Laura J. Dickie

Vitamin D3 down-regulates intracellular Toll-like receptor 9 expression and Toll-like receptor 9-induced IL-6 production in human monocytes

OBJECTIVE To determine whether vitamin D(3) modulates monocytic expression of intracellular Toll-like receptors (TLRs) 3, 7 and 9. METHODS Human monocytes were isolated from peripheral blood and cultured with 100 nM vitamin D(3) for 24, 48 and 72 h. Expression of CD14 and TLR2, TLR3, TLR4, TLR7 and TLR9 were examined by flow cytometry. Monocytes exposed to vitamin D(3) for 48 h were then stimulated with a TLR9 agonist for a further 24 h. The level of IL-6 secretion was measured by ELISA. RESULTS CD14 was up-regulated, whereas TLR2, TLR4 and TLR9 expression was down-regulated by vitamin D(3) exposure in a time-dependent manner. TLR3 expression was unaffected by vitamin D(3) and there was no measurable expression of TLR7 on the monocytes. TLR9-induced IL-6 production was impaired in monocytes treated with vitamin D(3) compared with untreated cells. CONCLUSION The intracellular TLRs are differentially regulated by vitamin D(3), with TLR9 being down-regulated by vitamin D(3) exposure whereas TLR3 was unaffected. This decreased TLR9 expression in monocytes had a downstream functional effect as these cells subsequently secreted less IL-6 in response to TLR9 challenge. This may have significant biological relevance and may be a factor in the association of vitamin D deficiency with susceptibility to autoimmune disease.

Familial Mediterranean fever and related periodic fever syndromes/autoinflammatory diseases.

Purpose of reviewThe spectrum of periodic fever syndromes (PFS)/autoinflammation diseases is continuously expanding. This review provides an overview of the primary research and an update on the main clinical developments in these disorders published in the past 12–18 months. Recent findingsIL-1&bgr; is pivotal to the pathogenesis of most of the PFS. In familial Mediterranean fever (FMF) MEFV mutations lead to gain of pyrin function, resulting in inappropriate IL-1&bgr; release that is dependent on ASC but not the NLRP3 inflammasome. Anti-IL-1 therapy is effective in tumour necrosis factor receptor-associated periodic syndrome (TRAPS), whilst both spontaneous and pathogen-associated molecular patterns (PAMPs) induced IL-1&bgr; release have been demonstrated in NLRP12-associated periodic syndrome (NAPS12). Somatic NLRP3/CIAS1 mosaicism is a significant cause of cryopyrin-associated periodic syndromes (CAPS). Close connections have also been established between metabolic and inflammatory pathways. In TRAPS increased reactive oxygen species (ROS) of mitochondrial origin leads to production of pro-inflammatory cytokines, whilst NLRP3 inflammasome activation in type 2 diabetes (T2D) is induced by oligomers of islet amyloid polypeptides (IAPP). SummaryCaspase 1 activation and IL-1&bgr; release is central to the pathogenesis of many autoinflammatory syndromes. This is supported by the effectiveness of anti-IL-1 biologics in treatment of these disorders.

An Integrated Classification of Pediatric Inflammatory Diseases, Based on the Concepts of Autoinflammation and the Immunological Disease Continuum

Historically, pediatric inflammatory diseases were viewed as autoimmune but developments in genetics of monogenic disease have supported our proposal that “inflammation against self” be viewed as an immunologic disease continuum (IDC), with genetic disorders of adaptive and innate immunity at either end. Innate immune-mediated diseases may be associated with significant tissue destruction without evident adaptive immune responses and are designated as autoinflammatory due to distinct immunopathologic features. However, the majority of pediatric inflammatory disorders are situated along this IDC. Innate immunity has been demonstrated in polygenic disorders, particularly Crohns disease (CD). A genetic overlap exists between CD and some major histocompatability complex (MHC) class I-associated diseases, including psoriasis; these diseases seem to represent a true intermediate between autoinflammation and autoimmunity. Conversely, classical autoimmune diseases, with autoantibody and MHC class II associations, including celiac disease and rheumatoid arthritis (RA), have adaptive immune genetic associations, including Cytotoxic T-Lymphocyte Antigen-4 (CTLA4) and PTPN22. This proposed classification is clinically relevant, because innate immune-mediated disorders may respond to cytokine antagonism whereas autoimmune-mediated diseases respond better to anti-T and B cell therapies. Furthermore, the etiopathogenesis of poorly defined “autoimmune” diseases, such as juvenile idiopathic arthritis, may be inferred to have substantial innate immune involvement, based on response to IL-1 antagonism.

The early phase of psoriatic arthritis

Evaluation of the preclinical phases of the classic autoimmune diseases including rheumatoid arthritis has been facilitated by the availability of autoantibody and genetic markers that point firmly towards the early dysregulation of the adaptive immune responses. The association of psoriatic disease with the human leucocyte antigen—Cw0602 (HLA-Cw0602) gene has likewise led to the perception that autoimmunity has a pivotal role in early psoriatic arthritis (PsA). However, this HLA-Cw0602 genetic association does not appear to hold for PsA or associated nail, scalp and intergluteal skin involvement. Of note, these three sites of psoriasis are predictive of PsA evolution. For initiation of both skin and nail disease there is a link with Koebnerisation, or site-specific trauma. Nail disease is most common in the dominant hand thumbnail, pointing towards local tissue factors as disease initiators Likewise, for PsA, there is also good evidence for a history of previous joint trauma and histological studies showing microdamage in normal entheses which are typical locations where PsA frequently occurs. Furthermore, subclinical enthesopathy including osteitis is common in subjects with psoriasis but without arthritis. Collectively, these findings indicate that the classic model of adaptive immune dysregulation does not generally hold for the early stages of PsA. The way in which knowledge pertaining to tissue-specific factors in PsA, combined with the emerging data relating to monogenic disorders and animal models, points towards perturbation in the healing response and dysregulation of innate immune responses in early PsA is discussed. The way in which this model explains the clinical disconnect between skin and joint disease and the emerging human data that support it are demonstrated.

TLR dependent XBP-1 activation induces an autocrine loop in rheumatoid arthritis synoviocytes.

X-box binding protein 1 (XBP1) is a central regulator of the endoplasmic reticulum (ER) stress response. It is induced via activation of the IRE1 stress sensor as part of the unfolded protein response (UPR) and has been implicated in several diseases processes. XBP1 can also be activated in direct response to Toll-like receptor (TLR) ligation independently of the UPR but the pathogenic significance of this mode of XBP1 activation is not well understood. Here we show that TLR-dependent XBP1 activation is operative in the synovial fibroblasts (SF) of patients with active rheumatoid arthritis (RA). We investigated the expression of ER stress response genes in patients with active RA and also in patients in remission. The active (spliced) form of (s)XBP1 was significantly overexpressed in the active RA group compared to healthy controls and patients in remission. Paradoxically, expression of nine other ER stress response genes was reduced in active RA compared to patients in remission, suggestive of a UPR-independent process. However, sXBP1 was induced in SF by TLR4 and TLR2 stimulation, resulting in sXBP1-dependent interleukin-6 and tumour necrosis factor (TNF) production. We also show that TNF itself induces sXBP1 in SF, thus generating a potential feedback loop for sustained SF activation. These data confirm the first link between TLR-dependent XBP1 activation and human inflammatory disease. sXBP1 appears to play a central role in this process by providing a convergence point for two different stimuli to maintain activation of SF.

Failure of sustained response to etanercept and refractoriness to anakinra in patients with T50M TNF-receptor-associated periodic syndrome

TNF-receptor-associated periodic syndrome (TRAPS) is an autoinflammatory condition and is the most common autosomal dominant hereditary periodic fever. Autoinflammatory syndromes are distinguished from autoimmune diseases by the fact that their disease immunopathology is largely played out by cells and molecules of the innate immune system.1 2 The pathogenesis of TRAPS is still not completely understood and treatment options are extremely limited. Prednisolone improves symptoms during attacks but the effect often wanes and patients may require increasing doses. Etanercept, a TNFR2 fusion protein, is reported to have a beneficial effect on many patients with TRAPS,3,–,6 whereas infliximab is not generally effective in most cases of TRAPS and may cause an overwhelming flare but occasional benefit has also been described.7,–,9 Anakinra, …

First Report of Circulating MicroRNAs in Tumour Necrosis Factor Receptor-Associated Periodic Syndrome (TRAPS)

Tumor necrosis factor-receptor associated periodic syndrome (TRAPS) is a rare autosomal dominant autoinflammatory disorder characterized by recurrent episodes of long-lasting fever and inflammation in different regions of the body, such as the musculo-skeletal system, skin, gastrointestinal tract, serosal membranes and eye. Our aims were to evaluate circulating microRNAs (miRNAs) levels in patients with TRAPS, in comparison to controls without inflammatory diseases, and to correlate their levels with parameters of disease activity and/or disease severity. Expression levels of circulating miRNAs were measured by Agilent microarrays in 29 serum samples from 15 TRAPS patients carrying mutations known to be associated with high disease penetrance and from 8 controls without inflammatory diseases. Differentially expressed and clinically relevant miRNAs were detected using GeneSpring GX software. We identified a 6 miRNAs signature able to discriminate TRAPS from controls. Moreover, 4 miRNAs were differentially expressed between patients treated with the interleukin (IL)-1 receptor antagonist, anakinra, and untreated patients. Of these, miR-92a-3p and miR-150-3p expression was found to be significantly reduced in untreated patients, while their expression levels were similar to controls in samples obtained during anakinra treatment. MiR-92b levels were inversely correlated with the number of fever attacks/year during the 1st year from the index attack of TRAPS, while miR-377-5p levels were positively correlated with serum amyloid A (SAA) circulating levels. Our data suggest that serum miRNA levels show a baseline pattern in TRAPS, and may serve as potential markers of response to therapeutic intervention.

Differential effects of infliximab on absolute circulating blood leucocyte counts of innate immune cells in early and late rheumatoid arthritis patients

Anti‐tumour necrosis factor (TNF) biologics have revolutionized therapy of rheumatoid arthritis (RA). We compared the effects of infliximab on numbers of circulating leucocyte subsets in early RA (disease/symptom duration of ≤1 year) and late RA patients (>1 year). A control group consisted of early RA patients treated with a combination of methotrexate (MTX) and methylprednisolone. Blood samples were obtained at baseline (pre‐therapy) from all RA patients, divided into three groups: (i) late RA receiving infliximab/MTX, (ii) early RA–infliximab/MTX, (iii) early RA–steroid/MTX, and also from follow‐up patients at 2 and 14 weeks. Significant differences in absolute counts of monocytes and granulocytes were observed between healthy controls and RA patients. At baseline CD14bright monocytes and CD16+ granulocytes were increased in both early RA and late RA patients. CD4+ T cells, CD8+ T cells and B cells were all increased at baseline in early RA, but not in late RA. At 2 weeks following infliximab treatment decreased granulocytes were observed in both early and late RA and decreased natural killer (NK) cells in late RA. CD16+ granulocytes and NK cells were also decreased at 14 weeks post‐infliximab in early RA. Biotinylated infliximab was used to detect membrane‐associated TNF (mTNF)‐expressing leucocytes in RA patients. CD16+ granulocytes, NK cells and CD14dim monocytes all expressed higher levels of mTNF in RA patients. In summary infliximab is associated with decreased CD16+ granulocyte and NK cell counts, possibly through binding of mTNF. Differential effects of infliximab between early and late RA suggest that pathogenic mechanisms change as disease progresses.

Periodic fever syndrome and autoinflammatory diseases

The concept of autoinflammatory disease as a new disease classification has resulted in a paradigm shift in our understanding of the the broad spectrum of immunological diseases. The effectiveness of interleukin-1 blockade in a variety of disorders has resulted in a marked reduction in suffering for many of these patients.

A1.52 TLR-Dependent XBP1 activation induces an autocrine loop in rheumatoid arthritis synovial fibroblasts

Background and Objectives Rheumatoid arthritis (RA) is a polygenic disorder usually arising from combined genetic predisposition and environmental influences with associated dysfunctional immune responses. The X-box binding protein 1 (XBP1) transcription factor is a central regulator of the endoplasmic reticulum (ER) stress response. It is induced via activation of the IRE1 stress sensor as part of the unfolded protein response (UPR) and has been implicated in several diseases processes including RA. XBP1 can also be activated in direct response to Toll-like Receptor (TLR) ligation independently of the UPR but the pathogenic significance of this mode of XBP1 activation is not well understood. Our aim was to investigate the relevance of interactions between UPR and TLR signalling pathways in the serum and synovial fibroblasts (SFs) of patients with RA, using samples from healthy individuals and patients with osteoarthritis (OA) as controls Materials and Methods Peripheral blood mononuclear cells (PBMC) were obtained from control groups: healthy individuals (n = 24) and RA patients, comprising active disease (n = 47) and remission (n = 12). SFs from RA and OA patients were isolated by digestion of synovial biopsies. Gene expression profiling was performed using qPCR for the detection of sXBP1 and enzyme-linked immunosorbent assays (ELISA) to quantify levels of pro-inflammatory cytokines, IL-6 and TNF. siRNA targeting of XBP1 was used for knockdown experiments in SFs. Results We investigated the expression of ER stress response genes in patients with active RA and patients in remission. We show that TLR-dependent XBP1 activation is operative in the SFs of patients with active RA. The active (spliced) form of (s)XBP1 was significantly overexpressed in the active RA group compared to healthy controls and patients in remission (p = 0.01). Paradoxically, expression of nine other ER stress response genes was reduced in active RA compared to patients in remission, suggestive of a UPR-independent process. However, sXBP1 was induced in SFs by TLR4 and TLR2 stimulation, resulting in sXBP1-dependent IL-6 and TNF production. We also show that TNF itself induces sXBP1 in SFs, thus generating a potential feedback loop for sustained activation of these cells. Conclusions sXBP1 plays a central role in two different cellular processes that may first appear unconnected. However, linking inflammatory pathways with ER stress provides SFs with a timely, coordinated and protective response. XBP1 activation may therefore be a suitable target in the treatment of RA, since it forms a cornerstone of two different molecular processes implicated in the pathogenesis of RA.