Prapaporn Pisitkun

IL-17 drives psoriatic inflammation via distinct, target cell-specific mechanisms

Significance Psoriasis is an inflammatory disease affecting the skin, a barrier site. The disease is characterized by abnormal growth of keratinocytes and infiltration of inflammatory cells. Clinical trials targeting the IL-17 cytokine have shown remarkable efficacy, and IL-17 also has been strongly implicated in the imiquimod-induced mouse model of psoriasis. However why IL-17 cytokines should be so central is not known, because target cells and their functions have not been clearly delineated. Here we demonstrate that IL-17 signaling into nonkeratinocytes, specifically dermal fibroblasts, induces mediators that further increase IL-17 production by innate γδT cells and promote cellular infiltration, whereas IL-17 signaling into keratinocytes aids proliferation and blocks their differentiation. These findings reveal the circuitry underpinning critical disease-driving effects of IL-17. Psoriasis is a chronic inflammatory skin disease characterized by abnormal keratinocyte proliferation and differentiation and by an influx of inflammatory cells. The mechanisms underlying psoriasis in humans and in mouse models are poorly understood, although evidence strongly points to crucial contributions of IL-17 cytokines, which signal via the obligatory adaptor CIKS/Act1. Here we identify critical roles of CIKS/Act1-mediated signaling in imiquimod-induced psoriatic inflammation, a mouse model that shares features with the human disease. We found that IL-17 cytokines/CIKS-mediated signaling into keratinocytes is essential for neutrophilic microabscess formation and contributes to hyperproliferation and markedly attenuated differentiation of keratinocytes, at least in part via direct effects. In contrast, IL-17 cytokines/CIKS-mediated signaling into nonkeratinocytes, particularly into dermal fibroblasts, promotes cellular infiltration and, importantly, leads to enhanced the accumulation of IL-17–producing γδT cells in skin, comprising a positive feed-forward mechanism. Thus, CIKS-mediated signaling is central in the development of both dermal and epidermal hallmarks of psoriasis, inducing distinct pathologies via target cell-specific effects. CIKS-mediated signaling represents a potential therapeutic target in psoriasis.

Reappraisal of cervical spine subluxation in Thai patients with rheumatoid arthritis

Subluxation of the cervical spine is one of a number of devastating complications of rheumatoid arthritis. In spite of this, the features of cervical spine subluxation in Thai patients with rheumatoid arthritis have never previously been studied. We enrolled 134 patients with rheumatoid arthritis who were being followed at the rheumatology clinic, Ramathibodi Hospital, during 1978–2001. Radiological examinations were made in lateral neck flexion, extension and open-mouth views. Symptoms of neck pain and the results of relevant neurological examinations were recorded at the time of imaging. Other data on clinical features and treatments since diagnosis were reviewed retrospectively. The overall prevalence of cervical spine subluxation was 68.7%, which can be categorised into anterior (26.9%), posterior (14.9%), lateral (17.2%), vertical (16.4%) atlantoaxial and subaxial subluxation (28.4%). The percentages of cervical subluxation in patients who had suffered from the disease for 1, 5, 10 or more than 10 years were 77.8%, 64.9%, 70% and 64.7%, respectively. None of the patients had neurological deficits. No correlation between neck pain and cervical spine subluxation was established. The number of patients treated with corticosteroids was significantly higher in the subluxation group than in the non-subluxation group (p=0.04). However, no difference in duration of treatment and cumulative dosages of steroids was displayed between the two groups. It was concluded that the prevalence of cervical spine subluxation in Thai patients with rheumatoid arthritis is much higher than the average, even in the early phase of the disease. Hence, radiological examination of the cervical spine should be included in the initial evaluation of Thai RA patients. Corticosteroid use was associated with cervical subluxation, regardless of dose and duration of treatment. The possible explanations are that steroids may directly cause ligament laxity, osteoporosis and decreasing muscle mass, which leads to accelerated subluxation, or that steroid treatments are used in more severe cases which have a higher tendency towards cervical subluxation.

Incidence and clinical correlation of anticentromere antibody in Thai patients.

Anticentromere antibodies (ACA) are useful in assessing and classifying patients with mild variant of systemic sclerosis called calcinosis, Raynauds phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias (CREST) syndrome. From their prognostic significance, we are interested in the prevalence and disease correlation in Thai patients. A total of 3,233 serum samples of patients with any musculoskeletal symptoms were sent for antinuclear antibody determination at Ramathibodi Immunology Laboratory Service between the years 1998 and 2001. Forty sera (1.23%) were ACA positive. These sera were from 27 patients with autoimmune diseases and 13 with nonautoimmune diseases. Among autoimmune group, scleroderma was the most common diagnosis (33.3%) with limited sclerosis being the most frequent variant. The percentages of autoimmune disease were almost the same among the low-titer (1:40) and the high-titer (1:640) groups. The study suggests that the prevalence of ACA in Thai patients is low. The presence of ACA detected in patients with vague musculoskeletal symptoms does not suggest a diagnosis of CREST syndrome. Even high-titer ACA can be found in nonautoimmune diseases.

FC Gamma Receptor IIB Deficient Mice: A Lupus Model with Increased Endotoxin Tolerance-Related Sepsis Susceptibility.

ABSTRACT Hyper-elevated immune response of FcGRIIb−/− mice, a lupus model with an inhibitory-signaling defect, can become exhausted (less subsequent immune-response than the first response) with sequential lipopolysaccharide (LPS) stimulation. Endotoxin tolerance-related modifications of inflammatory response were investigated in FcGRIIb−/− mice in both an in vivo sepsis model and in vitro using cultured macrophages. Serum cytokine concentrations, after the second LPS injection (at 5-fold higher levels than the first dose), did not exceed the first dose levels in either FcGRIIb−/− or wild-type mice. These data indicated an endotoxin-tolerance response in both genetic backgrounds. However, the difference of cytokine levels between the first and second LPS injection was more prominent in FcGRIIb−/− mice. More importantly, CLP-induced sepsis after LPS-preconditioning (two separated doses of LPS administration) was more severe in FcGRIIb−/− mice (as measured by mortality rate, bacteria count in blood, serum cytokines, creatinine, and alanine transaminase). An attenuated response was demonstrated after two sequential LPS stimulations of bone-marrow-derived macrophages. Cytokine production was reduced and lower bacterial killing activity occurred with macrophages from FcGRIIb−/− mice relative to wild-type macrophages. Thus, there is a more prominent effect of endotoxin-tolerance in FcGRIIb−/− macrophages relative to wild-type. In conclusion, repeated-LPS administrations induced quantitatively greater endotoxin-tolerance responses in FcGRIIb−/− mice both in vivo and in vitro. Endotoxin-tolerance in vivo was associated with more severe sepsis, at least in part, due to macrophage-dysfunction.

Biomarkers for Refractory Lupus Nephritis: A Microarray Study of Kidney Tissue.

The prognosis of severe lupus nephritis (LN) is very different among individual patients. None of the current biomarkers can be used to predict the development of refractory LN. Because kidney histology is the gold standard for diagnosing LN, the authors hypothesize that molecular signatures detected in kidney biopsy tissue may have predictive value in determining the therapeutic response. Sixty-seven patients with biopsy-proven severely active LN by International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification III/IV were recruited. Twenty-three kidney tissue samples were used for RNA microarray analysis, while the remaining 44 samples were used for validation by real-time polymerase chain reaction (PCR) gene expression analysis. From hundreds of differential gene expressions in refractory LN, 12 candidates were selected for validation based on gene expression levels as well as relevant functions. The candidate biomarkers were members of the innate immune response molecules, adhesion molecules, calcium-binding receptors, and paracellular tight junction proteins. S100A8, ANXA13, CLDN19 and FAM46B were identified as the best kidney biomarkers for refractory LN, and COL8A1 was identified as the best marker for early loss of kidney function. These new molecular markers can be used to predict refractory LN and may eventually lead to novel molecular targets for therapy.

The role of macrophages in the susceptibility of Fc gamma receptor IIb deficient mice to Cryptococcus neoformans

Dysfunctional polymorphisms of FcγRIIb, an inhibitory receptor, are associated with Systemic Lupus Erythaematosus (SLE). Cryptococcosis is an invasive fungal infection in SLE, perhaps due to the de novo immune defect. We investigated cryptococcosis in the FcγRIIb−/− mouse-lupus-model. Mortality, after intravenous C. neoformans-induced cryptococcosis, in young (8-week-old) and older (24-week-old) FcγRIIb−/− mice, was higher than in age-matched wild-types. Severe cryptococcosis in the FcγRIIb−/− mice was demonstrated by high fungal burdens in the internal organs with histological cryptococcoma-like lesions and high levels of TNF-α and IL-6, but not IL-10. Interestingly, FcγRIIb−/− macrophages demonstrated more prominent phagocytosis but did not differ in killing activity in vitro and the striking TNF-α, IL-6 and IL-10 levels, compared to wild-type cells. Indeed, in vivo macrophage depletion with liposomal clodronate attenuated the fungal burdens in FcγRIIb−/− mice, but not wild-type mice. When administered to wild-type mice, FcγRIIb−/− macrophages with phagocytosed Cryptococcus resulted in higher fungal burdens than FcγRIIb+/+ macrophages with phagocytosed Cryptococcus. These results support, at least in part, a model whereby, in FcγRIIb−/− mice, enhanced C. neoformans transmigration occurs through infected macrophages. In summary, prominent phagocytosis, with limited effective killing activity, and high pro-inflammatory cytokine production by FcγRIIb−/− macrophages were correlated with more severe cryptococcosis in FcγRIIb−/− mice.

The model of circulating immune complexes and interleukin-6 improves the prediction of disease activity in systemic lupus erythematosus

Systemic Lupus Erythematosus (SLE) is an autoimmune disease resulting in autoantibody production, immune complex deposition, and complement activation. The standard biomarkers such as anti-dsDNA and complements (C3 and C4) do not always correlate with active clinical SLE. The heterogeneity of SLE patients may require additional biomarkers to designate disease activity. Ninety SLE patients participated in this study. Evaluation of disease activity was achieved with the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) and modified SLEDAI-2K. The measured serum biomarkers were anti-dsDNA, C3, C4, ESR, interleukin-6 (IL-6), and circulating immune complexes (CIC). IL-6, ESR and CIC significantly increased in active clinical SLE. Complement, anti-dsDNA, ESR and CIC correlated with SLEDAI-2K while only anti-dsDNA, CIC, ESR and IL-6 correlated with modified SLEDAI-2K. A combination of biomarkers gave a higher odds ratio (OR) than any single biomarker. A combination of IL-6 or CIC exhibited the highest OR (OR = 7.27, 95%CI (1.99–26.63), p = 0.003) while either complement or anti-dsDNA showed a moderate odds ratio (OR = 3.14, 95%CI (1.16–8.48), p = 0.024) of predicting clinical active SLE. The combination of CIC and IL-6 strongly predicts active clinical SLE. CIC and IL-6 can be used in addition to standard biomarkers to determine SLE activity.

The Synergy of Endotoxin and (1→3)-β-D-Glucan, from Gut Translocation, Worsens Sepsis Severity in a Lupus Model of Fc Gamma Receptor IIb-Deficient Mice

We investigated the influence of spontaneous gut leakage upon polymicrobial sepsis in a lupus model with Fc gamma receptor IIb-deficient (FcGRIIb-/-) mice aged 8 and 40 weeks, as representing asymptomatic and symptomatic lupus, respectively. Spontaneous gut leakage, determined by (i) the presence of FITC-dextran, (ii) elevated serum endotoxin, and (iii) elevated serum (1→3)-β-D-glucan (BG), was demonstrated in symptomatic lupus but not in the asymptomatic group. In parallel, spontaneous gut leakage, detected by elevated serum BG without fungal infection, was demonstrated in patients with active lupus nephritis. Gut leakage induced by dextran sulfate solution (DSS) or endotoxin administration together with BG or endotoxin alone, but not BG alone, enhanced the severity of cecal ligation and puncture (CLP) sepsis more prominently in 8-week-old FcGRIIb-/- mice. Additionally, the bone marrow-derived macrophages of FcGRIIb-/- mice produced higher cytokine levels when coexposed to endotoxin and BG, when compared to wild-type mice. In summary, spontaneous gut leakage was demonstrated in symptomatic FcGRIIb-/- mice and the induction of gut permeability worsened sepsis severity. Gut translocation of endotoxin and BG had a minor effect on wild-type mice, but the synergistic effect of BG and endotoxin was prominent in FcGRIIb-/- mice. The data suggest that therapeutic strategies addressing gut leakage may be of interest in sepsis conditions in patients with lupus.

Cortical Bone Loss in a Spontaneous Murine Model of Systemic Lupus Erythematosus

Patients with systemic lupus erythematosus (SLE), a chronic inflammatory disease characterized by loss of T- and B-cell tolerance to autoantigens, are at increased risk for osteoporosis and fractures. Mice deficient in Fc gamma receptor IIb (FcγRIIB) exhibit spontaneous SLE and its restoration rescues the disease. To determine whether deleting FcγRIIB affects cortical bone mass and mechanical properties, we analyzed cortical bone phenotype of FcγRIIB knockouts at different ages. FACS analysis revealed that 6-month-old FcγRIIB−/− mice had increased B220lowCD138+ cells, markers of plasma cells, indicating active SLE disease. In contrast, 3-month-old FcγRIIB−/− mice did not develop the active SLE disease. µCT analysis indicated that FcγRIIB deletion did not affect cortical bone in 3-month-old mutants. However, 6- and 10-month-old FcγRIIB−/− males and females had osteopenic cortical bone and the severity of bone loss increased with disease duration. FcγRIIB deletion decreased cross-sectional area, cortical area, and marrow area in 6-month-old males. Cortical area and cortical thickness were decreased in 10-month-old FcγRIIB−/− males. Lack of FcγRIIB decreased cortical thickness without affecting cortical area in females. However, deletion of a single FcγRIIB allele was insufficient to induce cortical bone loss. The bending strength was decreased in 6- and 10-month-old FcγRIIB-deficient males compared to WT controls. A microindentation analysis demonstrated significantly decreased hardness in both 10-month-old FcγRIIB−/− males and females. Our data indicate that FcγRIIB contributes to the regulation of cortical bone homeostasis subsequent to SLE development and that deletion of FcγRIIB in mice leads to SLE-like disease associated with cortical bone loss and decreased bending strength and hardness.

349 Tmem173/sting is crucial for lupus development in fcgr2b-deficiency mice

Background and aims Type I interferon is one of the most critical cytokines involving in lupus pathogenesis. The activation of endosomal nucleic acid sensors leading to type I IFN production empowers the lupus phenotypes in several mouse models. The signalling of cytosolic DNA sensor also induces type I IFN production and the role in lupus disease are not clear. Stimulator of interferon genes (Sting), also known as transmembrane protein 173 (TMEM173) and MPYS/MITA/ERIS, is a cytosolic DNA sensor which recognised cyclic di-GMP and subsequently stimulated type I interferon production. The Fcgr2b-deficient mice develop spontaneous lupus phenotypes which are splenomegaly, the presence of anti-nuclear antibodies (ANA) and fatal glomerulonephritis. The polymorphisms of FCGR2B associates with the increases of lupus susceptibility in human. The goal of the study is to identify the role of Sting in lupus mouse model. Methods The Sting-deficient mice were bred with the Fcgr2b-deficient mice to create double deficient mice and control littermates. The mice were analysed for survival rate, autoantibodies production, severity of pathology, gene expression profiles, and immunophenotypes. Results In the absence of Sting, the Fcgr2b-deficient mice survived longer and the level of ANA and anti-dsDNA antibody considerably reduced. The glomerulonephritis in the double-deficient mice also ameliorated. The expression of interferon inducible genes such as Cxcl10, Mx1, and Irf5 in the kidneys of the double-deficient mice was significantly lower than the Fcgr2b-deficient mice. Conclusions Sting-mediated signalling pathway plays the substantial role in lupus pathogenesis of the Fcgr2b-deficient mice. Blocking Sting function may be the advantage for treatment in lupus patients.