Hsa_circ_0012919 promotes pyroptosis in CD4+T cells of systemic lupus erythematous by miR-125a-3p/GSDMD axis

The etiology of systemic lupus erythematous (SLE) remains unclear. Pyroptosis, a new model of programmed cell death, was poorly explored in the pathogenesis of SLE. We found cell pyroptosis in CD4+T cells of SLE patients and kidneys from MRL/lpr mice by examining caspase-1 and gasdermin D (GSDMD) in by RT-PCR, Western blot, and levels of IL-1β, IL-18 and TNF-α were detected by RT-PCR and Elisa. Expression of caspase-1 and GSDMD and levels of IL-1β, IL-18, TNF-α decreased significantly after downregulation of hsa_circ_0012919 (p < 0.05). Inhibition of miR-125a-3p enhanced expression of caspase-1 and GSDMD (p < 0.05), and increased the release of IL-1β, IL-18 and TNF-α (p < 0.05), thereby counteracting the effect of hsa_circ_0012919 knockdown on pyroptosis. Finally, we identified GSDMD as the target gene of miR-125a-3p. Silencing GSDMD reversed the effect of 5-aza-deoxycytidine in increasing release of IL-1β, IL-18, TNF-α and activating caspase-1, but it could be reversed by miR-125a-3p inhibitor. In conclusion, hsa_circ_0012919 regulated the pyroptosis in the CD4+ T cells of SLE patients by miR-125a-3p/GSDMD axis.     

Normal response to tumor necrosis factor-alpha and transforming growth factor-beta by keratinocytes in psoriasis

Abstract Normal and chronic plaque psoriatic keralinocyte cultures were tested for their in vitro response to 2–200 ng/nil TNF-α and 0.1–10 ng/ml TGF-β in a serum-free culture system. All normal and lesional psoriatic epidermal cell cultures showed a dose- and lime-dependent inhibition of growth in response to TNF-α and TGF-β. Inhibition in individual cultures was first seen at a concentration of 2 ng/ml for TNF-α and 0.1 ng/ml for TGF-β at day 2, but became significant at 20 ng/ml and 1 ng/ ml for TNF-α and TGF-β respectively at days 2-6. This effect was statistically significant at days 3–4 for the group of normal (TNF-α and TGF-β, n = 10, p<0.01 and psoriatic cultures (TNF-α. n = 9, p<0.0l; TGF-β, n = 7, p<0.05). Epidermal cells from normal and psoriatic skin were inhibited to the same extent at the same optimal concentrations by each cytokine. Inhibition was abolished by the addition of specific antibody to each cytokine, whilst antibody to a different cytokine had no effect. Nuclear and/or nuclear membrane staining was observed with antibody to the p55 TNF receptor both in cultured keralinocyles and in the tipper epidermal layers of both normal and psoriatic skin. In contrast, plasma membrane and cytoplasmic expression of the p55 TNF receptor was observed on macrophages and lymphocytes infiltrating psoriatic der-mis. This study has shown that the growth of normal and psoriatic keratinocytes was equally inhibited by TNF-α and TGF-βin vitro. The expression of TNF receptor by psoriatic keratinocytes in vivo may permit regulation of epidermal growth by administration of TNF-α in this disease.     

Effects of low concentrations of cis- and trans-urocanic acid on cytokine elaboration by keratinocytes

The urocanic acid cis isomer (cis-UCA) is a possible cutaneous photoreceptor for the immunomodulatory phenomena that follow ultraviolet B irradiation. Several experiments in animals show an inhibitory action of cis-UCA on cellular immunity. However, the action of cis-UCA on the synthesis of cytokines in keratinocytes remains unknown. Long-term cultures of normal human keratinocytes were prepared in a serum-free medium, and stimulated with 1 μg/ml of phorbol 12-myristate 13-acetate (TPA) and UCA or UVB-UCA (10–100 μg/ml). Synthesis of the following cytokines was measured using ELISA and Northern blot techniques: TNF-α, IL-1α, IL-1β, IL-6, IL-8 and TGF-β1. TPA increased TNF-α protein levels in culture supernatants. No changes in IL-1α and IL-1β protein levels were detected in basal culture supernatant after TPA stimulus. TPA augmented RNA expression for TNF-α, IL-1α, IL-1β and TGF-β1. UCA isomers did not induce cytokine changes in protein synthesis. Expression of IL-1α and IL-1β genes was increased after exposure to 100 μg/ml UVB-UCA (70 μg/ml cis-UCA). A slight increase in TNF-α RNA expression was detected when the dose of UVB-UCA reached 100 μg/ml. No effects on cytokine synthesis were found after UCA stimulus. These results suggest that low doses of cis-UCA do not effect cytokine synthesis by keratinocytes.     

Genetic support for the role of the NLRP3 inflammasome in psoriasis susceptibility

NACHT leucine‐rich repeat‐ and PYD‐containing (NLRP)3 protein controls the inflammasome by regulating caspase‐1 activity and interleukin (IL)‐1β processing. The contribution of IL‐1β in the pathogenesis of psoriasis is well recognized. Polymorphisms in NLRP3 and caspase recruitment domain–containing protein (CARD)8, a negative regulator of caspase‐1 activity, have been associated with susceptibility to common inflammatory diseases, such as Crohn's disease and rheumatoid arthritis. To investigate the role for genetic variants in the NLRP3 inflammasome in psoriasis susceptibility. In a patient sample comprising 1988 individuals from 491 families and 1002 healthy controls, genotypes for four selected single‐nucleotide polymorphisms (SNPs) in NLRP3 (three SNPs) and CARD8 (one SNP) were determined by TaqMan^® Allelic Discrimination. Using the transmission disequilibrium test (TDT), a significant increase in the transmission of the NLRP3 rs10733113G genotype to a subgroup of patients with more widespread psoriasis was demonstrated (P = 0.015). Using logistic regression analysis in 741 patients with psoriasis and 1002 controls, the CARD8 rs2043211 genotype was significantly different in cases and controls in overall terms [OR 1.3 (1.1–1.5), P = 0.004] and for both genders. Our data support the hypothesis that the inflammasome plays a role in psoriasis susceptibility.     

Tumour necrosis factor alpha is pro-inflammatory in normal human skin and modulates cutaneous adhesion molecule expression

Tumour necrosis factor a (TNF-α) is a potent immunoregulatory cytokine produced by many cutaneous cells, including kcratinocytes, mast cells and Langerhans cells. To explore its potential role in inflammatory skin disease, we have studied immunohistochemically the effects of intradermal recombinant human TNF-α (rHuTNF-α) on cutaneous inflammatory cells, adhesion molecules and Langerhans cells in normal human skin. Volunteers received rHuTNF-α 100U (group A), 5000 U (group B), or 100 U daily for 5 days (group C), and biopsies were taken at 6 h (groups A and B), or 6 h after the final injection (group C). An inflammatory cell infiltrate developed in all cases: following single injections of either 100 or 5000 U rHuTNF-α this was predominantly neutrophilic, whereas following multiple injections of 100 U few neutrophils were seen, although many lymphocytes (CD3⁺, CD4⁴) were present. In all groups there was an increase in cells of monocyte/macrophage lineage (CD36⁾⁺. TNF-α induced a dose- and time-dependent decrease in CDla⁺ epidermal Langerhans cell numbers and an increase in dermal CDla⁴ cells, suggesting migration of Langerhans cells away from the epidermis. TNF-α induced endothelial E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in all groups, and adhesion molecule expression by interstitial dermal dendritic cells (ICAM-1 and VCAM-1) and keratinocytes (ICAM-1) was observed. These findings indicate that TNF-α is a potent modulator of cutaneous immune function in vivo, and this central role in the cutaneous immune response suggests that TNF-α may be an attractive target for therapeutic inhibition.     

The Expression of Endothelin-1 and Its Binding Sites in Mouse Skin Increased after Ultraviolet B Irradiation or Local Injection of Tumor Necrosis Factor α

Endothelin (ET)-1 is a 21-amino acid peptide which has vasoconstrictor and growth regulatory activity. Recently, cultured keratinocytes have been reported to express ET-1 and its receptor when irradiated by ultraviolet (UV) B. In order to further understand the role of ET-1 in vivo during UVB-induced inflammation, we examined the localization, intensity and time course of the expression levels of ET-1 and its binding sites in UVB-exposed BALB/c mouse skin. Frozen and paraffin sections prepared from mouse skin 48 h after treatment with UVB irradiation (0.36 or 0.72 J/cm²) or after injection with tumor necrosis factor (TNF)-α (1.0 μg) or interleukin (IL)-1α (0.05 μg) were incubated with monoclonal anti-ET-1 IgG and then visualized by peroxidase staining. In normal skin, faint ET-1 immunoreactivity was observed in the epidermis, pilosebaceous structures and blood vessels. Upon exposure to UVB irradiation or administration of TNF-α injection or IL-1α injection, such immunoreactivity was found to be significantly enhanced. Subsequently, the frozen sections were incubated with ¹²⁵I ET-1 for 30 min, and visualized by autoradiographic technique. In normal skin, ET-1 weakly bound to the skin, while UVB irradiation and TNF-α injection significantly enhanced ET-1 binding in the epidermis, pilosebaceous structures and blood vessels. Time course experiments (1, 2, 4 and 7 days) indicated that ET-1 immunoreactivity and ET-1 binding peaked 1 or 2 days after UVB irradiation or TNF-α injection. These results suggest that the up-regulated expression of ET-1 and its binding sites in the epidermis and pilosebaceous structures may act as an autocrine/paracrine factor during UVB-induced inflammation.     

Muckle–Wells syndrome without mutation in exon 3 of the NLRP3 gene,identified by evidence of excessive monocyte production of functional interleukin 1β and rapid response to anakinra

We report a man with lifelong urticaria, night sweats, arthralgia and lethargy. He had high levels of inflammatory markers and serum amyloid A, but no identifiable mutation in exon 3 of the NLRP3 (NOD‐like receptor family, pyrin domain‐1 containing 3) gene, and no relevant family history. We found marked production of functional interleukin (IL)‐1 by the patient's monocytes at baseline and after stimulation with lipopolysaccharide. The patient made an immediate response to treatment with an IL‐1β receptor antagonist. We propose that this patient has Muckle–Wells syndrome without deafness, occurring de novo. Functional screening for IL‐1 production could aid diagnosis in future similar cases.     

Inhibitory effect of azelastine, a potent antiallergic agent, on release of tumor necrosis factor-α from activated human peripheral blood mononuclear cells and U937 cells

Abstract It is generally accepted that tumor necrosis factor-α (TNF-α) is a multifunctional cytokine which is involved in the regulation of inflammation as well as immunity. In the present study, we investigated whether azelastine, a potent antiallergic agent, affects release of TNF-α from peripheral blood mononuclear cells (PBMC) and U937 cell line in vitro. When human PBMC and U937 cells were stimulated by phytohemagglulinin (PHA) and 12-0-letradecanoyl-phorbol-13-acetate (TPA). respectively, the cells released significant amounts of TNF-α as determined by TNF-α-specific enzyme immunoassay. TNF-α levels in the culture supernatant of PHA-stimulated human PBMC and TPA-activated U937 cells decreased in a dose-dependent manner when these cells were cultured in the presence of azelastine. This inhibitory effect of azelastine was obtained at concentrations where the drug produced no toxicity. Moreover, azelastine also inhibited release of TNF-α from U937 cells which were already activated by TPA. These results suggest that the inhibitory effect of azelastine on TNF-α release plays an important role in its antiallergic action in addition to inhibition and/or antagonism of histamine and leukolriens, which has been previously reported.     

Contrasting effects of ultraviolet A1 and ultraviolet B exposure on the induction of tumour necrosis factor-α in human skin

Ultraviolet B (UVB) irradiation of the skin causes immunosuppression which is relevant to the induction of skin cancer. The mechanism of this immunomodulation is unclear but various regulatory molecules have been implicated, including cis-urocanic acid (cis-UCA) and the cytokines tumour necrosis factor-α (TNF-α) and interleukin 10 (IL-10). Whether ultraviolet A (UVA) induces similar changes has not been investigated fully. We studied the effect of in vivo UVB and long-wave UVA (UVA1) exposure on the induction of TNF-α, IL-10 and cis-UCA in human skin. Volunteers were irradiated with three minimal erythema doses (MED) of UVB or UVA1. At different times after irradiation, suction blisters were raised from irradiated and from non-irradiated (control) skin. The TNF-α and IL-10 protein concentration, and the percentage of cis-UCA in the blister fluid, were then determined. UVB irradiation of human skin led to a rapid and significant increase in TNF-α concentration in suction-blister fluid, with maximal values 6 h after irradiation (n = 6, P < 0.05). In contrast, UVA1 irradiation led to a decrease in TNF-α concentration in the suction-blister fluid compared with non-irradiated skin, with the lowest values 6 h after irradiation (n = 6, P < 0.05). Both UVB and UVA1 exposure of the skin induced a slight increase in IL-10 concentration. However, the increase in IL-10 was only significant after UVB irradiation (UVB, n = 6, P < 0.05; UVA, n = 7, P < 0.1). As previously shown, both UVB and UVA1 result in the photo-isomerization of trans-UCA and an increased percentage of cis-UCA was found in the suction-blister fluid. Thus the results show differential effects of UVB and UVA1 irradiation on the induction of immunoregulatory molecules, which may help to explain the variation in immune responses after UVB and UVA1 exposure of human skin.     

Presentation of cryopyrin-associated periodic fever syndrome as chronic,afebrile urticaria in a 12-month-old female

A healthy 12-month-old female presented with relapsing and remitting urticaria since birth that was resistant to treatment with antihistamines. A thorough history revealed extensive rheumatic disease on the father's side of the family, and subsequent genetic testing was positive for a missense variant of NLRP3, indicating cryopyrin-associated periodic fever syndrome (CAPS). CAPS encompasses a spectrum of diseases, all related to a defect in the same gene; manifestations vary in severity and presentation, but most are associated with recurrent rash and fever. Because the patient's only presenting symptom was rash, this case highlights the importance of having a high index of suspicion for cryopyrin-associated periodic fever syndrome in infants with persistent, early urticaria.     

Evaluation of plasma zonulin level and its relationship with inflammatory cytokines in patients with vitiligo

Background

It has been proven that there is an increase in intestinal permeability in some autoimmune diseases. In our study, we purposed to assess intestinal permeability in vitiligo disease by looking at zonulin levels. At the same time, we aimed to examine the correlation of inflammatory cytokines and lipopolysaccharide (LPS) levels with zonulin.

Methods

Forty-one patients and 41 healthy participants were involved in our study. Blood samples were taken from all patients and controls, and the levels of zonulin, tumor necrosis factor (TNF)-α, interleukin (IL)-6 and LPS were examined.

Results

The mean of zonulin in the patient group was found to be statistically higher than the control group (p < 0.05). A positive correlation was found between zonulin level and IL-6, TNF-α, and LPS levels (p < 0.05). TNF-α and LPS levels in the vitiligo group were significantly higher than in the control group, but there was no such significance in terms of IL-6 levels.

Conclusion

We think that serum zonulin level increases and intestinal permeability increases in vitiligo disease.     

Fas-FasL interaction in cytotoxic T cell-mediated vitiligo: The role of lesional expression of tumor necrosis factor-α and interferon-γ in Fas-mediated melanocyte apoptosis

Vitiligo is an acquired skin depigmentation disorder resulting from the selective loss of epidermal melanocytes, and previous studies have suggested that a T lymphocyte-mediated mechanism has a role in melanocyte loss. Although Fas-Fas ligand (FasL) interactions are important for T lymphocytes to mediate cytotoxicity, there are only few reports examining the involvement of the Fas-FasL pathway in vitiligo using in vivo mouse models. In addition, there have been no reports concerning Fas-mediated apoptosis in human melanocytes in vitro. In this study, we found that the Fas-mediated pathway is involved in cytotoxic T lymphocyte (CTL)-dependent vitiligo in a mouse model and FasL-induced apoptosis of human melanocytes. Tumor necrosis factor (TNF)-α and interferon (IFN)-γ, the expression levels of which have been reported to be elevated in lesional skin of patients with vitiligo, synergistically upregulated Fas expression on human melanocytes but inhibited the Fas-mediated apoptosis. Treatment with TNF-α and IFN-γ synergistically upregulated the expression of the anti-apoptotic genes, c-IAP2, c-FLIP and MCL1. A siRNA knock-down study showed that c-FLIP and MCL1, but not c-IAP2, were involved in inducing synergistic inhibitory effects on Fas-mediated apoptosis. Furthermore, we found that FasL and TNF-related apoptosis - inducing ligand (TRAIL) synergistically induced apoptosis on human melanocytes. In conclusion, our results suggest that the Fas-FasL pathway is involved in CTL-dependent vitiligo and the elevated expression levels of TNF-α and IFN-γ in lesional skin may act synergistically on melanocytes to suppress Fas-mediated apoptosis.     

Resistance of Sézary cells to TNF-α-induced apoptosis is mediated in part by a loss of TNFR1 and a high level of the IER3 expression

Failure to execute an apoptotic programme is one of the critical steps and a common mechanism promoting tumorogenesis. Immediate early responsive gene 3 (IER3) has been shown to be upregulated in several cancers. IER3 is a stress-induced gene, which upregulation leads to reduction in production of reactive oxygen species (ROS) protecting malignant cells from apoptosis. We observed that malignant lymphocytes from patients with Sézary syndrome (SzS) were resistant to pro-apoptotic dose of tumour necrosis factor-α (TNF-α). The aim of this study was to investigate the role of IER3 in the mechanism of such resistance. CD4+ CD26- lymphocytes from the peripheral blood of patients with SzS and healthy controls were negatively selected using CD4 and CD26 magnetic beads and analysed for expression of TNFR1, TNFR2, IER3 expression, and ROS production in response to TNF-α at an apoptotic dose. Sézary cells with a higher level of IER3 expression retained their viability to TNF-α. IER3 upregulation correlated with a decrease level of intracellular ROS and low TNFR1 expression on malignant cells. Targeting IER3 could be of interest for the development of future therapeutic strategies for patients with SzS.     

TLR4 and NLRP3 inflammasome activation in monocytes by N-propionyl cysteaminylphenol-maleimide-dextran (NPCMD)

BackgroundN-propionyl cysteaminylphenol-maleimide-dextran (NPCMD) is a toxic tyrosinase substrate developed to treat melanoma.ObjectiveWe investigated the effect of NPCMD on innate immune responses in monocytes.MethodsCD14⁺ monocytes and a monocytic cell line, THP-1, were stimulated with NPCMD in vitro. Cytokines in the culture supernatants were determined by ELISA and flow cytometry.ResultsNPCMD stimulated CD14⁺ monocytes and THP-1 cells to secrete TNFα, IL-6 and IL-8, but not IL-10 or IL-12. TNFα secretion from THP-1 cells stimulated with NPCMD was inhibited by addition of an anti-TLR4 mAb in culture. Moreover, NPCMD stimulated production of pro-IL-1β in CD14⁺ monocytes and monocytic cell line THP-1 cells and activated the NLRP3-inflammasome, resulting in production of mature IL-1β. Use of ASC and NLRP3-deficient THP-1 cell lines established involvement of the NLRP3 inflammasome in an IL-1β secretion in treatment with NPCMD. Inhibition of IL-1β secretion by an endocytosis inhibitor, cytochalasin B, and a lysosomal enzyme cathepsin B inhibitor, CA-074 Me, suggested the involvement of lysosomal rupture and leakage of cathepsin B into the cytosol in NLRP3 activation by NPCMD.ConclusionThe immunopotentiating effect of NPCMD mediated by TLR4 and NLRP3 inflammasome activation could be useful for eliciting effective adaptive immune responses against melanoma and other tumors.     

CIAS1 mutation in a patient with overlap between Muckle-Wells and chronic infantile neurological cutaneous and articular syndromes

The Muckle-Wells syndrome is a rare autosomal dominant disorder belonging to the group of hereditary fever syndromes. The chronic infantile neurological cutaneous and articular (CINCA) syndrome is a systemic inflammatory disorder of unknown etiology with neonatal onset. They are considered as two different entities. We report the case of a 36-year-old man suffering since birth from a nonpruritic generalized urticaria, with inflammatory flares, joint manifestations and progressive deafness requiring a bilateral hearing aid. An initial diagnosis of Muckle-Wells syndrome was made. However, the patient had an unusual clinical presentation with slightly dysmorphic facial appearance, clubbing of the fingers, mild mental retardation and papilledema. After a genetic advice, a diagnosis of CINCA syndrome was made. Search for mutations in the CIAS1 gene revealed a new mutation in a heterozygous state. This case report really raises the question of a link between these two inflammatory diseases. Further studies are needed to confirm the involvement of mutations of the CIAS1 gene in CINCA syndrome.     

Exacerbation of skin lesions during fever in a patient with chronic infantile neurologic cutaneous articular (CINCA) syndrome

Chronic infantile neurologic cutaneous articular (CINCA) syndrome is a serious chronic systemic inflammatory disease that presents at a young age and that is characterized by skin, joint, and central nervous system disease. Skin symptoms are the first to appear, in the form of a longstanding nonpruritic urticarial rash, with exacerbations coinciding with episodes of fever, arthritis, and enlarged lymph nodes. The findings of biopsy of skin lesions are extremely variable but characterized by perivascular neutrophilic infiltrate. With the discovery of mutations in the CIAS1 gene, which encodes a protein known as cryopyrin, this entity has been classified as one of the cryopyrin-associated autoinflammatory diseases, along with familial cold urticaria and Muckle-Wells syndrome. This discovery has also made available new therapeutic options. We present the case of a boy diagnosed with CINCA syndrome who presented with an outbreak of painful skin lesions and fever. These lesions were thought to be an exacerbation of underlying lesions during an episode of fever.