Transcriptomic profiling of programmed cell death 1 (PD-1) expressing T cells in early rheumatoid arthritis identifies a decreased CD4 + PD-1 + signature post-treatment.

Programmed cell death protein 1 (PD-1)-expressing T cells are expanded in individuals with established rheumatoid arthritis (RA). However, little is known about their functional role in the pathogenesis of early RA. To address this, we investigated the transcriptomic profiles of circulating CD4+ and CD8+ PD-1+ lymphocytes from patients with early RA (n = 5) using fluorescence activated cell sorting in conjunction with total RNA sequencing. Additionally, we assessed for alterations in CD4+PD-1+ gene signatures in previously published synovial tissue (ST) biopsy data (n = 19) (GSE89408, GSE97165) before and after six-months of triple disease modifying anti-rheumatic drug (tDMARD) treatment. Comparisons of gene signatures between CD4+PD-1+ vs. PD-1- cells identified significant upregulation of genes including CXCL13 and MAF, and in pathways including Th1 and Th2, cross talk between dendritic cells and NK cells, B cell development and antigen presentation. Gene signatures from early RA ST before and after six-month tDMARD treatment revealed downregulation of the CD4+PD-1+ signatures following treatment, identifying a mechanism through which tDMARDs exert their effect by influencing T cell populations. Furthermore, we identify factors associated with B cell help that are enhanced in the ST compared with PBMCs, highlighting their importance in driving synovial inflammation.

Differential expansion of T peripheral helper cells in early rheumatoid arthritis and osteoarthritis synovium.

OBJECTIVES: Programmed cell death protein 1 (PD-1)-expressing T cells are implicated in the pathogenesis of autoimmune inflammatory diseases such as rheumatoid arthritis. A subset of CXCR5- T cells, termed T peripheral helper (Tph) cells, which drive B cell differentiation, have been identified in ectopic lymphoid structures in established rheumatoid arthritis synovial tissue. Here, we aimed to characterise these in treatment-naïve, early rheumatoid arthritis to determine whether these cells accumulate prior to fully established disease. METHODS: Fresh dissociated tissue and peripheral blood mononuclear cell (PBMC) suspensions were stained with Zombie UV, followed by anti-CD45RO, PD-1, CD3, ICOS, CD8, CD4, CD20, CXCR5, TIGIT and CD38 antibodies prior to analysis. For histology, rheumatoid arthritis synovial sections were prepared for Opal multispectral immunofluorescence with anti-CD45RO, CD20, PD-1 and CXCR5 antibodies. Images were acquired on the Perkin Elmer Vectra V.3.0 imaging system and analysed using InForm Advanced Image Analysis software. RESULTS: Flow cytometry revealed T cell infiltration in the rheumatoid arthritis synovium with differential expression of PD-1, CD45RO, ICOS, TIGIT and CD38. We observed a higher frequency of PD1hiCXCR5- Tph in rheumatoid arthritis synovial tissue and PBMCs versus controls, and no significant difference in T follicular helper cell frequency. Microscopy identified a 10-fold increase of Tph cells in early rheumatoid arthritis synovial follicular and diffuse regions, and identified Tph adjacent to germinal centre B cells. CONCLUSIONS: These data demonstrate that PD-1hi Tph cells are present in early rheumatoid arthritis, but not osteoarthritis synovium, and therefore may provide a target for treatment of patients with early rheumatoid arthritis.

Distinct stromal and immune cell interactions shape the pathogenesis of rheumatoid and psoriatic arthritis.

OBJECTIVES: Immune and stromal cell communication is central in the pathogenesis of rheumatoid arthritis (RA) and psoriatic arthritis (PsA), however, the nature of these interactions in the synovial pathology of the two pathotypes can differ. Identifying immune-stromal cell crosstalk at the site of inflammation in RA and PsA is challenging. This study creates the first global transcriptomic analysis of the RA and PsA inflamed joint and investigates immune-stromal cell interactions in the pathogenesis of synovial inflammation. METHODS: Single cell transcriptomic profiling of 178 000 synovial tissue cells from five patients with PsA and four patients with RA, importantly, without prior sorting of immune and stromal cells. This approach enabled the transcriptomic analysis of the intact synovial tissue and identification of immune and stromal cell interactions. State of the art data integration and annotation techniques identified and characterised 18 stromal and 14 immune cell clusters. RESULTS: Global transcriptomic analysis of synovial cell subsets identifies actively proliferating synovial T cells and indicates that due to differential λ and κ immunoglobulin light chain usage, synovial plasma cells are potentially not derived from the local memory B cell pool. Importantly, we report distinct fibroblast and endothelial cell transcriptomes indicating abundant subpopulations in RA and PsA characterised by differential transcription factor usage. Using receptor-ligand interactions and downstream target characterisation, we identify RA-specific synovial T cell-derived transforming growth factor (TGF)-ß and macrophage interleukin (IL)-1ß synergy in driving the transcriptional profile of FAPα+THY1+ invasive synovial fibroblasts, expanded in RA compared with PsA. In vitro characterisation of patient with RA synovial fibroblasts showed metabolic switch to glycolysis, increased adhesion intercellular adhesion molecules 1 expression and IL-6 secretion in response to combined TGF-ß and IL-1ß treatment. Disrupting specific immune and stromal cell interactions offers novel opportunities for targeted therapeutic intervention in RA and PsA.

Attenuation of the BTLA/HVEM Regulatory Network in the Circulation in Primary Sjögren's Syndrome.

Primary Sjögren's syndrome (SjS) is an inflammatory autoimmune disorder which targets the lacrimal and salivary glands, resulting in glandular dysfunction. Currently, the immune drivers of SjS remain poorly understood and peripheral biomarkers of disease are lacking. The present study therefore sought to investigate the immune cell constituents of the SjS peripheral blood, and to assess the role of the BTLA/HVEM/CD160 co-stimulatory network by characterizing expression within the periphery. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of n = 10 patients with SjS and n = 10 age- and sex-matched healthy control donors. Cells were divided and stained with three panels of antibodies, allowing assessment of T, B, and myeloid cell subsets, and measurement of BTLA, HVEM, and CD160 surface expression by flow cytometry. We identified distinct alterations in proportions of peripheral T, B, and myeloid cell types in SjS compared with healthy controls. Expression of BTLA/CD160/HVEM and frequency of BTLA/CD160/HVEM-expressing cells were significantly altered in peripheral SjS lymphocytes. The proportion of T cells co-expressing BTLA/HVEM and CD160/HVEM were significantly reduced in SjS. We found decreased BTLA and HVEM levels on peripheral B and T cells of SjS patients, and decreased BTLA/HVEM and CD160/HVEM co-expression, demonstrating dysregulation of the BTLA/HVEM axis in the peripheral blood of SjS patients. These results indicate the potential of targeting the BTLA-HVEM axis for the treatment of SjS.

Discovery of LYC-55716: A Potent, Selective, and Orally Bioavailable Retinoic Acid Receptor-Related Orphan Receptor-γ (RORγ) Agonist for Use in Treating Cancer.

Retinoic acid receptor-related orphan receptor γ (RORc, RORγ, or NR1F3) is the nuclear receptor master transcription factor that drives the function and development of IL-17-producing T helper cells (Th17), cytotoxic T cells (Tc17), and subsets of innate lymphoid cells. Activation of RORγ+ T cells in the tumor microenvironment is hypothesized to render immune infiltrates more effective at countering tumor growth. To test this hypothesis, a family of benzoxazines was optimized to provide LYC-55716 (37c), a potent, selective, and orally bioavailable small-molecule RORγ agonist. LYC-55716 decreases tumor growth and enhances survival in preclinical tumor models and was nominated as a clinical development candidate for evaluation in patients with solid tumors.

Inhibiting Oxidative Phosphorylation In Vivo Restrains Th17 Effector Responses and Ameliorates Murine Colitis.

Integration of signaling and metabolic pathways enables and sustains lymphocyte function. Whereas metabolic changes occurring during T cell activation are well characterized, the metabolic demands of differentiated T lymphocytes are largely unexplored. In this study, we defined the bioenergetics of Th17 effector cells generated in vivo. These cells depend on oxidative phosphorylation (OXPHOS) for energy and cytokine production. Mechanistically, the essential role of OXPHOS in Th17 cells results from their limited capacity to increase glycolysis in response to metabolic stresses. This metabolic program is observed in mouse and human Th17 cells, including those isolated from Crohn disease patients, and it is linked to disease, as inhibiting OXPHOS reduces the severity of murine colitis and psoriasis. These studies highlight the importance of analyzing metabolism in effector lymphocytes within in vivo inflammatory contexts and suggest a therapeutic role for manipulating OXPHOS in Th17-driven diseases.

Programmed death-1 controls T cell survival by regulating oxidative metabolism.

The coinhibitory receptor programmed death-1 (PD-1) maintains immune homeostasis by negatively regulating T cell function and survival. Blockade of PD-1 increases the severity of graft-versus-host disease (GVHD), but the interplay between PD-1 inhibition and T cell metabolism is not well studied. We found that both murine and human alloreactive T cells concomitantly upregulated PD-1 expression and increased levels of reactive oxygen species (ROS) following allogeneic bone marrow transplantation. This PD-1(Hi)ROS(Hi) phenotype was specific to alloreactive T cells and was not observed in syngeneic T cells during homeostatic proliferation. Blockade of PD-1 signaling decreased both mitochondrial H2O2 and total cellular ROS levels, and PD-1-driven increases in ROS were dependent upon the oxidation of fatty acids, because treatment with etomoxir nullified changes in ROS levels following PD-1 blockade. Downstream of PD-1, elevated ROS levels impaired T cell survival in a process reversed by antioxidants. Furthermore, PD-1-driven changes in ROS were fundamental to establishing a cell's susceptibility to subsequent metabolic inhibition, because blockade of PD-1 decreased the efficacy of later F1F0-ATP synthase modulation. These data indicate that PD-1 facilitates apoptosis in alloreactive T cells by increasing ROS in a process dependent upon the oxidation of fat. In addition, blockade of PD-1 undermines the potential for subsequent metabolic inhibition, an important consideration given the increasing use of anti-PD-1 therapies in the clinic.

Sterol metabolism controls T(H)17 differentiation by generating endogenous RORγ agonists.

Retinoic acid receptor-related orphan receptor γ (RORγt) controls the differentiation of naive CD4(+) T cells into the TH17 lineage, which are critical cells in the pathogenesis of autoimmune diseases. Here we report that during TH17 differentiation, cholesterol biosynthesis and uptake programs are induced, whereas their metabolism and efflux programs are suppressed. These changes result in the accumulation of the cholesterol precursor, desmosterol, which functions as a potent endogenous RORγ agonist. Generation of cholesterol precursors is essential for TH17 differentiation as blocking cholesterol synthesis with chemical inhibitors at steps before the formation of active precursors reduces differentiation. Upon activation, metabolic changes also lead to production of specific sterol-sulfate conjugates that favor activation of RORγ over the TH17-inhibiting sterol receptor LXR. Thus, TH17 differentiation is orchestrated by coordinated sterol synthesis, mobilization and metabolism to selectively activate RORγ.

Anaplerotic metabolism of alloreactive T cells provides a metabolic approach to treat graft-versus-host disease.

T-cell activation requires increased ATP and biosynthesis to support proliferation and effector function. Most models of T-cell activation are based on in vitro culture systems and posit that aerobic glycolysis is employed to meet increased energetic and biosynthetic demands. By contrast, T cells activated in vivo by alloantigens in graft-versus-host disease (GVHD) increase mitochondrial oxygen consumption, fatty acid uptake, and oxidation, with small increases of glucose uptake and aerobic glycolysis. Here we show that these differences are not a consequence of alloactivation, because T cells activated in vitro either in a mixed lymphocyte reaction to the same alloantigens used in vivo or with agonistic anti-CD3/anti-CD28 antibodies increased aerobic glycolysis. Using targeted metabolic (13)C tracer fate associations, we elucidated the metabolic pathway(s) employed by alloreactive T cells in vivo that support this phenotype. We find that glutamine (Gln)-dependent tricarboxylic acid cycle anaplerosis is increased in alloreactive T cells and that Gln carbon contributes to ribose biosynthesis. Pharmacological modulation of oxidative phosphorylation rapidly reduces anaplerosis in alloreactive T cells and improves GVHD. On the basis of these data, we propose a model of T-cell metabolism that is relevant to activated lymphocytes in vivo, with implications for the discovery of new drugs for immune disorders.

Inflammasomes in inflammatory bowel disease pathogenesis.

PURPOSE OF REVIEW: Inflammasomes are molecular platforms assembled in response to infection or danger signals, and they regulate the activation of caspase-1 and the maturation of the inflammatory cytokines IL-1ß and IL-18. In this review, we will summarize the centrality of Nod-like receptor proteins that assemble inflammasomes and regulate intestinal homeostasis by controlling host defense responses, microbiota composition, intestinal inflammation and tissue damage. RECENT FINDINGS: In the intestine, the innate immune system evolved to tolerate commensal microorganisms while maintaining the capacity to trigger host defense responses to invading pathogens. Recent findings suggest that inflammasomes play a critical role in the intricate interplay between the local microbial community and the mucosal immune system by sensing commensal bacteria, regulating microbial ecology, establishing the host defense response that discriminates pathogenic from commensal microbes and preventing the emergence of pathobionts. A model to reconcile the conflicting results in the literature on the role of inflammasomes in experimental colitis will be discussed. SUMMARY: A better understanding of the relationship between inflammasome signaling and the intestinal microbiota might provide insight into the complex interaction of the innate immune system with the intestinal environment, and identify new approaches for the treatment of inflammatory bowel disease and gastrointestinal cancer.

DJ-1 is critical for mitochondrial function and rescues PINK1 loss of function.

Mutations or deletions in PARKIN/PARK2, PINK1/PARK6, and DJ-1/PARK7 lead to autosomal recessive parkinsonism. In Drosophila, deletions in parkin and pink1 result in swollen and dysfunctional mitochondria in energy-demanding tissues. The relationship between DJ-1 and mitochondria, however, remains unclear. We now report that Drosophila and mouse mutants in DJ-1 show compromised mitochondrial function with age. Flies deleted for DJ-1 manifest similar defects as pink1 and parkin mutants: male sterility, shortened lifespan, and reduced climbing ability. We further found poorly coupled mitochondria in vitro and reduced ATP levels in fly and mouse DJ-1 mutants. Surprisingly, up-regulation of DJ-1 can ameliorate pink1, but not parkin, mutants in Drosophila; cysteine C104 (analogous to C106 in human) is critical for this rescue, implicating the oxidative functions of DJ-1 in this property. These results suggest that DJ-1 is important for proper mitochondrial function and acts downstream of, or in parallel to, pink1. These findings link DJ-1, pink1, and parkin to mitochondrial integrity and provide the foundation for therapeutics that link bioenergetics and parkinsonism.

RNA binding activity of the recessive parkinsonism protein DJ-1 supports involvement in multiple cellular pathways.

Parkinson's disease (PD) is a major neurodegenerative condition with several rare Mendelian forms. Oxidative stress and mitochondrial function have been implicated in the pathogenesis of PD but the molecular mechanisms involved in the degeneration of neurons remain unclear. DJ-1 mutations are one cause of recessive parkinsonism, but this gene is also reported to be involved in cancer by promoting Ras signaling and suppressing PTEN-induced apoptosis. The specific function of DJ-1 is unknown, although it is responsive to oxidative stress and may play a role in the maintenance of mitochondria. Here, we show, using four independent methods, that DJ-1 associates with RNA targets in cells and the brain, including mitochondrial genes, genes involved in glutathione metabolism, and members of the PTEN/PI3K cascade. Pathogenic recessive mutants are deficient in this activity. We show that DJ-1 is sufficient for RNA binding at nanomolar concentrations. Further, we show that DJ-1 binds RNA but dissociates after oxidative stress. These data implicate a single mechanism for the pleiotropic effects of DJ-1 in different model systems, namely that the protein binds multiple RNA targets in an oxidation-dependent manner.

Short telomeres, even in the presence of telomerase, limit tissue renewal capacity.

Autosomal-dominant dyskeratosis congenita is associated with heterozygous mutations in telomerase. To examine the dosage effect of telomerase, we generated a line of mTR+/- mice on the CAST/EiJ background, which has short telomeres. Interbreeding of heterozygotes resulted in progressive telomere shortening, indicating that limiting telomerase compromises telomere maintenance. In later-generation heterozygotes, we observed a decrease in tissue renewal capacity in the bone marrow, intestines, and testes that resembled defects seen in dyskeratosis congenita patients. The progressive worsening of disease with decreasing telomere length suggests that short telomeres, not telomerase level, cause stem cell failure. Further, wild-type mice derived from the late-generation heterozygous parents, termed wt*, also had short telomeres and displayed a germ cell defect, indicating that telomere length determines these phenotypes. We propose that short telomeres in mice that have normal telomerase levels can cause an occult form of genetic disease.

Phosphorylation of H2AX at short telomeres in T cells and fibroblasts.

Eukaryotic cells undergo arrest and enter apoptosis in response to short telomeres. T cells from late generation mTR(-/-) mice that lack telomerase show increased apoptosis when stimulated to enter the cell cycle. The increased apoptosis was not inhibited by colcemid, indicating that the response did not result from breakage of dicentric chromosomes at mitosis. The damage response protein gamma-H2AX localized to telomeres in metaphases from T cells and fibroblasts from mTR(-/-) cells with short telomeres. These data suggest that the major mechanism for induction of apoptosis in late generation mTR(-/-) cells is independent of chromosome segregation and that loss of telomere function through progressive telomere shortening in the absence of telomerase leads to recognition of telomeres as DNA breaks.

Genomic instability in both wild-type and telomerase null MEFs.

To examine chromosome instability in the absence of telomerase, we established mouse embryonic fibroblast (MEF) lines from late generation mTR-/- and wild-type animals and examined metaphases using telomere fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY). In early passages, mTR-/- G6 cell lines showed more chromosome ends with no telomere signal, more chromosome end-to-end fusions and greater radiosensitivity than wild-type lines. At later passages, however, the rate of genomic instability in the wild-type MEFs increased to a level similar or higher than seen in the mTR-/- G6 cell lines. This high degree of instability in wild-type MEF lines suggests that post-crisis MEFs should not be considered genetically defined cell lines. Surprisingly, the increased radiosensitivity seen in early passage mTR-/- G6 cultures was lost after crisis. Both post-crisis mTR-/- G6 MEFs and wild-type MEFs showed loss of p53 and gamma-H2AX phosphorylation in response to irradiation, indicating a loss of DNA damage checkpoints.

Insulin-like growth factor-II induces hypertrophy of adult cardiomyocytes via two alternative pathways.

Insulin-like growth factor (IGF)-II is known to induce hypertrophy of isolated adult rat ventricular cardiomyocytes cultured in the absence of serum. However, it is not known how the growth factor exerts this hypertrophic effect. We show here that IGF-II induces hypertrophy of the cultured cardiomyocytes via two alternative pathways: (1) an IGF-I receptor-dependent pathway, or (2) a lysosome-dependent pathway when the IGF-I receptor-dependent pathway is blocked.

Hypertrophy of cultured adult rat ventricular cardiomyocytes induced by antibodies against the insulin-like growth factor (IGF)-I or the IGF-I receptor is IGF-II-dependent.

Antibodies against the insulin-like growth factor-I (IGF-I) or the IGF-I receptor (IGF-IR) directly initiate a rapid (within 6 h) hypertrophy of isolated adult rat ventricular cardiomyocytes cultured in the absence of serum. Further, cardiomyocytes treated with either of these agonistic antibodies upregulate the expression of their genes for insulin-like growth factor-II (IGF-II) and the IGF-II receptor (IGF-IIR). Genistein, an inhibitor of the tyrosine kinase IGF-IR, also induces the cardiomyocytes to hypertrophy. Anti-IGF-II antibody inhibits the cardiomyocyte hypertrophy induced by anti-IGF-I and anti-IGF-IR antibodies or by genistein. Results are consistent with a model in which local production of IGF-II is upregulated when the IGF-IR signaling pathway is blocked and in which an IGF-II-mediated pathway, likely involving the IGF-IIR, then stimulates hypertrophy of the cardiomyocytes.

Insulin-like growth factor-induced hypertrophy of cultured adult rat cardiomyocytes is L-type calcium-channel-dependent.

The insulin-like growth factors-I and -II are potent growth stimulators in vivo and for many different cultured cells in vitro. Here IGF-I and -II are shown to directly induce hypertrophy of adult rat ventricular cardiomyocytes in serum-free medium as demonstrated by their increased size, total protein synthesis, and transcription of muscle-specific genes. The cells hypertrophied within 1 day when exposed to as little as 10(-11) M IGF-I or 10(-10) M IGF-II. With 10(-8) M IGF-I, cell size was significantly increased 34% by 1 day of culture and 57% by 2 days. With 10(-8) M IGF-II, cell size was similarly increased 32% by day 1 and 57% by 2 days. During hypertrophy, total protein synthesis was increased 2.3-fold with IGF-I and 2-fold with IGF-II. Gene expression for myosin light chain 2 and troponin I was upregulated with either growth factor. Hypertrophy induced by IGF-I was blocked by IGF binding protein-3, which binds IGF-I, while that induced by IGF-II was blocked by antibodies against IGF-II. Nicardipine, an inhibitor of L-type Ca2+-channels, completely blocked the hypertrophy induced by either IGF showing for the first time that such voltage-dependent channels are necessary for the hypertrophic effects of the IGFs on adult cardiomyocytes.