Detailed Long-term Dynamics of Neutrophil-to-Lymphocyte Ratio under Biologic Treatment Reveals Differential Effects of Tumour Necrosis Factor-alpha and Interleukin 12/23 Antagonists.

Psoriasis is thought to be associated with a reduced life expectancy through systemic inflammation. A comparative, retrospective analysis of neutrophil-to-lympho-cyte ratio, a biomarker of systemic inflammation and cardiovascular risk, under 196 treatments with tumour necrosis factor-α and interleukin-12/23 antagonists was performed. Neutrophil-to-lympho-cyte ratio decreased significantly within 3 months of initiation of treatment and remained stable at reduced levels for at least 33 months. Dynamics were more pronounced and neutrophil-to-lympho-cyte ratio under treatment was lower in patients treated with tumour necrosis factor-α compared with interleukin-12/23 antagonists (geometric mean (95% confidence interval): 2.03 (1.9, 2.1) vs 2.63 (2.2, 3.2), respectively, p = 0.014). tumour necrosis factor-α antagonist treatment and baseline neutrophil-to-lympho-cyte ratio were independent predictors of a median low cardiovascular risk neutrophil-to-lympho-cyte ratio (< 2.15) during treatment (odds ratio (95% confidence interval): 0.53 (0.4-0.8) and 4.68 (1.0-19.1), p = 0.001 and p = 0.032, respectively). These results demonstrate a rapid and sustained reduction in biomarkers of systemic inflammation under biologic treatment. Furthermore, these data suggest class-specific effects on systemic inflammation, which may be relevant for the prevention of psoriasis co-morbidity by systemic treatment.

Evaluation of Psoriasis Area and Severity Index as a Proxy for Bio-markers of Systemic Disease under Treatment with Tumour Necrosis Factor-alpha and Interleukin 12/23 Antagonists in Patients with Psoriasis: A Retrospective Cohort Study of 186 Treatment Cycles.

The efficacy of psoriasis treatments is usually evaluated using the Psoriasis Area and Severity Index (PASI). However, there is a lack of systematic statistical assessments of PASI as a proxy for systemic disease in individual patients. Therefore, a retrospective study of 186 treat-ments with adalimumab, etanercept, and ustekinumab for psoriasis (341 patient-years) was performed. While PASI significantly and independently correlated with biomarkers of systemic inflammation (especially neutrophil-to-lymphocyte ratio, C-reactive protein), the strengths were only weak-to-moderate and varied considerably inter-individually. A decrease in PASI indicated a neutrophil-to-lymphocyte ratio decrease and a C-reactive protein decrease or stable low margin C-reactive protein in ≥ 80%. Sensitivity, specificity, and positive predictive value of PASI 0 and PASI 2.75 (optimal Youden Index) for low cardiovascular risk C-reactive protein were 24%, 92%, 85%, and 62%, 61%, 76%, respectively. Performance was similar using absolute thresholds and PASI 100 or PASI 75, and overall worse for low cardiovascular risk neutrophil-to-lympho-cyte ratio and if psoriasis arthritis was present. In conclusion, PASI allows robust low-order estimates of systemic inflammation, but cannot substitute for laboratory biomarkers for more precise assessments.

Regulatory T-cell deficiency leads to pathogenic bullous pemphigoid antigen 230 autoantibody and autoimmune bullous disease.

BACKGROUND: Autoimmune bullous diseases/dermatoses (AIBDs) are severe autoantibody-mediated skin diseases. The pathogenic relevance of autoreactive CD4+ T cells for the induction of autoantibody production remains to be fully evaluated. Scurfy mice lack functional regulatory T (Treg) cells, experience spontaneous activation of autoreactive CD4+ T cells, and display severe erosive skin lesions suggestive of AIBDs. OBJECTIVE: We sought to determine whether AIBDs develop in Treg cell-deficient scurfy mice. METHODS: Histology, indirect immunofluorescence (IF) microscopy, direct IF, and ELISA were used to prove the presence of AIBDs in scurfy mice. Monoclonal autoantibodies from sera of scurfy mice were screened by using indirect IF on murine skin, and immunoprecipitation and mass spectrometry were used for target antigen identification, followed by confirmation in modified human embryonic kidney cells and murine keratinocytes. Pathogenicity was determined by injecting the autoantibody into neonatal mice and transferring scurfy CD4+ T cells into nu/nu mice. RESULTS: Autoantibodies against different known autoantigens of AIBDs spontaneously develop in scurfy mice. Histology reveals subepidermal blisters, and direct IF of skin of scurfy mice shows a predominant linear staining pattern. The mAb 20B12 shows a linear staining pattern in indirect IF, recognizes the murine hemidesmosomal protein bullous pemphigoid antigen 230 (BP230) as the target antigen, and cross-reacts with human BP230. Purified mAb 20B12 induces subepidermal blisters in neonatal mice. Transfer of scurfy CD4+ T cells is sufficient to induce antibodies with reactivity to AIBD autoantigens and subepidermal blisters in the skin of recipient T cell-deficient nu/nu mice. CONCLUSION: We show that the absence of Treg cells leads to AIBDs by pathogenic autoantibodies targeting BP230.

Routine Laboratory Parameter Dynamics and Laboratory Adverse Events in Psoriasis Patients on Long-term Treatment with Adalimumab, Etanercept, and Ustekinumab.

Only limited data on laboratory parameter dynamics and safety under prolonged biologic treatment in a "real-world" scenario are available for recommendations on screening and monitoring. This study is a retrospective analysis of routine parameter dynamics and laboratory adverse events (LAE) in psoriasis patients on long-term treatment (n = 199) with tumour necrosis factor (TNF)-α-antagonists (adalimumab, etanercept), and the interleukin (IL)12/23-antagonist ustekinumab. Overall, neutrophil (PMN) counts (-11%) and triglycerides (+9%) changed considerably. TNF-α-antagonists and ustekinumab differentially affected lymphocyte counts (+13% and ±0%, respectively). Dynamics were pronounced during the first 180 days of treatment. In 340 treatment-years, 15 Common Terminology Criteria for Adverse Events (CTCAE) III-IV° LAE were recorded (11 involved liver enzymes). They prompted alteration of the biologic regime in only 2 cases. Age, sex, previous systemic treatments, and psoriatic arthritis did not significantly predict LAE. Liver enzyme and triglyceride screening may be warranted in some instances. Our data suggest that unguided monitoring of other routine laboratory parameters is unnecessary under long-term biologic treatment.

Interindividual variation of NETosis in healthy donors: introduction and application of a refined method for extracellular trap quantification.

Neutrophil extracellular trap (NET) formation is a mechanism of innate immune defence by which neutrophil (polymorphonuclear) granulocytes (PMN) produce net-like structures of decondensed chromatin decorated with antimicrobial peptides for trapping and possibly killing microorganisms. If this process leads to cell death, it is termed NETosis. Alterations of this particular mechanism have been reported to be involved in the pathogenesis of chronic inflammatory diseases including psoriasis and lupus erythematosus. Still, quantification of NETosis poses a considerable challenge. We report and test a refined protocol for morphological NET quantification in healthy human donors that encompasses isolation, stimulation, DNA staining, live imaging and semi-automated offline analysis. The results were highly reproducible and in good agreement with manual counting. The average intra-donor coefficient of variation of NETosis rates to phorbol myristate acetate (PMA) stimulation was low compared to the respective interdonor coefficient of variation (10% vs 82%, n=4, respectively, if experiments were repeated on the same day, and 38% vs 74%, n=6, respectively, if experiments were repeated on average 42±34 days apart). Overall, the interdonor coefficient of variation was 67% (n=10). These findings altogether support the existence of a distinct predisposition of PMN from different donors for undergoing NETosis. Picogreen fluorescence correlated stronger to cell death than to morphological NETosis (r2 =.89, P<.001, n=8, and r2 =.68, P=.012, n=8, respectively). This indicates that cytotoxicity may confound Picogreen fluorescence. Our results and the related protocol may help investigators with the quantification of NETosis and the design of respective basic and translational research studies.

Correlation of autoantibodies against BP180/BP230 in response to topical corticosteroids in patients with bullous pemphigoid.

Topical steroids are effective in treating bullous pemphigoid (BP). Autoantibodies against BP180 are related to disease activity, but correlation of these autoantibodies with response to topical steroid therapy has not yet been clearly evaluated. We investigate the usefulness of close and early monitoring of autoantibodies against BP180 and BP230 for assessment of response to therapy and early detection of therapeutic failure in BP patients treated topically. In eight BP patients under treatment with topical or systemic steroid therapy we retrospectively evaluated clinical course and autoantibodies against BP180 and BP230 as well as indirect immunofluorescence titres (IIF). Data were included at diagnosis, during hospitalization and follow-ups. Autoantibodies against BP180 parallel disease activity in all topically and as well as systemically treated patients. Autoantibodies against BP230 correlated in five out of eight patients. Autoantibodies directed against BP180 and, to a lesser degree, against BP230 correlate with the clinical course of topically treated BP patients. Monitoring autoantibodies against BP180 is a useful tool to evaluate the efficacy of topical therapy in BP.

Analysis of high-dose intravenous immunoglobulin therapy in 16 patients with refractory autoimmune blistering skin disease: high efficacy and no serious adverse events.

High-dose intravenous immunoglobulin (IVIG) therapy is used in patients with severe autoimmune blistering diseases that are refractory to standard immunosuppressive therapy. To determine the efficacy and frequency of adverse events of IVIG therapy, we retrospectively analysed data for 16 patients with pemphigus vulgaris, pemphigus foliaceus, paraneoplastic pemphigus, bullous pemphigoid and paraneoplastic bullous pemphigoid. Frequency of adverse reactions and efficacy of IVIG were analysed over time with a scoring system for every 6 months of IVIG therapy. Headache (43.8%) and fatigue (43.8%) were the most common side-effects recorded; serious adverse reactions did not occur. There was good overall efficacy, as measured by clinical response rates using a clinical score, as well as indicated by a mean reduction of 75.8% in the starting steroid dose.

Regulatory T-cell deficiency leads to features of autoimmune liver disease overlap syndrome in scurfy mice.

Introduction: Scurfy mice have a complete deficiency of functional regulatory T cells (Treg) due to a frameshift mutation in the Foxp3 gene. The impaired immune homeostasis results in a lethal lymphoproliferative disorder affecting multiple organs, including the liver. The autoimmune pathology in scurfy mice is in part accompanied by autoantibodies such as antinuclear antibodies (ANA). ANA are serological hallmarks of several autoimmune disorders including autoimmune liver diseases (AILD). However, the underlying pathogenesis and the role of Treg in AILD remain to be elucidated. The present study therefore aimed to characterize the liver disease in scurfy mice. Methods: Sera from scurfy mice were screened for ANA by indirect immunofluorescence assay (IFA) and tested for a wide range of AILD-associated autoantibodies by enzyme-linked immunosorbent assay, line immunoassay, and addressable laser bead immunoassay. CD4+ T cells of scurfy mice were transferred into T cell-deficient B6/nude mice. Monoclonal autoantibodies from scurfy mice and recipient B6/nude mice were tested for ANA by IFA. Liver tissue of scurfy mice was analyzed by conventional histology. Collagen deposition in scurfy liver was quantified via hepatic hydroxyproline content. Real-time quantitative PCR was used to determine fibrosis-related hepatic gene expression. Hepatic immune cells were differentiated by flow cytometry. Results: All scurfy mice produced ANA. AILD-associated autoantibodies, predominantly antimitochondrial antibodies, were detected at significantly higher levels in scurfy sera. CD4+ T cells from scurfy mice were sufficient to induce anti-dsDNA autoantibodies and ANA with an AILD-related nuclear envelope staining pattern. Liver histology revealed portal inflammation with bile duct damage and proliferation, as in primary biliary cholangitis (PBC), and interface hepatitis with portal-parenchymal necroinflammation, as found in autoimmune hepatitis (AIH). In scurfy liver, TNFα and fibrosis-related transcripts including Col1a1, Timp1, Acta2, Mmp2, and Mmp9 were upregulated. The level of proinflammatory monocytic macrophages (Ly-6Chi) was increased, while M2-type macrophages (CD206+) were downregulated compared to wildtype controls. Despite severe hepatic inflammation, fibrosis did not develop within 25 days, which is close to the lifespan of scurfy mice. Discussion: Our findings suggest that Treg-deficient scurfy mice spontaneously develop clinical, serological, and immunopathological characteristics of AILD with overlapping features of PBC and AIH.

Long-term safety of combination treatment with methotrexate and tumor necrosis factor (TNF)-α antagonists versus TNF-α antagonists alone in psoriatic patients.

Methotrexate, a folic acid analog, is the conventional systemic anti-psoriatic agent most commonly chosen for combination with biologics in the treatment of psoriasis. Real-world long-term safety data of this combination versus biologic treatment alone in dermatological practice are sparse. Here, we present results of a comparative retrospective study of laboratory dynamics and adverse events in psoriatic patients receiving a tumor necrosis factor (TNF)-α antagonist (adalimumab or etanercept) with and without concomitant methotrexate (176 treatment courses, mean duration of 629 days). Co-treatment with methotrexate significantly (P < 0.05) correlated with a decrease of leukocyte, neutrophil and erythrocyte counts and an increase of glutamate pyruvate transaminase (GPT) (Pearson correlation, n > 148). The relative risk for a Common Terminology Criteria for Adverse Events (CTCAE) grade 1-2 laboratory adverse event was significantly elevated to 1.11 for anemia and 1.16 for a GPT increase if the patients received concomitant methotrexate at the time the laboratory test was performed. Combination treatment was given for equal or more than 30% of the time (MTX≥30% ) during 12% of the treatment courses. During these treatment courses, dynamics of leukocyte (-8.1%), neutrophil (-8.1%), erythrocyte (-3.2%) counts and GPT (+16.9%) from baseline to average under treatment were significantly more pronounced. CTCAE grade 3-4 laboratory adverse events occurred in 9.5% and 5.2% of treatment courses with and without MTX≥30% , respectively (p = 0.70), and affected transaminases in 90% of the cases. Methotrexate was discontinued due to CTCAE grade 3-4 laboratory adverse events in 4.25% of the treatment courses with MTX of 30% or more. Elevated baseline γ-glutamyl transferase levels significantly predicted the occurrence of CTCAE grade 3-4 laboratory adverse events and should trigger investigations for pre-existing liver disease or alcohol abuse. In conclusion, our comparative data supplement previous short-term studies and support a tolerable long-term safety profile of the combination treatment. However, given the additional toxicities and low evidence for benefits, alternative options such as biologic monotherapy or switching to a different biologic should be considered in a dermatological setting.

Expression of p53 up-regulated modulator of apoptosis (PUMA) in non-melanoma skin cancer of long-term immunosuppressed solid organ transplant recipients compared to immunocompetent patients.

The risk of UV radiation (UVR)-induced non-melanoma skin cancer (NMSC) is dramatically increased in immunosuppressed organ transplant recipients compared to immunocompetent patients. In the skin, p53 up-regulated modulator of apoptosis (PUMA) is a central regulator of apoptosis in response to UVR damage and immune response regulation. Data on the expression of PUMA in patients with NMSC relative to immune status is limited To study differences in the expression and distribution of PUMA in cutaneous SCC and BCC by immunohistochemistry between immunocompetent patients and organ transplant recipients, and the effect of CsA-containing immunosuppressive maintenance regimens on this expression. PUMA expression in SCC (n = 34) and BCC (n = 20) was analysed comparatively by immunohistochemical staining in matched cohorts of 27 immunocompetent patients and 27 organ transplant recipients SCC and BCC showed unequivocal positive PUMA expression, however, there was no significant difference in NMSC between organ transplant recipients and immunocompetent patients. A 17% reduction in staining score for PUMA in SCC, but not in BCC, of organ transplant recipients treated with a cyclosporin (CsA)-containing regimen was noted compared to organ transplant recipients without chronic CsA intake (p = 0.0381) PUMA expression in SCC, but not BCC, is significantly reduced by CsA-containing therapy, suggesting a disturbance of apoptosis by iatrogenic immunosuppression with a divergent impact on SCC and BCC.

NHC-gold compounds mediate immune suppression through induction of AHR-TGFß1 signalling in vitro and in scurfy mice.

Gold compounds have a long history of use as immunosuppressants, but their precise mechanism of action is not completely understood. Using our recently developed liver-on-a-chip platform we now show that gold compounds containing planar N-heterocyclic carbene (NHC) ligands are potent ligands for the aryl hydrocarbon receptor (AHR). Further studies showed that the lead compound (MC3) activates TGFß1 signaling and suppresses CD4+ T-cell activation in vitro, in human and mouse T cells. Conversely, genetic knockdown or chemical inhibition of AHR activity or of TGFß1-SMAD-mediated signaling offsets the MC3-mediated immunosuppression. In scurfy mice, a mouse model of human immunodysregulation polyendocrinopathy enteropathy X-linked syndrome, MC3 treatment reduced autoimmune phenotypes and extended lifespan from 24 to 58 days. Our findings suggest that the immunosuppressive activity of gold compounds can be improved by introducing planar NHC ligands to activate the AHR-associated immunosuppressive pathway, thus expanding their potential clinical application for autoimmune diseases.

Scurfy Mice Develop Features of Connective Tissue Disease Overlap Syndrome and Mixed Connective Tissue Disease in the Absence of Regulatory T Cells.

Due to a missense mutation in the Foxp3 gene, scurfy mice are deficient in functional regulatory T cells (Treg). The consequent loss of peripheral tolerance manifests itself by fatal autoimmune mediated multi-organ disease. Previous studies have outlined the systemic inflammatory disease and demonstrated production of anti-nuclear antibodies (ANA) in scurfy mice. However, specific autoantibody targets remained to be defined. ANA are immunological markers for several connective tissue diseases (CTD) and target a large number of intracellular molecules. Therefore, we examined scurfy sera for the presence of different ANA specificities and further assessed the organ involvement in these animals. Indirect immunofluorescence was used as a screen for ANA in the sera of scurfy mice and dilutions of 1/100 were considered positive. Addressable laser bead immunoassays (ALBIA) were used to detect specific autoantibody targets. Subsequent histological tissue evaluation was verified by hematoxylin and eosin (H&E) staining. In our study, we observed that nearly all scurfy mice produced ANA. The most prevalent pattern in scurfy sera was nuclear coarse speckled, also known as the AC-5 pattern according to the International Consensus on ANA Patterns. U1-ribonucleoprotein (U1RNP) was found to be the most common target antigen recognized by autoantibodies in scurfy mice. Additionally, scurfy mice exhibited a mild myositis with histological characteristics similar to polymyositis/dermatomyositis. Myopathy-specific autoantibody profile revealed significantly increased levels of anti-SMN (survival of motor neuron) as well as anti-Gemin3 antibodies in scurfy sera. Overall, we demonstrate that the impaired peripheral tolerance in the absence of regulatory T cells in scurfy mice is associated with features of mixed connective tissue disease (MCTD). This includes, along with our previous findings, very high titers of anti-U1RNP antibodies and an inflammatory myopathy.

Regulatory T cell deficient scurfy mice exhibit a Th2/M2-like inflammatory response in the skin.

BACKGROUND: Scurfy mice have a functional defect in regulatory T cells (Treg), which leads to lethal multi-organ inflammation. The missing Treg function results in uncontrolled autoimmune cellular and humoral inflammatory responses. We and others have previously shown that during the course of disease scurfy mice develop severe skin inflammation and autoantibodies including anti-nuclear autoantibodies (ANA). OBJECTIVE: Autoimmune skin inflammation and ANA are hallmarks for the diagnosis of autoimmune connective tissue diseases; therefore we analyzed scurfy mice for typical signs of these diseases. METHODS: Indirect immunofluorescence was used to specify the ANA pattern in scurfy mice. Skin fibrosis was assessed by cutaneous collagen accumulation (Goldeners trichrome staining), collagen crosslinking/disorganization (Sirus red polarimetry) and quantitative PCR for fibrosis-related transcripts. The cellular components of the inflammatory infiltrates in scurfy skin were analyzed by flow cytometry and intracellular cytokine staining. RESULTS: The majority of scurfy mice developed ANA with a predominant AC-5 pattern typical for mixed connective tissue disease, especially scleroderma. Scurfy mice showed higher skin collagen content compared to WT controls with a significant tendency in upregulation of TIMP-1. CD3+CD4+ T cells in scurfy skin exhibited a strong Th2 deviation with a significant increase of IL-4, IL-5 and IL-13, and M2-polarized CD11b+MHCII+ macrophages compared to WT mice. CONCLUSION: We show that Scurfy mice show a predominant AC-5 ANA pattern typical for mixed connective tissue disease as in scleroderma. The autoimmune inflammation in scurfy skin mainly consists of CD4+ T cells with Th2 differentiation and alternatively-activated (M2) macrophages as it is found in scleroderma with advanced fibrosis.

Altered expression of keratin 14 in lesional epidermis of autoimmune skin diseases.

BACKGROUND: Keratin 14 (K14) is an intermediate filament protein that is mainly expressed in the basal layer of healthy stratified epithelia. K14 has been identified as an autoantigen in the autoimmune-mediated skin disease of Scurfy mice and patients with the "immune dysregulation polyendocrinopathy, enteropathy, and X-linked" syndrome. OBJECTIVES: To examine whether K14 is a target protein in autoimmune skin diseases (ASD), we analyzed the expression pattern of K14 in lesional skin of patients with lichen ruber, cutaneous lupus erythematosus, dermatomyositis, graft-versus-host disease, psoriasis, and pemphigus vulgaris, and evaluated the reactivity of patient sera with recombinantly expressed and epidermis-derived K14. METHODS: K14 expression was analyzed by immunohistochemistry on paraffin-embedded tissue sections of 17 healthy individuals and 58 patients with ASD. Sera from 10 healthy individuals and 41 patients with ASD were analyzed by Western blot for the presence of anti-K14 autoantibodies. RESULTS: In skin of patients with ASD, K14 expression is retained in suprabasal layers. In ASD with interface dermatitis, we observed focal loss of K14 within the basal layer and in hair follicles as well. A scattered dot-like K14 staining is seen in papillary dermis, most prominently in cutaneous lupus erythematosus and lichen ruber. Using Western blot, we show that sera of different patients with ASD recognize recombinantly expressed K14. CONCLUSION: We show focal loss of K14 in the basal epidermis correlating with interface dermatitis and hair follicle involvement. Moreover, enhanced reactivity of sera of patients with atopic dermatitis with K14 suggests K14 may function as an autoantigen in ASD.