Journal: Journal of Innate Immunity

Article Title: Advanced Glycation End Products-Induced Activation of Keratinocytes: A Mechanism Underlying Cutaneous Immune Response in Psoriasis

doi: 10.1159/000534639

Figure Lengend Snippet: AGEs facilitated the production of IL-36α from keratinocytes. a The mRNA level of IL-36α in NHKs treated with BSA or BSA-AGEs for 24 h was detected by qRT-PCR. b Secretion of IL-36α from NHKs treated with BSA or BSA-AGEs for 48 h was detected by ELISA. c Expression of IL-36α (red) in lesional samples from psoriasis patients ( n = 5), and skin samples from healthy controls ( n = 5) was detected by immunofluorescence. Representative samples were shown. Nuclei were counterstained with DAPI (blue). Isotype control was shown with a lesional sample. Bar graphs represent the mean ± SD of IL-36α fluorescence intensity. rMFI: relative mean fluorescence intensity. d Serum level of IL-36α in patients with psoriasis ( n = 30) and healthy controls ( n = 30) was detected by ELISA. e Correlation between serum levels of IL-36α and AGEs was analyzed by Spearman rank correlation test in patients with psoriasis ( n = 30). * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant.

Article Snippet: Human IL-36α Quantitative ELISA Kit (EHC057a, NeoBioscience, China), human IL-36β Quantitative ELISA Kit (EHC058b, NeoBioscience, China), human IL-36γ Quantitative ELISA Kit (EHC056g, NeoBioscience, China), and human IL-17A Quantitative ELISA Kit (E-EL-H5812c, Elabscience, China) were used to analyze serum or cell culture supernatant samples according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence, Control, Fluorescence

Journal: Journal of Innate Immunity

Article Title: Advanced Glycation End Products-Induced Activation of Keratinocytes: A Mechanism Underlying Cutaneous Immune Response in Psoriasis

doi: 10.1159/000534639

Figure Lengend Snippet: AGE-primed keratinocytes potentiate Th17 cell response via secreting IL-36α. The frequency of RORγt + T ( a ) or IL-17A + T ( b ) cells among CD4 + T cells was detected by flow cytometry analysis on PBMCs from psoriasis patients ( n = 5) incubated for 48 h by the supernatants from NHKs, with the addition of IL-36α N, IL-36R A, or rhIL-36α into the incubation system, respectively. c The secretion of IL-17A from PBMCs of psoriasis patients ( n = 5) treated with the supernatants from NHKs for 48 h with the addition of IL-36α N, IL-36 A, or rhIL-36α into the incubation system, respectively, was detected by ELISA. SN, supernatant. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant.

Article Snippet: Human IL-36α Quantitative ELISA Kit (EHC057a, NeoBioscience, China), human IL-36β Quantitative ELISA Kit (EHC058b, NeoBioscience, China), human IL-36γ Quantitative ELISA Kit (EHC056g, NeoBioscience, China), and human IL-17A Quantitative ELISA Kit (E-EL-H5812c, Elabscience, China) were used to analyze serum or cell culture supernatant samples according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Journal of Innate Immunity

Article Title: Advanced Glycation End Products-Induced Activation of Keratinocytes: A Mechanism Underlying Cutaneous Immune Response in Psoriasis

doi: 10.1159/000534639

Figure Lengend Snippet: STAT1/3 signaling pathways mediate AGE-induced expression of K17 and IL-36α in keratinocytes. Protein levels of STAT1, p-STAT1, STAT3, p-STAT3, K17, Rb, p-Rb, and p27 KIP1 in NHKs pre-transfected with STAT1 ( a ) or STAT3 ( b ) siRNA prior to BSA-AGEs stimulation for 24 h was determined by Western blot assay. The mRNA level of IL-36α in NHKs pre-transfected with STAT1 ( c ) or STAT3 ( d ) siRNA prior to BSA-AGEs stimulation for 24 h was determined by qRT-PCR. Secretion level of IL-36α from NHKs pre-transfected with STAT1 ( e ) or STAT3 ( f ) siRNA prior to BSA-AGEs stimulation for 48 h was determined by ELISA. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant.

Article Snippet: Human IL-36α Quantitative ELISA Kit (EHC057a, NeoBioscience, China), human IL-36β Quantitative ELISA Kit (EHC058b, NeoBioscience, China), human IL-36γ Quantitative ELISA Kit (EHC056g, NeoBioscience, China), and human IL-17A Quantitative ELISA Kit (E-EL-H5812c, Elabscience, China) were used to analyze serum or cell culture supernatant samples according to the manufacturer’s instructions.

Techniques: Protein-Protein interactions, Expressing, Transfection, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Innate Immunity

Article Title: Advanced Glycation End Products-Induced Activation of Keratinocytes: A Mechanism Underlying Cutaneous Immune Response in Psoriasis

doi: 10.1159/000534639

Figure Lengend Snippet: AGEs exert pro-proliferative and proinflammatory effects on keratinocytes via RAGE. a Protein levels of RAGE, STAT1, p-STAT1, STAT3, p-STAT3 in NHKs pre-transfected with RAGE siRNA prior to BSA-AGEs stimulation for 24 h were determined by Western blot assay. b Immunofluorescence analysis of p-STAT1 and p-STAT3 in NHKs pre-transfected with RAGE siRNA prior to BSA-AGEs stimulation for 24 h. c Protein levels of RAGE, K17, Rb, p-Rb, and p27 KIP1 in NHKs pre-transfected with RAGE siRNA before BSA-AGEs stimulation for 24 h was determined by Western blot assay. d The mRNA level of IL-36α in NHKs pre-transfected with RAGE siRNA prior to BSA-AGEs stimulation for 24 h was determined by qRT-PCR. e Secretion level of IL-36α from NHKs pre-transfected with RAGE siRNA prior to BSA-AGEs stimulation for 48 h was determined by ELISA. *** p < 0.001, ns: not significant.

Article Snippet: Human IL-36α Quantitative ELISA Kit (EHC057a, NeoBioscience, China), human IL-36β Quantitative ELISA Kit (EHC058b, NeoBioscience, China), human IL-36γ Quantitative ELISA Kit (EHC056g, NeoBioscience, China), and human IL-17A Quantitative ELISA Kit (E-EL-H5812c, Elabscience, China) were used to analyze serum or cell culture supernatant samples according to the manufacturer’s instructions.

Techniques: Transfection, Western Blot, Immunofluorescence, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Innate Immunity

Article Title: Advanced Glycation End Products-Induced Activation of Keratinocytes: A Mechanism Underlying Cutaneous Immune Response in Psoriasis

doi: 10.1159/000534639

Figure Lengend Snippet: Schematic representation of the role of AGEs in psoriatic inflammation. Accumulated AGEs in psoriatic epidermis bind to RAGEs on the membrane of keratinocytes and activate STAT1/3 signaling pathways. Subsequently, K17 is upregulated and thus causes nuclear export and degradation of p27 KIP1 , which leads to the cell cycle progression and ultimately proliferation of keratinocytes. Meanwhile, IL-36α is overexpressed and secreted from keratinocytes and promotes Th17 cell response, which could contribute to the formation of cutaneous immune response in psoriasis.

Article Snippet: Human IL-36α Quantitative ELISA Kit (EHC057a, NeoBioscience, China), human IL-36β Quantitative ELISA Kit (EHC058b, NeoBioscience, China), human IL-36γ Quantitative ELISA Kit (EHC056g, NeoBioscience, China), and human IL-17A Quantitative ELISA Kit (E-EL-H5812c, Elabscience, China) were used to analyze serum or cell culture supernatant samples according to the manufacturer’s instructions.

Techniques: Membrane, Protein-Protein interactions

Journal: Clinical & Translational Immunology

Article Title: Circular RNA TRAPPC6B inhibits intracellular Mycobacterium tuberculosis growth while inducing autophagy in macrophages by targeting microRNA‐874‐3p

doi: 10.1002/cti2.1254

Figure Lengend Snippet: Overexpression of circTRAPPC6B inhibited intracellular Mtb growth while activating autophagy in Mtb‐infected macrophages. (a, b) After transfection with circTRAPPC6B overexpressing plasmids or control plasmids for 24 h, THP‐1 macrophages (a) and macaque spleen macrophage (b) were infected with Mycobacterium H37Rv at MOI = 1 for 3 or 7 days. The growth of the bacilli was evaluated by CFU count. The data were obtained from six independent experiments. Data are expressed as mean ± SEM. * P < 0.05 vs day 0 ( n = 6). (c, d) After transfection with siRNA against circTRAPPC6B (c) or plasmids overexpressing circTRAPPC6B (d) for 24 h, THP‐1 macrophages were infected with Mycobacterium H37Rv at MOI = 1 for 24 h. Representative Western blot showing the change of the LC3‐I and LC3‐II protein expression in THP‐1 macrophages. The data were obtained from three independent experiments. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 vs uninfected and untransfected control. ## P < 0.01 vs H37Rv‐infected but untransfected control. (e) After transfection with circTRAPPC6B overexpressing vectors or control vectors (NC) for 24 h, THP‐1 macrophages were infected with GFP‐BCG at MOI = 10 for 24 h. THP‐1 macrophages were incubated with anti‐LC3 for 2h at room temperature and then fluorescently labelled secondary Ab for 1 h at room temperature. Representative immunofluorescence confocal image of THP‐1 macrophages showing the change of LC3B puncta and GFP‐BCG colocalisation. Scale bar, 20 μm. (f, g) Quantification assay of e. 100 cells were count in every independent experiment. The data were obtained from the mean number of LC3B puncta or colocalisation of LC3B with GFP‐BCG of three independent experiments. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01 vs NC ( n = 3). MOI, multiplicity of infection; CFU, colony‐forming unit; GFP, green fluorescence protein; BCG, Bacillus Calmette‐Guérin.

Article Snippet: After blocking with 5% skim milk in TBST buffer for 2 h, the membrane was probed at 4°C overnight with monoclonal antibody against LC‐3 (1: 1000, Proteintech, Rosemont, IL, USA) or ATG16L1 (1: 1000, Proteintech).

Techniques: Over Expression, Infection, Transfection, Control, Western Blot, Expressing, Incubation, Immunofluorescence, Fluorescence

Journal: Clinical & Translational Immunology

Article Title: Circular RNA TRAPPC6B inhibits intracellular Mycobacterium tuberculosis growth while inducing autophagy in macrophages by targeting microRNA‐874‐3p

doi: 10.1002/cti2.1254

Figure Lengend Snippet: miR‐874‐3p regulated intracellular Mtb growth and autophagy in Mtb‐infected macrophages. (a) After PBMCs were freshly isolated from blood by standard Ficoll density gradient centrifugation, miR‐874‐3p expression was analysed in human PBMCs from patients with 32 active TB and 31 HC by qRT‐PCR. U6 was used as a housekeeping gene for normalising changes in miRNA gene expression. Data are expressed as mean ± SEM. ** P < 0.01 vs HC. (b) Spearman’s correlation analysis between miR‐874‐3p and circTRAPPC6B expression in PBMCs of 32 TB patients. (c) After THP‐1 macrophages were infected with H37Rv at MOI = 1 at different time points (0, 1, 2, 3, 4, and 7 days), miR‐874‐3p expression was analysed by qRT‐PCR. The data were obtained from three independent experiments. Data are expressed as mean ± SEM. ** P < 0.01, *** P < 0.001 vs 0 d. (d, e) After transfection with miR‐874‐3p inhibitor or negative control (miR‐NC) for 24 h, THP‐1 macrophages (d) and macaque spleen macrophage (e) were infected with Mycobacterium H37Rv at MOI = 1 for 3 or 7 days. The growth of the bacilli was evaluated by CFU count. The data were obtained from six independent experiments. Data are expressed as mean ± SEM. * P < 0.05 vs day 0 ( n = 6). (f) After transfection with miR‐874‐3p inhibitor or negative control (miR‐NC) for 24 h, THP‐1 macrophages were infected with GFP‐BCG at MOI = 10 for 24 h. Then, THP‐1 macrophages were incubated with anti‐LC3B for 2h at room temperature and then fluorescently labelled secondary Ab for 1h at room temperature. Representative immunofluorescence confocal image of THP‐1 macrophages showing the change of LC3B puncta and GFP‐BCG colocalisation. Scale bar, 20 μm. (g, h) Quantification assay of f. 100 cells were count in every independent experiment. The data were obtained from the mean number of LC3B puncta or colocalisation of LC3B with GFP‐BCG of three independent experiments. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01 vs miR‐NC. (i, j) After transfection with miR‐874‐3p mimics (i) or inhibitor (j) for 24 h, THP‐1 macrophages were infected with Mycobacterium H37Rv at MOI = 1 for 24 h. Representative Western blot showing the change of the LC3‐I and LC3‐II protein expression in THP‐1 macrophages. The data were obtained from three independent experiments. Data are expressed as mean ± SEM. * P < 0.05, *** P < 0.001 vs uninfected and untransfected control; ### P < 0.001 vs H37Rv‐infected but untransfected cells. qRT‐PCR, quantitative real‐time polymerase chain reaction; PBMCs, peripheral blood mononuclear cells; TB, tuberculosis; HC, health control; MOI, multiplicity of infection; CFU, colony forming unit; GFP, green fluorescence protein; BCG, Bacillus Calmette‐Guérin.

Article Snippet: After blocking with 5% skim milk in TBST buffer for 2 h, the membrane was probed at 4°C overnight with monoclonal antibody against LC‐3 (1: 1000, Proteintech, Rosemont, IL, USA) or ATG16L1 (1: 1000, Proteintech).

Techniques: Infection, Isolation, Gradient Centrifugation, Expressing, Quantitative RT-PCR, Gene Expression, Transfection, Negative Control, Incubation, Immunofluorescence, Western Blot, Control, Real-time Polymerase Chain Reaction, Fluorescence

Journal: Clinical & Translational Immunology

Article Title: Circular RNA TRAPPC6B inhibits intracellular Mycobacterium tuberculosis growth while inducing autophagy in macrophages by targeting microRNA‐874‐3p

doi: 10.1002/cti2.1254

Figure Lengend Snippet: circTRAPPC6B regulated autophagy in Mtb‐infected macrophages via miR‐874‐3p. After transfection with circTRAPPC6B‐overexpressing vectors (pHBAd‐cir), miR‐874‐3p mimics, or corresponding negative controls as indicated for 24 h, THP‐1 macrophages were infected with BCG at MOI = 10 or Mycobacterium H37Rv at MOI = 1 for 24 h. (a) Representative Western blot showing the change of the LC3‐I and LC3‐II protein expression in H37Rv‐infected THP‐1 macrophages. The data were obtained from three independent experiments. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01 and *** P < 0.001. (b) THP‐1 macrophages were incubated with anti‐LC3B for 2h at room temperature and then fluorescently labelled secondary Ab for 1h at room temperature. Representative immunofluorescence confocal image of BCG‐infected THP‐1 macrophages showing the change of LC3B puncta. (c) Quantification of b. 100 cells were count in every independent experiment. The data were obtained from the mean number of LC3B puncta of three independent experiments. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01. BCG, Bacillus Calmette‐Guérin; MOI, multiplicity of infection.

Article Snippet: After blocking with 5% skim milk in TBST buffer for 2 h, the membrane was probed at 4°C overnight with monoclonal antibody against LC‐3 (1: 1000, Proteintech, Rosemont, IL, USA) or ATG16L1 (1: 1000, Proteintech).

Techniques: Infection, Transfection, Western Blot, Expressing, Incubation, Immunofluorescence