bluuebunny/crossref_metadata_2025_split huggingface dataset:
MicroRNA-10a negatively controls regulatory T cell suppression of colitis through inhibition of mitochondrial oxidation and Blimp1 expression
Authors: Yang Wenjing , Yu Tianming , Yao Suxia , Cong Yingzi 
Published: January 2023

Abstract
MicroRNAs play critical roles in maintaining intestinal homeostasis, and regulatory T cells (Tregs) restrict intestinal inflammation. However, how Tregs is regulated is still not completely understood. In this study, we investigate the mechanisms by which miR-10a controls Tregs’ suppressive function, thereby regulating intestinal inflammation. We found that miR-10a deficient Tregs expressed higher function markers in the intestine and were more potent in suppressing T cell proliferation and colitis induced by CD4+ CD45Rbhi T cells. Moreover, miR-10a did not affect Treg stability, which was evidenced by no differences in Foxp3 expression and methylation in Regulatory T-Cell-Specific Demethylated Region (TSDR). RNA sequencing data indicated that miR-10a regulates Tregs’ oxidative phosphorylation, which was verified by a higher level of oxygen consumption and mitochondrial ROS in miR-10a deficient Tregs. Pretreatment with a low dose of oligomycin, the ATP inhibitor which reduces mitochondrial respiration, impaired Tregs function in suppressing naïve T cell proliferation and colitis induced by CD4+ CD45Rbhi T cells in Rag−/− mice. Mechanically, miR-10a directly binds to uqcrq, the subunit VII of mitochondrial respiratory chain complex III, and Prdm1, which encodes Blimp1, a transcription factor that regulates Tregs’ functions. Furthermore, Blimp1-deficient Tregs showed decreased expression of function markers with the lower level of OCR and failed to restrict CD45Rbhi T cell-induced colitis. Collectively, miR-10a negatively regulates Tregs’ suppressive function by altering the mitochondrial metabolic process and inhibiting Blimp1, thereby controlling intestinal inflammation.
The autophagy gene Epg5 promotes susceptibility to enteric viral infection
Authors: Kalugotla Gowri , Lee Sanghyun , Schriefer Lawrence , Perez Luis Alberto Casorla , Huang Huiyan , Lu Qun , Orvedahl Anthony , Silverman Gary , Pak Stephen , Wang David , Baldridge Megan T 
Published: January 2023

Abstract
Mutations in the autophagy gene EPG5 are responsible for Vici syndrome, a multisystem human genetic disease with a combined immunodeficiency component. However, the mechanism(s) by which EPG5 regulates immune signaling are incompletely understood. Previously, we found that Epg5−/− mice are resistant to influenza and exhibit hyperinflammation in the lungs including elevated IL1B/IL-1β and TNF/TNFa. Here, we show that the disruption of Epg5 results in protection against multiple enteric viruses including norovirus and rotavirus. RNA sequencing revealed that IFN-λ responses are highly upregulated in the intestines of Epg5−/− mice. Further, mice lacking Epg5 exhibit substantial alterations of the intestinal microbiota. Surprisingly, germ-free Epg5−/− mice showed persistent inflammation of both the intestine and lung, suggesting a microbiota independent mechanism. Epg5−/− Ifnlr1−/− mice, which lack the receptor for IFN-λ, regained susceptibility to viral infection but maintained microbial dysbiosis, indicating that IFN-λ signaling is the primary mediator of resistance to enteric viruses but is dispensable for microbial dysbiosis in Epg5−/− mice. Intriguingly, epg-5-mutant Caenorhabditis elegans animals are also resistant to Orsay virus, which is an intestinal cell-tropic virus and the only known virus that naturally infects C. elegans. This study unveils an important role for autophagy gene Epg5 in protection of mammalian hosts by modulating intestinal IFN-λ responses, which is unexpectedly independent of the microbiota, and raises the possibility of a conserved viral resistance mechanism that is shared between C. elegans and mammals.
Intestinal epithelial autophagy protects from cytokine-driven mortality and IFNγ-dependent cell death in acute small intestinal injury
Authors: Foerster Elisabeth G , Streutker Catherine J , Maughan Heather , Tsang Derek K L , Robert Lukian M , De Sa Oliver , Girardin Stephen E , Philpott Dana J 
Published: January 2023

Abstract
Crohn’s disease (CD) is characterized by a breakdown of the intestinal mucosal barrier. A genetic polymorphism of the autophagy gene ATG16L1 is associated with increased risk of developing CD, and it impairs the protective and regenerative functions of the intestinal epithelium. Autophagy acts as a regulator of cell death and has been implicated in the protection from both necroptosis and apoptosis in the epithelium.
Our work showed that mice specifically deleted for Atg16l1 in intestinal epithelial cells (Atg16l1ΔIEC) had exacerbated T cell-mediated intestinal damage in the model of anti-CD3 enteropathy compared to wild-type mice, which was characterized by crypt epithelial cell death and heightened inflammation. Moreover, Atg16l1 deficiency delayed the recovery of the intestinal epithelium, and Atg16l1-deficient IECs were impaired in their proliferative response. Pathology and mortality were largely driven by microbiota-dependent expression of Ifng, Tnf, and type I IFN response genes in Atg16l1ΔIEC mice. Mechanistically, while survival was rescued by blocking these cytokine pathways independently, only anti-IFNγ treatment abrogated intestinal epithelial cell (IEC) death in Atg16l1ΔIEC mice, thereby decoupling IEC death and survival. In summary, our findings suggest differential roles for IFNγ, TNF, and type I IFN in acute enteropathy and IEC death in the context of autophagy deficiency.
Supported by grants from CIHR (MOP-12353, FDN-14333) and NSERC (RGPIN-201).
NAIP—NLRC4 inflammasome activation in tuft cells contributes to host defense against bacteria
Authors: Churchill Madeline J , Rauch Isabella 
Published: January 2023

Abstract
The small intestinal epithelium is exposed to various microbes, and intestinal epithelial cells (IECs) have developed an array of host defense mechanisms to distinguish commensals from pathogenic microbes and initiate an immune response against the later. NAIP—NLRC4 inflammasome induced-pyroptosis, which is activated when bacterial flagellin or type III secretion systems are detected in the cytosol of the host cell, is one such mechanism. Activation of NAIP—NLRC4 induces extrusion of IECs and release of the inflammatory mediators prostaglandin E2 and IL-18. Work on epithelial inflammasome activation thus far has not delineated if different subtypes of IECs have specific roles in the ensuing immune response. Tuft cells are a rare subset of IEC that is primarily known for its role in sensing parasites, and it is unknown whether they are involved in promoting antibacterial host responses. Here we show that upon activation of the NAIP—NLRC4 inflammasome specifically in tuft cells, they uniquely among IECs release prostaglandin D2, and within the small intestine, tissue IL-22 increases. NLRC4 expression only in tuft cells leads to better control of Salmonella Typhimurium compared to NLRC4 null controls. Taken together, these data suggest that NAIP— NLRC4 inflammasome activation in tuft cells leads to lipid mediator induced downstream inflammatory responses that are complementary to signaling pathways that are activated by other IEC subsets. Understanding how NAIP—NLRC4 activation in different epithelial subsets affects downstream inflammatory responses will inform on the different signaling pathways that synergize together to mount pathogen specific host defense mechanisms.
Contribution of the microbiome, environment, and genetics to mucosal Type 2 immunity and anaphylaxis in a murine food allergy model
Authors: Stark Kelsey , Falkowski Nicole R , Brown Christopher A , McDonald Roderick A , Huffnagle Gary B 
Published: January 2023

Abstract
Murine models of food allergy are useful tools for identifying potential pathways involved in the manifestation of anaphylactic reactions to food in humans. Individual mice exhibit inherent heterogeneity in their immune responses, including anaphylaxis, much like what is seen clinically in food allergic humans. The microbiome (MB) may be one major driver of this difference in individuals. MB dysbiosis by broad spectrum antibiotics and/or yeast colonization can alter systemic immunity and favor the development of mucosal Type 2 immunity to aeroallergens. Our objective was to use a well-characterized murine model of food allergies (chicken egg ovalbumin, OVA) and determine if dysbiosis could enhance the manifestation of food allergies and identify elements of the MB and host response associated with this heterogeneity. In our dataset, the intensity of anaphylaxis was most strongly associated with a disrupted MB that included C. albicans colonization, loss of a specific Lachnoclostridium species, development of a highly polarized Type 2 response in the intestines, and activation of mucosal mast cells. OVA-specific IgE serum levels were not predictive of the response and in germ-free mice the response was not fully recapitulated. Conventionalization of germ-free mice resulted in Akkermansia muciniphila outgrowth and more heterogeneity in the allergic response. We also observed that housing environment could induce MB changes that altered the response. Thus, our data recapitulate the heterogeneity in anaphylactic reactions, ranging from severe to none, seen in patients with circulating levels of food allergen-specific IgE and support the idea that alterations in the MB can be one factor underlying this heterogeneity.
Supported by grants from NIH NIAID R01AI138348, Mary H. Weiser Food Allergy Center (MHWFAC), and the Nina and Jerry D. Liptak Endowment of the MHWFAC
Exploiting commensal bacteria to strengthen host immunity and treat infections
Authors: Ramirez Zeni Elizia , Surana Neeraj K 
Published: January 2023

Abstract
As the rise of microbial resistance to antibiotics continues, there is a pressing need for new therapeutic strategies. One method includes augmenting host immunity and subsequent defense molecules to protect against pathogenic organisms. Antimicrobial peptides (AMPs) represent an ancient arm of the innate immune system that is critical for maintaining host–microbiome interactions. We previously identified that the commensal bacteria Ruminococcus gnavus and Lactobacillus reuteri induce ileal expression of Reg3γ, an AMP with bactericidal activity against gram-positive bacteria. We now find that R. gnavus and L. reuteri induced expression of these AMPs requires Myd88 and group 3 innate lymphoid cells but, interestingly, does not require persistent probiotic colonization. Consistent with this increase in AMP expression, R. gnavus- and L. reuteri-treated mice were protected against intestinal colonization by vancomycin-resistant enterococci (VRE). Finally, patients at high-risk for VRE domination were &gt;4 times less likely to have Enterococcus in their fecal microbiota if they also had Ruminococcus present, a finding that demonstrates the clinical translatability of R. gnavus mediated protection. Taken together, these results spotlight the potential for probiotic based therapies to combat antimicrobial resistant organisms.
Supported by grants from NIH (1F31AI161989-01)
Generation of large numbers of functional NK cells without feeders or serum
Authors: Le Fevre Tim , Ang Elaine , Wognum Albertus W , Szilvassy Stephen J , Eaves Allen C , Louis Sharon A , Tabatabaei-Zavareh Nooshin 
Published: January 2023

Abstract
Natural killer (NK) cells are innate lymphocytes that modulate immune responses by secreting proinflammatory cytokines, and provide antitumor and antivirus activity. The ability to generate therapeutically relevant numbers of functional NK cells is critical for the development of advanced cellular immunotherapies. We have developed a streamlined culture workflow that enables expansion of functional NK cells without using either feeder cells or serum. Fresh human NK cells were isolated from peripheral blood using EasySep™, then cultured in ImmunoCult™ NK Cell Expansion Medium in plates coated with ImmunoCult™ NK Cell Expansion Coating Material. NK cells were fed, harvested, and replated on days 7 and 10 – 11 prior to phenotyping and functional assessment on day 14. The average frequency of CD56+ CD3− NK cells was 88% (range 75 – 96%, n=34), 74% (range 49 – 92%) of which expressed CD16, with an average expansion of 88 fold (range 3 – 454). Expanded NK cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, or co-cultured with K562 cells. As detected by intracellular flow cytometry, the average frequency of interferon-γ+ among NK cells was 68% (range 41 – 90%, n=7) and 43% (range 12 – 55%) when stimulated by PMA/ionomycin or K562 cells, respectively. Similarly, the frequency of degranulated NK cells (CD107a+) was 81% (range 35 – 97%, n=7) and 64% (range 35 – 97%), respectively. The ability of expanded NK cells to kill target K562 cells was visualized by activated caspase in co-cultures with labeled K562 cells using the Incucyte® imaging system. At an effector:target ratio of 1:1, an average of 50% of K562 cells were killed (range 45 – 56, n=3). These results show large numbers of functional NK cells can be generated.
Characterization of a novel mouse model for deletion of a floxed gene in alternatively activated macrophages
Authors: Vo Thao T , Patial Sonika , Saini Yogesh 
Published: January 2023

Abstract
V-maf musculoaponeurotic fibrosarcoma oncogene family, protein B (MafB) promoter has been identified as macrophage-specific promoter, but it has never been characterized for pulmonary macrophages. Therefore, we hypothesized that MafB-driven Cre Recombinase expression will allow recombination in alveolar macrophages that contain floxed alleles. MafB-Cre+/− \Rosa-mTOMFlox/Flox/mEGFP (MafB-EGFP) mice was generated by crossbreeding MafB-Cre+/+ and Rosa-mTOMFlox/Flox/mEGFP parents. To determine the extent of macrophage-specific gene recombination, we performed bronchoalveolar lavage, fluorescent microscopy, and flow cytometry on BALF and lung digest of 42 days old MafB-EGFP mice. In addition, we assess the beneficial effect of in vitro and in vivo macrophage activation on recombination efficiency. Flow cytometry analyses on macrophages from MafB-EGFP offspring revealed that: 1) only 30–40% of the alveolar macrophages are amenable to the Cre-Lox mediated recombination, 2) majority of bone marrow-derived macrophages are amenable to the Cre-Lox mediated recombination, 3) Classical activation status did not affect the Cre-Lox recombination efficiency in freshly cultured alveolar macrophages. However, the alternative activation increased the recombination efficiency to ~80–90% in cultured alveolar macrophages. Intratracheal IL-33 treatment of MafB-EGFP mice also increased Cre-Lox mediated recombination of alveolar macrophages to ~80%. Our data suggest that while MafB-EGFP mice do not allow Cre-Lox recombination in the homeostatic lung and inflamed lungs with classically activated macrophages, these mice allow significant recombination in alternatively activated macrophages.
Supported by NIEHS (R01, ES030125)
RenMice™ HiTS platform enables identification of novel therapeutic antibodies
Authors: Frame Jenna , Zhang Xiaoqian , Jin James , Soto Rebecca , Zhang Shujin , Li Xin , Zhang Jing , Shen Yuelei 
Published: January 2023

Abstract
Despite an increase in approved cancer-targeting antibody drugs over the last decade, the process of identifying novel therapeutic antibodies is routinely hampered by limitations in the discovery process. Such barriers include immune tolerance of highly homologous genes, antibody sequence humanization, clone selection and models for drug efficacy/safety evaluation. To overcome these challenges and increase the diversity of antibody paratopes and sequences that recognize functional epitopes, we developed the RenMice™ HiTS (Hyperimmune Target Specific) Platform, which consists of chromosome engineered mice with fully human immunoglobulin variable domains replacing the mouse loci, each with a specific target gene knocked out. Immunization of target-specific RenMice™ generates a sizeable diversity of antibodies, including those that recognize conserved regions between the antigen and the endogenous proteins of the immunized species. The platform is ideal for challenging targets, such as proteins with high homology across species, or multi-pass transmembrane proteins, such as GPCRs/ion channels, and can be used to generate antibodies that cross-react with human, monkey, dog, and mouse targets using a hybrid immunization strategy with both human and mouse/dog antigen. Generation of these species cross-reactive antibodies can be used for high-throughput in vivo efficacy screening in wild-type mice, and the preliminary response and toxicity can be assessed in dogs. Altogether, the RenMice™ HiTS platform facilitates the generation of antibodies that recognize novel epitopes and challenging targets while simultaneously allowing for a streamlined and successful preclinical phase based on in vivo efficacy and safety.
Effects of sample storage using tumor and tissue preservation reagent on genomic data quality
Authors: Shi Xiaoshan , Tran Aimee , Widmann Stephanie , Tyznik Aaron J 
Published: January 2023

Abstract
In-depth understanding of the intrinsic properties and complexity of the tumor microenvironment is crucial to develop both diagnostic and therapeutic strategies for cancer treatment. The tumor microenvironment is composed of various cell types, all of which interact and collectively determine cancer progression and response to therapy. Additionally, tumor cells have a strong ability to adapt to environmental stimuli and can evade treatment modalities. As a result of those features, it is essential to understand the dynamic gene expression profile to decipher tumor heterogeneity and develop efficient and safe therapeutic strategies to fight cancer. Challenges are faced by downstream genomic assay design where tumor specimens cannot be processed immediately in the clinical site. In this case, samples need to be preserved properly to maintain good cell quality and transcriptomic profile. In this work, we propose a storage solution named BD® Tumor and Tissue Preservation Reagent (TTPR). We evaluated the RNA quality of sample storage in this solution up to three days. Expression of cellular stress-related genes were assessed over the storage time course. Importantly, samples stored in TTPR proved compatible with single cell capture workflow using BD RhapsodyTM system and the single-cell gene expression is comparable to that of cells from fresh sample. In conclusion, TTPR is a useful and convenient solution for short-term sample storage prior to downstream genomic assays including single-cell sequencing. For Research Use Only. Not for use in diagnostic or therapeutic procedures.
BD, the BD Logo and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2021 BD. All rights reserved. MSA Approved 1121
Supported by NA
Development of a cell free virus free beads-based SARS-Cov-2 virus neutralization assay
Authors: Hou Xiaohong , Sims Emily , Pan Wenjing , Brown Brittany , Steele Miranda , Song Stephanie , Han Jian 
Published: January 2023

Abstract
Development of a simple, fast, and safe method to detect neutralizing antibody levels to a pathogen is urgent to help understand the immune status of convalescent patients and the immune responses after vaccine. Here we report the development of a cell-free and virus-free beads-based virus neutralization assay. We focused on the detection of neutralization antibody (nAb) in blood circulation to SARS-Cov-2 spike protein receptor binding domain (RBD). This assay uses histidine tagged recombinant RBD protein to replace living virus and uses streptavidin coated polystyrene beads as a carrier of human angiotensin-converting enzyme 2 (hACE2). This bead serves as a surrogate for target cells. Neutralization was detected by the decrease or absence of fluorescence signal associated with RBD. The assay was compared to a cell-based neutralization assay and ELISA, and these results are reported. The bead-based assay is simple, fast, safe and importantly, can truly reflect the immune status of individuals. The result of the assay can help assess the response of vaccine recipients and assist in selecting serum from convalescent patients for therapeutic purposes.
Development of a multiplex microbead-based immunoassay for quantitative analysis of mouse immune checkpoint proteins
Authors: Lie Wen-Rong , Kornmeier Christine , Williams Sasha , Pfeifer Justin , Banerjee Munmun , Hoberg James 
Published: January 2023

Abstract
Immune checkpoint protein research plays a significant role in improving antitumor immune response as well as achieving a deeper understanding of the immune system and the disease states of inflammation, sepsis, and autoimmunity. Various mouse models have contributed to the clinical successes of immune checkpoint inhibitor immunotherapies and the understanding of checkpoint molecule-related inflammatory processes. Here we report the assay verifications and applications of a newly developed Luminex bead-based mouse immune-oncology checkpoint protein multiplex immunoassay for the quantitative profiling of 28 mouse immune checkpoint molecules and immune regulators (PD-1, PD-L1, PD-L2, CTLA-4/CD152, TIM-3, LAG-3, B7-H2/ICOSL, B7-H3/CD276, BTLA, GITR, HVEM, CD27, CD40, CD80/B7-1, CD226/DNAM-1, 4-1BBL/CD137L/TNFSF9, CD137/4-1BB, 5’-NT/CD73, BCA-1/CXCL-13, CD25/IL-2Rα, Granzyme B, E-cadherin, Galectin-1, Galectin-3, IFNγ, IL-10, TLR-2, and TNFα). We present quantitative protein biomarker profiling of mouse serum/plasma samples and conditioned media and lysates from mouse tumor cell lines, including LLC, B16-F10, MCA205, ID8, MB49, AT3, CT-2A, and YUMM1.7, as well as DC2.4 (dendritic cell line), C2C12 (myoblast cell line), and bEnd.3 (endothelial cell line). Our results demonstrate that this mouse immune checkpoint protein multiplex immunoassay is a powerful research tool for the quantitation of key immune regulator proteins and has potential application in biomarker discovery and translational research.
Linking TCR to its cognate antigens with high-throughput and high-dimensional Smart Immune Receptor Sequencing in single cells
Authors: Guo Yuwan , Malone Michael J , Jiang Ning 
Published: January 2023

Abstract
With advances in next generation sequencing, researchers are able to enjoy the ease of sequencing tens of thousands of single cells in one experiment. However, the large cell loss associated poses great challenges for immune receptor profiling from rare clinical samples. To address these challenges, we previously developed Smart Immune Receptor Sequencing (SmartIR-Seq) to simultaneously profile T cell receptor (TCR) sequences and whole transcriptome in single cells more efficiently. However, to better understand T cell functions, it is essential to develop a method to link TCR sequence information to its cognate antigens in the same assay. Here, we describe a high-dimensional SmartIR-Seq technology, SmartIR-SeqHD, which allows us to multiplex cognate antigen specificities, paired TCR sequences, and whole transcriptome in single cells.
In SmartIR-SeqHD, we in vitro transcribed and translated peptide encoding DNA oligos to generate a library of peptides, which were then loaded onto MHCs using a previously developed UV exchange method. By crosslinking streptavidin molecule with a universal oligo, we annealed a DNA oligo which serves as the DNA barcode for that antigen-specificity. Next, tetramer-stained cells were single-cell sorted into plates and lysed, enabling DNA barcodes to be amplified together with mRNAs, including TCR transcripts. We added cell barcodes during PCR amplification and included molecular identifier on the DNA barcodes. The addition of one more “dimension” to SmartIR-Seq using DNA-barcoded pMHC tetramers allows screening for large panels of antigens on primary T cells affordably and quickly, thus further improving the efficiencies and flexibility in the analysis of CD8+ T cells from rare clinical samples.
Supported by grants from NIH R33 IMAT