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Sexual dimorphism in the mast cell transcriptome and the pathophysiological responses to immunological and psychological stress

View Article: PubMed Central - PubMed

ABSTRACT

Background: Biological sex plays a prominent role in the prevalence and severity of a number of important stress-related gastrointestinal and immune-related diseases including IBS and allergy/anaphylaxis. Despite the establishment of sex differences in these diseases, the underlying mechanisms contributing to sex differences remain poorly understood. The objective of this study was to define the role of biological sex on mast cells (MCs), an innate immune cell central to the pathophysiology of many GI and allergic disorders.

Methods: Twelve-week-old C57BL/6 male and female mice were exposed to immunological stress (2 h of IgE-mediated passive systemic anaphylaxis (PSA)) or psychological stress (1 h of restraint stress (RS)) and temperature, clinical scores, serum histamine, and intestinal permeability (for RS) were measured. Primary bone marrow-derived MCs (BMMCs) were harvested from male and female mice and analyzed for MC degranulation, signaling pathways, mediator content, and RNA transcriptome analysis.

Results: Sexually dimorphic responses were observed in both models of PSA and RS and in primary MCs. Compared with male mice, female mice exhibited increased clinical scores, hypothermia, and serum histamine levels in response to PSA and had greater intestinal permeability and serum histamine responses to RS. Primary BMMCs from female mice exhibited increased release of β-hexosaminidase, histamine, tryptase, and TNF-α upon stimulation with IgE/DNP and A23187. Increased mediator release in female BMMCs was not associated with increased upstream phospho-tyrosine signaling pathways or downstream Ca2+ mobilization. Instead, increased mediator release in female MCs was associated with markedly increased capacity for synthesis and storage of MC granule-associated immune mediators as determined by MC mediator content and RNA transcriptome analysis.

Conclusions: These results provide a new understanding of sexual dimorphic responses in MCs and have direct implications for stress-related diseases associated with a female predominance and MC hyperactivity including irritable bowel syndrome, allergy, and anaphylaxis.

Electronic supplementary material: The online version of this article (doi:10.1186/s13293-016-0113-7) contains supplementary material, which is available to authorized users.

No MeSH data available.


Female BMMCs release more preformed mediators in response to IgE-mediated degranulation. BMMCs were sensitized with anti-DNP IgE (1 μg/mL) overnight and later stimulated with DNP-HSA. a Male and female BMMCs were stimulated with 0, 15, 31, and 62 ng/mL of DNP-HSA for 1 h and female BMMCs exhibited elevated β-hexosaminidase release at all DNP concentrations (P < 0.05; n = 6). b Compared with male BMMCs, female BMMCs had an increased release of β-hexosaminidase after 30, 45, 60, and 90 min of DNP-HSA stimulus (62 ng/mL). c Female BMMCs released 141.9 ng/106 cells of histamine into supernatant and male BMMCs released 83.7 ng/106 cells of histamine after 1 h DNP stimulus (62 ng/mL) (P < 0.001; n = 5). d Tryptic activity from female BMMCs increased to 5.3 after degranulation, and tryptic activity from male BMMCs was 1.9 after degranulation with 1 h DNP stimulus (62 ng/mL) (P < 0.05; n = 6). e Female BMMCs released 107.4 pg of TNF-α into supernatant and male BMMCs released 25.5 pg after 1 h DNP stimulus (62 ng/mL) (P < 0.001; n = 5). All mediator release was normalized to unstimulated cells of respective sex. Values represent mean ± SE. #P = 0.10 *P < 0.05, **P<0.01 ***P < 0.001 vs. males
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Fig3: Female BMMCs release more preformed mediators in response to IgE-mediated degranulation. BMMCs were sensitized with anti-DNP IgE (1 μg/mL) overnight and later stimulated with DNP-HSA. a Male and female BMMCs were stimulated with 0, 15, 31, and 62 ng/mL of DNP-HSA for 1 h and female BMMCs exhibited elevated β-hexosaminidase release at all DNP concentrations (P < 0.05; n = 6). b Compared with male BMMCs, female BMMCs had an increased release of β-hexosaminidase after 30, 45, 60, and 90 min of DNP-HSA stimulus (62 ng/mL). c Female BMMCs released 141.9 ng/106 cells of histamine into supernatant and male BMMCs released 83.7 ng/106 cells of histamine after 1 h DNP stimulus (62 ng/mL) (P < 0.001; n = 5). d Tryptic activity from female BMMCs increased to 5.3 after degranulation, and tryptic activity from male BMMCs was 1.9 after degranulation with 1 h DNP stimulus (62 ng/mL) (P < 0.05; n = 6). e Female BMMCs released 107.4 pg of TNF-α into supernatant and male BMMCs released 25.5 pg after 1 h DNP stimulus (62 ng/mL) (P < 0.001; n = 5). All mediator release was normalized to unstimulated cells of respective sex. Values represent mean ± SE. #P = 0.10 *P < 0.05, **P<0.01 ***P < 0.001 vs. males

Mentions: Our in vivo experiments in PSA and RS models demonstrated that females released greater amounts of histamine and exhibited heightened MC-associated pathophysiology. To gain further insight into this sexually dimorphic response, we determined whether male and female MCs exhibited different functional and (or) morphological characteristics, utilizing primary bone marrow-derived mast cells (BMMCs) derived from adult male and female mice. BMMCs were sensitized overnight with DNP-specific IgE (1 μg/mL), followed by DNP-HSA to induce FcεR1 cross-linking and degranulation after which MC granule mediators were measured in the supernatants. Compared with IgE/DNP-stimulated BMMCs derived from males, BMMCs derived from females exhibited a greater release of β-hexosaminidase (a MC granule marker) at multiple time points of stimulus (Fig. 3a; F(1,17) = 59.32, p < 0.0001 for sex effect, F(4,17) = 223.1, p < 0.0001 for time effect, interaction: F(4,17) = 3.23, p = 0.0383; Fig. 3a) and at multiple concentrations of IgE-DNP (Fig. 3b). Female BMMCs also released more histamine (by 1.7-fold), tryptase (by 2.8-fold), and TNF-α (by 4.2-fold) in response to IgE/DNP compared with male BMMCs (Fig. 3c–e). Spontaneous degranulation in unstimulated conditions was not different between male and female BMMCs with regard to any mediators except histamine where female BMMCs had slightly higher spontaneous release in unstimulated conditions (Additional file 1: Figure S1). All stimulated mediator release was corrected with unstimulated controls. Therefore, differences in spontaneous degranulation were not the cause of the increased mediator release found in female BMMCs. Also, there were no noticeable difference in the morphologic appearance between unstimulated male and female BMMCs upon visualization of toluidine blue-stained slides of BMMCs(Additional file 1: Figure S1). Despite an expected upregulation of mRNA expression for multiple cytokines following IgE/DNP stimulation in BMMCs, there were no differences between male and female BMMCs, with the exception of moderately increased expression of IL-1β in male-stimulated BMMCs (F(1,12) = 5.255, p = 0.0408 for effect, F(2,12) = 15.31, p = 0.0005 for time effect; Additional file 2: Figure S2A-F).Fig. 3


Sexual dimorphism in the mast cell transcriptome and the pathophysiological responses to immunological and psychological stress
Female BMMCs release more preformed mediators in response to IgE-mediated degranulation. BMMCs were sensitized with anti-DNP IgE (1 μg/mL) overnight and later stimulated with DNP-HSA. a Male and female BMMCs were stimulated with 0, 15, 31, and 62 ng/mL of DNP-HSA for 1 h and female BMMCs exhibited elevated β-hexosaminidase release at all DNP concentrations (P < 0.05; n = 6). b Compared with male BMMCs, female BMMCs had an increased release of β-hexosaminidase after 30, 45, 60, and 90 min of DNP-HSA stimulus (62 ng/mL). c Female BMMCs released 141.9 ng/106 cells of histamine into supernatant and male BMMCs released 83.7 ng/106 cells of histamine after 1 h DNP stimulus (62 ng/mL) (P < 0.001; n = 5). d Tryptic activity from female BMMCs increased to 5.3 after degranulation, and tryptic activity from male BMMCs was 1.9 after degranulation with 1 h DNP stimulus (62 ng/mL) (P < 0.05; n = 6). e Female BMMCs released 107.4 pg of TNF-α into supernatant and male BMMCs released 25.5 pg after 1 h DNP stimulus (62 ng/mL) (P < 0.001; n = 5). All mediator release was normalized to unstimulated cells of respective sex. Values represent mean ± SE. #P = 0.10 *P < 0.05, **P<0.01 ***P < 0.001 vs. males
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Fig3: Female BMMCs release more preformed mediators in response to IgE-mediated degranulation. BMMCs were sensitized with anti-DNP IgE (1 μg/mL) overnight and later stimulated with DNP-HSA. a Male and female BMMCs were stimulated with 0, 15, 31, and 62 ng/mL of DNP-HSA for 1 h and female BMMCs exhibited elevated β-hexosaminidase release at all DNP concentrations (P < 0.05; n = 6). b Compared with male BMMCs, female BMMCs had an increased release of β-hexosaminidase after 30, 45, 60, and 90 min of DNP-HSA stimulus (62 ng/mL). c Female BMMCs released 141.9 ng/106 cells of histamine into supernatant and male BMMCs released 83.7 ng/106 cells of histamine after 1 h DNP stimulus (62 ng/mL) (P < 0.001; n = 5). d Tryptic activity from female BMMCs increased to 5.3 after degranulation, and tryptic activity from male BMMCs was 1.9 after degranulation with 1 h DNP stimulus (62 ng/mL) (P < 0.05; n = 6). e Female BMMCs released 107.4 pg of TNF-α into supernatant and male BMMCs released 25.5 pg after 1 h DNP stimulus (62 ng/mL) (P < 0.001; n = 5). All mediator release was normalized to unstimulated cells of respective sex. Values represent mean ± SE. #P = 0.10 *P < 0.05, **P<0.01 ***P < 0.001 vs. males
Mentions: Our in vivo experiments in PSA and RS models demonstrated that females released greater amounts of histamine and exhibited heightened MC-associated pathophysiology. To gain further insight into this sexually dimorphic response, we determined whether male and female MCs exhibited different functional and (or) morphological characteristics, utilizing primary bone marrow-derived mast cells (BMMCs) derived from adult male and female mice. BMMCs were sensitized overnight with DNP-specific IgE (1 μg/mL), followed by DNP-HSA to induce FcεR1 cross-linking and degranulation after which MC granule mediators were measured in the supernatants. Compared with IgE/DNP-stimulated BMMCs derived from males, BMMCs derived from females exhibited a greater release of β-hexosaminidase (a MC granule marker) at multiple time points of stimulus (Fig. 3a; F(1,17) = 59.32, p < 0.0001 for sex effect, F(4,17) = 223.1, p < 0.0001 for time effect, interaction: F(4,17) = 3.23, p = 0.0383; Fig. 3a) and at multiple concentrations of IgE-DNP (Fig. 3b). Female BMMCs also released more histamine (by 1.7-fold), tryptase (by 2.8-fold), and TNF-α (by 4.2-fold) in response to IgE/DNP compared with male BMMCs (Fig. 3c–e). Spontaneous degranulation in unstimulated conditions was not different between male and female BMMCs with regard to any mediators except histamine where female BMMCs had slightly higher spontaneous release in unstimulated conditions (Additional file 1: Figure S1). All stimulated mediator release was corrected with unstimulated controls. Therefore, differences in spontaneous degranulation were not the cause of the increased mediator release found in female BMMCs. Also, there were no noticeable difference in the morphologic appearance between unstimulated male and female BMMCs upon visualization of toluidine blue-stained slides of BMMCs(Additional file 1: Figure S1). Despite an expected upregulation of mRNA expression for multiple cytokines following IgE/DNP stimulation in BMMCs, there were no differences between male and female BMMCs, with the exception of moderately increased expression of IL-1β in male-stimulated BMMCs (F(1,12) = 5.255, p = 0.0408 for effect, F(2,12) = 15.31, p = 0.0005 for time effect; Additional file 2: Figure S2A-F).Fig. 3

View Article: PubMed Central - PubMed

ABSTRACT

Background: Biological sex plays a prominent role in the prevalence and severity of a number of important stress-related gastrointestinal and immune-related diseases including IBS and allergy/anaphylaxis. Despite the establishment of sex differences in these diseases, the underlying mechanisms contributing to sex differences remain poorly understood. The objective of this study was to define the role of biological sex on mast cells (MCs), an innate immune cell central to the pathophysiology of many GI and allergic disorders.

Methods: Twelve-week-old C57BL/6 male and female mice were exposed to immunological stress (2&nbsp;h of IgE-mediated passive systemic anaphylaxis (PSA)) or psychological stress (1&nbsp;h of restraint stress (RS)) and&nbsp;temperature, clinical scores, serum histamine, and intestinal permeability (for RS) were measured. Primary bone marrow-derived MCs (BMMCs) were harvested from male and female mice and analyzed for MC degranulation, signaling pathways, mediator content, and RNA transcriptome analysis.

Results: Sexually dimorphic responses were observed in both models of PSA and RS and in primary MCs. Compared with male mice, female mice exhibited increased clinical scores, hypothermia, and serum histamine levels in response to PSA and had greater intestinal permeability and serum histamine responses to RS. Primary BMMCs from female mice exhibited increased release of &beta;-hexosaminidase, histamine, tryptase, and TNF-&alpha; upon stimulation with IgE/DNP and A23187. Increased mediator release in female BMMCs was not associated with increased upstream phospho-tyrosine signaling pathways or downstream Ca2+ mobilization. Instead, increased mediator release in female MCs was associated with markedly increased capacity for synthesis and storage of MC granule-associated immune mediators as determined by MC mediator content and RNA transcriptome analysis.

Conclusions: These results provide a new understanding of sexual dimorphic responses in MCs and have direct implications for stress-related diseases associated with a female predominance and MC hyperactivity including irritable bowel syndrome, allergy, and anaphylaxis.

Electronic supplementary material: The online version of this article (doi:10.1186/s13293-016-0113-7) contains supplementary material, which is available to authorized users.

No MeSH data available.