bluuebunny/crossref_metadata_2025_split huggingface dataset:
TGF-β receptor 3 (betaglycan) regulates Th1 differentiation and T dependent B cell responses.
Authors: Raman Chander , Sestero Christine M. , Orlandella Rachael M , Boland Molly T , De Sarno Patrizia , Kesterson Robert A , Soldevila Gloria 
Published: January 2023

Abstract
TGFβR3, also known as betaglycan, is a co-receptor for the TGFβ receptor and is predicted to have an important function in promoting high affinity binding of TGF-β to TGFβR1 or TGFβR2. TGFβR3 also binds inhibins with high affinity and antagonizes activin dependent activation of SMADs a function independent of TGFβR1/TGFβR2 signaling. Contrary to TGFβR1 and TGFβR2, our knowledge on the role of TGFβR3 in the immune system is very sparse. Its biology has been elusive because the knock-out of Tgfbr3 exhibit embryonic lethality. We developed the first Tgfbr3f/f mouse in which Cre mediated deletion of Tgfbr3 is very efficient. We found that deletion of TGFβR3 in thymic T cells (Tgfbr3f/f.CD4Cre) had no effect on CD4 or CD8 double positive (DP) or single positive (SP) T cell populations or peripheral T cell populations. Treg numbers in the thymus and periphery were unaltered and the mice did not develop spontaneous autoimmunity as observed when Tgfbr2 is deleted during thymic development. These observations indicated to us that TGFβR3 likely had a function independent of “classical” TGF-β signaling through TGFβR1/2 receptors. To test for this possibility we generated Tgfbr3f/f.dLckCre) mice in which is Tgfbr3 is selectively deleted in mature CD4 and CD8 T cells. We found that T dependent Ab responses, particularly of the IgG2c subclass, was greatly enhanced in mice with T-cell restricted loss of TGFβR3. We further determined that the differentiation of naïve CD4 T cells to IFN-γ expressing Th1 cells under polarizing and non-polarizing conditions was enhanced when TGFβR3 was deficient. These and other data indicate that regulation of Th1 differentiation by TGFβR3 is novel and independent of TGF-β signaling.
B cells have bifurcated Toll-like receptor signaling allowing both inflammatory cytokine and type 1 interferon responses.
Authors: Akkaya Munir , Rawat Mukul , Akkaya Billur , Chaturvedi Akanksha , Pena Mirna , Miozzo Pietro , Kim Ann S , Pierce Susan K 
Published: January 2023

Abstract
B cells express the endosomal, DNA sensing, Toll-like receptor 9 (TLR9) that, in response to CpG oligonucleotides, signals through the adaptor protein MyD88 and activates the NF-kB pathway leading to the transcriptional activation of proinflammatory cytokines. In dendritic cells TLR9-MyD88 signaling is bifurcated into two distinct pathways, the NF-kB pathway and an IRF7-dependent pathway that results in the activation of type 1 interferon (IFN) genes. In these cells, monomeric CpG-B activates the NF-kB-dependent pathway and multimeric CpG-A or CpG-A coupled to the liposomal transfection reagent, Dotap (CpG-A-Dotap), activates the IRF-7-dependent pathway. Here we provide evidence that the B cell TLR9 signaling pathway is similarly bifurcated. Both CpG-B and CpG-B-Dotap potently activate the NF-kB-dependent pathway leading to IL-6 synthesis but neither trigger an IFN response. In contrast, CpG-A-Dotap but not CpG-A alone stimulates an IFN response as shown by increases in the levels of mRNA encoding IFN and in the amount of IFN protein produced and also by the activation of B cells from IFN-reporter mice both in vitro and in vivo. By confocal microscopy CpG-A and CpG-B appear to traffic to different intracellular vesicles. However, the cellular machinery that mediates the IL-6 and IFN pathways seems to be linked in that pretreatment of B cells with CpG-B blocks the subsequent activation of the IFN pathway by CpG-A-Dotap. It will be of interest to determine if natural DNA containing ligands might trigger the two pathways in B cells.
The transcriptional co-factor PARP14 limits severity of allergic skin inflammation
Authors: Krishnamurthy Purna , Serezani Ana PM , Kaplan Mark H 
Published: January 2023

Abstract
Atopic dermatitis (AD) is a chronic relapsing inflammatory condition of the skin that is characterized by pruritic skin lesions, erythema, and swelling, resulting in an impaired skin barrier. Patients with AD and mice subjected to models of AD develop an inflammatory T helper 2 (Th2) immune response and reduced expression of epidermal differentiation complex (EDC) genes, which encode proteins needed for keratinocyte maturation and barrier integrity. We are exploring the molecular controls regulating Th2 cells and keratinocytes in a mouse model of AD. Poly-ADP ribose polymerse-14 (PARP14) is a transcription co-factor that enhances Th2 differentiation. Correspondingly, PARP14-deficient mice have reduced lung inflammation in a model of allergic airway disease; however, its potential role in AD has not been explored. In keratinocytes, PARP enzymatic activity does not alter expression of EDC genes. Surprisingly, PARP14 deficiency resulted in an increase in the severity and number of skin lesions in an experimental mouse model of AD. Disease severity and increasing age corresponded with increases in Th2 cytokine secretion and antibody production (IgE). Adoptive transfer of PARP14 deficient T cells from a mouse model of AD resulted in increase in alopecia, epidermal thickening and altered EDC gene expression. Thus, PARP14 serves opposing roles in allergic disease: promoting pathogenic Th2 immune responses in the lung while potentially limiting T cell-dependent inflammatory responses during skin inflammation.
Exon 2 of Foxp3 contributes to the peripheral tolerance of the immune system
Authors: Du Jianguang , Wang Qun , Wang Lina , Zhou Baohua 
Published: January 2023

Abstract
Foxp3 is essential for the development and function of CD4 regulatory T cells (Tregs) that maintain peripheral immune tolerance. A major difference of Foxp3 expression between human and mouse is the alternative splicing of exon 2 in human Tregs. In vitro studies showed that the exon 2 region of Foxp3 associates and inhibits the transcription factors RORα and RORγt, which are critical for Th17 differentiation. However the in vivo role of exon 2 is still unknown. By deleting exon 2 of mouse Foxp3 gene (dE2), we show here that peripheral and mesenteric lymph nodes of dE2 mice contained about twice amount of lymphocytes when compared with wildtype mice due to the higher level of CD4 T cell activation and proliferation. Exon 2 deletion resulted in more severe air way inflammation in OVA-induced chronic asthma, characterized by higher number of lymphocytes and eosinophils in bronchoalveolar lavage, and high level of OVA-specific serum IgE. CD4 T cells in the mediastinal lymph nodes of dE2 mice expressed higher IL-4, IL-9 and IFN-γ, but surprisingly not IL-17, compared with those in wild type mice. These data indicate that Foxp3 exon 2 is important in maintaining proper functions of peripheral Tregs, and thus contributes to the peripheral tolerance to dampen allergic inflammation against harmless antigens.
Using mass cytometry to identify novel B cell subsets in red meat allergy
Authors: Cox Kelly M , Commins Scott , Capaldo Brian , Solga Mike , McSkimming Chantel , Chew Claude , Schuyler Alexander , Lannigan Joanne , McNamara Coleen , Erickson Loren 
Published: January 2023

Abstract
Previous studies have identified a novel food allergy driven by IgE antibodies specific for galactose-α-1,3-galactose (alpha-gal), an oligosaccharide found in red meat. While it is known that B cells play an important role in allergy as the producers of IgE antibodies that drive the allergic response, little is known about the phenotype of these B cells. The number of markers used to identify the major human B cell subsets by flow cytometry has been limited to common B cell proteins and thus precludes high dimensional immune phenotyping of B cell subsets, including unique phenotypes present in allergic individuals. We have addressed this problem by using mass cytometry (CyTOF), which enables the simultaneous analysis of up to 40 markers in a single staining panel. Here we analyzed the expression of 23 cell surface markers in PBMCs from 19 alpha-gal-allergic patients and 20 non-allergic controls by CyTOF. Additionally, we combined our CyTOF data with clinical endpoints to identify markers that may correlate with allergic disease. Our data reveals substantial heterogeneity within major B cells subsets on an individual level. Furthermore, our analysis identifies a number of markers that vary significantly in their expression in allergic versus non-allergic B cells and correlate with serum alpha-gal IgE titers. We hypothesize that B cells with this phenotype play an important role in mediating alpha-gal allergy. These findings demonstrate the power of using CyTOF and analytical tools to extract a hierarchy from high dimensional cytometry data in an unsupervised manner to identify known B cell subsets as well as to find novel B cell populations that differ between alpha-gal allergic and non-allergic individuals.
Mechanisms and consequences of Siglec-8 internalization and toxic payload delivery in human eosinophils
Authors: O’Sullivan Jeremy , Cao Yun , Bochner Bruce 
Published: January 2023

Abstract
Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is a cell surface protein expressed selectively on human eosinophils, mast cells, and basophils that induces eosinophil apoptosis and inhibits mast cell mediator release. Hence, Siglec-8 is an ideal target for diseases involving these cells. However, the effective delivery of therapeutic agents to these cells through Siglec-8 requires an understanding of the dynamics of Siglec-8 surface expression, which have not been elucidated. We observed by flow cytometry that Siglec-8 endocytosis in eosinophils proceeds slowly and plateaus at 80% loss in response to antibody or polyvalent synthetic ligand. Surprisingly, based on sensitivity to pharmacological inhibition, Siglec-8 appears to be internalized in both a clathrin- and lipid raft-dependent manner. Actin cytoskeletal rearrangement and tyrosine kinase and PKC activity are necessary for endocytosis. Interestingly, Siglec-8 is shuttled to the cell surface in a manner dependent on actin rearrangement, microtubule polymerization, and the activities of tyrosine kinases, dynamin, and PI3K. Neither process is significantly affected by inhibitors of Src family kinases or PTPs. Internalized Siglec-8 localizes to the lysosome, and sialidase treatment of eosinophils enhances ligand binding, indicating the presence of masking sialylated cis ligands on the cell surface. Finally, conjugating the ribosome-inactivating protein saporin to an anti-Siglec-8 mAb enhanced cell death induction in unstimulated eosinophils. The dynamics of Siglec-8 surface expression therefore appear to be suitable for sustained targeting and may deliver drugs to effectively treat diseases involving these cell types, including malignancies.
Specific subsets of kinases mediate Siglec-8 engagement-induced ROS production and apoptosis in human eosinophils.
Authors: Janevska Daniela , O’Sullivan Jeremy A. , Cao Yun , Bochner Bruce S. 
Published: January 2023

Abstract
Siglecs are type I transmembrane proteins expressed primarily on leukocytes. Among them is Siglec-8, a CD33 subfamily member that is selectively expressed on the cell surface of human eosinophils. Siglec-8 has an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM), putatively responsible for signal transduction. In cytokine-activated eosinophils, Siglec-8 binding causes apoptosis with increased mitochondrial damage and ROS production, but exact signaling mechanisms are unknown. Using a mAb (2C4) against Siglec-8 in combination with small molecule inhibitors, we first examined Siglec-8-mediated apoptosis and ROS production by flow cytometry after 24 hr IL-5 priming (30 ng/mL) of human eosinophils. We observed that 2C4-mediated eosinophil apoptosis was inhibited by PP1 and SU6656 (Src kinase inhibitors), ibrutinib (Btk inhibitor), LY294002 (PI3K inhibitor), GF109203x (PKC inhibitor), and sodium orthovanadate (a protein phosphatase inhibitor) at IC50’s of 1.7 μM, 1.8 μM, 0.9 nM, 1.2 μM, 2.4 μM, and 22 μM respectively. Complete inhibition of ROS production occurred at expected IC90’s. Western blot analysis following Siglec-8 cross-linking with 2C4 showed increased phosphorylation of Src530, Csk, PI3Kδ, and Blk that was detectable within 15 min, and c-Abl phosphorylation that was detectable within 60 min. Additionally, co-immunoprecipitation data shows that Siglec-8 associates with SHP-2, a protein tyrosine phosphatase. While the sequence of signaling events is yet to be determined, Siglec-8 mediated apoptosis in eosinophils involves the unexpected recruitment of molecules normally associated with cell survival.
Epithelial cell IKKβ regulates eosinophil levels in the intestine and severity of allergic responses to ingested allergens
Authors: Kim Eunsoo , Lembert Melanie M. , Martin Tara L. , Rowe John C. , Steiner Haley E. , Cormet-Boyaka Estelle , Boyaka Prosper N. 
Published: January 2023

Abstract
Allergic sensitization to food allergens has subsequent potential to developing allergic responses in the gastrointestinal tract, but also to skin or lung. Our previous study showed that lack of IKKβ in intestinal epithelial cells regulates favors IgA responses to ingested allergen, which in turn limits the severity of allergic responses in the airway. In this study we investigated whether intestinal epithelial IKKβ also regulated allergic responses to oral antigens. Wild-type C57BL/6 and IKKβΔIEC mice, which lack IKKβ in intestinal epithelial cells, were orally sensitized to a food antigen in the presence of cholera toxin. Allergen-specific serum IgE responses and fecal IgA responses were similar between the groups. However, after oral allergen-challenge, IKKβΔIEC mice only developed minimal clinical and histological signs of allergy, including drop in body temperature and mucus in small intestinal villi and crypts. Interestingly, IKKβΔIEC mice expressed lower levels of CCL11 (eotaxin) and eosinophils than control wild-type mice and their levels were only weakly increased after oral allergen sensitization and challenge. In summary, this study reveals a new role of intestinal epithelial cells in the regulation of allergy in the GI tract through a NF-κB-CCL11 axis.
Spingosine-1-phosphate receptor 1 agonist alleviates hypersensitivity pneumonitis induced by Methanosphaera stadtmanae
Authors: Huppé Carole-Ann , Lecours Pascale Blais , Talbot Maude , Morissette Mathieu , Marsolais David 
Published: January 2023

Abstract
Hypersensitivity pneumonitis (HP) is characterized by the formation of B cell-rich tertiary lymphoid tissue (TLT) in the lung, and by a granulocytic response upon antigen rechallenge. Clinically, antigen avoidance is the only effective approach to manage HP symptoms and progression. Sphingosine-1-phosphate receptor 1 (S1PR1) regulates key mechanisms underlying the formation of TLTs such as lymphocyte trafficking and intercellular interactions. Given that TLTs are key to the antigen-specific response in the lung, we investigated the impact of an S1PR1 agonist on pulmonary TLTs accumulation and determined the effect of quantitative TLTs modulation on the granulocytic response to an antigen rechallenge. Experimental HP was induced in mice using intranasal instillations of an immunogenic archaea found in farm bioaerosol, Methanosphaera stadtmanae (MSS), for 3 weeks. After a 48 hours resting period, mice were treated for 7 days with the S1PR1 agonist, RP001 (1mg/kg/day), or its vehicle using osmotic pumps. Five days after treatment initiation, mice were either euthanized, rechallenged with saline or rechallenged with MSS. Mice were euthanized 48h later to quantify pulmonary TLTs and inflammatory cells. RP001 did not influence TLTs accumulation and lung inflammation prior to MSS rechallenge. After MSS rechallenge, RP001 reduced the accumulation of TLTs by 49% (p=0.02) and reduced neutrophil accumulation by 39% (p=0.002), when compared to vehicle. Thus, S1PR1 modulation, after the initiation of a HP-like disease, alleviated TLTs accumulation along with the neutrophilic response to an antigen rechallenge. Our results support the concept that S1PR1 modulation can interfere with mechanisms that sustain HP.
The ORMDL3-ceramide axis may be a novel therapeutic target for the control of allergic airway disease
Authors: Sturgill Jamie L , Conrad Daniel H , Oyeniran Clement , Spiegel Sarah 
Published: January 2023

Abstract
Asthma, defined as a chronic inflammatory condition characterized by episodic shortness of breath with expiratory wheezing and cough, is a serious health concern. The WHO estimates that asthma affects more than 230 million people worldwide. There is a strong genetic component to asthma and numerous genome-wide association studies have identified ORM (yeast)-like protein isoform 3 (ORMDL3) as an asthma associated gene. Surprisingly however, the mechanism by which ORMDL3 contributes to asthma pathogenesis is not well understood. The yeast ortholog of ORMDL3 is a negative regulator of serine palmitoyltransferase (SPT), the rate limiting step in de novo ceramide synthesis, yet elevations of ceramide rather than its reduction have been linked to lung inflammation. Thus, we examined the role of ORMDL3 in asthma immunopathology. Consistent with its role in yeast, we show that decreasing expression of ORMDL3 in lung epithelial cells and macrophages increases ceramide and conversely, modest increases in ORMDL3 decrease ceramide levels. In a house dust mite (HDM) mouse model of allergic airway disease, allergen challenge induced expression of ORMDL3 and resulted in a concomitant increase in lung ceramide. Intriguingly, the use of specific drugs, which inhibit ceramide synthesis, prevented HDM-induced airway hyperreactivity (AHR) and suppressed airway inflammation. Nasal administration of the orally available FDA approved prodrug FTY720/fingolimod reduced both ORMDL3 expression and ceramide production while mitigating airway inflammation, hyperreactivity, and mucus hypersecretion in HDM challenged mice. Thus the ORMDL3 -ceramide pathway may be a novel therapeutic target for the control of allergic asthma.
Sex differences in M2-polarization during allergic lung inflammation
Authors: Keselman Aleksander M , McCormick Sarah M , Fang Xi Jessie , Heller Nicola M 
Published: January 2023

Abstract
Asthma is a chronic airway inflammatory disease affecting over 300 million people world-wide. Asthma exhibits sex differences by affecting mostly women, suggesting that hormones like estrogen may participate in disease progression. Alveolar macrophages become activated in asthma, promoting pathology but also repairing tissue damage. Estrogen is known to enhance macrophage activation and function in a variety of systems although its role in asthma is poorly understood. We hypothesized that macrophages from female mice are intrinsically hyper-responsive to IL-4 and that estrogen signaling enhances M2-polarization. We assessed IL-4-induced M2-gene expression in bone marrow-derived macrophages (BMMs) from male and female C57BL/6 mice and found enhanced expression of two M2 genes, Ym1 and Arg1, in response to IL-4 in female compared to male BMMs. Histone H3 acetylation and K4-trimethylation was elevated in female BMMs, consistent with enhanced transcriptional activity. Pre-treatment of BMMs with an ERα-specific agonist further enhanced IL-4-induced M2-gene expression. Next, we assessed sex differences in a mouse model of allergic lung inflammation. We found that alveolar macrophages from female compared to male mice exhibited increased Ym1 expression following allergen challenge. Therefore, macrophages exhibit intrinsic sex differences in their ability to polarize to an M2-phenotype and estrogen augments IL-4-induced M2-gene expression. Together these data suggest that intrinsic and hormonal factors contribute to sex differences in macrophages in asthma. Understanding the role of estrogen signaling in M2-polarization is paramount for identifying novel therapeutic targets to better treat women with asthma.
Differences in Innate immune cell production of IFNα and IFNβ to cell activation in the Autoinflammatory Diseases CANDLE (Chronic Atypical Neutrophilic Dermatosis with Lipodystrophy and Elevated temperature) and SAVI (STING Associated Vasculopathy with Onset in Infancy)
Authors: Marrero Bernadette , Liu Yin , Biancotto Angelique , Calvo Katherine R , Huang Yan , De Jesus Adriana A , Goldbach-Mansky Raphaela 
Published: January 2023

Abstract
The Type I IFN mediated autoinflammatory diseases SAVI and CANDLE are caused by gain-of-function mutations in Stimulator of Interferon Genes (STING) and loss-of-function mutations in proteasome components. We evaluated the innate immune cellular origin of IFN Type I in SAVI and CANDLE.
We examined expression of IFN genes from FACS sorted T, B, NK and monocytes in SAVI (n=5) and CANDLE (n=3) normalized to healthy controls (HC). The Interquartile Range of Type I IFN expression was 78(4–151) and 481-fold(56-38,242) of IFNB1 and 60(3–116) and 0.4-fold(0.1–39) of IFNA7/IFNA17 in CANDLE and SAVI monocytes, respectively. Monocyte depletion reduced IFN message by 90% and FACS analysis confirmed that monocytes and not dendritic cells are the main source of IFNs. The overall mean ratio of IFNβ to IFNα was 989:1 in SAVI and 1.3:1 in CANDLE monocytes.
Mean IFNα plasma levels measured by pan IFNα ELISA with 31pg/ml ±58 SEM in CANDLE and 230pg/ml ±112 SEM in SAVI. IP10 serum levels were 2215pg/ml ±702 SEM and 3632pg/ml ±860 SEM in CANDLE and SAVI, respectively. STING pathway activation with cGAMP in the presence of cycloheximide (CHX), a blocker of translational elongation, led to IFNB1 but not IFNA1, 2, 7, 17, 21 while CHX + epoxomicin, a proteasome inhibitor led to transcription of IFNAs and IFNB1 suggesting secondary upregulation of IFNAs in SAVI.
Compared to healthy controls, CANDLE and SAVI had increased left-shifted immature CD13lo CD16+ neutrophils (9 and 4-fold, respectively), as well as higher expression of the activation marker CD36+ (6.9 and 6.9-fold, respectively) in total granulocytes.
In conclusion, Type I IFN is mainly produced in monocytes and analyses of the IFN signaling in CANDLE and SAVI will allow us to develop improved targeted therapies.