bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dampened inflammation with ruxolitinib treatment leads to CD8+ T cell exhaustion and induction of T regulatory cells in a murine model of HLH
Authors: Albeituni Sabrin H , Verbist Katherine , Tedrick Paige , Tillman Heather , Nichols Kim 
Published: January 2023

Abstract
Hemophagocytic lymphohistiocytoses (HLH) is a heterogeneous group of disorders characterized by severely damaging immune responses. In HLH, activated CD8+ T cells and macrophages overproduce pro-inflammatory cytokines, which contribute greatly to morbidity and mortality. In a murine preclinical model, HLH is recapitulated by infecting perforin knockout mice with lymphocytic choriomeningitis virus (LCMV). The inability of CD8+ T cells to kill infected cells results in a high production of inflammatory cytokines and ultimately death. We recently reported that treatment with the JAK1/2 inhibitor ruxolitinib significantly reduces the manifestations of HLH in this model. In this study, we sought to: 1) compare the effect of ruxolitinib treatment with IFN-γ blockade (currently in clinical trials) in the acute phase and 2) determine the long-term effects of treatment upon discontinuation. On day 9 p.i., ruxolitinib treatment was superior to IFN-γ blockade in reducing splenomegaly, numbers of inflammatory foci in the liver, LCMV-specific hepatic CD8+ T cells, and splenic IFN-γ+TNF-α+ CD8+ T cells. Surprisingly, on day 35 p.i., ruxolitinib treated mice achieved 100% survival, while only 20% of the IFN-γ Ab-treated mice survived. Since ruxolitinib dampens inflammation, we hypothesized that lower inflammation and viral persistence would induce T cell exhaustion. Indeed, CD8+ T cells stimulated with LCMV-specific peptide produced less IFN-γ and TNF-α. In addition, a significant population of CD8+CD44hi T cells co-expressed PD-1 and Tim-3. This was also correlated with enhanced percentages of Foxp3+CD4+ Tregs in treated mice. Overall, this study provides evidence and mechanistic insight for ruxolitinib as a novel treatment for HLH.
Blockade of critical immune regulators results in different roads to pathogenesis in Primary Sjögren’s syndrome
Authors: Voynova Elisaveta Ninova , Braley-Mullen Helen , Ettinger Rachel 
Published: January 2023

Abstract
Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease that affects exocrine glands resulting in severe dryness of mucosal surfaces, primarily the mouth and eyes. Female, but not male NOD.H-2h4 mice develop pSS-like manifestations in aged mice. Previously, we have shown that pSS disease that arise in these mice is driven by early peripheral dysregulation that manifests as spontaneous germinal centers (sGC) that are dependent on CD40/CD40L interactions (Mahmoud, T et al., Sci Transl Med 2016). We now examined if other key immune pathways also play a role in pathogenesis of pSS. We found that NOD.H-2h4 mice deficient in either CD28/IFNg or PD1/IFNg developed exacerbated pSS manifestations, compared to the NOD.H-2h4 wild type (WT) mice. As in the WT mice, a single treatment with anti-CD40L mAb in CD28/IFNg−/− mice diminished both sGC and salivary gland tertiary lymphoid structures (TLS). Unexpectedly, however, the aggressive disease noted in PD1/IFNg−/− animals was not similarly inhibited by treatment with anti-CD40L mAb. While anti-CD40L dissolved the sGC, it had no effect on the TLS that develop in the salivary glands of these PD1/IFNg−/− mice. The different results following anti-CD40L mAb treatment in CD28/IFNg−/−vs. PD1/IFNg−/− mice is likely not due to lack of functional Tregs in CD28/IFNg−/− mice, as while transient depletion of Tregs in PD1/IFNg−/− mice resulted increased salivary gland TLS, anti-CD40L still had minimal impact on disease.
A serum-free medium for differentiation of monocytes to macrophages
Authors: Tabatabaei-Zavareh Nooshin , Wang Tinya , Khan Fareeha , Karas Evan A , Le Fevre Tim A , Szilvassy Stephen J , Thomas Terry E , Eaves Allen C , Wognum Albertus W 
Published: January 2023

Abstract
Macrophages play an important role in defense against pathogens and in tissue homeostasis. They are classified into two main groups depending on how they are activated: 1) M1 (activated by IFN-γ and LPS) and 2) M2, which include M2a (activated by IL-4 or IL-13), M2b (immune complexes with IL-1β or LPS) and M2c (IL-10) subsets. It is difficult to control macrophage activation and polarization in cultures containing serum, in which variable amounts of M1- and M2-specific cytokines and other factors may be present. Here we describe a serum-free culture system that selectively supports the differentiation of monocytes into either M1 or M2a macrophages. Monocytes were isolated using EasySep immunomagnetic cell separation and cultured for 4 or 6 days in serum-free ImmunoCult medium with 50 ng/mL M-CSF. The cells were then stimulated by adding 10 ng/mL LPS plus 50 ng/mL IFN-γ for M1 or 10 ng/mL IL-4 for M2a macrophage polarization and cultured for 2 more days. The yields of M1 and M2a macrophage were 51±18% and 58±14% (mean±SD), respectively. M1 macrophages expressed high levels of CD80 and CCR7 (96±3% and 62±15%, n=57), but very low levels of CD206 and CD209. In contrast, M2a macrophages expressed CD206 and CD209 (90±10% and 92±13%, n=59), but were negative or low for CD80 and CCR7. M1 macrophages produced TNF-α and IL-12 (mean±SEM: 2821±396 and 656±86, pg/mL, n=24). M2a macrophages produced small amounts of IL-10 (29±6 pg/mL, n=21). Both M1 and M2a macrophages were functional as demonstrated in a phagocytosis assay using fluorescently-labeled E.coli. By selecting appropriate stimuli this culture method can be easily adapted to generate other macrophage subsets as well and should prove useful for the study of macrophage biology.
The Effects of Childhood, Adolescent and Adult Obesity on Epithelial T Cell Homeostasis in the Intestine
Authors: Limon Natalie M 
Published: January 2023

Abstract
According to the CDC, 30% of adults in the U.S population are obese, while 17% of children and adolescents are obese. Obesity has resulted in a wide array of complications including disruption of barrier permeability as well as problems with tissue repair. The epithelial layer of the intestines contains intraepithelial intestinal lymphocytes (IEL) that are important in maintaining epithelial homeostasis and repairing tissue. To outline the mechanism by which obesity disrupts intestinal epithelial function among different age groups, childhood, adolescent, and adult mice were placed in a high fat diet (HFD) for 7 weeks to observe IEL number and function. In all age groups, mice administered a HFD exhibit a significant decrease in IEL within the epithelial layer of the small intestine. Notably, in the childhood cohort, IEL seeding is disrupted resulting in limited IEL numbers during adolescence. Interestingly, T cells in the epidermis of the skin are not reduced in number after 7 weeks of HFD suggesting that the intestine is more sensitive to early obesity. This study shows the harmful impact of obesity on the immune system in the small intestine whether obesity occurs in youth or in adulthood.
IL-2 producing and non-producing effector CD8 T cells phenotypically and transcriptionally coalesce to form memory subsets with similar protective properties.
Authors: Kahan Shannon M , Bakshi Rakesh K , Luther Rita , Harrington Laurie E , Hendrickson R. Curtis , Lefkowitz Elliot J , Weaver Casey T , Zajac Allan J 
Published: January 2023

Abstract
Interleukin-2 (IL-2) acts as a differentiation factor that drives effector CD8 T cell formation and also programs robust memory recall responses. However, the roles of intrinsic IL-2 production in shaping effector and memory CD8 T cell differentiation remain uncertain. To address this, we utilized novel IL-2 and IFN-γ double cytokine reporter systems to trace the cytokine production profiles and fate decisions of effector and memory virus-specific CD8 T cells. Notably, effector and memory CD8 T cells segregate into populations that co-produce IL-2 and IFN-γ or populations that produce only IFN-γ. IL-2-producing effector CD8 T cells preferentially survive and develop memory traits, including the ability to mount protective recall responses. These cells also display transcriptional profiles more closely associated with memory formation. Additionally, cells that produce IL-2 appear refractory to signaling by IL-2 through their STAT5 pathways, which potentially directs their developmental choices. These data illustrate that the intrinsic ability of effector CD8 T cells to produce IL-2 can dictate their sensitivity to the cytokine milieu, predict their developmental fates, and forecast their ability to mount vigorous secondary responses, resist exhaustion, and protect the host. Despite having distinct properties during the effector phase of the response, IL-2-producing and non-producing CD8 T cells transcriptionally and phenotypically coalesce as memory matures to form subsets which respond similarly to secondary lymphocytic choriomeningitis virus challenge. Thus once memory is established, robust secondary responses are not limited to polyfunctional IL-2-producing virus-specific CD8 T cells.
The human γδ T cell repertoire is shaped by age, tissue compartmentalization and viral exposure
Authors: Sant Sneha Ashok , Dash Pradyot , Wang Zhongfang , Pizolla Angela , Ngyuen Oanh , Koutsakos Marios , Lappas Martha , Crowe Jane , Loudovaris Tom , Mannering Stuart I , Westal Glen P , Wakim Linda , Xu Jianqing , Thomas Paul G , Loh Liyen , Kedzierska Katherine 
Published: January 2023

Abstract
γδ T cells can provide immunity against foreign pathogens and internal tissue malignancies. Despite numerous studies in mice, the potential role of γδ T cells in human viral infections and their TCR profiles are understudied, especially at different tissue locations. We utilised a single-cell multiplex-nested-RT-PCR to dissect the TCRγδ repertoire (n=372 γδ pairs) in healthy individuals across three major age groups (neonates, adults and elderly donors) in peripheral blood and tissues (spleen, lymph node and lungs). Neonatal γδTCRs display diverse TCRγδ repertoires, while adults show an enrichment of γ9δ2 TCRs with diverse CDR3 regions. Strikingly, elderly donors displayed distinct TCRγδ profiles with large clonal CDR3 expansion. Tissue-associated TCRγδ usage differs markedly from γ9δ2 cells that dominate the adult peripheral blood with more restricted CDR3 regions. Using our established in vitro co-culture assay of influenza virus infection in lung epithelial cells, we found that human γδ T cells respond to influenza infection by IFN-γ production and enrichment of γ9δ2 T cells. Our findings further support the role of monocytes and IL-15 in γδ T cell activation after influenza infection. Our study provides novel insights into the composition and diversity of TCRγδ repertoires and demonstrates how human γδ TCRs are shaped by the age, tissue compartmentalization and viruses. Our findings have future implications for immunotherapies involving γδ T cells in patients across different ages.
Non-cytopathic bovine viral diarrhea virus 2 induce autophagy in MDBK cell
Authors: Du-Gyeong Han , Kyoung-Seong Choi 
Published: January 2023

Abstract

Objective
Bovine viral diarrhea virus (BVDV) is classified into two distinct species, BVDV1 and BVDV2. Both species consist of two biotypes, cytopathic (cp) and non-cytopathic (ncp). Autophagy is an essential control process by which cells degrade unnecessary or dysfunctional cellular components or organelles in the cytoplasm. In this study, we explored autophagy activity in ncp BVDV2-infected MDBK cells.

Materials and methods
Ncp BVDV 2 (12F001) strain was cultured in MDBK cell and titration of the 50% tissue culture infective dose (TCID50) was conducted using the Kärber method. Transfection with GFP-LC3 was performed using the LipofectamineTM 2000 reagent. MDBK cells (8′105) were seeded in 6-well plates, infected with ncp BVDV2 or treated with rapamycin or DMSO, and then incubated. Subsequently, cells were used for western blot for LC3B, p62/SQSTM1, ATG14, and β-Actin.

Results
Our results showed that LC3 protein expression levels were increased in ncp BVDV2-infected MDBK cells by western blot and confocal microscopy. This expression was high in rapamycin-treated cells, followed by ncp BVDV2 infection, and DMSO treatment. We determined autophagy activity in ncp BVDV2-infected MDBK cells by assessing the protein levels of SQSTM/p62, LC3 and ATG14 by western blot. The decreased levels of SQSTM/p62 were accompanied by an enhanced expression of LC3 and ATG14 in ncp BVDV2-infected MDBK cells.

Conclusion
These results suggest that ncp BVDV 2 infection activate autophagy in MDBK cells. We conclude that autophagy may be linked to the establishment of persistent infection through virus survival. Thus, further studies should be needed to determine the association between autophagy and virus replication.
Increased generation of regulatory T cells in the presence of reduced T cell receptor signaling capacity correlates with a reduction in positive selection of thymocytes
Authors: Palin Amy , Lee Jan , Oliva Miles , El Khoury Dalal , Love Paul E 
Published: January 2023

Abstract
The T cell antigen receptor (TCR) is the primary signal transducing structure expressed on thymocytes and mature T cells, transmitting signals necessary for maturation and activation. The TCRα and β chains are rearranged from germline gene segments to generate a highly variable repertoire of antigen receptors. Thymocytes undergo a selection process in the thymus to ensure presence of a functional, but not highly autoreactive, T cell receptor on their surface. These processes, and T cell function, depend on the invariant CD3 molecules to transduce intracellular signals from the surface TCRαβ chains. We have created a knock-in mouse with Cre-recombinase-inducible tyrosine (6Y)-to-phenylalanine (6F) mutations in the 6 Immunoreceptor Tyrosine Activation Motif (ITAM) tyrosines of the 2 CD3ζ chains of the TCR/CD3 complex, resulting in a theoretical 60% decrease in TCR signaling potential. We found that pre-selection, but not post-selection, switching of wild-type (6Y) CD3ζ to non-signaling (6F) CD3ζ results in a significant increase in the percentage of thymic and peripheral regulatory CD4+ T cells (Tregs) relative to control germline 6Y CD3ζ mice. Mice with a pre-selection 6Y to 6F CD3ζ switch also have increased CD4+ CD25+ Foxp3− Treg precursors. This increase in Tregs and thymic Treg precursors is cell autonomous, and correlates with a reduction in positive, but not negative, selection of CD4SP thymocytes. Tregs with reduced signaling capacity do not, however, have suppressive capacity equivalent to wild-type Tregs, despite expressing normal levels of transcription factors and surface proteins characteristic of Tregs. Collectively, the data support an instructive, TCR-signal-based model of Treg lineage commitment in the thymus.
Necrotic liver induces pyroptosis through caspase-1 in eosinophils.
Authors: Macapagal Daphne , Connor Jane , Mustelin Tomas Mikael , Ramalingam Thirumalai , Wynn Thomas , Davidson Todd 
Published: January 2023

Abstract
During sterile inflammatory reactions, damage associated molecular patterns (DAMPS) are released from necrotic cells and recruit immune cells to the sites of tissue injury. We found that necrotic liver homogenates, when injected intraperitoneally, can recruit large numbers of immune cells. Among these immune cells were activated eosinophils. While eosinophils are well known to accumulate in the lungs of asthmatics or in response to helminths and parasites, several studies have shown the presence of eosinophils in liver biopsies of drug-induced liver injury, primary biliary cirrhosis, chronic hepatitis C, hepatic allograft rejection, and liver fibrosis. The prevalence of eosinophilic infiltrates in the liver points to a correlation between infiltration and disease outcome. Our results demonstrated eosinophil activation upon exposure to necrotic liver components. Exposure to necrotic liver cells induced eosinophil degranulation as measured by expression of CD107a and eosinophil peroxidase release into the supernatant as measured by ELISA. Further study of eosinophil activation during necrotic liver exposure showed IL-1β and IL-18 release as well as cell death. All of these responses were dependent upon caspase-1 activation. Caspase-1 mediated cell death accompanied by IL-1β and IL-18 release are hallmarks of pyroptosis, an inflammatory cell death pathway. We verified eosinophil pyroptosis in vivo using a liver fibrosis model involving Schistosoma mansoni infected mice. Eosinophils extracted from S. mansoni infected livers showed caspase-1 activation and cytokine release indicating that these cells undergo pyroptosis in vivo in response to hepatocyte injury.
Lyn/Fyn-STAT5 cascade in IgG-mediated inflammation
Authors: Paranjape Anuya R , Ryan John J 
Published: January 2023

Abstract
The Src family kinases Lyn and Fyn provide critical and antagonistic controls over mast cell responses to IgE and IgG mediated activation. Similarly, Fyn activates, while Lyn impedes IgE-induced STAT5 phosphorylation, which is critical for IgE-activated cytokine production. However, the role of STAT5B, the paralog that selectively influences cytokine production downstream of IgE, remains uncovered in the context of IgG-induced responses. Therefore, we examined the importance of STAT5 in IgG signaling, and broadened these studies to test the role for Lyn, Fyn, and Stat5 in IgG-mediated inflammation in vivo. Consistent with IgE, STAT5B KO mast cells produced significantly less cytokines and chemokines after IgG-induced activation in vitro. On the other hand, IgG-induced cytokine production in STAT5B KO macrophages was unaffected, indicating that the non-redundant role of STAT5B might be lineage-restricted. To expand these studies, we employed the K/BxN model of inflammatory arthritis, in which macrophages do play a critical role. In this model, Fyn or STAT5B deficiency did not alter arthritic inflammation. By contrast, Lyn deficiency significantly exacerbated arthritis. These studies indicate clinically relevant, lineage-restricted role for the Lyn/Fyn-STAT5 cascade, showing that inhibitory effects of Lyn kinase are critical in macrophage-driven inflammatory responses.
Induced negative regulation decodes PAMP quality and quantity to generate bacteria-specific inflammatory responses
Authors: Gottschalk Rachel A , Dutta Bhaskar , Krauss Kathleen , Dorrington Michael G , Martins Andrew J , Uderhardt Stefan , Tsang John S , Torabi-Parizi Parizad , Fraser Iain D.C. , Germain Ronald N 
Published: January 2023

Abstract
Toll-like receptors (TLRs) expressed on macrophages are ligated in various combinations by diverse pathogen-associated molecular patterns (PAMPs), but how mixtures of signals through these receptors are integrated to mediate distinct biological responses to different pathogens remains unknown. By stimulating macrophages using a wide matrix of TLR2/TLR4 concentration combinations, we identified conditions resulting in non-linear responses. Transcriptomic and RNAi analyses identified negative regulators induced in a TLR-specific manner that regulated cytokine production dynamics and chemokine milieus across the combinatorial matrix. TLR4 ligand concentration explained much of the variation in production of many chemokines. However, combinations of sub-maximal amounts of either single ligand produced a zone of non-linear responses with sustained production of select inflammatory mediators, due to lack of negative regulation induced only at higher individual ligand concentrations. A similar sustained response was observed for Gram-positive bacteria, as compared to a panel of Gram-negative species, which express high amounts of the TLR4 ligand LPS. The novel TLR4-specific negative regulators we describe thus aid the innate immune system in discriminating Gram-negative from Gram-positive species, supporting qualitatively distinct inflammatory responses to these classes of bacteria.
This research was supported by the Intramural Research Program of the NIH, NIAID
The regulatory actions of retinoic acid on M2a macrophage polarization of porcine and human myeloid-derived cells.
Authors: Dawson Harry D , Chen Celine , Urban Joseph F. 
Published: January 2023

Abstract
Previously we demonstrated that the most bioactive vitamin A metabolite, all-trans retinoic acid (ATRA) increased T helper 2-associated responses induced by Ascaris infection of pigs. We also demonstrated, on a limited basis, that ATRA potentiated the mRNA expression of several IL-4 induced chemokines (chemokine (C-C motif) ligand 11 ((CCL11), CCL17, CCL22 and CCL26) associated with alternative activation (M2a) in porcine macrophages in vitro. Herein, we describe several mechanisms whereby ATRA affects IL-4 signaling and extend these findings to porcine monocytes/macrophages and human macrophages. In human THP-1 cells, IL-4 exposure led to an increase in the mRNA for the M2a macrophage-associated chemokines, CCL11, CCL13, CCL17, CCL18, CCL22 and CCL26 as well as the M2a-associated surface markers, CD206, CD209 and CD274. ATRA synergistically increased IL-4–induced CCL2, CCL13, CCL18, CCL22 and CCL26 mRNA and CCL13, CCL18 and CCL26 protein levels while having no effect or slightly down regulating MRC1, CD209 and CD274 mRNA expression. The effects of ATRA on mRNA levels were greater at 24 compared to 48 hrs; however, greater differences in protein levels were observed at 48 hrs for CCL13, CCL18 and CCL26. In contrast to porcine monocytes and macrophages, ATRA decreased IL-4 induced CCL17 mRNA and protein expression in THP-1 cells. Thus, in porcine monocytes and macrophages and in human macrophages, ATRA selectively increased signaling in response to IL-4. Given the prevalence of allergic and parasitic diseases worldwide and the close similarities in the porcine and human immune responses, these findings have important implications for the nutritional regulation of allergic inflammation at mucosal surfaces.
Common Patterns of Gene Expression in Tissues of Patients with Systemic Lupus Erythematosus Imply Similar Pathways of Molecular Pathogenesis
Authors: Heuer Sarah Elise , Catalina Michelle , Robl Robert , Geraci Nicholas , Lipsky Peter , Grammer Amrie 
Published: January 2023

Abstract
The mechanisms underlying tissue pathology in SLE are not completely understood. To gain additional insight, gene expression profiles from lupus affected skin, synovium, and kidney were obtained and analyzed for commonalities. More than 300 arrays from lupus patients and controls were analyzed to determine differentially expressed (DE) genes (8279 discoid lupus skin, 5465 synovium, 6381 glomerulus, 5587 kidney tubulointerstitum). Notably, the majority of lupus tissue DE genes were detected in more than one tissue and 439 were differentially expressed in all tissues. Curated STRING-based interaction analysis of these 439 DE genes identified a number of functions and pathways that were commonly activated in all four tissues such as p38 MAPK and Ras signaling, T cell signaling, the IFN-signature, and hallmarks of antigen presentation. IPA identified 65 pathways shared among these tissues, including PI3K signaling, B cell receptor signaling, TLR signaling, IL-6 signaling, and T cell signaling. When DE genes in at least 3 of 4 tissues were examined, 1,379 and 1,066 genes that were commonly up-regulated and down-regulated, respectively, were identified. Curated STRING-based interaction analysis of the up-regulated genes in at least three of the four tissues identified several additional cellular and molecular processes, including neutrophil chemotaxis, T cell adhesion, DNA damage response, cell proliferation, and hallmarks of CD8+ T cells, monocytes, and Th2 cells. These results indicate that common cellular and molecular pathways can be identified in all of the affected lupus tissues, implying that related processes might be involved in the pathology of multiple organs in this autoimmune disease.