Coevolutionary analysis of Forkhead box protein P3 and its physical binary interactors E3 ubiquitin-protein ligase CHIP, Zfp-90, and nuclear receptor ROR-α.

Forkhead box protein P3 (FOXP3) is known to orchestrate the development and maintenance of T regulatory cells, a cell population specialized in immune suppression and peripheral immune tolerance. FOXP3 activity is fine-tuned through its interaction with several protein-binding partners. By using IntAct database, we retrieved three physical binary interactors: E3 ubiquitin-protein ligase CHIP, Zfp-90, and nuclear receptor ROR-α. Coevolution clusters between FOXP3 and its interactors were identified with the use of iBIS2 algorithm, the iterative version of BIS/BIS2. Most of the coevolving pairs came from some species of monotremes and marsupials, as well as from a group of bats, thus suggesting that protein interactions of FOXP3 with its partners may be changed and/or modulated during mammalian speciation. Furthermore, our analysis would suggest the occurrence of a determinant role of FOXP3 in suppressing pregnancy alloreactions in placental mammals. Similarly, FOXP3, through its interaction with different protein interaction mechanisms, would explain the unique control of inflammatory response to infections in bats. By identifying several inter-protein clusters between the different protein pairs, our findings may provide a guide for new therapeutic approaches to modulate T regulatory suppression and/or enhance immune tolerance.

The S100A7 nuclear interactors in autoimmune diseases: a coevolutionary study in mammals.

S100A7, a member of the S100A family of Ca2+-binding proteins, is considered a key effector in immune response. In particular, S100A7 dysregulation has been associated with several diseases, including autoimmune disorders. At the nuclear level, S100A7 interacts with several protein-binding partners which are involved in transcriptional regulation and DNA repair. By using the BioGRID and GAAD databases, S100A7 nuclear interactors with a putative involvement in autoimmune diseases were retrieved. We selected fatty acid-binding protein 5 (FABP5), autoimmune regulator (AIRE), cystic fibrosis transmembrane conductance regulator (CFTR), chromodomain helicase DNA-binding protein 4 (CHD4), epidermal growth factor receptor (EGFR), estrogen receptor 1 (ESR1), histone deacetylase 2 (HDAC2), v-myc avian myelocytomatosis viral oncogene homolog (MYC), protection of telomeres protein 1 (POT1), telomeric repeat-binding factor (NIMA-interacting) 1 (TERF1), telomeric repeat-binding factor 2 (TERF2), and Zic family member 1 (ZIC1). Linear correlation coefficients between interprotein distances were calculated with MirrorTree. Coevolution clusters were also identified with the use of a recent version of the Blocks in Sequences (BIS2) algorithm implemented in the BIS2Analyzer web server. Analysis of pair positions identified interprotein coevolving clusters between S100A7 and the binding partners CFTR and TERF1. Such findings could guide further analysis to better elucidate the function of S100A7 and its binding partners and to design drugs targeting for these molecules in autoimmune diseases.

Contribution of Immunohistochemistry in Revealing S100A7/JAB1 Colocalization in Psoriatic Epidermal Keratinocyte.

The application of immunohistological methods provides an invaluable contribution in revealing the protein colocalization, which may reflect the occurrence of molecular interaction processes.This chapter describes comprehensive protocols for detection of S100A7/JAB1 colocalization by immunohistochemistry in archival formalin-fixed and paraffin-embedded skin biopsies from healthy and psoriatic subjects. In addition, we provide a protocol for immunocytochemical detection of S100A7/JAB1 colocalization in S100A7 CRISPR-activated human keratinocyte cell line.

S100A7 in Psoriasis: Immunodetection and Activation by CRISPR technology.

Psoriasis, an inflammatory autoimmune skin disease, is the result of a chronic interaction between hyperproliferative keratinocytes and infiltrating activated immune cells. The mechanisms underlying psoriasis pathogenesis remain largely unknown, although a combination of genetic and environmental factors plays an important role in its development. S100A7 is overexpressed in psoriasis, and there is growing evidence that S100A7 may be involved in the pathogenesis of psoriasis. Since the mechanisms underlying S100A7 regulation and function remain elusive, a better understanding of these mechanisms may be useful to uncover additional treatment approaches for psoriasis. Immunohistology provides invaluable tools for a better understanding of the pathogenetic mechanisms of psoriasis. Here, we describe basic methods for immunofluorescence and immunohistochemistry analysis of S100A7 expression in psoriatic patients as well as in S100A7 CRISPR-activated human keratinocyte cell line.

S100A7, Jab1, and p27kip1 expression in psoriasis and S100A7 CRISPR-activated human keratinocyte cell line.

Psoriasis, a chronic immune-mediated inflammatory skin disease, is characterized by dysregulated keratinocyte proliferation. The EF-hand calcium binding protein S100A7 has been found to be overexpressed in psoriatic keratinocytes. It is know that S100A7 may interact with Jab1, a cofactor that stabilizes c-Jun. Jab1 is known to downregulate the expression of the cell cycle inhibitor p27Kip1 in some cancer models. In this study, we aimed to investigate the possible interaction between S100A7 and Jab1 and the downstream effects on p27 Kip1 expression in normal human keratinocyte cells transfected with S100A7 CRISPR activation plasmid and in archival psoriatic skin samples. Our results showed that the upregulated S100A7 colocalizes with Jab1 at the nuclear level in transfected cells and psoriatic skin samples. We also showed a differential protein expression of Jab1 between cytoplasmic and nuclear compartments, thus suggesting Jab1 translocation from nucleus to cytoplasm. p27 Kip1 protein expression patterns would imply a translocation from nucleus and a subsequent degradation of this protein. The upregulation of S1007 and its interaction with Jab1 would contribute to the p27 Kip1 -dependent impaired proliferation that characterizes psoriatic skin.

FOXP3, ICOS and ICOSL gene polymorphisms in systemic sclerosis: FOXP3 rs2294020 is associated with disease progression in a female Italian population.

Systemic sclerosis (SSc), an autoimmune disorder, is characterized by vasculopathy, inflammation, progressive perivascular and interstitial fibrosis. Its pathogenesis is largely unknown, however strong evidences suggest that genetic predisposition may contribute to SSc development. Several gene polymorphisms involved in regulatory T cell function have been identified in many autoimmune diseases, including SSc. Moreover, dysregulation of co-stimulatory and/or co-inhibitory signals, including ICOS signalling, can lead to autoimmunity. The aim of the present study was to investigate the association of the FOXP3 rs2294020, ICOS rs6726035 and ICOSL rs378299 SNPs with both the susceptibility and the progression to SSc in an Italian case-series of patients. SNP genotyping results were successfully obtained from a total of 350 subjects including 166 individuals with SSc and 184 healthy controls. Although analysis tests did not show any significant associations between the SNPs under study and susceptibility to SSc, the occurrence of FOXP3 rs2294020 in female patients was associated with decreased time to progression from early to definite SSc (allelic model: HR=1.43; CI=1.03-1.99; p=0.03; dominant model: HR=1.54; CI=1.04-2.28; p=0.03). The inclusion of presence of ACA autoantibodies in the model did not significantly change the estimates. No conclusions can be drawn for the susceptibility to the disease or the time to progression in men due to the low statistical power. This study provides evidence of the association of rs2294020 with SSc evolution in female patients, modulating the time of progression from the diagnosis of early SSc to the diagnosis of definite SSc, while no effect on SSc susceptibility per se was found. rs2294020 may be considered a disease-modifying gene-variant rather than a disease-susceptibility SNP in SSc.

Obesity, Type 1 Diabetes, and Psoriasis: An Autoimmune Triple Flip.

Obesity, type 1 diabetes, and psoriasis are wide-ranging health problems. Genetics, epigenetics, and environmental factors together with immune disturbances are involved in these diseases. The white adipose tissue is an active endocrine organ, secreting a wide variety of soluble mediators called adipokines that have a central role in the relationship between adipose tissue and immune system. Inflammatory cytokines, including the IL-23/IL-17 and IL-18 axes, and microRNAs are involved in many processes, including immunity and inflammation, thus having a major role in the onset of these three diseases. In this review, we present an overview of the roles of adipokines, cytokines, and microRNAs in the pathogenesis and the progression of these three diseases.

Association between rs2294020 in X-linked CCDC22 and susceptibility to autoimmune diseases with focus on systemic lupus erythematosus.

Autoimmune diseases often share common susceptibility genes. Most genetic variants associated with susceptibility to systemic lupus erythematosus are also associated with other autoimmune diseases. The X-linked variant rs2294020 is positioned in exon 7 of the CCDC22 gene. The encoded protein functions in the regulation of NF-κB, a master regulator in immune response. The aim of this study is to investigate whether the rs2294020 polymorphism may be a general susceptibility factor for autoimmunity. We evaluated case-control association between the occurrence of rs2294020 and different autoimmune diseases, including new data for systemic lupus erythematosus and previous genome-wide association studies (GWAS) (though most did not analyse the X chromosome) of psoriasis, celiac disease, Crohn's disease, ulcerative colitis, multiple sclerosis, vitiligo, type-1 diabetes, rheumatoid arthritis, and ankylosing spondylitis. Cases from patients affected by amyotrophic lateral sclerosis and type-2 diabetes were also included in the study. We detected nominal significant associations of rs2294020 with systemic lupus erythematosus (additive model test: p=0.01), vitiligo (p=0.016), psoriasis (p=0.038), and in only one of two studies of multiple sclerosis (p=0.03). Our results suggest that rs2294020 is associated with the risk of several autoimmune diseases in European populations, specifically with diseases that present themselves, among else, in the skin.

S100A7: A rAMPing up AMP molecule in psoriasis.

S100A7 (psoriasin), an EF-hand type calcium binding protein localized in epithelial cells, regulates cell proliferation and differentiation. An S100A7 overexpression may occur in response to inflammatory stimuli, such in psoriasis, a chronic inflammatory autoimmune-mediated skin disease. Increasing evidence suggests that S100A7 plays critical roles in amplifying the inflammatory process in psoriatic skin, perpetuating the disease phenotype. This review will discuss the interactions between S100A7 and cytokines in psoriatic skin. Furthermore, we will focus our discussion on regulation and functions of S100A7 in psoriasis. Finally, we will discuss the possible use of S100A7 as therapeutic target in psoriasis.

Effects of adalimumab, etanercept and ustekinumab on the expression of psoriasin (S100A7) in psoriatic skin.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease. It is characterized by immune cell activation and altered epidermal differentiation. S100A7 (psoriasin) is overexpressed in psoriasis, suggesting a determinant role of this protein in inflammation and keratinocyte differentiation. OBJECTIVE: The purpose of this study was to investigate the expression of S100A7 in the skin from psoriatic patients undergoing biological therapy with adalimumab, etanercept or ustekinumab. METHODS: S100A7 expression and distribution were analyzed by immunohistochemistry. RESULTS: S100A7, overexpressed in epidermal keratinocytes of psoriatic lesions, was downregulated, under the biological therapy with adalimumab, etanercept or ustekinumab, only in patients achieving a PASI score<15. CONCLUSIONS: Dysregulation of S100A7 may represent a non-negligible player in the maintenance of psoriasis and the relative epidermal changes. Blockage of S100A7 may represent an additional therapeutic approach in the treatment of psoriasis.

Biological therapy induces expression changes in Notch pathway in psoriasis.

Psoriasis is a chronic inflammatory skin disease, characterized by hyperproliferation of keratinocytes and by skin infiltration of activated T cells. To date, the pathophysiology of psoriasis has not yet been fully elucidated. The Notch pathway plays a determinant role in cell fate determination, proliferation, differentiation, immune cell development and function. Many biological agents, used in the treatment of psoriasis, include TFN-α inhibitors, such as etanercept, adalimumab, and anti IL-12/IL-23 p40 antibody, such as ustekinumab. This study aimed to determine mRNA expression levels by real-time RT-PCR, and protein expression levels, analysed by Western blot and immunohistochemistry, of some components of the Notch pathway, such as NOTCH1, NOTCH2, JAGGED1, and HES1 after biological treatments in psoriatic patients. mRNA and protein levels of NOTCH1, NOTCH2, JAGGED1 and HES1 were upregulated in skin samples from untreated psoriatic patients compared with normal controls. Biological therapy showed to downregulate differently the protein expression levels of the molecules under study. Our study suggests that Notch pathway components might be a potential therapeutic target against psoriasis.

The sex bias in systemic sclerosis: on the possible mechanisms underlying the female disease preponderance.

Systemic sclerosis is a multifactorial and heterogeneous disease. Genetic and environmental factors are known to interplay in the onset and progression of systemic sclerosis. Sex plays an important and determinant role in the development of such a disorder. Systemic sclerosis shows a significant female preponderance. However, the reason for this female preponderance is incompletely understood. Hormonal status, genetic and epigenetic differences, and lifestyle have been considered in order to explain female preponderance in systemic sclerosis. Sex chromosomes play a determinant role in contributing to systemic sclerosis onset and progression, as well as in its sex-biased prevalence. It is known, in fact, that X chromosome contains many sex- and immuno-related genes, thus contributing to immuno tolerance and sex hormone status. This review focuses mainly on the recent progress on epigenetic mechanisms--exclusively linked to the X chromosome--which would contribute to the development of systemic sclerosis. Furthermore, we report also some hypotheses (dealing with skewed X chromosome inactivation, X gene reactivation, acquired monosomy) that have been proposed in order to justify the female preponderance in autoimmune diseases. However, despite the intensive efforts in elucidating the mechanisms involved in the pathogenesis of systemic sclerosis, many questions remain still unanswered.

Genetic polymorphisms of FOXP3 in Italian patients with systemic sclerosis.

A case control study to evaluate the possible influence of FOXP3 polymorphisms (rs3761548 and rs2280883) in the susceptibility of systemic sclerosis in an Italian Caucasian population. Subgroup analysis was also performed to test association between these SNPs and specific disease phenotypes. The study groups consisted of 467 individuals: 228 patients (194 with limited cutaneous form and 34 with diffuse cutaneous form of the disease) and 239 healthy control subjects. Genotyping was performed by high resolution melting analysis. Genotype distribution and allele frequency of the FOXP3 polymorphisms were analyzed statistically, using χ(2) or Fisher exact test. Single-marker analysis of allelic and genotype frequencies revealed that SNP rs3761548 was not associated with systemic sclerosis susceptibility. Analysis of genotype and allele distributions of the rs2280883 genetic variant was associated, only in female subjects with systemic sclerosis, its limited subtype, and anti-centromere autoantibodies. Although these findings require replication in a larger set and other populations, FOXP3 rs2280883 may represent a novel susceptibility locus for systemic sclerosis in female subjects.

Expression and localisation of osteopontin and prominin-1 (CD133) in patients with endometriosis.

In this study, we investigated the expression and localisation of the proteins, osteopontin (OPN) and prominin-1 (CD133), as well as the plasma OPN levels in the endometrium of patients with endometriosis. Samples of ectopic endometriotic lesions and normal endometrium were obtained from 31 women with endometriosis and 28 healthy control subjects. The mRNA and protein expression of OPN and CD133 was analysed by real­time RT-PCR and immunohistochemistry. The plasma levels of OPN were determined by ELISA. Our results revealed that OPN mRNA and protein expression, as well as its release in the blood, was significantly increased in the endometriotic lesions in comparison to normal tissue. Although the presence of CD133+ cells was detected in the normal endometrium, as well as in the endometriosis specimens, a significant quantitative variation of this protein was not demonstrated in the patients with endometriosis. In conclusion, our data indicate that OPN is involved in the development of endometriosis by enhancing the invasiveness, proliferation and survival of endometrial cells in ectopic lesions. CD133 cannot be used as a disease marker for endometriosis, although an involvement of this protein in the pathogenesis of endometriosis cannot be excluded.

Isoproterenol modulates matrix metalloproteinase-2 (MMP-2) and its tissue inhibitor-2 (TIMP-2) in rat parotid gland.

OBJECTIVE: Chronic isoproterenol treatment causes hypertrophy and hyperplasia of rodent salivary glands. Cell-extracellular matrix interactions play a critical role in salivary gland proliferation and matrix metalloproteinases (MMPs) are known to be involved in cell proliferation. The present study was undertaken to investigate the expression of MMP-2 and the tissue inhibitor metalloproteinase (TIMP)-2 in rat parotid gland following isoproterenol treatment. DESIGN: Female Wistar rats were daily treated with isoproterenol (25mg/kg body weight) for 0, 1, 3, and 7 days. Expression of parotid gland MMP-2 and its tissue inhibitor TIMP-2 was analysed by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: Our results suggest that isoproterenol modulates expression of MMP-2 and TIMP-2 mRNAs, as well as their protein expression levels in a time dependent-manner. Interestingly, at day 1 of treatment, MMP-2 and TIMP-2 expression were higher in comparison to untreated gland. At days 3 and 7, we can observe a gradual decrease of mRNA and protein levels of MMP-2 and TIMP-2. CONCLUSIONS: Our results suggest the presence of a isoproterenol-dependent modulation of extracellular matrix components. Such a modulation seems to be associated with ß-adrenergic agonist-induced hyperplasy, occurring during the first 24h of agonist treatment, and hypertrophy of the parotid gland.

Nectin like-5 overexpression correlates with the malignant phenotype in cutaneous melanoma.

NECL-5 is involved in regulating cell-cell junctions, in cooperation with cadherins, integrins and platelet-derived growth factor receptor, that are essential for intercellular communication. Its role in malignant transformation was previously described. It has been reported that transformation of melanocytes is associated with altered expression of adhesion molecules suggesting the potential involment of NECL-5 in melanoma development and prognosis. To shed light on this issue, the expression and the role of NECL-5 in melanoma tissues was investigated by bioinformatic and molecular approaches. NECL-5 was up-regulated both at the mRNA and the protein levels in WM35, M14 and A375 cell lines compared with normal melanocytes. A subsequent analysis in primary and metastatic melanoma specimens confirmed "in vitro" findings. NECL-5 overexpression was observed in 53 of 59 (89.8%) and 12 of 12 (100%), primary melanoma and melanoma metastasis, respectively; while, low expression of NECL-5 was detected in 12 of 20 (60%) benign nevi. A significant correlation of NECL-5 overexpression was observed with most of known negative melanoma prognostic factors, including lymph-node involvement (P = 0.009) and thickness (P = 0.004). Intriguingly, by analyzing the large series of melanoma samples in the Xu dataset, we identified the transcription factor YY1 among genes positively correlated with NECL-5 (r = 0.5). The concordant computational and experimental data of the present study indicate that the extent of NECL-5 expression correlates with melanoma progression.

Association of TIMP-1 +372 SNP with digital ulcer manifestation in female systemic sclerosis patients.

A candidate gene for TIMP-1 gene located on the X-chromosome (rs4898) was selected for a control case study to investigate a possible association of this SNP with the susceptibility to systemic sclerosis and its digit ulcer manifestation. A total of 461 individuals of Italian Caucasian origin (228 SSc patients and 233 healthy control subjects) were genotyped for TIMP-1 +372 T/C single nucleotide polymorphism rs4898. Subgroups were analyzed according to the presence or absence of digital ulcers. The CC genotype and C allele frequencies were significantly lower in female SSc patients than in controls (OR 0.53, CI 0.29-0.96, p=0.03 and OR 0.72, CI 0.53-0.98 p=0.04, respectively). CC genotypes frequency was lower also in female patients with ulcers than those without ulcers (OR 0.37, CI 0.14-1.00, p=0.03). Furthermore, CC genotype and C allele frequencies were lower also in female patients with ulcers in comparison to female healthy control subjects (OR 0.27, CI 0.10-0.70, p=0.004; OR 0.60, CI 0.40-0.89, p=0.01, respectively). The TIMP-1 rs4898 polymorphism may play a protective role in the susceptibility to SSC in females, and in particular to digital ulcer formation.

Evaluation of circulating CD4+CD25+ and liver-infiltrating Foxp3+ cells in HCV-associated liver disease.

In hepatitis C virus (HCV)-associated liver disease, the immune system is unable to clear the viral infection. Previous studies have raised the possibility of an involvement of regulatory T cells (Tregs). In this study, we analysed the peripheral blood from 30 patients with HCV-associated chronic liver disease and 20 healthy controls by flow cytometry for the evaluation of the Treg population [CD4⁺CD25hi forkhead box protein 3 (Foxp3)⁺], as well as the activated/effector CD4⁺ T cells (CD4⁺CD25low) and IFN-γ-secreting cells. We also analysed liver biopsies of the patients by immunohistochemical evaluation of Foxp3⁺ cells. Our results showed higher proportions of CD4⁺CD25low and IFN-γ⁺ cells in the patients than in the controls. By contrast, the proportions of peripheral CD4⁺CD25hi cells did not significantly differ. The 11 patients displaying Foxp3⁺ cells in the liver infiltrates showed significantly higher proportions of peripheral CD4⁺CD25low cells. Moreover, we found lower serum transaminase levels in the patients than in the controls, as shown by Foxp3⁺ immunohistochemistry, although these results were only statistically significant as regards alanine transaminase (ALT). In conclusion, these data suggest that the presence of Tregs infiltrating the liver is associated with high levels of activated/effector T cells in the peripheral blood and lower activity of hepatitis. Therefore, liver-infiltrating Tregs may play a role in limiting tissue damage and may thus support an effective immune response against HCV.

Insight into the role of microRNAs in brain tumors (review).

MicroRNAs (MiRNAs) are small non-coding RNAs able to regulate gene expression at a posttranscriptional level. Recent evidence indicates that they play a crucial role in the initiation and progression of human cancers. In this review we briefly describe microRNA biogenesis and function, giving a more detailed account of the current state of knowledge concerning the role of microRNAs in brain tumors and stem-like tumor cells. MicroRNAs control brain tumor development by regulating multiple biological characteristics such as proliferation, invasion, differentiation and angiogenesis. Research in this field is rapidly spreading and encourages potential applications of microRNAs as diagnostic and prognostic tools, in addition to therapeutic targets and tools, to grant clinical benefits to patients suffering of brain tumors.

Analysis of matrix metalloproteinase-9 gene polymorphism -1562 C/T in patients suffering from systemic sclerosis with and without ulcers.

The objective of this study was to determine whether the matrix metalloproteinase-9 (MMP-9) rs3918242 single nucleotide polymorphism may confer susceptibility to systemic sclerosis (SSc) with and without ulcers in an Italian Caucasian population. The MMP-9 rs3918242 functional polymorphism was genotyped in 461 subjects of Italian Caucasian origin: 228 patients with SSc (92 with and 136 without ulcers) and 233 unrelated healthy individuals. The SNP under study was in Hardy-Weinberg equilibrium in the control population. Genotype and allele distributions between SSc patients, with or without ulcers, were not statistically significant (p>0.05). A significant increase of the genotype C/T was observed in male SSc patients without ulcers when compared to patients with ulcers (P=0.04). The MMP-9 rs3918242 functional polymorphism is not associated with susceptibility to SSc. However, the presence of the polymorphism may have a protective effect on the development of ulcers in SSc male patients.