bluuebunny/crossref_metadata_2025 huggingface dataset:
In TH17-associated ileal inflammation, IFNγ and IL-17A support mucosal homeostasis and modulate self-tolerance
Authors: De Ciantis Christopher L. , Jeschke Jonathan C. , Mayne Christopher G. , Ziegelbauer Jennifer , Singh Selena , Suchi Mariko , Iwakura Yoichiro , Drobyski William R. , Salzman Nita H. , Williams Calvin B.
Published: January 2023

Crohn’s Disease is characterized by dysregulation of the immune response in the ileum. To study the perturbation of homeostasis within the ileum we utilize a “Bigenic” mouse model combining a TCR transgene and matching transgenic target antigen restricted to ileal crypt expression. Juvenile Bigenic mice experience TH17-associated ileitis and ileal hyperplasia. Antigen transcript is demonstrated to be localized exclusively in ileal crypts, as visualized with in situ hybridization of mRNA. Roughly 50% of Bigenic mice mature and acquire symptomatic phenotype (BigenicS) including colitis and suboptimal growth, while losing ileal hyperplasia. The remaining non-symptomatic Bigenic mice (BigenicNS) retain ileal hyperplasia and successfully build a supply of TH17 and ileal-reactive Treg cells.

Previous studies in our lab indicate that IL-17A and INFγ are protective in this Bigenic model – their depletion by antibody binding accelerates disease and hinders accumulation of ileal-reactive Tregs. Furthermore, INFγKO Bigenic mice demonstrate almost complete penetrance of the symptomatic phenotype, with near 100% fatality by 50 days of life. Manipulation of microbiota in juvenile INFγKO Bigenic mice can protect them from this dysregulated state. Administration of a streptomycin/bacitracin antibiotic cocktail ad libitum in drinking water upon initial manifestation of weight loss symptoms or initiated preemptively upon weaning rescues and prevents, respectively, the weight loss phenotype and colitis.
Identification of functionally unique CD4 T cells that are the circulating counterparts of epidermal resident memory T cells
Authors: Morawski Peter A. , Duhen Thomas , Klicznik Maria M. , Hoellbacher Barbara , Motley Samantha , Campbell Daniel J. , Gratz Iris K.
Published: January 2023

As a barrier organ, the skin contains specialized T cell populations that combat infection and also help maintain tissue homeostasis and promote wound repair. Circulating skin-tropic T cells can also be identified in the blood based on their expression of the cutaneous lymphocyte antigen (CLA), but the developmental and functional relationships between these circulating CLA+ T cells and tissue-resident T cells in the skin are not fully understood. Using 33-parameter Cytometry by time-of-flight (CyTOF) analysis we identified a novel population of skin-tropic CD4+ T cells in human blood that expresses the CD103 integrin and phenotypically and functionally resembles epidermal resident memory T cells (TRM) in the skin. RNA sequencing identified a set of signature genes shared by circulating CD103+CLAhi T cells in the blood and epidermal TRM cells in the skin, and suggested that CD103+CLAhiT cells contribute to normal skin function and response to tissue damage. Finally, using humanized NSG mice carrying full-thickness human skin grafts we demonstrated that, despite their identification as TRM, CD103+CLAhi T cells in the skin are capable of exiting the tissue and re-entering circulation. Thus, CD103+CLAhi T cells in the blood represent a circulating T cell population of cutaneous TRM, and this provides novel opportunities for the study and therapeutic manipulation of TRM cells in the contexts of cutaneous infection, inflammation, and tissue-repair.
Generation of IL-3 secreting T helper cells is associated with microbial challenge at barriers and IL-1 family of cytokines
Authors: Velayudhan Shajo Kunnath , Goldberg Michael F. , Saini Neeraj , Ng Tony , Johndrow Christopher , Johnson Alison Joy , Porcelli Steven A.
Published: January 2023

Naive CD4+ T cells differentiate into distinct T helper subsets upon T cell receptor stimulation in a unique cytokine environment. The cytokine environment leads to the expression of subset-specific master transcription factor which enables secretion of cytokines characteristic of that subset and suppression of cytokines associated with other T helper subsets. We recently described that cutaneous Mycobacterium bovis BCG vaccination in mice induces a subset of T helper cells characterized by the secretion of IL-3. Here, we report that the induction of this subset of T helper cells is route dependent; cutaneous but not intravenous immunization with M. bovis BCG induced these cells. Similar results were obtained with a model of cutaneous Herpes Simplex Virus II infection. Infection with Herpes Simplex Virus by vaginal route or with M. tuberculosis by aerosol route induced these cells suggesting mucosal sites are conducive to their generation. In these scenarios, IL-3 secreting cells co-expressed GM-CSF, IFNγ, TNF and IL-2. In vitro polarization experiments showed that IL-3 secretion by CD4+ T cells was facilitated by IL-1 family of cytokines and suppressed by cytokines that induce Th1 and Th2 helper cells. The characteristic cytokine expression pattern and the dependence on route and cytokine environment of these cells support the idea that IL-3 secreting CD4+ T cells may represent a unique T helper subset.
The immunological role of crypt olfactory neurons against neurotropic viruses in teleost fish
Authors: Sepahi Ali , Kraus Aurora , Galindo-Villegas Jorge , Kelly Cecilia , Garcia-Moreno Diana , Mulero Victoriano , Pau Mar Huertas , Salinas Irene
Published: January 2023

Vertebrate olfactory sensory neurons (OSNs) are located in direct contact with the external environment and therefore they directly exposed to microorganisms such as viruses. Teleost fish have four types of OSNs, including crypt neurons, a cell type with enigmatic function. Crypt neurons express tyrosine receptor kinase A (TrkA)-like molecule and ORA4. In this study, we hypothesized that TrkA expressing crypt neurons are fast sensors of viruses in the olfactory mucosa of fish serving as an ultra-rapid mechanism to elicit local antiviral immune responses. Our results show that, within minutes, nasal delivery of rhadboviruses induces caspase dependent apoptosis in crypt neurons along with neuronal activation in rainbow trout olfactory organ (OO) and olfactory bulb (OB). Induction of chemokine CCL19 and prostaglandin synthase expression was detected 15 minutes after nasal delivery of virus in OO; whereas, in parallel, significant downregulation of CCL19 expression was measured in the OB. Pharmacological blocking of TrkA abolished these responses. CD8+ T cells infiltrated the OO within minutes of nasal viral delivery and this response was abrogated when TrkA was blocked. We identified that the microvasculature surrounding the OB and not the peripheral blood is the source of the infiltrating CD8 T cells suggesting a rapid shunt of these immune cells from the OB microvasculature into the OO. In support, ablation of crypt neurons in zebrafish results in increased susceptibility to rhabdoviral challenge. Combined, our results indicate a novel function for a subset of teleost OSNs, crypt neurons, and the importance of sensory neurons as a first layer of danger detection in vertebrates.
The catalytic subunit of DNA-PK has a unique function in inflammation independently of Ku70 and DNA repair: a new opportunity to target the enzyme without interfering with DNA repair
Authors: Ghonim Mohamed Ahmed , Pyakurel Kusma , Luu Hanh , Okpechi Samuel , Ju Jihang , Boulares Hamid
Published: January 2023

Our laboratory demonstrated a critical role for DNA-dependent protein kinase (DNA-PK) in asthma pathogenesis via modulating the pertinent immune responses. DNA-PK is DNA repair enzyme composed of a catalytic subunit (DNA-PKcs) and two DNA-binding subunits (Ku70 and Ku80). Human cells express high levels of DNA-PK, surprisingly such high levels do not confer increased ability to repair DNA damage. Here we show that the role of DNA-PK in promoting inflammatory responses is independent of its function in the DNA repair. We examined the effect(s) of partial depletion of Ku70 on Ovalbumin (OVA) induced lung inflammation in mouse model of the disease. Of note, depletion of Ku70 by gene heterozygosity causes deficiency in DNA-PK-dependent DNA repair. Unlike the protective effects provided by DNA-PKcs gene heterozygosity, Ku70 heterozygosity did not alter the OVA-induced eosinophilia, mucus hypersecretion, Th2 cytokines production, or OVA-specific IgE upon OVA challenge. Interestingly, Ku70 heterozygosity enhanced methacholine-induced AHR over that of WT mice. Using a cell culture system, we demonstrate that while IL-4 and TNF-α are potent inducers of DNA-PKcs, such activation did not coincide with any detectable DNA damage or repair responses. Remarkably, while Ku70−/− blocked DNA-PKcs autophosphorylation in response to the DNA damage agent, etoposide, it did not affect the kinase in response to TNFα. Our findings suggest that the mechanism by which DNA-PK functions in inflammation is completely unrelated to its role in DNA repair, thus, unraveling a completely novel function for the kinase. More importantly, this provides a window of opportunity to target DNA-PK in inflammatory diseases without interfering with DNA repair processes.
Generation of specialized CD4 subsets: TFH and ThCTL, requires signals from local antigen & infection during the effector phase.
Authors: Devarajan Priyadharshini , Vong Allen M. , Castonguay Catherine H. , Bautista Bianca L. , Swain Susan L.
Published: January 2023

Certain key specialized tissue-restricted CD4 effector subsets such as cytotoxic T cells (ThCTL) in the infected tissue and follicular helper T cells (TFH) in secondary lymphoid organs reach their peak responses after other CD4 effectors (Th1, Th17). Using an influenza A virus (IAV) infection model, we find that generation of ThCTL and TFH require that CD4 effectors recognize specific Ag/MHC Class II, 5–7 days after initial infection, a phase we have termed the late Ag checkpoint. We used TcR transgenic CD4 donor T cells and host mice in which Ag presentation is restricted to specific APC, as well as addition of various APC subsets at the checkpoint, to study which APC drive TFH vs. ThCTL generation. While tissue-restricted antigen presentation is crucial, several APC subsets were able to present Ag effectively to drive TFH and ThCTL generation. However, we found that host infection during the late Ag checkpoint was needed for optimal generation of donor ThCTL and TFH subsets. This contrasts with most CD4 memory generation that is not dependent on infection. Thus multiple CD4 fate decisions are made at the late Ag checkpoint. Both optimal specialized effector and most memory generation require continuing Ag presentation, while further tissue-specific effector differentiation requires local Ag presentation and we think, signals from pathogen recognition pathways (PRR). We suggest that to induce optimum protective immune responses, vaccines and immunotherapies may need to be designed taking into account these requirements for additional Ag presentation and PRR pathways at the late Ag checkpoint.
Immortalization of splenic and peripheral blood macrophages using a multi-cistronic v-Raf/v-Myc lentivirus.
Authors: Beauregard Chelsi Ryan , Broder Christopher C. , Schaefer Brian C.
Published: January 2023

We have created an improved system for immortalizing mature macrophages by modifying the original v-raf/v-myc (J2) virus. Previously, these J2 viruses required a producer co-culture condition and maintained replicating virus. This provided more complexity, reagents, and biosafety risks. Our improved virus is replication deficient. Its lentiviral genome contains the v-raf/v-myc oncogenes and a thy1.1 surface marker, each of which is separated by a self-cleaving peptide. This virus, therefore, eliminates the issues described above. We transduced our modified virus into total murine PBMC isolated from murine peripheral blood or spleen. We found the transduction and successful cell line production can be done with as few as 1×105 PBMC. The resulting cytokine independent macrophage cell line from both spleen and blood expressed mature macrophage markers (F4-80+CD11b+) but lacked early myeloid progenitor markers (Sca1−c-kit−). In addition, we found these macrophages were functional as demonstrated by their ability to undergo phagocytosis and ability to upregulate MHCII in response to IFNγ. These findings demonstrate that the v-raf and v-myc oncogenes in our new system can immortalize macrophages from spleen but uniquely, and perhaps more importantly, can immortalize macrophages from blood. This advance provides a non-invasive method to obtain the starting tissue needed to make these macrophage cell lines. Having the v-raf/v-myc oncogenes in a lentivirus means it is likely this method will work on blood from humans and other species. Additionally, the cytokine independence associated with cell lines made with our virus make it ideal for creating macrophage cell lines from mammals without commercially available growth factors.
Using machine learning and RNA-seq to increase the accuracy and decrease the invasiveness of diagnosing eosinophilic esophagitis.
Authors: Lin Edwin , Flygare Steven , Peterson Kathryn , Clayton Frederic , Yandell Mark
Published: January 2023

Eosinophilic esophagitis (EoE) is an esophageal disease characterized by dysphagia and food impaction triggered by food or drug hypersensitivity with a prevalence of ~1/1,000 people. A conclusive diagnosis of EoE requires endoscopy and multiple biopsies, followed by eosinophil counts in multiple high-power fields. This procedure is painful and expensive, and due to the focal nature of the lesions, misdiagnoses are possible if biopsies do not sample enough diseased tissue. RNA sequencing allows the entire transcriptome within a biological sample to be assayed, but due to the vastness of the data, bioinformatics tools are required for meaningful interpretation. We have trained different machine learning classification algorithms and characterized their performance. We show that accurate classification is generalizable to biopsies that were not controlled for depth and were sequenced using different protocols and machines. Furthermore, we assess whether EoE status can be predicted using buccal epithelial tissue biopsies, which can be collected non-invasively but lack histological change. Together, these results show that machine-learning algorithms based on RNA-seq data could enable more accurate diagnosis of EoE using less invasive and lower-cost biopsy protocols.
Tetracysteine tagged OVA (323–339) peptide as a novel tool for visualizing peptide-MHCII complexes in primary mouse cells
Authors: Al Souz Jafar A. , Akkaya Munir , Kamdar Rahul , Kamenyeva Olena , Shevach Ethan M. , Akkaya Billur
Published: January 2023

Professional APCs present peptides to CD4+ T cells in the context of MHCII. To understand the dynamics of peptide-MHCII complexes in T cell-APC interaction, real-time visualization of these complexes is crucial. However, current reagents such as peptide specific MHCII antibodies do not allow one to probe peptide-MHCII dynamics during live interactions in vivo, as they also block T cell-APC interactions. Fluorescent tags such as green fluorescent protein (GFP) consist of more than 200 amino acids and have the potential of disrupting the functionality of the antigenic peptides. Therefore, we constructed an OVA(323–339) peptide containing a pro-fluorescent tetracysteine tag (CCPGCC) at the C-terminus with aminocaproic acid linker (OVACACA). First, we tested whether the addition of tag had an impact on T cell stimulation and found that OVACACA pulsed APCs could activate OTII cells to same degree as OVA(323–339). Secondly, we treated the APCs with an organoarsenic compound to switch on the fluorescence and measured the signal by flow cytometry. We showed that the fluorescent signal specifically was derived from OVACACA-MHCII, as neither MHCII−/− APCs pulsed with OVACACA, nor MHCII+/+ APCs pulsed with OVA(323–339) produced the same signal. We also tracked the fluorescent signal in OTII-DC cultures in vitro and detected that it was concentrated at the synapse over time. Altogether our findings suggest that tetracysteine tagged peptides can be used for tracking peptide-MHCII complexes during the interactions of primary mouse T cells and APCs and have the potential to be an alternative to MHCII tetramers to detect antigen specific T cells.
Serum IGFBP2, IGFBP4, and sTNFR2 are predictive of concurrent clinical and histological nephritis in SLE
Authors: Haque Anam , Soliman Samar , Henderson Jared , Stanley Samantha , Saxena Ramesh , Mohan Chandra
Published: January 2023

Background

Systemic Lupus Erythematosus (SLE) is an autoimmune disorder that targets various body systems, including the kidneys, leading to Lupus Nephritis (LN). Present diagnostic methods for LN include the renal biopsy, which is invasive. This project takes advantage of a large cohort of LN samples where the serum samples were obtained at the time of renal biopsy. Our goal is to identify serum protein markers that are predictive of concurrent clinical and histological nephritis.

Methods

Serum samples from 10 healthy controls and 66 SLE patients with active renal disease obtained at the time of renal biopsy were ELISA-tested for levels of IGFBP2, IGFBP4, and sTNFR2, selected because they were elevated in LN patients in previous screening studies.

Results

Serum sTNRF2 (p < 0.0001), IGFBP2 (p < 0.0001) and IGFBP4 (p < 0.05) were all elevated in LN patients compared to healthy controls. Concurrently measured Glomerular Filtration Rate was negatively correlated with serum IGFBP4 (p < 0.0001), IGFBP2 (p < 0.05) and sTNFR2 (p < 0.001). Serum IGFBP4 correlated with SLEDAI (p < 0.01), and Renal SLEDAI (p < 0.01). All 3 serum proteins showed a positive correlation with Renal SLICC scores with IGFBP4 being the strongest (p < 0.0001). There was also a significant correlation between various concurrent renal biopsy pathology lesions with the assayed serum proteins, notably IGFBP4 (p < 0.0001).

Conclusion

Serum IGFBP2, IGFBP4, and sTNFR2 are predictive of concurrent clinical nephritis activity in LN as well as histological changes on renal biopsy. Whereas the biopsy is subject to sampling errors, the assayed proteins may represent more stable biomarkers of renal pathology changes in LN.
Tracking Plasmodium Invasion of Red Blood Cells by Simplified Flow Cytometry
Authors: Tran Kimvan Thi , Mulry James , Greco Beatrice , Tyagarajan Kamala
Published: January 2023

Malaria is an acute global problem with large number of deaths and infection on an annual basis. Study of malarial parasites, infection mechanism, action of antimalarial drugs and understanding disease dynamics requires culturing of red blood cells (RBC’s) with plasmodium strains followed by quantifying extent of invasion. The current “gold standard” methods for quantifying parasitemia utilizes microscopic examination of blood smears are laborious, time-consuming and extremely subjective in obtaining quantitative data and dependent on the skills of users. We have developed novel methods using simplified sample prep with analysis on the small footprint, touch-screen based Muse® Cell Analyzer that uses microcapillary cytometry to analyze thousands of RBC’s. The approaches utilize nucleic acid based staining to provide quantitation of infected red blood cells or parasitemia level or quantify parasite density in samples. The methods provide good sensitivity and high reproducibility across a wide parasitemia range and show valuable applicability to screening and monitoring of cultures. Approaches were applied to RBC’s infected with different P. falciparum strains followed by treatment with different doses and times with Chloroquine and Dihydroartemisinin. The studies demonstrate early and sensitive detection of parasitemia changes with treatment and showcase utility of the assays for studying action of anti-malarial drugs. The capability to obtain parasitemia results on infected RBC’s rapidly and easily on a small, accessible platform like Muse® Cell Analyzer can greatly increase ability of academic and clinical malarial researchers to obtain critical information relevant to status and progress of malarial disease
New Flow-Based Multiplex Assays for Simultaneous Quantification of 13 Human or Mouse Macrophage/Microglia Related Cytokines
Authors: Sun Binggang , Rughwani Priyanka , Chow Kay , Lehmann Jason , Ji Shaoquan
Published: January 2023

Immune responses are mediated by innate immune cells (e.g., macrophages) and subsequently, adaptive immune cells (e.g., T and B cells). Macrophages secrete an array of inflammatory mediators to affect their host defense, tissue repair, and immunoregulatory functions. Pro-inflammatory macrophages (M1) produce cytokines, such as IL-12p70, TNF-α, IL-6, IL-1β, IL-12p40, IL-18, IL-23, IFN-γ, and IP-10, while anti-inflammatory and tissue repairing macrophages (M2) release a different set of factors, such as IL-4, IL-10, IL-6, Arginase, TARC, MDC, IL-1RA and TGF-β1. We have developed new assay panels targeting human or mouse macrophage/microglia cells, using fluorescence–encoded beads that are suitable for use on commonly available flow cytometers. Each panel allows for the simultaneous quantification of 13 analytes related to macrophage function in humans and mice. These panels have been validated using biological samples to quantify analyte concentration changes in response to various stimuli. These high quality, low cost, easy-to-use panels provide an alternative multiplex solution to the biomedical research community.
Highly robust digital gene expression profiling of 96 gene targets for IO Lymphocyte and Myeloid activity in 96 Colorectal FFPE samples without any enzyme (RT or amplification) reactions
Authors: Merritt Christopher , Kharkia Anisha , Ong Giang
Published: January 2023

The NanoString nCounter Analysis platform uses a novel molecular barcoding technology to measure multiplexed gene expression of up to 800 targets. The assay counts fluorescent barcodes hybridized to targets to provide precise digital data. The standard nCounter gene expression assay can be used to process 12 samples per run. We recognize an additional need for a higher sample-throughput assay which would enable researchers to quickly evaluate tens of multiplexed targets in hundreds of samples. Here we present data from a new nCounter assay, nCounter PlexSet Reagents for gene expression, which is performed in a 96-sample format and is capable of digitally profiling up to 96 gene targets per sample. We used two preselected pathway panels for IO Lymphocyte Activity (96 gene targets) and IO Myeloid Activity (79 gene targets), chosen from our 400+ preselected pathway panels list, in order to evaluate the presence of immune signatures in 44 colorectal cancer (CRC) samples. Each CRC sample was ran in duplicates across the two panels in addition to a Reference Sample to calibrate for variability between PlexSet reagents. The results show that the PlexSet reagents for gene expression provides highly reproducible data with an average R2 > 95% between duplicate samples.
Sample sparing platform for microscopy shows that CD45RB ligation increases Treg sensitivity to activation signals in vitro
Authors: Lee Joanne Haeun , Lee Jounghyun H. , Kam Lance C.
Published: January 2023

Small subpopulations of cells, e.g. regulatory T cells, often play key roles in the function of the entire immune system. While their smaller number makes them difficult to work with in conventional immunocytochemistry assays or flow cytometry, microscopy can provide functional and spatial information on a single cell basis. Combined with surface modification technology such as synthetic lipid bilayers and micropatterned antibodies, we are in a unique position to be able to study the spatial mobility of intracellular signals (e.g. show that mobility of Lck differs between mouse and human T cells). Even still, conventional microscopy platforms such as 96-well plates or flow cell systems require a minimum of 10k to 1 million cells and does not allow for an interchangeable surface. Conical wells with a 1mm bottom and 5mm top diameter (microwells), laid out in a 96-well configuration were mass manufactured out of polypropylene from Protolabs, and adhered to surface modified glass coverslips. 1k cells seeded and centrifuged at 100g for 10 mins resulted in 81% of the cells landing on an image-able area. Based on the report that anti-CD45RB mAb MB23G2 enhances graft-survival, we tested the hypothesis that anti-CD45RB ligation in vitro increases Treg sensitivity to activation signals. 3 different activation signal levels (1, 5, and 20 μg/mL), 2 cell types, and 2 treatment conditions conventionally required at least 150k cells per sample, or 1.8M cells total. On the microwell, we performed the experiment with 8k cells per sample, with quadruplicates for a total of 384k cells, to show that the ratio of phosphorylation of Lck at Tyr 394 to 505 sites, and alignment to micropatterned anti-CD3 dots are increased in Tregs treated with anti-CD45RB.
High throughput screening of IRAK4 small molecule inhibitors in TLR ligand stimulated whole blood
Authors: Sujatha-Bhaskar Swathi , Huang Zhiyu , Francis George , Bryan Callie , Zarrin Ali A. , Kiefer James , Ghilardi Nico , Brightbill Hans D.
Published: January 2023

The IRAK4 kinase is a key regulator of TLR and IL1R signaling. Small molecule inhibitors of IRAK4 represent an important advance in targeting these pathways. We developed a high throughput whole blood assay to identify potent IRAK4 inhibitors through the inhibition of TLR ligand (R848, LPS, and Gardiquimod) induced inflammatory cytokine production (IL6, TNF, IFNa). Extensive optimization was carried out to ensure rigorous measurement of small molecule potencies, percent maximum inhibition, and cytokine endpoint correlations to clearly distinguish compounds from one another in the context of human whole blood variance. Whole blood treated with IRAK4 inhibitors resulted in partial inhibition of TLR induced cytokines. Whole blood is a complex mixture of cell types and serum proteins. Our analysis shows various dependency of TLR signaling on IRAK4 kinase activity varies among various immune cells allowing for residual inflammatory outputs. Ex vivo whole blood stimulation assays have proven to be an important tool in drug discovery to identify potent IRAK4 small molecule clinical candidates and further define the role of IRAK4 signaling in immune responses.
Enforcing anergy of autoreactive B cells through inhibition of the PI3K pathway
Authors: Franks S. Elizabeth , Getahun Andrew , Cambier John C.
Published: January 2023

Variations in expression and/or function of a number of genes confer increased risk of development of autoimmunity. Among these variations is the expression of negative regulators of the PI3K pathway, whose activity is critical in maintaining B cell anergy. Anergy, in which autoreactive B cells are unresponsive to antigen, is maintained via upregulation of the inositol phosphatases PTEN and/or SHIP-1, which hydrolyze PI(3,4,5)P3, the product of PI3K, yielding PI(4,5)P2 and PI(3,4)P2, respectively. Levels of these negative regulators are reduced in SLE due to aberrant regulation by miRNA-7 and miRNA-155. Deletion of miRNA155, which raises SHIP-1 expression, has been shown to reduce autoantibody responses in the lpr autoimmune mouse model of Lupus. Acute deletion of SHIP-1 or PTEN in autoreactive B cells leads to autoimmunity. We report that it is possible to pharmacologically correct defects in PI3K pathway regulation which confer risk of autoimmunity, thus preventing autoimmunity while conferring minimal disruption of adaptive immune function. This intervention does not prevent autoimmunity caused by compromise of other risk allele emulating conditions, such as reduced SHP-1 expression. We hypothesize that PI3K inhibitors can be used therapeutically to compensate for decreased negative regulation of the PI3K pathway. The knowledge gained from these studies will aid in the development of precision treatments for autoimmunity that act by correcting effects of specific risk alleles.
Fingolimod ameliorates neuropsychiatric lupus without reducing circulating autoantibodies or brain cytokine expression.
Authors: Mike Elise V. , Putterman Chaim
Published: January 2023

Although the pathogenesis of neuropsychiatric systemic lupus erythematosus (NPSLE) is still not well understood, autoantibodies, loss of brain barrier integrity, and pro-inflammatory cytokines have all been implicated in disease progression. Fingolimod (FL), an approved treatment for multiple sclerosis, improves neurologic disease through S1PR modulation and reducing passage of autoaggressive lymphocytes into the CNS. In initial studies, we found that FL treatment reduces lymphocytic infiltration of the choroid plexus in MRL/lpr lupus mice and attenuates memory impairment and depressive-like behavior. Here, we determined the effects of FL on systemic disease and pro-inflammatory cytokine expression in the CNS. Brain, spleen, lymph node, and serum samples were evaluated in MRL/lpr mice treated with FL three times weekly for 10 weeks starting at 10 weeks of age and from untreated controls (n=16/group).

FL-treated mice exhibited a significant reduction in splenomegaly and lymphadenopathy. However, there was no change in serum titers of anti-double stranded DNA antibodies. Moreover, immunofluorescence staining revealed no difference in IgG deposition between treated and control mice, suggesting that autoantibody reduction is not critical for FL-mediated disease improvement. Surprisingly, quantification of brain cortical cytokine levels revealed a significant increase in IL-1a, IL-1b, IFNg, IFNb and IL-12p70 in FL-treated mice, while IL-1a, IL-1b and MCP-1 were increased in the hippocampus.

These findings suggest that FL attenuates NPSLE, but the mechanism of improvement is not mediated by a reduction of autoantibodies or pro-inflammatory cytokine signaling.
A(1-7) reduces pathologies associated with SLE in MRL-lpr mice
Authors: Soto Maira , Gaffney Kevin J. , Jadhav Sachin S. , Rodgers Kathleen E.
Published: January 2023

Patients with Systemic Lupus Erythematosus (SLE) suffer from a chronic inflammatory autoimmune disease that causes damage to various organ systems. Even with the array of current treatments, patients with lupus are still in need of effective therapies to treat this complex, multi-system disease. Recent studies of the Renin Angiotensin System (RAS), widely known for its role in blood pressure regulation, have uncovered immunomodulatory effects. One of the more recently discovered components of RAS, angiotensin (1-7) [A(1-7)], is a bioactive peptides that affects various systems. In our study, MRL-lpr mice were treated daily with saline or A(1-7) for 6 weeks starting at 8 weeks of age. Proteinuria was monitored throughout the duration of the study. MRL-lpr mice treated with saline had higher levels of proteinuria compared to the MRL/MpJ- control mice, and A(1-7) treated MRL-lpr mice. A glomerulonephritis score revealed that the MRL-lpr mice had significant glomerular pathologies compared to MRL-MpJ mice of the same age. A(1-7) treatment significantly reduced the glomerular pathologies in the MRL-lpr mice. Treatment with A(1-7) reduced the size of both inguinal and axillary lymph nodes in MRl-lpr mice, and levels of anti-dsDNA IgG compared to the saline treated MRL-lpr mice. Treatment with A(1-7) also significantly reduced the severity of cutaneous pathologies resulting in only mild inflammation and hair loss compared to the saline treated MRL-lpr mice. Daily systemic treatment with A(1-7) significantly ameliorated disease in one of the most severe mouse models of SLE, making Mas agonists a potential target for future SLE therapies.
Sex hormones and gender influence the expression and function of regulatory T cells differentially in SLE patients
Authors: Singh Ram Pyare , Hahn Bevra H.
Published: January 2023

Regulatory T cells have been implicated in the regulation and maintenance of immune homeostasis. Whether gender and sex hormones differentially influence the expression and function of regulatory T cell phenotype and their influence on Foxp3 expression remain obscure. We provide evidence in this study that in humans the number and percent of regulatory T cells expressing CD4+ and CD8+ are significantly reduced in healthy females compared to healthy males. Both CD4+CD25+hiCD127low and CD8+CD25hi+CD127low subsets in healthy males have a 3–4-fold increase in Foxp3 mRNA expression compared to healthy females. Incubation of CD4+ regulatory T cells with 17b- estradiol at physiological levels either maintains or decreases FoxP3 expression in females with SLE, in contrast to inducing a significant increase in CD4+ Treg in healthy females. Of significance, testosterone significantly increases FoxP3 expression in CD4+CD25hiCD127low cells from females with SLE. Furthermore, plasma concentrations of testosterone positively correlate in those females with the expression of FoxP3 in their CD4+CD25+CD127lowT cells. These data demonstrate that females may be more susceptible than males to SLE and other autoimmune diseases in part because healthy females have fewer regulatory T cells and less FoxP3 expression within those cells. In addition, females with SLE, compared to healthy females, have less ability to generate CD4+Treg in response to physiologic concentrations of 17b-estradiol, whereas an active testosterone metabolite increases the generation of CD4+Tregs. These data clearly suggest that gender and sex hormones influence susceptibility to SLE via their effects on regulatory T cells and Foxp3 expression.
Apoptotic cell induced, TLR9-dependent AhR activity is a critical driver of tolerance induction and suppression of lupus
Authors: Shinde Rahul Suresh , Hezaveh Kebria , Halaby Marie Jo , Kloetgen Andreas , Lamorte Sara , Munn David , Tsirigos Aristotelis , Madaio Michael , Gabrielsson Sussane , Wither Joan , De Carvalho Daniel , McGaha Tracy
Published: January 2023

The Aryl hydrocarbon receptor (AhR) can potently modulate immunity at multiple levels, but its mechanistic role in immune regulation is still being elucidated. Here we show phagocytes exposed to apoptotic cells exhibit rapid activation of AhR driving IL-10 that antagonizes inflammatory cytokine production. AhR activation was dependent on apoptotic cell DNA-TLR9 interactions and reactive oxygen species (ROS) production in phagocytes that promoted nuclear accumulation and transcriptional activity. In vivo, apoptotic cell-induced AhR signals in myeloid cells were required for prevention of inflammatory innate and adaptive immunity against DNA and histones. Moreover, disease progression in lupus correlated with AhR signal strength, and disease course could be reduced or exacerbated by modulation of AhR activity. Finally, we observed myeloid specific deletion of AhR in mice resulted in systemic autoimmunity, and in SLE patients an increased AhR transcriptional signature correlated with disease. Thus, our findings suggest AhR activity influenced by apoptotic cell death is a key mechanism in maintenance of peripheral tolerance reducing inflammation and thereby retarding systemic autoimmune disease progression.
siRNA-mediated c-Rel knockdown ameliorates collagen-induced arthritis in mice
Authors: Ruan Qingguo , Fan Tingting , Zhong Fuhua , Liu Ruiling , Chen Youhai H. , Wang Ting
Published: January 2023

Previous studies have shown that inflammatory mediators involved in the development of rheumatoid arthritis (RA) are regulated by the Rel/nuclear factor-κB (Rel/NF-κB) transcription factor family. c-Rel, a member of the Rel/NF-κB family that is preferentially expressed by immune cells, is a risk factor for several inflammatory diseases including RA. In the current study, we investigated whether targeting c-Rel can be used to treat collagen-induced arthritis, an animal model for RA. c-Rel specific siRNA (siRel) delivered by nanoparticles was used to knockdown the expression of c-Rel. Our results showed that siRel treatment significantly ameliorated collagen-induced arthritis. Further study revealed that c-Rel expression in the dendritic cells and macrophages from mice treated with siRel was significantly down-regulated. Consistent with the phenotypical result, the expression of inflammatory cytokines TNF-α, IL-1β, IL-6, IL-12 and IL-23 by peritoneal macrophages and splenocytes were significantly decreased. In addition, attenuated systemic and collagen-specific Th1 and Th17 immune responses were observed. Furthermore, we found that the expression of inflammatory cytokines was significantly down-regulated and the infiltration of CD3+ T cells and F4/80+ macrophages was markedly reduced in hind paws of mice treated with siRel. Collectively, our study provides strong evidence that siRNA-mediated c-Rel knockdown can suppress the development of collagen-induced arthritis in mice. Therefore, blocking c-Rel may represent an attracting strategy for the treatment of human rheumatoid arthritis.
Protein phosphatase 2A promotes IL-2R signaling in human regulatory T cells
Authors: Ding Ying , Yu Aixin , Malek Thomas R.
Published: January 2023

Low-dose IL-2 therapy is a direct approach to boost Tregs and promote immune tolerance in autoimmune patients. The selective response of Tregs to low-dose IL-2 has been suggested to be due to high level of CD25 and activity of the Ser/Thr phosphatase, PP2A. Here we directly assessed the contribution of these two parameters in promoting IL-2 signaling in human Tregs. IL-2-induced tyrosine phosphorylation of STAT5 (pSTAT5) was proportional to CD25 levels on human CD4+ T cells, directly demonstrating that high IL-2R levels promote the Treg response to low-dose IL-2. To evaluate the role of PP2A, expanded human Tregs were transduced with lentivirus to overexpress or knock down the PP2A catalytic subunit (PP2Ac). Overexpression of PP2Ac increased the level of all IL-2R subunits and promoted phosphorylation of Jak3 at Tyr980/981. Compared to control-transduced cells, PP2Ac-overexpressed Tregs were 3-fold more sensitive to IL-2 based on pSTAT5 activation. Increased expression of CD25 only partially accounted for this enhanced activation of pSTAT5. Consistent with these findings, knockdown of PP2Ac in Tregs significantly reduced the pSTAT5 responses to IL-2. Overexpression of PP2Ac in Tregs also increased the expressions of proteins related to growth, survival, activation, and immunosuppressive function, and included several IL-2-regulated genes. Collectively, these findings indicate that PP2A promotes IL-2R signaling through multiple mechanisms, one related to control of IL-2R expression and another that may directly regulate IL-2R signaling.
PLGA-microsphere delivery of insulin and IGRP epitopes as a therapy for type 1 diabetes
Authors: Racine Jeremy J. , Leeth Caroline M. , Serreze David V.
Published: January 2023

Autoreactive CD8+ T-cells are the ultimate effectors destroying pancreatic β–cells during type 1 diabetes (T1D), revealing a critical role for HLA class I molecules in disease pathogenesis. The HHD transgene contains the α1 and α2 domains of the T1D susceptible variant HLA-A*02:01, covalently linked to human β2m with the α3, transmembrane, and cytoplasmic domains being of murine H2-Db origin. Introduction of HLA-A2.1 into NOD mice via the HHD transgene restores T1D development to NOD.β2m−/− mice. Using these NOD.β2m−/−-A2 mice, we previously identified four epitopes (INS1/2A2–10, INS1B5–14, IGRP265–273 and IGRP228–236) that are targets of CD8+ T-cells in both NOD mice and humans. When coupled to a clinically relevant delivery-mechanism, poly(lactic-co-glycolic acid) microspheres, delivery of these peptides during both early and late-prodromal stages of T1D can ameliorate progression to overt diabetes in NOD.β2m−/−-A2 mice. Delivery of all four peptides was capable of ameliorating T1D and insulitis when delivered every three weeks whether the injections were continued throughout the study (started at six or ten weeks of age) or whether the injections were only given three times (starting at ten weeks of age). After three injections of all four peptides, CD8+ T-cell recall responses to IGRP265–273 and IGRP228–236 were reduced. Attenuation of CD8+ T-cell responses correlated with a reduction in PD-1+ effector CD8+ T-cells as well as a small increase in PD1− CXCR3− central memory CD8+ T-cells in the popliteal LN after footpad priming. Work is ongoing to determine the effectiveness and dosing in 2nd generation HLA- “humanized” NOD-cMHCI−/−-A2 mice that retain murine FcRn functionality.
Tailoring a new generation of chimeric antigen receptors for regulatory T cells
Authors: Ferreira Leonardo , Kaul Anupurna , Guerrero-Moreno Ryan , Fontenot Jason D. , Bluestone Jeffrey A. , Tang Qizhi
Published: January 2023

Chimeric antigen receptors (CARs) direct T cells to target antigens with an extracellular antigen-binding domain and simultaneously deliver TCR signaling and costimulation, resulting in potent antigen-specific T cell activation. This technology has revolutionized our capacity to create tumor-specific effector T (Teff) cells. CAR-Treg engineering is a promising strategy to create antigen-specific Tregs for treating autoimmune and inflammatory diseases. Yet, there are stark differences in signaling between Tregs and Teff cells. Here, we interrogated CAR-mediated signaling in human Tregs and Teff cells using twenty-four CARs with distinct signaling architectures, including CD28 mutants, TNFR family members, and inhibitory receptors. CD28 signaling plays a key role in Treg development and function. Accordingly, CAR-mediated CD28 signaling was indispensable for robust CAR Treg expansion, requiring intact PI3K, ITK, and LCK binding. Similar dependence on CD28 signaling was observed in Teff cells. Treg maturation has been shown to be accompanied by the upregulation of TNFR family members. CARs incorporating TNFR signaling generally resulted in delayed activation kinetics in both Tregs and Teff cells. Strikingly, however, CAR-mediated CD30 signaling led to constitutive activation in Tregs in the absence of antigen, whereas in Teff cells it resulted in delayed activation. Inhibitory receptor expression, a hallmark of Tregs, triggers Teff cell exhaustion. CAR-mediated inhibitory receptor signaling, except for PD1, specifically hampered activation of Teff cells. Altogether, our results indicate that CAR engineering can uncover differences in signaling between human Tregs and Teff cells and maximize CAR-Treg therapies.
Development of citrullinated-vimentin-specific CAR for targeting Tregs to treat autoimmune rheumatoid arthritis
Authors: Raffin Caroline , Zhou Yu , Piccoli Luca , Lanzavecchia Antonio , Sadelain Michel , Tareen Semih U. , Fontenot Jason D. , Bluestone Jeffrey A.
Published: January 2023

Rheumatoid arthritis (RA) is one of the most common chronic autoimmune diseases that is characterized by an aberrant inflammation of the synovial membrane that causes irreversible joint and bone damage. Regulatory T cells (Tregs) are defective in RA patients while adoptive transfer of expanded autologous Tregs efficiently reversed disease in collagen-induced arthritis (CIA), an animal model of RA. These studies strongly suggest that Treg therapy may be effective in the treatment of RA patients by reducing joint inflammation and inducing immune tolerance. However, clinical treatment with polyclonal Tregs may lead to general immunosuppression. Thus, the development of precise RA Ag-specific Tregs would increase specific activity, promote selective migration to the site of the abnormal inflammation and enhance their function. Thus, we engineered chimeric antigen receptor (CAR)-expressing Tregs specifically targeting an antigen present in the joint of RA patients to induce a localized and effective immunosuppressive response. We developed a single chain Fv directed against a posttranslational modified intermediate filament protein, citrullinated vimentin (CV), an antigen found, almost exclusively, in the extracellular matrix of the synovial tissue of the RA patients and implicated in the pathogenesis of the disease. The scFv was grafted into a functional CAR construct, transduced into human Tregs and shown to react with CV expressed in RA patient synovial fluid and CV-expressing cells. Studies are underway to demonstrate the functional activity of these CAR-expressing Tregs in in vivo mouse models a validation step in the development of a promising therapeutic tool to treat RA and potentially other autoimmune disorders.
IL36-Mediated Skin Inflammation Requires Signaling Through Chemokine Receptor CCR6.
Authors: Campbell James J. , Ebsworth Karen , Ertl Linda S. , McMahon Jeffrey P. , Yau Simon , Wang Yu , Liu Shirley , Dang Ton , Zhang Penglie , Schall Thomas J. , Singh Rajinder
Published: January 2023

Generalized pustular psoriasis (GPP) is a rare inflammatory skin disorder with an etiology distinct from common plaque psoriasis. GPP patients often do not respond to therapeutic agents used routinely to treat plaque psoriasis. Genetic evidence suggests that GPP arises from dysfunction in the IL36/IL36Ra/IL36R signaling axis, and many aspects of GPP can be re-created in the mouse by intradermal (ID) injection of pre-activated IL36. We have used this ID-IL36 model to study the leukocyte populations that accumulate within GPP skin. In a previous study, we reported that a small molecule CCR6 antagonist, CCX2553, ameliorates inflammation in an imiquimod-induced model of plaque psoriasis, and we now find this is to be the case in the GPP model as well. However, in contrast to the imiquimod model, a conventional CD4+ αβ T cell population accumulates in the ID-IL36-treated skin instead of the characteristic γδT17 population found in imiquimod-treated skin. CCX2553 reduces IL36-induced inflammation while preventing skin accumulation of this CD4+ αβT cell population. Thus, although disparate T cell populations are associated with each model, inflammation is ameliorated by CCR6 antagonism in both models. These findings suggest that CCR6 may constitute a novel target for a mechanistically distinct approach towards GPP therapy.
Deletion of adenosine A2A receptor blocks the beneficial effects of Lactobacillus reuteri on Treg-deficiency-induced autoimmunity
Authors: Liu Yuying , He Baokun , Hoang Thomas K. , Tran Dat Q. , Rhoads J Marc
Published: January 2023

The lack of Foxp3+ regulatory T cells (Tregs) causes a lethal, CD4+T cell-driven autoimmune disease in scurfy (SF) mice and humans characterized by immunodysregulation, polyendocrinopathy, enteropathy, with X-linked inheritance (or IPEX syndrome). We observed that adenosine 2A receptor (A2A) activation limits inflammation and tissue damage, and that Lactobacillus reuteri DSM 17938 (LR) modulates the abnormal fecal microbial community associated with the disease, stimulating the production of bioactive metabolites, especially inosine by activating A2A expressed on T cells. To conclusively provide evidence of a central role of A2A in the actions of LR, we generated A2A null SF mice (SF·A2A−/−). Male mice were given daily by gavage culture media (MRS) or LR in MRS (107CFU/day), starting at d8 to d22 for plasma and tissue analysis or long-term survival We found that A2A receptor deletion in SF mice did not affect early life events, the development of a lymphoproliferative disorder, or hyper-production of proinflammatory cytokines. LR treatment prolonged survival and reduced multi-organ inflammation in SF mice. In marked contrast, A2A receptor deletion reversed the effect of LR in prolonging lifespan in SF mice and negated the effects of LR on inflammation in the liver and lungs of SF mice. A2A deletion also inhibited LR-mediated reduction of TH1/TH2 cells and reversed the effect of LR on plasma pro-inflammatory cytokines. In conclusion, (a) the absence of the A2A receptor does not affect the development of disease in SF mice, and (b) the A2A receptor plays a critical role in immunomodulation produced by LR treatment. Lactobacillus reuteri and A2A receptor activation may have a role in other Treg dysfunction-mediated autoimmune diseases.