bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expansion of the intraepithelial lymphocyte (IEL) compartment results in an increased bioenergetic profile and reduced IFNγ production in γδ IELs
Authors: Alonso Sara , Jia Luo , Laguerta Alyssa , Edelblum Karen 
Published: August 2023

Abstract
Intestinal γδ intraepithelial lymphocytes (γδ IEL) serve as the first line of defense against pathogen invasion. We recently identified a transmissible hyperproliferative γδ IEL (γδHYP) phenotype that protects against enteric infection, and these mice have no signs of overt intestinal pathology. Given that IELs are metabolically constrained to prevent aberrant activation, we hypothesized that the bioenergetics of γδHYP IELs may regulate their effector function. Electron microscopy revealed a 70% increase in mitochondrial number (p=0.005) and 24% increase in mitochondrial area (p=0.04) in γδHYP IELs relative to WT. Seahorse mitochondrial stress assays on γδ IELs showed a 50% increase in spare respiratory capacity in γδHYP IELs compared to WT (p=0.014). Mitochondrial metabolism was shown to influence γδ T cell cytokine production, and similarly, we found that stimulation with PMA and ionomycin reduced the frequency of IFNγ+ γδ IELs by 59% in γδHYP relative to WT (p&lt;0.0001). We also observed that γδHYP IELs exhibit higher CD39 expression compared to WT (p&lt;0.0001), and a CD39hi subset produces less IFNγ compared to CD39neg and CD39int cells (p&lt;0.0001) The addition of the ATP-synthase inhibitor oligomycin during IEL stimulation increased IFNγ production by both WT and γδHYP IELs by 66% and 44%, respectively (p&lt;0.0001). Together, our data demonstrate that the shift towards high CD39 expression and elevated mitochondrial metabolism contribute to decreased IFNγ production in γδHYP IELs. Further understanding of the mechanisms regulating γδ IEL homeostasis and effector function may ultimately allow fine tuning of mucosal surveillance to confer protection against intestinal injury or infection while limiting aberrant cytotoxicity.
Supported by NJCCR (COCR23PRF024)
Myeloid cells and sphingosine-1-phosphate are required for TCRαβ+ intraepithelial lymphocyte recruitment to the colon epithelium
Authors: Danielson Sarah Mann , Lefferts Adam , Kuhn Kristine A 
Published: August 2023

Abstract
Intraepithelial lymphocytes (IELs) of the colon are essential for barrier maintenance and repair after injury. As bacterial depletion or global MyD88 deficiency results in a significant reduction of colon IELs and impaired barrier integrity, we aimed to connect the signals of how bacteria recruit colon IELs to the epithelium using conditional knockouts of MyD88 to target T cells (Lck-Cre), IECs (Villin-Cre), and myeloid cells (LysM-Cre). Only in the colons of LysM-Cre+ mice were TCRαβ+ IELs reduced, at 3.3-fold (p&lt;0.005 compared to Cre− littermate controls) without effect on lamina propria T cells or myeloid cells. TCRγδ+ IELs were unaffected in all knockouts, suggesting they are recruited via different mechanisms. We then examined upregulated cell surface gene expression of IELs during recruitment, identifying sphingosine-1-phosphate receptor 1 (S1PR1) as a candidate for further study. Indeed, S1PR1 expression is significantly increased at 3.7-fold in IELs (p&lt;0.05 compared to lamina propria T cells). In accordance with these findings, we demonstrated that TCRαβ+ IEL recruitment is blocked by administration of the S1PR1 receptor agonist FTY720 (p&lt;0.05 compared to vehicle treatment). Furthermore, myeloid cell production of sphingosine-1-phosphate (S1P) was required for colon TCRαβ+ IEL recruitment in a MyD88-dependent manner. In the TNFΔARE/+ model of spontaneous colitis, loss of myeloid MyD88 resulted in protection from disease (p&lt;0.05 compared to Cre-littermate controls). Overall, these data demonstrate the requirement for myeloid-produced S1P for TCRαβ+ IEL recruitment to the colon and suggest that TCRαβ+ IELs can contribute to intestinal inflammation under a specific genetic predisposition.
Weight cycling may induce lipid handling in mast cells
Authors: Caslin Heather L , Cottam Matthew A , Betjemann Alexa , Mashayekhi Mona , Silver Heidi J , Hasty Alyssa 
Published: August 2023

Abstract
Weight cycling worsens diabetes risk in both humans and mice. We recently found that weight loss and weight cycling induce many immunological changes in the adipose tissue of mice by single cell RNA-sequencing. We also found a substantial increase in adipose mast cells with weight cycling, but not weight loss. The purpose of this study was to confirm and characterize the changes in mast cells with weight cycling. However, using flow cytometry (cKit+FcER1+) and Toluidine blue staining, we found that adipose mast cells are greatest in the weight loss group, in contrast to our prior study using single cell RNA-sequencing. Upon further analysis, we found two unique mast cell populations in our single cell sequencing dataset: one that we consider traditional mast cells and one with lower expression of mast cell surface and protease genes and elevated expression of lipid handling and antigen presentation genes. The second fraction appeared similar to lipid-associated macrophages and accounted for most of the mast cells in the weight cycled group. By flow cytometry, we confirmed the %CD9+, MHCII (I-A/I-E)+, and CD74+ mast cells were highest in weight cycled mice. We also found two mast cell populations in human subcutaneous adipose tissue that appear similar to the murine populations in our dataset, with one population significantly correlating with weight variability. Together, these data suggest that weight cycling may induce a population of lipid-associated mast cells. We next aim to to induce lipid handling in bone marrow derived mast cells by repeated exposure to adipose conditioned media in order to understand the mechanisms which induce lipid handling.
This project was funded by a Veterans Affairs Merit Award 5I01BX002195 to AHH. AHH is also supported by a Career Scientist Award from the Veterans Affairs (IK6 BX005649). HLC was funded by an American Heart Association Postdoctoral Fellowship (20POST35120547), MAC was funded by an NIH F31 Predoctoral Fellowship (1F31DK123881), and both were previously supported by the Molecular Endocrinology Training Program (T32 DK07563). Additionally, the human mast cell work was funded by VICTR grants to HLC and MM. The Vanderbilt Institute for Clinical and Translational Research (VICTR) is funded by the National Center for Advancing Translational Sciences (NCATS) Clinical Translational Science Award (CTSA) Program, Award Number 5UL1TR002243-03.
Dietary whey protein increases brain leukocytes in mice regardless of their hypersensitivity status
Authors: Ekanayake Dilini Vimarshani , Nookala Suba , Nagamoto-Combs Kumi 
Published: August 2023

Abstract
Cow’s milk allergy (CMA) often manifests as milder reactions and may be linked to neurological problems. Previously, we demonstrated that C57BL/6J mice sensitized to a bovine whey allergen, β-lactoglobulin (BLG, Bos d 5), moderately increased BLG-specific IgE levels and exhibited behavioral changes without severe allergic reactions. When these non-anaphylactic CMA mice were placed on a whey-protein (WP)-containing diet for 2 weeks to simulate continuous dairy consumption, we found neuropathology indicative of neuroinflammation and cortical demyelination. Since immune cells migrate to the central nervous system (CNS) and promote neuroinflammation in demyelinating conditions such as multiple sclerosis, we hypothesized that the number of leukocytes would increase in BLG-sensitized mouse brains to orchestrate neuropathology. To test this hypothesis, we used flow cytometry to determine the number and phenotypes of leukocytes in the brains of naïve, sham, and BLG-sensitized mice after the 2 weeks of the WP diet. The frequencies of cells expressing common leukocyte marker CD45, pan T cell marker CD3, cytotoxic T cell marker CD8, integrin CD11b, myeloid cell marker CD14, and co-stimulatory marker CD86 significantly increased, regardless of the sensitization status. The percentages of these cells were low in mice that never received WP. This result indicated that WP diet consumption alone increased CNS leukocyte populations. Additional immunophenotyping is needed to determine whether the identified cells can be differentiated among the experimental groups. Detailed characterization of CNS leukocyte phenotypes and dynamics will help elucidate the mechanism of CMA-induced neuroinflammation and cortical demyelination.
Continuous consumption of whey protein affects motor and cognitive functions in a mouse model of cow’s milk hypersensitivity
Authors: Verma Geetika , Nagamoto-Combs Kumi 
Published: August 2023

Abstract
Hypersensitization to a food allergen can occur in the absence of immediate allergic reactions, a condition referred to as asymptomatic sensitization. Using mice asymptomatically sensitized to a bovine whey allergen, β-lactoglobulin (BLG, Bos d 5), we have previously shown that chronic exposure to the allergen results in behavior changes with increased neuroinflammation and cortical demyelination. However, whether allergen avoidance can resolve these pathologies is unknown. Thus, we aimed to compare immunological and behavioral changes in sham- or BLG-sensitized mice with or without the removal of dietary whey after the mice had consumed either a control diet (CTL) or a whey-protein-containing diet (WP) for 2 weeks. As previously reported, BLG-sensitized mice did not display overt physical reactions during the 2-week allergen exposure period, while their plasma levels of BLG-specific IgE and IgG1 were elevated compared to the respective sham groups. Following an additional 2 weeks on either the CTL or WP diet, these antibodies remained elevated in some of the sensitized mice in both diet groups. The grip-strength test indicated that sham mice that switched to the CTL diet after the 2-week WP diet increased limb strengths. In contrast, BLG-sensitized mice on either diet, as well as sham mice that stayed on the WP diet, showed a decline in limb strengths, suggesting that WP consumption, whether during sensitization or as part of the daily diet, affected their motor function. Furthermore, removing dietary WP improved spatial memory performance with the cross-maze test in both sham and BLG-sensitized mice. These results suggested that continuous exposure to a dietary allergen may influence motor and cognitive functions.
Supported by grants from NIH (R01 AI168563)
B cell intrinsic Toll-like receptor 9 signaling regulates class switching to IgE
Authors: Chang Karen , Nelson Alexander J , Wu Yee Ling 
Published: August 2023

Abstract
Chronic allergic diseases such as allergic asthma are driven by excessive production of allergen specific IgE. B cells produce IgE by class switch recombination (CSR) in response to T cell help and Th2 cytokines. However, development of allergic diseases can also be influenced by factors such as early-life exposure to microbial products. In vivo, stimulation of Toll-like receptor 9 (TLR9) with microbial DNA reduces IgE levels, largely thought to be a response to Th1 cytokines. However, the direct effects of TLR9 signaling on B cell class switching to IgE remain unclear. To delineate the B cell intrinsic effects of TLR9 stimulation, we treated mouse splenic B cells in vitro with IL-4 and anti-CD40 antibody with or without a synthetic TLR9 agonist, CpG oligodeoxynucleotides (CpG). Compared to stimulation with IL-4 and anti-CD40 alone, the addition of CpG decreased IgE production. Using B cells from reporter mice that track germline transcription, a prerequisite for CSR, we found that CpG inhibited Iɛ but increased Iγ1 germline transcription. By Western blot analyses, we probed the expression of transcription factors known to negatively regulate Iɛ and positively regulate Iγ1 transcription. These transcription factors were synergistically upregulated in the presence of CpG with IL-4 and anti-CD40. Together, these data illustrate a molecular mechanism by which microbial products directly perturb the production of IgE and influence the development of allergic disease.
R01 HL165120
Nutritional factors govern gut homeostasis via oral tolerance
Authors: Nagai Motoyoshi , Okawa Takuma , Nakata Kazuaki , Kawamura Yuki I , Hase Koji 
Published: August 2023

Abstract
Oral tolerance is one of the most critical systems to regulate allergic disorders. However, how dietary factors regulate the induction and maintenance of oral tolerance remains to be elucidated. To address this, we temporarily omitted the food supply by fasting before oral ovalbumin (OVA) administration. We observed that fasting mitigated the expansion of OVA-specific Treg cells. This abnormality mainly resulted from functional defects in CX3CR1high macrophages responsible for the uptake of luminal OVA and the reduction of tolerogenic CD103+dendritic cells (DCs). Eventually, fasting impaired the preventive effect of orally administrated OVA in the development of asthma and allergic rhinitis. We further explored the food ingredients essential for the induction of oral tolerance and found that the intake of dextrin and a basic amino acid is indispensable for oral tolerance induction. These observations illustrate that nutritional signals are critical in maintaining immune homeostasis via induction of tolerance to ingested food antigens.
Maternal-neonatal model of immune protection to type 2 pulmonary inflammation
Authors: Krishnamoorthy Nandini , Smith Ethan , Bruggemann Thayse , Walker Katherine , Chiang Nan , Serhan Charles N , Levy Bruce D 
Published: August 2023

Abstract
Asthma tends to “run in families” is a concept supported by several research studies of asthma genetics, but this only explains a relatively small fraction of asthma inheritance. We are now standing this paradigm on its head by asking the opposite question: Can asthma protection be passed on from one generation to the next? Epidemiological and experimental studies have determined that the early postnatal period offers a critical window for the establishment of lung protection. Maternal milk represents a potentially important transgenerational mechanism by which offspring may acquire early-life immunity from their mothers. To study this transfer of protection we have developed a new preclinical animal model of maternal transference of immune protection. In this model, maternal exposure to endotoxin postpartum resulted in infant protection from allergic inflammation to the common indoor allergen house dust mite and to a clinical strain of Respiratory Syncytial Virus (RSV). The overall downregulation of type 2 immune responses to an allergen and a virus indicated that the mechanism of protection was common for different antigens. Metabololipidomics analysis of maternal milk from endotoxin-exposed mothers revealed elevated levels of select specialized pro-resolving mediators (SPMs) from essential fatty acids. Young animals from endotoxin-exposed mothers had overall lower allergic immune responses, which was related to altered pulmonary dendritic cell (DC) subsets. Importantly, these protective effects of asthmatic inflammation reduction were also recapitulated with the direct administration of SPMs. This study provides a novel transgenerational immunological conduit of protection in a heretofore-unexamined process
The role of NFAT in CD8+Foxp3+ Tregs
Authors: Patino Liliana C , Ortega-Recalde Oscar , Gauthier Thierry , Liu Na , Jin Wenwen , Chen WanJun 
Published: August 2023

Abstract
CD8+Foxp3+ regulatory T cells (CD8 Treg) correspond to a very low frequency population which can be observed during GVHD at early stage or in some cancer types. However, in vitro, around 30–60% of CD8 can express Foxp3 after induction culture with TCR stimulation and TGF-β (iCD8Treg). Why in healthy mice and humans this cell population has very low or non-frequency been still an unresolved question. In vitro, iCD8Tregs can suppress effector cells in a similar way as iCD4Treg. However, in vivo, in the IBD model, we observed that in contrast to iCD4Treg, iCD8Treg lose the Foxp3 expression and consequently their suppressive function. Also, iCD8Treg increase the IFN-γ production. These results suggest that iCD8Treg are not able to maintain the Foxp3 expression in comparison with iCD4Treg cells. What signaling pathway is responsible for the Foxp3 losing in iCD8Treg? We found a positive correlation between Foxp3 maintenance and cyclosporin A (CSA) concentration, and a reduction of IFN-γ in culture in iCD8Treg, but not for iCD4Treg. We evaluated the effect of CSA, an NFAT inhibitor, on the Tregs induction culture (with TGF-β) in the CD8 cells and found that there was no increase of Foxp3 expression. These results suggest that NFAT signaling plays a role in the Foxp3 maintenance but not in the induction of the iCD8Treg and this role is different from iCD4 Tregs.
A STAT5 activator protects CD8+ T cells in immunosuppressive environments
Authors: Shudde Claire E , Gu Zehui , Piper Taylor L , Zheng Yating , Courtney Adam H 
Published: August 2023

Abstract
CD8+ T cells are central mediators of an anti-tumor immune response because they can recognize and kill cancerous cells. However, cancer can evade this attack by establishing a tumor microenvironment (TME) that suppresses CD8+ T cells by depriving them of pro-survival cytokines, such as IL-2. IL-2 has been directly administered to cancer patients as an immunotherapy, but was found to cause broad immune activation that could result in multi-organ failure and possessed poor pharmacologic properties. To address these limitations, we have devised a strategy to activate the intracellular mediator of IL-2 signaling – STAT5. The activator of endogenous STAT5 that we developed was evaluated for its capacity to maintain T cell survival and function. To recapitulate a niche devoid of pro-survival cytokines like the TME, we activated CD8+ T cells in the absence of IL-2 and evaluated their viability in culture. We determined that direct activation of STAT5 could maintain CD8+ T cell viability up to 10 days post-activation, whereas control cells died off by day 3. To investigate whether STAT5 activation could be used to retain CD8+ T cell function, we assessed the potential of these cells to become activated by antigen and kill cancerous cells. We found that T cells that persisted in the absence of IL-2 due to STAT5 activation could upregulate a reporter of T cell activation (Nur77-GFP) and kill cancer cells ex vivo. Moreover, STAT5 activation enabled CD8+ T cells to produce IFNγ upon restimulation by antigen presenting cells. Our data demonstrate that a STAT5 activator can be used to maintain T cell survival, activation, and cytotoxic capacity in cytokine-depleted environments, suggesting its potential to counteract an immunosuppressive TME.
Supported by grants from NIH (T32GM140223), V Foundation for Cancer Research, and the Concern Foundation
The role of the ubiquitin ligase Cul4b in B lymphocytes
Authors: Davis Destiny K , Elliott Gabrielle , Renshaw Lindsay , Moser Emily 
Published: August 2023

Abstract
Vaccination or infection induces antibody secretion by long-lived plasma cells, creating durable immune protection against secondary infections. These plasma cells develop from B cells that have undergone affinity maturation, which requires somatic hypermutation in proliferating B cells, forming single strand DNA breaks that are repaired by mismatch repair machinery. Cullin 4b (Cul4b) is an E3 ubiquitin ligase found in most cells, including B cells, but its function in B cells is unknown. Cul4b is one of two docking proteins forming the backbone of the Cullin RING Ligase 4 (CRL4) complex, which ubiquitinates substrate proteins for degradation. Previous studies have shown that Cul4b promotes DNA repair with cell proliferation, and high Cul4b is linked to aggressive colon cancer. Because proliferation and DNA repair are essential for humoral immunity, we hypothesized that Cul4b also functions in B cells. To test this, we generated mice that lack Cul4b in B cells. We immunized these mice with an mRNA vaccine against an H1N1 Influenza A virus, and measured virus-specific serum antibody. We found that Influenza-specific antibodies were dramatically reduced in mice serum that lacked Cul4b in B cells. To determine if Cul4b promotes DNA damage repair in B cells, we induced single-strand DNA breaks with UV irradiation and then stimulated the cells to proliferate in vitro. We found that Cul4b promoted B lymphocyte proliferation after UV exposure. Understanding the Cul4b-DNA damage repair pathway in B cells will identify new drug targets to transiently amplify this pathway to improve vaccines and encourage stronger antibody responses, or to inhibit this pathway to prevent tumor progression in cancer.
Phenotypic Evaluation of TREX1 Null Mice as a Model for Lupus-Like Autoimmune Disease
Authors: Li Zhiying , Liang Juan , Chen Zhong , Xu Shuxin , Chen Yingjiao , Tong Huan , Ku Cunxiang , Zhao Jing , Xiang Gao 
Published: August 2023

Abstract
TREX1 (DNase III) is the major mammalian 3’-5’ DNA exonuclease and thought to play a major role in cell death and genomic DNA degradation. Dysfunction of Trex1 has been suggested to activate immune response by self DNA, as TREX1 null mice develop inflammatory myocarditis similar to autoimmune cardiomyopathy and produce type 1 IFN. TREX1 D18N causes a monogenic cutaneous form of lupus called familial chilblain lupus and exhibits dysfunctioned dsDNA-degrading activity, providing a link between dsDNA degradation and nucleic acid-mediated autoimmune diseases. Using CRISPR-Cas9, we generated a Trex1−/− mouse by knocking out exon 2 and performed comprehensive phenotypic analyses to evaluate the loss of function of TREX1 and its potential application in SLE research.
Both female and male TREX1 null mice showed dramatically increased post-wean morbidity and significantly increased serum ALT, AST and LDH levels, compared to wildtype and heterozygotes from 6 to 24 weeks of age, indicating liver and heart injuries. Pathohistological analysis showed inflammatory immune cell infiltration in liver, heart, spleen, kidney, lung and skin among TREX1 null mice. Serum anti-dsDNA antibody levels were significantly increased and remained high from 7 to 25 weeks of age, consistent with Trex1’s being the major DNA exonuclease. Urine protein to creatinine ratio was dramatically increased at 28 weeks of age among TREX1 null mice.
In conclusion, the Trex1−/− mice we generated in this work showed lupus-like inflammatory autoimmune responses in multiple organs, providing an urgently needed tool to study Trex1-mediated autoimmunity and to evaluate potential therapeutic treatments for lupus.