Journal: Frontiers in Immunology

Article Title: Histamine Regulates the Inflammatory Profile of SOD1-G93A Microglia and the Histaminergic System Is Dysregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fimmu.2017.01689

Figure Lengend Snippet: SOD1-G93A microglia express histaminergic receptors. (A) Primary microglia from SOD1-G93A mice were stained with anti-CD11b (red) and anti-H1R, anti-H2R, anti-H3R, and anti-H4R (green). Höechst 33258 was used for nuclei (scale bar 50 µm). In inserts, wild-type (WT) microglia (scale bar 50 µm). (B) Equal amounts of WT and SOD1-G93A primary microglia total lysates were subjected to western blotting with anti-H1R, anti-H2R, anti-H3R, and anti-H4R. Anti-GAPDH was used for protein normalization. Data represent mean ± SEM of n = 3 independent experiments. Statistical significance was calculated by Student’s t -test, as referred to WT cells, * p < 0.05.

Article Snippet: Antibodies for WB and IF were rabbit anti-HDC (1:500 WB; 1:200 IF, Abcam, USA); rabbit anti-HNMT (1:200 WB, IF, Atlas, Sweden); rabbit anti-DAO (1:200 WB and IF, Bioss, USA); rabbit anti-H1R (1:500 WB, 1:200 IF, Alomone, Israel); rabbit anti-H2R (1:500 WB and 1:200 IF, Alomone); rabbit anti-H3R (1:500 WB, 1:200 IF, Alomone); rabbit anti-H4R (1:500 WB, 1:200 IF, Santa Cruz); mouse anti-NOX2/gp91 phox (1:1,000 WB, BD Transduction Laboratories, USA); rabbit anti-phospho-NF-κB p65 (Ser536) (1:500 WB, CST, USA); rabbit anti-NF-κB p65 (1:500 WB, CST); rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1,000 WB, CST); mouse anti-p44/42 MAPK (ERK1/2) (L34F12) (1:1,000 WB, CST); rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) (1:500 WB, CST); mouse anti-p38 MAPK (A-12) (1:500 WB, Santa Cruz); rabbit anti-Arginase 1 (ARG1) (1:700 WB, Abcam); rabbit anti-P2Y12 (1:200 WB, Anaspec, USA); rat anti-CD11b (1:200 IF, AbD Serotec, USA); rabbit anti-Iba1 (1:500, WB, IF, Wako, USA); rat anti-CD68 (1:500, WB, IF, AbD Serotec); rabbit anti-CD163 (1:100 WB, Santa Cruz); rabbit anti-CD206 (1:500 WB, Abcam); mouse anti-GAPDH (1:2,500 WB, Calbiochem, USA).

Techniques: Staining, Western Blot

Journal: Frontiers in Immunology

Article Title: Histamine Regulates the Inflammatory Profile of SOD1-G93A Microglia and the Histaminergic System Is Dysregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fimmu.2017.01689

Figure Lengend Snippet: MAPKs are activated by histamine in SOD1-G93A microglia. Wild-type (WT) and SOD1-G93A primary microglia were treated with histamine (100 µM) for 15 min–1 h–6 h and equal amounts of total lysates were subjected to SDS-PAGE, western blotting, and immunoreactions with anti-p-p38 and anti-p-38 (A) or with anti-pERK and anti-ERK (B) . (C) SOD1-G93A microglia stimulated with histamine (100 µM) for 1 h in the presence of specific antagonists for H1R (orphenadrine, 10 µM), H2R (ranitidine, 10 µM), H3R (thioperamide, 5 µM), or H4R (JNJ-7777120, 5 µM) were subjected to immunoreactions with anti-pERK and anti-ERK. Anti-GAPDH was used for protein normalization. HA, histamine. Data represent mean ± SEM of n = 4 independent experiments. Statistical significance was calculated by ANOVA followed by Post Hoc Tukey’s test, as referred to ctrl cells, * p < 0.05 or to histamine-treated cells, # p < 0.05.

Article Snippet: Antibodies for WB and IF were rabbit anti-HDC (1:500 WB; 1:200 IF, Abcam, USA); rabbit anti-HNMT (1:200 WB, IF, Atlas, Sweden); rabbit anti-DAO (1:200 WB and IF, Bioss, USA); rabbit anti-H1R (1:500 WB, 1:200 IF, Alomone, Israel); rabbit anti-H2R (1:500 WB and 1:200 IF, Alomone); rabbit anti-H3R (1:500 WB, 1:200 IF, Alomone); rabbit anti-H4R (1:500 WB, 1:200 IF, Santa Cruz); mouse anti-NOX2/gp91 phox (1:1,000 WB, BD Transduction Laboratories, USA); rabbit anti-phospho-NF-κB p65 (Ser536) (1:500 WB, CST, USA); rabbit anti-NF-κB p65 (1:500 WB, CST); rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1,000 WB, CST); mouse anti-p44/42 MAPK (ERK1/2) (L34F12) (1:1,000 WB, CST); rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) (1:500 WB, CST); mouse anti-p38 MAPK (A-12) (1:500 WB, Santa Cruz); rabbit anti-Arginase 1 (ARG1) (1:700 WB, Abcam); rabbit anti-P2Y12 (1:200 WB, Anaspec, USA); rat anti-CD11b (1:200 IF, AbD Serotec, USA); rabbit anti-Iba1 (1:500, WB, IF, Wako, USA); rat anti-CD68 (1:500, WB, IF, AbD Serotec); rabbit anti-CD163 (1:100 WB, Santa Cruz); rabbit anti-CD206 (1:500 WB, Abcam); mouse anti-GAPDH (1:2,500 WB, Calbiochem, USA).

Techniques: SDS Page, Western Blot

Journal: Frontiers in Immunology

Article Title: Histamine Regulates the Inflammatory Profile of SOD1-G93A Microglia and the Histaminergic System Is Dysregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fimmu.2017.01689

Figure Lengend Snippet: NF-κB pathway is modulated by histamine in SOD1-G93A microglia. (A) Total RNA was extracted from SOD1-G93A microglia stimulated with histamine (100 µM) for 6–18 h and the expression profiles of IL-10, IL-6, and IL-1β were examined by qRT-PCR. (B) Wild-type (WT) and SOD1-G93A microglia exposed to 100 µM histamine for 1–6–18 h were subjected to immunoreactions with anti-pNF-κB and anti-NF-κB. (C) SOD1-G93A microglia stimulated with histamine (100 µM) for 6 h in the presence of H1R–H2R–H3R–H4R antagonists were subjected to immunoreactions with anti-pNF-κB and anti-NF-κB. (D) SOD1-G93A microglia treated with histamine (100 µM) for 6 h in the presence of specific inhibitors of pERK (PD98059, 100 µM) and pp38 (SB203580, 20 µM) were subjected to immunoreactions with anti-pNF-κB and anti-NF-κB. Anti-GAPDH was used for protein normalization. HA, histamine. Data represent mean ± SEM of n = 4 independent experiments. Statistical significance was calculated by ANOVA followed by Post Hoc Tukey’s test, as referred to ctrl cells, * p < 0.05 or to histamine-treated cells, # p < 0.05.

Article Snippet: Antibodies for WB and IF were rabbit anti-HDC (1:500 WB; 1:200 IF, Abcam, USA); rabbit anti-HNMT (1:200 WB, IF, Atlas, Sweden); rabbit anti-DAO (1:200 WB and IF, Bioss, USA); rabbit anti-H1R (1:500 WB, 1:200 IF, Alomone, Israel); rabbit anti-H2R (1:500 WB and 1:200 IF, Alomone); rabbit anti-H3R (1:500 WB, 1:200 IF, Alomone); rabbit anti-H4R (1:500 WB, 1:200 IF, Santa Cruz); mouse anti-NOX2/gp91 phox (1:1,000 WB, BD Transduction Laboratories, USA); rabbit anti-phospho-NF-κB p65 (Ser536) (1:500 WB, CST, USA); rabbit anti-NF-κB p65 (1:500 WB, CST); rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1,000 WB, CST); mouse anti-p44/42 MAPK (ERK1/2) (L34F12) (1:1,000 WB, CST); rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) (1:500 WB, CST); mouse anti-p38 MAPK (A-12) (1:500 WB, Santa Cruz); rabbit anti-Arginase 1 (ARG1) (1:700 WB, Abcam); rabbit anti-P2Y12 (1:200 WB, Anaspec, USA); rat anti-CD11b (1:200 IF, AbD Serotec, USA); rabbit anti-Iba1 (1:500, WB, IF, Wako, USA); rat anti-CD68 (1:500, WB, IF, AbD Serotec); rabbit anti-CD163 (1:100 WB, Santa Cruz); rabbit anti-CD206 (1:500 WB, Abcam); mouse anti-GAPDH (1:2,500 WB, Calbiochem, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Histamine Regulates the Inflammatory Profile of SOD1-G93A Microglia and the Histaminergic System Is Dysregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fimmu.2017.01689

Figure Lengend Snippet: Histamine modulates inflammation in SOD1-G93A microglia. Wild-type (WT) and SOD1-G93A primary microglia were exposed to 100 µM histamine for 1–6–18 h and equal amounts of total lysates subjected to western blotting (WB) and immunoreactions with anti-NADPH oxidase 2 (NOX2) (A) , anti-ARG-1 (B) . SOD1-G93A microglia stimulated with histamine (100 µM) in the presence of H1R–H2R–H3R–H4R antagonists for 6 h were subjected to immunoreactions with anti-NOX2 (C) or anti-ARG-1 (D) . SOD1-G93A primary microglia were exposed to 100 µM histamine for 1–6–18 h and equal amounts of total lysates subjected to WB and immunoreactions with anti-CD163 (E) or anti-CD206 (F) . Anti-GAPDH was used for protein normalization. HA, histamine. Data represent mean ± SEM of n = 4 independent experiments. Statistical significance was calculated by ANOVA followed by Post Hoc Tukey’s test, as referred to ctrl cells, * p < 0.05 or to histamine-treated cells, # p < 0.05.

Article Snippet: Antibodies for WB and IF were rabbit anti-HDC (1:500 WB; 1:200 IF, Abcam, USA); rabbit anti-HNMT (1:200 WB, IF, Atlas, Sweden); rabbit anti-DAO (1:200 WB and IF, Bioss, USA); rabbit anti-H1R (1:500 WB, 1:200 IF, Alomone, Israel); rabbit anti-H2R (1:500 WB and 1:200 IF, Alomone); rabbit anti-H3R (1:500 WB, 1:200 IF, Alomone); rabbit anti-H4R (1:500 WB, 1:200 IF, Santa Cruz); mouse anti-NOX2/gp91 phox (1:1,000 WB, BD Transduction Laboratories, USA); rabbit anti-phospho-NF-κB p65 (Ser536) (1:500 WB, CST, USA); rabbit anti-NF-κB p65 (1:500 WB, CST); rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1,000 WB, CST); mouse anti-p44/42 MAPK (ERK1/2) (L34F12) (1:1,000 WB, CST); rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) (1:500 WB, CST); mouse anti-p38 MAPK (A-12) (1:500 WB, Santa Cruz); rabbit anti-Arginase 1 (ARG1) (1:700 WB, Abcam); rabbit anti-P2Y12 (1:200 WB, Anaspec, USA); rat anti-CD11b (1:200 IF, AbD Serotec, USA); rabbit anti-Iba1 (1:500, WB, IF, Wako, USA); rat anti-CD68 (1:500, WB, IF, AbD Serotec); rabbit anti-CD163 (1:100 WB, Santa Cruz); rabbit anti-CD206 (1:500 WB, Abcam); mouse anti-GAPDH (1:2,500 WB, Calbiochem, USA).

Techniques: Western Blot

Journal: Frontiers in Immunology

Article Title: Histamine Regulates the Inflammatory Profile of SOD1-G93A Microglia and the Histaminergic System Is Dysregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fimmu.2017.01689

Figure Lengend Snippet: Histamine induces migration in SOD1-G93A microglia. (A) Wild-type (WT) and SOD1-G93A primary microglia were exposed to 100 µM histamine for 1–6–18 h and equal amounts of total lysates subjected to western blotting and immunoreactions with anti-P2Y12. Anti-GAPDH was used for protein normalization. (B) SOD1-G93A microglia were stimulated with 100 µM histamine in the presence or absence of H1R–H2R–H3R–H4R antagonists or P2Y12 antagonist (MRS2395, 10 µM) for 18 h and stained with anti-CD11b (green). Scale bar 50 µm. The number of migrating cells was then quantified. (C) WT microglia were stimulated with 100 microM histamine in the presence or absence of H1R-H2R-H3R-H4R antagonists for 18 h and the number of migrating cells was quantified, HA, histamine. Data represent mean ± SEM of n = 3 independent experiments. Statistical significance was calculated by ANOVA followed by Post Hoc Tukey’s test, as referred to ctrl cells, * p < 0.05 or to histamine-treated cells, # p < 0.05.

Article Snippet: Antibodies for WB and IF were rabbit anti-HDC (1:500 WB; 1:200 IF, Abcam, USA); rabbit anti-HNMT (1:200 WB, IF, Atlas, Sweden); rabbit anti-DAO (1:200 WB and IF, Bioss, USA); rabbit anti-H1R (1:500 WB, 1:200 IF, Alomone, Israel); rabbit anti-H2R (1:500 WB and 1:200 IF, Alomone); rabbit anti-H3R (1:500 WB, 1:200 IF, Alomone); rabbit anti-H4R (1:500 WB, 1:200 IF, Santa Cruz); mouse anti-NOX2/gp91 phox (1:1,000 WB, BD Transduction Laboratories, USA); rabbit anti-phospho-NF-κB p65 (Ser536) (1:500 WB, CST, USA); rabbit anti-NF-κB p65 (1:500 WB, CST); rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1,000 WB, CST); mouse anti-p44/42 MAPK (ERK1/2) (L34F12) (1:1,000 WB, CST); rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) (1:500 WB, CST); mouse anti-p38 MAPK (A-12) (1:500 WB, Santa Cruz); rabbit anti-Arginase 1 (ARG1) (1:700 WB, Abcam); rabbit anti-P2Y12 (1:200 WB, Anaspec, USA); rat anti-CD11b (1:200 IF, AbD Serotec, USA); rabbit anti-Iba1 (1:500, WB, IF, Wako, USA); rat anti-CD68 (1:500, WB, IF, AbD Serotec); rabbit anti-CD163 (1:100 WB, Santa Cruz); rabbit anti-CD206 (1:500 WB, Abcam); mouse anti-GAPDH (1:2,500 WB, Calbiochem, USA).

Techniques: Migration, Western Blot, Staining

Journal: Frontiers in Immunology

Article Title: Histamine Regulates the Inflammatory Profile of SOD1-G93A Microglia and the Histaminergic System Is Dysregulated in Amyotrophic Lateral Sclerosis

doi: 10.3389/fimmu.2017.01689

Figure Lengend Snippet: Protein expression levels of H1-H4R in cortex and lumbar spinal cord from wild-type (WT) and SOD1-G93A mice. Cortex (A,C,E,G) and lumbar spinal cord (B,D,F,H) from WT (~20 weeks) and SOD1-G93A mice at different stages of disease (100, 140 days, and end stage) ( n = 4/group) were subjected to SDS-PAGE, western blotting, and immunoreactions with anti-H1R, anti-H2R, anti-H3R, and anti-H4R. Anti-GAPDH was used for protein normalization. Data represent mean ± SEM. Statistical significance was calculated by ANOVA followed by Post Hoc Tukey’s test, as referred to WT mice, * p < 0.05.

Article Snippet: Antibodies for WB and IF were rabbit anti-HDC (1:500 WB; 1:200 IF, Abcam, USA); rabbit anti-HNMT (1:200 WB, IF, Atlas, Sweden); rabbit anti-DAO (1:200 WB and IF, Bioss, USA); rabbit anti-H1R (1:500 WB, 1:200 IF, Alomone, Israel); rabbit anti-H2R (1:500 WB and 1:200 IF, Alomone); rabbit anti-H3R (1:500 WB, 1:200 IF, Alomone); rabbit anti-H4R (1:500 WB, 1:200 IF, Santa Cruz); mouse anti-NOX2/gp91 phox (1:1,000 WB, BD Transduction Laboratories, USA); rabbit anti-phospho-NF-κB p65 (Ser536) (1:500 WB, CST, USA); rabbit anti-NF-κB p65 (1:500 WB, CST); rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1,000 WB, CST); mouse anti-p44/42 MAPK (ERK1/2) (L34F12) (1:1,000 WB, CST); rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) (1:500 WB, CST); mouse anti-p38 MAPK (A-12) (1:500 WB, Santa Cruz); rabbit anti-Arginase 1 (ARG1) (1:700 WB, Abcam); rabbit anti-P2Y12 (1:200 WB, Anaspec, USA); rat anti-CD11b (1:200 IF, AbD Serotec, USA); rabbit anti-Iba1 (1:500, WB, IF, Wako, USA); rat anti-CD68 (1:500, WB, IF, AbD Serotec); rabbit anti-CD163 (1:100 WB, Santa Cruz); rabbit anti-CD206 (1:500 WB, Abcam); mouse anti-GAPDH (1:2,500 WB, Calbiochem, USA).

Techniques: Expressing, SDS Page, Western Blot