Journal: Frontiers in Medicine

Article Title: Promoter Methylation Leads to Decreased ZFP36 Expression and Deregulated NLRP3 Inflammasome Activation in Psoriatic Fibroblasts

doi: 10.3389/fmed.2020.579383

Figure Lengend Snippet: (A–D) Relative expression of selected genes, obtained from a publicly available database. Expression of each gene is shown in healthy skin and in lesional psoriatic skin. Boxplots report ±SEM (*** p < 0.001). (A) ZFP36 expression in psoriatic skin samples is significantly reduced, compared to healthy skin samples [Healthy donor: n = 21, median 10.83, range 8.59–12.85; Lesional psoriatic fibroblasts (PSO): n = 33, median 9.23, range 8.79–9.78]. (B) NLRP3 levels show no significant variation among the two groups of healthy and psoriatic skin (Healthy donor: n = 21, median 2.73, range 2.58–3.13; Lesional PSO: n = 33, median 2.80, range 2.56–3.13). (C) CASP1 expression results increased in lesional psoriatic samples compared to normal skin samples (Healthy donor: n = 21, median 6.88, range 5.13–8.52; Lesional PSO: n = 33, median 8.00, range 5.45–8.77). (D) IL1B is significantly more expressed in lesional skin samples from psoriatic patients than skin from healthy donors (Healthy donor: n = 21, median 4.04, range 3.69–6.67; Lesional PSO: n = 33, median 5.05, range 4.06–7.03). (E,F) Relative quantity of mRNA measured by real-time (RT) PCR in healthy and psoriatic fibroblasts samples. (E) Differential expression of TTP mRNA in psoriatic and healthy fibroblasts, measured through qRT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as endogenous control. Results are represented as means of three experiments (±SEM) (* p < 0.05). (F) IL1β mRNA levels in psoriatic and healthy fibroblasts, measured through qRT-PCR. GAPDH was used as endogenous control. Results are represented as the means of three experiments (±SEM) (** p < 0.01).

Article Snippet: Antibodies used were actin (MAB1501, clone 4, Millipore Corporation, Billerica, MA, USA), vinculin (AB6039, Millipore Corporation, Billerica, MA, USA), NLRP3 (bs10021R, Bioss, Thermo Fisher Scientific, Waltham, MA, USA), IL-1β (ab34837, Abcam, Cambridge, UK), caspase-1 (#2225S, Cell Signaling Technologies, Danvers, MA, USA), TTP (#71632S Cell Signaling Technologies, Danvers, MA, USA), ASC (AF3805, R&D systems, Minneapolis, MN, USA), and tubulin (MAB1864, clone YL1/2, Millipore Corporation, Burlington, MA, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in Medicine

Article Title: Promoter Methylation Leads to Decreased ZFP36 Expression and Deregulated NLRP3 Inflammasome Activation in Psoriatic Fibroblasts

doi: 10.3389/fmed.2020.579383

Figure Lengend Snippet: (A) Tristetraprolin (TTP) protein levels in healthy and psoriatic fibroblasts. Two different healthy samples and two different psoriatic samples were divided into two series. In “Fibroblasts II series,” psoriatic cell samples from the same donor are distinguished in lesional and non-lesional. Vinculin was used as loading control. (B) NLRP3, pro-IL1β, and IL1β (p17) protein levels in healthy and psoriatic fibroblasts. (C) Pro-CASP1 and active CASP1 (p20) protein levels in healthy and psoriatic fibroblasts. Vinculin was used in (B,C) as a loading control.

Article Snippet: Antibodies used were actin (MAB1501, clone 4, Millipore Corporation, Billerica, MA, USA), vinculin (AB6039, Millipore Corporation, Billerica, MA, USA), NLRP3 (bs10021R, Bioss, Thermo Fisher Scientific, Waltham, MA, USA), IL-1β (ab34837, Abcam, Cambridge, UK), caspase-1 (#2225S, Cell Signaling Technologies, Danvers, MA, USA), TTP (#71632S Cell Signaling Technologies, Danvers, MA, USA), ASC (AF3805, R&D systems, Minneapolis, MN, USA), and tubulin (MAB1864, clone YL1/2, Millipore Corporation, Burlington, MA, USA).

Techniques:

Journal: Frontiers in Medicine

Article Title: Promoter Methylation Leads to Decreased ZFP36 Expression and Deregulated NLRP3 Inflammasome Activation in Psoriatic Fibroblasts

doi: 10.3389/fmed.2020.579383

Figure Lengend Snippet: (A) qRT-PCR shows a decrease in NLRP3 mRNA in HaCaT cells transfected with a TTP-expressing vector (pcDNA3.1-ZFP36), compared to control transfection (pcDNA3.1). GAPDH was used as endogenous control. Results are represented as the means of three experiments (±SEM) (** p < 0.01). (B) Luciferase reporter assay shows a decrease in luciferase activity in HEK293T cell line cotransfected with luciferase reporter vector and the TTP-expressing vector, compared to the control cotransfection with the empty vector. Measured luciferase activity was normalized over β-gal signals. Results are represented as the means of three experiments (±SEM) (* p < 0.05). (C) Human NLRP3 3′UTR displaying TTP binding site (highlighted). (D) NLRP3 mRNA levels measured by qRT-PCR in HaCaT cells transfected at time 0 with a TTP-overexpressing vector or an empty vector. After transfection (48 h), cells were treated with Act.D to block transcription. NLRP3 mRNA levels were recorded at the moment of ActD treatment (Ctr), after 1 h and after 3 h. Results are represented as the means of three experiments (±SEM). Statistical analysis was performed comparing ZFP36-transfected and empty vector-transfected cells at each timepoint (* p < 0.05). GAPDH was used as endogenous control.

Article Snippet: Antibodies used were actin (MAB1501, clone 4, Millipore Corporation, Billerica, MA, USA), vinculin (AB6039, Millipore Corporation, Billerica, MA, USA), NLRP3 (bs10021R, Bioss, Thermo Fisher Scientific, Waltham, MA, USA), IL-1β (ab34837, Abcam, Cambridge, UK), caspase-1 (#2225S, Cell Signaling Technologies, Danvers, MA, USA), TTP (#71632S Cell Signaling Technologies, Danvers, MA, USA), ASC (AF3805, R&D systems, Minneapolis, MN, USA), and tubulin (MAB1864, clone YL1/2, Millipore Corporation, Burlington, MA, USA).

Techniques: Quantitative RT-PCR, Transfection, Expressing, Plasmid Preparation, Luciferase, Reporter Assay, Activity Assay, Cotransfection, Binding Assay, Blocking Assay

Journal: Frontiers in Medicine

Article Title: Promoter Methylation Leads to Decreased ZFP36 Expression and Deregulated NLRP3 Inflammasome Activation in Psoriatic Fibroblasts

doi: 10.3389/fmed.2020.579383

Figure Lengend Snippet: (A) TTP silencing achieved by lentiviral infection (sample labeled shZFP36) was verified by Western blot, in comparison to endogenous TTP levels measured after infection with a vector expressing an aspecific scrambled short hairpin (labeled EV). Actin was used as loading control. (B) Western blot displaying the expression levels of inflammasome components. Inflammasome interactor levels vary after lentiviral infection with a short hairpin directed against TTP mRNA. Vinculin is included as loading control. (C) NLRP3, IL1β, and TTP mRNA levels have been analyzed by qRT-PCR in both cell samples. GAPDH was used as endogenous control. Results are represented as the means of three experiments (±SEM).

Article Snippet: Antibodies used were actin (MAB1501, clone 4, Millipore Corporation, Billerica, MA, USA), vinculin (AB6039, Millipore Corporation, Billerica, MA, USA), NLRP3 (bs10021R, Bioss, Thermo Fisher Scientific, Waltham, MA, USA), IL-1β (ab34837, Abcam, Cambridge, UK), caspase-1 (#2225S, Cell Signaling Technologies, Danvers, MA, USA), TTP (#71632S Cell Signaling Technologies, Danvers, MA, USA), ASC (AF3805, R&D systems, Minneapolis, MN, USA), and tubulin (MAB1864, clone YL1/2, Millipore Corporation, Burlington, MA, USA).

Techniques: Infection, Labeling, Western Blot, Plasmid Preparation, Expressing, Quantitative RT-PCR

Journal: Frontiers in Medicine

Article Title: Promoter Methylation Leads to Decreased ZFP36 Expression and Deregulated NLRP3 Inflammasome Activation in Psoriatic Fibroblasts

doi: 10.3389/fmed.2020.579383

Figure Lengend Snippet: (A) NLRP3 inflammasome protein levels are displayed by Western blot in three different fibroblast cell populations: healthy fibroblasts (Healthy), psoriatic fibroblasts (PSO), and psoriatic fibroblasts infected with a pRRL-TTP-overexpressing vector (PSO TTP). Tubulin was used as loading control. (B) TTP overexpression is shown in psoriatic fibroblast sample. Actin was used as loading control. (C) Inflammasome components variation pattern already seen in primary fibroblasts is maintained in the HaCaT keratinocyte cell line. Actin was used as loading control. (D) pRRL-empty vector was used to obtain the overexpression vector pRRL-TTP, by substitution of ΔNGFR sequence with TTP cDNA.

Article Snippet: Antibodies used were actin (MAB1501, clone 4, Millipore Corporation, Billerica, MA, USA), vinculin (AB6039, Millipore Corporation, Billerica, MA, USA), NLRP3 (bs10021R, Bioss, Thermo Fisher Scientific, Waltham, MA, USA), IL-1β (ab34837, Abcam, Cambridge, UK), caspase-1 (#2225S, Cell Signaling Technologies, Danvers, MA, USA), TTP (#71632S Cell Signaling Technologies, Danvers, MA, USA), ASC (AF3805, R&D systems, Minneapolis, MN, USA), and tubulin (MAB1864, clone YL1/2, Millipore Corporation, Burlington, MA, USA).

Techniques: Western Blot, Infection, Plasmid Preparation, Over Expression, Sequencing

Journal: Aging Cell

Article Title: Lysosomal storage and impaired autophagy lead to inflammasome activation in G aucher macrophages

doi: 10.1111/acel.12409

Figure Lengend Snippet: Caspase‐1 activation and increased ubiquitinated protein apoptosis‐associated speck‐like proteins ( ASC ) in Gaucher macrophages ( GM s). (A) ELISA or (B) Total supernatants or total protein lysates from treated macrophages from patients (P) and controls (C) were probed for IL ‐1β. (C) Control (C) and GM s (P) were immuno‐stained for LC 3 (red) and NLRP 3 (green) after treatment with lipopolysaccharide ( LPS ) (100 ng) and ATP (5 m m ) for 1 h. Cells were imaged using a confocal microscope (Z‐stack with 0.5 μm thickness). Co‐localization was evaluated using Imaris software. Merged channels are shown as yellow. Pictures represent seven independent experiments performed on cells from seven different patients. Single channels are presented in the supplements. (D) Caspase‐1 was analyzed in total lysate and supernatant by immunoblotting, after treatment with LPS alone or LPS (100 ng)+ ATP (5 m m ) or rapamycin (25 n m ). Data represent five independent experiments. (E) Total protein from control and Gaucher macrophages was immunoblotted for ASC after treatment with LPS (100 ng) and ATP (5 m m ), and then rapamycin (25 n m ), 3 MA (5 m m ) or Baf.1A (10 μ m ) for 40 min. ASC expression was normalized to β‐actin. The blot is representative of seven independent experiments performed on samples from seven different patients and controls. P < 0.05(*) and P < 0.01(**), P < 0.001(***). (F) Control and GM s were immunostained for p62 (red) and ASC (green) in the absence and presence of LPS (100 ng) and ATP (5 m m ) and then imaged by confocal microscopy. Merged channel volumes are shown in yellow, and insets showing surface renderings of three‐dimensional reconstruction using Imaris software are shown in the far right panels.

Article Snippet: Human macrophages were plated on glass chamber slides and, after various treatments, were fixed with 4% paraformaldehyde for 30 min. For LC3 staining, cells were fixed with cold acetone for 10 min. After one‐step washing with PBS, cells were blocked in PBS containing 0.1% saponin, 100 μ m glycine, and 2% donkey serum (2 h) followed by incubation with antibodies against LC3B (Sigma), SQSTM1/p62 (Cell signaling), ASC (Enzo Life science), cathepsin D (R&D systems), Lamp2 (Hybridoma, Columbia, MD, USA), p65‐NFkB (Origene) for 2 h. Then, cells were washed with PBS three times for 5 min followed by incubation with secondary antibodies.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Staining, Microscopy, Software, Western Blot, Expressing, Confocal Microscopy

Journal: Aging Cell

Article Title: Lysosomal storage and impaired autophagy lead to inflammasome activation in G aucher macrophages

doi: 10.1111/acel.12409

Figure Lengend Snippet: Inflammasome activation due to impaired autophagy in Gaucher macrophages ( GM s) (1) In both control and GM s, lipopolysaccharide ( LPS ) priming induces activation of p65‐ NF ‐ kB , which is translocated to the nucleus, leading to production of pro‐ IL ‐1β in the cytosol. In control macrophages (1a), LPS stimulates accumulation of ubiquitinated p65‐ NF ‐ kB , which is further recognized by p62, delivered to autophagosomes, and degraded in the lysosome (1a, solid line). In GM s (1b, dashed line), impaired autophagy prevents degradation of p65‐ NF ‐ kB through autophagy and results in its activation (1b). (2) In both control and GM s, stimulation by both LPS and extracellular ATP leads to inflammasome complex formation ( NLRP 3, apoptosis‐associated speck‐like proteins ( ASC ) and caspase1) and activation. (3) Activated inflammasomes undergo ubiquitination of ASC , leading to p62‐mediated engulfment of inflammasomes by autophagosomes. Pro‐ IL ‐1β conversion to active IL ‐1β is limited due to the destruction of activated inflammasomes by autophagolysosomes. In GM s, defective autophagy and lysosomal dysfunction inhibit the elimination of active inflammasomes through autophagy, resulting in the upregulation and secretion of IL ‐1β (3).

Article Snippet: Human macrophages were plated on glass chamber slides and, after various treatments, were fixed with 4% paraformaldehyde for 30 min. For LC3 staining, cells were fixed with cold acetone for 10 min. After one‐step washing with PBS, cells were blocked in PBS containing 0.1% saponin, 100 μ m glycine, and 2% donkey serum (2 h) followed by incubation with antibodies against LC3B (Sigma), SQSTM1/p62 (Cell signaling), ASC (Enzo Life science), cathepsin D (R&D systems), Lamp2 (Hybridoma, Columbia, MD, USA), p65‐NFkB (Origene) for 2 h. Then, cells were washed with PBS three times for 5 min followed by incubation with secondary antibodies.

Techniques: Activation Assay