bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple layers of heterogeneity and subset diversity in human MAIT cell responses to distinct microorganisms, and to innate cytokines
Authors: Dias Joana , Leeansyah Edwin , Sandberg Johan K
Published: January 2023

Mucosa-associated invariant T (MAIT) cells are a large innate-like T cell subset in humans defined by invariant TCR Vα7.2 usage and expression of CD161. MAIT cells recognize microbial riboflavin metabolites of bacterial or fungal origin presented by the MR1 molecule. The extraordinary level of evolutionary conservation of MR1 and the limited known diversity of riboflavin metabolite antigens have suggested that MAIT cells are relatively homogeneous and uniform in responses against diverse microbes carrying the riboflavin biosynthesis pathway. The ability of MAIT cells to exhibit microbe-specific functional specialization has not been thoroughly investigated. Here, we found that MAIT cell responses against Escherichia coli and Candida albicans displayed microbe-specific polyfunctional response profiles, antigen-sensitivity and response magnitudes. MAIT cell effector responses against E. coli and C. albicans displayed differential MR1-dependency and TCR β-chain bias, consistent with possible divergent antigen sub-specificities between these microorganisms. Finally, while the MAIT cell immuno-proteome was overall relatively homogenous and consistent with an effector-memory-like profile, it still revealed diversity in a set of NK cell-associated receptors. Among these, CD56, CD84 and CD94 defined a subset with higher expression of the transcription factors promyelocytic leukemia zink finger (PLZF), Eomesodermin and T-bet and enhanced capacity to respond to IL-12 and IL-18 stimulation. Thus, the conserved and innate-like MAIT cells harbor multiple layers of functional heterogeneity as they respond to microorganisms or innate cytokines, and adapt their response patterns in a stimulus-specific manner.
Glucose metabolism regulates reactive oxygen species and nitric oxide production of macrophages in response to IFN-γ and Trypanosoma cruzi
Authors: Koo Sue , Szczesny Bartosz , Garg Nisha
Published: January 2023

Macrophages (mϕ) act as one of the prime barriers to Trypanosoma cruzi infection yet fail to achieve complete clearance of the parasite. The phenotypic diversity of mϕ include pro-inflammatory and immunomodulatory activated states which are supported by cytosolic glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), respectively, for their differential functions.

We previously reported that murine mϕ (RAW264.7 or bone marrow-derived) infected with T. cruzi Sylvio strain (SYL) produce TNF-α, but low levels of reactive oxygen species (ROS) and nitric oxide (NO) and fail to switch to the glycolysis observed in the pro-inflammatory LPS/IFNG-induced mϕ. Pre-blockage of OXPHOS with the ATP synthase inhibitor, oligomycin, resulted in further suppression of NO in mϕ responding to SYL. These data suggested that SYL-infected mϕ may utilize mitochondrial metabolism as an initial activator of NO production.

Providing interferon-γ (IFNG) during SYL infection of mϕ shut down OXPHOS and produced ROS and NO as observed in pro-inflammatory controls by 18 hours posttreatment. We hypothesized that glycolysis was required for the activation of IFNG/SYL- and LPS/IFNG-induced ROS and NO in mϕ. We found that eliminating glucose from the culture media or inhibiting glucose uptake resulted in the failure of ROS and NO production, but sustained TNF-α release. The protein expression of the ROS-producing NADPH oxidase (NOX2) subunit, gp91phox, was not detected in SYL-infected mϕ although abundant in pro-inflammatory mϕ. These data suggest that glycolysis may regulate NOX2 activation in mϕ responding to inflammatory stimuli. How NOX2 function and NO release is controlled by glucose and mitochondrial metabolism in mϕ will be further discussed.
Reciprocal regulation of germinal center and regulatory B cell responses by IFNγ receptor-STAT1 signaling in TLR7 mediated autoimmunity
Authors: Chodisetti Sathi Babu , Domeier Phillip P. , Soni Chetna , Schell Stephanie L. , Fasnacht Melinda J. , Rahman Ziaur S.M.
Published: January 2023

Toll like receptor 7 (TLR7) plays a critical role in systemic autoimmunity. The mechanisms that fine-tune germinal center versus regulatory B cell response in TLR7 mediated autoimmunity remain unclear. Regulatory B cells that produce IL-10 (B10 cells) negatively regulate autoimmunity and infections. We report that TLR7 deficiency in non-autoimmune B6 and autoimmune prone B6.Sle1b mice showed increased IL-10 producing B10 cells. In contrast, B6.Yaa and B6.Sle1b.Yaa mice expressing an extra copy of TLR7 in the Yaa locus showed decreased percentage of B10 cells. TLR7 deficiency in B cells inhibited whereas an extra copy of TLR7 enhanced proliferation and cytokine expression of cocultured antigen specific CD4 T cells. Interestingly, B10 cells displayed highest IFNγR expression compared to other subsets of T and B cells. Whereas B6.Sle1b.Yaa mice lacked B10 cells and marginal zone compartment (MZ), B6.Sle1b.Yaa mice deficient in IFNγR (B6.Sle1b.Yaa/IFNγR−/−) restored these populations. Further, B6.Sle1b.Yaa/IFNγR−/− mice showed diminished spontaneous germinal center (Spt-GC) and serum autoantibody responses, and renal pathology compared to B6.Sle1b.Yaa control mice. Similar results were observed when B6.Sle1b and B6.Sle1b.IFNγR−/− mice were treated with TLR7 ligand, imiquimod. B10 and GC B cells exhibited higher STAT1 and T-bet expression than follicular B cells. Mice deficient in STAT1 and T-bet had significantly reduced GC and IgG2c Ab responses while retained intact B10 and MZ compartments upon imiquimod treatment, contrary to what was observed in wild type mice. Together, these data define the critical differential role of IFNγR, STAT1, and T-bet in GC B cell and B10 cell development in TLR7 mediated autoimmunity.
ENU-induced C-terminal truncation of Runx3 results in Th17-associated autoimmune colitis
Authors: Su Yu-Chia , Chen Yi-Ting , Chen Yu-Ling , Cheng Bing-Ho , Kung John T
Published: January 2023

Inflammatory bowel disease (IBD) is a common, chronic, progressive inflammatory intestinal disease in the world. The etiology of IBD is due to the imbalance of host immune responses to intestinal flora. Many human genes play the important role to induce improper immune responses, including IL-10, STAT3, IRGM, AGT16L1, NOD2 and RUNX3. It has been reported that homozygous Runx3-ko mice develop colitis spontaneously. However, homozygous Runx3-ko mice show very high mortality rate in the first 2 week of life, and are hard to study the role of Runx3 in the spontaneous colitis. A mouse pedigree (spontaneous colitis, SC) carried an ENU-induced Tyr301stop point mutation of Runx3, which resulted in a short form truncated form of Runx3. SC mice started to show spontaneous diarrhea after weaning. SC mice exhibited prominant enlargement of colon that revealed large amounts of inflammatory cells infiltration from mucosal to muscular layers. Immunofluorescent staining showed large amounts of CD4+ T cells involved in the inflammatory colon. Moreover, inflammatory colons of SC mice expressed higher mRNA levels of IL-17A, and serum IL-17A level were also upregulated in SC mice. In addition, SC CD4+ T cells expressed stronger IL-17A than WT CD4+ T cells after in vitro activation. Transfer of total SC CD4+ T cells into Rag1-ko host mice resulted in autoimmune colitis. In summary, C-terminal truncated Runx3 results in autoimmune colitis through elevated Th17 immune responses. Thus, the SC mouse model is a valuable tool for investigations on the effect of immune system upon spontaneous colitis.
Thiol isomerase ERp72 enhances the development of antibody-induced arthritis
Authors: Zhao Zhenzhen , Chen Fengwu , Wu Yi
Published: January 2023

Thiol isomerases control labile disulfide bonds that are important in cell activation, cytokine signaling and in a variety of diseases. However, their role in autoimmune diseases such as arthritis remains unknown. ERp72, a thiol isomerases which contains three highly conserved CGHC active motifs, is capable of oxidizing, reducing and isomerizing disulfide bonds. In this study, we generated a new knockout mouse strain deficient of ERp72 (ERp72−/−). Using K/BxN serum-transfer-induced arthritis model (STIA), we determined the role of ERp72 in the pathogenesis of arthritis. To induce STIA, ERp72+/+and ERp72−/− mice received intraperitoneal injection of 200μl K/BxN serum twice on day 0 and day 2. Severity of arthritis was evaluated by daily measurements of joint diameter and clinical scores. Compared with ERp72+/+ mice, ERp72−/− mice developed much more severe arthritis, their joint swelling diameter was significantly increased. Clinical scoring of hind paw edema and hyperemia showed similar results. As indicated by H&E and Safranin O staining, the joint tissue from ERp72−/− mice exhibits more severe synovial hyperplasia, inflammatory cell infiltration and bone erosion on day 12. To determine the role of ERp72 in regulation of cytokine production, we measured the profiles of proinflammatory cytokines. In the joint tissue of ERp72−/− mice bearing STIA, both IL-1β and IL-6 levels were markedly increased by more than 50% than that of ERp72+/+ mice. Interestingly, the basal level of proinflammatory cytokine TNF-α in ERp72−/− mice was significantly higher in that of ERp72−/− mice. Taken together, our observation demonstrates that ERp72 is a negative regulator of innate immunity and plays an important role in the development of arthritis.
CD11b activation protects against lupus nephritis by suppressing TLR-dependent IFN responses via AKT-FOXO3-IRF7
Authors: Khan Samia Q , Faridi Mohd. Hafeez , Trim Wenpu , Khaliqdina Shehryar , Cimbaluk David J. , Kaplan Mariana J. , Gupta Vineet
Published: January 2023

Genetic variations in the ITGAM gene (coding for CD11b) produce defective CD11b and associate with a risk for systemic lupus erythematosus (SLE, lupus). Elevated level of IFN I in circulation plays a pathogenic role and is a heritable risk factor for SLE. Whether variations in ITGAM are linked to high IFN I and whether CD11b activation could be a therapeutic strategy is unknown and is explored here. We measured serum IFN I activity in 171 SLE patients and report that three ITGAM variants significantly associate with elevated levels of IFN I in lupus, suggesting a direct link between reduced CD11b activity and elevated inflammation in patients. Given that ITGAM SNPs result in functionally deficient CD11b, we tested whether partial CD11b activation with small molecule agonist, leukadherin-1 (LA1) reduces IFN I responses and determined the underlying mechanistic pathways. CD11b activation with LA1 reduced TLR-dependent pro-inflammatory signaling in leukocytes and suppressed IFN I signaling via an AKT-FOXO3-IRF7 pathway. TLR-stimulated macrophages from CD11b SNP carriers showed increased basal expression of IRF7 and IFNB, as well as increased nuclear exclusion of FOXO3, which was suppressed by LA1. To test the efficacy of LA1 in vivo, we used the MRL/lpr mice that develop IFN I-dependent multi-organ lupus. LA1 treatment reduced IFN I responses and protected lupus-prone MRL/lpr mice from kidney injury. LA1-treated mice had reduced proteinuria, IgG renal immune complex deposition, and glomerular damage as compared to controls. LA1 suppresses TLR-stimulated overproduction of cytokines in vivo, which drives lupus pathogenesis. These findings suggest that pharmacological CD11b activation is a promising therapeutic strategy in human SLE.
Deficiency in Interleukin-1 receptor-associated kinase (IRAK) 4 activity attenuates manifestations of murine Lupus
Authors: Medvedev Andrei , Manavalan Tissa , Pattabiraman Goutham , Murphy Michael Bradford
Published: January 2023

IL-1R-associated kinase (IRAK) 4 mediates host defense against infections. As an active kinase, IRAK4 elicits full spectra of MyD88-dependent responses, while kinase-inactive IRAK4 induces a subset of cytokines and negative regulators whose expression is not regulated by mRNA stability. IRAK4 kinase activity is critical for resistance against Streptococcus pneumonia, but its role in autoimmunity is incompletely understood. In this study, we determined the role of IRAK4 kinase activity in murine lupus. Lupus development in BXSB male mice expressing the Y chromosome autoimmunity accelerator (Yaa) increased basal and Toll-like receptor (TLR) 4/7-induced phosphorylation of IRAK1, mitogen-activated protein kinases, p65 nuclear factor-κB (NF-κB), enhanced TNF-α and CCL5 gene expression in splenic macrophages, but decreased levels of Toll-interacting protein (Tollip) and IRAK-M, without affecting IRAK4 or IRAK1 expression. Mice harboring kinase-inactive IRAK4 on the lupus-prone Yaa background had reduced glomerulonephritis, splenomegaly, serum anti-nuclear antibodies, decreased numbers of splenic macrophages, total and TNF-a+ dendritic cells, activated T- and B-lymphocytes, and lower TNF-α expression in macrophages compared to lupus-prone mice with functional IRAK4. Furthermore, mice expressing kinase-inactive IRAK4 had decreased proteinuria and increased survival in the chemically-induced (pristine) lupus model. Thus, IRAK4 kinase activity significantly contributes to murine lupus and could represent a new target for therapeutic interventions.
Ectopic expression of innate immune protein Lipocalin-2 in probiotic Lactococcus lactis protects against environmental and gut inflammatory stressors
Authors: SAHA PIU , Chassaing Benoit , Yeoh Beng San , Viennois Emilie , Xiao Xia , Singh Vishal , Kennett Mary J , VIJAY-KUMAR MATAM
Published: January 2023

Overexpression of lipocalin-2 (Lcn2), an acute phase protein, has been demonstrated to enhance cellular resistance to oxidative stress in various mammalian cell lines, in addition to mediating anti-bacterial effects by sequestering bacterial siderophores (iron chelators). To investigate whether Lcn2 could be exploited to augment the stability and efficacy of probiotic bacteria, we have engineered the recombinant Lactococcus lactis (MG1363) that expresses the murine Lcn2 (LactisLcn2) and the empty plasmid (LactisCon). The expression of Lcn2 did not impair the growth of LactisLcn2, especially since L. lactis do not require iron for its growth. In contrast, the L. Lactis-derived Lcn2 was effective in mitigating the bioactivity of enterobactin (E. coli-derived siderophore) and inhibited the growth of E. coli in vitro. The sequestration of iron in LactisLcn2 could, in part, explain its ability to inhibit the iron-dependent growth of E. coli. Moreover, the Lcn2 expression also augments the capacity of LactisLcn2 to withstand various environmental stressors in vitro, including extreme pH, toxicity of bile salts, H2O2-, and myeloperoxidase-mediated bacterial killing. When administered into mice with DSS-induced acute colitis, LactisLcn2 was observed to survive better in the inflamed gut and provide superior mucoprotection against intestinal inflammation when compared with LactisCon. Overall, our findings suggest that Lcn2 expression can be exploited to augment the iron-scavenging capability and survivability of probiotic bacteria against adverse conditions, which could further enhance their efficacy to treat inflammatory bowel diseases.
Identification and characterization of a novel association between dietary potassium and risk of crohn’s disease and ulcerative colitis
Authors: Awasthi Amit , Malik Sakshi , Khalili Hamed
Published: January 2023

Crohn’s disease (CD) and Ulcerative Colitis (UC) that constitute IBD are caused by imbalance between Th1 and Th17 immune cells, and Foxp3+ T-regulatory immune cells. However, in response to environmental and gut microbial triggers, T cell immune balance is disrupted which lead to inflammation in IBD. It is suggested that smoking, diet and hygiene affect T cells functions in IBD.

It is shown that salt (sodium) can alter the balance between pathogenic Th17 and Treg cells. We identified that extracellular potassium induces the generation of Foxp3+ Tregs in human by enhancing TGF-β1-signaling. We found that extracellular potassium activates Smad 2 and 3 while suppresses Smad7 and SGK1 in human T cells. We found that extracellular potassium not only induces Foxp3 expression in human T cells but also inhibited generation of human Th1 and Th17 cells.

We further translated these findings in a prospective cohort of nearly 170,000 US women who had been followed up for over 20 years. Among a total of 194,711 women over a follow-up of 3,220,247 person-years, we documented 273 cases of CD and 335 cases of UC. Dietary intake of potassium (Ptrend = 0.005) but not sodium (Ptrend = 0.44) was inversely associated with risk of CD. Although, both dietary potassium and sodium were not significantly associated with risk of UC, there was a suggestion of an inverse association with dietary potassium (Ptrend = 0.08). The association of potassium with risk of CD and UC appeared to be modified by loci involved in the TH17 pathway that have previously been associated with susceptibility to CD, particularly SNP rs7657746 (IL21). Dietary potassium is inversely associated with risk of CD potentially by regulating immune tolerance through its effect on Tregs and Th17 pathway.
Mechanisms by which IFN-γ mitigates lipid peroxidation that leads to progressive experimental autoimmune encephalomyelitis
Authors: Robinson Rachel R , Rojano Elba Mirelle , Forsthuber Thomas
Published: January 2023

Multiple sclerosis (MS) is the most common demyelinating autoimmune disease of the central nervous system affecting over 2.3 million people worldwide. Although most patients are initially diagnosed with relapsing-remitting MS, the vast majority of these will eventually develop progressive MS. Importantly, the mechanisms that contribute to the progression of MS are largely unknown. IFN-γ, a well-studied pro-inflammatory cytokine, has long been implicated in the pathogenesis of MS and its preclinical model, experimental autoimmune encephalomyelitis (EAE). Although pro-inflammatory, disease-promoting effects are well documented for IFN-γ, paradoxically, protective functions have also been demonstrated for this cytokine. We are interested in protective mechanisms of IFN-γ. Previous studies in our lab revealed that mice lacking IFN-γ signaling developed severe and progressive EAE due to increased presence of myelin debris and lipid peroxidation in the CNS, the cause of which is yet to be elucidated. We hypothesize that IFN-γ helps regulate expression of scavenger receptors, antioxidant molecules, and pro-oxidant molecules in order to promote resolution of lipid peroxidation and clearance of myelin debris in the CNS during EAE. We are currently investigating key scavenger receptor, antioxidant, and pro-oxidant molecule expression in IFN-γR−/− vs. wild type mice during EAE. Our results will contribute to a better understanding of the underlying mechanisms and may lead to new treatments to prevent progression of MS.
Nutraceutical Apigenin: mechanism of action associated with its anti-inflammatory activity and regulation of dendritic cell metabolism
Authors: Moore Patrick , Ginwala Rashida , Revuri Nikil , Kranz Victoria A , Houle John D , Khan Zafar K , Jain Pooja
Published: January 2023

Flavonoids have a range of biological activities, including anti-inflammatory, anti-carcinogenic, and neuroprotective effects. Apigenin, one such flavonoid abundant in certain plants, has been employed for centuries in the treatment of Parkinson’s, neuralgia, asthma and other diseases owing to the positive health effects attributed to this group of compounds. Due to Apigenin’s relative abundance and low intrinsic toxicity amongst flavonoids, it can be considered as a potential natural treatment for inflammatory disorders of the central nervous system (CNS), such as multiple sclerosis. However, there is distinct lack of information regarding the molecular mechanism of action of Apigenin leading to its modulatory effects on dendritic cells (DC), a key cell type responsible for maintaining immune balance especially in specialized locations such as the CNS. Here, we investigate the effect of Apigenin on RelB, a NF-kB family protein, which is vital to DC maturation and function. Apigenin reduced cytoplasmic RelB levels in LPS-treated DCs isolated from normal peripheral blood of a healthy donor. Gene and protein expression of downstream targets of RelB activation, IL-12, IL-23, IL-6, IL-1β and TNF-α was also reduced upon Apigenin treatment. Further, RelB is also known to engineer a metabolic switch in immune cells upon inflammation to meet the increased energy requirements. We observed a decrease in glucose uptake and lactate production (glycolysis), and an increase in mitochondrial activity when LPS-induced DCs were treated with Apigenin. These results provide key information about the molecular events controlled by Apigenin in its regulation of DC activity marking its potential as a therapy for neuroinflammatory disease.
CD6 as a potential target for treating multiple sclerosis
Authors: Li Yan , Singer Nora , Whitbred Joy , Bowen Michael , Fox David A , Lin Feng
Published: January 2023

CD6 was established as a marker of T cells more than three decades ago, and recent studies have identified CD6 as a risk gene for multiple sclerosis (MS), a disease in which autoreactive T cells are integrally involved. Nevertheless, the precise role of CD6 in regulating T cell responses is controversial and its significance in the pathogenesis of various diseases remains elusive, partly due to the lack of animals engineered to alter expression of the CD6 gene. In this report, we found that CD6 KO mice showed decreased pathogenic T cell responses, reduced spinal cord T-cell infiltration and attenuated disease severity in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. CD6-deficient T cells exhibited augmented activation, but also significantly reduced survival and proliferation after activation, leading to overall decreased Th1 and Th17 polarization. Activated CD6-deficient T cells also showed impaired infiltration through brain microvascular endothelial cell monolayers. Furthermore, by developing CD6 humanized mice, we identified a mouse anti-human CD6 monoclonal antibody that is highly effective in treating established EAE without depleting T cells. These results suggest that 1) CD6 is a negative regulator of T cell activation, 2) at the same time, CD6 is a positive regulator of activated T cell survival/proliferation and infiltration; and 3) CD6 is a potential new target for treating MS and potentially other T cell-driven autoimmune conditions.
Engineered myelin basic protein (MBP)-specific human T regulatory cells ameliorate myelin oligodendrocyte glycoprotein (MOG) peptide-induced Experimental Autoimmune Encephalomyelitis in vivo
Authors: Kim Yong Chan , Zhang Ai-Hong , Yoon Jeongheon , Wucherpfennig Kai W , Scott David W
Published: January 2023

Application of expanded polyclonal T regulatory cells (Tregs) offers great promise for the treatment of undesirable immune responses. To increase efficacy and reduce possible non-specific side effects, we elected to create antigen-specific Tregs, based on chimeric antigen receptor (CAR) therapy in leukemia. Therefore, we have engineered a myelin basic protein-specific TCR in a retroviral vector and transduced it to human naïve CD4+ T cells and Tregs. The TCR V genes came from an MBP-specific T-cell clone derived from an MS patient [1–3](Ob2F3). We observed that transduced Tregs were activated in vitro in response to MBP peptide on DR15 APC and upregulated Foxp3, LAP and Helios expression. As expected, these engineered MBP-specific Tregs suppressed proliferation and cytokine production by MBP-specific T effector cells in the presence of MBP peptide. Mechanistic studies demonstrated that suppression may involve transfer of soluble mediators but is enhanced by cell contact. Furthermore, transfer of human MBP-specific Tregs into MOG-immunized mice was able to ameliorate EAE in HLA DR15-transgenic mice. MBP-specific Tregs migrated into CNS in greater numbers than irrelevant FVIII-specific Tregs in day 2 after infusion, and H&E staining indicated that infusion of MBP-specific Tregs prevented perivascular inflammatory infiltration in CNS. Thus, we propose that engineered MBP-specific Tregs may be able to control pathologic inflammation in MS in vivo.
Nutraceutical Apigenin regulates DC function in a RelB-dependent manner during neuroinflammation
Authors: Ginwala Rashida , McTish Emily , Moore Patrick , Revuri Nikil , Raman Chander , Singh Narendra Prasad , Nagarkatti Mitzi , Nagarkatti Prakash , Kranz Victoria A , Houle John D , Jain Pooja , Khan Zafar K
Published: January 2023

Apigenin, a natural flavonoid, found in several plants is known to have anti-oxidant and anti-inflammatory properties indicated by its use for centuries as a medicinal approach to treat inflammatory disorders. However, there are significant gaps in knowledge regarding its effect on dendritic cell (DC) function in maintaining an immune balance in immunospecialized locations like the central nervous system (CNS). In order to establish the potential utility of Apigenin as a therapeutic agent against neuroinflammatory diseases, we tested and found that Apigenin treatment ameliorated severity of disease progression and relapse after onset of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 and SJL mouse models of multiple sclerosis. An increased retention of DCs and other myeloid cells in the periphery correlated with decreased immune cell infiltration and reduced demyelination in the CNS in treated mice. Mechanistically, Apigenin treatment reduced RelB expression in presence of LPS in human peripheral blood DCs, which is central to DC maturation, its antigen presentation capabilities and DC-mediated T cell activation. IL-12A and IL-23, downstream pro-inflammatory targets of RelB were reduced upon Apigenin treatment in these cells. Further, RelB causes a metabolic switch in immune cells upon inflammation, which was seen as a decrease in glucose uptake and lactate production (glycolysis), and an increase in mitochondrial activity when LPS-induced DCs were treated with Apigenin. These results indicate a protective role of Apigenin against DC-regulated neurodegenerative effects through a probable RelB mediated pathway thus implicating a potential therapy for neuroinflammatory disease.
Combination of cannabinoids, Δ9- tetrahydrocannabinol (THC) and cannabidiol (CBD), mitigate experimental autoimmune encephalomyelitis (EAE) by altering the gut microbiome.
Authors: Al-Ghezi Zinah Zamil , Alghetaa Hasan F , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

Multiple Sclerosis (MS), is a common autoimmune disease that affects the central nervous system. Currently, there is no cure for MS, and most treatments involve the use of immunosuppressive drugs that can have adverse effects or increased toxicity. Cannabis, commonly known as marijuana, is a product of the Cannabis sativa, and for several centuries has been used as an alternative medicine in many cultures. Cannabis sativa produces over 421 chemical compounds, including about 80 terpenophenols named phytocannabinoids, and include both psychotropic THC and non-psychoactive CBD. In the currently study, we show that a combination therapy using THC and CBD results in amelioration of EAE, an animal model of MS, by reducing hind limb paralysis, decreasing immune cellular infiltration into the brain, and mitigating the presence of inflammatory biomarkers, including gram negative bacteria-associated lipopolysaccharide (LPS). Interestingly, the gut microbiome plays an important role in immune function and studies have shown that it is altered significantly in MS patients. Inasmuch, we performed 16S rRNA sequencing on experimental groups to investigate the gut microbiome composition after using a combination of THC and CBD compared to disease controls. Interestingly, we found that EAE mice showed increase in the mucin degrading bacterial species, Akkermansia municiphila, which was significantly reduced in disease mice treated with THC+CBD. Collectively, our data suggests that THC+CBD can ameliorate EAE by preventing accumulation of mucin-degrading bacteria that would lead to increased gut microbial dysbiosis.
Using the cooling agent icilin and synthetic analogs to attenuate autoreactive T cell function during experimental autoimmune encephalomyelitis.
Authors: Ewanchuk Benjamin , Allan Euan R. O. , Yates Robin M.
Published: January 2023

Targeting inflammatory function of autoreactive T lymphocytes defines a key facet of therapeutics for autoimmune diseases such as multiple sclerosis (MS). The super-cooling drug icilin has recently been shown to limit systemic inflammation through activation of the TRPM8 calcium channel in murine models of colitis. We thus aimed to define an anti-inflammatory mechanism for the drug candidate icilin and TRPM8 in T cell-mediated adaptive neuroinflammation.

Using experimental autoimmune encephalomyelitis (EAE), a CD4+ T cell-driven model of MS, we assayed the influence of icilin on CD4+ T cell activation, proliferation, and effector function. Significant reductions in clinical disease were observed in WT EAE mice receiving icilin treatment, coinciding with fewer infiltrating leukocytes in the central nervous system. Unexpectedly, TRPM8−/− mice treated with icilin also displayed protection from EAE, showing similar reductions in disease score and leukocyte infiltration. Both WT and TRPM8−/− mice receiving icilin treatment showed significant delays in initial disease onset, attributed in vitro to reduced T cell proliferation via icilin-mediated G1 cell cycle arrest. A library of chemically and structurally related icilin analogs was screened to identify novel drug candidates targeting autoimmune T cell function. Amongst the screened drug analogs, a small fraction potently attenuated T cell proliferation in vitro.

These results suggest that TRPM8-independent ablation of T cell function drives the anti-inflammatory properties of icilin during EAE. Further, novel drug candidates structurally related to icilin represent a promising class of molecules for limiting T cell responses in MS and other autoinflammatory diseases.
Expression of MHC I and II and antigen processing genes following INF-γ treatment in fibroblastic reticular cells and double negative cells
Authors: Marti Luciana C , Agostini Fernanda , Palomino Diana Torres , Severino Patricia
Published: January 2023

Lymph node (LN) is a secondary lymphoid organ with highly organized structure. Fibroblastic reticular cells (FRCs) integrate LN structure, and are characterized by podoplanin expression (PDPN, gp38) and lack of CD31. FRCs are involved in several immune response processes but the mechanism is still under investigation. Another cell population present in LN is a fibroblastic cell lacking PDPN and CD31 expression, called double negative cells (DNCs). FRCs and DNCs are described to control migratory pattern and positioning of immune cells during homeostasis or inflammatory response. We evaluate in human derived FRCs and DNCs the gene expression after IFN-γ-treatment and found up-regulated genes related to antigen processing and MHC class I and II up-regulated. MHC class I is expressed by all nucleated cells and MHC class II constitutive expression is confined to the immune system antigen-presenting cells, but can be induced in several cells types. MHC class II genes control has been described to play a role in eliminting foreign antigens, while minimizing autoreactive responses against self-antigens. MHC class II molecules can fulfill additional functions by triggering a variety of signaling pathways, they can regulate proliferation and maturation to apoptosis. In addition, we were able to demonstrate that HLA-ABC and HLA-DR were increased in the membrane of both FRCs/DNCs derived from different patients/lymph nodes after treatment with IFN-γ. FRCs seem to be more responsive to IFN-γ than DNCs. Furthermore, we have tested the ability of FRCs to internalize particles, showing a functional MHC II pathway.
Identification of the genetic determinants for the frequency of regulatory T cells using system genetics
Authors: Maazi Hadi , Hartiala Jaana , Suzuki Yuzo , Crow Amanda , Lam Jonathan , Patel Nisheel , Rigas Dimanda , Sankaranarayanan Ishwarya , Gilliland Frank D , Allayee Hooman , Akbari Omid
Published: January 2023

Regulatory T (Treg) cells are indispensable components of immune regulation contributing to the maintenance of respiratory tolerance and preventing autoimmunity and exaggerated immune responses. The frequency and absolute number of Treg cells in lymphoid and non-lymphoid tissues are tightly regulated through incompletely understood mechanisms. In this study, we sought to understand the pulmonary immune regulation through identification of the genetic determinants of the frequency and absolute number of CD4+FOXP3+T cells in mice using a genome-wide association study (GWAS) approach. We quantified the frequency and absolute number of CD4+FOXP3+T cells in the lungs and spleen of 102 inbred strains from the hybrid mouse diversity panel under steady state conditions and after exposure to house dust mite (HDM).

Among strains of mice examined, the absolute number of pulmonary CD4+FOXP3+ T cells varied by 312-fold at the steady state and 210-fold after HDM exposure. A GWAS analysis results revealed several loci that contribute to the regulation of CD4+FOXP3+ T cells in the lungs and spleen of mice, including a locus on chromosome 5 harboring the homeodomain-only protein (Hopx) gene. Characterization of Hopx−/− mice validated the GWAS findings by demonstrating that the frequency and number of CD4+FOXP3+ T cells are higher in the lungs of Hopx−/− compared to WT mice at the steady state and after HDM exposure. We found that in contrast to the lungs, the number of CD4+FOXP3+ T cells is lower in the spleen of Hopx−/− compared to WT mice at the steady state. Our findings suggest that Hopx is a novel genetic determinant of the frequency and number of CD4+FOXP3+ T cells in mice with opposing directional effects in mucosal and secondary lymphoid organs.
Amelioration of pulmonary inflammation through T cell activation in murine asthma model
Authors: Alvarez Carlos A , Jones Mark B. , Cobb Brian A
Published: January 2023

Asthma is an increasingly common chronic inflammatory airway disorder with phenotypic heterogeneity driven by age, gender, atopy, lung function, lifestyle, body mass indexes, and the underlying cellular and molecular pathways. In asthma, T helper (Th) cells drive inflammatory cascades via cytokine/inflammatory mediators and recruit leukocytes into the airway. While treatments are available, such as inhaled corticosteroids, individuals with severe asthma are often steroid resistant and have a poor quality of life. It is therefore important to develop our understanding of the underlying T cell driven mechanisms leading to pulmonary inflammation such that novel therapies may be developed. Previous work from our lab has demonstrated the importance of commensal microbiota in the development and maintenance of peripheral homeostasis. Exposure to bacterial polysaccharides, such as PSA from Bacteroides fragilis, inhibits Th1 and Th2 activity in murine models by activation of CD4+ FoxP3− CD45Rblo CD62Llo CD44hi T effector memory (Rblo Tem) cells in an IL-10 dependent fashion. Here, we show that activated Rblo Tem cells can reverse established pulmonary inflammatory disease in a murine house dust mite (HDM) model. Adoptive transfer of these cells nearly eliminates lung pathology and severely limits tissue infiltration by leukocytes. These results provide a rationale for the use of autologous cell transfer as a means to prevent and reverse ongoing inflammatory lung disease.
Characterizing childhood resistance to deadly immune dysfunction in severe infections
Authors: Joachim Rose Bernadette , Suber Freeman , Kobzik Lester
Published: January 2023

Epidemiological data show that pre-pubertal children possess a relative resistance to death from severe infections—particularly those characterized by “cytokine storm” and profound immune dysfunction (e.g. the 1918 pandemic flu, measles, bacteremia). To assess the role that pubertal status may have on an animal’s ability to appropriately regulate inflammation, we employed an endotoxemia model of sterile sepsis in mice. Following acute endotoxin injection (i.p., 23 μg/g), pre-pubertal mice (PND 25) exhibited significantly better survival than post-pubertal mice (PND 35) over a 72-hour period (78 vs. 29%, p<0.0001, n>50). Serum endotoxin concentrations were similar in both age groups at 2 and 20hr time points, suggesting that the difference in mortality was not due to differential endotoxin absorption. Surprisingly, improved survival among pre-pubertal animals did not correlate with a decreased inflammatory response. At 2hr pre-pubertal mice had higher absolute white blood cell counts, elevated numbers of Ly6G+CD11B+ granulocytic cells in the peritoneal cavity, and increased levels of several pro-inflammatory cytokines, compared to post-pubertal animals. In contrast, by 20hr pre-pubertal mice showed significantly decreased expression of pro-inflammatory cytokines, and increased expression of the anti-inflammatory cytokine IL-10 (1100 ± 290 vs. 750 ± 225 pg/ml, p<0.001). Blockade of puberty with leuprolide substantially improved survival compared to age-matched post-pubertal mice (75 vs. 32%, p<0.0001, n>30), and treatment of pre-pubertal mice with estradiol increased subsequent mortality, suggesting a causal role for sex hormone(s) in the increased susceptibility of post-pubertal mice.
Allergy and asthma in inner-city children: possible roles of MAIT cells and variation in the home environment
Authors: Chandra Shilpi , Wingender Gerhard , Greenbaum Jason A , Khurana Archana , Gholami Amin Moghaddas , Rosenbach Michael , Jaffee Katy , Gern James E , Wood Robert , O’Connor George , Sandel Megan , Kattan Meyer , Bacharier Leonard , Togias Alkis Togias , Horner Anthony A. , Kronenberg Mitchell
Published: January 2023

Humans have two populations of innate-like T lymphocytes with an invariant T cell antigen receptor (TCR) a chain: invariant natural killer T (iNKT) cells and mucosal associated invariant T (MAIT) cells. iNKT cell involvement in human asthma has been suggested, but is controversial, while there has been little analysis of MAIT cells. Using peripheral blood cells from the urban environment and childhood asthma (URECA) birth cohort study, we carried out a comprehensive investigation to determine if iNKT cell frequency or MAIT cell frequency early in life is correlated with the cytokine polarization of mainstream CD4+T cells and/or the development of asthma by age seven. We also determined if iNKT cell antigenic activity in house dust samples was associated with environmental endotoxin or clinical outcomes including aeroallergen sensitization and recurrent wheeze at 3 and asthma at age 7. We also analyzed the correlation between cytokines released by activated iNKT and MAIT cells and various clinical outcomes. Our results shows that the frequency of MAIT cells was associated with increased production of IFNg by activated CD4+ T cells from children that did not go on to develop asthma. Moreover, an increased MAIT cell frequency early in life was associated with a decreased risk of asthma by age seven. In contrast, iNKT cell frequency early in life was not correlated with the development of asthma or allergic sensitization. iNKT cell antigen content in house dust varied greatly, and increased antigen was associated not only with endotoxin levels in the surroundings but also with protection from asthma. Together, these data suggest that MAIT cell frequency and iNKT cell antigen content at one year correlates with protection from asthma development.
Epigenetic regulation of proinflammatory chemokines in Cystic Fibrosis lung epithelium
Authors: Chen Kong , Kolls Jay K
Published: January 2023

Functional loss of CFTR in lung epithelial cells leads to pulmonary disease in Cystic Fibrosis (CF) patients. Our preliminary data indicates that proinflammatory chemokines and cytokines are spontaneously released by Human Bronchial Epithelial (HBE) cells established from CF patients and the spontaneous release can be abolished by a bromodomain inhibitor, a small molecule that inhibits epigenetic enhancement of gene transcription. Epigenetics alter gene expression in many other diseases, thus, this mechanism could also be responsible for the dysregulation of inflammatory mediators observed in the CF airway. We hypothesize that the enhanced production of proinflammatory mediators observed in CF airways are in part controlled by an epigenetic regulatory program. RNA-seq analysis revealed a dramatic upregulation of chemokine genes in the airway epithelium from CF patients, which correlated with downregulation of histone deacetylases, suggesting chemokine expression in CF airway can be modified by histone acetylation. In vitro, chemokine productions in polarized HBE cells from CF donors are also enhanced and are sensitive to BET inhibition. ATAC-seq analysis on HBE cells derived from control and CF donors was conducted and more peaks were observed in CF cells at the promoter regions of several chemokine genes suggesting these loci are more accessible compared to control HBE cells. These data strongly implicate that pro-inflammatory environment in CF lungs epigenetically enhances chemokines production through repressing histone deacetylases, suggesting that therapeutic approaches aiming to reverse the consequence of losing histone deacetylases can be beneficial to CF patients through reducing lung inflammation.
CD4+ T cell receptor repertoire in sarcoidosis patients
Authors: Mitchell Angela M , Kaiser Ylva , Falta Michael T , Eklund Anders , Grunewald Johan , Fontenot Andrew P
Published: January 2023

Sarcoidosis is a multisystem disorder that most commonly affects the lungs and involves granuloma formation with the accumulation of CD4+ T cells at sites of disease. Considerable evidence indicates that CD4+ T cells are involved in the initiation and perpetuation of sarcoidosis. Particular T cell clones within the bronchoalveolar lavage (BAL) are preferentially expanded in patients with sarcoidosis, and these oligoclonal T cell populations accumulate within the lung and disappear with disease resolution. A link between the expansions of CD4+T cells expressing the T cell receptor (TCR) α-chain variable (V) region Vα2.3 and the presence of the HLA-DRB1*0301 (DR3) allele in sarcoidosis patients has been seen. We hypothesize that sarcoidosis-specific CD4+ T cells accumulate and expand in the lungs of sarcoidosis patients in response to a specific antigen. To determine whether these expanded CD4+T cell clones play a pathogenic role in disease progression, as well as to address their antigen specificity, we analyzed the TCR repertoire of CD4+ T cells isolated from the BAL of sarcoidosis patients utilizing iRepertoire, emulsion PCR, and single cell PCR analyses. We identified TCR Vα2.3/Jα42 CDR3 motifs that were found exclusively in HLA-DR3+ sarcoidosis patients (the motifs were not found in DR3− or DR3+ healthy control patients), suggesting a public TCR repertoire in this subset of patients. Importantly, these α-chains were paired with differing β-chains, suggesting a critical role of the α-chains in recognizing the HLA-DR3-peptide complex.
Blocking the TNF Superfamily member LIGHT to control lung and skin fibrosis
Authors: Herro Rana , Croft Michael
Published: January 2023

Anti-inflammatory drugs have so far failed to reduce fibrosis and tissue remodeling in the clinic. Alternatively, 5 TNF inhibitors have been FDA approved to treat different inflammatory diseases and are safe to administer to patients. In human, LIGHT and its receptors have been linked to numerous autoimmune and inflammatory diseases with fibrotic components. We show that blocking LIGHT signaling in isolation is efficient in reducing fibrosis in the skin and lung of mice induced with bleomycin to mimic human Systemic Sclerosis and Idiopathic pulmonary fibrosis, as well as in 2 severe allergen-driven models of asthma and atopic dermatitis. Neutralizing LIGHT signaling through its receptors by genetic deletion or antibody blocking, dramatically decreased collagen and smooth muscle deposition in the lungs and skins. Moreover the specific deletion of LIGHT-receptor HVEM on keratinocytes or the therapeutic blocking of LIGHT-HVEM interaction post-disease onset, abrogated skin fibrosis induced by house dust mite in a model of atopic dermatitis. LIGHT seems to play a central role in lung and skin fibrosis since it can control the expression of major pro-fibrotic factors such as TSLP, IL-13, TGF-b and Periostin. Also LIGHT alone can induce a fibro-proliferative disorder that mimics human SSc, in the lung and skin when administered alone, by increasing the accumulation of both fibers collagen and smooth muscle actin. Lastly we observed an upregulation of LIGHT and its receptors in lung biopsies of patients suffering from asthma, scleroderma and idiopathic pulmonary fibrosis. The relevance of this work to fibrosis therapies associated with asthma, atopic dermatitis, scleroderma and idiopathic pulmonary fibrosis are tremendous.
An aberrant immune response to an ocular commensal results in disease in a mouse model of Muckle-Wells Syndrome
Authors: Raychaudhuri Kumarkrishna , St Leger Anthony , Almaghrabi Fatimah , Fuss Ivan J. , Strober Warren , Caspi Rachel R
Published: January 2023

Appropriate regulation of host immunity by commensal bacteria at mucosal tissues, including the ocular surface, can prevent infection and limit disease. Our recent data (St. Leger et al, this conference) suggest that Corynebacterium mastitidis (=C. mast), an ocular commensal, elicits production of protective IL-17 from conjunctival gdT cells. What is not known is whether this relationship, when dysregulated, can lead to pathology and disease. In this study, we used a mouse model of Muckle-Wells Syndrome (MWS), which is a human autoinflammatory disease that results in spontaneous inflammation of the conjunctiva, skin and joints. This is caused by a mutation in the NLRP3 gene (cias1) that leads to an overactive NLRP3 inflammasome and results in production of multiple proinflammatory cytokines, including, most prominently, IL-1b. Here, we show that ocular colonization with a commensal bacterium, C. mast, induces ocular inflammation in MWS mice, but not in WT controls. Dendritic cells from MWS mice produced increased amounts of IL-1b upon stimulation with C. mast lysates. In keeping with this, DCs harvested from MWS mice more efficiently activated gdT cells (in particular, Vγ4+ cells) in co-culture experiments, compared to DCs from WT mice. Our results suggest that the commensal C. mast can act as a pathobiont and trigger ocular inflammation in mice with an overactive NLRP3 inflammasome. Thus, an aberrant immune response to commensal microbes in humans with this mutation may be the cause of recurrent conjunctivitis in patients with MWS or similar autoinflammatory diseases.