bluuebunny/crossref_metadata_2025 huggingface dataset:
Select targeting of intracellular Cysteine in Toll-Interleukin-1 receptor resistance domains for protection against Influenza.
Authors: Snyder Greg A , Shirey Kari Ann , Brown Lindsey J
Published: January 2023

Regulation of Toll-like receptor (TLR) signaling using small molecule agonists and antagonists is widely sought after for controlling inflammation and disease. Recently, small molecule and TIR-based-decoy-peptide inhibitors targeting intracellular Toll-Interleukin-1 receptor (TIR) resistance domains have been identified and functionally characterized to protect against lethality in animal models of infection, inflammation and disease. We observe multiple instances of targeting, modification or involvement of conserved Cysteine residues within intracellular TIR domains by functionally characterized small molecule and decoy peptide inhibitors. We hypothesize that cysteine interactions among intracellular Toll and Interleukin-1 receptors may possess therapeutic value. We have used molecular modeling, X-ray crystallography and in vitro and in vivo functional characterization to characterize TIR cysteine interactions in molecular detail using previously functionally verified derived small molecule and decoy-peptide inhibitors. As a proof of concept, we have used the small molecule receptor antagonist TAK242, which selectively binds Cys747 located in the intracellular TIR domain of TLR4. We show that intracellular targeting of TLR4 TIR domain cysteine using TAK242 is able block LPS activated TLR4 NF-kB signaling in cells and protects mice against lethal influenza (PR8) infection at a lower dose than the ectodomain TLR4 antagonist Eritoran. Structural analysis shows this Cysteine residue to be highly conserved among other surface receptor TLRs 1,2,4,5,6 and 10. Additionally, surface plasmon resonance binding of cysteine containing TIR-decoy peptides, is sensitive to reduction using 2-Mercaptoethanol.
Lactic acid suppresses LPS-induced cytokine production in vitro and in vivo.
Authors: Caslin Heather , Abebayehu Daniel , Qayum Amina Abdul , Hoeferlin L. Alexis , Chalfant Charles E. , Ryan John J.
Published: January 2023

Sepsis is a considerable health burden worldwide with no molecular-based drug treatments. Following the cytokine storm, a hypoinflammatory phase is observed in patients, characterized by impaired cytokine production and glycolysis in immune cells. Lactic acid levels are associated with mortality in patients; however, any regulatory role of lactic acid is currently unknown. We hypothesized that lactic acid contributes to the late phase of sepsis and is not solely a consequence of bacterial infection. As a product of glycolysis, lactic acid may feedback to inhibit glycolysis and overall energy production. Therefore, our purpose was to examine the effects of lactic acid on LPS-induced cytokine production and glycolysis in vitro and on LPS-induced septic shock in vivo. In bone marrow derived and peritoneal mast cells, lactic acid significantly suppressed cytokine and chemokine production at concentrations ≥ 6mM (IL-6) and 12.5 mM (TNF and MCP-1). These effects were dependent upon pH and MCT-1 transport. Additionally, lactic acid attenuated glucose uptake, lactate production, and the expression of the glycolytic enzyme hexokinase 2 following LPS-activation. Extending our in vitro results, lactic acid significantly suppressed IL-6, TNF-α, and MCP-1 induction in multiple in vivo experiments using LPS-induced septic shock. Interestingly, lactic acid did not affect temperature change or observational score. These findings suggest that elevated lactic acid levels may contribute to reduced cytokine concentrations and impaired immune cell metabolism observed in sepsis patients. This information may improve our understanding of immunosuppression in the late phase of sepsis and could reveal new molecular targets for treatment.
15-deoxy-Δ12,14-prostaglandin J2 enhances the interaction between Keap1 and p62/SQSTM1
Authors: Joo Myungsoo , Lee Ji Yeon , Kim Kyun Ha
Published: January 2023

15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) can suppress inflammation by activating Nrf2, an anti-inflammatory transcription factor. 15d-PGJ2 forms an adduct to Keap1 and disrupts Keap1 ubiquitinating Nrf2, resulting in the accumulation and activation of Nrf2 in the nucleus. Here, we investigated a fate of the Keap1 disabled by15d- PGJ2. Our results show that while suppressing the ubiquitination of Nrf2, 15d-P GJ2 did not affect the ubiquitination of Keap1. Unlike prostaglandin E2, 15d-PGJ2 increased the binding between Keap1 and p62/SQSTM1 and enhanced the degradation of Keap1 in a cycloheximide chase experiment. 15d-PGJ2 increased the amount of Keap1, when treated with lysosomal protease inhibitors, E-64d and pepstatin, but not with MG132, a proteasome inhibitor, suggesting that Keap1 is preferably removed via autophagosome. Together, these results suggest that 15d-PGJ2 increases the binding of Keap1 to p62/SQSTM1, facilitating the removal of the dysfunctional Keap1 via autophagy. Our findings suggest that 15d-PGJ2 regulates the anti-inflammatory activity of Nrf2 by not only inactivating the function of Keap1 but clearing the dysfunctional Keap1.
T Follicular Regulatory (TFR) Cells Regulate B Cell Responses By Targeting TF Helper (TFH) Cells
Authors: Lopez-Ocasio Maria D , Shevach Ethan M
Published: January 2023

Interaction of TFH cells with B cells not only helps TFH to fully commit to this lineage, but also provides B cells with survival and differentiation cues. A GC response is critical for the development of an immune response against pathogens, but a dysregulated response could lead to autoantibody production. TFR cells, a subset of Foxp3+ Tregs, localize into follicles to regulate GC responses. To elucidate the mechanism (s) and cellular target (s) utilized by TFR cells to suppress GC responses, we quantitated TFR function in vitro. In the absence of TFR cells, TFH from primed mice induce both naive B cell differentiation into GC B cells and class switching in the presence of anti-CD3 alone or anti-IgM/anti-CD3 in a contact dependent manner. Addition of TFR cells from primed mice efficiently suppressed GC B cell proliferation, differentiation and class switching in the anti-CD3 alone cultures, but only moderately suppressed BCR-stimulated B cells. Under anti-CD3 conditions, IL-4-deficient TFH cells did not promote B cell differentiation and class switching. In contrast, IL-21R-deficient B cells differentiated into GC B cells with a reduced number of IgG1+ cells. When IL-4 deficient TFH cells were co-cultured with IL-21R-deficient B cells, both GC differentiation and class switching were reduced. Under anti-IgM/anti-CD3 conditions, IL-4-deficient TFH cells promoted B cell differentiation, but class switching was reduced, while IL-21R-deficient B cells differentiated normally into GC B cells, but class switching was reduced. These studies suggest that at least in vitro, TFR cells regulate the GC responses by acting directly on TFH cells most likely by inhibiting cytokine production.
Remission induction of established nephritis by therapeutic administration of a novel BTK inhibitor in an inducible model of lupus nephritis
Authors: Chalmers Samantha A. , Doerner Jessica L , Bosanac Todd , Khalil Sara , Smith Dustin , Harcken Christian , Dimock Janice , Herlitz Leal , Webb Deborah , Seccareccia Elise , Feng Di , Fine Jay S , Ramanujam Meera , Klein Elliott , Putterman Chaim
Published: January 2023

Lupus nephritis (LN) patients currently lack highly effective and safe treatment options. Bruton’s tyrosine kinase (BTK) is a tyrosine kinase which is important for B cell and macrophage function. Based on preliminary studies in an inducible model of nephritis that highlighted the importance of BTK dependent macrophage effector function in the pathogenesis of disease, we extended our studies to explore the effect of BTK inhibition on remission induction of established, proliferative nephritis.

Nephritis was induced in female 129 sv/J mice (10 weeks of age) via the passive transfer of nephrotoxic serum (NTS). Once a mouse developed severe nephritis (>300 mg/dl proteinuria for two consecutive days, or a single measurement of >2000 mg/dl), treatment begun via daily oral gavage with 3 mg/kg of BI-BTK-1, a novel, highly selective and potent BTK inhibitor. Each treated mouse was matched to a control mouse that received daily gavage of the vehicle alone.

Within two days of beginning treatment, mice treated with BI-BTK-1 had a significant reversal of the proteinuria compared to vehicle control treated mice (p<0.05). Terminal urine revealed a significantly reduced A:C ratios (p <0.001). Additionally, terminal serum revealed normalized BUN levels. Histological assessment revealed significant structural protection in the treated compared to control treated mice, including both glomerular (p<0.001) and tubular compartments (p < 0.001). Kidney RT-PCR and RNA-seq analysis are currently in progress.

Therapeutic treatment with BI-BTK-1 reversed severe, established nephritis induced by pathogenic antibodies. These exciting results indicate the novel therapeutic potential of BTK inhibition for treating LN patients with established disease.
Male microbiota-associated metabolite protects female BWF1 mice from lupus possibly by restoring tolerogenic CD103+ DC function.
Authors: Harder James W , Chhabra Anita Y. , Alard Pascale , Zhang Xiang , Hua Yuan , Ferrill Rachel , Kosiewicz Michele M
Published: January 2023

Alterations in microbiota composition are associated with autoimmune diseases. We have found previously that microbiota composition differs significantly between female and male BWF1 mice, and transfer of male microbiota to females suppresses disease and increases survival. Protection appears to be mediated, at least in part, through male microbiota action on CD103+ dendritic cells (CD103DC) that induce Tregs in the periphery (pTregs). Female BWF1 CD103DC appear to have a defect in their ability to synthesize retinoic acid (RA; critical for Treg conversion and synthesis requires retinaldehyde dehydrogenase, RALDH2, activity) that is restored by male microbiota transfer. How the male microbiota protects against disease is unknown, but may be mediated through differential production of metabolites. We have performed multiple metabolomic analyses on feces from female and male BWF1 mice and from female recipients of male microbiota. We have identified >90 metabolites that are differentially produced, including those that can act as HDAC inhibitors, fatty acid synthesis inhibitors, and retinoid X receptor agonists (i.e., may enhance RALDH2 activity). We found that feeding the male microbial metabolite with RXR agonist activity to female BWF1 mice significantly increased CD103DC RALDH2 activity and the levels of pTregs in vivo, and delayed the onset of kidney disease. Taken together, these data suggest that at least one male microbiota-associated metabolite may be capable of suppressing disease directly, and may do so by enhancing tolerogenic CD103DC activity. Experiments are currently underway to test the effect of other male microbial metabolites on the immune mechanisms involved in lupus.
Selective HDAC6 inhibitor decreases lupus in mice
Authors: Ren Jingjing , Liao Xiaofeng , Vieson Miranda , Eden Kristin , Chen Miao , Scott Reilly , Kazmierczak Jillian , Luo Xin M , Reilly Christopher M
Published: January 2023

Histone deacetylase 6 (HDAC6) is a Class IIb HDACs enzyme primarily residing in the cytoplasm. HDAC6 alters gene transcription by removing acetyl groups from lysine residues on transcription factors. We, and others, have previously demonstrated that HDAC6 expression is increased in systemic lupus erythematosus (SLE) patients and animal models of lupus and that inhibition of HDAC6 decreased disease. In our current studies, we tested if an orally active selective HDAC6 inhibitor would decrease disease pathogenesis in a lupus mouse model with established early disease. Importantly, we sought to delineate the cellular and molecular mechanism(s) of action for the HDAC6 inhibitor. We treated 20-week-old early-diseased NZB/W F1 (lupus) female mice with two different doses of the selective HDAC6 inhibitor (ACY-738) for five weeks. At the termination of the study, our results showed a reduced germinal center B cell response, decreased T follicular helper cells and diminished IFN-g production from T helper cells in splenic tissue. Additionally, we found the IFNa-producing ability of plasmacytoid dendritic cells was decreased along with immunoglobulin isotype switching and the generation of pathogenic autoantibodies. Renal tissue showed decreased immunoglobulin deposition and reduced inflammation as judged by glomerular and interstitial inflammation. Taking together, these studies show selective HDAC inhibition decreased several parameters of disease pathogenesis in lupus prone mice. The decrease was in part due to inhibition of autoreactive B cell differentiation. Studies are currently underway to define further the mechanism of how HDAC6 inhibits auto-reactive B cell differentiation.
R428 as a novel therapeutic agent for the treatment of experimental immune-mediated nephritis
Authors: Shao Wenhai , Zhen Yuxuan
Published: January 2023

Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease. Glomerulonephritis is a major cause of morbidity in SLE. Glomerular proliferation is essential for the pathogenesis and progression of glomerular diseases. Nephrotoxic serum-mediated anti-glomerular basement membrane (anti-GBM) disease is a well-studied mouse model of immune-mediated glomerulonephritis. Axl receptor tyrosine kinase mediates glomerular cell survival signaling, resulting in proliferative responses. We previously showed that Axl-KO mice developed significantly reduced nephritis with improved kidney function and survival rate compared to the WT mice under the same treatment. In this study, we explore the therapeutic effect of Axl-specific small molecular inhibitor, R428, in anti-GBM nephritis. Our results show that R428 effectively prevents disease progression and exacerbation. Mesangial cell proliferation was greatly reduced in the R428-treatment mice. Mechanistic studies revealed that R428 suppresses the expression of pro-inflammatory cytokines and reduces Akt phosphorylation in the kidney. In summary, these results indicate that R428, currently under clinical trial in cancer patients, may have clinical therapeutic application in lupus nephritis patients.
A double-blind, placebo-controlled, crossover study of magnesium supplementation in patients with XMEN syndrome: preliminary results.
Authors: Ravell Juan C , Price Susan , Kanellopoulou Chrysi , Matthews Helen F , Cohen Jeffrey , Lenardo Michael
Published: January 2023

Introduction

X-linked immunodeficiency magnesium defect, Epstein-Barr virus (EBV) infection and neoplasia (XMEN) syndrome is a primary immunodeficiency caused by the loss of expression of the magnesium transporter 1 (MAGT1). This syndrome is associated with CD4 lymphopenia, chronic EBV infection in most patients, and EBV related lympho proliferative disorders. The loss of MAGT1 leads to impaired T cell activation and decreased expression of the activator receptor, NKG2D on natural killer (NK) cells and CD8 T cells, leading to decreased EBV-specific cytolytic function of these cells. Results of previous studies suggest that magnesium supplementation may be a viable therapeutic option for patients with XMEN.

Methods

Patients ≥6 years of age with XMEN syndrome and persistent EBV viral load of ≥5,000 copies/mL by whole blood PCR are randomized to receive escalating doses of either placebo or oral magnesium L-threonate for 12 weeks. This is followed by crossover treatment for an additional 12 weeks. For patients who experience a 0.5-log decrease in the number of EBV-infected B cells by FISH with oral magnesium as compared to placebo, the study is complete. Patients who do not meet this outcome then undergo a 2-week washout period and proceed to Part II, an open-label, non-randomized evaluation of intravenous magnesium sulfate (MgSO4) followed by oral magnesium L-threonate.

Results

here we report the preliminary results for the first patient enrolled in the clinical trial, including changes in the number of EBV infected B cells and NKG2D expression with magnesium treatment.
Detection of naive B cells expressing potentially protective antibodies
Authors: Bancroft Tara A , McGuire Andrew , Gray Matthew , Feng Junli , Pancera Marie , Steach Holly , Debuysscher Blair , Stamatatos Leonidas , Taylor Justin
Published: January 2023

Most effective vaccines work through the induction of protective antibodies able to neutralize or eradicate infection. Unfortunately, the induction of protective antibodies has proved difficult for a number of infections, such as HIV/AIDS, or the protective antibodies that are elicited through vaccination do not bind all strains of the virus, such as with influenza. Broadly neutralizing antibodies against these viruses, displaying protective potentials in experimental animal models, have been isolated from infected individuals. The detailed characterization of these antibodies revealed that they are derived from specific, but infrequent, VH/VL gene pairing. To better characterize the rare naïve B cells that express the germline (gl) forms of these antibodies (gl B cell receptors, glBCRs) we developed unique anti-idiotype antibodies, that specifically recognize the gl form of HIV-1 and influenza broadly neutralizing antibodies, b12 and FI6, respectively. The abilities of anti-idiotypic antibodies against gl-b12 and -FI6 IgG to specifically identify the rare naïve human B cells that express gl-b12 or -FI6 were evaluated. In the case of FI6, we generated an anti-idiotype that bound to 5 closely related alleles within the VH3 family. Using this anti-idiotype in conjugation with a Flu protein that can bind to gl-FI6, we have begun to characterize a family of gl-FI6-like antibodies. For b12, one anti-idiotype displayed superior ability to target naïve B cells expressing gl-b12-like BCRs. The availability of this novel reagent allows for the high-throughput, in-depth, characterization of the rare B cells that are the precursors of broadly neutralizing antibodies, and will assist efforts to elicit such antibodies through vaccination
CTL-based vaccine-induced control is associated with induction of high follicular to extra-follicular ratios of virus-specific CD8+ T cells.
Authors: Mohammad Hadia Mohammad Abdelaal , Li Shengbin , Sawahata Ryoko , Mwakalundwa Gwantwa , Fraser Kathryn , Mylvaganam Geetha , Martins Mauricio , Masopust David , Amara Rama Rao , Watkins David , Connick Elizabeth , Skinner Pamela J
Published: January 2023

There is a need to develop an effective HIV vaccine. We earlier showed that during chronic HIV-1/SIV infections, HIV/SIV replication is concentrated within B cell follicles, whereas HIV/SIV-specific CD8 T cells are largely excluded from these sites suggesting that the inability of HIV/SIV-specific CD8 T cells to fully suppress virus replication may be due to their deficiency in B cell follicles. We hypothesize that a successful HIV vaccine will either prevent HIV seeding B cell follicles or induce high levels of virus-specific CD8 T cells in B cell follicles. Here we investigated whether control associated with three CTL inducing SIV vaccines was associated with levels of SIV-specific CD8T cells in follicular (F) and extrafollicular (EF) areas in lymph nodes of vaccinated animals after challenge with pathogenic SIV relative to a cohort of unvaccinated chronically infected animals using in situ tetramer staining combined with immunohistochemistry. We defined control as a set point pVL of ≤104. We found lower levels of tet+ SIV-specific CD8 T cells in F compared to EF areas of non-controller animals (P<0.0001), but not in controller animals (P=0.1). Although we found similar levels of tet+ SIV-specific CD8T cells in F and EF areas between the controller and non-controller animals (P=0.6) and (p=0.5) respectively, the controller animals had significantly higher F: EF ratios of tet+ SIV-specific CD8T cells (P=0.028). Also, we found a significant inverse correlation between F: EF ratio of tet+ SIV-specific CD8 T cells and pVL in the controller (r=− 0.75, P=0.0046) but not in non-controller animals (r=0.01, P=0.97). These results support developing CTL-based HIV vaccines that augment relative levels of virus-specific CTL within B cell follicles.
SIV-specific CD8 T cells with perforin granules located exclusively within cell membranes correlate with the maturation of SIVmac239Δnef vaccine induced protection
Authors: Mwakalundwa Gwantwa L , Wagstaff Reece , Mohamed Hadia , Johnson Paul R , Skinner Pamela J
Published: January 2023

Vaccination with live-attenuated SIVmac239Δnef protects most animals challenged with a pathogenic SIV. In this model, there is a maturation of protection which occurs between 5 and 20 weeks post-vaccination. We previously showed that this maturation of protection did not correlate with distribution or density of SIV-specific CD8 T cells in lymphoid or genital tissues. In this study, we investigated whether the maturation of protection correlated with levels of the effector protein perforin in SIV-specific CD8 T cells in lymphoid or vaginal tissues. Using in situ tetramer staining with MHC tetramers combined with immunohistochemistry, and quantitative image analysis, we quantified perforin levels in SIV-specific CD8 T cells; Perforin negative cells are characterized as central memory T cells (TCM), perforin low cells are effector memory (TEM), and perforin high/med are effector T cells (TEFF). We also quantified a unique population of SIV-specific CD8 T cells with perforin localized exclusively within the cell membrane, which are likely to be a subset of TEM. From 5 to 20 weeks post-infection, we found increase in perforin negative and decrease in perforin high tetramer binding CD8 T cells. We also found an increase in SIV-specific CD8 T cells expressing low levels of perforin located exclusively in the cell membrane that correlated with the maturation of protection. These findings indicate that the maturation of protection induced by SIVmac239Δnef vaccine correlates with a shift from a primary response dominated by effector CD8 T cells, to a memory response, which includes an increase in SIV-specific CD8 T cells with perforin located exclusively in the cell membrane. These findings help inform the development of a successful HIV vaccine.
Rubella/SIV vectors for immunotherapy of SIV infection in rhesus macaques
Authors: Berkower Ira , Virnik Konstantin
Published: January 2023

Currently available anti-retroviral therapy (ART) can fully suppress viremia after SIV infection in macaques, but the drugs do not cure infection. When ART drugs are withdrawn, the virus rebounds vigorously. Apparently, ART cannot control or eliminate viral reservoirs. We postulate that early ART, combined with induction of strong T-cell immunity via live viral vectors, can achieve a functional cure by reducing or eliminating long-lived viral reservoirs. This could be detected as lack of viral rebound when ART is stopped.

We have developed the rubella vaccine strain (RA27/3)as a live attenuated viral vector that was safe and highly immunogenic in rhesus macaques. Rubella vectors-expressing SIV Gag replicated vigorously in vivo while eliciting potent T cellimmunity to gag. The vectors alsoelicited anti-Gag antibodies that were equal to SIV infection, durable for >1 year, and were boosted by re-exposure to the vector. We are testing immunotherapy in a three stage experiment: 1st stage, SIV infection followed by starting ART on day 3; 2nd, Immunizing with SIV/Gag vectors while on ART; 3rd Stopping ART and measuring viral rebound. So far, we have shown complete viral suppression while on ART and a good immune response to the rubella vector and to the gag insert.

Pending viral loads will indicate whether all control animals were infected by SIV, and any reduction or delay in viral rebound due to immunotherapy effects on the viral reservoir. The results in primate models may predict the effect of immune therapy on recent HIV infection of humans. This approach could be ideal for treating HIV infection in infants, since the viral reservoir is small, and the immune system is still intact and capable of responding to the rubella/gagvector.
The effects of direct-fed microbial supplementation, as an alternative to antibiotics, on growth performance, intestinal immune status and epithelial barrier protein expression in broiler chickens
Authors: Oh Sungtaek , Gadde Ujvala Deepthi , Lee Youngsub , Davis Ellen , Zimmerman Noah , Rehberger Tom , Lillehoj Hyun S.
Published: January 2023

This study was conducted to investigate the effects of Bacillus subtilis supplementation in broiler chicken diets on growth performance, feed efficiency, intestinal cytokine and tight junction (TJ) protein mRNA expression. Day-old broiler chicks (n = 140) were to one of five dietary treatments: basal diet (CON), basal diet supplemented with antibiotic (BMD) or probiotics, namely B. subtilis strain 1781 (PB1), a combination of B. subtilis strain 1104+strain 747 (PB2) or B. subtilis strain 1781+strain 747 (PB3). At 14 days of age, ileal samples were collected and used for intestinal cytokine, TJ protein and mucin gene expression analysis using qRT-PCR. The chickens fed with antibiotic (BMD) and B. subtilis strain 1781 alone (PB1) had significantly higher body weights compared to controls of the same age. Dietary supplementation with antibiotic (BMD) or probiotics (PB1, PB2, PB3) significantly improved the feed efficiency as evidenced by decreased FCR compared to controls. No differences were observed in the expression of IL1β, IL17F, IFNγ and MUC2 gene among the different treatment groups. However, elevated expression of IL6 (BMD, PB1, PB2), IL8 (PB2) and TNFSF15 (PB1, PB2, PB3) compared to controls was observed in the ileum. IL2 and IL10 expression was upregulated in chicks in the PB2 and PB3 groups, and IL4 was elevated in the PB1 group. IL13 was elevated in all probiotic-fed groups. Probiotic supplementation was also shown to significantly increase the expression of TJ proteins JAM2, ZO1 (PB2, PB3) and occludin (PB1, PB2). Taken together, B. subtilis supplementation altered intestinal immune activity and influenced gut barrier integrity through increased expression of TJ proteins.
Infection with Porcine Reproductive and Respiratory Syndrome virus (PRRSv) impacts immune cell metabolism without evidence of cell-mediated immune response.
Authors: Loving Crystal L , Olson Zahra F , Schweer Wes P , Gabler Nicholas K
Published: January 2023

Specific metabolic pathways affect immune cell differentiation and function, and the metabolic state of leukocytes may provide a signature associated with a particular condition or treatment. Health challenge in food production animals may warrant dietary changes to meet metabolic demand of the immune system. Porcine reproductive and respiratory syndrome virus (PRRSV) infection negatively impacts pig performance, and the PRRSV-specific immune response is inefficient at providing protection. Given the poor cell-mediated response induced by infection, we hypothesized that the metabolic profile of peripheral blood mononuclear cells (PBMC) from PRRSV-infected pigs would not show a shift to glycolysis, which is a hallmark of T effector cells. Using an Extracellular Flux Analyzer (Seahorse Biosciences) we interrogated the glycolytic and mitochondrial function of PBMC following infection of pigs with PRRSV, and PRRSV-specific cell-mediated immunity. Basal glycolysis of PBMC was not significantly impacted by PRRSV infection, but contrary to our hypothesis, glycolytic capacity and reserve were significantly increased in PBMC from PRRSV-infected pigs over non-infected pigs. The effect of PRRSV infection on PBMC mitochondrial respiration was minimal, with a slight increase in spare respiratory capacity detected. Despite the increased glycolytic capacity of PBMC, there was not a detectable PRRSV-specific cell-mediated immune response. Thus, while there was metabolic signature of lymphocyte activation, it was not associated with a functional response.
Diclazuril treatment ineffective at preventing Equine Protozoal Myeloencephalitis relapse in established mouse model.
Authors: Wagner Alayna , Kasmark Leah , Witonsky Sharon , Lindsay David , Leeth Caroline McPhee
Published: January 2023

The debilitating and potentially fatal neurologic disease, Equine Protozoal Myoencephalitis (EPM), is one of the most common neurologic diseases seen in the equine population of the United States. Disease develops as a result of unintentional ingestion of the pathogenic parasite, Sarcocystis neurona. Treatment options consist of the anticoccidial drug Diclazuril and numerous other antiprotozoal drugs. Weeks to months after cessation of antiprotozoal drug treatment and corresponding neurologic improvement, horses may present with clinical disease symptoms again. Little is known whether this reoccurrence of symptoms is from relapse or reinfection. We sought to understand if relapse was possible following appropriate treatment with diclazuril, a commonly used medication for the treatment of EPM. Using a mouse model of equine EPM, we subjected infected mice to treatment with or without diclazuril for 30 and 60 days. All untreated mice developed neurologic symptoms consistent with S. neurona infection within 30 days post infection. All diclazuril treated mice developed neurologic symptoms cessation of treatment. Cerebellum samples were examined for lesions characteristic of those associated with S. neurona infection and immunohistochemically for presences S. neurona. Sera immunoglobulin levels were analyzed to evaluate treatment’s effect on humoral immunity. In conclusion horses treated appropriately with 60 days of diclazuril may be at risk for relapse and more effective treatment options should be explored.
Kinetic Analysis of ZAP70 Phosphorylation in Human T cells: p-Y292, p-Y319, and p-Y493
Authors: Soper David M
Published: January 2023

The 70-kDa zeta chain-associated protein (ZAP-70) is a Syk family protein tyrosine kinase normally expressed in T and NK cells. Patients deficient in ZAP-70 have no functional T cells in the periphery, are prone to repeated infections, and suffer from ZAP-70-related severe combined immunodeficiency (SCID). In some cases of chronic lymphocytic leukemia (CLL), ZAP-70 is aberrantly expressed in B cells and used a prognostic marker to monitor disease progression and therapeutic intervention. In normal T cells, ZAP-70 is required for thymic development, T cell receptor (TCR) mediated signaling, and T cell activation. ZAP-70 consists of two tandem Src homology 2 (SH2) domains, two linker regions, and a kinase domain. Following TCR engagement, several tyrosine residues within ZAP-70 are phosphorylated and serve to both positively and negatively regulate kinase activity. A kinetic analysis was performed using human peripheral blood mononuclear cells (PMBCs) stimulated with hydrogen peroxide (H2O2), anti-CD3/CD28, anti-CD3/PD-1, or anti-CD3/TIGIT. Using in-house generated clones, T cells were analyzed for three distinct ZAP-70 phospho-epitopes: p-Y292, p-Y319, and p-Y493.
Coalescence of Nanoscale Cytoplasmic Signalosomes Contributes to T Cell Receptor Signaling to NF-κB
Authors: Traver Maria K , Campanello Leonard , Paul Suman , Shroff Hari , Losert Wolfgang , Schaefer Brian C
Published: January 2023

T cell receptor (TCR) activation of the transcription factor NF-κB is a crucial determinant of effector T lymphocyte function. The complex regulatory network surrounding this pathway remains poorly understood, particularly at time periods further removed from initial TCR triggering. We have previously demonstrated that following activation of the TCR, the proteins p62, Bcl10, and Malt1 rapidly combine to form a cytoplasmic filamentous signalosome called POLKADOTS, which recruits further signaling proteins and initiates the terminal steps in activation of NF-κB. Here, we examine the fate of POLKADOTS signalosomes following initial activation of NF-κB. We demonstrate via super-resolution and confocal microscopy techniques that POLKADOTS filaments converge on the microtubule-organizing center via microtubule transport, aggregating in a peri-nuclear aggresomal structure. Aggresomes are poorly understood structures thought to be depots of misfolded protein destined for degradation; however, our data suggest that the aggresomal accumulations of POLKADOTS continue to promote T cell activation. We show that the formation and maintenance of this aggresome corresponds with a secondary increase in NF-κB translocation. Additionally, the aggresomal structure prolongs phosphorylation of IKK and promotes the proteolytic activity of Malt1. Together, these results demonstrate that TCR signaling to NF-κB directs the orchestrated assembly, transport, and stable coalescence of nanoscale cytoplasmic signaling complexes. More broadly, our findings provide evidence that aggresomes can serve as stable platforms of signal transduction, thereby proposing a new role for these little understood structures.
Molecular mechanisms for adenosine regulation of helper T cell activation
Authors: Cekic Caglar , Akdemir Imran , Koyas Altay , Kayhan Merve , Savas Ali Can
Published: January 2023

Polarization of T helper subsets into different functional phenotypes strongly affects the progression of immune-related diseases. One of the hallmarks of immune cell activation and inflammation is the elevation of extracellular adenosine and increased expression of adenosine A2A receptors in activated immune cells. Adenosine strongly inhibits activation of helper T cells by elevating cAMP concentrations, which activate PKA and EPAC proteins to regulate cellular responses. Here we showed that adenosine strongly inhibits T cell accumulation rather than differentiating into functional subsets in the presence of polarizing conditions. Different aspects of adenosine mediated T cell suppression were phenocopied by specific activation of PKA and/or EPAC pathways. Adenosine can suppress phospho-activation of Akt pathway to promote T cell quiescence and to inhibit immediate downstream events after TCR stimulation. One of the targets for Akt is Foxo1. Foxo1 is known to suppress T cell proliferation. Our results showed that adenosine receptor stimulation reduced inhibitory phosphorylation of Foxo1 downstream of Akt. Foxo1 inhibitor, AS1842856, completely reversed the inhibition of T cell accumulation by adenosine signaling. Our results suggest that adenosine by activating PKA and/or EPAC pathways and by sustaining Foxo1 activation suppresses T cell activation and accumulation. These findings have important implications to develop novel interventions to regulate helper T cell responses in different pathological conditions.
Molecular mechanisms regulating CXCR4 mediated cytokine mRNA stability
Authors: Dinkel Brittney A , Kremer Kimberly , Hedin Karen E
Published: January 2023

T cell cytokine production has been shown to be dependent not only on T cell antigen receptor (TCR), but also the chemokine receptor CXCR4. Evidence in the literature suggests that, in addition to stimulating migration, CXCR4 might also more directly regulate T cell immune activation. Recent studies from our lab have revealed that TCR stimulation in the absence of SDF-1 induces association of TCR and CXCR4, and that this is inhibited by pretreating cells with the specific CXCR4 antagonist AMD3100. Cytokine production, particularly cytokines IL-2, IL-4, and IL-10, by TCR-stimulated cells was inhibited by using either AMD3100 or CXCR4 siRNA to prevent TCR-CXCR4 formation. Interestingly, TCR-CXCR4 was not required for activation of many TCR-stimulated signaling pathways or for cytokine gene transcription. Instead, TCR-CXCR4 formation during TCR-mediated T cell immune activation was required for the post-transcriptional stabilization of cytokine mRNAs through a Rac-1 mediated pathway. Our results suggest that targeting the TCR-CXCR4 complex formation or downstream signaling pathways may provide therapeutic targets to regulate cytokine production.
Leupaxin is serine phosphorylated and recruited to the immunological synapse upon TCR engagement in cytotoxic T lymphocytes (CTL)
Authors: Yao Shugang , Ostergaard Hanne
Published: January 2023

Background

Leupaxin is a member of paxillin family proteins that function as adaptor proteins in integrin signaling. Compared to other members, leupaxin is mainly expressed in leukocytes. However, the role of leupaxin in T cells is unknown.

Results

Leupaxin has been shown to be tyrosine phosphorylated in cells including tumor cells, fibroblasts and B cells. However we detected no leupaxin tyrosine phosphorylation in T cells. Instead, leupaxin showed mobility shift in response to TCR signaling. We demonstrated that the mobility shift was caused by serine phosphorylation and was ERK dependent. Although leupaxin is downstream of integrin receptors, LFA-1 stimulation did not induce leupaxin phosphorylation. Several potentially phosphorylated residues were mutated into alanine individually. When Ser54 was mutated, leupaxin no longer showed mobility shift, suggesting that leupaxin is phosphorylated at Ser54. Leupaxin was recruited to the immunological synapse and colocalized with LFA-1. Leupaxin has N-terminal leucine-aspartic acid (LD) domains and C-terminal LIM domains that contain four double zinc finger motifs. The LIM domains were dispensable for leupaxin localization at the contact zone. Leupaxin failed to be recruited to the immunological synapse after deleting LD2–4 domains, suggesting the importance of LD2–4 domains for the recruitment.

Conclusions

Leupaxin is not tyrosine phosphorylated but serine phosphorylated at Ser54 in response to TCR engagement. Leupaxin is recruited to the immunological synapse through LD2–4 domains. Studies are underway to determine the contribution of leupaxin to CTL adhesion, polarization and degranulation.
PKCθ Signaling in CD8 T cells regulates Notch1 and C-rel localization
Authors: Sherman Heather L , Ozay E. Ilker , Garrigan Daniel T. , Minter Lisa M.
Published: January 2023

The NF-kB family of transcription factors regulate many functions essential to T cells, including activation, proliferation, and survival. In resting T cells, certain NF-kB subunits are retained in inactive complexes in the cytosol. Following stimulation, phosphorylation events mediated by the IKK complex allows nuclear import of NF-kB and binding to target promoters. Using Jurkat CD4 T cells, it was shown that the NF-kB subunit, c-Rel, interacts with PKCq and anchors it to chromatin. Previous work in our lab demonstrated a physical and functional interaction between PKCq and the transmembrane receptor Notch1, in primary CD4 T cells, upstream of NF-kB activation. We further showed that inhibiting Notch1 signaling can attenuate symptoms in a model of acute graft-vs-host disease. Using this same model revealed that CD8 T cells require intact PKCq signaling to mediate disease, and data from the literature suggest CD8 T cells lacking PKCq are phenotypically similar to c-Rel-deficient CD8 T cells. Furthermore, we have discovered that PKCq, Notch1, and c-Rel localize differentially in CD4 and CD8 T cells following activation. Here we show that PKCq cellular localization regulates a novel interaction between Notch1 and c-Rel in CD8 T cells, which may constitute a crucial driver of graft-vs-host disease.
A Subset of Human Autoreactive CD1c-Restricted T Cells Preferentially Express TRBV4-1+ TCRs and Recognize Phospholipids
Authors: Guo Tingxi , Koo Edward M.Y. , Hirano Naoto
Published: January 2023

Unlike the highly polymorphic, peptide-presenting conventional MHC molecules, CD1 consists of a family of monomorphic, lipid-presenting proteins. Recent studies have revealed the molecular basis of mycobacterial lipid recognition by CD1a-c-restricted T cells. In addition to foreign lipids, subsets of CD1a-c-restricted T cells recognize self-lipids, which may have implications for human diseases such as autoimmunity and cancer. And yet, the molecular identity of these self-reactive T cells remains largely elusive. In this study, using a novel CD1c+ artificial antigen-presenting cell (aAPC)-based system, we have isolated human CD1c-restricted autoreactive T cells and characterized them at the molecular level. By employing the human cell line K562, deficient in MHC class I/II and CD1 expression, as a backbone, we generated an aAPC expressing CD1c as the sole antigen-presenting molecule with costimulatory molecules, CD80 and CD83. When stimulated with this CD1c+ aAPC endogenously presenting self-lipids, a subpopulation of primary human CD4+ T cells from multiple donors consistently upregulated CD154 (CD40L) in a CD1c-specific manner. These activated CD4+ T cells preferentially expressed TRBV4-1+ TCRs. Interestingly, TRAV usage and CDR3 sequences of these TRBV4-1+ T cells were diverse. Clonotypic analyses of the reconstituted TRBV4-1+ TCRs demonstrated that the heterogeneity of the CDR3 sequences greatly impacted the strength of CD1c-restricted autoreactivity. Furthermore, cell-free assays using recombinant CD1c loaded with distinct lipids identified several phospholipid species as potential self-ligands. These data provide new insights into the molecular identity of human autoreactive CD1c-restricted T cells.
Inhibition of Phosphatidylinositol-3-Kinase by Hibiscone C in T cells
Authors: Rawlings Jason S , Besley Caroline , Goess Brian
Published: January 2023

Inhibitors of Phosphatidylinositol-3-Kinase (PI3K) are of significant interest due to their ability to target an intracellular pathway, which regulates cell proliferation, growth, and migration. Mutations in PI3K can lead to an upregulation of the pathway, resulting in tumorigenesis, autoimmunity, and leukemia in the immune system, while a nonfunctional PI3K can result in immunodeficiency. Wortmannin, a known and potent PI3K inhibitor, possesses many unfavorable characteristics as a chemotherapeutic and immunotherapy drug such as its insolubility in neutral buffers, limited supply, and high toxicity in vivo, which have prevented its clinical application. The small molecule inhibitor Hibiscone C, synthesized from the readily available precursors acetone and isobutylaldehyde, shares the same structural chemistry at the binding site as Wortmannin, suggesting that it shares similar biological activity. Via analysis of phosphorylation of the downstream effector molecule Akt in activated T cells, we demonstrate that Hibiscone C also can irreversibly inhibit PI3K activity due to the presence of the furanosteroid ring, to which PI3K covalently binds in each molecule. As predicted by the lack of functional groups in Hibisconce C that are present in Wortmannin, Hibiscone C was found to be less potent of an inhibitor. Despite this reduced inhibitory potency, Hibiscone C serves as a minimalist structure with which to study the requirements for PI3K binding affinity and inhibition. This reduced structure can be manipulated in order to synthesize derivatives that can maximize potency, specificity, and solubility.
S1P1 regulation of pulmonary tertiary lymphoid tissues
Authors: Huppé Carole-Ann , Lecours Pascale Blais , Lechasseur Ariane , Morissette Mathieu C. , Marsolais David
Published: January 2023

Pulmonary tertiary lymphoid tissues (TLT) emerge as central regulators of antigen-specific responses in the lung and they have been associated with the severity of chronic respiratory diseases such as hypersensitivity pneumonitis (HP). The sphingosine-1-phosphate receptor S1P1 is a potent modulator of lymphoid organ biology and of the TH17 response, which is often associated with HP and TLT formation in the lung. We recently identified a novel immunogenic agent commonly found in the bioaerosols of working environments that induces an HP-like disease and potently stimulates TLT formation in the lung. We first aimed to determine if the ability of this agent to induce TLTs was related to their propensity to trigger a TH17 response in the lung. Secondly, we determined whether or not S1P1 acts as a modulator of pulmonary TLTs. Mice were exposed intra-nasally to Methanosphaera stadtmanae (MSS) for a 3-weeks time period in order to induce TLT formation. Upon MSS rechallenge, Il-17 expression was increased by 3 fold in the lung supporting the involvement of this cytokine in the current model. Among five homeostatic chemokines/proinflammatory cytokines tested, we found that CXCL13 was the most significantly increased by the MSS rechallenge (4.9-fold), supporting its involvement in TLT formation in response to MSS. S1P1 pharmacological agonism led to a 50% inhibition of Il-17 and Cxcl13 expression in the lung, which translated into a full inhibition of TLT expansion in response to the MSS rechallenge; and into a 77% alleviation of the pulmonary neutrophilic response. This study unravels a role for the S1P pathway in the regulation of TLTs as well as its interplay with the TH17 response in the lung.
Leptin Enhances TH2 and ILC2 Responses in Allergic Airway Disease
Authors: Zheng Handong , Zhang Xing , Castillo Eliseo F , Luo Yan , Liu Meilian , Yang Xuexian
Published: January 2023

Allergic asthma and obesity are the leading health problems in the world. Many studies have shown that obesity is a risk factor of development of asthma. However, the underlying mechanism has not been well established. In this study, we demonstrate that leptin, an adipokine elevated in obese individuals, promoted proliferation and survival of pro-allergic type 2 helper T cells and group 2 innate lymphoid cells and production of type 2 cytokines, which together contribute to allergic responses. Leptin activates mTORC1, MAPK and STAT3 pathways in TH2 cells. The effects of leptin on TH2 cell proliferation, survival and cytokine production are dependent on the mTORC1 and MAPK pathways as revealed by specific inhibitors. In vivo, leptin-deficiency led to attenuated experimental allergic airway inflammation. Our results thus support that obesity associated elevation of leptin contributes to the increased susceptibility of asthma via modulation of pro-allergic lymphocyte responses.
Accumulation of pulmonary vacuolated eosinophils in experimental allergic asthma that synergize with dendritic cells to induce Th-17 cells
Authors: Laumonnier Yves , Wiese Anna Valeska , Ender Fanny , Quell Katharina , Vollbrandt Tillman , König Peter , Köhl Jörg
Published: January 2023

Eosinophils are considered a homogenous cell population, contributing to the allergic asthma phenotype by promoting mucus hypersecretion and airway hyperresponsiveness. Here, we identified a previously unrecognized vacuolated CD11cdim eosinophilic population (vEOS) which accmulates exclusively in the lung tissue and in mediastinal lymph nodes but not in the airways in a house dust mite-mediated allergic asthma model and an IL-33 mediated pulmonary inflammation. In addition to the vEOS, we found classical SiglecF+CD11c− eosinophils (EOS) in the airways and the lung. Both cell types shared typical structural features of eosinophils and expressed CCR3. In contrast to EOS, vEOS expressed higher levels of the CD11a, CD11b and CD18 integrins. Immunofluorescence microscopy revealed that vEOS were located exclusively near arteries and airways, where they were in close contact with T cells. Moreover, vEOS took up antigen and expressed costimulatory molecules more efficiently than EOS. Consequently, in co-culture experiments with OVA-TCR transgenic T cells, vEOS and dendritic cells (DCs) drove T cell proliferation. However, in the presence of vEOS, T cells divided more frequently than in co-culture with DCs. Further, T cell activation in response to co-culture with vEOS together with DCs markedly enhanced IL-17 but not IL-13 production.

Collectively, our data demonstrate that vEOS are an important subset of eosinophils, which differ from EOS in their location and their ability to activate T cells. Further, they suggest that cross-talk between vEOS and DCs might be a crucial step for the induction of Th17 cells in allergic asthma. Thus, vEOS may serve as an important target for Th17-associated steroid-resistant asthma.