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Increased expression of the NLRP3 inflammasome components in patients with Behçet's disease.

Kim EH, Park MJ, Park S, Lee ES - J Inflamm (Lond) (2015)

Bottom Line: Also, increased expression of NLRP3 and ASC was observed in 25 BD skin lesions compared to 25 erythema nodosum patients.Compatible with this, secretion of IL-1β by PBMCs stimulated with LPS alone or LPS plus ATP was increased in BD compared to healthy controls, which was suppressed by caspase-1 inhibitor.Our findings suggest the possible link between increased IL-1β secretion and increased expression of NLRP3 inflammasome components in BD patients with skin manifestations.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Ajou University School of Medicine, 164 Worldcup-ro, Yeongtong-Gu, Suwon, 443-380 South Korea ; Present address: Department of Dermatology, Cheil General Hospital and Women's Healthcare Center, Dankook University College of Medicine, Cheonan, South Korea.

ABSTRACT

Background: Behçet's disease (BD) is a systemic inflammatory disease with manifestations including recurrent oral and genital ulcerations, and vasculitis involving the skin, mucosa, joints, eyes, veins, arteries, nervous and gastrointestinal systems. BD is seen as a disease at the crossroad between autoimmune and autoinflammatory syndromes, possibly triggered by an aberrant response to infectious stimuli. The relevance of Gram negative bacteria-mediated oral inflammation with the increased expression of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3), leading to systemic inflammation, prompted us to investigate the expression of NLRP3 inflammasome components and its link with IL-1β hypersecretion.

Findings: When peripheral blood mononuclear cells (PBMCs) from 15 active, 15 stable BD patients and 15 healthy volunteers were stimulated, the basal and LPS-induced expressions of NLRP3 inflammasome components were significantly increased at both mRNA and protein levels in BD patients compared to healthy controls. Also, increased expression of NLRP3 and ASC was observed in 25 BD skin lesions compared to 25 erythema nodosum patients. Compatible with this, secretion of IL-1β by PBMCs stimulated with LPS alone or LPS plus ATP was increased in BD compared to healthy controls, which was suppressed by caspase-1 inhibitor.

Conclusion: Our findings suggest the possible link between increased IL-1β secretion and increased expression of NLRP3 inflammasome components in BD patients with skin manifestations.

No MeSH data available.


Related in: MedlinePlus

The protein and mRNA expression of NLRP3, ASC, caspase-1 is increased in Behçet’s disease (BD). a and b Representative western blot analysis (lane 1–3: healthy control, lane 4–6: stable BD, lane 7–9: active BD) and quantitation of NLRP1, NLRP3, ASC and caspase-1 from cell lysates of freshly isolated PBMCs. β-actin was used as loading control (n = 5 per group). c The mRNA expression of NLRP1, NLRP3, ASC and caspase-1 was measured from freshly isolated PBMCs by real time PCR and normalized against the expression levels of glyceraldehyde 3-phosphate-dehydrogenase. The relative values are shown as a fold change to HC with no treatment (n = 8 per group). d Immunohistochemistry staining of skin lesions of BD patients and control EN lesions with anti-NLRP3 (left) and anti-ASC (right) antibody showed NLRP3 and ASC expression in inflammatory cells infiltrating the subcutaneous tissue (×200). Computer assisted image analysis showed significant increase in NLRP3 and ASC stained area in BD patients. The values are shown as a fold change to EN. The smaller box is a magnified region (×400) (n = 25 per group). Data are represented as mean ± S.D. (*p < 0.05), (−) no treatment. NLRP3: NACHT, LRR, and PYD domains-containing protein 3; PBMCs: peripheral blood mononuclear cells; HC: healthy volunteers; EN: erythema nodosum
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Fig1: The protein and mRNA expression of NLRP3, ASC, caspase-1 is increased in Behçet’s disease (BD). a and b Representative western blot analysis (lane 1–3: healthy control, lane 4–6: stable BD, lane 7–9: active BD) and quantitation of NLRP1, NLRP3, ASC and caspase-1 from cell lysates of freshly isolated PBMCs. β-actin was used as loading control (n = 5 per group). c The mRNA expression of NLRP1, NLRP3, ASC and caspase-1 was measured from freshly isolated PBMCs by real time PCR and normalized against the expression levels of glyceraldehyde 3-phosphate-dehydrogenase. The relative values are shown as a fold change to HC with no treatment (n = 8 per group). d Immunohistochemistry staining of skin lesions of BD patients and control EN lesions with anti-NLRP3 (left) and anti-ASC (right) antibody showed NLRP3 and ASC expression in inflammatory cells infiltrating the subcutaneous tissue (×200). Computer assisted image analysis showed significant increase in NLRP3 and ASC stained area in BD patients. The values are shown as a fold change to EN. The smaller box is a magnified region (×400) (n = 25 per group). Data are represented as mean ± S.D. (*p < 0.05), (−) no treatment. NLRP3: NACHT, LRR, and PYD domains-containing protein 3; PBMCs: peripheral blood mononuclear cells; HC: healthy volunteers; EN: erythema nodosum

Mentions: Previous reports show NLRP3 inflammasome expression is increased in inflammatory diseases [5]. We investigated the protein and mRNA levels of the different components of NLRP3 inflammasomes, and NLRP1 for comparison. The protein expression of NLRP3 inflammasome components in PBMCs of BD patients was analyzed by western blotting (Fig. 1a and b). The mean values of normalized NLRP3, ASC and caspase-1 protein levels were significantly up-regulated in active and stable BD compared with HC. NLRP3, ASC and caspase-1 mRNA expression was significantly up-regulated in active and stable BD compared with HC (Fig. 1c). Next, we examined skin lesions for in situ expression of NLRP3 and ASC to correlate our in vitro findings. NLRP3 and ASC in skin lesions of BD and control EN patients were detected by immunohistochemistry. NLRP3 and ASC was mostly expressed by CD68+ macrophages/monocytes (Additional file 3). Image analysis showed that NLRP3 and ASC expression was significantly increased in BD skin lesions (Fig. 1d). These findings show that mRNA and protein levels of NLRP3 inflammasome components are increased in BD patients.Fig. 1


Increased expression of the NLRP3 inflammasome components in patients with Behçet's disease.

Kim EH, Park MJ, Park S, Lee ES - J Inflamm (Lond) (2015)

The protein and mRNA expression of NLRP3, ASC, caspase-1 is increased in Behçet’s disease (BD). a and b Representative western blot analysis (lane 1–3: healthy control, lane 4–6: stable BD, lane 7–9: active BD) and quantitation of NLRP1, NLRP3, ASC and caspase-1 from cell lysates of freshly isolated PBMCs. β-actin was used as loading control (n = 5 per group). c The mRNA expression of NLRP1, NLRP3, ASC and caspase-1 was measured from freshly isolated PBMCs by real time PCR and normalized against the expression levels of glyceraldehyde 3-phosphate-dehydrogenase. The relative values are shown as a fold change to HC with no treatment (n = 8 per group). d Immunohistochemistry staining of skin lesions of BD patients and control EN lesions with anti-NLRP3 (left) and anti-ASC (right) antibody showed NLRP3 and ASC expression in inflammatory cells infiltrating the subcutaneous tissue (×200). Computer assisted image analysis showed significant increase in NLRP3 and ASC stained area in BD patients. The values are shown as a fold change to EN. The smaller box is a magnified region (×400) (n = 25 per group). Data are represented as mean ± S.D. (*p < 0.05), (−) no treatment. NLRP3: NACHT, LRR, and PYD domains-containing protein 3; PBMCs: peripheral blood mononuclear cells; HC: healthy volunteers; EN: erythema nodosum
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4487834&req=5

Fig1: The protein and mRNA expression of NLRP3, ASC, caspase-1 is increased in Behçet’s disease (BD). a and b Representative western blot analysis (lane 1–3: healthy control, lane 4–6: stable BD, lane 7–9: active BD) and quantitation of NLRP1, NLRP3, ASC and caspase-1 from cell lysates of freshly isolated PBMCs. β-actin was used as loading control (n = 5 per group). c The mRNA expression of NLRP1, NLRP3, ASC and caspase-1 was measured from freshly isolated PBMCs by real time PCR and normalized against the expression levels of glyceraldehyde 3-phosphate-dehydrogenase. The relative values are shown as a fold change to HC with no treatment (n = 8 per group). d Immunohistochemistry staining of skin lesions of BD patients and control EN lesions with anti-NLRP3 (left) and anti-ASC (right) antibody showed NLRP3 and ASC expression in inflammatory cells infiltrating the subcutaneous tissue (×200). Computer assisted image analysis showed significant increase in NLRP3 and ASC stained area in BD patients. The values are shown as a fold change to EN. The smaller box is a magnified region (×400) (n = 25 per group). Data are represented as mean ± S.D. (*p < 0.05), (−) no treatment. NLRP3: NACHT, LRR, and PYD domains-containing protein 3; PBMCs: peripheral blood mononuclear cells; HC: healthy volunteers; EN: erythema nodosum
Mentions: Previous reports show NLRP3 inflammasome expression is increased in inflammatory diseases [5]. We investigated the protein and mRNA levels of the different components of NLRP3 inflammasomes, and NLRP1 for comparison. The protein expression of NLRP3 inflammasome components in PBMCs of BD patients was analyzed by western blotting (Fig. 1a and b). The mean values of normalized NLRP3, ASC and caspase-1 protein levels were significantly up-regulated in active and stable BD compared with HC. NLRP3, ASC and caspase-1 mRNA expression was significantly up-regulated in active and stable BD compared with HC (Fig. 1c). Next, we examined skin lesions for in situ expression of NLRP3 and ASC to correlate our in vitro findings. NLRP3 and ASC in skin lesions of BD and control EN patients were detected by immunohistochemistry. NLRP3 and ASC was mostly expressed by CD68+ macrophages/monocytes (Additional file 3). Image analysis showed that NLRP3 and ASC expression was significantly increased in BD skin lesions (Fig. 1d). These findings show that mRNA and protein levels of NLRP3 inflammasome components are increased in BD patients.Fig. 1

Bottom Line: Also, increased expression of NLRP3 and ASC was observed in 25 BD skin lesions compared to 25 erythema nodosum patients.Compatible with this, secretion of IL-1β by PBMCs stimulated with LPS alone or LPS plus ATP was increased in BD compared to healthy controls, which was suppressed by caspase-1 inhibitor.Our findings suggest the possible link between increased IL-1β secretion and increased expression of NLRP3 inflammasome components in BD patients with skin manifestations.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Ajou University School of Medicine, 164 Worldcup-ro, Yeongtong-Gu, Suwon, 443-380 South Korea ; Present address: Department of Dermatology, Cheil General Hospital and Women's Healthcare Center, Dankook University College of Medicine, Cheonan, South Korea.

ABSTRACT

Background: Behçet's disease (BD) is a systemic inflammatory disease with manifestations including recurrent oral and genital ulcerations, and vasculitis involving the skin, mucosa, joints, eyes, veins, arteries, nervous and gastrointestinal systems. BD is seen as a disease at the crossroad between autoimmune and autoinflammatory syndromes, possibly triggered by an aberrant response to infectious stimuli. The relevance of Gram negative bacteria-mediated oral inflammation with the increased expression of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3), leading to systemic inflammation, prompted us to investigate the expression of NLRP3 inflammasome components and its link with IL-1β hypersecretion.

Findings: When peripheral blood mononuclear cells (PBMCs) from 15 active, 15 stable BD patients and 15 healthy volunteers were stimulated, the basal and LPS-induced expressions of NLRP3 inflammasome components were significantly increased at both mRNA and protein levels in BD patients compared to healthy controls. Also, increased expression of NLRP3 and ASC was observed in 25 BD skin lesions compared to 25 erythema nodosum patients. Compatible with this, secretion of IL-1β by PBMCs stimulated with LPS alone or LPS plus ATP was increased in BD compared to healthy controls, which was suppressed by caspase-1 inhibitor.

Conclusion: Our findings suggest the possible link between increased IL-1β secretion and increased expression of NLRP3 inflammasome components in BD patients with skin manifestations.

No MeSH data available.


Related in: MedlinePlus