bluuebunny/crossref_metadata_2025 huggingface dataset:
Trafficking of lung-migratory cDC2s into the spleen following influenza virus infection
Authors: Jenkins Meagan M. , Bachus Holly , Leon-Ruiz Beatriz , Ballesteros-Tato Andre
Published: January 2023

Trafficking of antigen-bearing, migratory conventional dendritic cells type 1 (cDC1) and type 2 (cDC2) from the lung into the lung-draining mediastinal LN (mLN) is essential for priming T cell responses to influenza virus. In the absence of cDC1s and cDC2s, or when these are unable to traffic from the lung into the mLN, T cell responses are compromised, which highlights the importance of these DC subsets in the generation of T-cell-dependent immunity to influenza. Importantly, it is generally considered that cDCs shortly die after reaching the mLN and therefore do not recirculate back to the blood. Nevertheless, DCs purified from the spleen of intransally influenza-infected mice efficiently prime CD8+ T cell responses. Paradoxically, the two locations are not connected by the lymphatic vasculature and the intransally-administered antigens do not freely reach the blood circulation. Thus, is not clear how DCs in the spleen acquire influenza antigens following intranasal infection. Here we show that a fraction of the lung-migratory cDC2s that reach the mLN following influenza virus infection egress from the mLN, reenter the blood circulation and subsequently migrate into the spleen to prime CD8+ T cell Responses. Collectively, our results demonstrate a new paradigm of DC migration, offer new insights into how systemic T cell responses to influenza are initiated, and will ultimately reveal new strategies to elicit systemic responses to vaccination.
Mono-(2-ethylhexyl) phthalate (MEHP)-induced neutrophil and macrophage infiltration into the testicular interstitium of peripubertal rats does not exacerbate MEHP-induced germ cell apoptosis.
Authors: Voss Jorine J. L. P. , Stermer Angela R. , Ghaffari Rashin , Tiwary Richa , Richburg John H.
Published: January 2023

The testis is an immune-privileged organ that maintains an immune suppressive environment. We have previously shown that exposure of postnatal day (PND) 28 Fischer rats to an acute dose of the Sertoli cell toxicant MEHP leads to an accumulation of CD11b+ cells in the testicular interstitium that correlates with a robust incidence of germ cell (GC) apoptosis. Here we hypothesize that the infiltrating immune cells contribute to GC apoptosis. PND 28 Fischer rats that received an oral dose of 700 mg/kg MEHP showed a significant infiltration of both CD11bc+/CD68+/CD163− macrophages and neutrophils, which peaked at 12 hours but had reduced by 48 hours. Testicular macrophages from MEHP-treated cells showed significantly upregulated expression of Tnfa and Il6, and the Arg1/Nos2 ratio was reduced. However, small increases in anti-inflammatory genes Il10 and Tgfb1 were also observed. Depletion of circulating monocytes with clodronate liposomes prior to MEHP-treatment reduced the macrophage influx, but did not lower GC apoptosis. Additionally, depletion of neutrophils using an anti-polymorphonuclear cell antibody prevented both macrophage and neutrophil infiltration, but also did not affect germ cell apoptosis. Together, these results show that exposure to MEHP leads to a rapid and temporary influx of pro-inflammatory monocytes and neutrophils in the interstitium of the testis. However, with this dosing paradigm, these infiltrating leukocytes do not exacerbate MEHP-induced testicular GC apoptosis. Current experiments are targeted to further investigate the functional significance of these infiltrating cells in the pathogenesis of MEHP-induced testicular injury, considering both a destructive and resolving/protective role.
Anti-sulfated-glycan monoclonal antibody S2 inhibits lymphocyte recruitment to nasal-associated lymphoid tissues and attenuates allergic rhinitis
Authors: Hirakawa Jotaro , Kawashima Hiroto
Published: January 2023

Nasal-associated lymphoid tissue (NALT) is a mucosal lymphoid tissue that functions as a border of defense against inhaled antigens. Lymphocyte recruitment to NALT is mediated by the interaction between L-selectin on lymphocytes and sulfated-glycans on high endothelial venules (HEV) in NALT. HEV are specialized post capillary venules regulating lymphocyte entry form blood into parenchyma of lymphoid organs. It has been previously reported that lymphocyte recruitment to NALT was markedly blocked in HEV-expressed N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) −1 and −2 double-deficient (DKO) mice. In the DKO mice, immune responses against nasally administered ovalbumin (OVA) including OVA-specific IgE, sneezes and nose scratches were significantly diminished.

We previously generated an anti-sulfated-glycan monoclonal antibody S2 which recognizes 6-sulfo sialyl Lewis X (sLeX) and 6-sulfo sialyl N-acetyllactosamine expressed on HEV in NALT by immunizing GlcNAc6ST-1/2 DKO mice with CHO-K1 cells stably expressing 6-sulfo sLeX. S2 administration inhibited lymphocyte recruitment to NALT and allergic rhinitis induced by intranasal administration of OVA. S2 administration to OVA-immunized mice resulted in the reduction of OVA-specific serum IgE, and counts of sneezes and nose scratches after nasal immunization. Total cell numbers in NALT and expression of Th2-related cytokines such as IL-5 and IL-13 were also diminished after S2 administration. Taken together, these results suggest that S2 would serve as a novel therapeutic agent useful for the treatment of allergic rhinitis.
The earliest B-1 precursor cells are present in E10.5 embryos more than HSC precursor cells.
Authors: Yoshimoto Momoko , Tarnawskey Stefan , Mishra Akansha , Kobayashi Michihiro , Bernstein Irwin , Hadland Brandon , Yoder Mervin C.
Published: January 2023

The peritoneal B-1 cells are innate-like B cells, secreting natural antibodies without T cell help. Cell transfer experiments suggest that CD5+ B-1a cells are of fetal origin because adult bone marrow stem/progenitors do not repopulate B-1a cells efficiently, while fetal liver cells do. We have previously reported the B-1 cell potential of extra-embryonic yolk sac (YS) and para-aortic region of E9.5 embryos appearing before the emergence of hematopoietic stem cells (HSCs), and that B-1 progenitors develop in the absence of HSCs. These data imply that at least a part of B-1a cells are HSC independent and derived from early YS/embryos. In addition, it has been reported that fetal liver HSCs failed to repopulate B-1a cells after transplantation. This raises a question whether HSCs at any stage of the embryo or post-natal life produce B-1a cells. Our recent report has demonstrated that E11.5 HSC precursors repopulated both B-1a and B-2 cells at a clonal level after co-culturing with Akt-overexpressing endothelial cells.

In order to test whether the first HSC repopulates B-1a cells, we directly transplanted Ter119− VE-cadherin+ c-kit+ “HSC precursor cells” from E10.5 embryos into sublethally irradiated immune-deficient neonatal mice. Our precise analysis of transplantation assays revealed that “HSC precursor cells” contained both neonatal HSCs that repopulated multi-lineage blood cells and B-1 specific precursors that repopulated only peritoneal B-1a, B-1b, and splenic marginal zone B cells. Surprisingly, the frequency of B-1 precursors is more than that of neonatal HSCs. Thus, the earliest transplantable B-1 specific precursor cells are present in E10.5 embryos showing the same surface markers with cells display HSC activity.
The ion channel TRPM7 is required for B cell lymphopoiesis
Authors: Treanor Bebhinn , Buhari Fathima Hifza Mohamed , Zhao Tiantian , Brauer Patrick M. , Burrows Kyle M. , Cao Eric , Moxley-Paquette Vincent , Mortha Arthur , Zúñiga-Pflücker Juan Carlos , Krishnamoorthy Mithunah
Published: January 2023

The ion channel, transient receptor potential subfamily melastatin, member 7 (TRPM7) was first identified as critical for cell viability in the B cell line, DT40, by regulating cellular Mg2+ homeostasis. However, specific deletion of TRPM7 in T cells results in only a minor defect in thymocyte development and nearly normal numbers of peripheral T cells, raising the question of whether TRPM7 is in fact important for B cell development in vivo. Here, we generated a murine model in which TRPM7 is specifically deleted in B cells under control of the Mb1 promotor. We show that Mb1-Cre mediated deletion of TRPM7 results in a complete loss of peripheral B cells and a developmental block at the pro B cell stage in the bone marrow, coincident with the expression of Mb1. Importantly, we demonstrate using the in vitro OP9 stromal cell system of lymphopoiesis that supplementation with high levels of extracellular Mg2+ can partially support B cell development in TRPM7-deficient mice. Thus, our findings identify a critical role for TRPM7 in B cell lymphopoiesis.
ABCB7 is required for B lymphocyte development
Authors: Lehrke Michael Jonathan , Chung Ji Young , Schwab Aaron , Shapiro Michael , Shapiro Virginia Smith
Published: January 2023

ABCB7 is a mitochondrial iron transporter critical for cellular iron homeostasis, as its loss results in mitochondrial iron accumulation. ABCB7 has been demonstrated to be critical for embryogenesis and hematopoiesis, though its role in specific hematopoietic lineages is unexplored. We found that ABCB7 is required for lymphopoiesis, as CD2-iCre ABCB7 cKO mice have a severe block in both T and B lymphocyte development. The block in B cell development occurs at the Fr. C to Fr. C’ transition, where we found reduced expression of intracellular μ and pre-BCR, as well as reduced proliferation. We observed reduced expression of E2A, Ikaros, and Pax5, transcription factors essential for B cell lineage development. Previous literature has shown that deletion of ABCB7 results in mitochondrial iron accumulation. Because this is toxic to cells, we hypothesized that B cells increase heme synthesis in order to detoxify accumulating iron. In support of this, we found that HO-1, a surrogate marker for intracellular heme concentration, was elevated in B cells lacking ABCB7. Interestingly, heme levels regulate Bach2, a transcriptional repressor important for B cell development as it suppresses myeloid program in lymphocytes. Bach2 expression was found to be decreased in ABCB7-deficient B cells. We are currently examining the role of Bach2 and the mechanism responsible for the block in B cell development upon loss of ABCB7.
mTORC1 and mTORC2 differentially regulate the development of NK cells
Authors: Yang Chao , Siebert Jason , Thakar Monica , Malarkannan Subramaniam
Published: January 2023

Natural killer (NK) cells are innate lymphoid cells that play essential roles in both innate and adaptive immunity. Kinase mTOR is critical for NK cell development; however, the unique roles of mTORC1 or mTORC2 in regulating this process remain unknown. Ncr1iCre-mediated conditional deletion of Rptor or Rictor (genes encoding defining component of mTORC1 or mTORC2, respectively) results in altered homeostatic NK cellularity and impaired development at distinct maturation stages. The transition from CD27+CD11b− to CD27+CD11b+ stage is impaired in Rptor cKO mice, while, the terminal maturation from CD27+CD11b+ to CD27−CD11b+ stage is compromised in Rictor cKO mice. Mechanistically, Raptor deficiency renders large alteration of the gene expression profile including transcription factors governing early NK cell development. Comparatively, loss of Rictor causes a restricted transcriptome changes. The reduced expression of T-bet correlates with the terminal maturation defects and results from impaired mTORC2-AktS473-FoxO1 signaling. To further explore the development of murine NK cells and the effects of Raptor or Rictor deficiency, single cell RNA sequencing analyses were performed. The Lin−NK1.1+ cells from BM of adult WT mice are unbiasedly classified into six different populations including CD3+ cells, ILC1, immature NK cells, two intermediate mature NK cells, and terminal mature NK cells. Most Raptor-deficient NK cells form new cluster that is not seen in either WT or Rictor cKO mice. Rictor deficiency affects the terminal maturation of NK cells and potentially the development of ILC1. Collectively, our results reveal the divergent roles of mTORC1 and mTORC2 in NK cell development.
Characterizing hematopoietic precursors of Langerhans Cell Histiocytosis (LCH) using a humanized mouse model
Authors: Mancusi Rebecca , Bigenwald Camille , Merad Miriam
Published: January 2023

Langerhans Cell Histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by the accumulation of immature dendritic cells in inflammatory lesions. LCH pathogenesis is not fully understood, however recent findings demonstrate that misguided differentiation programs of dendritic cell precursors drive LCH. Importantly, expression of the somatic mutation BRAFV600E in these precursor dendritic cells indicates a “high-risk” LCH diagnosis. Previous studies have utilized mouse models to further investigate the role BRAFV600E in LCH pathogenesis. However, the lack of a human immune compartment in mice prevents a comprehensive analysis on the function of human dendritic cells in LCH. This study aims to study dendritic cell commitment, maturation, and function using a humanized mouse model of LCH. We describe a series of experiments in which we reconstitute GFP+ BRAFV600E+ transduced human hematopoietic precursors in irradiated, immunodeficient mice. Additionally, transduction and transplantation protocols were optimized to improve novel strategies in the development of a humanized mouse model of LCH. Initial results suggest successful human engraftment in vivo based on flow cytometry analysis of GFP expression in bone marrow 7 and 14 days post-transplantation. We expect to observe an LCH-like phenotype in these mice with continued examination. Our study ultimately develops Mount Sinai’s first humanized mouse models of LCH and enables efficient long-term research on the role of BRAFV600E in the human immune compartment of LCH.
Altered TGFβ signaling in non-hematopoietic cells leads to eosinophilic esophagitis.
Authors: Laky Karen , Kinard Jessica , Guerrerio Anthony , Frischmeyer-Guerrerio Pamela
Published: January 2023

Eosinophilic esophagitis (EoE) is a chronic immune-mediated disease characterized by esophageal inflammation and dysfunction. Among the top ten single nucleotide polymorphisms (SNPs) that are associated with EoE susceptibility, approximately one third are in genes that encode proteins involved in transforming growth factor beta (TGFβ) signaling. Patients and mice heterozygous for a mutation in the kinase domain of TGFβR1 (TGFBR1M318R+/−) exhibit cardiovascular, skeletal, pulmonary, and immune manifestations of Loeys-Dietz Syndrome (LDS). Immune manifestations of LDS include elevated serum IgE against food antigens and an increased likelihood of developing EoE that is characterized by esophageal dilation, food impaction, basal cell hyperplasia, and an inflammatory infiltrate composed of eosinophils, mast cells, T cells, type 2 innate lymphoid cells (ILC2), and antigen presenting cells. How altered TGFβ signaling leads to EoE is unknown. We made bone marrow chimeras to determine which cells are responsible for the development of EOE. We found that altered TGFβ signaling in radio-resistant non-hematopoietic cells was both necessary and sufficient to cause EoE. Moreover, the development of EoE was not predicated on altered TGFβ signaling in the hematopoietic effector cells. Using RNASeq and immuno-histochemistry analyses we show that altered TGFβ signaling in esophageal epithelial cells results in the expression of cytokines, chemokines, and adhesion molecules that facilitate the recruitment, activation, and accumulation of eosinophils, mast cells, T cells, and ILC2. Targeting these inflammatory mediators in vivo should have therapeutic potential for patients suffering with EoE.
Transcription factor signal transducer and activator of transcription 3 (STAT-3) serves as a negative regulator controlling IgE class switching
Authors: Dascani Paul Lorenzo , Ding Chuanlin , Kong Xiangyu , Yan Jun
Published: January 2023

A mutation in the Signal Transducer and Activator of Transcription 3 (STAT-3) has been linked to incidence of Autosomal Dominant Hyper Immunoglobulin E Syndrome (AD-HIES), a disease characterized by elevated serum IgE antibody and susceptibility to respiratory and skin infection. However, how this genetic mutation leads to the phenotype has not been fully understood. In this study we investigated the specific role of STAT-3 in the germinal center (GC) B cells and plasma cells for IgE class switching. Through the use of CD2-Cre or CD19-Cre driven STAT-3 conditional knockout (cKO) mice in a Th2-type immunization model, we aimed to determine the specific cell subset(s) and signaling mechanism by which STAT-3 mediated class switching is regulated. We demonstrated in vivo and in vitro that CD2-Cre driven STAT-3 cKO mice showed elevated IgE and decreased IgG1 in the serum, and a reduction in GC formation. Within the GC, IgG1+ GC B cells were decreased while IgE+ GC B cells were more prevalent. Additionally, these mice exhibited reduced IgG1 and elevated IgE populations of antibody-producing plasma cells. Subsequent experiments using a CD19-Cre B-cell specific cKO mouse established this effect to be B-cell intrinsic. Mice with STAT-3 deficiency in B cells have also demonstrated an increase in lung inflammation when used in an asthma-like disease model. Our data also revealed other transcription factors, including Bcl-6 and Aicda, function as downstream signals of STAT-3 regulation. This model suggests a negative role for STAT-3 in regulating class switching of the GC B cells from the IgG1 to the IgE producing state, which may serve as a therapeutic target for treatment of AD-HIES and other immune disorders.
Contribution of the glycosyltransferase FucT-VII to allergic skin inflammation
Authors: Khan Ibrahim Mohammmed , Ulrich Benjamin Joseph , Kansas Geoffrey S. , Kaplan Mark H.
Published: January 2023

The development of E and P selectin ligands on the surface of immune cells requires α1,3 fucosyltransferase FucT-VII, an enzyme encoded by the Fut7 gene. T cell activation and cytokine environments regulate glycosyltransferase expression, eventual glycan formation and cell migration. The T helper 2 (Th2) subset are mediators of allergic inflammation, but the requirement for FucT-VII-dependent migration in allergic inflammation is less clear. In this study we determined if Fut7 expression was required for allergic inflammation in the constitutively active STAT6VT model in which mice are predisposed to a spontaneous inflammatory phenotype similar to that of atopic dermatitis. STAT6VT mice were crossed with Fut7−/− animals and compared for the development of disease and inflammation. Decreased incidence of disease was observed in Fut7−/− STAT6VT mice, compared to STAT6VT mice. Interestingly, if disease did occur in Fut7−/− animals, the inflammation was not attenuated and in some cases was more severe. One factor that impacted disease development was related to offspring who inherited the STAT6VT allele from their maternal parent. The dams would have experienced inflammation throughout pregnancy and might have promoted a mechanism of migration independent of Fut7 gene expression. Together, these data indicate that Fut7 promotes, but is not strictly required for, allergic skin inflammation.
Forward genetic screen of allergy reveals genetic basis for allergic disease
Authors: SoRelle Jeffrey , Chen Zhe , Wang Jianhui , Tang Miao , Li Xiaohong , Beutler Bruce
Published: January 2023

Although much is known about how mammals respond to allergens, it is still unclear how allergies are acquired. Allergies arise from environmental triggers, but the genetic causes are not clearly understood. We undertook a forward genetic approach to discover genes with non-redundant function in allergic sensitivity. Our screen measured antigen-specific IgE antibody response to a model cysteine protease allergen, papain, in N-ethyl-N-nitrosourea (ENU) mutagenized mice. Males heterozygous for ENU-induced mutations (G1) were bred to wild-type females, and their daughters (G2) were backcrossed to the G1 to produce homozygous mutations in G3 mice, which were screened for IgE responses to papain. Whole exome sequencing of the G1 detects single nucleotide mutations, which are genotyped in all G3 progeny. This method allows for real-time mapping, where phenotype (papain-specific IgE) and genotype are statistically compared using dominant, additive, and recessive models of inheritance to determine linkage. 14,694 G3 mice in 636 pedigrees were screened with estimated genomic saturation of 7.6% of all genes damaged or destroyed in 3 homozygous mice. Mutations with altered allergen-specific IgE segregated into two pathways: class switch recombination (Increased IgE: Itch, Itk. Decreased IgE: Jak3, Stat6, and Ikbkb) and B cell receptor signaling (increased IgE: Blk, Plcg2, Rasgrp3, CD19, and Pik3r1). Interestingly, many BCR mutations were dominant or semi-dominant and did not cause significant immunodeficiency. This first forward genetic screen of allergic disease unveiled several genes with unknown roles in allergy and provides a potential genetic explanation for some patients who present with allergy without immunodeficiency.
Type 2 innate lymphoid cells contribute to a mixed-type allergic response following topical exposure to the quaternary ammonium compound didecyldimethylammonium chloride.
Authors: Shane Hillary L. , Lukomska Ewa , Anderson Stacey E.
Published: January 2023

Didecyldimethylammonium chloride (DDAC) is an antimicrobial dialkyl-quaternary ammonium compound used in numerous industrial and commercial products. Clinical and animal data suggest that DDAC exposure elicits multiple types of hypersensitivity reactions. To examine the immunological mechanism behind this mixed type response and the potential involvement of ILCs, we assessed early immune responses in the skin following dermal DDAC exposure (0.25% and 0.5%) in a BALB/c murine model. DDAC exposure resulted in a rapid and dramatic increase in the Th2-skewing and ILC2 activating cytokine TSLP in the skin. Correspondingly, dermal ILC2s were activated 24 hours after DDAC exposure, resulting in increased levels of CD25, ICOS and KLRG1, and decreased CD127 on a per cell basis throughout a 7 day period of exposure. Following ILC2 activation, the Th2 cytokine IL-4 was found to be elevated compared to control mice in total ear protein lysate in the 0.5% DDAC treated mice. To determine a functional role for ILC2s in DDAC induced sensitization we used Rag2−/− mice, which lack mature T and B cells. ILC2s from Rag2−/− were similarly activated by DDAC and importantly, produced significant levels of IL-4 (0.5%DDAC) and IL-5 (0.25% and 0.5%) in the skin. These data indicate that ILC2s contribute to Th2 immune responses early after DDAC exposure. ILC2s have been previously implicated allergic responses, but to our knowledge have not been thoroughly investigated in chemical sensitization. These results indicate that skin resident ILC2s become activated and produce TH2 cytokines following exposure to DDAC, providing a possible mechanism for the development of a mixed-type allergic response commonly observed with chemical sensitizers.
The B cell IL-10 receptor suppresses antibody production.
Authors: Dooley Abigail , Quintana Montserrat , Cheung Michael , Sun Li Lauren , Gupta Neetu
Published: January 2023

Generation of high affinity, broadly neutralizing antibodies has remained a holy grail in influenza and malaria. Appropriate B cell responses are essential for producing neutralizing antibodies to combat these infections. Additionally, cytokines and their receptors are known to orchestrate the immune response to infections on several levels. However, the regulation of antibody production by cytokine receptors on B cells is not well understood. The anti-inflammatory cytokine, IL-10 activates the JAK-STAT pathway upon binding to the IL-10 receptor (IL-10R), and inhibits production of the pro-inflammatory cytokines. IL-10 is also thought to regulate antibody production but the specific role of IL-10R expressed on B cells is unclear. We hypothesized that the B cell-expressed IL-10R regulates B cell antibody response to infections. To address this, we generated mice with conditional deletion of IL-10R in the B cell lineage. Using these mice, we first examined B cell antibody production in response to LPS and B cell receptor stimulation. Surprisingly, the loss of IL-10R in B cells increased antibody production. Both IgM and IgG antibody secreting cells were significantly increased when the IL-10R was deleted in B cells. Further, higher IL-10 levels were detected in culture supernatants of IL-10R-deficient B cells stimulated with LPS. Our data suggest that the IL-10R on B cells negatively regulates antibody production. Our ongoing experiments are aimed at addressing the molecular basis of this inhibitory influence of the B cell IL-10R on antibody production. This study has implications in the treatment of seasonal infectious diseases such as influenza and malaria, in which a robust antibody response is essential for protective immunity.
Deletion of the highly conserved functional region of the Cd4 enhancer NCE in RLM11 cells using CRISPR/Cas9 reduces CD4 expression
Authors: Sarafova Sophia D. , Futral Cameron , Brennan Paul , Guzynski Mia
Published: January 2023

The glycoprotein cell surface marker CD4 is a key mediator in the adaptive immune response and T-cell development. Signaled CD4+ CD8+ double positive cells (DP) upregulate CD4 on the cell surface, allowing for a persistent TCR signal in the thymus and differentiating into the CD4+ single positive helper lineage. Previously, we have demonstrated that NCE (novel cis element), located in the first intron of Cd4, acts as an enhancer in RLM11 cells (CD4+, CD8−, Th-Pok− thymoma, arrested at the intermediate stage of development), but not in AKR-1G1 or VL3-3M2 cells (DP thymomas). We have identified a highly conserved region within NCE, labeled core-NCE, which may be vital to the enhancer’s function. To investigate the contribution of core-NCE to the level of CD4 expression, we utilized CRISPR/Cas9 technology to target the region for deletion. Here we describe the construction of plasmids containing guide sequences for targeted deletion of core-NCE by Cas9 cutting and NHEJ repair. We have successfully generated and sequence verified plasmids with three upstream and three downstream guides and introduced them in RLM11 cells. After single cell sorting, we obtained one clone that has the 300bp core-NCE deleted on one Cd4 allele, which resulted in 66% reduction in CD4 expression from that allele. As we removed the core-NCE in cells that have already established CD4 expression, we conclude that the core NCE has an enhancer function at the intermediate/transitional stage of thymocyte development that is separate from the previously described epigenetic function of the same intronic region, making NCE a good candidate for the regulatory element responsible for CD4 upregulation during differentiation into the CD4 lineage.
Thymic selection of innate self-specific T cells: Both Timing and Signal Strength matter
Authors: Verstichel Greet , Thiault Nicolas , Chen Angeline , Cheroutre Hilde
Published: January 2023

Naïve CD4 and CD8 T cells are selected in the thymus in response to low-avidity TCR interactions. Such weak TCR signals are crucial for installing a quiescent program that guides them to the lymph nodes yet allows for immune activation upon encounter of high-affinity ligands in the periphery. In contrast, agonist-selected T cells receive strong self-based TCR signals in the thymus that imprint them to guard the mucosal borders and maintain quiescence at steady state. Although the TCR signal strength is a decisive factor in instructing these different outcomes, the mechanism that generates protective self-specific T cells remains largely enigmatic. Previous work suggests that the self-specific double negative (DN) T cell lineage already diverges early during T cell development. We have explored the role of developmental timing by Cre-mediated deletion of components of the TCR signaling pathway. The Lck proximal promoter drives Cre expression before b-selection and the CD4 promoter drives Cre expression after b-selection but before positive selection. As TCR targets we investigated the lack of Themis expression, essential for the generation of conventional naïve T cells, but dispensable for agonist selection. Vice versa, we analyzed the requirement for Stim1 and Stim2, driving agonist selection, but not conventional selection. Both Lck prox Cre- and CD4 Cre-driven deletion of Themis lead to a drastic decrease in naïve T cells. However, DN T cells are particularly dependent on early strong TCR signals at the Pre-TCR stage, as only Lck prox Cre- but not CD4 Cre-driven deletion of Stim1/2 eliminates this subset. Our results suggest that strong signals at the Pre-TCR checkpoint are crucial for the generation of innate self-specific T cells.
MicroRNA miR-155 promotes medullary thymic epithelial cell maturity and regulatory T cell development
Authors: Dong Jiayi , Warner Lindsey , Chen Mei-Chi , Lin Ling-Li , Lu Li-Fan
Published: January 2023

MicroRNA-155 (miR-155) is a small non-coding RNA highly upregulated in regulatory T (Treg) cells, an immune cell population specialized in maintaining immunological tolerance. While the importance of miR-155 in conferring Treg cell competitive fitness has been well established, how miR-155 regulates Treg cell development remains unknown. By taking cell-type specific gene targeting approaches, we demonstrate that miR-155 controls thymic Treg (tTreg) cell differentiation in both T cell-intrinsic and -extrinsic manners. Specifically, we show that mature CD80hiMHCIIhi medullary thymic epithelial cells (mTECs) crucial for the generation of tTreg cells express high levels of miR-155, where TEC-specific deletion of miR-155 diminishes this population and impairs tTreg cell development. Previously, the size of the mature mTEC compartment has been shown to be regulated by TGF-β signaling. Here, our results identify SMAD3, a key molecule downstream of TGF-β signaling, is targeted by miR-155 in mTECs. Thus, we propose a miR-155-SMAD3-TGF-β axis in the thymic medulla to determine mTEC maturity and, consequently, the quantity of tTreg cells.
Analysis of immune cells from mice with Chagas disease treated with NPOx-B
Authors: MANZANO OSCAR ARTURO ANGELES , Galicia Viridiana Galicia , Castro Ma Natividad Flores , Camacho Mitzi , Baeza Isabel , Wong Carlos , Wong-Baeza Carlos , Reséndiz-Mora Albany
Published: January 2023

Chagas disease is a parasitic disease caused by Trypanosoma cruzi it is endemic in Latin America, where is a major health problem since it affects 8–10 million people, causes 50,000 deaths per year and about 25% of the population is at risk of acquiring the disease. It remains practically incurable, mainly because of the limited interest in developing new drugs and because the drugs available for its treatment, Benznidazole (Bz) and Nifurtimox (Nx) are inefficient to cure patients. The acute phase of the disease appears shortly after infection, the chronic phase appears after a silent asymptomatic period that may last several years. During the chronic phase, the heart, esophagus, colon and PNS are irreversibly affected, patients usually die from heart failure. The chronic phase may be established by the evasion of the immune response by the parasite, secondly because Bz and Nx generate free radicals that can affect the cells of the immune system (CIS) which could favour the chronic phase. In our research group, we design and synthesized the benzyl ester of Npropyl oxamic (NPOxB) that is an effective inhibitor of the alpha-hydroxy acid dehydrogenase II enzyme, which is a specific enzyme in the glycolytic pathway of the parasite. In this work we treated with NPOxB or Bz infected mice with NINOA or INC5 strains, we evaluate the parasitaemia, the macrophages, B, T, plasma and dendritic cells. The NPOx-B decreased more the parasitaemia of both T. cruzi strains than the Bz. The mice infected and treated with Bz showed a decrease in B, T and plasma cells compared to the infected mice treated with NPOxB. So NPOxB is more effective in decrease the parasitaemia and lacks the toxicity of Bz, it doesn’t affect in CIS, the cure of the disease can be achieved more efficiently.
Multidimensional phenotypic immune profiling of Chlamydia trachomatis infected women using mass cytometry platform.
Authors: Kollipara Avinash , Iannone Marie A. , Poston Taylor , Lee De’Ashia E. , O’Connell Catherine , Zheng Xiaojing , Hillier Sharon , Wiesenfeld Harold , Darville Toni
Published: January 2023

Chlamydia trachomatis (CT) is the most frequently reported bacterial sexually transmitted infection in the U.S., with 1 in 20 sexually active young women being infected. Infections are easily curable with antibiotics, but reinfections are frequent and infection can lead to reproductive tract sequelae. Adaptive immune responses generated during infection likely limit extent of infection, but frequently fail to prevent reinfection. We developed a 35 marker CyTOF panel to phenotype PBMCs. We examined PBMCs from women infected with CT alone (N=8), women with CT co-infected with gonorrhea or Mycoplasma genitalium (N=6), and from these same women 1 month post antibiotic treatment, and compared them to 11 healthy uninfected women. Compared to uninfected women, the frequency of B cells was increased in CT-infected women and remained elevated at 1 month, with no effects observed on various B cell subsets. Among T cells, Th1 and Tc1 percentages were reduced in CT-infected women and remained low at 1 month. Th2 and Th17 phenotype percentages were not different than uninfected women. Th1/Th2/Th17 cells expressing CD107a were reduced in CT-infected women, but rebounded towards normal at 1 month. Myeloid DCs were reduced in CT-infected women, but rebounded towards normal at 1 month. All of these differences were present regardless of coinfection. CD107a+ NK cells were reduced in CT-only infected women and failed to rebound at one month. The reductions in these various cell types is likely due to their trafficking to the infected tissue. CyTOF analysis of PBMCs from additional patients and of endometrial biopsy samples from these same subjects should provide important data related to the female host response to CT.
Loss of acid ceramidase activity in a murine model of Farber disease leads to an early and profound immuno-phenotype that reflects alterations in both the innate and adaptive immune cell populations
Authors: Coquery Christine Morel , Sampey Brante , Solyom Alexander , Gaukel Eric J. , He Xingxuan , Zhu Changzhi , DeAngelis Victor , Schuchman Edward H. , Wring Stephen A.
Published: January 2023

Farber disease is an ultra-rare lysosomal storage disorder where a deficiency of acid ceramidase activity leads to the accumulation of ceramide and the formation of granulomas which, in conjunction with a chronic inflammatory state, likely results in tissue damage. A rapidly progressive murine knock-in Asah1P361R/P361R model of Farber disease recapitulates many aspects of the human disease, including an increase in ceramide and pronounced tissue inflammation. The focus of this study was to describe the aberrant immune cells in this disease model. Blood, livers, lungs, and spleens were collected from 4- to 8-week old litter-matched Farber and wild-type mice for characterization by flow cytometry. Characterization of the monocyte population revealed a marked increase in the frequency of MHCII−CD11bhiLy6C+ cells (3–6 fold vs WT). These cells were highly activated as evidenced by CD86 expression, and skewed toward a pro-inflammatory M1 phenotype, based on CD38 expression. A concurrent decrease in CD206+ anti-inflammatory M2 macrophages was identified. In addition to the macrophage compartment, a profound increase in neutrophils in the spleen, liver, and lung was evident by 4 weeks of age (2–7 fold vs. wild-type). Marked differences in the adaptive immune compartment also were noted, with a clear increase in the frequency of plasmablasts (precursors to immunoglobulin-producing plasma cells), likely secondary to the increase in pro-inflammatory monocytes. Importantly, the alterations observed in the tissues were reflected in the blood of Farber mice. In summary, these data expand the characterization of the murine Farber disease model and provide insight into the immuno-phenotype of Farber disease.
Gain-of-function STING mutations induce T cell death in the SAVI mouse
Authors: Wu Jianjun , YAN NAN
Published: January 2023

STING is ER-localized protein that plays a central role in the innate immune response to microbial DNA, cyclic di-nucleotide and aberrant host DNA. We previously established a STING-associated vasculopathy with onset in infancy (SAVI) mouse model by introducing the N153S mutation in the mouse STING (N154S in the human STING). The SAVI mouse recapitulates several abnormal phenotypes observed in human patients, including inflammatory lung disease, T cell cytopenia and premature death; and these disease phenotypes surprisingly occur independent of IRF3. To further investigate the SAVI disease mechanism, we focused on the role of STING in T cell survival. We found that both mature CD4 and CD8 T cells in STING N153S mice are undergoing apoptosis. Inducible expression of STING N154S but not WT STING in Jurkat T cells also caused robust apoptotic cell death. Mechanistically, we defined a novel structural motif in the STING protein as well as an IFN-independent signaling cascade that are required for STING gain-of-function mutants to drive T cell death. We hope that genetic or pharmacological targeting of the STING-mediated cell death signaling pathway would be therapeutically useful for treating SAVI patients.
Extracellular ASC in bronchoalveolar lavage (BAL) fluid exists primarily in the soluble form and not in a speck complex
Authors: McAndrew Christian C. , Tsai MuChun , Napolitano Nina M. , Song Min-Ae , Shields Peter G. , Gavrilin Mikhail A. , Wewers Mark D.
Published: January 2023

Rationale

Apoptosis-associated speck-like protein containing a CARD (ASC) serves as an adaptor to link NOD-like receptors to caspase-1 during inflammasome activation. The oligomerization of ASC into a large speck-like complex appears to be crucial to this process. Furthermore, extracellular ASC specks may propagate an inflammatory response. Although extremely high levels of ASC exist in BALF, little is known about whether BALF ASC exists in the soluble form or as part of a speck complex.

Methods

Subjects (normal volunteers and patients with pneumonia) underwent IRB approved BAL. BALF was removed from cells by centrifugation at 400g and ASC was quantified by ELISA. Additionally, BALF was spun at 16,000g to pellet ASC specks and separate them from soluble ASC. Samples were crosslinked with DSS, separated on a denaturing gel, and immunoblotted with an anti-ASC antibody to visualize ASC oligomers.

Results

Extracellular levels of ASC were very high in the BALF when measured by ELISA and immunoblot. However, although pelletable ASC oligomers could easily be observed in stimulated THP-1 cell supernatants, no oligomers of ASC were found in BALF samples from healthy donors or pneumonia patients, and negligible amounts of ASC pelleted down when BALF was spun at high speed.

Conclusion

In contrast to inflammasome activated THP-1 cells, extracellular ASC in BALF exists primarily in the soluble non-oligomeric state. This observation held true for healthy donors and donors with lung infections.
Observed increases in CD4:CD8 and TIGIT expression during sofosbuvir-based therapy in a longitudinal hepatitis C cohort
Authors: Stevenson Timothy J. , McMahon Brian , Townshend-Bulson Lisa , Hewitt Annette , Barbour Youssef , Espera Hannah , Homan Chriss , Varney Danielle , Gove Jim , Rhodes Wileina , Snowball Mary , Otto Evon , Plotnik Julia , Negus Susan , Simons-Petrusa Brenna
Published: January 2023

Chronic hepatitis C virus (HCV) infection reduces immune function through exhaustion and repertoire reduction. Sofosbuvir-based direct-acting antiviral (DAA) therapies offer >90% sustained virologic response (SVR) for HCV infection, but the extent of immune recovery and relationship with clinical factors are unknown. The longitudinal Alaska Hepatitis C Cohort provides an opportunity to evaluate immune recovery following sofosbuvir-based DAA and determine the influence of an extensive clinical and laboratory history. Forty-two persons (genotype 1; mean age 50 ± 12 years; 69% female) received 8 or 12-week sofosbuvir-based DAA therapy. Mean HCV RNA detection date was 2007 (range: 1994–2017). Pre-treatment HCV RNA levels averaged 4.9×106 IU/L (range: 4.9×104–2.4×107), were not detectable in any person after 4-weeks of therapy, and all achieved SVR. 18% (n=7) of persons had a fibrosis score of ≥F2 (moderate fibrosis to cirrhosis). Peripheral blood mononuclear cells (PBMCs) were evaluated at four time points: Pre-treatment (Pre), 4-weeks treatment (4wk), End of Treatment (EOT), and SVR post 12-weeks EOT (SVR-12) by flow cytometry to evaluate memory, exhaustion, and functional immune markers. Initial analysis revealed a significant increase in the CD4:CD8 from 4wk to EOT (p=0.007). Regulatory T-cells significantly increased at EOT and SVR-12 compared to 4wk (p=0.04 and 0.03, respectively). TIGIT showed a significant increasing trend in T-cells and NK cells throughout sampling timeline. Preliminary analysis indicates sofosbuvir-based DAA supports immune recovery to some extent. Additional analysis of immune recovery markers, determination of estimated date of HCV infection, and correlation of morbidity factors continues.
Sex differences in Vitamin D alter inflammation during heart disease
Authors: Bruno Katelyn A. , Mathews Jessica , Douglass Erika , Hill Anneliese , Cooper Leslie T. , Hoyne Jonathan , Fairweather DeLisa
Published: January 2023

An estimated 1 billion people worldwide have deficient or insufficient levels of vitamin D (VitD). Considerable evidence indicates that VitD deficiency is associated with an increased risk of autoimmune and cardiovascular disease (CVD), yet it remains unclear whether low VitD is simply a biomarker or has a true pathologic role. In this study we examined how sex differences in VitD influences various CVDs. We found that patients with CVD had significantly lower VitD than healthy controls (no ICD9/10 codes) (p=4E-6). In myocarditis patients low VitD correlated to poor ejection fraction (EF) in women with myocarditis (p=0.02), but high VitD correlated with low EF in men (p=0.04). In VDR knockout mice we found that VDR decreased myocarditis in females (p=2E-8) but increased inflammation in males (p=0.03) with an increase in total immune cells (p=0.02), CD3, CD4 and CD8 T cells (p=0.04), and the inflammasome in females. VDR altered Treg (p=0.04), mast cells (p=0.04) and cytokine profiles in males. Additionally, sera VitD levels were significantly higher in WT male mice with myocarditis compared to females. Our findings in VDR mice indicate that VitD/VDR reduces myocarditis in females, but increases disease in males. These data suggest that VitD sufficiency may increase inflammation in men, while VitD deficiency may make disease worse in women. These findings suggest that Vitamin D may have a role in the pathogenesis of inflammatory and cardiovascular disease.
Prevention of myocarditis using regenerative medicine therapy
Authors: Fairweather DeLisa , Bruno Katelyn A. , Hill Anneliese , Stalboerger Paul G. , Cooper Leslie T. , Behfar Atta
Published: January 2023

Myocarditis is an important cause of acute and chronic heart failure. We wanted to determine whether purified exosome product (PEP) can improve and/or prevent myocarditis using a preclinical mouse model of myocarditis. Because estrogen protects women from myocarditis and heart failure we also tested premenopausal PEP (pmPEP). To determine the appropriate dose of PEP/pmPEP, we administered PEP and pmPEP vs. PBS controls ip to male BALB/c mice at day −1, 0 and +1 after viral infection to induce myocarditis and harvested mice at day 10 post infection (pi) during the peak of acute myocarditis. We found that the dose and exposure route was successful. We found that pmPEP, but not PEP, significantly decreased acute myocarditis based on histology (ANOVA p=0.0009). and decreased total immune cells (CD45 p=0.008), macrophages, neutrophils and mast cells (CD11b p=0.04), macrophages (F4/80 p=0.009), and T cells (CD3 p=0.02, CD4 p=0.01) but no other immune cell populations in the heart. Markers for both M1 and M2 macrophages were also significantly decreased with pmPEP treatment, indicating a “global” decrease in inflammation. Importantly, CR1, the central inhibitor of the complement cascade, was significantly increased by pmPEP but not PEP (p=0.004). Next we treated male BALB/c mice with PEP, pmPEP or PBS ip on day 8, 9 and 10 pi (a clinically relevant timepoint) and harvested on day 11 pi during acute myocarditis. Here we found that both PEP and pmPEP significantly reduced acute myocarditis compared to controls (ANOVA PEP p=0.006, pmPEP p= 0.005). These findings suggest that PEP/pmPEP could be given to patients who come in with acute onset myocarditis to decrease the severity of disease and potentially prevent sudden death.
Anti-inflammatory miR-146a is protective against diet-induced obesity and regulates macrophage metabolism
Authors: Nelson Morgan , Runtsch Marah Clarice , O’Connell Ryan
Published: January 2023

MicroRNAs (miRNAs) have been recognized for their ability to regulate many aspects of mammalian immunity. miR-146a is one such miRNA that restricts inflammation by dampening pro-inflammatory signaling pathways. Here we investigate the role of miR-146a in protecting against diet-induced obesity (DIO), an inflammatory and metabolic disease that affects one-third of adults in the United States and increases risk for developing other health-related diseases. In this study, we placed miR-146a−/− and WT mice on a high-fat diet (HFD) or standard diet and observed significant weight gain, increased fat content, adipocyte hypertrophy, and increased serum glucose levels in miR-146a−/− mice compared to WT mice on HFD. Furthermore, we observed increased macrophage infiltration into adipose tissue and elevated inflammatory gene expression in metabolic tissues in the absence of miR-146a.

In WT mice, miR-146a expression was higher in adipose-resident immune cells than in adipocytes, prompting us to perform RNA-seq on adipose tissue macrophages (ATMs) from miR-146a−/− and WT mice. This revealed that in addition to regulating pro-inflammatory gene expression, miR-146a also regulates AKT activation and the metabolic capacity of macrophages through a mechanism involving its direct target Traf6. Overall, this work identifies a critical role for miR-146a in repressing DIO and associated metabolic and inflammatory symptoms, and suggests that miR-146a mimics may present an effective therapy to treat such conditions in the clinic. The future directions of this project include determining the cell intrinsic role of miR-146a in this disease phenotype and working out the mechanism whereby miR-146a regulates macrophage metabolism.