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

Journal: mBio

Article Title: Mycobacterium tuberculosis Utilizes Host Histamine Receptor H1 to Modulate Reactive Oxygen Species Production and Phagosome Maturation via the p38MAPK-NOX2 Axis

doi: 10.1128/mbio.02004-22

Figure Lengend Snippet: Histamine-activated HRH1 signaling enhanced the intracellular survival of M. tuberculosis in macrophages. (A) Colony forming unit (CFU) analysis of intracellular mycobacteria in PMA-differentiated THP-1 macrophages infected with H37Ra or H37Rv (MOI = 10:1) for 6 h, and then treated with 10 μM histamine for another 72 h. (B) Western blotting of protein expression levels of the four histamine receptors in THP-1 macrophages infected with H37Ra or H37Rv (MOI = 10:1) for 24 h. (C) RT-qPCR analysis of THP-1 macrophages infected with H37Ra or H37Rv (MOI = 10:1) for 24 h. (D) Flow cytometric analysis of HRH1 expression in THP-1 macrophages infected with H37Ra or H37Rv (MOI = 10:1); data represent HRH1 + cell counts and HRH1 mean fluorescence intensity (MFI). (E) Representative immunofluorescence confocal microscopy images (total 80 to 100 images, scale bars: 5 μm) of HRH1 expression in THP-1 macrophages infected with GFP labeled H37Ra (MOI = 10:1). All data represent the mean ± SD of at least three experiments. * , P < 0.05; * * , P < 0.01; ** * , P < 0.001; *** * , P < 0.0001; ns, not significant.

Article Snippet: Differentiated THP-1 cells (2 × 10 5 cells/mL) treated with siRNA and/or fluorescent GFP-H37Ra/RFP-H37Rv (MOI = 10) for 24 h were then washed twice with 1× PBS, fixed with 4% formaldehyde for 15 min, and permeabilized with 0.3% Triton X-100 for 10 min. After blocking with 3% BSA in 1× PBS for 2 h at RT, the cells were incubated overnight at 4°C with anti-HRH1 (Immunoway, 1:100) and anti-LAMP-1 (lysosomal associated membrane protein 1, Abcam, ab25630, 1:200) antibodies followed by incubation for 1 h at RT with a fluorophore-conjugated secondary antibody (anti-rabbit-Alexa fluor 488/555, Invitrogen, USA,1:200).

Techniques: Infection, Western Blot, Expressing, Quantitative RT-PCR, Fluorescence, Immunofluorescence, Confocal Microscopy, Labeling

Journal: mBio

Article Title: Mycobacterium tuberculosis Utilizes Host Histamine Receptor H1 to Modulate Reactive Oxygen Species Production and Phagosome Maturation via the p38MAPK-NOX2 Axis

doi: 10.1128/mbio.02004-22

Figure Lengend Snippet: Inhibition or knockdown of HRH1 attenuated M. tuberculosis survival in macrophages. (A) Colony forming unit (CFU) analysis of intracellular mycobacteria in PMA-differentiated THP-1 macrophages infected with H37Ra or H37Rv for 6 h, and then treated with 10 μM 2-PE (HRH1 agonist) for 72 h. (B) Colony forming unit (CFU) analysis of intracellular mycobacteria in PMA-differentiated THP-1 macrophages infected with H37Ra or H37Rv (MOI = 10:1) for 6 h before treatment with 10 μM cetirizine (HRH1 antagonist) for 72 h. (C) Western blotting of histamine receptor (HRH1 to 4) protein expression level in THP-1 cells transfected for 48 h with gene-targeted siRNAs. (D) Colony forming unit (CFU) analysis of intracellular mycobacteria in PMA-differentiated THP-1 macrophages transfected for 48 h with gene-targeted siRNAs before infection with H37Ra (MOI = 10:1) for 72 h. (E) Colony forming unit (CFU) analysis of intracellular mycobacteria in PMA-differentiated THP-1 macrophages transfected for 48 h with gene-targeted siRNAs before infection with H37Rv (MOI = 10:1) for 72 h. All data represent the mean ± SD of at least three experiments. * , P < 0.05; * * , P < 0.01; ** * , P < 0.001; *** * , P < 0.0001; ns, not significant.

Article Snippet: Differentiated THP-1 cells (2 × 10 5 cells/mL) treated with siRNA and/or fluorescent GFP-H37Ra/RFP-H37Rv (MOI = 10) for 24 h were then washed twice with 1× PBS, fixed with 4% formaldehyde for 15 min, and permeabilized with 0.3% Triton X-100 for 10 min. After blocking with 3% BSA in 1× PBS for 2 h at RT, the cells were incubated overnight at 4°C with anti-HRH1 (Immunoway, 1:100) and anti-LAMP-1 (lysosomal associated membrane protein 1, Abcam, ab25630, 1:200) antibodies followed by incubation for 1 h at RT with a fluorophore-conjugated secondary antibody (anti-rabbit-Alexa fluor 488/555, Invitrogen, USA,1:200).

Techniques: Inhibition, Infection, Western Blot, Expressing, Transfection

Journal: mBio

Article Title: Mycobacterium tuberculosis Utilizes Host Histamine Receptor H1 to Modulate Reactive Oxygen Species Production and Phagosome Maturation via the p38MAPK-NOX2 Axis

doi: 10.1128/mbio.02004-22

Figure Lengend Snippet: HRH1 suppressed NOX2-mediated cROS production. (A) PMA-differentiated THP-1 macrophages were transfected with HRH1 or control siRNA for 48 h before infection with H37Ra (MOI = 10:1) for 24 h. Cytoplasmic ROS (cROS) was analyzed by flow cytometry. (B) PMA-differentiated THP-1 macrophages were transfected with HRH1 or control siRNA for 48 h before infection with H37Rv (MOI = 10:1) for 24 h. Cytoplasmic ROS (cROS) was analyzed by flow cytometry. (C) Western blotting of the protein expression level of NOX family members following either infection with H37Ra and H37Rv alone or in combination and with or without HRH1 knockdown. (D) PMA-differentiated THP-1 macrophages were transfected with HRH1 or control siRNA for 48 h before infection with H37Ra (MOI = 10:1) for 24 h. NADPH oxidase activity was analyzed by measuring the absorbance at 450 nm using the EpochTM 2 microplate spectrophotometer. (E) Flow cytometric analysis of cROS expression following infection with H37Ra or H37Rv and in the presence or absence of 25 μM NOX2 inhibitor. (F) Colony forming unit (CFU) analysis of intracellular mycobacteria in THP-1 macrophages with or without 25 μM NOX2 inhibitor. All data represent the mean ± SD of at least three experiments. * , P < 0.05; * * , P < 0.01; ** * , P < 0.001; *** * , P < 0.0001; ns, not significant.

Article Snippet: Differentiated THP-1 cells (2 × 10 5 cells/mL) treated with siRNA and/or fluorescent GFP-H37Ra/RFP-H37Rv (MOI = 10) for 24 h were then washed twice with 1× PBS, fixed with 4% formaldehyde for 15 min, and permeabilized with 0.3% Triton X-100 for 10 min. After blocking with 3% BSA in 1× PBS for 2 h at RT, the cells were incubated overnight at 4°C with anti-HRH1 (Immunoway, 1:100) and anti-LAMP-1 (lysosomal associated membrane protein 1, Abcam, ab25630, 1:200) antibodies followed by incubation for 1 h at RT with a fluorophore-conjugated secondary antibody (anti-rabbit-Alexa fluor 488/555, Invitrogen, USA,1:200).

Techniques: Transfection, Infection, Flow Cytometry, Western Blot, Expressing, Activity Assay, Spectrophotometry

Journal: mBio

Article Title: Mycobacterium tuberculosis Utilizes Host Histamine Receptor H1 to Modulate Reactive Oxygen Species Production and Phagosome Maturation via the p38MAPK-NOX2 Axis

doi: 10.1128/mbio.02004-22

Figure Lengend Snippet: M. tuberculosis infection inhibits phagosome maturation and acidification via the HRH1-NOX2 axis. (A) PMA-differentiated THP-1 macrophages were transfected with HRH1 or control siRNA for 48 h before infection with H37Ra or H37Rv (MOI = 10:1) for another 24 h. Flow cytometric analysis of intracellular phagosome acidification levels. (B) PMA-differentiated THP-1 macrophages were transfected with HRH1 or control siRNA for 48 h before infection with H37Ra or H37Rv (MOI = 10:1) and cultured with or without the NOX2 inhibitor (25 μM) for another 24 h. Flow cytometric analysis of intracellular phagosome acidification levels. (C) Representative immunofluorescence confocal microscopy images (total 80 to 100 images) to illustrate the staining of phagosome maturation in RFP-H37Rv infection macrophages; scale bars: 5 μm. (D) Quantification of phagosome maturation using LAMP-1 as an indicator. All data represent the mean ± SD of at least three experiments. * * , P < 0.01; ** * , P < 0.001; *** * , P < 0.0001; ns, not significant.

Article Snippet: Differentiated THP-1 cells (2 × 10 5 cells/mL) treated with siRNA and/or fluorescent GFP-H37Ra/RFP-H37Rv (MOI = 10) for 24 h were then washed twice with 1× PBS, fixed with 4% formaldehyde for 15 min, and permeabilized with 0.3% Triton X-100 for 10 min. After blocking with 3% BSA in 1× PBS for 2 h at RT, the cells were incubated overnight at 4°C with anti-HRH1 (Immunoway, 1:100) and anti-LAMP-1 (lysosomal associated membrane protein 1, Abcam, ab25630, 1:200) antibodies followed by incubation for 1 h at RT with a fluorophore-conjugated secondary antibody (anti-rabbit-Alexa fluor 488/555, Invitrogen, USA,1:200).

Techniques: Infection, Transfection, Cell Culture, Immunofluorescence, Confocal Microscopy, Staining

Journal: mBio

Article Title: Mycobacterium tuberculosis Utilizes Host Histamine Receptor H1 to Modulate Reactive Oxygen Species Production and Phagosome Maturation via the p38MAPK-NOX2 Axis

doi: 10.1128/mbio.02004-22

Figure Lengend Snippet: HRH1 inhibited p47 phox phosphorylation to suppress NOX2 activity via the p38MAPK signaling pathway. (A) Western blotting of the protein levels of p47 phox and p-p47 phox (Ser345) in PMA-differentiated THP-1 cells transfected with HRH1 or control siRNA for 48 h before infection with H37Rv (MOI = 10:1) for another 24 h. (B) Western blotting of the protein levels of p47 phox and p-p47 phox (Ser345) in the presence or absence of cetirizine (0 μM, 5 μM, 10 μM). (C) Western blotting of the protein levels of p47 phox and p-p47 phox (Ser345) in the presence or absence of 2-PE (0 μM, 5 μM, 10 μM). (D) Western blotting of the protein levels of p38MAPK and p-p38MAPK in PMA-differentiated THP-1 cells transfected with HRH1 or control siRNA for 48 h before infection with H37Rv (MOI = 10:1) for another 24 h. (E) Western blotting of the protein levels of p38MAPK and p-p38MAPK in PMA-differentiated THP-1 cells transfected with HRH1 or control siRNA for 48 h and cultured in the presence or absence of cetirizine (0 μM, 5 μM, 10 μM). (F) Western blotting of the protein levels of p38MAPK and p-p38MAPK in the presence or absence of 2-PE (0 μM, 5 μM, 10 μM). (G-H) PMA-differentiated THP-1 cells were transfected with HRH1 or control siRNA for 48 h before infection with H37Rv (MOI = 10:1) for another 24 h in the presence or absence of 10 μM p-p38MAPK inhibitor. (G) Western blotting of the protein levels of p38MAPK, p-p38MAPK, p47 phox , and p-p47 phox (Ser345). (H) Colony forming unit (CFU) analysis of intracellular bacteria. All data represent the mean ± SD of at least three experiments. * , P < 0.05; *** * , P < 0.0001.

Article Snippet: Differentiated THP-1 cells (2 × 10 5 cells/mL) treated with siRNA and/or fluorescent GFP-H37Ra/RFP-H37Rv (MOI = 10) for 24 h were then washed twice with 1× PBS, fixed with 4% formaldehyde for 15 min, and permeabilized with 0.3% Triton X-100 for 10 min. After blocking with 3% BSA in 1× PBS for 2 h at RT, the cells were incubated overnight at 4°C with anti-HRH1 (Immunoway, 1:100) and anti-LAMP-1 (lysosomal associated membrane protein 1, Abcam, ab25630, 1:200) antibodies followed by incubation for 1 h at RT with a fluorophore-conjugated secondary antibody (anti-rabbit-Alexa fluor 488/555, Invitrogen, USA,1:200).

Techniques: Activity Assay, Western Blot, Transfection, Infection, Cell Culture

Journal: mBio

Article Title: Mycobacterium tuberculosis Utilizes Host Histamine Receptor H1 to Modulate Reactive Oxygen Species Production and Phagosome Maturation via the p38MAPK-NOX2 Axis

doi: 10.1128/mbio.02004-22

Figure Lengend Snippet: HRH1 interacts with GRK2 to negatively regulate the p38MAPK signaling pathway. (A) PMA-differentiated THP-1 cells were infected with H37Rv (MOI = 10:1) for 24 h and immunoprecipitated (IP) with anti-GRK2 or anti-HRH1 antibodies. Immunoprecipitates were then immunoblotted (IB) with anti-GRK2 or anti-HRH1 antibodies, accordingly. (B) Western blotting of GRK2 protein levels in PMA-differentiated THP-1 cells transfected with HRH1 siRNA for 48 h and infected with H37Rv (MOI = 10:1) for 24 h. (C) Western blotting of GRK2 protein levels in the presence or absence of 2-PE (0, 5 μM, 10 μM) with H37Rv (MOI = 10:1) infection for 24 h. (D) Western blotting of GRK2 protein levels in the presence or absence of cetirizine (0, 5 μM, 10 μM) with H37Rv (MOI = 10:1) infection for 24 h. (E) Western blotting of GRK2 protein levels in PMA-differentiated THP-1 cells transfected with HRH1 siRNA for 48 h and infected with H37Rv (MOI = 10:1) for 24 h in the presence or absence of 10 μM p-p38MAPK inhibitor. (F) Western blotting of the protein levels of p47 phox /p-p47 phox (Ser345), p38MAPK, and p-p38MAPK in PMA-differentiated THP-1 cells transfected with GRK2 siRNA for 48 h and infected with H37Rv (MOI = 10:1) for 24 h. (G) Western blotting of the protein levels of p47 phox /p-p47 phox (Ser345), p38MAPK, and p-p38MAPK in the presence or absence of GRK2i (inhibitor, 0, 1, or 5 μM) with H37Rv (MOI = 10:1) infection for 24 h.

Article Snippet: Differentiated THP-1 cells (2 × 10 5 cells/mL) treated with siRNA and/or fluorescent GFP-H37Ra/RFP-H37Rv (MOI = 10) for 24 h were then washed twice with 1× PBS, fixed with 4% formaldehyde for 15 min, and permeabilized with 0.3% Triton X-100 for 10 min. After blocking with 3% BSA in 1× PBS for 2 h at RT, the cells were incubated overnight at 4°C with anti-HRH1 (Immunoway, 1:100) and anti-LAMP-1 (lysosomal associated membrane protein 1, Abcam, ab25630, 1:200) antibodies followed by incubation for 1 h at RT with a fluorophore-conjugated secondary antibody (anti-rabbit-Alexa fluor 488/555, Invitrogen, USA,1:200).

Techniques: Infection, Immunoprecipitation, Western Blot, Transfection

Journal: mBio

Article Title: Mycobacterium tuberculosis Utilizes Host Histamine Receptor H1 to Modulate Reactive Oxygen Species Production and Phagosome Maturation via the p38MAPK-NOX2 Axis

doi: 10.1128/mbio.02004-22

Figure Lengend Snippet: Mycobacterium tuberculosis utilized host HRH1 to modulate reactive oxygen species production and phagosome maturation via the GRK2-p38MAPK-NOX2 axis, a summary model. Uninfected macrophages displayed normal regulation of HRH1 and p38MAPK pathway (left). After mycobacteria infection and endocytosis by macrophages (middle panel), HRH1 expression was enhanced and interacted with GRK2, which then inhibited the p38MAPK signaling pathway. The inhibited p38MAPK signaling further decreased the level of phosphorylated oxidase subunit p47phox (Ser345), leading to the inability of the oxidase subunit p47 phox (Ser345) to bind NOX2 (gp91 phox ) on the phagosome membrane, and thus inhibited the NOX2 (gp91 phox ) oxidase complex formation. This reduced cROS production, which further prevented phagosome maturation and acidification. This process provided a beneficial environment that favored the intracellular survival of M. tuberculosis in macrophages. However, HRH1 inhibitor treatment blocked the inhibitory effect of GRK on p38MAPK signaling in M. tuberculosis -infected macrophages. As a result, NOX2 could function normally to promote phagosome maturation and acidification, which was detrimental to the survival of M. tuberculosis in macrophages (right).

Article Snippet: Differentiated THP-1 cells (2 × 10 5 cells/mL) treated with siRNA and/or fluorescent GFP-H37Ra/RFP-H37Rv (MOI = 10) for 24 h were then washed twice with 1× PBS, fixed with 4% formaldehyde for 15 min, and permeabilized with 0.3% Triton X-100 for 10 min. After blocking with 3% BSA in 1× PBS for 2 h at RT, the cells were incubated overnight at 4°C with anti-HRH1 (Immunoway, 1:100) and anti-LAMP-1 (lysosomal associated membrane protein 1, Abcam, ab25630, 1:200) antibodies followed by incubation for 1 h at RT with a fluorophore-conjugated secondary antibody (anti-rabbit-Alexa fluor 488/555, Invitrogen, USA,1:200).

Techniques: Infection, Expressing