bluuebunny/crossref_metadata_2025 huggingface dataset:
Establishment and characterization of a porcine lymphoma cell line
Authors: Rahe Michael C , Borgerding Anthony J , Wiseman Barry , Cenatti Leonardo , Martin Daniel , Murtaugh Michael P
Published: January 2023

The lack of well characterized, established domestic porcine cell lines hinders advancement of porcine cellular immunology understanding in disease resistance and anti-viral immunity. Numerous cases of multicentric lymphoma were diagnosed in pigs at the time of slaughter. Affected organs were harvested and submitted for single cell isolation and analysis. Culture of disaggregated single cell suspensions in RPMI 1640 with weekly passage resulted in clusters of dividing cells in about 2% of attempted isolations. In one pig, cells grew in approximately 50% of wells. Cell lines were established by limiting dilution repeated 3 times from splenic and subiliac lymph node lymphomas. Initial flow cytometry analysis showed a population of CD3+, CD79a+, CD21+, CD4−, and CD8− cells which have grown and been maintained in culture for more than 7 months and more than 10 subcultures. To further characterize the nature of this paradoxical homogenous population of CD3+ and CD79a+ cells, transcriptome analysis is being carried out to validate initial cytometric findings and identify key gene pathways. This new porcine lymphoma cell line will be a valuable resource for more in-depth cellular investigations into the porcine immune system, as well as providing a potential tool for the growth of lymphotropic viruses of pigs and humans.
A novel role for Osteopontin in facilitating West Nile virus neuroinvasion
Authors: Paul Amber M , Acharya Dhiraj , Le Linda , Stokic Dobrivoje S , Leis A Arturo , Bai Fengwei
Published: January 2023

West Nile virus (WNV) is a positive sense, single-stranded RNA flavivirus that can cause human neuroinvasive diseases, including encephalitis and meningitis. The mechanisms by which WNV enters the central nervous system (CNS) and the host-factors that are involved in WNV neuroinvasion are not completely understood. In this study, we found increased production of soluble osteopontin (OPN) in human plasma following WNV infection compared to uninfected controls, indicating a role for OPN in WNV pathogenesis. In a mouse model, after challenge with WNV (2000 plaque forming units, PFU), OPN deficient (Opn−/−) mice exhibited a higher survival percentage (80%) than wild-type (WT) mice (20%). However, both groups had comparable levels of virus in circulating blood and peripheral organs. Interestingly, viral burden in the brains of Opn−/− mice were significantly lower than WT mice, which was coupled to reduced polymorphonuclear neutrophil (PMN) infiltration. Soluble OPN has been previously described as a chemokine for PMN recruitment, and intracerebral supplement of recombinant OPN (50 ng/mouse) followed by an intraperitoneal challenge of WNV (2000 PFU), resulted in increased PMN infiltration and WNV load within the CNS. Furthermore, we also confirmed that infected-PMNs carried WNV into the CNS. Together, these data suggest OPN plays a negative role in recruiting WNV-infected PMNs into the CNS, thus mediating WNV neuroinvasion.
Porcine reproductive and respiratory syndrome virus non-structural protein Nsp2TF down-regulates Swine Leukocyte Antigen class I (SLA class I) expression
Authors: Cao Qian , Subramaniam Sakthivel , Cao Dianjun , Meng Xiang-Jin
Published: January 2023

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is arguably the most economically-important global swine pathogen. The virus typically induces sub-optimal immune responses especially a gradual increase of cellular immune-mediated (CMI) responses in the host. In this study, we demonstrated that PRRSV reduced SLA-I surface expression on porcine alveolar macrophages, monocyte-derived dendritic cells and a porcine kidney epithelial cell line, which may partly explain for the impaired CMI responses observed against PRRSV in pigs. To identify the PRRSV proteins responsible for SLA-I down-regulation, we screened all 22 PRRSV proteins by transient transfection assays. Our results indicated that Nsp1α, Nsp2TF and GP3 significantly down-regulated SLA-I surface expression of which Nsp2TF showed the greatest effect. To further validate whether Nsp2TF directly contributes to the PRRSV-induced SLA-I down-regulation, a panel of mutant viruses were generated with abolished Nsp2TF expression by disrupting −2 ribosomal frameshifting elements essential for the Nsp2TF protein synthesis as well as by introducing stop codons in TF domain of the protein. The infections with these mutant PRRSV strains fully restored the SLA-I surface expression in the infected cells. Additionally we demonstrated that the last 68 amino acids of TF domain are critical for this function. Our study for the first time uncovered an immune-modulatory function of PRRSV Nsp2TF in down-regulating SLA-I expression. Further studies are warranted to generate Nsp2TF-knockout vaccine strains and investigate their efficacies in inducing better CMI responses against the virus in pigs.
Regulatory role of neutrophils in the inflammatory responses to paramyxovirus infection in mice
Authors: Cheemarla Nagarjuna R , Banos-Lara Rocio , Guerrero-Plata Maria Antonieta
Published: January 2023

Neutrophils are the most abundant leukocytes (50% to 70%) in humans and are the first immune cell population recruited to the sites of infection. They are known to act in the first line of innate immune defense against invading pathogens, and more recently, to playing a crucial role in orchestrating adaptive immune responses. However, the role of neutrophils in the control of adaptive immune response during respiratory viral infections is still largely unknown. In order to elucidate the role of neutrophils in respiratory antiviral defense, we employed an experimental mouse model of human metapneumovirus (hMPV) infection. HMPV is a member of the Paramyxoviridae family, and a leading respiratory pathogen causing severe symptoms such as bronchiolitis and pneumonia in the young, elderly and immunocompromised patients. Our findings demonstrate that neutrophils are rapidly recruited in high numbers to the lungs of infected mice (>80%) during the acute phase of infection. Specific depletion of neutrophil in vivo using a monoclonal antibody and simultaneous infection with hMPV exhibited significantly higher levels of inflammatory cytokines, pulmonary inflammation and severe clinical disease (as indicated by an enhanced body weight loss and clinical illness) compared to hMPV-infected competent mice. Interestingly, the lack of neutrophils altered the T cell responses in the lung, particularly the γδ-T-cell subset. Finally, we also observed that the recruitment of neutrophils to the lung was controlled by the expression of the hMPV attachment protein. These novel findings demonstrate that neutrophils regulate T cell responses and are critically important for the control of the hMPV-induced inflammatory responses in the respiratory tract.
Insight into the role of immunosenescence during varicella zoster virus infection (shingles) in the aging cell model
Authors: Kim Ji-Ae , Park Seul-Ki , Shin Ok Sarah
Published: January 2023

Varicella zoster virus (VZV) is the etiological agent of shingles, a painful skin rash that affects a significant proportion of the elderly population. In the present study, we used two aging cell models, Hutchinson-Gilford progeria syndrome (HGPS) fibroblasts and stress or replicative senescence-induced normal human dermal fibroblasts (NHDFs), to investigate age-associated susceptibility to VZV infection. VZV infectivity titers were significantly associated with donor age in HGPS fibroblasts and senescence induction in NHDFs. High throughput RNA-sequencing (RNA-seq) analysis was performed to assess global and dynamic changes in the host transcriptomes of VZV-infected aging cells. Analysis of differentially expressed genes (DEGs) indicated that VZV infection in aged HGPS fibroblasts resembled that in senescent NHDFs, particularly in terms of genes associated with pattern recognition receptors in virus sensing network, providing novel insights into the mechanisms of senescence associated susceptibility to VZV infection. Additionally, we identified stimulator of interferon genes (STING) as a potential VZV sensing receptor. Knockdown of STING expression resulted in increased viral replication in primary fibroblasts, whereas STING overexpression led to suppression of VZV plaque formation. In conclusion, our findings highlight the important role of immunosenescence following VZV infection and provide significant insights into the mechanisms underlying cellular sensing of VZV infection and the induction of immune responses in aged skin cells.
Pathobiology of West Nile Virus infection in the Collaborative Cross mouse model
Authors: Ireton Renee C. , McMillan Aimee , Thomas Sunil , Treuting Piper , Voss Kathleen , Hendrick Duncan , Wilkins Courtney , Green Rich , Mooney Michael , McWeeney Shannon , Gale Michael J
Published: January 2023

West Nile Virus (WNV) is an emerging neuroinvasive flavivirus that causes serious encephalitis disease that is lethal in 6% of human cases. The immunopathology of WNV infection has been well-studied in C57BL/6J mice, but the model lacks the diversity in susceptibility and outcomes that are observed in humans, thus limiting its usefulness as a model to understand the host genetics and overall breadth of WNV infection outcomes and immune response. We are using the Collaborative Cross (CC) mouse model to evaluate the pathobiology and immune regulation that underlies WNV disease progression and neuroinvasion. Our studies reveal that the CC model captures the diversity in susceptibility and outcomes of WNV observed in humans. We generated a standardized histology scoring system to evaluate viral localization and immunopathology of WNV infection in tissue including brain isolates from CC lines. Our studies show a diverse array of histological phenotypes that link with the susceptibility and disease outcomes of the CC mice with WNV infection. Time course studies also showed variation in the temporal progression of viral induced-pathology and immunopathogenesis among different CC lines. Our findings show that the CC model of WNV is a relevant and powerful mouse model for mechanistic studies of WNV infection and neuropathology. Application of the CC model to genetic mapping of quantitative trait loci that impart immune regulation of WNV infection will be presented.
IL-36 cytokines regulate antiviral immunity
Authors: Jensen Liselotte E , Milora Katelynn A
Published: January 2023

Viruses may succeed as pathogens by deploying immune evasion mechanisms aimed at limiting detection by the host. If the host has developed counter strategies to ensure viral clearance is less clear. HSV-1 blocks inflammatory signaling from infected cells by preventing extracellular release of IL-1β. We found that HSV-1 infected keratinocytes retain the ability to signal danger to the surrounding tissue through secretion of IL-1α. This inflammatory signal appeared essential in vivo for early recruitment of immune cells to infected sites. Furthermore, mice deficient of the common receptor for IL-1α and IL-1β were more susceptible to lethal outcome of HSV-1 infection. Hence, IL-1α may have evolved to counteract an HSV-1 immune evasion strategy targeting IL-1β. We here extend these studies to the novel IL-1 family members IL-36α, IL-36β and IL-36γ, which signal via their common receptor IL-36R. We previously showed that poly(I:C), a double stranded RNA analogue often used to simulate viral infections, promoted expression and release of IL-36γ through a cell death mechanism called pyroptosis, suggesting a role for IL-36γ in immunity against viruses. Surprisingly, using the flank skin HSV-1 infection model, IL-36γ knockout (KO) mice developed disease indistinguishable from that observed in wild type mice. However, IL-36β KO mice progressed to more severe disease as reflected by larger skin lesions and increased mortality. Interestingly, inflammation induced by the anti-viral drug imiquimod was dependent upon IL-36α. Hence, the immune system appears to have several layers of redundancies that ensure a productive immune response.
Interferon-gamma inducible GTPases disrupt viral replication complex upon their targeting by the LC3 conjugation system of autophagy
Authors: Hwang Seungmin , Biering Scott , Choi Jayoung , Park Sungwoo , Williams Lelia , Dominici Erin
Published: January 2023

Viruses with a single strand (+) sense RNA genome replicate within vacuole-like structures in cytoplasm, called replication complex (RC). RC is made through reorganization of cellular organelle membranes, and it provides a favorable microenvironment for the viruses to replicate. How the host immune system counteracts viral RCs has been obscure. Recently, we found that interferon-gamma (IFNγ) inhibits the replication of murine norovirus (MNV) at the stage of RC formation. Intriguingly, this anti-viral activity of IFNγ depends on a protein complex involved in cellular autophagy. Autophagy is an evolutionarily conserved pathway that sequesters cytoplasmic materials in membrane-bound autophagosomes and delivers them to lysosome for degradation. To form an autophagosome, the microtubule-associated-protein-1-light-chain-3 (LC3) conjugation system (Atg7, Atg3, Atg12-Atg5-Atg16L1) is essential. Surprisingly, only the LC3 conjugation system of autophagy, but not lysosomal degradation through autophagy, was required for IFNγ to inhibit MNV RC formation. Through a comparative mechanism study, we found that the LC3 conjugation system was required to target IFNγ inducible GTPases - immunity related GTPases (IRGs) and guanylate binding proteins (GBPs) - to the RC. Both IRGs and GBPs are known to be targeted to the membrane of vacuoles containing bacteria, protozoa, or fungus. The targeted membranes are vesiculated and vacuoles get ruptured, leading to the death of exposed pathogens. Similarly, the GTPases disrupt the membrane structures of the RC and thus the replication of MNV. These data suggest that IFNγ utilizes the effectors against vacuolar pathogens for the control of viruses replicating in cytosolic membranous structures.
Bias-free imaging of antigen degradation using bioorthogonal antigens
Authors: Pawlak Joanna B. , Hos Brett , Megantari Otty A , Ossendorp Ferry A , van Kasteren Sander I
Published: January 2023

Degradation of antigen into short MHC-loadable polypeptides by antigen presenting cells presents a Heisenbergian conundrum: reporter strategies for imaging these processes affect the processes itself to such an extent that the information obtained from reporter antigen strategies may not be representative of the process itself.

For example fusion constructs of antigens are only imagable until they are degraded or separated from the antigen, which is essential if a peptide from the antigen is to be loaded on an MHC and reach the surface for sampling by T-cells. This results in an imaging bias to only those parts of the pathway in which reporters remain intact. Further complications can arise from the fact that reporter-constructs can alter the rate of proteolysis and routing by virtue of changing overall physiochemical properties of the antigens.

I will present our recent developments of ‘bioorthgonal antigens’, a bias free antigen labelling strategy that allows the imaging of antigen routing with minimal interference in the pathway. These antigens carrying amino acids with tiny chemical modifications (2 or 3 atoms in size) in their side-chains that are stable to proteolysis and other conditions found in the cell, can be selectively visualised using click chemistry. This allows, for example the localization of degrading antigen inside an APC or the determination of the rate of epitope appearance on the cell surface without the need for peptide-MHC-specific antibodies. This offers the first opportunity to have a detectable group that does not affect processing and presentation
Type I interferon signaling acts directly on transitional T2 B cells to promote RNP-Ag tolerance loss in BXD2 mice
Authors: Hamilton Jennie A , Wu Qi , Yang PingAr , Luo Bao , Li Jun , Liu Shanrun , Hsu Hui-Chen , Mountz John D
Published: January 2023

In lupus, autoreactive B cells fail to be removed at the transitional B cell stage. Lupus prone BXD2 mice exhibit transitional B cell tolerance loss and production of autoAb against RNA binding proteins (RNP). The purpose of this study was to determine direct effects of IFNα on developing transitional B cells. Analysis of IFNαR expression in B6 and BXD2 mice revealed the highest expression levels on transitional B cells with lower levels on mature FO, MZ, and GC B cells. Interestingly, both in vitro and in vivo, peripheral B cell responsiveness to IFNα started at the IgMhiCD23lo T1 stage and was significantly upregulated at the T2 stage, as measured by CD69 induction (76±5% in B6 vs 89±3% in BXD2 T2). In vivo treatment of B6 and BXD2 mice with IFNα promoted B cell upregulation of CD69 and CD86 on T2 B cells. Using an antigen (Ag) tetramer approach for isolation and characterization of La and RNP (La13-27 and snRNP357-373) autoreactive B cells, we found that IFNα stimulation increased maturation of transitional RNP autoreactive B cells and expansion of the autoantigen engaged IgMlo T3 compartment in BXD2 mice. Consistent with abnormal signaling and activation in vivo, transitional B cells from spleens of BXD2 mice exhibited elevated basal phospho-tyrosine and BCR induced activation, beginning at the T2 stage. Single cell analysis of transitional T2 B cells from BXD2 mice revealed a heterogeneity of gene expression within this population and a negative correlation between the expression of IFNαR gene and Id3, an important transcription factor for B-cell anergy. Our results suggest that B-cell tolerance loss in BXD2 mice occurs at the transitional stage, and IFNα exhibits the potential to break tolerance to RNP in transitional B cells.
An autoimmune risk variant in PTPN22 alters tolerance at multiple checkpoints during naive B cell development
Authors: Metzler Genita , Dai Tony , Thouvenel Chris , Khim Jit , Rawlings David J
Published: January 2023

A missense risk variant (R620W) in the tyrosine phosphatase PTPN22 is strongly associated with multiple autoimmune diseases. To model this variant in vivo, we generated knock-in mice expressing the analogous variant (R619W) in murine Ptpn22. Expression of the variant significantly altered lymphocyte function and led to the development of systemic autoimmunity. Further, B lineage-restricted variant expression was sufficient to promote autoimmunity (Dai et al. JCI 2013). In the current study, we performed a detailed assessment of the impact of the variant on B cell tolerance, including its impact on the establishment of the naive repertoire. Utilizing a bone marrow (BM) chimera model, variant expressing B cells exhibited a competitive advantage over wild-type (WT) cells at multiple checkpoints, beginning in the BM and continuing in the periphery. Interestingly, while follicular (FM) B cells maintain this competitive advantage, marginal zone (MZ) B cells do not. In addition, using single cell BCR cloning, we observed a similar differential phenotype, where FM B cells are highly enriched for self-reactivity, while MZ B cells are less autoreactive than WT controls. Using a range of analyses to assess cell signaling, surface receptor expression, cell cycle and gene expression, we provide evidence that the risk variant regulates a complex interplay between the BCR and CD40 co-receptor pathways, culminating in a uniquely autoreactive pre-GC repertoire with important implications for subsequent development of autoimmunity.
Targeting antigen experienced B lymphocytes to retard type 1 diabetes progression in NOD mice
Authors: Leeth Caroline McPhee , Hasham Muneer , Ratiu Jeremy , Zhu Jing , Chapman Harry , Branca Jane , Mills Kevin , Serreze David
Published: January 2023

While CD4+ and CD8+ T lymphocytes are ultimately responsible for the destruction of insulin-producing beta cells resulting in clinical type 1 diabetes (T1D), B lymphocytes are also critical pathogenic players in T1D development in NOD mice and likely also in humans. The role of B lymphocytes in this disease is primarily through antigen presentation, presenting diabetogenic peptide to autoreactive T lymphocytes. It is not known if changes in antigen affinity of plasma membrane bound immunoglobulin molecules are important for B lymphocyte diabetogenicity. We generated NOD mice deficient in AID, an enzyme crucial for affinity maturation, and found that T1D development was greatly inhibited in these novel mutant mice. While insulitis was still evident, progression to glucosuria did not occur. The phenotype of these AIDnull B lymphocytes differed from standard NOD as did the T lymphocyte populations. With the knowledge that affinity maturation is important to the development of T1D, we targeted maturing B lymphocytes using a small molecule inhibitor of the RAD51 complex, DIDS. NOD mice treated with this agent were protected from T1D development: Total B lymphocyte numbers were maintained with a reduction in the marginal zone subset. Insulitis was reduced in the treated mice. Circulating insulin autoantibodies (IAA) are indicative of impending diabetes development; however, IAA positive mice receiving DIDS treatment did not develop T1D. These results suggest that targeting antigen-experienced B lymphocytes during their maturation process may be a viable approach in T1D.
BANK1 Controls IgG Production Through TLR7-Dependent STAT1 Activation in a Lupus Model
Authors: Wu Ying-Yu , Kumar Ramesh , Iida Ryuji , Bagavant Harini , Alarcon-Riquelme Marta
Published: January 2023

The purpose of our study was to investigate the effects of the adaptor Bank1 in TLR7 signaling using the B6.Sle1.yaa mouse, a lupus model that develops disease through exacerbated TLR7 expression. Crosses of B6.Sle1.yaa with Bank1−/−mice maintained several B and myeloid cell phenotypes close to normal levels in wild-type mice. Most striking was the reduction in total serum IgG antibodies, but not of IgM, and reduced serum levels of autoantibodies, IL-6, and BAFF. Bank1 deficiency did modify frequencies of MZ B cells and splenic B cells, as well as expression of CXCR4 by follicular helper T cells. Bank1 deficiency did not modify frequencies of germinal center B cells or plasma cells or clinical disease outcomes. Purified B cells from B6.Sle1.yaa.Bank1−/−mice had lost strong induction of Ifnb, Ifna4, Irf1, 7, 9, Aicda and Stat1 gene expression following TLR7 agonist stimulation, compared with B6.Sle1.yaa mice. Interestingly, phosphorylation of Tyr701, but not of Ser727 of STAT1, was impaired in splenic B cells from B6.Sle1.yaa.Bank1−/− mice, as was the nuclear translocation of IRF7 in response to TLR7 agonist stimulation. Further, Bank1 deficiency in B6.Sle1.yaa mice reduced the production of IgG2c after in vitro TLR7 agonist stimulation. Our results demonstrate that Bank1 controls TLR7-mediated type I interferon production, leading to positive feedback loop on IFNAR and IFN I-mediated gene expression, including Tlr7 gene. Combined with the control of the nuclear translocation of IRF7, and the modulation of transcription and STAT1 phosphorylation, Bank1 contributes to IgG production during development of autoimmune disease.
Identification of Trex1 interacting proteins and endogenous DNA substrates
Authors: Crowl John T , Brunette Rebecca , Stetson Daniel B
Published: January 2023

Detection of DNA by cytosolic nucleic acid sensors activates the interferon stimulatory DNA (ISD) pathway, an antiviral response mediated by the production of type I interferons (IFN). This process is critical for host defense against viral pathogens but must be tightly regulated to avoid the chronic production of interferon, which can lead to autoimmunity. Three-prime repair exonuclease 1 (Trex1) is a crucial regulator of the ISD pathway, and the absence of functional Trex1 in both mice and humans leads to the development of autoimmune disease. Multiple DNA species have been proposed as the endogenous, cell-intrinsic substrates of Trex1. However, the in vivo identity of Trex1 substrates and the biological processes that generate them remain unknown. In order to definitively identify DNA that uniquely accumulates in the absence of Trex1, we have used a high-throughput, unbiased deep sequencing method. Additionally, to explore the functional requirements for Trex1 we have used a yeast two-hybrid screen to identify novel Trex1 interacting proteins.
Cytokine-mediated regulation of oligodendrocyte metabolism
Authors: Minchenberg Scott B , Massa Paul T
Published: January 2023

Multiple Sclerosis (MS) is a debilitating autoimmune disease characterized by inflammatory-mediated demyelination in the central nervous system. Our primary goal is to understand how inflammatory processes can lead to oligodendrocyte dysfunction, axonal degeneration, and demyelination in the CNS. Our lab discovered that the protein tyrosine phosphatase SHP-1, a major negative regulator of the immune system, is expressed in oligodendrocytes and plays an essential role in regulating STAT1 and STAT3 activation in oligodendrocytes. Importantly, it has recently been discovered that both STAT1 and STAT3 are important transcriptional regulators of metabolic genes. Metabolic regulation is particularly important for oligodendrocytes because lipid synthesis is required for the production and maintenance of the myelin sheath and energetic support for axons. Using freshly isolated O4+ oligodendrocytes, we determined that SHP-1 deficient oligodendrocytes have significantly reduced glycolysis and oxidative phosphorylation (OXPHOS) relative to oligodendrocytes of wild type mice. We were able to show that in vivo treatment with IFN-γ, a potent activator of STAT1, is able to down regulate wild type oligodendrocyte metabolism without diminishing oligodendrocyte viability. Further, when oligodendrocytes of SHP-1-deficient mice were treated with IL-10, a potent STAT3 activator, we were able to rescue metabolic deficits in SHP-1-deficient oligodendrocytes suggesting that SHP-1 is a major regulator of oligodendrocyte metabolism via regulation of STAT1 and STAT3 activation.
Dysregulation of TCR signal fine-tuning molecules due to increased ubiquitination in systemic autoimmune disease
Authors: Singh Ram Raj , Bui Viet , Prasad Priti , Ranganath Veena , Halder Ramesh , Pinkhasov Julia
Published: January 2023

T cell selection is primarily determined by the avidity of T cell receptor (TCR) for self-ligand-MHC. Evidence suggests that besides the TCR, there are other signal regulating molecules that play a role in establishing signal threshold in T cells during the process known as TCR tuning, which modulates the intensity of TCR signaling. Here, we determined the expression of putative TCR signal fine-tuning molecules (TFT) in mice and humans with systemic autoimmune diseases, and investigated their role in T-cell signaling defects and mechanisms of their alteration. We found the expression levels of a battery of TFTs to be reduced on thymocytes in young MRL/lpr and MRL+/+ mice compared to C3H controls. MRL Thymocytes displayed signaling defects, which were corrected by TFT agonists. In the periphery, T-cells in the diseased MRL mice exhibited an unexpectedly drastic reduction in the TFTs, which correlated with an increased responsiveness to TCR stimulation and to weak antigenic ligands. TFT expression was also reduced in T cells from patients with rheumatoid arthritis as compared to controls, particularly in elderly patients who experience more severe disease than younger patients. The reduced expression of TFTs in MRL peripheral T cells was due to their increased ubiquitination, which led to degradation of these molecules. Blocking proteasomal degradation using MG132 restored their expression. Finally, MRL-lpr mice had increased E3 ubiquitin ligases that associated with the TFTs expression. Thus, the ability of T cells to alter their signal threshold by altering the expression of TFTs via their post-translational modification mediated by E3 ligases may be a novel mechanism for T cells to escape tolerance and trigger autoimmune disease.
Endosome-to-cytoplasm pathway via Sec61 and proteasomal degradation as an essential component of antigen cross-presentation and the lupus tissue injury in systemic lupus erythematosus (SLE): Extension of our self-organized criticality theory
Authors: Tsumiyama Ken , Shiozawa Shunichi
Published: January 2023

Objective

We propose that SLE can be induced upon repeated immunization with any antigens in the mice normally not prone to SLE, in which newly generated autoantibody-inducing CD4 T cell (aiCD4 T cell) helps the generation of CTL via antigen cross-presentation and induces lupus tissue injury. Here we dissect the molecular mechanism of antigen cross-presentation.

Methods

Bone marrow-derived dendritic cell (BMDC) or splenic DC (spDC) was cultured with OVA, and inhibitor of Sec61 Exotoxin A (Exo A) or inhibitor of proteasome MG132. Mice were repeatedly immunized with OVA to induce SLE as in PLoS ONE 4:e8382, 2009 with/without Exo A or MG132, and immunoprecipitation or immunoblotting studies were performed.

Results

We show that accumulated antigen in the cytoplasm up-regulates the amount of cross-presentable antigen, thereby enhancing tissue injuries of SLE, in which Sec61 is responsible for endosome-to-cytoplasm transport of antigen: in the first, the amount of endosomal Sec61 and the amount of cytoplasmic OVA were increased in spDC after repeated immunization with OVA. Second, cytoplasmic OVA was increased after co-culture with inbibitor of proteasome MG132 in BMDC, but disappeared after adding inhibitor of Sec61 Exo A. Third, the generation of CTL and the development of lupus nephritis were both inhibited in vivo after repeated immunization with OVA in the presence of Sec61 inhibitor Exo A or proteasome inhibitor MG132.

Conclusion

For antigen cross-presentation, antigen must be transported from endosome to cytoplasm via Sec61 and then degraded through proteasome. The increase of endosomal Sec61 facilitates antigen cross-presentation, thereby contributing to the pathology of SLE.
Constitutive expression of the NKG2D ligand RAE1ɛ within pancreatic islets ameliorates autoimmune diabetes development in non-obese diabetic mice
Authors: Sharma Neekun , Markiewicz Mary A.
Published: January 2023

Type 1, or autoimmune, diabetes is driven by T cell-mediated destruction of the insulin-producing β cells in pancreatic islets. The natural killer cell activating receptor NKG2D and its ligands are implicated in this process. However, the mechanism by which NKG2D-NKG2D ligand interaction affects diabetes development is poorly understood. Transgenic expression of the NKG2D ligand retinoic acid early transcript 1ɛ (RAE1ɛ) in β-islet cells of the pancreas induces the recruitment of cytotoxic T lymphocytes (CTLs) to the islets in mice. Surprisingly, however, we report here that the severity of spontaneous autoimmune diabetes is decreased in non-obese diabetic (NOD) mice expressing RAE1ɛ in islets (RIP-RAE1ɛ NOD). Interestingly, NKG2D expression is reduced on the surface of CD8T cells within the pancreas of RIP-RAE1ɛ NOD mice compared with control NOD mice. We further demonstrate that the NKG2D ligand H60a is expressed on T cells within the pancreas of wild-type NOD mice. Together, these results demonstrate that transgenic expression of RAE1ɛ in islets leads to down-regulation of NKG2D surface expression on CD8T cells, likely resulting in decreased ability of the cells to respond to NKG2D engagement by H60a naturally expressed in the pancreas. These findings suggest that interaction between NKG2D and NKG2D ligands, both expressed on islet-infiltrating T cells, plays an important role in the pathology associated with autoimmune diabetes.
Blood relatives with incomplete lupus are distinguished from unaffected relatives and relatives with classified disease by altered autoantibodies, soluble inflammatory and regulatory mediators, and clinical features
Authors: Munroe Melissa , Young Kendra A , Norris Jill M , Aberle Teresa , Roberts Virginia C , Guthridge Joel M , Kamen Diane L , Gilkeson Gary S , Weisman Michael H , Ishimori Mariko L , Wallace Daniel J , Karp David R , Sivils Kathy L , Harley John B , James Judith A
Published: January 2023

Blood relatives (Rel) of lupus patients have increased risk of systemic lupus erythematosus (SLE). Some have autoantibodies and clinical features, but do not meet ≥ 4 ACR criteria needed to reach SLE classification (incomplete lupus, ILE). This study examined individuals enrolled in the Lupus Family Registry and Repository who only met 3 ACR classification criteria during medical record review. ILE patients (n=77) were matched to unaffected Rel and unrelated controls (Ctls), with a subset of ILE patients (n=55) matched to medical record-confirmed SLE patients. ILE patients had significantly higher SLE-specific screening questionnaire (CSQ) scores than unaffected Rel and Ctls (p≤0.0001). A number of soluble mediators that positively correlated with CSQ scores were highest in ILE patients, including SCF (p=0.0001), BLyS (p=0.0018), MCP-3 (p=0.0167), and TNFRI (p=0.0196), as well as ANA titer (p<0.01) and number of lupus-associated autoantibodies (p<0.01). Comparing ILE vs. SLE patients, SLE patients had higher rates of arthritis, serositis, and renal disease (p≤0.004), number of autoantibody specificities (p<0.004), and levels of BLyS (p=0.0138), IL-2Rα (p=0.0201), IP-10 (p=0.0269), and TNFRII (p=0.0309). Yet, ILE patients had higher levels of the regulatory mediator TGF-β (p=0.0454). Identification of factors which discern relatives at increased risk of transitioning to classified SLE may be beneficial to curtail inflammatory damage.
Reduced IL-2R signaling in T1D subjects is pervasive in the memory CD4 T effector compartment and is mediated through intrinsic mechanisms
Authors: Thorpe Jerill Z , Schwedhelm Katharine , Pickett Melissa , Greenbaum Carla J , Cerosaletti Karen , Buckner Jane H , Long S. Alice
Published: January 2023

The IL-2/IL-2R pathway is strongly implicated in type 1 diabetes (T1D). Yet, identifying the functional significance and underlying mechanisms of altered IL-2R signaling in T1D has been challenging due to variance in the subjects, cell types and the assays utilized. We performed a large, well-matched, cross-sectional study of control and T1D subjects (n>75 subjects per group), with an optimized IL-2/pSTAT5 assay (internal control CoV <7%). We found reduced responses to IL-2 in memory CD4 Treg and Teff cell populations of T1D subjects relative to controls. Known genetic risk alleles accounted for some, but not all, of the reduction in pSTAT5. To determine additional factors underlying reduced IL-2R signaling, we interrogated CD4 Teff cells of control and T1D subjects held constant for risk alleles associated with IL-2R signaling. IL-2 signaling in T1D memory CD4 Teff cells remained significantly lower than controls after in vitro activation and proliferation, suggesting a role for intrinsic factors. These intrinsic factors were not caused by divergence to MAPK or Akt signaling pathways, as measured by western blot. Instead, differences in basal activation state prior to in vitro activation correlated with IL-2 response, as measured by targeted flow and qPCR of activation and negative regulation. Thus, multiple factors including genetics, disease and activation state contribute to natural variation in IL-2 responsiveness, requiring precise assays and well-defined cohorts to reach solid conclusions. These findings offer a better understanding of the mechanisms contributing to reduced IL-2R signaling in T1D and have implications for development of more targeted IL-2-based therapies and stratification of patients for therapy.
Immune cells encounter with α-synuclein fuels neurodegeneration in Parkinson’s disease
Authors: Pandey Manoj K , Gross Christina , Caballero Madison , McKay Mary A , Tiwari Durgesh , Schroeder Lindsay M , Gurtler Michael A , Burrow Thomas A
Published: January 2023

GBA1 mutations and increases of neuronal α-synulcein protein (α-syn) are the hallmark of Parkinson’s disease (PD). However, the mechanism, by which Gba1 mutation and or excess release of α-syn trigger neurodegeneration in PD is still wanted. Using Gba1 mouse model (D409V/null), we have observed increased level of α-syn and enhanced brain recruitment of inflammatory subsets of CD11chiCD11b+ CD86+DCs and CD3+CD4+ CD69+ T cells and their association with increased loss of CD45−CD11b− MAP2+ neurons and cognitive dysfunction. Performing adoptive transfer experiments, (e.g., WT cells → WT mice and Gba1 cells → Gba1 mice), augmented brain invasion of DCs and T cells, and neuronal cells destruction have been observed in Gba1 mouse. Searching the mechanism by which infiltrated immune cells could propagate brain inflammation in PD, DCs and CD4+T cells purified from WT and Gba1 mouse were used to perform their ex-vivo stimulation with α-syn and cytokines were measured. These data showed that α-syn treated Gba1 mouse immune cells cause marked increased generation of pro - inflammatory cytokines (e.g., IFNγ, TNFα, and IL6). Supernatant obtained from α-syn stimulated WT and Gba1 mouse DCs and CD4+T cells were used to perform in vitro stimulation of cortical neurons. These data showed that α-syn treated Gba1 mouse cells trigger increased neuronal death suggesting that increased brain penetration of immune cells and their encounter with α-syn propagate neurodegeneration in PD. Targeting DCs and / or CD4+ T cell function could be potential therapy to stop inflammation and neurodegeneration in PD.
TLR7 signaling drives development of a unique population of hemophagocytic macrophages associated with anemia
Authors: Akilesh Holly Marie , Buechler Matthew , Sun Xizhang , Whalen Elizabeth , Mason Michael , Elkon Keith B , Hamerman Jessica A
Published: January 2023

Toll-like receptor (TLR) 7 activity is associated with myeloid expansion in the autoimmune disease Systemic Lupus Erythematosus (SLE), yet how this contributes to severe anemia, often seen in SLE complication, is unclear. Transcriptional and functional assays indicate that TLR7 signaling in the common myeloid progenitor (CMP) preferentially skews lineage choice towards a hemophagocytic macrophage. Transgenic mice that overexpress Tlr7 and develop SLE-like disease exhibited increased hemophagocytic macrophages and progressively developed anemia. Interestingly, in these mice, we observed not only conventional hemophagocytic macrophage populations known as red pulp macrophages (RPM), but also an inflammation-induced hemophagocytic macrophage population (HPM). Unlike RPM that are primarily yolk sac-derived and require the transcription factor Spi-C for their development, we have found that HPM are monocyte-derived and do not require Spi-C. Additionally, TLR7 drives an expansion of hemophagocytic CD11bhi bone marrow macrophages (BMM) that are not found in the absence of TLR7 signaling. Although it has been shown that pathological conditions including excess heme overload can drive monocytes to differentiate to hemophagocytic macrophages, this is the first account of inflammation driving this process. These findings uncover a novel mechanism by which TLR7 signaling participates in hemophagocytic macrophage differentiation from monocytes and may account for one of the mechanisms driving anemia during chronic inflammation and chronic autoimmune diseases such as SLE.
‘Innate and T lymphocyte developmental pathways independent of Common lymphoid progenitors’
Authors: Ghaedi Maryam , Steer Catherine Anna , Martinez-Gonzalez Itziar , Halim Timotheus You Fu , Abraham Ninan , Takei Fumio
Published: January 2023

Lymphocytes are believed to develop from a population of committed lymphoid progenitors termed common lymphoid progenitors (CLPs). However, upstream lymphoid progenitors termed lymphoid-primed multi-potent progenitors (LMPPs) are more efficient than CLPs in differentiating into group 2 innate lymphoid cells (ILC2s) and T cells, suggesting that alternative pathways for the development of these cells exist. In this study, we divided LMPPs into CD127− (LMPP-s) and CD127+ (LMPP+s) subsets and compared them with Ly6D+ and Ly6D− CLPs. Adult LMPP+s differentiated into ILCs and T cells more rapidly and efficiently than other progenitors in transplantation assays. Development of ILC2s and T cells is highly active during neonatal period. In this period CLPs are rare and, unlike prominent neonatal LMPP+s, do not efficiently differentiate into ILC2s and T cells. ILC2s generated in neonates persist in adults. These results suggest that the majority of ILCs and T cells develop from LMPP+s via CLP-independent pathways.
Altered colonic intraepithelial lymphocyte composition and function in Crohn’s Disease
Authors: Ohri Neha , Gerich Mark E , Fennimore Blair P , Kuhn Kristi
Published: January 2023

Inflammatory bowel disease (IBD), which is subdivided into Ulcerative Colitis (UC) and Crohn’s Disease (CD), is a chronic inflammatory disease of the intestine with an incompletely understood pathogenesis. Studies to date have indicated that gender, microbiome, epithelial biology, and host immune responses contribute to disease. Intraepithelial lymphocytes (IELs) are T cells within the intestinal epithelium that are in close contact with bacterial populations; they are prime candidates to study the interaction between intestinal microbiota and host immunity. Thus, we evaluated IELs in pinch biopsies of uninflamed colon tissue from subjects with biopsy-proven IBD and controls that were undergoing routine colonoscopies. Using flow cytometry, we observed a significantly increased percentage of TCRγδ+ CD8αα and CD44+ IELs in CD compared to controls, suggesting an increase in activated IELs in individuals with CD. Mitogen-stimulated IELs from subjects with CD showed significantly increased TNF-α secretion. Furthermore, while none of our controls secreted IL-17, a subset of CD patients, all premenopausal women, had IELs that produced large amounts of this cytokine. Thus, our data indicate differences in IEL populations between CD, UC, and controls, which support the pathophysiologic differences between CD and UC. The different phenotypes suggest a role for these cells in the diagnosis of IBD. Additionally, given the inflammatory cytokine profile of IELs in subjects with CD, especially women, we propose that IL-17+ IELs are an interesting population of effector T cells that deserve further study to increase our understanding of this complex disease.
DNA sensing via STING regulates immune responses in a lung cancer model via IDO
Authors: Mohamed Eslam , Lemos Henrique , Huang Lei , Ou Rong , Pacholczyk Gabriela , Arbab Ali , Munn David , Mellor Andrew
Published: January 2023

The Stimulator of Interferon Genes (STING) is a signaling adaptor associated with the endoplasmic reticulum. STING is activated by cytosolic DNA sensors to stimulate IFN type I expression (IFN-I). Recent reports revealed that DNA sensing to activate STING in dendritic cells (DCs) that engulfed dying tumor cells impeded growth of immunogenic tumors and potentiated responses to therapy. Consistent with these findings, synthetic STING agonists enhanced tumor regression. Paradoxically, Lewis Lung Carcinoma (LLC) grew slower in STING-deficient mice, revealing that STING is required for optimal LLC growth. Mechanistically, STING ablation abolished upregulation of the immunoregulatory enzyme, indoleamine 2,3 dioxygenase (IDO) in DCs located in tumor draining lymph nodes during LLC growth. Also, expression of the regulatory cytokine IL-10 and infiltration of myeloid derived suppressor cells (MDSCs) into the tumor microenvironment (TME) was diminished in mice lacking STING. In contrast, STING was not required for optimal growth of LLC tumors expressing neo-antigens, revealing a pivotal role for tumor antigenicity in influencing responses to DNA in the TME. These findings support the hypothesis that DNA from dying cells are sensed in the TME to activate STING, which induces dominant regulatory responses via IDO when tumor antigenicity is low and dominant immunogenic responses when tumor antigenicity is enhanced. Thus, cytosolic DNA sensing to activate STING has dichotomous roles in the TME, and tumor antigenicity is a pivotal factor influencing immune responses to DNA at sites of chronic inflammation associated with tumor growth.
Immune Enhancing Effects of Aqueous Extracted Black Raspberry in Diet-induced Hyperlipidemia Mice
Authors: Lee Youngjoo , Kim Jiyeon , An Jinho , Lee Sungwon , Lee Heetae , Kong Hyunseok , Song Youngcheon , Choi Hye Ran , Shin Daekeun , Kwon Ji-Wung , Kim Kyungjae
Published: January 2023

As obese population has increased today, metabolic diseases accompanied with obesity have been issued as well. Hyperlipidemia, one of those metabolic abnormalities, is commonly diagnosed with obesity. It is also associated with inflammation and immune dysfunctions, which can be led to global immune decline as the host ages. In this study, the potential effects of black raspberry on the diet-induced hyperlipidemia were examined in terms of immunology. We first induced hyperlipidemia in mice by feeding with 45kcal high fat diet (HFD) for 23 weeks and the progress of disease was traced by blood lipid profiling. Oral administration of black raspberry then proceeded for 16 weeks. As a result, the lymphocyte subset populations such as CD4+, CD8+ and CD11c+ were quantitatively restored by black raspberry treatment. The proliferation and cytokine production of lymphocytes were also normalized in black raspberry treated mice. Regarding specific immunity, oral administration of black raspberry significantly restored both cellular and humoral immune function as determined by in vivo cytotoxic T lymphocytes (CTLs) assay and specific immunoglobulin G (IgG) production level. Lastly the lipid indices including total cholesterol and HDL were considerably improved by black raspberry treatment, indicating the therapeutic effect of black raspberry on hyperlipidemia. That is, not only practical improvement in hyperlipidemia, but also the immune-enhancing effect of black raspberry was newly elucidated with quantitative and qualitative evidences.
Distribution of CD6 on B cells and its role in regulating B1a cell self-renewal
Authors: Enyindah-Asonye Gospel M , Li Yan , Singer Nora , Gupta Neetu , Fung John , Lin Feng
Published: January 2023

CD6 has been established as a marker and an important regulator for T cells. However, despite a previous report suggesting that CD6 is present on human B1a cells that are the primary source of natural antibodies, the precise distribution and potential role of CD6 on B cells remain unclear. In this report, by studying wild type and CD6 knockout (KO) mice, we found that murine CD6 is not detectable on any B2 cells, but is differentially expressed on B1a cells, and its expression is regulated by the activated state and anatomical location of the B1a cells. CD6 KO mice had significantly reduced numbers of B1a cells in different tissue compartments as a result of impaired ability to undergo self-renewal, and these mice had reduced levels of serum natural antibodies including levels of natural IgM. Consistent with previous reports showing that B1a cells produced natural IgM is critical in the pathogenesis of intestinal ischemia/reperfusion-induced injury, we also found that intestinal inflammation and tissue damage was significantly reduced in CD6 KO mice in this model. These findings clarified the distribution of CD6 on B cells and its expression regulation, and revealed a critical role of CD6 in regulating B1a function