bluuebunny/crossref_metadata_2025 huggingface dataset:
Developmental queues dictate IL-15 expression in γδ T cells (115.4)
Authors: Colpitts Sara , Stoklasek Thomas , Lefrançois Leo
Published: January 2023

Interleukin-15 (IL-15) is a homeostatic cytokine largely produced by myeloid cells. Surprisingly, analysis of a recently generated transgenic reporter mouse using emerald GFP expression as a surrogate for IL-15 promoter activity (EmGFP/IL-15) revealed unexpected expression in lymphoid γδ T cells. Similar to myeloid cells, in which EmGFP/IL-15 is developmentally regulated in the bone marrow, we found that EmGFP/IL-15 expression was downregulated as EmGFP/IL-15high thymic progenitors committed to a mature T cell fate. EmGFP/IL-15 was significantly reduced between DN2 and DN3 cells and silenced in DN4 and single positive thymocytes. However, γδ T cells and their precursors (CD25+ pre-γδ T cells) maintained low levels of EmGFP/IL-15, which was similar to DN3 cells. Interestingly, we found that intestinal intraepithelial γδ T cells were EmGFP/IL-15neg while a small subset of γδ T cells located in the peripheral lymph nodes expressed elevated levels of EmGFP/IL-15. These cells had a CD44highCD62LlowCD27lowCCR6+ phenotype, which corresponded to the Vγ2+ subset of IL-17-producing γδ T cells. These findings suggest that EmGFP/IL-15 is regulated concomitantly with the waves of γδ T cell development occurring perinatally and that transcriptional regulation at the IL-15 locus may overlap with that of IL-17-inducing machinery. Current studies aim to determine if the novel production of IL-15 by γδ T cells contributes to the overall homeostasis of IL-15-dependent cells in vivo.
Properties of TNFR2, PDL-1 dependent human CD8+ regulatory cells generated ex-vivo that can be strongly protective in vivo (116.4)
Authors: Horwitz David , Pan Stephanie , Wang Ju Hua , Lan Qin , Zheng Song Guo , Gray John Dixon
Published: January 2023

In addition to CD4+CD25+Foxp3+ Tregs, natural and induced CD8+ Tregs have been described. Here we report our progress in generating human CD8+ Tregs ex-vivo. Naïve CD8+ cells were activated with anti-CD3/28 beads with IL-2 ± TGF-β for 1 to 9 days. As with CD4+ cells, TGF-β markedly enhanced expression of Foxp3. TGF-β also enhanced CD122, CD103, and PD-1, but decreased granzymes, HLA-DR, CD80 and CD86 expression. Sustained Foxp3 expression and suppressive function was IL-2 dependent. Secondarily stimulated CD8+ cells proliferated weakly, and suppressed activation, proliferation and cytokine production by CD4+ cells in vitro. In vivo functional activity was assessed in NOD SCID IL-2R cγ chain-/- mice injected with allogeneic human PBMC. While human CD4+Foxp3+ Tregs doubled survival from GVHD, CD8 Regs, remarkably, quadrupled survival. Only previously activated CD8+ cells had suppressive activity and protection was unrelated to Foxp3 expression. Adding retinoic acid or rapamycin to TGF-β did not improve survival. By contrast, these activated CD8+ cells rapidly expressed TNFR2 and PDL-1 within one day, and at all time points studied only cells expressing these markers had suppressive activity. Suppressive activity in vivo was IL-10 dependent and partially TGF-β dependent. Thus, in agreement with functional CD8regs described in lupus, diabetes and multiple sclerosis, protective CD8 regs can be induced ex-vivo, and the therapeutic potential of TNFR2, PDL-1+ cells should be carefully explored.
Anti-CD52 therapy in experimental autoimmune encephalomyelitis (116.5)
Authors: Turner Mike , Chretien Nathalie , Havari Evis , LaMorte Michael , Roberts Bruce , Kaplan Johanne , Siders William
Published: January 2023

Alemtuzumab is an anti-CD52 humanized monoclonal antibody that alters the circulating lymphocyte pool and is under clinical investigation for relapsing-remitting multiple sclerosis. A recently developed depleting murine CD52 (muCD52)-specific antibody was evaluated in a standard mouse EAE model to expand our understanding of anti-CD52 therapy. C57BL/6 mice were immunized with MOG35-55 peptide to induce EAE, treated with anti-muCD52 antibody, and characterized with respect to development and severity of disease as well as mechanism of action of the antibody. Results from these studies demonstrated a profound impact of anti-muCD52 therapy. Specifically, prophylactic treatment effectively prevented the development of disease while therapeutic intervention inhibited disease progression and reversed existing symptoms. Longitudinal flow cytometric evaluation demonstrated a decrease in the number of MOG35-55 specific CD4+ T cells as determined by IFNγ intracellular cytokine staining. Simultaneous histological evaluation also showed a reduction in the level of lymphocytic infiltrate in the CNS of anti-muCD52 treated animals compared to control animals that received the vehicle. Collectively, these results demonstrated the therapeutic efficacy of anti-muCD52 treatment in association with a reduction in MOG-reactive pathogenic T cells and diminished lymphocyte infiltration in the CNS of mice with EAE.
CD14 deficiency enhances Th2 responses and alternative activation of macrophages in response to schistosome infection or IL-4. (117.10)
Authors: Tundup Smanla , Srivastava Leena , Nagy Tamas , Harn Donald
Published: January 2023

Prior to exposure to helminth antigens or helminth infection, the resting state of antigen presenting cells (APCs) plays a crucial role in their function in driving CD4+ Th2 immune bias. Whether or not APC innate receptors regulate alternative activation of APCs is not well understood. To test this, we injected schistosome eggs, or schistosome infected wild-type (WT) and CD14 -/- mice and examined evolution of Th2-type responses and alternative activation of macrophages. In response to schistosome infection, or exposure to schistosome eggs, CD14 negatively regulates Th2 responses and alternative activation of macrophages (AAMϕ). CD14-/- mice had enhanced production of CD4+ specific IL-4, IL-5 and IL-13 and increased numbers of regulatory T-cells. Livers of schistosome infected CD14-/- mice had elevated levels of AAMϕs, increased granuloma size with increased collagen deposition compared to WT counterparts. Q-PCR analysis showed significantly increased levels of Ym1, Fizz1 and Arg1 in livers of schistosome infected CD14-/- mice compared to WT controls. CD14 -/- mice injected with Lewis X glycoconjugate or IL-4 also showed significant increase in expansion of AAMϕs. Furthermore, in-vitro studies showed that enhanced alternative activation in response to IL-4 was independent of STAT-6 mediated signaling, suggesting that CD14 regulates both glycan and IL-4 induced alternative activation of macrophages through an unknown mechanism/pathway.
Macrophages and neutrophils in necrotic granulomas from cynomolgus macaques and humans localize to distinct microenvironments and express nitric oxide synthase and arginase enzymes. (117.11)
Authors: Mattila Joshua , Ojo Olabisi , Lin Philana , Flynn JoAnne
Published: January 2023

Macrophages are abundant in granulomas, the hallmark lesion of tuberculosis (TB), where they engage in activities critical for mycobacterial control. The assortment of macrophage functions, ranging from being the primary anti-mycobacterial effector cell to the primary mycobacterial host cell, underscores the complexity of macrophages in this system. Despite their importance, the molecular phenotypes and functions of granuloma macrophages in human TB are not well understood. In this study, we examined a variety of macrophage markers in non-human primate and human granulomas to better describe macrophage diversity and spatial organization. We identified three myeloid cell markers, including CD68, CD163, and HAM56, that stained populations of macrophages occupying discrete positions in necrotic granulomas that may be relevant to granuloma function. Neutrophils expressed high levels of calprotectin, a small bacteriostatic protein sometimes associated with macrophages, and also localized to specific positions in necrotic granulomas. In addition to phenotypic markers, we identified cell-specific expression of nitric oxide synthase isoforms (iNOS and eNOS) and arginase isoforms (arg1 and arg2) expression in granulomas by biochemical, molecular and immunohistochemical techniques. Both macrophages and neutrophils were determined to express NOS and arg enzymes, including co-expression, suggesting these enzymes with opposing effects act in concert to maintain mycobacterial control.
Understanding the role of FTT0831c encoded protein of F. tularensis in modulation of macrophage function (117.15)
Authors: Malik Meenakshi , Schiewe Rachel , Mora Vanessa , Atianand Maninjay , Harton Jonathan , Bakshi Chandra Shekhar
Published: January 2023

Francisella tularensis (Ft), a category A select agent, cripples innate immune defenses of the host to create an environment permissive for its growth. The objective of this study was to understand the mechanisms of innate immune evasion by Ft LVS (Type B) and the highly virulent SchuS4 (Type A) strain. We report that proteins encoded by FTL_0325/FTT0831c genes of Ft LVS and SchuS4 strains respectively, are required for intramacrophage survival, virulence and suppression of proinflammatory cytokines. Our in vitro studies showed that over-expression of FTT0831c suppressed the canonical NF-κB pathway by inhibiting nuclear translocation of the p65 protein resulting in blockade of NF-κB-dependent activation of proinflammatory cytokines. Further studies showed that FTL_0325/FTT0831c gene products are also required for suppression of caspase-1 activation and secretion of IL-1β and IL-18. In conclusion, this study establishes FTL_0325/FTT0831c of Francisella as a key virulence factor that functions as immunosuppressant to facilitate bacterial persistence. Suppression of key signaling pathways via FTT0831c by virulent F. tularensis SchuS4 explains one of the mechanisms adopted by this pathogen to down-modulate the host’s innate immune response.
Anti-inflammatory role of IL-10 producing macrophages during early Brucella abortus infection (117.20)
Authors: Xavier Mariana , Winter Maria , Atluri Vidya , Godinez Ivan , Silva Teane , Barton Greg , Santos Renato , Tsolis Renee
Published: January 2023

Chronic bacterial diseases such as Brucellosis may result from the pathogen’s ability to evade the initial immune response. Moreover, since Brucella spp. is known to target phagocytes, we investigated its effects on macrophage (mø) phenotype and induction of anti-inflammatory responses. Brucella did not induce alternative activation of mø in cultured bone marrow-derived mø (BMM) or in splenic CD11b+ cells isolated from mice during early infection. However, upregulation of interleukin 10 (IL-10) in serum, spleen and splenic mø from B. abortus infected mice was observed as early as 3 days post infection. Infection of BMM from MyD88-/-, TLR2-/-, TLR4-/-, TLR2/4-/- and TLR9-/- mice demonstrated that IL-10 induction was MyD88 dependent and was partially dependent on pathogen recognition by TLR2 and TLR4. Functionally, in vitro IL-10-/- BMM infection resulted in significantly higher pro-inflammatory cytokine production and decreased bacterial intracellular survival. In vivo 9 days infection of IL-10-/- mice confirmed a lower ability of B. abortus to survive in absence of IL-10, as well as higher induction of pro-inflammatory cytokines, resulting in increased pathology in liver and spleen of infected mice. Taken together, our results suggest that early IL-10 induction by infected mø contributes to an initial balance between pro-inflammatory and anti-inflammatory cytokines that is beneficial to the pathogen, thereby leading to enhanced bacterial survival and persistent infection.
Rapid induction of lipid mediators is a novel effector function of the inflammasome in vivo (117.24)
Authors: von Moltke Jakob , Moayeri Mahtab , Trinidad Norver , Kintzer Alexander , Wang Samantha , van Rooijen Nico , Krantz Bryan , Leppla Stephen , Gronert Karsten , Vance Russell
Published: January 2023

Detection of microbial products by host inflammasomes is critical for innate immune surveillance, while eicosanoids, signaling lipids that include the prostaglandins and leukotrienes, are rapid triggers of inflammation. Here we report for the first time a link between inflammasome activation and eicosanoid synthesis. Using a novel protein toxin, we show that systemic cytosolic delivery of bacterial flagellin in vivo leads to loss of vascular fluid into the intestine and peritoneal cavity, resulting in rapid (<30 min) death in mice. This unexpected response is dependent on the inflammasome components NAIP5, NLRC4, and CASP1, but interestingly, is independent of the known inflammasome effector functions, namely IL-1β/-18 production and pyroptotic cell death. Instead, inflammasome activation in resident peritoneal macrophages results, within minutes, in the release of prostaglandins and other eicosanoids that induce vascular fluid loss. Mice deficient in cyclooxygenase-1, a critical enzyme in prostaglandin biosynthesis, are resistant to the pathological effects of systemic inflammasome activation. Compared to other macrophage populations, resident peritoneal macrophages are specifically primed for inflammasome-dependent production of eicosanoids by high expression of Cox1 and other eicosanoid biosynthetic enzymes. Thus, our results identify eicosanoid production as a novel cell type-specific effector function of the inflammasome with dramatic physiological consequences in vivo.
CASPASE-1 SIGNALING IS REQUIRED FOR CONTROL OF TRYPANOSOMA CRUZI INFECTION IN VIVO. (117.27)
Authors: Da Silva Grace , Silveira Tatiana , Sesti-Costa Renata , Gutierrez Fredy , Guedes Paulo , Caetano Braulia , Gazzinelli Ricardo , Zamboni Dario , Silva João
Published: January 2023

The Toll-like receptors (TLRs), 2, 4 and 9 are important to immunity against Trypanosoma cruzi, etiologic agent of Chagas disease. A new family of pattern recognition receptors, Nod-like receptors (NLRs) is described, including NLRP3, which associates to ASC forming a complex that active caspase-1, called inflammasome. This complex is required for cleavage of the active forms of the IL-1β and IL-18. Here, we investigated the role of caspase-1 in the immune response against T. cruzi. First we verify if T. cruzi triggers caspase-1 activation in murine bone marrow-derived macrophages (BMMs). We found high caspase-1 activation in BMMs from WT mice, but not from ASC-/- mice. The blockage of potassium efflux, oxygen radicals reactive (ROS) and B catahepsin (activators of caspase-1 via ASC) resulted in inhibition of caspase-1 activation in BMMs from WT mice. To understand the role of caspase-1, WT, caspase-1-/- and ASC-/- mice were infected with Y strain of T.cruzi. Our results showed that ASC-/- and caspase-1-/- mice presented higher mortality, whereas WT are resistant to infection. In addition, caspase-1-/- mice presented higher myocarditis, increased cardiac damages and more parasites in this tissue on day 17 p.i. Together, these results suggest that caspase-1 dependent of ASC is crucial to infection against T. cruzi. These results reveal new mechanisms involved in the innate immunity against this parasite, which could contribute to new approaches in the control of this disease.
γ-Ray-associated XBP-1+ macrophages as an inducer of M2 macrophages (117.3)
Authors: Kogiso Mari , Nakamura Kiwamu , Kita Katsuhiro , Cornforth Michael , Kobayashi Makiko , Suzuki Fujio
Published: January 2023

The susceptibility of mice to infections with opportunistic pathogens is strongly influenced by M2MΦ appeared in association with whole body γ-irradiation. Therefore, in this study, we investigated why M2MΦ were predominantly generated in mice exposed to whole body γ-irradiation. F4/80+ MΦ were isolated from peritoneal exudates of mice 6-24 h after 5 Gy of γ-irradiation. The expressions of XBP-1 in these MΦ preparations were detected by Western Blot. The production and intracellular CCL2 expression by XBP-1+ MΦ were assayed by ELISA and flow cytometry. In some experiments, resident MΦ were cultured with media supplemented with culture fluids of XBP-1+ MΦ (33%, v/v). MΦ with the ability to produce IL-10 and not produce IL-12 were considered as M2MΦ. In the results, XBP-1+ MΦ were demonstrated in peritoneal MΦ preparations isolated from mice irradiated with 5 Gy of γ-rays. CCL2 was detected in culture fluids of XBP-1+ MΦ. Also, intracellular CCL2 was detected in XBP-1+ MΦ by flow cytometry. The conversion of MΦ from resident MΦ to M2MΦ was demonstrated when resident MΦ were cultured with media supplemented with the culture fluids of XBP-1+ MΦ. Resident MΦ did not change to M2MΦ when they were cultured with XBP-1+ MΦ culture fluids previously depleted of CCL2. These results indicate that the MΦ conversion from resident MΦ to M2MΦ is stimulated by CCL2 released from XBP-1+ MΦ that appear in association with whole body γ-irradiation.
Suppression of the NF-κB Pathway by Diesel Exhaust Particles Impairs Human Antimycobacterial Immunity (117.4)
Authors: Schwander Stephan , Sarkar Srijata , Song Youngmia , Sarkar Somak , Kipen Howard , Laumbach Robert , Zhang Junfeng , Ohman-Strickland Pamela , Gardner Carol
Published: January 2023

Chronic exposure to air pollution increases susceptibility to respiratory infections including tuberculosis in humans. We hypothesized that exposure to diesel exhaust particles (DEP), a major component of urban fine particulate matter, suppresses antimycobacterial human immune effector cell functions by modulating TLR-signaling pathways and NF-κB activation. To examine the effects of DEP on M.tb-induced host immunity, PBMC from 20 healthy persons [13 m, 7 f, mean age 33.5 years) were stimulated with DEP and M.tb or PPD (purified protein derivative). We show by TEM studies that both DEP and H37Ra, an avirulent laboratory strain of M.tb, were taken up by the same peripheral CD14+CD3- blood monocytes. M.tb and PPD-induced IFN-γ, TNF-α, IL-1β, and IL-6 production was reduced in a DEP dose-dependent manner. In contrast, the production of anti-inflammatory IL-10 remained unchanged. DEP stimulation prior to M.tb infection altered the expression of mRNAs encoding TLR 3, 4, 5, 7 and 10 and a subset of M.tb-induced host genes including the inhibition of many NF-κB and IRF pathway target genes. We propose that DEP down-regulate M.tb-induced host gene expression via MyD88-dependent (IL6, IL1A, PTGS2) and MyD88-independent (IFNA, IFNB) pathways. Pre-stimulation of PBMC with DEP suppressed the expression of proinflammatory mediators upon M.tb infection inducing a hypo-responsive cellular state. These findings provide possible mechanism by which air pollutants alter antimicrobial immunity.
Slamf1 controls monocyte / macrophage migration in experimental colitis. (117.5)
Authors: van Driel Boaz , Liao Gongxian , Romero Javier , Faubion William , Wang Guoxing , Berger Scott , O'Keeffe Michael , Magelky Erica , Manocha Monika , Azcutia Veronica , de Waal Malefyt Rene , Grisham Matthew , Luscinskas F. , Mizoguchi Emiko , Bhan Atul , Wang Ninghai , Terhorst Cox
Published: January 2023

Signaling lymphocyte activation molecule (Slamf1) is a T cell co-stimulatory molecule and a regulator of cytokine production by macrophages and dendritic cells. Because Slamf1 positively regulates microbicidal mechanisms in macrophages, we evaluate whether Slamf1 would affect enterocolitis. To induce colitis, mouse CD45RBhi CD4+ T cells were transferred into RAG-/- or Slamf1-/- RAG-/- recipient mice. In a second set of experiments, an agonistic αCD40 antibody was administered to the same recipient strains to induce colitis. Whilst the absence of Slamf1 in RAG-/- recipients mitigated colitis, the function of disease-causing effector or regulatory T cells was not affected by the ablation of the receptor. Surprisingly, in Slamf1-/- mice monocyte / macrophage migration into the inflamed tissue was impaired not only in αCD40-induced colitis but also in two other in vivo models of inflammation: thioglycolate induced peritonitis and in an “air pouch” model in response to TNFα. Administering αSlamf1 to RAG-/- mice ameliorated enterocolitis and altered this migration. Slamf1 is a key contributor to the innate immune responses during experimental colitis by affecting the migration of monocytes / macrophages to the sites of inflammation. As αSlamf1 also mitigates the pathogenesis of colitis, SLAMF1 should be a plausible therapeutic target in treating inflammatory bowel diseases.
Kupffer cells regulate the biological response of inflammatory mononuclear phagocytes to biliary obstruction in mice. (117.6)
Authors: Duwaerts Caroline , Gehring Stephan , Van Rooijen Nico , Gregory Stephen
Published: January 2023

Background: Biliary obstruction is a serious complication of liver diseases. Most studies fail to differentiate the contribution of resident hepatic macrophages (Kupffer cells, KC) and inflammatory mononuclear phagocytes (iMNP) that infiltrate the liver subsequent to injury. This study was undertaken to examine the distinct roles and interactions of these mononuclear phagocyte populations in liver injury attending cholestasis. Methods: Female, C57Bl/6 mice were injected with magnetic beads 3 days prior to sham operation or bile duct ligation (BDL) to facilitate subsequent KC isolation. Three days post-surgery, bead-containing KC and iMNP were separated, purified and analyzed. To examine the ability of KC to modulate iMNP activity, iMNP were isolated from intact and KC-depleted livers on day 3 post-surgery and compared. Results: Following BDL, iMNP exhibited increased expression of Ly-6C and elevated production of chemokines/ cytokines compared to KC. In the absence of KC, iMNP exhibited significant decreases in Ly-6C expression and inflammatory chemokine/cytokine production. Conclusions: KC play a key role in regulating the influx and biological response of iMNP to biliary obstruction and cholestasis.
Recovery of the impaired production of alpha-defensins (HNPs) in cultures of burn patient peripheral blood neutrophils treated with glycyrrhizin (GL) (118.12)
Authors: Yoshida Shohei , Lee Jong , Okita Yamato , Kobayashi Makiko , Herndon David , Suzuki Fujio
Published: January 2023

HNPs produced by neutrophils at the bacterial translocation site play an important role on controlling gut bacteria-associated infections. However, these peptides were not produced by burn patient neutrophils. CCL2 released from burn patient neutrophils was shown to be inhibitory on HNP production. In this study, we tried to recover the impaired production of HNPs in cultures of neutrophils isolated from severely burned patients. Neutrophils (1 x 106 cells/ml), isolated from peripheral blood of severely burned patients and healthy donors, were cultured in media supplemented with burn patient sera (10% v/v) and/or various doses of GL (Minophagen Pharmaceutical Co., Ltd., Tokyo, Japan). Culture fluids were harvested 18 hrs after cultivation and assayed for HNPs by ELISA. In the results, a 28 to 37 ng/ml of HNPs was detected in culture media of healthy donor neutrophils, while these peptides were not produced by burn patient neutrophils. However, HNPs were detected in cultures of burn patient neutrophils treated with 10 to 100 μg/ml of GL. HNPs were not produced by healthy donor neutrophils in cultures supplemented with burn patient sera, and CCL2 was detected in these sera. This chemokine was shown to be inhibitory on HNP production by healthy donor neutrophils. CCL2 was not produced by burn patient neutrophils treated with GL. These results indicate that GL improves HNP production by burn patient neutrophils through the control of CCL2 production.
SerpinB1 is essential for regulation of neutrophil extracellular trap (NET) generation (118.3)
Authors: Farley Kalamo , Stolley James , Zhao Picheng , Cooley Jessica , Remold-O'Donnell Eileen
Published: January 2023

NETosis, a programmed death pathway induced in mature neutrophils by pathogens and inflammatory mediators, can be either a protective process that prevents spread of microbial infection or a pathological process that causes severe tissue damage and inflammation. Little is known about the mechanisms that regulate such a diverse functional spectrum. Previously we demonstrated serpinB1 null mice are highly susceptible to pulmonary Pseudomonas aeruginosa infection due to increased pathology mediated neutrophilic death. Using in vitro and in vivo approaches we examine the regulatory role of SerpinB1 in NETosis. We found bone marrow or LPS-derived serpinB1 null neutrophils are remarkably prone to NETosis in both NADPH-dependent and independent manner. A regulatory role for SerpinB1 in NETosis is also supported by the increased number of neutrophils with expanded nuclei, likely representing chromatin decondensation, and by finding that exogenous rSERPINB1 abrogates NET production. Importantly, during P. aeruginosa lung infection 4 times more NETs were harvested from serpinb1-/- mice than WT mice. The total number of in vitro activated serpinB1-/- neutrophils that died was not different from wild-type, indicating that the primary protective role of SerpinB1 is determining whether activated neutrophils die by the highly injurious NETs mechanism or by a less phlogistic mode, generating apoptotic-like cells with shrunken appearance and small nuclei.
Treatment of Atopic Dermatitis by Bone Marrow-derived Clonal Mesenchymal Stem Cells (119.3)
Authors: Jeon Myung-Shin , Na Kwangmin , Yoo Hyun Seung , Choi Mi-Sook , Lee Hyeon Sook , Kim Hyo Jin , Lim Hyun-Ja , Yi TacGhee , Choi Gwang Seung , Song Sun U
Published: January 2023

Mesenchymal stem cells (MSCs) can perform immunomodulatory activities, including suppression of T- and B-cell proliferation. MSCs have been primarily studied for their suppression of pro-inflammatory Th1 responses, but their therapeutic effects on atopic dermatitis (AD), which is a Th2-dominant disease, have not been reported. In this study, using an ovalbumin-induced mouse AD model, we investigated whether MSCs reduce the allergic inflammation in AD. We used mouse bone marrow-derived clonal mesenchymal stem cells (mcMSCs) isolated by our newly established isolation protocol termed the subfractionation culturing method (SCM). When injected intravenously into animals between antigen challenges, mcMSCs were found to inhibit the majority of AD-specific pathological changes by decreasing cell infiltration in skin lesions and reducing serum level of IgE. To explore the mechanism of this effect, we investigated the effects of mcMSCs on IgE production in B cells. We found that mcMSCs significantly inhibited IgE production when B cells were stimulated either by lipopolysaccharide (LPS) or anti-CD40 antibodies in the presence of interleukin (IL)-4. In addition, allogeneic mcMSCs can inhibit both isotype class switching and terminal differentiation of B cells probably by inhibiting NF-κB activation. These results suggest that mcMSC-based cell therapy is a promising therapeutic modality for AD treatment.
Context dependent differentiation of resident and inflammatory intestinal macrophages from a common monocyte precursor (120.1)
Authors: Mowat Allan , Scott Charlotte , Bain Calum
Published: January 2023

Macrophages (mϕ) are essential for homeostasis and protective immunity in the intestine, but also drive pathology in inflammatory bowel disease. It is not clear whether these different functions are due to distinct populations of mϕ, or if the same cell change. We show here that two populations of mϕ exist within the mouse colon, based on the levels of CX3CR1 expression. Most resting mϕ express CX3CR1 at much higher levels than any other tissue mϕ and also express CD11c. These resident mϕ produce a balanced mixture of TNFα and IL10 constitutively, but respond poorly to stimulation via TLR, despite expressing all TLR. During acute DSS colitis, mϕ expressing lower levels of CX3CR1 come to dominate. These “inflammatory” mϕ produce TNFα predominantly and respond via TLR. Using a combination of gene profiling, immunophenotyping and adoptive transfer models, we show that CX3CR1int mϕ in both healthy and inflamed intestine are derived from Ly6Chi CCR2+ monocytes that upregulate CX3CR1 after entering the intestine, sequentially acquiring F4/80 and class II MHC expression. Under resting but not inflammatory conditions, the newly arrived monocytes differentiate further, becoming CX3CR1hi, producing IL10, expressing CD163 and CD206 and becoming TLR unresponsive. These results provide novel evidence that “resident” and “inflammatory” mϕ in the intestine derive from the same monocyte precursor whose ultimate fate is determined locally and depends on the presence or absence of inflammation
Retinoic acid is a specific IgA isotype switching factor (120.11)
Authors: Kim Pyeung-Hyeun , Seo Goo-Young , Kim Hyun-A , Lee Mi-Ra , Park Mi-Hee
Published: January 2023

Retinoic acid (RA) is known to have several activities which lead to potent mucosal IgA response. In the present study, we obtained several evidences that RA can act as a specific IgA isotype switching factor. RA, though less effective than TGF-β1, significantly increased IgA secretion and Ig germ-line α transcripts (GLTα) by LPS-activated mouse spleen B cells, while concurrently decreasing other isotypes secretion and GL transcripts. Based on limiting dilution analysis, RA increased the frequency of IgA secreting B cell clones by two-fold. This was not accompanied by increased numbers of IgA secreting cells/clone. Further, RA increased the activity of GLα promoter reporter by 3-fold and this was further increased by TGF-β1. Herein, RA-induced GLα expression was diminished by a TGF-β receptor I inhibitor (SB431542) and overexpression of DN-Smad3. Nevertheless, site-directed mutagenesis revealed that putative Smad3 binding elements (SBEs) are not relevant to RA-inducible promoter activity. Interestingly, RA-induced GLα transcription and IgA secretion were virtually disappeared by LE540, a RA receptor (RAR) antagonist. In addition, p38 inhibitor (SB203580) and ERK inhibitor (PD98059) diminished RA-induced GLα transcription and IgA secretion. In addition, we found that RA can induce p38MAPK/ERK phosphorylation. Taken together, these results suggest that RA induces GLα transcription through diverse pathways including RAR, Smad3, and p38MAPK/ERK, leading to IgA isotype switching.
Alcohol consumption is associated with tissue-specific alterations of myeloid-derived suppressor cells. (120.19)
Authors: Brand Rhonda , Stottlemyer J. Mark , Falo Louis
Published: January 2023

Alcohol consumption results in defects in immune function. The role of myeloid-derived suppressor cells (MDSC) and regulatory T-cells (Treg) in suppression of immune responses has been established. MDSCs in particular have been implicated in the suppression of both adaptive and innate immune responses. To develop a better understanding of the mechanisms by which alcohol effects immune function, we evaluated the effects of alcohol suppression specifically on MDSC and Treg populations. Previous studies suggest that ethanol consumption can result in tissue-specific inhibition of the antigen presentation function of dendritic cells and other APC populations. We used the Lieber-DeCarli liquid ethanol diet feeding model, which generates oxidative stress and liver steatosis, to examine the effect of alcohol consumption on MDSC and regulatory T cell populations in the spleen and serum. We found that the Lieber-DeCarli liquid ethanol diet had no effect on either circulating or splenic Treg populations, a result consistent with previous reports using a non-liver damaging ethanol feeding model (Meadows-Cook diet). On the other hand, we observed a specific increase in splenic MDSCs after ethanol exposure. These results suggest that alcohol ingestion induces regional changes in MSDC populations that may correlate with observed regional immune effects.
Monocyte-derived inflammatory dendritic cells expressing CX3CR1hi control urinary tract infection in mice. (120.25)
Authors: Partida-Sanchez Santiago , Horvath Dennis , Cortado Hanna , Li Birong , Flaño Emilio , Justice Sheryl
Published: January 2023

Phagocytes are essential for protecting the urinary tract from bacterial infections. Although neutrophils are required for clearing of bacteria during urinary tract infection (UTI), the function of monocyte-derived macrophages and dendritic cells (DCs) is less clear. To determine the role of monocyte-derived phagocytes, we used CD11b-DTRTg mice and selectively depleted CD11b+ blood monocytes prior to inoculation of uropathogenic Escherichia coli (UPEC) into the bladder. We then monitored monocyte-derived macrophages and DCs during the course of UTI. We found that conditional CD11b ablation effectively depleted a population of CXC3CR1+high, but not CX3CR1+low cells in the bladder and kidney. Interestingly, CX3CR1+high cells expressed low levels of F4/80 but expressed CD11c, and other DC markers, suggesting they were monocyte-derived DCs. UPEC induced bacterial burden was significantly elevated in the kidneys of CD11b-depleted mice at 48 hrs post-infection, when compared to controls. Furthermore, CX3CR1 KO mice were unable to clear UPEC 48 hrs post-infection; instead, bacteria spread from the bladder to the kidney, similarly to CD11b monocyte depleted mice. CX3CR1+high DCs, but not CX3CR1+low macrophages isolated from infected bladders produced abundant amounts of IL-6 and TNF-α pro-inflammatory cytokines. Our studies indicate that a monocyte-derived novel DC subset expressing CX3CR1 is required for preventing bacterial spreading, and the clearance of bacteria in the kidney.
Hsp90 and autoantibodies expression in periodontal disease (120.29)
Authors: Aguilera-Galaviz Luis , Hernandez-Montoya Martha , Lopez Luna Maria , Garcia Iris , Aceves-Medina Maria
Published: January 2023

In Periodontal disease (PD)several studies suggest, that exaggerated host immune and inflammatory response is considered as a potential risk for developing cardiovascular diseases, alter the course of diabetes, osteoporosis and premature babies during pregnancy. Objetives: the baseline of this study was to detect the presence of auto-antibodies and Hsp90 expression in healthy and systemic compromised patient. Methods: 32 patients with periodontitis were evaluated and 20 healthy people, blood sample to set up IFI for ANCA, antiepithelial antibodies, ANA and WB to determine the molecular profile using keratinocyte extract RT-PCR for Hsp 90 was performed in periodontal tissue. Results: 57% were positives for ANCA, the total of sera were positives for nuclear and/or cytoplasmic antibodies and 57% presented anti-epithelial antibodies, the sera from these patients recognized proteins in a wide range of molecular weight keratinocytes extract, and 30 and 70 kD in mouse skin extract, Hsp90 expression was considerably highest in PD patient and systemic compromise. Conclusions: The immune response in periodontal disease is influenced by the microflora, the production of antibodies against self structures, could be involved in the pathogenic mechanism; these autoantibodies are associated with necrotic or apoptotic phenomena. Metabolic activity of microorganism should be involved in cross reactions and antigen procesesing in situ could stimulate self reactive lymphocytes.
GITR on the surface of antigen presenting cells, but not on T cells, regulates the pathogenesis of CD4+ T cell-mediated experimental colitis (120.3)
Authors: Liao Gongxian , Berger Scott , Detre Cynthia , de Waal Malefyt Rene , Herzog Roland , Bhan Atul , Terhorst Cox
Published: January 2023

The receptor GITR is thought to function on the surface of regulatory and activated effector CD4+ T cells. To better understand the role of GITR in in vivo immune responses, we use an established experimental colitis model. Chronic enterocolitis was induced by the transfer of wt or GITR−/− CD4+ T cells into either GITR−/− x Rag−/− or Rag−/− recipients. When non-fractionated CD4+ cells from either wt or GITR−/− donors were transferred, colitis unexpectedly developed in GITR−/− x Rag−/−, but not in Rag−/− recipients. In these mice, the percentage of Treg and Th17 cells is decreased, whilst that of Th1 cells increased. Furthermore, Tregs fail to prevent colitis in GITR−/− x Rag−/− recipients. This is not due to an aberrant function of GITR−/− Treg or Teff cells, but is caused by an imbalance in the number of tolerogenic CD103+ and PDCA1+ plasmacytoid dendritic cells in the GITR−/− mouse. This in turn impairs Treg development and expands the Th1 population in GITR−/− x Rag−/− recipients upon the transfer of non-fractionated CD4+ cells. We conclude that for the induction of colitis, GITR is dispensable on the surface of Treg and Teff cells, nor do GITR-L / GITR interactions on T cells play a role in controlling the disease. GITR, however, controls in vivo DC and monocyte development and in its absence aggravated chronic enterocolitis is caused by an imbalance of colitogenic Th1 cells and regulatory T cells.
Tonsil-derived mesenchymal progenitor cells acquire a follicular dendritic cell phenotype under cytokine stimulation (120.34)
Authors: Woo So-Youn , Kim Ji-Yon , Cho Kyung-Ah , Kim Han Su , Ryu Kyung-Ha
Published: January 2023

Tonsils comprise part of the mucosal immune system and contain lymphocytes, macrophages, and follicular dendritic cells (FDCs). FDCs are located in the B cell area of the follicles of secondary lymphoid organs, such as the spleen, tonsils, or lymph nodes, and they trap and retain immune complexes on their surfaces to regulate B cell activation and maturation. Stromal cells from the palatine tonsils are often used for FDC in vitro studies, and it has been reported that human palatine tonsils may be a good source of multipotent mesenchymal cells. Therefore, we assessed whether tonsil-derived mesenchymal stromal cells could differentiate into a FDC-like phenotype. We discovered that stromal cells isolated from human tonsils not only had the potential to differentiate into various cell types of mesenchymal origin, but they also could differentiate into FDC-like cells under cytokine stimulation in vitro.
Regenerating islet-derived 3-gamma regulates pulmonary Th17 immunity by altering the gut microbiome (120.37)
Authors: McAleer Jeremy , Choi Sun Mi , Chen Kong , Binnie Matthew , Sheppard Dean , Qin Shulin , Reinhart Todd , Davila Eduardo , Hooper Lora , Ouyang Wenjun , Kolls Jay
Published: January 2023

Regenerating islet-derived 3-gamma (Reg3g) is a 17kDa lectin produced by intestinal paneth cells and has bactericidal activity against gram-positive organisms. Here, we find that lung infection with Staphylococcus aureus induces the expression of reg3g and reg3b in a MyD88-dependent manner. Expression of reg3b in the lung was localized to large airway cells, and increased following treatment with IL-17 and IL-22. Further, recombinant Reg3g had strong binding to S. aureus and growth inhibition in vitro. In vivo studies indicated that reg3g-/- mice were capable of clearing S. aureus infection, and produced significantly more IL-17 than control B6 mice. This was associated with increased segmented filamentous bacteria colonization in the gut of Reg3g-/- mice. To test the hypothesis that Reg3g regulates lung Th17 immunity by altering the gut microbiome, mice were treated with antibiotics prior to immunization with ovalbumin plus cholera toxin. Antibiotic treatment reduced lung Th17 numbers in Reg3g-/- mice to the level observed in B6 mice. Finally, gut vaccination with heat-killed Klebsiella pneumoniae increased Th17 cytokine expression in the lung and protected against live challenge, demonstrating the intestinal niche can augment pulmonary immunity. These data demonstrate dual roles for Reg3g in host defense: 1) direct anti-microbial activity against S. aureus, and 2) decreasing SFB levels in the gut, which could negatively impact Th17-mediated host defense.
Increased localization of Regulatory T cells in Hilar Lymph Node during the resolution of an ovalbumin induced - murine model of Allergic Airway Disease (120.38)
Authors: Natarajan Prabitha , Guernsey Linda , Secor Eric , McNamara Jeffery , Schramm Craig , Thrall Roger
Published: January 2023

In our biphasic ovalbumin (OVA)-induced allergic airway disease (AAD) model, short-term exposure of mice for 7 to 14 days to OVA via aerosol results in AAD, whereas continued daily exposure for 42 days leads to resolution of disease and development of local inhalational tolerance (LIT). In our earlier studies, we have shown from adoptive transfers that hilar lymph node (HLN) B cells isolated from LIT mice suppress development of AAD via TGF-β expression and had regulatory function. We also showed, in-vitro, that these cells induced conversion of effector T cells (Teffs) to regulatory T cells (Tregs) in a TGF-β dependent manner. In the present study, we investigated the association of Tregs and B cells within HLN of AAD and LIT mice. We demonstrated a significant increase in number of Tregs localized in B cell zones and T-B border in HLN of mice at LIT as compared to AAD qualitatively from confocal images. Quantification of Tregs using Imaris software in HLN yielded similar results. We studied the expression of chemokine receptors: CXCR4 and CXCR5 on Tregs via flow cytometry and showed a significant increase in number of Tregs expressing CXCR4 and CXCR5 in HLN from mice at AAD and LIT as compared to naive and sensitized groups. This observation correlated with the results obtained using confocal microscopy. Hence, we conclude that HLN regulatory B cells directly interact with Teffs converting them into Tregs in the B cell zones and contribute to the resolution of disease.
Inducible nitric oxide synthase inhibition protects mice from chlorine-induced airway dysfunction and inflammation. (120.39)
Authors: O'Koren Emily , Potts Erin , Foster W. , Gunn Michael
Published: January 2023

Inhalation of toxic chlorine (Cl2) gas causes an oxidative injury to the upper airways that results in epithelial damage, elevated airway hyperreactivity (AHR), and hypoxia in both humans and mice. We hypothesize that activated resident immune cells, specifically alveolar macrophages, respond to Cl2 gas injury by releasing effector molecules like TNF-alpa and iNOS, and that blocking the excess buildup of harmful effector molecules after injury will decrease inflammation and improve overall lung function. We targeted iNOS, an inflammation-induced enzyme that catalyzes the release of nitric oxide (NO) from L-arginine. We observed that Cl2-exposed iNOS-/- mice were completely protected from elevated AHR and hypoxia and exhibited decreased cytokine inflammation, BAL inflammatory infiltrates, and alveolar leakage. We then evaluated the effect of a small molecule inhibitor of iNOS, 1400w, on lung function and inflammation after Cl2 exposure. Mice treated with 1400w, starting 3hr post Cl2 exposure, exhibited the same protected responses as iNOS-/- deficient mice. AHR was decreased by 98%; arterial blood oxygen saturation levels were almost at normal levels, and pro-inflammatory cytokine levels, alveolar leakage, and BAL inflammatory infiltrate numbers were approximately reduced by half compared to PBS-treated Cl2-exposed control mice. Our findings demonstrate that iNOS inhibition is a promising therapeutic target for Cl2 injury.
Commensal bacteria regulate Treg abundance in a gnotobiotic model of food allergy. (120.6)
Authors: Feehley Taylor , Stefka Andrew , Tripathi Prabhanshu , Antonopoulos Dionysios , Chang Eugene , Theriault Betty , Nagler Cathryn
Published: January 2023

Increasing evidence points to a central role for commensal bacteria in the regulation of immune-mediated disease. We postulated that microbiota-derived signals govern susceptibility to allergic responses to food. We find that CD4+Foxp3+Tregs are significantly reduced in the colonic lamina propria (cLP) (but not the spleen or MLN) of mice that have been rederived germ free. Peanut specific IgE and IgG1 responses are significantly increased in response to intragastric sensitization of germ free mice. Depletion of the microbiota by treatment with an antibiotic cocktail also alters the response to allergic sensitization; increased Bacteroidetes (Bacteroideaceae) and reduced Firmicutes (Lachnospiraceae) correlate with the depletion of colonic Tregs and susceptibility to allergic sensitization in antibiotic-treated mice. We used selective media to culture bacteria representative of each of these families from the feces of SPF mice to examine their influence on allergic susceptibility. We hypothesized that certain bacterial families promote Treg induction, increasing protection against allergy. We find that Tregs are not induced in mice monocolonized with Bacteroides uniformis; sensitization of these mice results in PN specific IgE and IgG1 responses similar to germ free mice. Ongoing experiments are examining whether dual colonization with Lachnospiraceae (Coprococcus spp) restores the Treg compartment in the cLP and/or if Treg transfer protects against allergic sensitization.
Identifying the function of Notch1 in T-cell activation (121.1)
Authors: Dongre Anushka , Osborne Barbara
Published: January 2023

The Notch receptor is activated by ligands that belong to the Delta or Jagged family. Ligand binding is followed by proteolytic cleavages resulting in the release of intra-cellular Notch which translocates to the nucleus and functions as a transcriptional activator. While the role of Notch in thymocyte maturation has been studied, the precise role of Notch in T-cell activation and cellular metabolism remains incompletely understood. Evidence from our lab has suggested that TCR mediated activation leads to the generation of active Notch and inhibition via Gamma Secretase Inhibitors (GSI) decreases T cell activation and proliferation. However, since GSI’s are known to have multiple substrates, whether such a decrease in activation is indeed dependent on Notch or is simply a GSI mediated effect needs to be examined. To determine the role of Notch in T cell activation we conditionally deleted Notch1 in peripheral T cells. Our data demonstrate that CD4 cells have reduced expression of activation markers accompanied by a decrease in IL-2 and IFN-Gamma secretion as well as impaired proliferation in the absence of Notch1. Notch1 was also required for polarization towards Th1. Furthermore, absence of Notch1 decreased pAkt-S473 implying reduced mTORC2 activity. Our data implicate the importance of Notch1 in T cell function. Future studies are aimed to delineate the role of non-canonical Notch1 signaling in T cells and how it may influence molecular pathways downstream of the TCR.
A protective role for human IL-10-expressing CD4+ T cells in colitis (121.11)
Authors: Bream Jay , Ramakrishnan Amritha , Wang Fengying , Zhang Hao , Margolick Joseph , Brayton Cory , Ranatunga Dilini
Published: January 2023

Little is known about the relationship between cell- or tissue-specific IL-10 expression and disease susceptibility in humans. Here, we utilized the previously described hIL10BAC transgenic model to examine the role of human IL-10 (hIL-10) in maintaining intestinal homeostasis. Genomically-controlled hIL-10 expression rescued Il10−/− mice from Helicobacter-induced colitis and was associated with control of pro-inflammatory cytokine expression and TH17 cell accumulation in gut tissues. Resistance to colitis was associated with an accumulation of hIL-10-expressing CD4+Foxp3+ T regulatory cells specifically within the lamina propria but not other secondary lymphoid tissues. Co-transfer of CD4+CD45RBlo cells from Il10-/-/hIL10BAC mice rescued Rag1-/- mice from colitis further suggesting that CD4+ T cells represent a protective source of hIL-10 in the colon. In concordance with an enhanced capacity to express IL-10, CD4+CD44+ T cells isolated from the lamina propria exhibited lower levels of the repressive histone mark H3K27Me3 and higher levels of the permissive histone mark AcH3 in both the human and mouse IL10 locus compared to the spleen. These results provide experimental evidence verifying the importance of T cell-derived human IL-10 expression in controlling inflammation within the colonic mucosa. We also provide molecular evidence suggesting the tissue microenvironment influences IL-10 expression patterns and chromatin structure in the human (and mouse) IL10 locus.