bluuebunny/crossref_metadata_2025_split huggingface dataset:
Superior isolation of antigen-specific brain infiltrating T cells using manual homogenization technique
Authors: Garcia Luz Milbeth Cumba , Huseby April M , Johnson Aaron J 
Published: January 2023

Abstract
Effective recovery of activated brain infiltrating lymphocytes is critical for research investigations involving murine neuroimmunological disease models. To optimize lymphocyte recovery, we compared isolation methods using brains harvested from 7 day Theiler’s virus infected mice. Brains were processed using either a mechanical dounce based approach or were enzymatically digested using type IV collagenase. The resulting cell suspensions from these two techniques were transferred to a Percoll gradient, centrifuged, and lymphocytes were recovered. Flow cytometric analysis of CD45hi cells showed greater percentage of CD44hiCD62loactivated lymphocytes using dounce method. Also, a 3-fold greater recovery of activated virus specific CD8 T cells for the immunodominant virus epitopes Db: VP2121-130 and Kb: OVA257-264 was achieved through mechanical dounce homogenization approach as compared to collagenase digest. Greater percentage of viable cells was also achieved with the dounce homogenization. We therefore conclude that manual homogenization is a superior approach to isolate activated T cells from the mouse brain.
The role of the RIG-I like receptors in mediating CD8+ T cell immunity during virus infection
Authors: Aguilar-Valenzuela Renan A , Ma Daphne Y , Suthar Mehul S 
Published: January 2023

Abstract
West Nile virus (WNV) is a mosquito-borne Flavivirus that is a major cause of virus-induced encephalitis in the US. Upon virus infection, pattern recognition receptors (PRRs) recognize viral PAMPs to trigger potent antiviral defense programs. In particular, the RIG-I-like receptors (RLRs) are a family of cytosolic PRRs that comprise of RIG-I, MDA5 and LGP2 and are expressed in nearly every host cell, including antigen-presenting cells. We have previously demonstrated that RLRs as well as their central adaptor protein mitochondria antiviral signaling (MAVS) are critical for mediating immune protection against WNV infection. Recently, we discovered that the RLRs function in a non-canonical manner within CD8+ T cells to promote T cell expansion, survival and effector function during virus infection. Specifically, we found that the RLR LGP2 plays an essential role in promoting virus-specific CD8+ T cell survival and effector functions. To better understand this novel RLR pathway in T cells, we evaluated the cell-intrinsic roles of RIG-I and MAVS, two known interacting partners of LGP2. Following in vitro CD8+T cell stimulation, we found that RIG-I−/−, but not MAVS−/−, CD8+ T cells displayed higher proliferation rates than wild type (WT) cells. Adoptive transfer studies into RAG−/− mice revealed that WNV-specific CD8+ T cells from RIG-I−/− or MAVS−/− mice were present in lower percentages than WT CD8+ T cells during virus infection. To define the non-canonical RLR signaling, we are undertaking a genetics approach to screen proteins associated with the RLR interactome in CD8+ T cells. Altogether, our data indicate that RLRs function in a noncanonical manner within CD8+ T cells to regulate antigen-specific responses during virus infection.
The length of the N-terminal region of a photocleavable peptide bound to DR1 determines the kinetics of photocleavable peptide fragment release
Authors: Negroni Maria Pia , Stern Lawrence J 
Published: January 2023

Abstract
MHC class II molecules (MHCII) bind peptides and present them to CD4+ T cells. As prepared as a recombinant protein, DR1 (a MHCII) is in equilibrium between peptide-receptive and peptide-averse conformations. In order to dissect which step of the peptide binding reaction is being affected in DR1 mutants with altered peptide affinity, we wanted to generate DR1 fully in the peptide-receptive conformation. To generate this form, we used a peptide carrying the photocleavable 3-amino-3-(2-nitrophenyl)-propionic acid residue, an approach previously used for other MHCI and MHCII proteins. We used variants of a well-characterized peptide derived from influenza hemagglutinin (HA), with different truncations at the N-terminal end, expecting to generate peptides with different affinity for DR1. This was confirmed by higher IC50 values obtained for the shorter peptides compared to the longer ones. All of the studied peptides were cleaved as expected after UV treatment as shown by mass spectrometry. However, when DR1-photocleavable peptide complexes were exposed to UV light, they generated different amounts of peptide-receptive DR1 measured by their ability to bind fluorescently-labeled HA peptide. The shorter the peptide, the more peptide-receptive DR1 was generated. This suggests that minimizing the interaction between photocleavable peptide and DR1 allows faster dissociation of the photo-generated fragments, and thus faster association of an incoming peptide. These results showed that the kinetics of photocleavable peptide fragment release can interfere with the kinetics of subsequent reactions.
Brain infiltrating CD8+ T lymphocytes in lupus-prone mice
Authors: Morawski Peter A , Qi Chen-Feng , Bolland Silvia 
Published: January 2023

Abstract
Systemic lupus erythematosus (SLE) is an autoimmune disorder affecting multiple organ systems including the skin, kidneys, spleen, lungs, and brain. Severe SLE often manifests with multiple psychiatric and neurological sequelae, and may be accompanied by CNS vasculitis. However, the cellular contributors to CNS disease in lupus patients remain poorly defined. Our data demonstrate a unique lymphocyte bias in the CNS of mice expressing multiple copies of Toll-like receptor 7 (TLR7[Tg], FcgRIIB-Yaa), which present with a lupus-like disease. Lupus-prone mice have a substantial population of infiltrating CD8+ T cells in the CNS, a bias that is not present in other peripheral lymphoid or non-lymphoid tissues. These animals exhibit blood-brain barrier damage and neuropathology in the presence of the infiltrating lymphocytes. However, genetic ablation of CD8+ T cells in lupus-prone mice by removal of the major histocompatibility complex (MHC) adaptor protein b2m results in aggravation of disease, suggesting the removal of a protective population. Circulating CD8+ T cells in lupus-prone mice display an activated phenotype, express a number of surface trafficking and adhesion molecules, and can home to the CNS. The endothelium of the brain, but not of other peripheral organs in these mice is activated, expressing adhesion markers complementary to those found on infiltrating CD8+ T cells. Our ongoing studies aim to elucidate specifically how these CD8+ T cells enter the CNS and further clarify their role in neuropathology. Our findings suggest a possible mechanism regulating CNS pathology that could have applicability to human neuropsychiatric manifestations of SLE.
Augmented expression of the autoimmune regulator (AIRE) induces paradoxical development of polymyositis-like autoimmunity
Authors: Matsumoto Mitsuru 
Published: January 2023

Abstract
Autoimmunity is prevented by the function of transcriptional regulator Aire, which promotes the expression of wide varieties of tissue-restricted antigens from medullary thymic epithelial cells (mTECs) and from a subset of peripheral antigen-presenting cells (APCs). We hypothesized that augmented expression of Aire would ameliorate autoimmune diabetes in NOD mice. We observed, in contrast, that mice overexpressing Aire developed muscle-specific autoimmunity associated with incomplete maturation of mTECs. This led to failure of deletion of autoreactive T cells together with dramatically reduced production of Tregs in the thymus. In peripheral APCs, costimulatory molecule expression was augmented by the additive Aire expression. Our results highlight the importance of coordinated action between central tolerance and peripheral tolerance under the common control of Aire. We suggest that autoimmune diseases develop not only by the loss of function of Aire but also by its augmented expression, which could occur in some forms of human autoimmune disease.
Adipose-resident group 1 innate lymphoid cells promote obesity-associated insulin resistance
Authors: O’Sullivan Timothy Edward , Rapp Moritz , Bhardwaj Priya , Fan Xiying , Dannenberg Andrew , Sun Joseph Chai-Yuen 
Published: January 2023

Abstract
Innate lymphoid cells (ILCs) are the most recently identified arm of the innate immune system that function to protect epithelial barriers against pathogens and maintain tissue homeostasis. While ILCs can also promote pathology at these mucosal sites, it remains unknown whether aberrant activation of tissue-resident ILCs can contribute to disease in non-barrier tissues. Here, we identify a subset of long-term adipose-resident ILC1 that are dependent on the transcription factors Nfil3 and T-bet, but phenotypically and functionally distinct from circulating mature natural killer (NK) cells. Diet-induced obesity drove early proinflammatory cytokine production in adipose tissue depots, leading to selective proliferation and accumulation of adipose-resident ILC1s. ILC1-derived IFN-γ was both necessary and sufficient to drive proinflammatory macrophage polarization to promote obesity-associated insulin resistance. Therefore, these findings suggest that adipose-resident ILC1s contribute to obesity-associated pathology in response to dysregulated local proinflammatory cytokine production.
The Ron receptor tyrosine kinase regulates inflammatory response in an experimental model of multiple sclerosis
Authors: Dey Adwitia , Snyder Lindsay , Weaver Veronika , Cantorna Margherita T , Hankey-Giblin Pamela A. 
Published: January 2023

Abstract
In the experimental autoimmune encephalitis (EAE) murine model of multiple sclerosis (MS), both central nervous system (CNS)-resident macrophages (microglia) and infiltrating monocyte-derived macrophages contribute to disease progression. The Ron receptor tyrosine kinase is expressed on tissue resident macrophages including microglia and is involved in regulating inflammatory responses. An in vivo deletion of Ron (Ron KO) promotes inflammatory macrophage activation (M1) and limits a reparative macrophage phenotype (M2). Herein we investigated whether Ron expression plays a critical role in regulating disease progression in EAE. Ron KO mice exhibit delayed onset of EAE (day 14) compared to Wild-Type (WT) (day 10), however Ron KO mice exhibit greater peak severity at onset. Ron KO mice display T-cell mediated peripheral inflammation, as demonstrated by the significant increase in the secretion of interferon gamma (IFNγ) but not IL-17 and IL-10. At day 14, cultured lymph node cells from Ron KO mice exhibit increased expression of M1-macrophage mediated biomarkers iNOS, COX-2, IL-6, IL12B, IL1β and TNF-α. A likely cause of this increased inflammatory response can in part be attributed to a T-cell mediated increase in IFNγ. Furthermore, CNS tissues from Ron KO mice at day 14 have increased gene expression of hallmark inflammatory biomarkers iNOS, IL12B and COX-2. The results indicate an interplay between the innate and adaptive immune system in fostering an M1-macrophage mediated inflammatory state, as the underlying factor for the observed increase in disease severity in Ron KO mice. Maintenance of Ron expressing macrophages could then be a potential therapeutic approach to treating MS symptoms.
Specific transcription factors distinctly regulate kinetics of IL1B and TNF gene expression
Authors: Pulugulla Sree H , Adamik Juraj , Grillini Andrea N , Galson Deborah L , Auron Philip E 
Published: January 2023

Abstract
Interleukin 1β (IL-1β) and Tumor Necrosis Factor α (TNFα) expression by monocytes is stringently regulated, whereas sustained expression is associated with chronic inflammation. Transcriptional kinetics of IL1B gene mRNA in THP-1 cells treated with either Lipopolysaccharide (LPS) or Phorbol 12-Myristate 13-Acetate (PMA) revealed two distinct phases, an early vigorous transient (0–4 hours) followed by a decreased continuous expression (4–24 hours). In contrast, the TNF gene showed only the early transient induction (0–4 hours) followed by complete shutdown (4–24 hours). Distinctly, PMA generated only a single delayed, but sustained, lower level of IL1B expression (7–24 hours), with negligible induction of TNF. To address possible mechanisms, mRNA expression kinetics of these genes in the presence of informative transcription factor inhibitors were compared. These revealed that the initial IL1B transient was dependent on both NF-κB and C/EBPβ. In contrast, late continuous expression required both HIF-1α and C/EBPβ, but was independent of NF-κB. The early phase expression of TNF was almost exclusively dependent on NF-κB, with minimal involvement of C/EBPβ. These results suggest a role for HIF-1α in replacing NF-κB during sustained late expression of IL1B. LPS re-stimulation of THP-1 cells 24-hours after the first LPS treatment generated an IL1B mRNA kinetic similar to that of the initial LPS challenge, i.e., an early transient followed by decreased continuous expression. In contrast, the TNF gene was completely refractive to LPS re-stimulation (Endotoxin Tolerance). Our results also argue that extracellular succinate plays a role in IL1B gene re-stimulation, suggesting a possible macrophage quorum response.
Transcriptional regulation of Osteopontin (OPN)-induced immunomodulation in RSV infected infants
Authors: Qureshi Nagib , Clayton Elijah , Durre Kainath , Qureshi Mahboob Hossain 
Published: January 2023

Abstract
RSV bronchiolitis and pneumonia in infancy is known to be associated with increased susceptibility to asthma. This phenomenon is characterized by a hyper reactive airway with a predominant Th2 response and a skewed dendritic cell (DC) polarization, which can be modulated by exogenous OPN administration. OPN is an immunomodulatory agent inducing a strong Th1 polarization in RSV infected lungs. In our present study, we used a neonatal mouse model to examine the transcriptional regulation of this immunomodulation in RSV-infected pups. RSV-infected pups were intranasally administered with or without different doses of OPN, and messenger RNA (mRNA) expression of GATA-3 and STAT-6 were examined by PCR and RTPCR methods. At day 3 post-infection, GATA-3 expression was significantly higher in the OPN treated lungs compared to that in the PBS treated lungs, contradicting the enhanced Th1 cytokine responses observed after OPN treatment. Interestingly, an enhanced expression of STAT-6 was also observed at the same time point in the OPN treated and RSV infected pups compared to that in the PBS treated pups. Of note, STAT-6 influences IL4 transcription which has a prominent role in Th2 differentiation. On the other hand, IL-4 instructs DC to promote Th1 differentiation which is STAT-6 dependent. Thus, the transcriptional regulation of OPN-induced immunomodulation is not one-dimensional and will need further investigations into the dynamics of other transcriptional factors (e.g. T-bet and Foxp3) to establish OPN as a novel therapeutic in the prevention of RSV-induced susceptibility to asthma.
LIGHT and IL-1β contribute to airway remodeling in a mouse model of recurrent rhinovirus infection
Authors: Mehta Amit Kumar , Doherty Taylor A , Croft Michael 
Published: January 2023

Abstract
Airway remodeling is defined by several structural changes including mucus metaplasia, increased peribronchial smooth muscle thickness and subepithelial fibrosis. Rhinoviruses (RVs) have been demonstrated to upregulate many airway inflammatory and remodeling factors that are implicated in the pathogenesis of asthma. Studies from our lab previously identified a tumor necrosis factor family member, LIGHT (TNFSF14), as an essential mediator of airway remodeling in an allergen-driven mouse model of severe asthma. In the present study, we examined effect of RV infection on induction of LIGHT and airway remodeling. We observed increased LIGHT mRNA after RV infection in an A549 cell line as well as in vivo after infection in the lung, and furthermore neutrophils were found to be another potential source of LIGHT. Recurrent infection of RV induced salient features of airway remodeling resembling those found in severe asthmatics. LIGHT−/− mice showed reduced neutrophilia and impairment in fibrosis and smooth muscle mass and lung collagen. Blocking LIGHT signaling with LTβRFc also inhibited subepithelial fibrosis and smooth muscle thickness. Recurrent infection of RV in the presence of house dust mite allergen further exacerbated the smooth muscle thickness and subepithelial fibrosis and this was again significantly reduced in LIGHT−/− mice. Recurrent infection of RV also induced IL-1β mRNA and neutralizing IL-1β signaling further reduced the subepithelial collagen deposition and smooth muscle thickness. This study extends our present knowledge of how viruses might cause worsening of allergic airway inflammation and promote airway remodeling in asthma by identifying LIGHT and IL-1β as being active during the response to rhinovirus.
Hapten-induced generation of a novel myeloid-derived FasL+ perforin+cell population in the bone marrow is induced by TNFa
Authors: Kish Danielle , Fairchild Robert L 
Published: January 2023

Abstract
Contact hypersensitivity (CHS) is a CD8 T cell mediated response to hapten skin sensitization and challenge. Hapten-primed CD8 cell infiltration into the skin challenge site during elicitation of CHS requires prior infiltration and activation of a population of Gr-1+CXCR2+ cells expressing FasL and perforin. The goal of these studies was to further characterize this novel population of myeloid-derived cells and to determine mechanisms generating the cells in the bone marrow during elicitation of CHS. The myeloid cells were purified from the bone marrow of hapten sensitized mice 3 hrs after happen challenge by depletion of CD3+, B220+, NK1+, F4/80+, class II MHC+ and Ly6G+ cells. Flow cytometry analyses indicate &gt;90% of the remaining bone marrow cells were CD11c+, Ly6C+, CCR2+, CX3CR1+ and FasL+ and express perforin and granzyme B. This cell population was at low numbers in the bone marrow of naïve mice but increased to peak numbers at 2 and 6 hrs. after hapten challenge of sensitized WT and Rag−/− mice and T-cell depleted WT mice. Whereas generation of neutrophils is inhibited by anti-G-CSF antibody, this treatment further increased numbers of these myeloid cells in the bone marrow following hapten challenge. Generation of the myeloid cells was absent in hapten sensitized and challenged TLR4- and IL-1 receptor-deficient mice and was inhibited by treatment with anti-TNFa mAb at the time of hapten challenge. These results indicate that generation of bone marrow-derived Ly6C+CXCR2+ cells expressing FasL and perforin/granzyme B in response to inflammation requires TNFa, IL-1 and TLR4 signaling but occurs independently of T cell activation.
Long-term safety of Crisaborole Topical Ointment, 2%, in children and adults with mild- to-moderate atopic dermatitis
Authors: Zane Lee Thomas , Eichenfield Lawrence F. , Call Robert S. , Forsha Douglass W. , Fowler Joseph F. , Hebert Adelaide A. , Spellman Mary , Stein Gold Linda F. , Van Syoc Merrie , Tschen Eduardo H. 
Published: January 2023

Abstract
Atopic dermatitis (AD) is a chronic inflammatory skin disease that often requires long-term topical treatment. Crisaborole Topical Ointment, 2% (Anacor Pharmaceuticals, Inc., Palo Alto, CA), a novel nonsteroidal, topical, anti-inflammatory phosphodiesterase 4 (PDE4) inhibitor, is currently being investigated for the treatment of AD. Herein we present the long-term safety results of patients ≥2 years of age with mild-to-moderate AD. A multicenter, open-label, long-term, 48-week, extension safety study was conducted in patients (N = 517) who opted to continue treatment after completing a 28-day Phase 3 pivotal study. Patients were assessed for AD severity every 4 weeks and treated with 4-week cycles of crisaborole as needed (Investigator’s Static Global Assessment ≥2 [Mild]). During the open-label extension and the pivotal studies, 65% of patients reported at least 1 treatment-emergent adverse event (TEAE), most of which were mild (51.2%) or moderate (44.6%) in severity and considered unrelated to treatment (93.1%). Treatment-related AEs occurred in 10.2% of patients; the most frequently reported events were atopic dermatitis (3.1%), application site pain (burning/stinging, 2.3%), and application site infection (1.2%). None of the 7 treatment-emergent serious AEs that occurred in the extension study were considered treatment related. During the long-term study, only 9 patients (1.7%) discontinued the study because of TEAEs. No cutaneous adverse reactions such as application site atrophy, telangiectasia, or hypopigmentation were reported. The safety profile of crisaborole was similar across age groups. Crisaborole Topical Ointment, 2%, has a favorable safety profile for the long-term treatment of patients with AD.
Regulation of T-cell receptor downmodulation by DENND1B in TH2 cells and allergic disease
Authors: Yang ChiaoWen , Hojer Caroline D , Zhou Meijuan , Wu Xiumin , Wuster Arthur , Lee Wyne P , Yaspan Brian L , Chan Andrew C 
Published: January 2023

Abstract
The DENN-domain is an evolutionary conserved protein module found in all eukaryotes and serves as an exchange factor for Rab-GTPases to regulate diverse cellular functions. Variants in DENND1B are associated with development of childhood asthma and other immune disorders. To understand how DENND1B may contribute to diseases, Dennd1b−/− mice were generated and exhibit hyper-allergic responses following antigen challenge. Dennd1b−/− TH2, but not other TH cells, exhibit delayed receptor-induced TCR downmodulation, enhanced TCR signaling, and increased production of effector cytokines. As DENND1B interacts with AP-2 and Rab35, TH2 cells deficient in AP-2 or Rab35 also exhibit enhanced TCR-mediated effector functions. Moreover, human TH2 cells carrying asthma-associated DENND1B variants express less DENND1B and phenocopy Dennd1b−/− TH2 cells. These results provide a molecular basis for how DENND1B, a previously unrecognized regulator of TCR downmodulation in TH2 cells, contributes to asthma pathogenesis and how DENN-domain containing proteins may contribute to other human disorders.
Investigate the role of PKCλ/ι on Th17 cells and allergic airway inflammation
Authors: Yang Jun-Qi , Guo Fukun , Zheng Yi 
Published: January 2023

Abstract
We have reported that atypical protein kinase PKCλ/ι plays key roles for Th2 differentiation and cytokine secretion both in cell culture systems and in allergic airway inflammation. To determine the potential role of PKCλ/ι on Th17 cells, we did investigations both in vivo and in vitro with the use of conditional PKCλ/ι-deficient mice. We found that PKCλ/ι-deficient CD4+ T cells showed impaired ability to differentiate into Th17 cells in vitro under Th17-skewed culture conditions. This impairment for Th17 differentiation was associated with the down-regulation of Stat3 and IL-23R in PKCλ/ι-deficient CD4+ T cells in culture. The secretion of IL-17, IL-17F, IL21 and IL-22, Th17 effector cytokines, was reduced from PKCλ/ι-deficient T cells either under non-skewed or Th17-skewed conditions. Furthermore, allergic airway inflammations induced by ovalbumin (OVA) or house dust mite (HDM) were applied to study the role of PKCλ/ι-Th17 axis in vivo. PKCλ/ι-deficiency significantly inhibited airway inflammations as showed reduced infiltrating cells and Th17 effector cytokines in the lung and bronchoalveolar lavage (BAL) fluid. These findings suggest that PKCλ/ι might be one of novel regulators for Th17 cells. Future studies will focus on underlying molecular mechanisms how PKCλ/ι control Th17 cells.