High Interferon Signature Leads to Increased STAT1/3/5 Phosphorylation in PBMCs From SLE Patients by Single Cell Mass Cytometry.

The establishment of an "interferon (IFN) signature" to subset SLE patients on disease severity has led to therapeutics targeting IFNα. Here, we investigate IFN signaling in SLE using multiplexed protein arrays and single cell cytometry by time of flight (CyTOF). First, the IFN signature for SLE patients (n=81) from the Stanford Lupus Registry is determined using fluidigm qPCR measuring 44 previously determined IFN-inducible transcripts. IFN-high (IFN-H) patients have increased SLE criteria and renal/CNS/immunologic involvement, and increased autoantibody reactivity against spliceosome-associated antigens. CyTOF analysis is performed on non-stimulated and stimulated (IFNα, IFNγ, IL-21) PBMCs from SLE patients (n=25) and HCs (n=9) in a panel identifying changes in phosphorylation of intracellular signaling proteins (pTOF). Another panel is utilized to detect changes in intracellular cytokine (ICTOF) production in non-stimulated and stimulated (PMA/ionomycin) PBMCs from SLE patients (n=31) and HCs (n=17). Bioinformatic analysis by MetaCyto and OMIQ reveal phenotypic changes in immune cell subsets between IFN-H and IFN-low (IFN-L) patients. Most notably, IFN-H patients exhibit increased STAT1/3/5 phosphorylation downstream of cytokine stimulation and increased phosphorylation of non-canonical STAT proteins. These results suggest that IFN signaling in SLE modulates STAT phosphorylation, potentially uncovering possible targets for future therapeutic approaches.

[Hypothalamic-pituitary dysfunction in a patient with neurosarcoidosis].

This is a case report of a 64-year-old man with pulmonary sarcoidosis also affecting the joints. He was admitted to an emergency department following 21 days of fatigue, visual disturbances and headache. Initial blood tests revealed hypothalamic-pituitary dysfunction including acute adrenal insufficiency, and an MRI scan of the cerebrum showed a neurosarcoidosis tumour involving the hypothalamus-pituitary gland. Neurosarcoidosis is a condition with widespread clinical variation and early, and correct diagnosis is important.

Effects of Anti-TNFα Treatment on Mucosal Expression of IL-17A, IL-21, and IL-22 and Cytokine-Producing T Cell Subsets in Crohn's Disease.

T helper 17 (Th17) cells produce interleukin (IL) 17-A. In addition, Th17 cells produce IL-21 and IL-22. Th17 cells have a disease-promoting role in Crohn's disease (CD). We investigated the effects of anti-TNFα treatment on mucosal gene expression (qPCR) of IL-17A, IL-21, and IL-22 as well as on the frequency of lamina propria (LP) T cell subsets producing these cytokines (flow cytometry) in 12 active CD patients before and after 4 weeks of anti-TNFα treatment with adalimumab. At baseline, in inflamed mucosa we found increased gene expression of IL-17A and IL-22 but not IL-21 when compared to noninflamed mucosa. There were increased frequencies of IL-21-producing LP T cells but no differences in the frequencies of IL-17A- or IL-22-producing LP T cells when comparing inflamed versus noninflamed mucosa at baseline. There were no changes in the mucosal gene expression of IL-17A, IL-21, and IL-22 or the frequencies of IL-17A-, IL-21- and IL-22-producing LP T cell subsets between baseline and following 4 weeks of adalimumab initiation. Our results do not support the hypothesis that anti-TNFα treatment has an early effect on the mucosal levels of IL-17A, IL-21, and IL-22 or LP T cell production of these cytokines in CD.

Immune responses and parasitological observations induced during probiotic treatment with medicinal Trichuris suis ova in a healthy volunteer.

Ingestion of eggs (ova) of the porcine nematode parasite Trichuris suis (TSO) may reduce the severity of autoimmune disorders, however the development of TSO treatment as a useful therapy for autoimmune diseases is hampered by a lack of knowledge on the development of the parasite and the nature of the local immune responses in humans. Here, we used colonoscopy to investigate the development of T. suis and related mucosal and systemic immune responses during TSO treatment in an intestinally healthy male volunteer. TSO treatment induced T. suis-specific serum antibodies, a transient blood eosinophilia, and increases in IFNγ+ and IL4+ cells within the circulating CD4+ T-cell population. Increased expression of genes encoding cytokines (IL4, IL10, IL17 and TGF-ß), and transcription factors (FOXP3, GATA3 and RORC) were apparent in the ascending and transverse colon (the predilection site of the worms), whereas only limited changes in gene expression were observed proximally (ileum) and distally (descending colon) to the infected tissue. We further show that T. suis is able to colonise the human colon, with a number of worms developing to a similar size and morphology observed in the natural pig host, and a small number of unembryonated eggs were passed in the faeces, indicating patent infection. Notably, the volunteer experienced a substantial improvement in psoriasis during the course of TSO treatment. Thus, TSO treatment induced a mixed Th1/Th2/T regulatory response at the local site of infection, which was also reflected to some extent in the peripheral circulation. These results, together with the first definitive observations that T. suis can mature to adult size and reproduce in humans, shed new light on the interaction between the human immune system and probiotic helminth treatment, which should facilitate further development of this novel therapeutic option.

STAT2 is involved in the pathogenesis of psoriasis by promoting CXCL11 and CCL5 production by keratinocytes.

The JAK/STAT signaling pathway is suggested to play an important role in the pathogenesis of psoriasis, and recently JAK/STAT inhibitors have shown promising results in psoriasis treatment. The present study aimed to characterize the role of STAT2 in psoriasis. We demonstrated an increased expression of STAT2 and an increased level of phosphorylated/activated STAT2 in lesional compared with nonlesional psoriatic skin. Gene silencing of STAT2 by siRNA in human keratinocytes revealed that upon IFNα stimulation CXCL11 and CCL5 were the only two cytokines, among 102 analyzed, found to be regulated through a STAT2-dependent mechanism. Moreover, the regulation of CXCL11 and CCL5 depended on IRF9, but not on STAT1 and STAT6. The CXCL11 and CCL5 expression was increased in lesional compared with nonlesional psoriatic skin, and analysis demonstrated positive correlation between the expression of CXCL11 and IFNγ and between the expression of CCL5 and IFNγ in lesional psoriatic skin. In contrast, no correlation between the expression of CXCL11 and IL-17A and the expression of CCL5 and IL-17A in lesional psoriatic skin was found. Our data suggest that STAT2 plays a role in the psoriasis pathogenesis by regulating the expression of CXCL11 and CCL5, and thereby attracting IFNγ-producing immune cells to the skin.

Follicular T helper cells and IL-21 in rheumatic diseases.

Rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) are lifelong diseases with increased mortality and chronic pains. They are both characterized by immunological imbalances causing the immune system attack and destroy the bodies own tissues (called autoimmune disease). The best treatment, we are currently able to offer these patients, cause significant side-effects and can not prevent significant loss of quality of life. At the heart of the disease mechanisms in RA and SLE are subsets of immune cells called T and B cells. These cell types produce proteins (called antibodies), which under normal circumstances protect the body against disease. In RA and SLE these cells produce antibodies that are directed at the bodies own tissues (called autoantibodies), causing inflammation and tissue damage. The cause of this loss of tolerance is still unknown. Interleukin 21 (IL-21) is thought to exert key functions in controlling and directing the T and B cell responses leading to formation of antibodies and autoantibodies alike. IL-21 is a signaling molecule secreted by a subpopulation of T cells called follicular T helper (Tfh) cells. IFNα is another signaling molecule of key importance in autoimmune disease. Stratification of SLE patients by their responsiveness to IFNα has proven a crucial tool in stratifying patients in terms of disease development and treatment response. The aim of this PhD study is to investigate the role of IL-21 and IFNα, and their effects on Tfh cells and B cells and the formation of autoantibodies in RA and SLE. The first part of this study addresses whether plasma levels of IL-21 influence disease activity in rheumatic disease. We further investigate the distribution of IL-21-producing Tfh cells in these patients. We find that IL-21 plasma levels correlate to disease activity and radiological progression in RA, and that the IL-21-producing Tfh cell are increased in the blood and synovial fluid of these patients. These findings support the idea that IL-21 and Tfh cells are linked to the development and perpetuation of these diseases. In the second part of this we investigate how small RNA molecules, called microRNAs, can regulate immunological processes. We find that microRNA-155 can regulate IL-21's capacity to signal, while microRNA-21 is important for survival of T cells. The third, and last part of this, concerns IFNα signaling and its impact on the development of SLE and the formation of autoantibodies. We find that IFNα signaling is altered in a murine model of SLE, and that inhibition of this signaling pathway leads to severe kidney disease. The latter is of key importance as inhibition of IFNα is currently in early trial as a new treatment form for SLE patients. In SLE patients, we find that IFNα responsiveness, as measured by a so-called IFN signature, is crucial in terms of development of the disease as well as serious complications such as kidney disease and involvement of the central nervous system. Interferon alpha does this by affecting intracellular signaling responses and the formation of autoantibodies. The data presented in this thesis supports that IL-21 and Tfh cells have a key role in the disease processes characterizing RA and SLE. We further describe a novel mechanism for microRNA-155 and microRNA-21 in regulating immunological processes in these diseases. Finally we show, that IFNα has important functions in the formation of autoantibodies in SLE. In conclusion, this thesis adds new and important knowledge on the interplay between Tfh cells and B cells and their formation of autoantibodies in rheumatic disease. This knowledge will guide and further the development of new treatment strategies to better patient outcome.

A disintegrin and metalloprotease-17 and galectin-9 are important regulators of local 4-1BB activity and disease outcome in rheumatoid arthritis.

OBJECTIVE: Co-stimulatory T cell cytokines are important in the progression of RA. This study investigates the interplay between 4-1BB, a disintegrin and metalloprotease-17 (ADAM17) and galectin-9 (Gal-9) in RA. METHODS: Stimulated mononuclear cells from patients with chronic RA (n = 12) were co-incubated with tissue inhibitor of metalloproteinase, 4-1BB ligand and Gal-9. Plasma samples were examined for soluble 4-1BB (s4-1BB) in newly diagnosed, treatment-naïve patients with RA (n = 97). The 28-joint DAS with CRP (28DAS-CRP), total Sharp score, erosion score and joint space narrowing were used to evaluate treatment outcome serially over a 2-year period. RESULTS: RA CD4(+) and CD8(+) synovial T cells express high levels of 4-1BB. The addition of TNF-α to cultured synovial mononuclear cells increased shedding of 4-1BB. 4-1BB ligand only increased TNF-α shedding in combination with Gal-9. RNA interference-mediated knockdown of ADAM17 or the addition of an ADAM17 inhibitor reduced the 4-1BB shedding. Shedding of 4-1BB was not influenced by Gal-9. Plasma levels of s4-1BB were increased in early RA and correlated with the number of swollen joints at baseline. After 3 months of treatment, the plasma levels of s4-1BB were equal to those of the controls. Baseline plasma levels of s4-1BB were inversely correlated with DAS28-CRP after 2 years of treatment, but not with total Sharp score, erosion score or joint space narrowing. CONCLUSION: ADAM17 induces 4-1BB shedding in RA. Gal-9 is pivotal for the function of 4-1BB and induction of TNF-α. Furthermore, high plasma levels of s4-1BB were associated with the number of swollen joints, but also with a low DAS28-CRP after 2 years treatment in early RA.

Overexpression of microRNA-155 increases IL-21 mediated STAT3 signaling and IL-21 production in systemic lupus erythematosus.

INTRODUCTION: Interleukin (IL)-21 is a key cytokine in autoimmune diseases such as systemic lupus erythematosus (SLE) by its regulation of autoantibody production and inflammatory responses. The objective of this study is to investigate the signaling capacity of IL-21 in T and B cells and assess its possible regulation by microRNA (miR)-155 and its target gene suppressor of cytokine signaling 1 (SOCS1) in SLE. METHODS: The signaling capacity of IL-21 was quantified by stimulating peripheral blood mononuclear cells (PBMCs) with IL-21 and measuring phosphorylation of STAT3 (pSTAT3) in CD4+ T cells, B cells, and natural killer cells. Induction of miR-155 by IL-21 was investigated by stimulating purified CD4+ T cells with IL-21 and measuring miR-155 expression levels. The functional role of miR-155 was assessed by overexpressing miR-155 in PBMCs from SLE patients and healthy controls (HCs) and measuring its effects on STAT3 and IL-21 production in CD4+ and CD8+ T cells. RESULTS: Induction of pSTAT3 in CD4+ T cells in response to IL-21 was significantly decreased in SLE patients compared to HCs (p < 0.0001). Further, expression levels of miR-155 were significantly decreased and SOCS1 correspondingly increased in CD4+ T cells from SLE patients. Finally, overexpression of miR-155 in CD4+ T cells increased STAT3 phosphorylation in response to IL-21 treatment (p < 0.01) and differentially increased IL-21 production in SLE patients compared to HCs (p < 0.01). CONCLUSION: We demonstrate that SLE patients have reduced IL-21 signaling capacity, decreased miR-155 levels, and increased SOCS1 levels compared to HCs. The reduced IL-21 signaling in SLE could be rescued by overexpression of miR-155, suggesting an important role for miR-155 in the reduced IL-21 signaling observed in SLE.

CXCL13 predicts disease activity in early rheumatoid arthritis and could be an indicator of the therapeutic 'window of opportunity'.

INTRODUCTION: A key phenomenon in rheumatoid arthritis is the formation of lymphoid follicles in the inflamed synovial membrane. C-X-C motif chemokine 13 (CXCL13) is central in this process as it attracts C-X-C chemokine receptor type 5 (CXCR5)-expressing B cells and T follicular helper cells to the follicle. We here examine the role of CXCL13 and its association with disease in patients with treatment-naïve early rheumatoid arthritis. METHODS: Plasma samples from patients in the OPERA trial were examined for CXCL13 at treatment initiation and after 6 months of treatment with either methotrexate plus placebo (DMARD) (n = 37) or methotrexate plus adalimumab (DMARD + ADA) (n = 39). Treatment outcome was evaluated after 1 and 2 years. CXCL13 plasma levels in healthy volunteers (n = 38) were also examined. RESULTS: Baseline CXCL13 plasma levels were increased in early rheumatoid arthritis patients in comparison with healthy volunteers. Also, plasma CXCL13 correlated positively with disease activity parameters; swollen joint count 28 (rho = 0.34) and 40 (rho = 0.39), visual analog score (rho = 0.38) and simplified disease activity index (rho = 0.25) (all P <0.05). CXCL13 levels decreased a significantly twofold more in the DMARD + ADA group than in the DMARD group. Baseline CXCL13 plasma levels in the DMARD group correlated inversely with disease activity parameters; disease activity score in 28 joints, four variables, C-reactive protein based (DAS28CRP) (rho = 0.58, P < 0.05) at 12 months. High baseline CXCL13 was associated with remission (DAS28CRP less than 2.6) after 2 years. CONCLUSIONS: In treatment-naïve early rheumatoid arthritis patients, plasma CXCL13 levels were associated with joint inflammation. Furthermore, patients with high baseline plasma CXCL13 levels had an improved chance of remission after 2 years. We propose that high CXCL13 concentrations indicate recent onset of inflammation that may respond better to early aggressive treatment. Thus, high levels of CXCL13 could reflect the 'the window of opportunity' for optimal treatment effect. TRIAL REGISTRATION: Clinicaltrial.gov NCT00660647. Registered 10 April 2008.

Highest frequencies of interleukin-22-producing T helper cells in alcoholic hepatitis patients with a favourable short-term course.

BACKGROUND: Alcoholic hepatitis (AH) has a severe prognosis due to hepatic inflammatory injury. The cytokine interleukin-22 (IL-22) is reported to exert anti-apoptotic and proliferative effects, but IL-22 has not been studied during the course of AH. IL-22 is mainly produced by CD4(+) (helper) T cells, including Th17 cells. In addition, Th17 cells produce the proinflammatory cytokine IL-17A, which has been implicated in AH. AIMS: We aimed to study the levels of circulating IL-22- and IL-17A-producing T helper cells and plasma cytokines in patients with AH and to examine the observations in relation to the short-term disease course. METHODS: We collected blood samples from 21 consecutive patients with severe AH on days 0, 14 and 30 after diagnosis, and included 10 stable alcoholic cirrhosis patients and 10 healthy subjects as controls. Analyses were performed using flow cytometry and ELISA. RESULTS: We found higher frequencies of IL-22-producing T helper cells in AH patients (median 1.7%) than in cirrhosis patients (1.0%, p = 0.03) and healthy controls (1.0%, p = 0.01), and a 1.5-fold increase in the plasma concentration of IL-17A in AH compared with healthy controls (p<0.01). Those patients who markedly improved their Glasgow Alcoholic Hepatitis Score demonstrated a 2-fold higher frequency of IL-22-producing T helper cells at baseline and during follow-up than patients whose condition deteriorated (p = 0.04). CONCLUSIONS: The frequency of IL-22-producing T helper cells was increased in AH patients and most so in those whose condition seemed to improve. T cell differentiation toward an IL-22-producing phenotype may thus be favourable in AH.

Increased plasma levels of IL-21 and IL-23 in spondyloarthritis are not associated with clinical and MRI findings.

We have investigated the role of the Th17-related cytokines interleukin-17A (IL-17A), IL-21, and IL-23 in spondyloarthritis (SpA) by examining their association with disease activity and magnetic resonance imaging (MRI) findings in patients with SpA (n = 80). Furthermore, to investigate the cellular origins of the cytokines, paired mononuclear cells from blood and synovial fluid were examined for the expression of IL-17A, IL-21, and IL-23R using multicolor flow cytometry. Both IL-21 and IL-23 levels were increased in plasma from SpA patients compared with healthy volunteers (P < 0.05), whereas IL-17A was not. A significant correlation was observed between individual levels of IL-21 and IL-23 (r = 0.7, P < 0.001). No association between individual levels of IL-17A, IL-21, and IL-23 with C-reactive protein (CRP), MRI changes, and clinical scoring (BASMI, BASFI, and BASDAI) were observed. The frequency of CD4+CD45RO+ T cells expressing IL-21 and IL-23R was increased in the inflamed SpA joint compared to peripheral blood (P < 0.05). This study demonstrate that the plasma levels of the Th17-related cytokines IL-21 and IL-23, but not IL-17A, are increased in SpA patients, but we did not find evidence that the level of these cytokines reflect disease activity in SpA.

Increased interleukin 21 (IL-21) and IL-23 are associated with increased disease activity and with radiographic status in patients with early rheumatoid arthritis.

OBJECTIVE: To investigate the levels of the T helper (Th)17-related cytokines interleukin 17A (IL-17A), IL-21, and IL-23 and their association with disease activity in rheumatoid arthritis (RA). METHODS: In a longitudinal sample set from patients with early RA (< 6 months; n = 40), we measured the plasma cytokine levels of IL-17A, IL-21, and IL-23 and analyzed for correlation with disease activity in 28 joints (Disease Activity Score 28-joint count; DAS28), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and total Sharp score (TSS). In a transverse sample set of patients with chronic RA (> 8 years), using paired peripheral blood mononuclear cells and synovial fluid mononuclear cells, we investigated the cellular expression of IL-17A, IL-21, and IL-23R. RESULTS: Patients with early-stage RA had significantly increased plasma levels of IL-21 and IL-23, but not IL-17A, compared to patients with chronic RA and healthy volunteer controls. Plasma levels of IL-21 and IL-23 after 12 months of treatment correlated with DAS28 and ESR, but not to TSS. Changes in IL-23 plasma levels from time of diagnosis to 12 months correlated with change in DAS28 and with TSS scores at 2 years. The numbers of CD4+ T cells producing IL-21 were significantly increased in the synovial fluid of patients with chronic RA, with only marginal coexpression of IL-21 and IL-17A. CONCLUSION: Our results show a significant association between plasma levels of IL-21 and IL-23 and disease activity in RA, supporting the hypothesis that IL-21 and IL-23 are important pathogenic factors of this disease.