Flow cytometric detection of type 1 (IL-2, IFN-gamma) and type 2 (IL-4, IL-5) cytokines in T-helper and T-suppressor/cytotoxic cells in rheumatoid arthritis, allergic asthma and atopic dermatitis.

Type 1 cytokines (a.o. IL-2 and IFN-gamma) play an important role in the pathogenesis of rheumatoid arthritis. On the other hand, IgE-mediated diseases such as allergic asthma and atopic dermatitis show a type 2 cytokine (amongst others IL-4 and IL-5) profile.This study examined simultaneously the intracellular production of IL-2, IFN-gamma, IL-4 and IL-5 in T-lymphocytes of patients with rheumatoid arthritis during treatment with methotrexate or salazopyrin, patients with allergic asthma or atopic dermatitis under stable treatment, compared to healthy controls.A three-colour flow cytometric analysis was used for cytokine detection in T-helper cells and T-suppressor/cytotoxic cells.Compared to controls, patients with symptomatic atopic dermatitis showed an increased number of IL-4-producing T-helper lymphocytes in basal circumstances (P=0.001), in contrast to asymptomatic allergic asthma patients. Compared to controls, rheumatoid arthritis patients, treated with salazopyrin, showed an increased number of IL-2-producing T-helper and T-suppressor/cytotoxic lymphocytes after in vitro stimulation with PMA and ionomycin (P=0.01). In contrast, rheumatoid arthritis patients, treated with methotrexate, a more potent disease modifying drug, did not show this type 1 cytokine profile. A positive correlation was found between the number of IFN-gamma producing T-helper cells and disease activity (Ritchie Index and number of swollen joints) in both rheumatoid arthritis patient groups.Active atopic dermatitis patients showed a type 2 cytokine profile, whereas stable asthma patients with lower disease activity did not show a predominance of type 2 cytokines. Rheumatoid arthritis patients under treatment with salazopyrin had a type 1 cytokine profile, which could not be demonstrated in patients treated with methotrexate. This imbalance between type 1 and type 2 cytokines in different immune mediated disorders can be related with treatment and the grade of disease activity. These results stress the need for further investigation of the influence of therapy on cytokine profiles.     

Old drugs,new tricks: Emerging role of drug repurposing in the management of atopic dermatitis

Atopic dermatitis is a chronic recurring pruritic inflammatory skin disease manifested by increased pro-inflammatory mediators which lead to dry, thickened, cracked, scaly skin. The current treatment options for atopic dermatitis management comprise drawbacks and leave unmet effective clinical needs. So, the approach for repurposing existing drugs for atopic dermatitis management may potentially overcome these unmet needs. Diseases that share the common pathophysiological pathways with atopic dermatitis can serve as a foundation for the repurposing of drugs. Drugs used in the management of cancer, rheumatoid arthritis, and other immune-mediated diseases such as psoriasis are under investigation to know the potential in atopic dermatitis management by utilizing repurposing strategies for a novel therapeutic indication. This review mainly envisages the probable repurposing of drugs for the management of atopic dermatitis disease; the barriers and regulatory aspects involved in the repurposing of existing drugs.     

Genetically programmed differences in epidermal host defense between psoriasis and atopic dermatitis patients

In the past decades, chronic inflammatory diseases such as psoriasis, atopic dermatitis, asthma, Crohn's disease and celiac disease were generally regarded as immune-mediated conditions involving activated T-cells and proinflammatory cytokines produced by these cells. This paradigm has recently been challenged by the finding that mutations and polymorphisms in epithelium-expressed genes involved in physical barrier function or innate immunity, are risk factors of these conditions. We used a functional genomics approach to analyze cultured keratinocytes from patients with psoriasis or atopic dermatitis and healthy controls. First passage primary cells derived from non-lesional skin were stimulated with pro-inflammatory cytokines, and expression of a panel of 55 genes associated with epidermal differentiation and cutaneous inflammation was measured by quantitative PCR. A subset of these genes was analyzed at the protein level. Using cluster analysis and multivariate analysis of variance we identified groups of genes that were differentially expressed, and could, depending on the stimulus, provide a disease-specific gene expression signature. We found particularly large differences in expression levels of innate immunity genes between keratinocytes from psoriasis patients and atopic dermatitis patients. Our findings indicate that cell-autonomous differences exist between cultured keratinocytes of psoriasis and atopic dermatitis patients, which we interpret to be genetically determined. We hypothesize that polymorphisms of innate immunity genes both with signaling and effector functions are coadapted, each with balancing advantages and disadvantages. In the case of psoriasis, high expression levels of antimicrobial proteins genes putatively confer increased protection against microbial infection, but the biological cost could be a beneficial system gone awry, leading to overt inflammatory disease.     

Phosphodiesterase 4 inhibitors as novel anti-inflammatory agents.

Preclinical and clinical studies of phosphodiesterase 4 inhibitors have shown that these agents may find utility in a wide range of inflammatory disorders, including asthma, chronic obstructive pulmonary disease, atopic dermatitis, rheumatoid arthritis, multiple sclerosis and various neurological disorders. The future of this class of drugs will depend upon the ability to demonstrate a reasonable safety margin against emesis and other typical phosphodieserase (PDE4) side effects, as well as in identification of the inflammatory disorder(s) most relevant to PDE4 inhibition.     

Long non‐coding RNAs in rheumatoid arthritis

Rheumatoid arthritis, a disabling autoimmune disease, is associated with altered gene expression in circulating immune cells and synovial tissues. Accumulating evidence has suggested that long non‐coding RNAs (lncRNAs), which modulate gene expression through multiple mechanisms, are important molecules involved in immune and inflammatory pathways. Importantly, many studies have reported that lncRNAs can be utilized as biomarkers for disease diagnosis and prognostication. Recently, dysregulation of lncRNAs in rheumatoid arthritis and other autoimmune diseases has been revealed. Experimental studies also confirmed their crosstalk with matrix metalloproteinases, nuclear factor‐κB signalling and T‐cell response pertinent to autoimmunity and inflammation. Circulating lncRNAs, such as HOTAIR, differentiated patients with rheumatoid arthritis from healthy subjects. Taken together, lncRNAs are good candidates as biomarkers and therapeutic targets in rheumatoid arthritis. Further investigation on in vivo delivery of these regulatory molecules and large‐cohort validation of their clinical applicability may be useful.     

T cells of atopic asthmatics preferentially infiltrate into human bronchial xenografts in SCID mice

T cells play an important role in the pathogenesis of bronchial asthma. However, it is not completely known how circulating lymphocytes infiltrate into the airways of asthmatic patients. Because SCID mice are unable to reject xenogenic transplants, many xenotransplant models using various human tissues have been developed. Therefore, to examine the interaction between bronchi and T lymphocytes of asthma, it may be possible to use the human bronchial xenograft and PBMC xenograft in SCID mice. We transplanted human bronchi into the subcutaneum of SCID mice and i.p. injected PBMCs that were obtained from patients with atopic asthma, atopic dermatitis and rheumatoid arthritis, and normal subjects (asthmatic, dermatitis, rheumatic, and normal huPBMC-SCID mice). There was no difference in the percentage of CD3-, CD4-, CD8-, CD25-, CD45RO-, CD103-, and cutaneous lymphocyte Ag-positive cells in PBMCs among the patients with asthma, dermatitis, rheumatoid arthritis, and normal subjects, and CD3-positive cells in peripheral blood of asthmatic, dermatitis, rheumatic, and normal huPBMC-SCID mice. The number of CD3-, CD4-, and CD8-positive cells in the xenografts of asthmatic huPBMC-SCID mice was higher than those of dermatitis, rheumatic, and normal huPBMC-SCID mice. IL-4 mRNA and IL-5 mRNA were significantly higher in the xenografts of asthmatic huPBMC-SCID mice than those in the xenografts of normal huPBMC-SCID mice, but there were no significant differences in the expressions of IL-2 mRNA or IFN-gamma mRNA between them. These findings suggest that T cells, especially Th2-type T cells, of asthmatics preferentially infiltrate into the human bronchi.     

Variations in the human Th2-specific chemokine TARC gene

Th2-specific chemokine thymus and activation-regulated chemokine (TARC)/CC chemokine ligand (CCL)17 is highly implicated in the pathogenesis of Th-2-dominated allergic diseases such as bronchial asthma (BA) and atopic dermatitis (AD). We performed polymorphism screening of the coding and promoter regions of the TARC gene. We found two rare variations in the coding region of exon 3 (2134C>T and 2037G>A) and a single nucleotide polymorphism (SNP) in the 5'-flanking region (-431C>T). Individuals carrying the 431T allele showed significantly increased serum levels of TARC compared with those not carrying the 431T allele, suggesting that this SNP has functional significance. However, when the genotypes at the SNP site were determined for 158 healthy individuals, 105 patients with BA and 148 patients with AD, we observed no significant association of the SNP with susceptibility to BA or AD.     

Small intestinal immune-environmental changes induced by oral tolerance inhibit experimental atopic dermatitis

Atopic dermatitis is a chronic skin inflammatory disease mediated by Th2-type immune responses. Although intestinal immune responses have been shown to play a critical role in the development or prevention of atopic dermatitis, the precise influence of intestinal immunity on atopic dermatitis is incompletely understood. We show here that orally tolerized mice are protected from experimental atopic dermatitis induced by sensitization and epicutaneous (EC) challenge to ovalbumin. Although the expression of Th2-type cytokines in the small intestine of orally tolerized and EC-challenged mice did not change significantly, these mice showed decreased inflammatory responses in the small intestine with restoration of microbial change elicited by the EC challenge. Interestingly, an increase in small intestinal eosinophils was observed with the EC challenge, which was also inhibited by oral tolerance. The role of small intestinal eosinophils and microbiota in the pathogenesis of experimental atopic dermatitis was further substantiated by decreased inflammatory mediators in the small intestine and attenuated Th2-type inflammation in the skin of eosinophil-deficient and microbiota-ablated mice with EC challenges. Based on these data, we propose that the bidirectional interaction between the skin and the intestine has a role in the pathogenesis of atopic dermatitis and that modulation of the intestinal microenvironments could be a therapeutic approach to atopic dermatitis.Subject terms: Allergy, Mucosal immunology     

Genetic susceptibility to autoimmune disorders: clues from gene association and gene expression studies

Susceptibility to autoimmune disorders results from the interaction of multiple genetic factors that regulate the threshold of autoreactivity. Genome-wide microsatellite screens and large-scale single nucleotide polymorphism (SNP) association studies have identified chromosomal loci that are associated with specific disorders including systemic lupus erythematosus, rheumatoid arthritis, juvenile arthritis, multiple sclerosis, and diabetes. Numerous candidate gene association studies have in turn investigated the association of specific genes within these chromosomal regions, with susceptibility to autoimmune diseases (e.g. FcgammaReceptors, TYK2 and systemic lupus). More recently, large-scale differential gene expression studies performed on selected tissues from patients with autoimmune disorders, have led to the identification of gene signatures associated with the activation of specific pathways in these diseases (e.g. interferon signature in lupus). In the future, integrated analyses of gene (and protein) expression together with SNP data will allow us to sketch an intelligible picture of the genesis of autoimmunity in humans. This review sets out to illustrate how the most recent advances in the field of systemic lupus erythematosus, rheumatoid arthritis and juvenile arthritis have led to a better understanding of these disorders.     

Elevated expression and genetic association links the SOCS3 gene to atopic dermatitis

In a systematic analysis of global gene-expression patterns, we found that SOCS3 messenger RNA was significantly more highly expressed in skin from patients with atopic dermatitis than in skin from healthy controls, and immunohistochemical analysis confirmed a similar elevation of SOCS3 protein. Furthermore, we found a genetic association between atopic dermatitis and a haplotype in the SOCS3 gene in two independent groups of patients (P<.02 and P<.03). These results strongly suggest that SOCS3, located in a chromosomal region previously linked to the disease (17q25), is a susceptibility gene for atopic dermatitis.     

Modulation of innate and adaptive immune responses by tofacitinib (CP-690,550)

Inhibitors of the JAK family of nonreceptor tyrosine kinases have demonstrated clinical efficacy in rheumatoid arthritis and other inflammatory disorders; however, the precise mechanisms by which JAK inhibition improves inflammatory immune responses remain unclear. In this study, we examined the mode of action of tofacitinib (CP-690,550) on JAK/STAT signaling pathways involved in adaptive and innate immune responses. To determine the extent of inhibition of specific JAK/STAT-dependent pathways, we analyzed cytokine stimulation of mouse and human T cells in vitro. We also investigated the consequences of CP-690,550 treatment on Th cell differentiation of naive murine CD4(+) T cells. CP-690,550 inhibited IL-4-dependent Th2 cell differentiation and interestingly also interfered with Th17 cell differentiation. Expression of IL-23 receptor and the Th17 cytokines IL-17A, IL-17F, and IL-22 were blocked when naive Th cells were stimulated with IL-6 and IL-23. In contrast, IL-17A production was enhanced when Th17 cells were differentiated in the presence of TGF-β. Moreover, CP-690,550 also prevented the activation of STAT1, induction of T-bet, and subsequent generation of Th1 cells. In a model of established arthritis, CP-690,550 rapidly improved disease by inhibiting the production of inflammatory mediators and suppressing STAT1-dependent genes in joint tissue. Furthermore, efficacy in this disease model correlated with the inhibition of both JAK1 and JAK3 signaling pathways. CP-690,550 also modulated innate responses to LPS in vivo through a mechanism likely involving the inhibition of STAT1 signaling. Thus, CP-690,550 may improve autoimmune diseases and prevent transplant rejection by suppressing the differentiation of pathogenic Th1 and Th17 cells as well as innate immune cell signaling.     

Antibody-induced arthritis: disease mechanisms and genes involved at the effector phase of arthritis

During the development of rheumatoid arthritis (RA) autoantibodies to IgG-Fc, citrullinated proteins, collagen type II (CII), glucose 6 phosphoisomerase (G6PI) and some other self-antigens appear. Of these, a pathogenic effect of the anti-CII and anti-G6PI antibodies is well demonstrated using animal models. These new antibody mediated arthritis models have proven to be very useful for studies involved in understanding the molecular pathways of the induction of arthritis in joints. Both the complement and FcgammaR systems have been found to play essential roles. Neutrophils and macrophages are important inflammatory cells and the secretion of tumour necrosis factor-alpha and IL-1beta is pathogenic. The identification of the genetic polymorphisms predisposing to arthritis is important for understanding the complexity of arthritis. Disease mechanisms and gene regions studied using the two antibody-induced arthritis mouse models (collagen antibody-induced arthritis and serum transfer-induced arthritis) are compared and discussed for their relevance in RA pathogenesis.     

Chronically inflamed human tissues are infiltrated by highly differentiated Th17 lymphocytes

Chronic inflammatory diseases are characterized by local tissue injury caused by immunocompetent cells, in particular CD4(+) T lymphocytes, that are involved in the pathogenesis of these disorders via the production of distinctive sets of cytokines. Here, we have characterized single CD4(+) T cells that infiltrate inflamed tissue taken from patients with psoriasis, Crohn's disease, rheumatoid arthritis, or allergic asthma. Results from a cytokine production and gene profile analysis identified a population of in vivo differentiatedretinoid-related orphan receptor gamma-expressing T cells, producing high levels of IL-17, that can represent up to 30% of infiltrating T lymphocytes. Activated Th17 cells produced IL-26, TNF-alpha, lymphotoxin-beta, and IL-22. IL-17 and IL-22 concentrations secreted by tissue infiltrating Th17 cells could reach up to 100 nM and were inversely correlated with the production of Th1- and Th2-associated cytokines. In addition, tissue-infiltrating Th17 cells are also characterized by high cell surface expression of CCR6, a chemokine receptor that was not expressed by Th1 and Th2 cells, isolated from the same lesions, and by the production of CCL20/MIP3alpha, a CCR6 ligand, associated with tissue infiltration. Culture supernatants of activated Th17 cells, isolated from psoriatic lesions, induced the expression of gene products associated with inflammation and abnormal keratinocyte differentiation in an IL-17 and IL-22-dependent manner. These results show that tissue-infiltrating Th17 cells contribute to human chronic inflammatory disease via the production of several inflammatory cytokines and the creation of an environment contributing to their migration and sequestration at sites of inflammation.     

A possible role for TSLP in inflammatory arthritis

Thymic stromal lymphopoietin (TSLP) is an IL-7-like cytokine that triggers dendritic cell-mediated Th2-type inflammatory responses and is considered as a master switch for allergic inflammation. In this study, we found increased levels of TSLP and, also TNF-alpha as previously reported, in synovial fluid specimens derived from patients with rheumatoid arthritis (RA) when compared with those from patients with osteoarthritis (OA). In addition, TNF-alpha up-regulated TSLP expression in RA- and OA-derived synovial fibroblasts, which was inhibited by IFN-gamma. Furthermore, anti-TSLP neutralizing antibody ameliorated a TNF-alpha-dependent experimental arthritis induced by anti-type II collagen antibody in mice. Collectively, these results suggest that TSLP, as a downstream molecule of TNF-alpha, may be involved in the pathophysiology of inflammatory arthritis. TSLP might thus play a role not only in allergic diseases but also in inflammatory arthritis such as RA.     

The Crosstalk between IL-22 Signaling and miR-197 in Human Keratinocytes

The interaction between the immune system and epithelial cells is tightly regulated. Aberrations of this balance may result in inflammatory diseases such as psoriasis, inflammatory bowel disease and rheumatoid arthritis. IL-22 is produced by Th17, Th22 and Th1 cells. Putative targets for IL-22 are cells in the skin, kidney, digestive and respiratory systems. The highest expression of IL-22 receptor is found in the skin. IL-22 plays an important role in the pathogenesis of T cell-mediated inflammatory diseases such as psoriasis, inflammatory bowel disease and rheumatoid arthritis. Recently, we found that miR-197 is down regulated in psoriatic lesions. In the present work we show that miR-197 over expression inhibits keratinocytes proliferation induced by IL-22 and keratinocytes migration. In addition, we found that IL-22 activates miR-197 expression through the binding of phosphorylated STAT3 to sequences in the putative promoter of miR-197. Finally we found that IL-22 receptor subunit IL22RA1 is a direct target of miR-197. Hence, we identified a novel feedback loop controlling IL-22 signaling, in which IL-22 induces miR-197, which in turn, negatively regulates IL-22 receptor and attenuates the biological outcome of such signaling. Regulation of this pathway may be important in inflammatory skin disorders such a psoriasis and in wound healing.     

Elevated levels of circulating pancreatic polypeptide in inflammatory and infectious disorders

Circulating levels of pancreatic polypeptide (PP) were found to be elevated when compared to healthy controls in 54% of patients with chronic inflammatory connective tissue disorders (SLE, rheumatoid arthritis, scleroderma, mixed connective tissue disease and temporal arteritis) and in 96% of patients with acute viral or bacterial infections. Significant positive correlations were obtained between the serum values of PP and those of haptoglobin or orosomucoid. Accompanying successful anti-inflammatory treatment of patients with autoimmune disorders, a reduction of PP levels was observed. The findings suggest that the magnitude of increase in PP was associated with the degree of the inflammatory activity. Raised PP levels may contribute to the alterations in carbohydrate and lipid metabolism observed during active inflammatory diseases in man.     

Antibody-induced arthritis: disease mechanisms and genes involved at the effector phase of arthritis

During the development of rheumatoid arthritis (RA) autoantibodies to IgG-Fc, citrullinated proteins, collagen type II (CII), glucose 6 phosphoisomerase (G6PI) and some other self-antigens appear. Of these, a pathogenic effect of the anti-CII and anti-G6PI antibodies is well demonstrated using animal models. These new antibody mediated arthritis models have proven to be very useful for studies involved in understanding the molecular pathways of the induction of arthritis in joints. Both the complement and FcγR systems have been found to play essential roles. Neutrophils and macrophages are important inflammatory cells and the secretion of tumour necrosis factor-α and IL-1β is pathogenic. The identification of the genetic polymorphisms predisposing to arthritis is important for understanding the complexity of arthritis. Disease mechanisms and gene regions studied using the two antibody-induced arthritis mouse models (collagen antibody-induced arthritis and serum transfer-induced arthritis) are compared and discussed for their relevance in RA pathogenesis.