Reactive arthritis and undifferentiated peripheral spondyloarthritis share human leucocyte antigen B27 subtypes and serum and synovial fluid cytokine profiles.

OBJECTIVES: Peripheral SpA (pSpA) is comprised of ReA, PsA, enteritis-associated arthritis and undifferentiated pSpA (upSpA). ReA and upSpA share T cell oligotypes and metabolomics in serum and SF. We investigated HLA-B27 subtypes and cytokines in serum and SF that were compared between ReA and upSpA. METHODS: ReA and upSpA were compared in two cohorts. In cohort I (44 ReA and 56 upSpA), HLA-B27 subtyping was carried out. In cohort II (17 ReA and 21 upSpA), serum and SF cytokines were compared using a multiplex cytokine bead assay (27 cytokines). A total of 28 healthy controls with similar age and sex to cohort II were included for comparison of serum cytokine levels. RESULTS: In cohort I, HLA-B27 was positive in 81.8% (36/44) of ReA and 85.71% (48/56) of upSpA patients. HLA-B27 typing was successful in 70 patients (30 ReA and 40 uSpA). HLA-B*2705 was the most common, followed by HLA-B*2704 and HLA-B*2707. Frequencies were the same between ReA and upSpA. In cohort II, 14 cytokines were detectable in the serum of patients. The levels of eight cytokines were higher than in the controls. The cytokine levels of ReA and upSpA were similar. Sixteen cytokines were detectable in the SF of patients. There was no statistical difference in the levels between ReA and upSpA. The cytokine profiles in sera and SF were also similar among HLA-B27-positive and negative patients. CONCLUSION: ReA and upSpA have similar HLA-B27 subtype associations and similar cytokine profiles. They should be considered as a single entity during studies as well as clinical management.

LincRNA-immunity landscape analysis identifies EPIC1 as a regulator of tumor immune evasion and immunotherapy resistance.

Through an integrative analysis of the lincRNA expression and tumor immune response in 9,626 tumor samples across 32 cancer types, we developed a lincRNA-based immune response (LIMER) score that can predict the immune cells infiltration and patient prognosis in multiple cancer types. Our analysis also identified tumor-specific lincRNAs, including EPIC1, that potentially regulate tumor immune response in multiple cancer types. Immunocompetent mouse models and in vitro co-culture assays demonstrated that EPIC1 induces tumor immune evasion and resistance to immunotherapy by suppressing tumor cell antigen presentation. Mechanistically, lincRNA EPIC1 interacts with the histone methyltransferase EZH2, leading to the epigenetic silencing of IFNGR1, TAP1/2, ERAP1/2, and MHC-I genes. Genetic and pharmacological inhibition of EZH2 abolish EPIC1's immune-related oncogenic effect and its suppression of interferon-γ signaling. The EPIC1-EZH2 axis emerges as a potential mechanism for tumor immune evasion that can serve as therapeutic targets for immunotherapy.

Nuclear magnetic resonance-based targeted profiling of urinary acetate and citrate following cyclophosphamide therapy in patients with lupus nephritis.

OBJECTIVE: Metabolomics, the study of global alterations in small metabolites, is a useful tool to look for novel biomarkers. Recently, we reported a reprogramming of the serum metabolomic profile by nuclear magnetic resonance (NMR) spectroscopy following treatment in lupus nephritis (LN). This study aimed to compare the urine excretory levels of citrate and acetate in patients with biopsy-proven LN before and six months after cyclophosphamide induction therapy and to evaluate their correlation with the Systemic Lupus Erythematosus Disease Activity Index 2K (SLEDAI 2K) and renal SLEDAI. METHODS: Urine obtained from LN patients (N = 18, 16 female) at diagnosis and six months following induction therapy with cyclophosphamide and healthy controls (HC; N = 18, median age = 35 years, all female) were stored at -80°C. Metabolomic profiling was done using high resolution 800 MHz 1D 1H NMR spectroscopy. The urinary ratio of metabolites was calculated as (metabolite×1000)/creatinine. Disease activity was measured using the SLEDAI. Metabolomic profiles were compared between groups and correlated with clinical parameters. RESULTS: Compared to HC, LN patients had significantly lower median urinary citrate/creatinine levels (LN = 18.26, range 12.80-27.62; HC = 107.7, range 65.39-138.4; p < 0.0001) which significantly increased after six months of cyclophosphamide treatment (51.05, range 11.51-170.2; p = 0.03). LN patients also differed from HC by having a higher mean urinary acetate/creatinine ratio (LN = 17.44, range 11.6-32.7; HC = 9.61, range 7.97-13.71; p = 0.054) with a non-significant fall in values after six months of treatment. The Area under curve for differentiating LN from HC for urinary citrate was 0.9136, and urinary acetate was 0.6883. The urinary acetate levels correlated with SLEDAI (r = 0.337, p = 0.048). Urinary citrate levels correlated positively with C3 (r = 0.362, p = 0.03) and negatively with urine protein/creatinine (r = -0.346, p = 0.039). CONCLUSIONS: Urinary citrate, which reflects dampened aerobic glycolysis and oxidative phosphorylation, improved significantly and is a potential non-invasive biomarker for diagnosis and monitoring treatment response in LN.

Metabolomics analysis revealed significantly higher synovial Phe/Tyr ratio in reactive arthritis and undifferentiated spondyloarthropathy.

OBJECTIVE: To compare the synovial phenylalanine/tyrosine (Phe/Tyr) ratio between ReA/uSpA and RA and OA by NMR spectroscopy. METHODS: Paired SF and serum of 30 patients with ReA/uSpA were collected and analysed using a 1D 1H Carr Purcell Meiboom Gill NMR spectra recorded on 800 MHz NMR spectrometer equipped with a TCI Cryoprobe (at 300 K). Phe and Tyr were quantified. SF from 25 patients with RA fulfilling ACR classification criteria and 21 patients with OA were taken as inflammatory and non-inflammatory controls. RESULTS: The synovial Phe/Tyr ratio was significantly higher in ReA/uSpA compared with RA and OA. Synovial Phe/Tyr ratios were comparable in RA and OA patients. Compared with serum, the Phe/Tyr was significantly higher in the SF in ReA/uSpA. The Phe/Tyr ratio was also found to be positively correlated between serum and SF samples, with a regression coefficient (r2) of 0.287. CONCLUSIONS: This NMR-based metabolomics study demonstrates that the synovial Phe/Tyr ratio is specifically elevated in ReA/uSpA.

Nuclear magnetic resonance-based metabolomics reveals similar metabolomics profiles in undifferentiated peripheral spondyloarthritis and reactive arthritis.

INTRODUCTION: After exclusion of reactive, psoriatic and enteritis-associated arthritis, a group of "undifferentiated" peripheral spondyloarthritis (pSpA) remains. This group shares genetics, T-cell repertoire, and cytokines with reactive arthritis (ReA). ReA is preceded by gut or urogenital infection. Otherwise the two may be similar. We know little of the metabolic pathways driving undifferentiated pSpA or ReA. Nuclear magnetic resonance (NMR)-based metabolomics presents a hypothesis-free approach to study and compare metabolic pathways driving undifferentiated pSpA and ReA. METHODS: Serum and synovial fluid metabolomes of 19 ReA and 13 undifferentiated pSpA, and serum metabolome of 18 controls were profiled using 1 H-based NMR. Partial least square-discriminant analysis (PLS-DA) identified metabolites different in patients as compared to controls. Multivariate analysis confirmed these. Altered metabolic pathways were identified using metabolites set enrichment analysis (MSEA). The serum and synovial fluid metabolomes of ReA and undifferentiated pSpA were compared. RESULTS: Ten serum metabolites of ReA/undifferentiated pSpA were different from those of controls. Six metabolites were different between serum and synovial fluid of these patients. MSEA identified five pathways different between patients and controls, and five pathways different between serum and synovial fluid of patients. PLS-DA showed no difference between the metabolomes of serum or of synovial fluid between ReA and undifferentiated pSpA. DISCUSSION: Identified metabolic pathways may be explored further to understand the pathogenesis and to target therapeutics. The similar immuno-metabolic pathways suggest similar pathogenesis of ReA and undifferentiated pSpA. Thus, they should be studied as a single disease entity.

Metabolite assignment of ultrafiltered synovial fluid extracted from knee joints of reactive arthritis patients using high resolution NMR spectroscopy.

Currently, there are no reliable biomarkers available that can aid early differential diagnosis of reactive arthritis (ReA) from other inflammatory joint diseases. Metabolic profiling of synovial fluid (SF)-obtained from joints affected in ReA-holds great promise in this regard and will further aid monitoring treatment and improving our understanding about disease mechanism. As a first step in this direction, we report here the metabolite specific assignment of 1 H and 13 C resonances detected in the NMR spectra of SF samples extracted from human patients with established ReA. The metabolite characterization has been carried out on both normal and ultrafiltered (deproteinized) SF samples of eight ReA patients (n = 8) using high-resolution (800 MHz) 1 H and 1 H─13 C NMR spectroscopy methods such as one-dimensional 1 H CPMG and two-dimensional J-resolved1 H NMR and homonuclear 1 H─1 H TOCSY and heteronuclear1 H─13 C HSQC correlation spectra. Compared with normal SF samples, several distinctive 1 H NMR signals were identified and assigned to metabolites in the 1 H NMR spectra of ultrafiltered SF samples. Overall, we assigned 53 metabolites in normal filtered SF and 64 metabolites in filtered pooled SF sample compared with nonfiltered SF samples for which only 48 metabolites (including lipid/membrane metabolites as well) have been identified. The established NMR characterization of SF metabolites will serve to guide future metabolomics studies aiming to identify/evaluate the SF-based metabolic biomarkers of diagnostic/prognostic potential or seeking biochemical insights into disease mechanisms in a clinical perspective.

NMR-Based Serum Metabolomics Revealed Distinctive Metabolic Patterns in Reactive Arthritis Compared with Rheumatoid Arthritis.

Reactive arthritis (ReA) is a member of seronegative spondyloarthropathy (SSA), which involves an acute/subacute onset of asymmetrical lower limb joint inflammation weeks after a genitourinary/gastrointestinal infection. The diagnosis is clinical because it is difficult to culture the microbes from synovial fluid. Arthritis patients with a similar clinical picture but lapsed history of an immediate preceding infection that do not fulfill the diagnostic criteria of other members of SSA, such as ankylosing spondylitis, psoriatic arthritis, and arthritis associated with inflammatory bowel disease, are labeled as peripheral undifferentiated spondyloarthropathy (uSpA). Both ReA and uSpA patients show a strong association with class I major histocompatibility complex allele, HLA-B27, and a clear association with an infectious trigger; however, the disease mechanism is far from clear. Because the clinical picture is largely dominated by rheumatoid-arthritis (RA)-like features including elevated levels of inflammatory markers (such as ESR, CRP, etc.), these overlapping symptoms often confound the clinical diagnosis and represent a clinical dilemma, making treatment choice more generalized. Therefore, there is a compelling need to identify biomarkers that can support the diagnosis of ReA/uSpA. In the present study, we performed NMR-based serum metabolomics analysis and demonstrated that ReA/uSpA patients are clearly distinguishable from controls and further that these patients can also be distinguished from the RA patients based on the metabolic profiles, with high sensitivity and specificity. The discriminatory metabolites were further subjected to area under receiver operating characteristic curve analysis, which led to the identification of four metabolic entities (i.e., valine, leucine, arginine/lysine, and phenylalanine) that could differentiate ReA/uSpA from RA.

NMR-Based Serum Metabolomics of Patients with Takayasu Arteritis: Relationship with Disease Activity.

Takayasu arteritis (TA) is a large vessel vasculitis of unknown pathogenesis. Assessment of disease activity is a challenge, and there is an unmet need for relevant biomarker(s). In our previous study, NMR based serum metabolomics had revealed distinctive metabolic signatures in TA patients compared with age/sex matched healthy controls and systemic lupus erythematosus (SLE). In this study we investigate whether the metabolites correlate with disease activity. Patients with TA fulfilling American College of Rheumatology (ACR) criteria were enrolled, and disease activity was assessed using Indian Takayasu Clinical Activity Score using acute phase reactant-erythrocyte sedimentation rate [ITAS-A (ESR)]. Sera were analyzed using 800 MHz NMR spectrometer to identify metabolites [based on partial least squares discriminant analysis (PLS-DA) VIP (variable importance in projection) score > 1.0 and permutation test, p-value <0.01]. 45 active and 53 inactive TA patients with median age 27 [(IQR) 22-35 years] and 27 [(IQR) 23-37 years], female to male ratio 3.5:1 and 4.9:1, and median duration of illness 5 [(IQR) 2-9 years] and 3 [(IQR) 1-6 years], respectively, were enrolled. The key metabolites with highest discriminatory potential in active TA (ITAS-A ≥ 4) were glutamate and N-acetyl glycoprotein (NAG), both elevated, with area under the curve 0.775 and 0.769 ( p-value <0.001). On follow up assessment, metabolic spectra started to differ with change in disease activity. This large cohort of patients revealed metabolic profiles discriminating between clinically active and inactive TA patients. It suggests glutamate and NAG have strong potential as biomarkers for disease activity in TA and may serve as a guide to therapy. We are now working to further validate these results in longitudinal studies.

NMR-Based Serum Metabolomics Reveals Reprogramming of Lipid Dysregulation Following Cyclophosphamide-Based Induction Therapy in Lupus Nephritis.

Lupus nephritis (LN) is a major cause of morbidity and mortality in lupus. Renal biopsy is the gold standard for classification of nephritis, but because of its impracticality, new approaches for improving patient prognostication and monitoring treatment efficacy are needed. We aimed to evaluate the potential of metabolic profiling in identifying biomarkers to distinguish disease and monitor treatment efficacy in patients with LN. Serum samples from patients with LN ( n = 18) were profiled on NMR-based metabolomics platforms at diagnosis and after 6 months of treatment. LN patients had a different metabolomic fingerprint as compared with healthy controls, with increased lipoproteins and lipids and reduced acetate and amino acids. Using multivariate statistical analysis, we found that the metabolic changes observed in naïve LN patients at diagnosis displayed a variation in the opposite direction upon responding to treatment. Increased levels of lipid metabolites including low- and very-low-density lipoproteins (LDL/VLDL) in LN patients significantly decreased after 6 months of treatment, whereas the serum levels of acetate increased. These levels correlated significantly with SLE Disease Activity Index (SLEDAI 2K), renal SLEDAI, and serum C3 and C4 levels. The result presented in this pilot longitudinal study revealed the reprogramming of metabolome in LN patients on immunosuppressive therapy using NMR-based metabolomics, and thus this approach may be used to monitor the response to treatment.

Serum BAFF in Indian patients with IIM: a retrospective study reveals novel clinico-phenotypic associations in children and adults.

We studied the serum levels of B cell survival factors BAFF and APRIL in patients with idiopathic inflammatory myositis (IIM) and their relation with clinical and autoantibodies. Seventy-five patients (51 females and 24 males) with IIM (Bohan and Peter's criteria 1975) and 25 healthy adults were analyzed for BAFF, APRIL and IL-17 by ELISA, and myositis-specific and associated antibodies (MSA and MAA) using line immunoblot assay. Of the 75 patients, 59 were adults, 42 had Dermatomyositis (DM), and 17 had Polymyositis. Median disease duration was 5 (3-12) months. BAFF levels were higher in IIM than healthy controls [p = 0.001], and in children with jDM than adults [p = 0.026]. BAFF levels were higher in adults with arthritis [p = 0.018], weight loss [p = 0.007], and PAH [p = 0.004]. Among the various MSAs, lowest levels were seen in those with anti-SRP [p = 0.043]. Median follow-up duration was 145 patient years. Twelve patients relapsed, while nine were in drug-free remission. BAFF were similar between these groups. Serum APRIL levels were elevated in limited number of patients with myositis, and the levels did not differ amongst the clinico-serologic phenotypes. IL-17 levels were higher in individuals positive for anti-SRP [p = 0.028]. Serum BAFF levels are elevated in IIM, more so in children. BAFF levels may be useful as biomarker for PAH and arthritis. Anti-SRP positivity is associated with elevated IL-17 levels suggesting role in pathogenesis.

Effect of induction therapy on circulating T-helper 17 and T-regulatory cells in active proliferative lupus nephritis.

INTRODUCTION: T-helper 17 (Th17) cells and T-regulatory (Treg) cells have been suggested to play pathogenic roles in lupus nephritis. The in vivo effects of current therapies for lupus nephritis (LN) on these cells have not been adequately studied. METHODS: We conducted a prospective observational study among patients with active proliferative lupus nephritis (LN) who received Eurolupus induction therapy and assessed them as per the European League Against Rheumatism criteria for renal response. Peripheral circulatory Th17 and Treg cell numbers were enumerated at start of therapy, at 3 and 6 months follow-up periods using flow cytometry. Baseline values were compared with inactive lupus patients (iSLE) and healthy controls (HC). RESULTS: Thirty patients with LN, 20 iSLE and 22 HC were enrolled into the study. In LN, Th17 frequency was significantly higher compared to HC, and Treg frequency significantly lower compared to both iSLE and HC. Nineteen patients fulfilled criteria for response (partial or complete) at 6 months. Responder group showed a significant decline in Th17 frequency and an increasing trend in Treg frequency compared to baseline after 6 months of therapy. CONCLUSION: Circulating Th17 cells were significantly raised in patients with active proliferative LN and showed a significant reduction in responder patients following therapy.

IL-17 and IFN-γ producing NK and γδ-T cells are preferentially expanded in synovial fluid of patients with reactive arthritis and undifferentiated spondyloarthritis.

The IL-17/1L-23 axis is important in the pathogenesis of spondyloarthropathy. Innate cells produce IL-17 in addition to Th17 cells. We studied the frequencies of natural killer (NK) (total, CD56bright, CD56dim, perforin+ and granzyme+), NK-T, γδ-T, and IFN-γ+, IL-17+ NK and γδ-T cells in peripheral blood (PB) and synovial fluid (SF) of ReA/uSpA patients. PB from 45 patients and paired SF from 39 patients were studied, together with PB from 18 healthy controls (HC). The frequency of γδ-T cells was decreased (p<0.05) while IL-17 producing NK and γδ-T cells were increased (p<0.05) in PB of patients as compared to HC. In SF, CD56bright NK cells were increased (p<0.001) but had reduced expression of perforin and granzyme (p<0.0001) as compared to PB. Frequency of IL-17+, IFN-γ+ NK and γδ-T cells was higher in SF as compared to PB (p<0.05). We suggest that innate cells by producing pro-inflammatory cytokines may contribute to pathogenesis.

NMR based serum metabolomics reveals a distinctive signature in patients with Lupus Nephritis.

Management of patient with Lupus Nephritis (LN) continues to remain a challenge for the treating physicians because of considerable morbidity and even mortality. The search of biomarkers in serum and urine is a focus of researchers to unravel new targets for therapy. In the present study, the utility of NMR-based serum metabolomics has been evaluated for the first time in discriminating LN patients from non-nephritis lupus patients (SLE) and further to get new insights into the underlying disease processes for better clinical management. Metabolic profiling of sera obtained from 22 SLE patients, 40 LN patients and 30 healthy controls (HC) were performed using high resolution 1D 1H-CPMG and diffusion edited NMR spectra to identify the potential molecular biomarkers. Using multivariate analysis, we could distinguish SLE and LN patients from HC and LN from SLE patients. Compared to SLE patients, the LN patients had increased serum levels of lipid metabolites (including LDL/VLDL lipoproteins), creatinine and decreased levels of acetate. Our results revealed that metabolic markers especially lipids and acetate derived from NMR spectroscopy has high sensitivity and specificity to distinguish LN among SLE patients and has the potential to be a useful adjunctive tool in diagnosis and clinical management of LN.

Increased Circulating Th17 Cells, Serum IL-17A, and IL-23 in Takayasu Arteritis.

Introduction. Th17, γδT, NK, and NKT cells in peripheral blood and serum IL-17 and IL-23 in Takayasu arteritis (TA) were measured and correlated with disease activity. Methods. Th17 (anti-CD3APC, CD4PECy7, and IL-17PE), NKT, NK (anti-CD3APC, CD56FITC), and γδT (anti-CD3FITC and γδTCRAPC) cells were enumerated by flow cytometry in peripheral blood of 30 patients with TA (ACR1990 criteria) and 20 healthy controls, serum IL-17 and IL-23 measured by ELISA. Relation with disease activity (NIH criteria, ITAS2010) was analyzed (using nonparametric tests, median with interquartile range). Results. Mean age of patients was 33.47 ± 11.78 years (25 females); mean symptom duration was 7.1 ± 5.3 years. 13 were not on immunosuppressants; 12 were active (ITAS2010 ≥ 4). The percentage of Th17 cells was significantly expanded in TA (patients 2.1 (1.5-3.2) versus controls 0.75 (0.32-1.2); p < 0.0001) with no differences in other cell populations. Serum IL-17 and IL-23 (pg/mL) in patients (6.2 (4.6-8.5) and 15 (14.9-26.5), resp.) were significantly higher (p < 0.001) than controls (3.9 (3.9-7.3) and undetectable median value, resp.). Subgroup analysis revealed no correlation of Th17 cells, serum IL-17, and IL-23 with disease activity or medications, nor any significant difference before and after medication. Conclusions. There is significant expansion of Th17 cells and elevated serum IL-17 and IL-23 levels in TA patients compared to healthy controls.

Identification of immunogenic HLA-B*27:05 binding peptides of salmonella outer membrane protein in patients with reactive arthritis and undifferentiated spondyloarthropathy.

OBJECTIVE: Salmonella outer membrane proteins (OMP) are major immunogenic targets to synovial fluid lymphocytes of patients with reactive arthritis (ReA)/undifferentiated spondyloarthropathy (uSpA). Because these patients have genetic predisposition to HLA-B*27 and its subtype HLA-B*27:05, we sought to identify immunogenic HLA-B*27:05-binding salmonella OMP peptides in patients with ReA/uSpA. METHODS: A total of 125 HLA-B*27:05-binding salmonella OMP peptides identified using ProPred-I software were synthesized and grouped in 23 pools. The peptide pools, along with crude enteric bacterial lysates and salmonella OMP, were cultured with synovial fluid (SF) or peripheral blood mononuclear cells (PBMC) from 23 patients with ReA/uSpA, 10 with rheumatoid arthritis (RA), and 10 healthy individuals in 96-well culture plates. Proliferation was measured by tritiated thymidine uptake and interferon-γ (IFN-γ) levels in culture supernatant. Individual peptides from pools having significant responses were retested with cryopreserved cells. Immunogenic peptides thus identified were further tested in 5 additional new patients with ReA/uSpA by flow cytometry. A Basic Local Alignment Search Tool program was used to search for similar peptides from a protein bank of arthritogenic bacteria and human protein. RESULTS: Nineteen of 23 SFMC from ReA/uSpA showed a significant proliferative response to salmonella OMP, with minimal response of PBMC (1/10) from ReA/uSpA, SFMC from RA (1/10), or PBMC from controls (1/10). Nine salmonella OMP peptides, QRAEMLPTL, SRSGLNIAL, LRFLYAKSL, RLEGTWVKL, ARCIAPYAL, KLFLTTAAL, YRNSDFFGL, QRPAVRVKL, and YRVGPGDVL, were identified. Response to QRAEMLPTL was seen in 6/7 HLA-B*27:05-positive patients. All immunogenic peptides had sequence similarity with peptides from arthritogenic bacterial proteins, while 5 had similarity with peptides from human proteins. CONCLUSION: Nine novel immunogenic OMP peptides binding to HLA-B*27:05 were identified that showed sequence similarity with other arthritogenic bacteria.