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

Abstract

Amyotrophic lateral sclerosis (ALS) is a late-onset motor neuron disease where activated glia release pro-inflammatory cytokines that trigger a vicious cycle of neurodegeneration in the absence of resolution of inflammation. Given the well-established role of histamine as a neuron-to-glia alarm signal implicated in brain disorders, the aim of this study was to investigate the expression and regulation of the histaminergic pathway in microglial activation in ALS mouse model and in humans. By examining the contribution of the histaminergic system to ALS, we found that particularly via H1 and H4 receptors, histamine promoted an anti-inflammatory profile in microglia from SOD1-G93A mice by modulating their activation state. A decrease in NF-κB and NADPH oxidase 2 with an increase in arginase 1 and P2Y12 receptor was induced by histamine only in the ALS inflammatory environment, but not in the healthy microglia, together with an increase in IL-6, IL-10, CD163, and CD206 phenotypic markers in SOD1-G93A cells. Moreover, histaminergic H1, H2, H3, and H4 receptors, and histamine metabolizing enzymes histidine decarboxylase, histamine N-methyltransferase, and diamine oxidase were found deregulated in spinal cord, cortex, and hypothalamus of SOD1-G93A mice during disease progression. Finally, by performing a meta-analysis study, we found a modulated expression of histamine-related genes in cortex and spinal cord from sporadic ALS patients. Our findings disclose that histamine acts as anti-inflammatory agent in ALS microglia and suggest a dysregulation of the histaminergic signaling in ALS.

Keywords: SOD1-G93A; amyotrophic lateral sclerosis; histamine; microglia; neuroinflammation.

Figure 1

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.

Figure 2

SOD1-G93A microglia express histaminergic enzymes. (A) Microglia from SOD1-G93A mice were stained with anti-CD11b (red) and anti-histidine decarboxylase (HDC), anti-histamine N-methyltransferase (HNMT), and anti-diamine oxidase (DAO) (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-HDC, anti-HNMT, and anti-DAO. 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.

Figure 3

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.

Figure 4

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.

Figure 5

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.

Figure 6

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.

Figure 7

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.

Figure 8

Protein expression levels of histidine decarboxylase (HDC), histamine N-methyltransferase (HNMT), and diamine oxidase (DAO) in cortex and lumbar spinal cord from wild-type (WT) and SOD1-G93A mice. Cortex (A,C,E) and lumbar spinal cord (B,D,F) from WT (~20 weeks) and SOD1-G93A mice at different stages of the disease (100, 140 days, and end stage) (n = 4/group) were subjected to SDS-PAGE, western blotting, and immunoreactions with anti-HDC, anti-HNMT, and anti-DAO. 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.

Figure 9

Histaminergic-related potential candidate genes for amyotrophic lateral sclerosis (ALS). The interaction pathway map represents histamine-related genes differentially deregulated in cortex of two subgroups of sporadic ALS (sALS) patients (sALS1 and sALS2). The map was created using MetaCore Pathway Map Creator tool (GeneGo). Gene expression values are presented on the map as “thermometer-like” figures (red for upregulation, blue for downregulation, and thermometer height is relative to fold-change) with sALS1 patients’ data represented as thermometer #1, and sALS2 patients as thermometer #2.