) knockout mice. Here, to address the role of B cell-expressed PD-L1 in antibody response and tumor development, we have generated Aicdacre Cd274fl/fl mice, in which PD-L1 was ablated only in activated B cells. As compared to their wild-type littermates, Aicdacre Cd274fl/fl mice showed normal development and maintenance of B cells, T cells, DCs and macrophages, while mounting a stronger T-dependent high affinity NP-binding IgM, IgG1 and IgG2a titers upon immunization with NP-CGG plus alum. Such phenotype was copied by C57 mice treated with a blocking αPD-L1 antibody. Tumor growth from engrafted E0771 breast cancer cells in Aicdacre Cd274fl/fl mice was significantly delayed, in association with increased CD8+ T cell infiltration and activities. Moreover, B cells isolated from Aicdacre Cd274fl/fl mice or those treated with aPD-L1 displayed higher levels of proliferation and differentiation to plasma cells upon in vitro stimulation with the T-dependent B-cell stimulus CD154. This along with the altered gene expression in Aicdacre Cd274fl/fl mice and αPD-L1-treated cells suggests a B cell-intrinsic role of PD-L1 in regulating B cell differentiation. Furthermore, PD-L1 expressed on B cell surface was tightly regulated during differentiation and internalized upon αPD-L1 treatment, suggesting a possible new mechanism on how PD-L1+ B cells modulate host immune responses.

Supported by grants from NIH: The Ruth L. Kirschstein NRSA Institutional Research Training Grant (to MK), NIH T32 Graduate Research in Immunology Program (to CC), R01 AI153506 and R21 AI135599 (to ZX). Mays Cancer Center Dew Foundation Pilot Grant (to ZX) and DoD BCRP BC170448 (to ZX)
Activation of C-type lectin receptor DC-SIGN increases human B cell survival
Authors: Arias Ivonne Nannette , Valentín-Acevedo Anibal José
Published: January 2023

DC-SIGN is a C-type lectin that plays an important immune role as a pattern recognition receptor (PRR). It is mainly expressed in dendritic cells, macrophages, and activated B cells where it can bind several pathogens including HIV and SARS-CoV-2. Interestingly, some of these pathogens use DC-SIGN to facilitate infection of target cells and support their survival within the host. While the role of DC-SIGN as a PRR is well established, little is known about the cellular outcomes induced upon ligand binding, particularly in B cells. Here, we demonstrate that when we activate DC-SIGN with a mannose ligand (mannan) on a lymphoblastic human B cell line with constitutive DC-SIGN expression (Ramos-DC), the cell’s survival increases. Moreover, analysis of cell death by flow cytometry showed that anti-IgM induced death was partially prevented by pre-treating cells with mannan. While mannan can bind to other lectins (e.g., Mannose Receptor, Dectin-1), preliminary immunophenotype analysis of these receptors confirmed their absence from Ramos-DC cells supporting the specificity of our observation. Finally, treating cells with an anti-DC-SIGN specific antibody before inducing death also prevented cells from dying, suggesting that the survival response observed is indeed mediated by DC-SIGN ligation. Our data suggest that DC-SIGN activation in human B cells induces a signaling pathway that results in cell survival even in the presence of death-inducing stimulation. Our future goal is to characterize the signaling events leading to this response in B cells. Our results contribute to the understanding of the role of DC-SIGN in immune function and how this knowledge could lead to the development of novel anti-viral therapies.

The project described was supported by Award Number R25GM110513 from the National Institute of General Medical Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health.
MICRORNA-10A NEGATIVELY REGULATES INTESTINAL IGA RESPONSE THROUGH SUPPRESSING REGULATORY T CELLS CONVERSION TO T FOLLICULAR REGULATOR CELLS
Authors: Yu Tianming , Yang Wenjing , Yao Suxia , Cong Yingzi
Published: January 2023

Intestinal IgA response is a key immune regulation between host and gut microbiota. Regulatory T cells (Tregs) have been recently shown to promote IgA responses through conversion to T follicular regulator cells (TFRs). However, how Treg converts to TFR to promote intestinal IgA response remain unclear. Tregs express microRNA (miR)-10a at high levels, however, whether it regulates TFR generation and function is unknown. We found that Treg cell-specific miR-10a deficient Foxp3YFP-cre miR-10af/f (Foxp3ΔmiR-10a) mice displayed higher levels of gut IgA compared to wild type (WT) Foxp3YFP-cre miR-10af/+ mice. TFRs in Peyer’s patches were also significantly increased while there were no differences in germinal center B cells, Tregs, and TFH cells. When orally immunized with cholera toxin (CT), Foxp3ΔmiR-10a mice showed more CT-specific fecal IgA and TFRs. miR-10a deficient Tregs induced more IgA compared to WT Tregs when co-cultured with naïve B cells in vitro. When transferred to TCRβxδ−/− mice, miR-10a deficient Tregs induced more gut IgA and higher levels of TFRs compared to WT Tregs in vivo. However, gut microbiota composition in Foxp3ΔmiR-10a mice was not altered by 16S rRNA gene sequencing. RNA-sequencing analysis showed that miR-10a was enriched in the oxidative phosphorylation (OXPHOS) process in Tregs. Consistently, miR-10a deficient Tregs displayed higher OXPHOS metabolic levels by seahorse analysis. Pretreatment of Tregs with oligomycin, which inhibits OXPHOS, inhibited Treg induction of IgA production in vitro, indicating miR-10a suppressed Tregs induction of IgA ability by inhibiting OXPHOS. Thus, miR-10a negatively regulates TFRs conversion through inhibition of OXPHOS to control intestinal IgA response.
Burns are more than skin deep: Heightened neuroinflammation after cutaneous injury in aged mice
Authors: Walrath Travis Michael , Najarro Kevin M , McMahan Rachel H , Idrovo Juan-Pablo , Quillinan Nidia , Kovacs Elizabeth J
Published: January 2023

By 2050, 1 in 6 people will be 65 or older. As the aging population grows so too does the importance of understanding how injuries in this age group lead to a greater incidence of neurological complications and mortality. Neurological complications may be driven by elevated pro-inflammatory cytokines and astrocyte activation within the hippocampus (HPC), which reduces levels of phosphorylated cyclic-AMP response binding element (pCREB), leading to cognitive decline and memory impairment. To examine the effects of injury on neuroinflammation in advanced age, young and aged mice were anesthetized and subjected to a 15% total body surface area scald or sham injury. As an index of neuroinflammation, we observed an 85-fold (p<0.05) increase in ccl2 and a 35-fold (p=0.05) elevation in tnfa 24 hours post injury in the brains of aged mice compared to young groups and aged sham mice. We also saw a 13-fold (p<0.05) increase in ccl2 within isolated hippocampus of aged injured mice, when compared to aged shams. Interestingly, when investigating specific HPC regions, immunofluorescence staining showed a 20% rise (p<0.05) in glial fibrillary acidic protein (GFAP) positive astrocytes within the cornu ammonis 3 (CA3) of aged injured mice when compared to sham groups. GFAP+ astrocytes are associated with neurotoxicity and cognitive decline. Finally, there was a 20% decrease (p<0.05) in pCREB staining by immunohistochemistry in the dentate gyrus of aged mice compared to young regardless of injury. These data suggest that remote injury in aged, but not young mice, causes neuroinflammation and astrocyte activation, which paired with an age related reduction in pCREB, could contribute to cognitive decline.

Supported in part by NIH R01 AG018859 (EJK), R35 GM131831 (EJK), T32 AG000279 (KLM), and K08 134185 (JPI)
Clonal and Diversity Differences of Extravascular B cells in the Influenza-Experienced Lung
Authors: Breen Michael P , Feng Feng , Aihara Fumiaki , Etesami Neelou , Fearns Rachel , Mizgerd Joseph , Kepler Thomas B
Published: January 2023

Memory B cells are a vital arm of the antiviral response. Recent literature has characterized a population of murine lung resident memory B cells after influenza A virus (IAV) infection capable of in situ differentiation into anti-influenza antibody secreting cells. Separately, PDL2+/CD80+ B cells appear to have a “memory-like” phenotype while the double-negative counterparts are “naïve-like” with low mutation frequency and class-switching. Additionally, our group has shown that PDL2+ EBCs impact Streptococcus pneumoniae immunity but their role in anti-viral immunity is understudied.

While extravascular B cells (EBCs) may play a critical immune function, it’s unclear if the PDL2+/CD80+ population is clonally distinct from the PDL2-/CD80- one as diversity can impact immune capabilities. We hypothesize PDL2+/CD80+ EBCs have a signature clonal repertoire influenced by infection history and tissue localization.

We infected BALB/cJ mice twice with related IAVs separated by four weeks. After a six-week rest period, we isolated PDL2-/CD80- and PDL2+/CD80+ EBCs from the lung and spleen. Immunoglobulin sequencing (IgSeq) was performed on IgM and IgG libraries that were analyzed with in-house clonal partitioning software (Cloanalyst).

There was significant increase in the number of EBCs in the lung and the proportion of PDL2+/CD80+ EBCs increased dramatically. Mutation frequency of PDL2+/CD80+ EBCs within the IAV-exposed lung rose, suggesting a role of tissue localization on B cell signature. Furthermore, diversity analysis using Shannon and Simpson estimators suggests differences between tissues and PDL2/CD80 expression. Together, our data highlight a unique clonal niche occupied by PDL2+/CD80+ EBCs.

Supported by grants from NIH (R33 HL-137081, T32 AI-007309)
Lung tissue resident memory CD8 T cells rely on lipid-rich environments for differentiation and display unique metabolic profiles
Authors: Sportiello Michael John , Geber Adam , Poindexter Alexis , Reilly Emma C , Emo Kris Lambert , Topham David J
Published: January 2023

Tissue resident memory CD8 T cells (TRM) protect against infections. In the lungs, TRM offer critical protection against influenza. Skin TRM display a unique metabolic profile compared to other memory cell subsets. They undergo enhanced fatty acid oxidation and oxidative phosphorylation as compared with other cells.

It is unclear if this programming holds true in the lung. We hypothesize that lung CD8 T cells with different integrin profiles will utilize unique metabolic programs. Differential expression and pathway analyses were conducted on RNAseq datasets, demonstrating an increased reliance on fatty acid oxidation in TRM compared to circulating memory CD8 T cells. This was most clear when a novel pathway analysis software ‘fluximplied’ that we created was used.

Metabolic models were constructed to verify these findings, functional assays were conducted, and the levels of oxidative phosphorylation, mitochondrial mass, and neutral lipids were measured in addition to fatty acid uptake, glucose uptake, and fatty acid oxidation Seahorse assays, confirming a lipid-centric metabolism of TRM.

It is unclear if this lipid-centric nature of TRM is a correlate of TRM phenotype or essential to TRM development. We developed an ex vivo model where naive T cells were differentiated with cytokines and metabolites into integrin expressing subsets. Higher amounts and more time with lipid-rich media increased expression of TRM markers. Lastly, in a T cell transfer model, we demonstrate that naive T cells that uptake more fatty acids have more TRM progeny than their low-uptake counterparts after transfer and influenza infection. In summary we demonstrate lung TRM have unique metabolic profiles and that lipids impact TRM differentiation patterns.

This work was funded by a Program Project grant through the National Institutes of Health, National Institute of Allergy and Infectious Diseases P01-AI102851 and through the National Institutes of Health training grant T32GM007356-46 and the National Institutes of Health training grant T32HL066988-20.
Influence of age, sex, and location on the diversity of the murine gut microbiome and/or expression of pro-inflammatory cytokines
Authors: Webster Sarah E , Vos Duncan , Rothstein Thomas L , Holodick Nichol E
Published: January 2023

The microbiome and immune system have a unique interplay, which influences homeostasis within the organism. Both the microbiome and immune system play important roles in health and in diseases of the aged. Various groups have demonstrated divergent changes in gut microbiota during aging, but these studies have largely ignored the compounding factor of biological sex in this aging, and little is known about the effect of institutional environment on the composition of gut microbiota in normal healthy mice. To better understand the roles of sex, aging, and environment in influencing the gut microbiome, we obtained normal healthy BALB/cByJ mice from a single source and aged male and female mice in two different geographical locations. A 16s rRNA microbiome analysis indicated that both age and sex play a role in microbiome composition; additionally, location plays a role in the diversity present as well. Interestingly, these microbiome changes occurred with changes in serum expression of several different pro-inflammatory cytokines including IL-10 and IL-6, which were also both differentially regulated in context to sex and aging. B-1a cells are known to produce mucosal IgA as well as IL-10 and IL-6, which play roles in regulating the microbiome. Furthermore, we found both IL-10 and IL-6 play a role in the constitutive expression of pSTAT-3 in B-1a cells. Together these results demonstrate sex and age shape the gut microbiome as well as systemic cytokine expression, which can influence, and be influenced by B-1a cells. In addition, location of housing can also influence the microbiome despite common animal sourcing. These results emphasize the importance of sex, age, and housing location in the mechanism of development of the gut microbiome.

Supported by NIAID of the NIH (R01AI154539)
I3C-MEDIATED PROTECTION AGAINST COLITIS DEPENDENT ON AHR EXPRESSION ON VIL1-EXPRESSING COLONIC EPITHELIAL CELLS
Authors: Saxena Archana , Mitchell Chandani , Wilson Keisha , Rutkovsky Alex , Wisniewski P J , Dopkins Nicholas , Nagarkatti Mitzi , Nagarkatti Prakash , Busbee Brandon
Published: January 2023

Many current treatments for ulcerative colitis (UC) often causes side effects warranting safe therapeutic strategies to control the pathogenesis of colitis. Current study was aimed to ascertain the possible role of aryl hydrocarbon receptor (AhR) in vil1-expressing colonic epithelial cells (CECs) during I3C-mediated protection against colitis. We investigated how intestinal regulatory mediators were altered in the absence or presence of AhR in CECs during I3C treatment under colitis or colitis-like conditions. We generated conditional AhR knockout mice in vil1-expressing CECs using the cre-flox system and induced colitis using the dextran sodium sulfate (DSS) model. Results showed that the mice with AhR deficiency in CECs (AV mice) lost the protective effects of I3C treatment during colitis and had a higher disease score with increased inflammation in the colon compared to the controls. Also, after treatment with I3C during DSS-induced colitis, AV mice were not able to prevent colitis-associated gut microbial dysbiosis even though flow cytometry analysis revealed AV mice were still capable of increasing IL-22 production by ILC3s in the colon. IL-22-ILC3 immune cell response was not the only major mechanism involved in I3C-mediated protection against colitis and regulation of the gut microbiome. Transcriptome analysis of RNA isolated from enriched CECs of experimental mice showed significant altered expression of several microRNAs, mucins (muc3 and muc13), and tight junction proteins in AV mice compared to controls. PCR gene expression data and direct effects of I3C on CECs using colonic organoids validated these results. In summary, AhR expression in CECs play a critical role in I3C-mediated prevention of colitis.
Ingestion of subtoxic doses of cadmium induces persistent dysbiosis and enhances susceptibility to DSS-induced colitis long after exposure
Authors: Kim Eunsoo , Cormet-Boyaka Estelle , Boyaka Prosper N
Published: January 2023

Health officials often focus on intoxication by high doses of heavy metal pollutants. However, most people will more likely be exposed to subtoxic doses via contaminated water or food. We addressed the effects exposure to subtoxic doses of heavy metal pollutants could have on the gut immune homeostasis. We report that chronic ingestion of environmentally relevant subtoxic doses of cadmium does not impair the barrier function of the intestine in mice. On the other hand, chronic exposure to subtoxic doses of this heavy metal stimulates the growth of intestinal epithelial cells including Paneth cells, and increases the production of antimicrobial peptides by Paneth cells. We also found that subtoxic doses of cadmium induce a dysbiosis of the gut microbiota that persist months after the end of the exposure. Importantly, 12–14 months after exposure to cadmium, the cadmium-exposed mice weighed more and had more fat content than age-matched controls mice suggesting that they have developed a state of chronic inflammation. Cadmium is also known to accumulate in soft tissues with a half-life of 10–20 years. Thus, to investigate the long-term effect of exposure to subtoxic doses of cadmium, control and post-menopausal mice (> 15 months old) previously exposed to cadmium were treated with dextran sodium sulfate (DSS) in drinking water. Post-menopausal mice previously exposed to cadmium developed more severe signs of DSS-induced colitis including weight loss, shortened large intestine. Furthermore, they exhibited enhanced Th17 responses in mucosal tissues. Our results provide new insights into the impact of chronic exposure to subtoxic doses of cadmium, and possibly other heavy metals on gut homeostasis and colitis or colon cancer.

Supported by Ohio State University NIH
A Prospectively Created Living Organoid Biobank of Crohn’s Disease Patients Enables Integrative Therapeutics
Authors: Katkar Gajanan Dattatray , Tindle Courtney , Fonseca Ayden , Taheri Sahar , Lee Jasper , Sayed Ibrahim M , Ibeawuchi Stella-Rita , Rama Pranadinata , Fuller Mackenzie , Stec Dominik , Anandachar Mahitha Shree , Goheen-Holland Vanae , Ear Jason , Boland Brigid , Sandborn William , Sahoo Debashis , Das Soumita , Ghosh Pradipta
Published: January 2023

Adult stem cells from various organs can be propagated as epithelial organoids that not only retain their tissue identity, but also their genetic and epigenetic disease-driving traits. We report the establishment of Crohn’s Disease (CD) patient-derived organoid cultures (PDOs) using biopsied tissues from colons of 50 consecutive subjects representing all clinical subtypes (Montreal Classification B1–B3 and perianal disease). Organoids were also generated from healthy subjects. The spectrum of phenotypic and genotypic changes within the “living biobank” agrees well with many observations reported in CD tissues. Gene expression analyses show that despite the heterogeneity of clinical subtypes, there are two major molecular subtypes, each with unique phenotypic features: immune-deficient infectious-CD [IDICD] and senescence-induced fibrostenotic-CD [SIFCD]. CD-PDOs are amenable to high-throughput drug screens allowing for the detection of phenotype reversal. As examples, defective barrier integrity in B1-PDOs was restored with pre-/pro-biotics, impaired microbial clearance in IDICD was reversed using agonists for nuclear receptors, and SIFCD was rectified using senolytics and - morphics. Phenotyped-genotyped CD-PDOs may fill the gap between basic biology and patient trials by enabling pre-clinical Phase ‘0’ human trials for personalized and integrative therapeutics.