bluuebunny/crossref_metadata_2025 huggingface dataset:
Peripheral Tolerance Regulates RBC-specific CD4+ T Cells in a Novel Mouse
Authors: Wong Sut Ling Andrea , Richards Amanda L , Zimring James C , Hudson Krystalyn
Published: January 2023

T cell tolerance mechanisms towards tissue-restricted antigens are well described and include deletion, anergy, or regulatory cells; however tolerance mechanisms against RBC-restricted antigens are poorly understood. Herein, we utilized the HOD mouse to investigate T cell tolerance to RBC-specific antigens. The HOD mouse expresses a fusion protein consisting of hen egg lysosyme (HEL), ovalbumin (OVA), and human blood group, Duffy driven by an RBC-specific promoter. The HOD mouse was crossed to TCR transgenic OTII mouse to generate HOD mice with autoreactive CD4+ T cells (OTII+HOD+). Young OTII+HOD+ mice do not have detectable autoantibodies, suggesting immune tolerance. Analysis of thymii 8 week-old mice revealed similar numbers of OTII CD4+ SP thymocytes between OTII+HOD+ and OTII+HOD−, suggesting that central tolerance plays little role in RBC specific CD4+ T cells. However, in the periphery, several tolerance mechanisms were identified in OTII+HOD+ mice. Autoreactive splenic CD4+ T cells in OTII+HOD+ mice underwent deletion, expressed higher levels of inhibitory markers PD-1, LAG3, CD5, and had higher numbers of CD25+Foxp3+ regulatory T cells, when compared to OTII+HOD−. Furthermore, CD4+ T cells from OTII+HOD+ had significantly reduced proliferation in response to cognate antigen, suggesting anergy or functional inactivation. Analysis of antigen presenting cells in OTII+HOD+ revealed higher frequencies of myeloid DC, known to express high levels of MHC II and propensity to activate CD4+ T cells. Finally, a break in tolerance was observed in 30% of OTII+HOD+ at 6 months, as indicated by anti-HOD antibodies. Future studies will focus on identifying defective tolerance mechanism that contributed to autoantibody genesis.
Gut intraepithelial myelin reactive CD4+T cells suppress central nervous system autoimmunity via LAG3
Authors: Kadowaki Atsushi , Miyake Sachiko , Saga Ryoko , Chiba Asako , Yamamura Takashi
Published: January 2023

Objective

Multiple sclerosis (MS) is an autoimmune disease that targets the myelin of the central nervous system (CNS). Increasing incidence of MS in Japan let us question which components of the immune system are altered by environmental factors. Gut mucosa is by far the largest immune organ that interacts with the external environment. Since T cells are pivotal in controlling CNS inflammation, we investigated the existence of myelin reactive T cells that might be induced in the gut and their influence on CNS autoimmunity.

Methods

Gut-T cells from MOG(35–55) specific T cell receptor transgenic (2D2) mice were analyzed by FACS. Sorted gut-T cells were adoptively transferred to experimental autoimmune encephalomyelitis (EAE) induced by immunization with MOG(35–55). Blocking antibodies were used for functional analysis. Gut microbiota was modulated by oral antibiotics treatment. Diets including poor or rich aryl hydrocarbon receptor ligands (AHRL) were fed to mice.

Results

Here we demonstrate that the gut epithelium of 2D2 mice is inhabited by intraepithelial T-lymphocytes (IEL) that can inhibit EAE upon transfer. The regulatory IEL had the phenotype of CD4+‘induced’IEL (CD2+CD5+), exhibited TH17-like profile and had ability to infiltrate the inflamed CNS tissue. The IEL constitutively expressed Ctla4 and Tgfb1, and markedly upregulated Lag3 in the CNS, thereby inhibiting the inflammation. We also demonstrated that the IEL are induced by gut microbiota and AHRL.

Conclusions

Gut environment favors the generation of myelin reactive T cells that have unique regulatory function. Dysbiosis or poor AHRL in diet may reduce the induction of such regulatory cells, thus promoting the development of MS.
Autoimmune demyelinating disease results from the combined effects of T cell affinity and increased bond lifetime for self antigen.
Authors: Blanchfield Jennifer Lori , Liu Baoyu , Evavold Brian D
Published: January 2023

A fundamental question for autoimmunity is how T cells become activated to attack self in the periphery. Antigen recognition by T cells is often characterized by affinity, defined as the strength and therefore likelihood of interaction between TCR and peptide:MHC (pMHC), with the assumption that higher affinity T cells will be more pathogenic. However, our analyses of polyclonal and monoclonal CD4+ T cells reveal a wide range of affinities for myelin oligodendrocyte glycoprotein (MOG) responsive T cells in EAE. We argue a finer degree of detail than affinity as the sole predictor of function because high and low affinity T cells promote paralytic disease. Affinity for antigen becomes a key factor in the kinetics of T cell activation, but is just one parameter. T cells also generate dynamic forces during this process and force adds a level of mechanical regulation to the TCR-pMHC bond by shortening or enhancing bond lifetimes. This is important because the lifetime contributes to functional potency of the response. For the first time, we report the bond lifetime of MOG specific T cells under applied force and demonstrate that upon activation, the T cells biophysically change, exhibiting similar lifetimes regardless of high or low affinity designation. This is novel because high, not low, affinity TCRs are predicted to exhibit long lifetimes. Comparatively, activated myelin specific T cells generate bond lifetimes similar to cells with foreign antigen specificity. In all, our data support a kinetic proofreading model of T cell activation linking antigen discrimination, actin remodeling and availability of intracellular chemical messengers where with time, high and low affinity T cells are essentially equipotent contributors to autoimmunity.
Type I interferon protects CD4 T cells from NK cell killing in a cGVHD model of SLE
Authors: Klarquist Jared , Hennies Cassandra M , Janssen Edith M
Published: January 2023

Myriad studies have linked type I interferon (IFN) to the pathogenesis of autoimmune diseases, including systemic lupus erythematosus (SLE). While increased levels of type I IFN are found in patients with SLE, and IFN blockade ameliorates disease in most mouse models of lupus, its precise roles in driving SLE pathogenesis remain largely unknown. Here, we dissected the role of type I IFN in disease development using the bm12 cGVHD model of SLE, where IAb (C57BL/6) mice develop SLE-like disease upon transfer of IAbm12 (bm12) CD4 T cells. Our data show increased serum levels of type I IFN over time, concomitant with the development of T follicular helper cells, germinal center (GC) B cells, plasmablasts, and anti-nuclear antibodies (ANA). However, these disease parameters are significantly reduced in IFN-α receptor-deficient (Ifnar−/−) recipient mice, indicating an important role for type I IFN sensing by non-T cells. Surprisingly, transfer of highly purified Ifnar−/− bm12 CD4 T cells into WT C57BL/6 hosts resulted in poor expansion of the transferred T cells, and limited development of GC B cells, plasmablasts, and ANA. Importantly, NK cell depletion restored the expansion of Ifnar−/− CD4 T cells and downstream sequelae. These findings suggest that one novel mechanism by which type I IFN contributes to autoimmune disease is by regulating the susceptibility of pathogenic CD4 T cells to NK cell killing, thus providing further rationale for the development of anti-IFNAR therapeutics for the treatment of SLE.
Experimental induction of autoantibodies to IgG by pigeon IgY
Authors: Aranda Ivan Sammir , Ortega Enrique , Martinez Erasmo
Published: January 2023

Background

IgG is a frequent target of autoantibodies in a variety of experimental and clinical settings. Interestingly, the possible antigens (Ag) that may induce such a break of tolerance have not been fully studied. Since our previous observations showed the frequent co-occurrence of antibodies (Abs) to pigeon IgY and self-reactivity to IgG in hypersensitivity pneumonitis secondary to avian exposure, we decided to evaluate experimentally the role of IgY in this kind of autoimmune reactivity.

Objective

To study whether pigeon IgY induces anti-IgG reactivity in an experimental model in which foreign and self-immunoglobulins are phylogenetically related.

Methods

IgY was purified from pigeon (Columba livia) eggs, prepared in Freund’s adjuvant and immunized to Balb/c mice. Abs production to foreign (IgY) and self-antigen (mouse IgG) were evaluated by ELISA and Western blot. Tregs (CD4+CD25+Foxp3+) were also studied by flow cytometry since the first evidences suggested a T-cell dependent Ab reactivity.

Results

IgG with anti-IgY activity occurred 2 weeks after immunization. Autoantibodies were predominantly IgG and bind Fc fractions of IgG. Immunoglobulins to self-IgG showed a gradual increase and they persisted for 3 weeks. A correlation between high levels of autoantibodies and elevated Tregs was also found.

Conclusions

Pigeon IgY induced autoantibody production, it was predominantly IgG, follows a kinetic similar to soluble immune complexes formation, and bind the Fc fraction of IgG. T-cell analyses suggested a Tregs dependent reactivity. The break of tolerance to self-IgG in mammals has not been previously studied using a phylogenetically related Ag.
Suppressive modifiers of autoimmunity: Identification and characterization of genes within the Sles1 interval
Authors: Khan Shaheen , Belobrajdic Katherine , Hwang Sun-Hee , Subramanian Srividya , Casco Jose , Wakeland Edward
Published: January 2023

Sle1 is a potent susceptibility locus for spontaneous systemic autoimmunity derived from the NZM2410 mouse strain. The NZW-derived suppressive modifier locus, Sles1, specifically prevents the spontaneous loss in tolerance mediated by the B6.Sle1 congenic. Fine mapping combined with phenotypic characterization of series of recombinant mouse strains have mapped Sles1 to an approximately 638 KB segment on murine chromosome 17. This revealed extensive epistatic gene interactions within the Sles1 region and suggested Btnl2 and H2 genes as the strongest candidates. We have performed targeted resequencing on B6 and B6.Sles1 mice and have identified functional polymorphisms within the Sles1 interval between the two mice strains. We further generated a series of BAC transgenic mice and have performed RNA-seq analysis and cell surface expression studies on B cells, Bone-marrow derived macrophages and Bone-marrow derived dendritic cells. We are in the process of aging BAC transgenic mice and will be phenotyping all the mice at 7 months with a goal to identify one or more genes driving the pathogenesis of autoimmunity.
Bowman birk protease inhibitor suppresses GM-CSF, Th17 cells and CNS autoimmune inflammatory demyelination
Authors: Safavi Farinaz , Li Zichen , Wang Limei , Ciric Bogoljub , Rasouli Javad , Zhang Guang-Xian , Rostami Abdolmohamad
Published: January 2023

Serine proteases play numerous roles in immune system and their inhibition modulates immune and autoimmune responses. Bowman Birk inhibitor (BBI), a soybean-derived serine protease inhibitor potently suppresses experimental autoimmune encephalitis (EAE) after oral administration. In this study, we show that BBI inhibits development of Th17 cells and reduces Th17 cell encephalitogenicity by suppressing their GM-CSF production and decreasing their numbers in the CNS of EAE mice. We also demonstrate that BBI induces IFN-γ production in Th17 cell culture, and that lack of IFN-γ or IFN-γR abrogates Th17 suppressive effect of BBI. In addition, BBI requires IL-27R, Stat-1, T-bet, and IL-10 to inhibit Th17 development. Taken together, our data demonstrate that BBI suppresses pathogenic Th17 cells through IFN- γ dependent pathway and ameliorates EAE. This suggests a novel immunomodulatory effect for serine protease inhibitors in immunity. In addition, BBI has potential to be safe oral therapy for autoimmune diseases, such as multiple sclerosis.
Oligonol Induces the Expression of the Anti-aging Molecule, SIRT1, via the AMPK-Autophagy Pathway
Authors: Park Seul-Ki , Seung Rak-Kyun , Kim Ji-Ae , Shin Ok Sarah
Published: January 2023

Oligonol, mainly found in lychee fruit, is an antioxidant polyphenolic compound which has been shown to have anti-inflammatory and anti-cancer properties. The detailed mechanisms by which oligonol may act as an anti-aging molecule have not been determined. In this study, we evaluated the ability of oligonol to modulate sirtuin (SIRT) expression in human lung epithelial (A549) cells. Oligonol was added to A549 cells and reactive oxygen species (ROS) production, mitochondrial superoxide formation, and p21 protein levels were measured. Signaling pathways activated upon oligonol treatment were also determined by western blotting. Furthermore, the anti-aging effect of oligonol was evaluated ex vivo in mouse splenocytes and in vivo in Caenorhabditis elegans. Oligonol specifically induced the expression of SIRT1, whose activity is linked to gene expression, metabolic control, and healthy aging. In response to influenza virus infection of A549 cells, oligonol treatment significantly up-regulated SIRT1 Oligonol specifically induced the expression of SIRT1, whose activity is linked to gene expression, metabolic control, expression and down-regulated viral hemagglutinin expression. Oligonol treatment also resulted in the activation of autophagy pathways and the phosphorylation of AMP-activated protein kinase (AMPK). Furthermore, oligonol-treated spleen lymphocytes from old mice showed increased cell proliferation, and mRNA levels of sirt1 in the lungs of old mice were significantly lower than those in the lungs of young mice. Additionally, in vivo lethality assay revealed that oligonol extended the lifespan of C. elegans infected with lethal Vibrio cholerae.
Comparative evaluation of inflammatory responses in mice exposed to gold nanoparticles of biological and chemical origins
Authors: Sharma Nilesh , Sadrinia Cyrus , Sahi Shivendra
Published: January 2023

Gold nanoparticles have found an ever-increasing application in diagnostics and therapeutics in recent times. In vivo and in vitro toxicity studies reveal often contradictory results. It has been shown that the toxicity, if caused, depends on the shape, size and crystal characteristics of the nanomaterial. In this background, nanoparticles–unconventionally synthesized from plant cell culture (ccAuNPs)–were compared to chemically-synthesized nanoparticles (csAuNPS) after administering repeated doses of (IP) 200 μL of each (40nm size) to c57bl/6 mice for 5 and 30 days. Control was maintained with an equal dose of PBS. Differential patterns of cytokine activities were observed between the two categories and two periods of exposure. Levels of Leptin, IL-1b, IL-10 and GCSF were significantly (p<0.05) elevated for both ccAuNPS and csAuNPS groups with respect to control in case of short exposure. Under long exposure, both nanomaterials induced significantly (p<0.05) lower activities of TNF-α and IL-6 than control, but for ccAuNPs, the levels of IL-1b, IGF-1 and GCSF were significantly lower than in csAuNPS and control both. There were no significant changes in body weight or liver function enzymes, ALT and AST, with respect to control for both groups of nanomaterials.
Cell division cycle 42 (Cdc42) regulates functions of plasmacytoid dendritic cells
Authors: Stutte Susanne , Schulz Anna M. , Kugler Ina , Imhof Axel , Brakebusch Cord , Brocker Thomas
Published: January 2023

Cell division cycle 42 (Cdc42) is a member of the Rho guanosine triphosphatase family and has pivotal functions in actin organization, cell migration and proliferation. We recently demonstrated that conventional dendritic cells with a Cdc42 deficiency show a significant reduction in lysosomal MHC class II–processing proteins, such as cathepsins, as they are lost by enhanced secretion.

Similarly, we found that Cdc42-deficient plasmacytoid dendritic cells (pDCs) are phenotypically more mature as they up-regulate MHCII and the co-stimulatory molecule CD86. Furthermore, their homing capacity is strongly impaired in steady state causing strongly reduced pDC cell numbers in peripheral organs. In addition, after inflammatory stimulation pDC infiltration is impaired when Cdc42 is absent. We analyzed Cdc42 deficient pDCs in more detail to understand the molecular contribution of Cdc42 in pDC functions and their migratory behavior.
Dietary Poly Unsaturated Fatty Acid (PUFA) regulation of Organ and Tissue Microenvironments in an Iso-Caloric Pair-Fed Mouse Model
Authors: Talmadge James E , Khadge Saraswoti , Thiele Geoffrey M , Klassen Lynnell W , Sharp John Graham , Duryee Michael J , McGuire Timothy R , Dafferner Alicia , Britton Holly , Beck Jordan , Black Paul , DiRussio Concetto
Published: January 2023

The majority of omega-3 (ω3) and ω3 PUFA diet studies have not differentiated dietary PUFA content from obesity. Our studies used pair-fed iso-caloric diets containing 35.5% of calories from fat with an ω6:ω3 ratio in ω6 and ω3 diets of 42:1 and 1:1 respectively with DeCarli diets and fish oil substituting for 70% of olive oil. The ω3 diet contained eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid at a 3:1 ratio, while the ω6 diet contained linoleic acid and a low level of linolenic acid. Female BALB/c mice were monitored for weight gain, diet consumption and sacrificed (12 wks) to examine mammary gland, spleen, bone and hepatic histopathology, leukocyte phenotypes and organ and tissue lipidomics. In association with pair feeding there were no differences in diet consumed and weight gain. The ω6 cohort had decreased numbers of progenitor cells and an increase in splenic subcapsular extramedullary hematopoiesis and hepatocyte lipidosis, hypertrophy and multinucleation and increased hepatic vascularity with thicker intima. The ω-6 cohort also had mammary fat pads (MFPs) with increased adipocytes in the tubular epithelium, stromal cellularity and epithelial tissue density. These changes and subclinical inflammation were accompanied by organ specific lipodemic changes, including increases in arachidonic acid (AA) in the plasma, spleen and liver, but not MFPs, of the ω-6 mice, and an increase in EPA and DHA in the plasma, spleen, MFPs and liver of the ω-3 mice. Further, B and T cells and macrophages had an increase in the % of phospho NFκβ+ cells and the liver and MFPs had an increase in the ω3 PUFA, docosapentaenoic acid. In summary, the dietary ω6:ω3 ratio, independent of obesity, regulates mammary gland, spleen and hepatic inflammation.
NAFLD induces neuroinflammation and accelerates Alzheimer’s disease
Authors: Bynoe Margaret S
Published: January 2023

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease afflicting about one third of the world’s population and 30% of the US population. It is induced by consumption of high lipid diets and is characterized by liver inflammation and subsequent liver pathology leading to steatosis and steatohepatitis. We fed mice with high fat/lipid diet (HFD) or a standard diet (SD) and monitored them for signs of systemic or central nervous system inflammation. We found that NAFLD induced acute liver inflammation and accelerates signs of Alzheimer’s disease (AD) in an AD-Transgenic model. Moreover, chronic NAFLD induced advanced signs of AD in WT mice. In the chronic disease stage, we observed neuronal apoptosis and decreased brain expression of low-density lipoprotein receptor-related protein-1 that is involved in beta-amyloid clearance, in both WT and APP-Tg mice. HFD fed mice harbored numerous activated and proliferating microglial cells in their brain. Removal of mice from HFD during acute disease reversed liver pathology and neuroinflammation and decreased beta-amyloid plaque load. Concomitant with microglial cell activation, we observed profound changes in microbiota, resulting in a more pathogenic microglial phenotype. Interestingly, removal of mice from HFD completely abrogated microglial cell activation and proliferation and changes in gut. Based on these findings, we conclude that inflammation induced by HFD induces profound pathogenic changes in the gut concomitant with pathogenic changes in the CNS leading to neurodegeneration.
Myocardial Ischemia/Reperfusion injury is mediated by polymorphonuclear neutrophils through direct activation of Phospholipase D (PLD) and mTOR
Authors: Ganesan Ramya , Henkels Karen M , Pavlov Vasile I , Stahl Gregory L , Gomez-Cambronero Julian
Published: January 2023

During ischemia/reperfusion injury (I/RI), leukocytes release cytokines and exacerbate inflammation causing cardiac dysfunction, while neutrophils produce oxygen radicals that worsen the initial myocardial infarction. Phospholipase D is a cell membrane remodeling and signaling protein implicated in the pathology of I/RI. We analyzed I/RI by ejection fraction, infarct size and serum Troponin levels in wild-type, PLD1−/− and PLD2−/− mice. PLD-deficient mice or inhibition of PLD in WT mice protected against I/RI. Removing PLD2 yielded a slightly better outcome, suggesting PLD2 contributed more to the injury than PLD1, particularly at longer (>4 h) reperfusion times. Three cell signaling mechanisms were investigated: mTOR pathway, Aurora Kinase A and Cyclin-D3. Gene expression of the mTOR pathway was increased in response to I/RI in WT mice and abrogated in PLD-KO mice suggesting PLD augmented mTOR pathway during IR/I. PLD1−/− mice have lower expression of CCND3/Aurora Kinase A, while PLD2−/− mice expressed more phospho-CyclinD3. We detected neutrophil-associated Ly6G after adoptive transfer of bone marrow neutrophils during I/RI. Myocardial PMN accumulation was increased following I/R and PLD inhibitors abrogated PMN accumulation. Less myocardial PMN accumulation was observed in PLD-KO mice following IR/I. Adoptive transfer of fluorescently labeled PLD2−/− or PLD1−/−PMN into PLD1−/− or PLD2−/− mice, respectively, demonstrated that PLD1 containing (PLD2−/−) PMNs accumulated more significantly in the myocardium following I/RI. This study demonstrates for the first time the specific mechanistic contribution of mammalian PLD1 and PLD2 to MI/R injury in a murine model and their role in neutrophil infiltration.
Antigen-specific transendocytosis of CD40 ligand accompanies T cell help for B cells
Authors: Gardell Jennifer L , Parker David C
Published: January 2023

It has been known for decades that the delivery of T cell help to B cells is antigen-specific, MHC-restricted, and depends on CD40L. However the mechanisms by which CD40L, a transmembrane cytokine, is delivered to the T cell surface and engages CD40 on antigen-presenting B cells remains to be determined. It has been thought that when a T cell recognizes an antigen-presenting B cell, CD40L expressed on the T cell surface engages with CD40 on the surface of B cells as long as the cells remain conjugated. We show for the first time that CD40L does not remain on the surface of the T cell, but is transferred to and endocytosed by B cells receiving T cell help. Transfer of CD40L is nearly absent from bystander B cells that are not presenting antigen. It was recently discovered that peptide-MHC engaged TCRs are deposited on B cells in microvesicles at the immunological synapse. Our data suggest that CD40L might be deposited in a similar manner as a secreted vesicle. CD40-engaged CD40L on vesicles could allow for sustained signaling in antigen-presenting B cells despite the brief interactions with helper T cells that occur in vivo in germinal centers.
RGC-32 regulates TGF-β extracellular matrix production in multiple sclerosis.
Authors: Tatomir Alexandru , Tegla Cosmin A , Cudrici Cornelia D , Boodhoo Dallas , Martin Alvaro , Mekala Armugam , Rus Violeta , Badea Tudor C , Rus Horea
Published: January 2023

Extracellular matrix (ECM) deposition in demyelinating multiple sclerosis (MS) lesions may impede axonal regeneration and modify immune responses. RGC-32 plays an important role in mediation of TGF-β downstream effects, but its role in ECM deposition and gliosis has not been investigated. To gain more insight into the role played by RGC-32 in gliosis, we examined the role of RGC-32 in mediation of ECM production and reactive astrocyte marker α-smooth muscle actin (α-SMA) expression. In MS lesions, collagen I, IV and V were found to be expressed by astrocytes which were also expressing RGC-32. In cultured astrocytes, α-SMA, collagens I, IV, V and fibronectin were significantly induced at 18 h of stimulation with TGF-β. Next, we silenced RGC-32 expression by transfecting astrocytes with siRGC-32 and compared the effect of this treatment to that of control siRNA. The RGC-32 siRNA effectively decreased the mRNA and protein RGC-32 expression by 90% when compared to astrocytes transfected with control siRNA. RGC-32 silencing resulted in a significant reduction in TGF-β- induced collagens I, IV, V and fibronectin expression. Using astrocytes isolated from RGC-32 knockout (KO) mice, we found that TGF-β- induced collagen IV and α-SMA expression were significantly reduced in RGC-32 KO when compared with wild type mice. Our data also indicate that RGC-32 plays an important role in the TGF-β-mediated induction of ECM and is also required for the transition of astrocytes to a reactive state. Therefore, RGC-32 may represent a new target for therapeutic intervention in MS.
The role of ILC2s in neonatal type 2 immunity bias and allergen sensitization
Authors: Steer Catherine A. , Allinger Patricia , Takei Fumio
Published: January 2023

Neonatal immunity is largely type 2, potentially dampening damaging inflammatory type 1 immunity. Without early correction from gradual microbial type 1 stimulation, type 2 immunity may become overactive, resulting in allergic disease. Exposure to inhaled allergens during the neonatal period may sensitize individuals resulting in the development of allergic lung disease. Our lab has characterized group 2 innate lymphoid cells (ILC2s) in mouse lungs, which upon exposure to inhaled allergens, produce IL-5 and IL-13 and drive type 2 lung inflammation. We investigated whether ILC2s play a role in neonatal type 2 bias and allergen sensitization. Mouse lung ILC2s rapidly develop and 10 day old (D10) pups had more ILC2s than adults. D10 ILC2s appeared to be activated due to intracellular IL-13 and IL-5 expression and eosinophil lung infiltration, not observed in ILC2-deficient pups. ILC2 numbers are high in D10 lung-draining lymph nodes but not in the spleen or liver. ILC2s may be driving Th2-biased cell differentiation, as intranasal (IN) OVA-antigen treatment into D10 OTII pups results in significantly more IL-4+IL-13+ CD4+ T cells in the lung-draining LN after 6 days compared to adult OTII mice. Moreover, upon IN protease-allergen papain treatment at D10 and then again 4 weeks later, higher numbers of LN Th2 cells were present compared to similarly treated adults. Importantly, IL-33-deficient D10 pups, had fewer activated ILC2s and little eosinophil lung infiltration, suggesting that neonatal ILC2s activated by endogenous IL-33, produce type 2 cytokines resulting in type 2-biased lung immunity. Therefore, ILC2s may play a role in the neonatal sensitization of individuals, causing the development of allergic lung inflammation in adults.
Characterizing immune responses in severe T-cell mediated adverse drug reaction.
Authors: Konvinse Katherine Chanel , White Katie , Pavlos Rebecca , Redwood Alec , Trubiano Jason , Ergen Elizabeth , Mallal Simon Alexander , Phillips Elizabeth J
Published: January 2023

Severe immune-mediated adverse drug reactions (IM-ADRs) significantly impact patient outcome and inflict substantial cost on healthcare and drug development. Many of these T-cell mediated reactions are now known to be class I and/or class II HLA restricted, however, the minority of patients carrying an HLA risk allele will develop an IM-ADR. To help define the specific immunopathogenesis of these T-cell mediated drug reactions, a biobank of cryopreserved PBMCs and blister fluid/skin biopsies has been developed from HLA class I and II genotyped patients with IM-ADRs (Stevens-Johnson Syndrome/Toxic epidermal necrolysis (SJS/TEN), abacavir hypersensitivity, and drug reaction with eosinophilia and systemic symptoms (DRESS)) at various time points from their acute reaction. Causative drugs included antibiotics, antiretrovirals, anticonvulsants and allopurinol. Overnight gamma interferon ELISpot assay proved sensitive to identify dose-dependent drug responses at different time points during and following the acute IM-ADR. We used intracellular cytokine staining and cell sorting to identify drug specific CD137+/CD69+ activated CD8+ and CD4+ T cells. Single cell and bulk TCR sequencing was performed to compare activated and non-activated populations of T cells in the presence and absence of drug. Immune responses differed according to the drug, clinical phenotype/tissue specificity, time from drug reaction, CD4+ and/or CD8+ dependency, and the oligoclonality of drug specific T-cell populations.
Eosinophil lineage commitment and IL-5-dependent expansion is regulated by IL-33 in mice
Authors: Johnston Laura K , Hsu Chia-Lin , Krier-Burris Rebecca A. , Chhiba Krishan D. , Chien Karen B. , McKenzie Andrew , Berdnikovs Sergejs , Bryce Paul J.
Published: January 2023

Eosinophils are important in the pathogenesis of many diseases, including asthma, eosinophilic esophagitis and eczema. While IL-5 is necessary for the maturation of eosinophil progenitors (EoP) into mature eosinophils (EoM), the signals that promote commitment to the eosinophil lineage are unknown. The IL-33 receptor, ST2, is expressed on several inflammatory cells, including eosinophils, and is best characterized for its role during the initiation of allergic responses in the peripheral tissues. Recently, ST2 expression was described on hematopoietic stem cells, where its function remains unclear. Here, we sought to determine whether IL-33 and ST2 contribute to hematopoietic lineage decisions. We found that both IL-33- and ST2-deficient mice exhibited diminished peripheral blood eosinophils at baseline. Correspondingly, IL-33 administration increased EoM as well as IL-5 in the blood and bone marrow in WT and IL-33-deficient but not ST2-deficient mice. Blocking IL-5 with a neutralizing antibody prevented IL-33-expanded EoP from maturing into EoM, while transgenic overexpression of IL-5 in ST2-deficient mice resulted in significantly lower hypereosinophilia than transgenic IL-5 mice. Finally, we observed that IL-33, but not IL-5, specifically expanded EoP and upregulated IL-5Rα on EoP as well as increased IL-5 after bone marrow was cultured for three days. Our findings identify a basal defect in eosinophilopoiesis in IL-33- and ST2-deficient mice. Furthermore, we establish unappreciated roles for IL-33 and ST2 in eosinophil development via progenitor regulation and define a mechanism whereby IL-33 licenses commitment into the eosinophil lineage by driving both responsiveness to IL-5 and IL-5 production.
Blockade of the Neogenin-RGMb-BMP signaling hub inhibits allergen-induced airway hyperreactivity
Authors: Yu Sanhong , leung Krystle , Kim Hye-Yeung , Xiao Yanping , Albacker Lee A , Lee Hyun-Jun , Umetsu Dale T , Freeman Gordon J , Dekruyff Rosemarie H
Published: January 2023

Bronchial asthma is associated with type 2 immune responses induced by components of adaptive as well as innate immunity. Although innate cytokines such as IL-25 have been shown to play a key role in development of airway hyperreactivity (AHR), little is known of innate molecules that regulate IL-25-mediated airway inflammation. We found that blockade of repulsive guidance molecule b (RGMb) in an experimental murine model of asthma blocked the development of AHR, a cardinal feature of asthma, and that RGMb is expressed on F4/80+CD11b+CD11cneg macrophages (RGMb+ macrophages), which accumulated in the lungs of OVA-sensitized and challenged mice, but not in naïve mice. Moreover, we found that a large fraction of the RGMb+ macrophages expressed the IL-25 receptor, IL-17RB, and produced IL-13. IL-25 was critical for the development of AHR in our model, since mice deficient in IL-17RB did not develop AHR. Finally, treatment with anti-RGMb mAb during the challenge phase of the protocol after allergen sensitization effectively prevented the development of AHR and airway inflammation, suggesting for the first time that RGMb+ cells, including RGMb+ macrophages, play critical roles in allergen-induced asthma.
Targeting the immune system with subtype-selective GABAA receptor modulator to alleviate asthma symptoms.
Authors: Nieman Amanda N , Forkuo Gloria S. , Guthrie Margaret L. , Yuan Nina Y. , Yu Olivia B. , Kodali Revathi , Zahn Nicolas M. , Jahan Rajwana , Stephen Michael R. , Poe Michael M. , Hartzler Benjamin , Emala Charles W. , Cook James M. , Arnold Leggy A.
Published: January 2023

Inflammation is one of the main characteristics of asthma in addition to mucus metaplasia and airway hyperresponsiveness due to muscle constriction. Therefore, current asthma treatments include corticosteroids, which are associated with many negative side effects1. Recently, small molecule modulators of the γ-aminobutyric acid receptor (GABAAR) have been shown to reduce inflammation and induce airway smooth muscle relaxation2,3. GABAARs are expressed in the neurons to moderate neuronal firing. However other cell types have distinct arrangements of GABAAR subtypes as well, which can be targeted with subtype-selective GABAAR modulators. Our study hypothesizes that compounds targeting GABAARs bearing the α4 subunit will predominantly target airway smooth muscle cells and immune cells. We have developed two subtype-selective GABAAR modulators, Xhe-III-74EE and Xhe-III-74A. Investigations with T cells showed reduced IL-2 levels for treated cells. In addition, both compounds reduced cellular rapid cytoplasmic calcium release upon activation. In vivo studies showed that XHE-III-74A was able to reduce eosinophilia in an ovalbumin sensitized and challenged (Ova S/C) model.
Memory ILC2s and type 2 lung inflammation
Authors: Martinez-Gonzalez Itziar , Mathae Laura , Gahedi Maryam , Steer Catherine , Poon Grace FT , Takei Fumio
Published: January 2023

Group 2 innate lymphoid cells (ILC2s) are critical mediators of allergic lung inflammation. Upon protease allergen encounter, epithelial-derived IL-33 activates ILC2s to produce large amounts of type 2 inflammatory cytokines. ILC2-derived cytokines induce T helper 2 (Th2) cell-independent allergic lung inflammation. Upon activation a subset of lung ILC2s becomes long-lived memory cells. Memory ILC2s are more sensitive and respond stronger to unrelated allergen encounters. Gene expression analysis and transfer experiments show that the high responsiveness is due to intrinsic changes in memory ILC2s. Indeed, memory ILC2s not only respond to IL-33 but also become responsive to the alarmin IL-25. Finally, memory ILC2s promote early Th2 differentiation in the draining lymph node, which leads to a stronger memory T cell response. On the other hand, the generation and maintenance of memory ILC2s is T-cell independent. Understanding the regulation of memory ILC2s may lead to the development of novel therapies for allergic diseases.
IRF5 as an upstream regulator of severe steroid-refractory asthma.
Authors: Oriss Timothy B , Morse Christina M , Raundhal Mahesh Ramdas , Huff Rachael E , Hannum Rachel , Ray Prabir , Ray Anuradha
Published: January 2023

Severe, corticosteroid (CS)-refractory asthma is a significant problem both clinically and in terms of economic impact. A CS-responsive T helper-type 2 (Th2)/Type 2 response is a feature of milder asthma. However, we recently reported heightened airway type 1 (IFN-γ) and type 17 (IL-17) immune responses along with detectable Th2 response in >50% of clinically diagnosed severe asthma cases in adults in the context of CS treatment. In macrophages, the transcription factor IRF5 was previously shown to promote the expression of Type 1/17-inducing cytokines. However, its role in promoting the expression of these cytokines in dendritic cells (DCs), which would impact T helper cell differentiation, has not been studied. Thus, to determine the role of IRF5 in eliciting a CS-refractory severe asthma phenotype, we utilized our newly established model of severe asthma, in which IFN-γ plays an important role in increased airway hyperresponsiveness (AHR), and which is poorly responsive to CS mimicking human disease. Using WT and IRF5−/− mice subjected to the severe asthma model, we show that IRF5 is required to promote Th1 and Th17 immune responses and importantly, also plays a role in increased AHR. In the absence of IRF5, reduced Th1/Th17 responses were associated with decreased expression of the type-1/17-promoting cytokines IL-12 and IL-6 in lung DCs. Also, using bronchoalveolar lavage (BAL) cells, we detected higher IRF5 expression in human severe versus milder asthma. Therefore, IRF5 may represent a potential target for therapeutic intervention in CS-refractory human severe asthma with a strong Th1/Th17 immunobiologic component.
Effects of Complement C4 Gene Copy-Number Variations, Gene Size Dichotomy and C4A-Deficiency on Genetic Risk and Clinical Presentation of East-Asian and European Systemic Lupus Erythematosus (SLE)
Authors: Yu Chack Yung , Chen Ji Yih , Wu Yee Ling , Mok Mo Yin , Wu Yeong-Jian Jan , Lintner Katherine E. , Wang Chin-Man , Chung Erwin K. , Yang Yan , Zhou Bi , Wang Huanyu , Yu Denise J.H.C. , Alhomosh Alaaedin , Jones Karla , Spencer Charles H. , Nagaraja Haikady N. , Lau Yu Long , Lau Chak-Sing
Published: January 2023

Human complement C4 is complex with multiple layers of diversity. This study aims to elucidate the copy-number variations (CNVs) of C4A and C4B in disease risk of SLE, and compare the basis of race-specific C4A-deficiency in East-Asians (EA) and Europeans. Our study-populations included (a) 999 EA-SLE patients and 1,347 healthy subjects; and (b) 232 European SLE patients and 500 healthy subjects. Variations in gene copy-numbers (GCNs) for total C4, C4A, C4B, long and short genes were determined and validated by independent genotyping technologies. Genomic regions with C4B96 were investigated to determine the basis of the most basic C4B protein that is concurrent with C4A-deficiency. In EA, strong protective effects for high GCNs of total C4 and C4A against SLE were notable; low and medium GCNs of total C4 and C4A, and the absence of short genes were risk factors for SLE. Homozygous C4A-deficiency was infrequent but had an odds-ratio (OR) of 12.4 (p=0.0015) in SLE. Low serum complement levels were strongly associated with low GCNs of total C4 (OR=3.27, p=7.0×10−7) and C4B (OR=2.55, p=2.5×10−5). Patients with low complement had high frequencies of anti-dsDNA (OR=4.96, p=9.7×10−17), hemolytic anemia (OR=3.89, p=3.6×10−10) and renal disease (OR=2.18, p=8.5×10−6). The monomodular-short haplotype with C4A-deficiency and in linkage-disequilibrium with HLA-DRB1*0301 prevalent in Europeans was scarce in EA. Instead, most EA-subjects with C4A-deficiency shared a recombinant haplotype with bimodular-LS encoding C4B1 and C4B96, which was linked to HLA-DRB1*1501. DNA-sequencing revealed the E920K polymorphism for C4B96. In conclusion, C4 CNVs and C4A-deficiency are important in the risk and manifestations of East-Asian and European SLE.
TRAF1 in regulation of T cell responses and inflammation
Authors: Edilova Maria , Abdul-Sater Ali A. , Clouthier Derek , Watts Tania H
Published: January 2023

TNFR associated factor one (TRAF1) is a signaling adaptor that links a subset of tumor necrosis factor receptors to downstream survival signaling through NF-kB and MAP kinases. TRAF1 is critical for survival signaling downstream of 4-1BB but its role downstream of TNFR2 has been controversial. Genome-wide association studies have identified a single nucleotide polymorphism (SNP), rs3761847, in an intronic segment of the TRAF1 gene as contributing to susceptibility to and severity of Rheumatoid arthritis. However, the effect of this SNP on TRAF1 expression or T cell biology have not been examined to date. To avoid the complications of chronic inflammation, we chose to examine the effect of this common SNP on TRAF1 levels and function in healthy donors. Samples were collected from 80 healthy donors and TRAF1 levels and cytokines measured by intracellular flow cytometry on T cells from resting and anti-CD3/CD28 treated PBMC. Individuals homozygous for the risk polymorphism (GG) exhibited significantly lower TRAF1 protein levels in T cells than donors with the disease resistant (AA) genotype. However, the frequency of naïve, memory and regulatory T cell subsets was indistinguishable between AA and GG donors. T cells from the disease susceptible donor T cells produced less IFNγ, TNFα and IL-2 upon stimulation. Work is in progress to investigate how TRAF1 specifically impacts TNFR2 signaling in T cells. Taken together, our data suggest that the TRAF1 SNP results in lower TRAF1 protein and lower cytokine production by T cells, supporting the evidence that TRAF1 has a net positive role in T cell cytokine production. However, these data also present a paradox of how lower TRAF1 protein levels contribute to increased inflammatory disease.
Epithelial ErbB2 activity determines thymus homeostasis and T cell maturation
Authors: Agrawal Tanvi , Chan Win-yan , Sage Elizabeth , Kolluri Krishna , Sladen Paul , Sturges Gabrielle , Vallath Sabarinath , Janes Samuel , Coles Mark , Ambler Carrie , Brown Jeremy , Giangreco Adam
Published: January 2023

Thymus involution, the age-dependent loss in organ structure and functionality, promotes increased immune dysfunction in humans, causing increased susceptibility to opportunistic infections, peripheral autoimmunity and reduced vaccine efficacy. Despite this, the mechanisms that regulate thymus involution remain incompletely understood. This has resulted in a lack of clinically approved therapies capable of reversing age-associated thymus involution. Here, we identify that thymic epithelial cell (TEC) specific ErbB2 activity determines thymus homeostasis and T cell maturation.

In these studies, we use a conditionally regulated bitransgenic mouse model in which a dominant-active ErbB2 transgene is expressed in keratin14 positive TECs following doxycycline administration. We show that increased ErbB2 expression causes rapid onset of thymus atrophy that resembles age-associated involution. Specific changes include destruction of thymic cortico-medullary boundaries, abnormal TEC differentiation, reduced thymus size and cellularity, and abnormal T cell differentiation. Using foetal thymus organ cultures derived from wild type and bitransgenic animals, we confirm that these phenotypes are due to TEC autonomous ErbB2 expression. Finally, we establish that treating aged mice with the clinically approved Erbb2 inhibitor Lapatinib restores thymus function, improves Streptococcus pneumoniae vaccine efficacy, and promotes infection resistance.

Overall, these results demonstrate a previously unknown role for ErbB2 in adult thymus homeostasis and suggest the potential translational relevance of ErbB2 inhibition as a means to reduce or reverse age-associated thymus involution.
B cell-intrinsic production of CCL3/4 promotes direct sampling of Germinal Center B cells by T follicular regulatory cells and limits non-foreign antigen specific B cell response.
Authors: Benet Zachary , Wu Rita , Marthi Matangi , Turner Jackson , Grigorova Irina
Published: January 2023

A hallmark of adaptive humoral immunity is the germinal center (GC) reaction. GCs are the primary site of antigen-dependent clonal expansion, immunoglobulin diversification, and affinity maturation and give rise to memory cells and long-lived plasma cells that secrete high-affinity antibodies. These coordinated processes in GCs are highly dependent on cognate follicular T cells. Previous studies and our findings suggest increased expression of proinflammatory chemokines CCL3/4 in GC centrocytes. However, the role of CCL3/4 for centrocyte interactions with follicular T cells and regulation of humoral immunity is poorly understood. We found that B cell intrinsic production of CCL3/4 is required for control of B cell expansion within GCs and restrict the development of class-switched anti-nuclear antibodies following immunization with foreign antigen. We determined that follicular regulatory T cells (Tfr) respond to CCL3/4 chemokines ex vivo and interact more frequently with WT compared to CCL3-deficient GC B cells in vivo using two photon microscopy. Finally, we have identified a subpopulation of GC centrocytes that have significantly increased expression levels of CCL3/4. These data suggests that CCL3/4 secreted by a subset of GC B cells promotes their direct interactions with Tfr cells and plays a role in the elimination of potentially self-reactive B cell clones within GCs.
miRNA regulation of macrophage inflammatory phenotype
Authors: Guerau-De-Arellano Mireia , Jablonski Kyle , Amici Stephanie A. , Gaudet Andrew , Popovich Phillip G. , Webb Lindsay
Published: January 2023

Classically (M1) and alternatively activated (M2) macrophages play distinct roles in various physiological and disease processes. M1 responses can cause inflammatory disease and tissue damage while M2 macrophages can limit inflammation and promote tissue repair. Therefore, understanding key players driving M1 or M2 phenotype may provide novel targets for therapeutic intervention. MicroRNA are small RNAs that bind to messenger RNA and post-transcriptionally modify gene expression and miR-155 has been associated with inflammatory phenotype. miR-155 was up-regulated more than 100-fold in M1, but not M2 macrophages, and inflammatory genes and proteins that define the M1 phenotype (e.g., iNOS, IL-1b, TNF-a) were reduced up to 72% in miR-155 knockout mouse macrophages. In contrast, miR-155 deficiency did not affect expression of genes associated with M2 macrophages (e.g., Arginase-1). Comparative transcriptional profiling of macrophages derived from wild-type and miR-155 knockout mice revealed that approximately half of the wild-type M1 signature was dependent on miR-155. Among miR-155-dependent M1 inflammatory effectors we found IL-6, iNOS, IL-1b, TNF-a and IL-12 as well as the recently described M1 marker CD38. miR-155 targets Bat5, Mafb, c-Maf, Bach1, Ptprj and Il6ra were increased in miR-155 KO M1 macrophages. Importantly, treatment with a miR-155 oligonucleotide inhibitor suppressed iNOS and TNF-a gene expressions in wild-type M1 macrophages, lending support to the idea that miR-155 targeting may be beneficial in inflammatory disease. Overall, these data indicate that miR-155 plays an essential role in the differentiation and effector potential of inflammatory M1 macrophages.
Loss of TRAF3 Promotes a Premalignant Metabolic State in B cells.
Authors: Mambetsariev Nurbek , Lin Wai W , Stunz Laura L , Bishop Gail A
Published: January 2023

TRAF3 is a signaling protein that acts as a tumor suppressor in B cells. B cell conditional TRAF3−/− mice display a remarkable expansion of B cell compartments and parenchymal B cell infiltration, attributed to enhanced B cell survival without increased proliferation. When aged, these mice develop lymphomas, and TRAF3 mutations are common in human B cell lymphoma and multiple myeloma. The mechanism of how loss of TRAF3 promotes B cell survival phenotype is not well understood; this particular role for TRAF3 is not seen in other immune cell types. We find that loss of TRAF3 leads to increased uptake of glucose in B cells in vitro and in vivo. This is accompanied by increased oxidative phosphorylation and glycolysis. In the absence of TRAF3, expression of Glut1 and Hexokinase II, two molecules important for glucose metabolism, is increased. Treatment of B cells with Glut1 inhibitor or the competitive glycolysis inhibitor 2-DG attenuates cell survival, and B cell viability is also substantially reduced in a glucose-free environment. Mechanistically, loss of TRAF3 leads to the induction of Pim2 kinase. Pim2 in turn promotes the protein stability of the oncogenic transcription factor c-Myc, known to drive expression of glycolytic genes. Our study shows that B cell deficiency of TRAF3 is sufficient for altered metabolic programming, consistent with a premalignant abnormal survival phenotype.