CD7 activation regulates cytotoxicity-driven pathology in systemic sclerosis, yielding a target for selective cell depletion.

OBJECTIVES: Cytotoxic T cells and natural killer (NK) cells are central effector cells in cancer and infections. Their effector response is regulated by activating and inhibitory receptors. The regulation of these cells in systemic autoimmune diseases such as systemic sclerosis (SSc) is less defined. METHODS: We conducted ex vivo analysis of affected skin and blood samples from 4 SSc patient cohorts (a total of 165 SSc vs 80 healthy individuals) using single-cell transcriptomics, flow cytometry and multiplex immunofluorescence staining. We further analysed the effects of costimulatory modulation in functional assays, and in a severely affected SSc patient who was treated on compassionate use with a novel anti-CD3/CD7 immunotoxin treatment. RESULTS: Here, we show that SSc-affected skin contains elevated numbers of proliferating T cells, cytotoxic T cells and NK cells. These cells selectively express the costimulatory molecule CD7 in association with cytotoxic, proinflammatory and profibrotic genes, especially in recent-onset and severe disease. We demonstrate that CD7 regulates the cytolytic activity of T cells and NK cells and that selective depletion of CD7+ cells prevents cytotoxic cell-induced fibroblast contraction and inhibits their profibrotic phenotype. Finally, anti-CD3/CD7 directed depletive treatment eliminated CD7+ skin cells and stabilised disease manifestations in a severely affected SSc patient. CONCLUSION: Together, the findings imply costimulatory molecules as key regulators of cytotoxicity-driven pathology in systemic autoimmune disease, yielding CD7 as a novel target for selective depletion of pathogenic cells.

Severe Immune-Related Enteritis after In Utero Exposure to Pembrolizumab.

Immune checkpoint blockade has become standard treatment for many types of cancer. Such therapy is indicated most often in patients with advanced or metastatic disease but has been increasingly used as adjuvant therapy in those with early-stage disease. Adverse events include immune-related organ inflammation resembling autoimmune diseases. We describe a case of severe immune-related gastroenterocolitis in a 4-month-old infant who presented with intractable diarrhea and failure to thrive after in utero exposure to pembrolizumab. Known causes of the symptoms were ruled out, and the diagnosis of pembrolizumab-induced immune-related gastroenterocolitis was supported by the results of histopathological assays, immunophenotyping, and analysis of the level of antibodies against programmed cell death protein 1 (PD-1). The infant's condition was successfully treated with prednisolone and infliximab.

Nailfold capillaroscopy and candidate-biomarker levels in systemic sclerosis-associated pulmonary hypertension: A cross-sectional study.

Objectives: Pulmonary hypertension is one of the leading causes of death in systemic sclerosis. Early detection and treatment of pulmonary hypertension in systemic sclerosis is crucial. Nailfold capillaroscopy microscopy, vascular autoantibodies AT1R and ETAR, and several candidate-biomarkers have the potential to serve as noninvasive tools to identify systemic sclerosis patients at risk for developing pulmonary hypertension. Here, we explore the classifying potential of nailfold capillaroscopy microscopy characteristics and serum levels of selected candidate-biomarkers in a sample of systemic sclerosis patients with and without different forms of pulmonary hypertension. Methods: A total of 81 consecutive systemic sclerosis patients were included, 40 with systemic sclerosis pulmonary hypertension and 41 with no pulmonary hypertension. In each group, quantitative and qualitative nailfold capillaroscopy microscopy characteristics, vascular autoantibodies AT1R and ETAR, and serum levels of 24 soluble serum factors were determined. For evaluation of the nailfold capillaroscopy microscopy characteristics, linear regression analysis accounting for age, sex, and diffusing capacity of the lungs for carbon monoxide percentage predicted was used. Autoantibodies and soluble serum factor levels were compared using two-sample t test with equal variances. Results: No statistically significant differences were observed in quantitative or qualitative nailfold capillaroscopy microscopy characteristics, or vascular autoantibody ETAR and AT1R titer between systemic sclerosis-pulmonary hypertension and systemic sclerosis-no pulmonary hypertension. In contrast, several serum levels of soluble factors differed between groups: Endostatin, sVCAM, and VEGFD were increased, and CXCL4, sVEGFR2, and PDGF-AB/BB were decreased in systemic sclerosis-pulmonary hypertension. Random forest classification identified Endostatin and CXCL4 as the most predictive classifiers to distinguish systemic sclerosispulmonary hypertension from systemic sclerosis-no pulmonary hypertension. Conclusion: This study shows the potential for several soluble serum factors to distinguish systemic sclerosis-pulmonary hypertension from systemic sclerosis-no pulmonary hypertension. We found no classifying potential for qualitative or quantitative nailfold capillaroscopy microscopy characteristics, or vascular autoantibodies.

Molecular mechanisms and treatment responses of pulmonary fibrosis in severe COVID-19.

BACKGROUND: Coronavirus disease 2019 (COVID-19) patients can develop pulmonary fibrosis (PF), which is associated with impaired outcome. We assessed specific leukocytic transcriptome profiles associated with PF and the influence of early dexamethasone (DEXA) treatment on the clinical course of PF in critically ill COVID-19 patients. METHODS: We performed a pre-post design study in 191 COVID-19 patients admitted to the Intensive Care Unit (ICU) spanning two treatment cohorts: the pre-DEXA- (n = 67) and the DEXA-cohort (n = 124). PF was identified based on radiological findings, worsening of ventilatory parameters and elevated circulating PIIINP levels. Longitudinal transcriptome profiles of 52 pre-DEXA patients were determined using RNA sequencing. Effects of prednisone treatment on clinical fibrosis parameters and outcomes were analyzed between PF- and no-PF-patients within both cohorts. RESULTS: Transcriptome analyses revealed upregulation of inflammatory, coagulation and neutrophil extracellular trap-related pathways in PF-patients compared to no-PF patients. Key genes involved included PADI4, PDE4D, MMP8, CRISP3, and BCL2L15. Enrichment of several identified pathways was associated with impaired survival in a external cohort of patients with idiopathic pulmonary fibrosis. Following prednisone treatment, PF-related profiles reverted towards those observed in the no-PF-group. Likewise, PIIINP levels decreased significantly following prednisone treatment. PF incidence was 28% and 25% in the pre-DEXA- and DEXA-cohort, respectively (p = 0.61). ICU length-of-stay (pre-DEXA: 42 [29-49] vs. 18 [13-27] days, p < 0.001; DEXA: 42 [28-57] vs. 13 [7-24] days, p < 0.001) and mortality (pre-DEXA: 47% vs. 15%, p = 0.009; DEXA: 61% vs. 19%, p < 0.001) were higher in the PF-groups compared to the no-PF-groups within both cohorts. Early dexamethasone therapy did not influence these outcomes. CONCLUSIONS: ICU patients with COVID-19 who develop PF exhibit upregulated coagulation, inflammation, and neutrophil extracellular trap-related pathways as well as prolonged ICU length-of-stay and mortality. This study indicates that early dexamethasone treatment neither influences the incidence or clinical course of PF, nor clinical outcomes.

Validation of non-invasive point of care blood content analysis using the TensorTip™ MTX device: a method comparison study.

OBJECTIVES: The TensorTip™ MTX is a non-invasive device designed to determine several physiological parameters with additional analysis of haemoglobin, haematocrit and blood gas analysis by interpreting blood diffusion colour of the finger skin based on spectral analysis. The aim of our study was to investigate the accuracy and precision of the TensorTip MTX in a clinical setting in comparison with routine analysis of blood samples. METHODS: Forty-six patients, scheduled for elective surgery, were enrolled in this study. Placement of an arterial catheter had to be part of the standard of care. Measurements were performed during the perioperative period. The measurements obtained with the TensorTip MTX were compared with the results of routine analysis of the blood samples as a reference using correlation, Bland-Altman analysis and mountain plots. RESULTS: No significant correlation was present in the measurements. Measurement of haemoglobin with the TensorTip MTX had a mean bias of 0.4 mmol/L, haematocrit's bias was 3.0 %. Bias of partial pressure of carbon dioxide and oxygen was 3.6 and 66.6 mmHg, respectively. Calculated percentage errors were 48.2 , 48.9, 39.9 and 109.0 %. Proportional bias was present in all Bland-Altman analyses. Less than 95 % of the differences fell within the pre-set limits of allowable error. CONCLUSIONS: Non-invasive blood content analysis with the TensorTip MTX device is not equivalent to and did not correlate sufficiently with conventional laboratory analysis. None of the parameters measured showed results within the limits of allowable error. Therefore, the use of the TensorTip MTX is not recommended for perioperative care.

Evidence-based rationale for low dose nivolumab in critically ill patients with sepsis-induced immunosuppression.

A substantial part of critically ill patients suffer from sepsis-induced immunosuppression. Reversal of immunosuppression through PD-1 checkpoint inhibition has been proposed as a treatment strategy to overcome immunosuppression in these patients. The PD-1 inhibitor nivolumab, currently used in treatment of cancer, has been evaluated in phase I/II studies in patients with sepsis, demonstrating tolerability and signs of clinical efficacy. No proper dose finding was performed in these studies and, after a single high dose of 480 mg or 960 mg nivolumab, PD-1 inhibition persisted beyond 90 days in the majority of cases. As the duration of sepsis is ~7-10 days, prolonged PD-1 inhibition may unnecessarily induce longer-term immune-related side effects. Based on previously published pharmacokinetic and pharmacodynamic data of nivolumab, a thorough in silico dose finding study for nivolumab in critically ill patients was performed. We found that volume of distribution and clearance of nivolumab were not higher in patients with sepsis compared to the cancer population for which nivolumab is currently approved and showed profound variability. We found that with a single dose of 20 mg nivolumab, the PD-1 receptor occupancy is predicted to stay above the 90% threshold for a median of 23 days (90% prediction interval of 7-78 days). We propose to investigate this dose in critically ill patients as a potential safe and cost-effective pharmacotherapeutic intervention to treat sepsis-induced immunosuppression.

Trio-based whole exome sequencing in patients with suspected sporadic inborn errors of immunity: A retrospective cohort study.

Background: De novo variants (DNVs) are currently not routinely evaluated as part of diagnostic whole exome sequencing (WES) analysis in patients with suspected inborn errors of immunity (IEI). Methods: This study explored the potential added value of systematic assessment of DNVs in a retrospective cohort of 123 patients with a suspected sporadic IEI that underwent patient-parent trio-based WES. Results: A (likely) molecular diagnosis for (part) of the immunological phenotype was achieved in 12 patients with the diagnostic in silico IEI WES gene panel. Systematic evaluation of rare, non-synonymous DNVs in coding or splice site regions led to the identification of 14 candidate DNVs in genes with an annotated immune function. DNVs were found in IEI genes (NLRP3 and RELA) and in potentially novel candidate genes, including PSMB10, DDX1, KMT2C, and FBXW11. The FBXW11 canonical splice site DNV was shown to lead to defective RNA splicing, increased NF-κB p65 signalling, and elevated IL-1ß production in primary immune cells extracted from the patient with autoinflammatory disease. Conclusions: Our findings in this retrospective cohort study advocate the implementation of trio-based sequencing in routine diagnostics of patients with sporadic IEI. Furthermore, we provide functional evidence supporting a causal role for FBXW11 loss-of-function mutations in autoinflammatory disease. Funding: This research was supported by grants from the European Union, ZonMW and the Radboud Institute for Molecular Life Sciences.

Autoantibodies in the disease criteria for systemic sclerosis: The need for specification for optimal application.

The ACR/EULAR classification criteria for systemic sclerosis (SSc) entail three autoantibodies: anti-centromere antibodies (ACA), anti-topoisomerase I antibodies (ATA), and anti-RNA-polymerase III antibodies (ARA). The importance of ACA and ATA in the classification criteria is evidence based, but the diagnostic value is overestimated by clinicians. Fortunately, these autoantibodies are characterized by good agreement between different immuno-assays. Inclusion of ARA, however, is based on limited evidence and is related to limited agreement between different immuno-assays. Harmonization of immuno-assays in terms of interpretation based on likelihood ratio's may improve future classification criteria for SSc and this needs to be achieved by close collaboration between clinicians, laboratory specialists and the diagnostic industry.

Blood-Based Immune Profiling Combined with Machine Learning Discriminates Psoriatic Arthritis from Psoriasis Patients.

Psoriasis (Pso) is a chronic inflammatory skin disease, and up to 30% of Pso patients develop psoriatic arthritis (PsA), which can lead to irreversible joint damage. Early detection of PsA in Pso patients is crucial for timely treatment but difficult for dermatologists to implement. We, therefore, aimed to find disease-specific immune profiles, discriminating Pso from PsA patients, possibly facilitating the correct identification of Pso patients in need of referral to a rheumatology clinic. The phenotypes of peripheral blood immune cells of consecutive Pso and PsA patients were analyzed, and disease-specific immune profiles were identified via a machine learning approach. This approach resulted in a random forest classification model capable of distinguishing PsA from Pso (mean AUC = 0.95). Key PsA-classifying cell subsets selected included increased proportions of differentiated CD4+CD196+CD183-CD194+ and CD4+CD196-CD183-CD194+ T-cells and reduced proportions of CD196+ and CD197+ monocytes, memory CD4+ and CD8+ T-cell subsets and CD4+ regulatory T-cells. Within PsA, joint scores showed an association with memory CD8+CD45RA-CD197- effector T-cells and CD197+ monocytes. To conclude, through the integration of in-depth flow cytometry and machine learning, we identified an immune cell profile discriminating PsA from Pso. This immune profile may aid in timely diagnosing PsA in Pso.

Autoantibody profiles in systemic sclerosis; a comparison of diagnostic tests.

OBJECTIVES: Autoimmune antibody profiling plays a prominent role in both classification and prognosis of systemic sclerosis (SSc). In the last years novel autoantibodies have been discovered and have become available in diagnostic assays. However, standardization in autoimmune serology is lacking, which may have a negative impact on the added value of autoantibodies in diagnosis and prognosis of SSc. In this paper we describe the comparison of commercially available diagnostic assays for the detection of SSc-associated autoantibodies and explored the coexistence of multiple SSc-associated autoantibodies within patients. METHODS: Serum samples of 347 patients from the Nijmegen Systemic Sclerosis Cohort were included in this study. All patients fulfilled the ACR/EULAR 2013 classification criteria for SSc and were classified as DcSSc or LcSSc according to the Leroy and Medsger criteria. All samples were evaluated on standard laboratory diagnostic tests for detection of SSc-specific autoantibodies CENPA and CENPB (ACA), Scl-70 (ATA), RNA Polymerase III (rp11/155) (ARA), and SSc-associated autoantibodies Fibrillarin, Th-To, PM-scl75, PM-Scl100, RNP68/A/C, Ku, NOR90, and PDGFR from suppliers EUROIMMUN, D-tek and Thermo Fisher Scientific. RESULTS: We found that 79% of the patients was positive for one or more of the SSc autoantibodies. Overall, a high agreement was observed between the diagnostic methods for the SSC-specific autoantibodies listed in the ACR/EULAR criteria (ATA, ACA, and ARA) (Cohen's kappa 0.53-0.97). However, a lower agreement was found for SSc-associated autoantibodies PM-Scl, and Ku, as well as for the SSc-specific autoantibodies fibrillarin and Th-To. Furthermore, the data revealed that the presence of ATA, ARA and ACA is predominantly mutually exclusive, with only a fraction of the patients testing positive for both ATA and ARA. CONCLUSION: Our data showed high concordance of prevalent SSc-specific autoantibodies between different diagnostic assays. Further standardisation for low prevalent SSc-specific and SSc-associated autoantibodies is needed.

Dysregulated Innate and Adaptive Immune Responses Discriminate Disease Severity in COVID-19.

The clinical spectrum of COVID-19 varies and the differences in host response characterizing this variation have not been fully elucidated. COVID-19 disease severity correlates with an excessive proinflammatory immune response and profound lymphopenia. Inflammatory responses according to disease severity were explored by plasma cytokine measurements and proteomics analysis in 147 COVID-19 patients. Furthermore, peripheral blood mononuclear cell cytokine production assays and whole blood flow cytometry were performed. Results confirm a hyperinflammatory innate immune state, while highlighting hepatocyte growth factor and stem cell factor as potential biomarkers for disease severity. Clustering analysis revealed no specific inflammatory endotypes in COVID-19 patients. Functional assays revealed abrogated adaptive cytokine production (interferon-γ, interleukin-17, and interleukin-22) and prominent T-cell exhaustion in critically ill patients, whereas innate immune responses were intact or hyperresponsive. Collectively, this extensive analysis provides a comprehensive insight into the pathobiology of severe to critical COVID-19 and highlights potential biomarkers of disease severity.

Diagnostic profiles for precision medicine in systemic sclerosis; stepping forward from single biomarkers towards pathophysiological panels.

Systemic sclerosis (SSc) is an autoimmune disease which is characterized by vasculopathy, tissue fibrosis and activation of the innate and adaptive immune system. Clinical features of the disease consists of skin thickening and internal organ involvement. Due to the heterogeneous nature of the disease it is difficult to predict disease progression and complications. Despite the discovery of novel autoantibodies associated with SSc, there is an unmet need for biomarkers for diagnosis, disease progression and response to treatment. To date, the use of single (surrogate) biomarkers for these purposes has been unsuccessful. Combining multiple biomarkers in to predictive panels or ultimately algorithms could be more precise. Given the limited therapeutic options and poor prognosis of many SSc patients, a better understanding of the immune-pathofysiological profiles might aid to an adjusted therapeutic approach. Therefore, we set out to explore immunological fingerprints in various clinically defined forms of SSc. We used multilayer profiling to identify unique immune profiles underlying distinct autoantibody signatures. These immune profiles could fill the unmet need for prognosis and response to therapy in SSc. Here, we present 3 pathophysiological fingerprints in SSc based on the expression of circulating antibodies, vascular markers and immunomodulatory mediators.

TNFα-Signaling Modulates the Kinase Activity of Human Effector Treg and Regulates IL-17A Expression.

Maintenance of regulatory T cells CD4+CD25highFOXP3+ (Treg) stability is vital for proper Treg function and controlling the immune equilibrium. Treg cells are heterogeneous and can reveal plasticity, exemplified by their potential to express IL-17A. TNFα-TNFR2 signaling controls IL-17A expression in conventional T cells via the anti-inflammatory ubiquitin-editing and kinase activity regulating enzyme TNFAIP3/A20 (tumor necrosis factor-alpha-induced protein 3). To obtain a molecular understanding of TNFα signaling on IL-17 expression in the human effector (effTreg, CD25highCD45RA-) Treg subset, we here studied the kinome activity regulation by TNFα signaling. Using FACS-sorted naïve (naïveTreg, CD25highCD45RA+) and effTreg subsets, we demonstrated a reciprocal relationship between TNFα and IL-17A expression; effTreg (TNFαlow/IL-17Ahigh) and naïveTreg (TNFαhigh/IL-17Alow). In effTreg, TNFα-TNFR2 signaling prevented IL-17A expression, whereas inhibition of TNFα signaling by clinically applied anti-TNF antibodies led to increased IL-17A expression. Inhibition of TNFα signaling led to reduced TNFAIP3 expression, which, by using siRNA inhibition of TNFAIP3, appeared causally linked to increased IL-17A expression in effTreg. Kinome activity screening of CD3/CD28-activated effTreg revealed that anti-TNF-mediated neutralization led to increased kinase activity. STRING association analysis revealed that the TNF suppression effTreg kinase activity network was strongly associated with kinases involved in TCR, JAK, MAPK, and PKC pathway signaling. Small-molecule-based inhibition of TCR and JAK pathways prevented the IL-17 expression in effTreg. Together, these findings stress the importance of TNF-TNFR2 in regulating the kinase architecture of antigen-activated effTreg and controlling IL-17 expression of the human Treg. These findings might be relevant for optimizing anti-TNF-based therapy and may aid in preventing Treg plasticity in case of Treg-based cell therapy.

Single CD28 stimulation induces stable and polyclonal expansion of human regulatory T cells.

CD4+FOXP3+ Treg are essential for immune tolerance. Phase-1 clinical trials of Treg-therapy to treat graft-versus-host-disease reported safety and potential therapeutic efficacy. Treg-based trials have started in organ-transplant patients. However, efficient ex vivo expansion of a stable Treg population remains a challenge and exploring novel ways for Treg expansion is a pre-requisite for successful immunotherapy. Based on the recent finding that CD28-signaling is crucial for survival and proliferation of mouse Treg, we studied single-CD28 stimulation of human Treg, without T cell receptor stimulation. Single-CD28 stimulation of human Treg in the presence of recombinant human IL-2(rhIL-2), as compared to CD3/CD28/rhIL-2 stimulation, led to higher expression levels of FOXP3. Although the single-CD28 expanded Treg population was equally suppressive to CD3/CD28 expanded Treg, pro-inflammatory cytokine (IL-17A/IFNγ) production was strongly inhibited, indicating that single-CD28 stimulation promotes Treg stability. As single-CD28 stimulation led to limited expansion rates, we examined a CD28-superagonist antibody and demonstrate a significant increased Treg expansion that was more efficient than standard anti-CD3/CD28-bead stimulation. CD28-superagonist stimulation drove both naïve and memory Treg proliferation. CD28-superagonist induction of stable Treg appeared both PI3K and mTOR dependent. Regarding efficient and stable expansion of Treg for adoptive Treg-based immunotherapy, application of CD28-superagonist stimulation is of interest.

Targeting PKC in human T cells using sotrastaurin (AEB071) preserves regulatory T cells and prevents IL-17 production.

Regulatory T-cells (Treg) are crucial for immune homeostasis and prevention of immune pathology. Yet, Treg may lose Foxp3 and start secreting IL-17, dependent on environmental cues. Our previous data revealed that Treg from severe psoriasis patients are particularly prone to such conversion. The question of how to maintain Treg stability in the context of inflammation awaits immediate resolution. The pan-protein kinase C (PKC) inhibitor sotrastaurin has shown efficacy in clinical trials of psoriasis. Here, we show that sotrastaurin inhibited effector T-cell responses, whereas the regulatory response was enhanced. Sotrastaurin prevented TCR/CD28-induced T-cell activation and pro-inflammatory cytokine production, but preserved a stable Treg phenotype as evidenced by maintenance of suppressive capacity, high Foxp3 and CD25 expression, and lack of IL-17A and IFNγ production. Moreover, in both circulating and dermal psoriatic Treg, prone to rapid induction of IL-17, sotrastaurin enhanced Foxp3 expression and prevented IL-17A and IFNγ production even when stimulated in the presence of the helper T 17-enhancing cytokines IL-1ß or IL-23. Thus, pharmacological inhibition of PKC may serve as a powerful tool to concurrently inhibit effector T cells and to facilitate Treg, thereby showing therapeutic potential for the treatment of psoriasis.

Smartphone-based analysis of biochemical tests for health monitoring support at home.

In the context of home-based healthcare monitoring systems, it is desirable that the results obtained from biochemical tests - tests of various body fluids such as blood and urine - are objective and automatically generated to reduce the number of man-made errors. The authors present the StripTest reader - an innovative smartphone-based interpreter of biochemical tests based on paper-based strip colour using image processing techniques. The working principles of the reader include image acquisition of the colour strip pads using the camera phone, analysing the images within the phone and comparing them with reference colours provided by the manufacturer to obtain the test result. The detection of kidney damage was used as a scenario to illustrate the application of, and test, the StripTest reader. An extensive evaluation using laboratory and human urine samples demonstrates the reader's accuracy and precision of detection, indicating the successful development of a cheap, mobile and smart reader for home-monitoring of kidney functioning, which can facilitate the early detection of health problems and a timely treatment intervention.

Org 214007-0: a novel non-steroidal selective glucocorticoid receptor modulator with full anti-inflammatory properties and improved therapeutic index.

Glucocorticoids (GCs) such as prednisolone are potent immunosuppressive drugs but suffer from severe adverse effects, including the induction of insulin resistance. Therefore, development of so-called Selective Glucocorticoid Receptor Modulators (SGRM) is highly desirable. Here we describe a non-steroidal Glucocorticoid Receptor (GR)-selective compound (Org 214007-0) with a binding affinity to GR similar to that of prednisolone. Structural modelling of the GR-Org 214007-0 binding site shows disturbance of the loop between helix 11 and helix 12 of GR, confirmed by partial recruitment of the TIF2-3 peptide. Using various cell lines and primary human cells, we show here that Org 214007-0 acts as a partial GC agonist, since it repressed inflammatory genes and was less effective in induction of metabolic genes. More importantly, in vivo studies in mice indicated that Org 214007-0 retained full efficacy in acute inflammation models as well as in a chronic collagen-induced arthritis (CIA) model. Gene expression profiling of muscle tissue derived from arthritic mice showed a partial activity of Org 214007-0 at an equi-efficacious dosage of prednisolone, with an increased ratio in repression versus induction of genes. Finally, in mice Org 214007-0 did not induce elevated fasting glucose nor the shift in glucose/glycogen balance in the liver seen with an equi-efficacious dose of prednisolone. All together, our data demonstrate that Org 214007-0 is a novel SGRMs with an improved therapeutic index compared to prednisolone. This class of SGRMs can contribute to effective anti-inflammatory therapy with a lower risk for metabolic side effects.

Molecular pathway profiling of T lymphocyte signal transduction pathways; Th1 and Th2 genomic fingerprints are defined by TCR and CD28-mediated signaling.

BACKGROUND: T lymphocytes are orchestrators of adaptive immunity. Naïve T cells may differentiate into Th1, Th2, Th17 or iTreg phenotypes, depending on environmental co-stimulatory signals. To identify genes and pathways involved in differentiation of Jurkat T cells towards Th1 and Th2 subtypes we performed comprehensive transcriptome analyses of Jurkat T cells stimulated with various stimuli and pathway inhibitors. Results from these experiments were validated in a human experimental setting using whole blood and purified CD4+ Tcells. RESULTS: Calcium-dependent activation of T cells using CD3/CD28 and PMA/CD3 stimulation induced a Th1 expression profile reflected by increased expression of T-bet, RUNX3, IL-2, and IFNγ, whereas calcium-independent activation via PMA/CD28 induced a Th2 expression profile which included GATA3, RXRA, CCL1 and Itk. Knock down with siRNA and gene expression profiling in the presence of selective kinase inhibitors showed that proximal kinases Lck and PKCθ are crucial signaling hubs during T helper cell activation, revealing a clear role for Lck in Th1 development and for PKCθ in both Th1 and Th2 development. Medial signaling via MAPkinases appeared to be less important in these pathways, since specific inhibitors of these kinases displayed a minor effect on gene expression. Translation towards a primary, whole blood setting and purified human CD4+ T cells revealed that PMA/CD3 stimulation induced a more pronounced Th1 specific, Lck and PKCθ dependent IFNγ production, whereas PMA/CD28 induced Th2 specific IL-5 and IL-13 production, independent of Lck activation. PMA/CD3-mediated skewing towards a Th1 phenotype was also reflected in mRNA expression of the master transcription factor Tbet, whereas PMA/CD28-mediated stimulation enhanced GATA3 mRNA expression in primary human CD4+ Tcells. CONCLUSIONS: This study identifies stimulatory pathways and gene expression profiles for in vitro skewing of T helper cell activation. PMA/CD3 stimulation enhances a Th1-like response in an Lck and PKCθ dependent fashion, whereas PMA/CD28 stimulation results in a Th2-like phenotype independent of the proximal TCR-tyrosine kinase Lck. This approach offers a robust and fast translational in vitro system for skewed T helper cell responses in Jurkat T cells, primary human CD4+ Tcells and in a more complex matrix such as human whole blood.

Increased expression of interleukin-22 by synovial Th17 cells during late stages of murine experimental arthritis is controlled by interleukin-1 and enhances bone degradation.

OBJECTIVE: Interleukin-22 (IL-22) is a mediator in antimicrobial responses and inflammatory autoimmune diseases. Although IL-22 and its receptor, IL-22R, have been identified in the synovium of rheumatoid arthritis patients, the source of IL-22 and its contribution to disease pathogenicity remain to be established. This study was undertaken to investigate the regulation of IL-22 by Th17 cells in vitro and to evaluate the potential for IL-22 depletion in an experimental arthritis model using mice deficient in the IL-1 receptor antagonist (IL-1Ra-/-). METHODS: Naive murine T cells were cultured under conditions leading to polarization of the cells into subsets of Th1, Th2, induced Treg, and Th17. Cytokines were measured in the culture supernatants, and the cells were analyzed by fluorescence-activated cell sorting. Tissue samples from the inflamed ankle synovium of IL-1Ra-/- mice were isolated, and messenger RNA levels of marker genes were quantified. IL-1Ra-/- mice were treated with neutralizing anti-IL-22 antibodies. Synovial cells were isolated from the inflamed tissue and sorted into fractions for analysis of cytokine production. RESULTS: In vitro tests showed that Th17 cells produced high levels of IL-22 after stimulation with IL-1 or IL-23. Interestingly, a synergistic increase in the production of IL-22 was observed after combining IL-1 and IL-23. In vivo, IL-1Ra-/- mice displayed a progressive erosive arthritis, characterized by up-regulation of IL-17 in mildly and severely inflamed tissue, whereas the levels of IL-22 and IL-22R were increased only in severely inflamed synovia. Anti-IL-22 treatment of IL-1Ra-/- mice significantly reduced the inflammation and bone erosion. Analysis of isolated single cells from the inflamed synovia revealed that IL-22 was mainly produced by IL-17-expressing T cells. CONCLUSION: These findings suggest that IL-22 plays an important role in IL-1-driven chronic joint destruction.

Splenic suppressor of cytokine signaling 3 transgene expression affects T cell responses and prevents development of collagen-induced arthritis.

OBJECTIVE: Members of the suppressor of cytokine signaling (SOCS) family are key negative intracellular regulators of cytokine and growth factor responses, including those that regulate immune responses in autoimmune disorders, such as rheumatoid arthritis (RA). The aim of this study was to investigate modulation of T cell immunity for the treatment of experimental arthritis, via enhanced expression of SOCS-3 in splenic antigen-presenting cells (APCs) obtained after intravenous injection of adenovirus encoding SOCS-3. METHODS: DBA/1 mice were immunized with type II collagen, and adenovirus vectors were administered by intravenous injection before the clinical onset of collagen-induced arthritis (CIA). Splenic cellular responses were analyzed by measuring cytokine production, using Luminex multi-analyte technology. Th cell populations were analyzed by flow cytometry. RESULTS: Systemic delivery of adenovirus encoding SOCS-3 resulted in enhanced transgene expression in splenic APCs, which led to decreased production of interleukin-23 (IL-23), IL-6, and tumor necrosis factor alpha, but significantly higher production of antiinflammatory IL-10, by these cells. Fluorescence-activated cell sorting analysis showed increased numbers of splenic CD4+ T cells after SOCS-3 treatment. In the presence of SOCS-3-transduced APCs, however, purified splenic CD3+ T cells showed reduced antigen-specific proliferation and a significant reduction in the production of interferon-gamma (-43%), IL-4 (-41%), and IL-17 (-70%). Interestingly, the altered splenic cellular responses were accompanied by a protective effect on CIA development, and histologic analysis of knee joints showed reduced joint inflammation and connective tissue destruction. CONCLUSION: This study demonstrates effective prevention of CIA after intravenously induced overexpression of SOCS-3; this is probably caused by the generation of tolerogenic APCs, which have an inhibitory effect on Th1, Th2, and especially, Th17 cell activity.