Characterization of macrophage phenotypes in three murine models of house-dust-mite-induced asthma

Abstract

In asthma, an important role for innate immunity is increasingly being recognized. Key innate immune cells in the lungs are macrophages. Depending on the signals they receive, macrophages can at least have an M1, M2, or M2-like phenotype. It is unknown how these macrophage phenotypes behave with regard to (the severity of) asthma. We have quantified the phenotypes in three models of house dust mite (HDM-)induced asthma (14, 21, and 24 days). M1, M2, and M2-like phenotypes were identified by interferon regulatory factor 5 (IRF5), YM1, and IL-10, respectively. We found higher percentages of eosinophils in HDM-exposed mice compared to control but no differences between HDM models. T cell numbers were higher after HDM exposure and were the highest in the 24-day HDM protocol. Higher numbers of M2 macrophages after HDM correlated with higher eosinophil numbers. In mice with less severe asthma, M1 macrophage numbers were higher and correlated negatively with M2 macrophages numbers. Lower numbers of M2-like macrophages were found after HDM exposure and these correlated negatively with M2 macrophages. The balance between macrophage phenotypes changes as the severity of allergic airway inflammation increases. Influencing this imbalanced relationship could be a novel approach to treat asthma.

Figure 1

Experimental design of the study: three models of HDM-induced allergic inflammation. HDM, house dust mite.

Figure 2

(a) HDM exposure induced higher percentages of eosinophils in BALF of males (♂) and females (♀) as compared to control, but no differences were found between the HDM protocols. Combining all models, higher percentages of eosinophils after HDM exposure were found in females as compared to males (P < 0.01). (b) The 21-day protocol increased HDM-specific IgE in serum of male and female mice as compared to control (trend, P < 0.1). Again combining all models, HDM exposure induced higher levels of HDM-specific IgE in females than in males (P < 0.05). ^### P < 0.001 compared to control.

Figure 3

(a) HDM exposure induced higher percentages of effector T cells in lung tissue of males (♂) and females (♀) as compared to control. Higher percentages of effector T cells were found in the 24-day protocol as compared to the 14- and 21-day protocol of HDM exposure. Combining all models, HDM exposure induced higher percentages of effector T cells in females than in males (P < 0.05). (b) HDM exposure induced higher percentages of regulatory T cells in lung tissue of males and females as compared to control. Higher percentages of regulatory T cells were found in the 24-day protocol as compared to the 14-day protocol of HDM exposure. Combining all models, HDM exposure induced higher percentages of regulatory T cells in females than in males (P < 0.05). (c) The 24-day protocol had higher ratios of effector T cells to regulatory T cells in lung tissue of males and females as compared to control and the 14- and 21-day protocols of HDM exposure. Combining all models, no differences were found between males and females. ^# P < 0.05, ^## P < 0.01 and ^### P < 0.001 compared to control. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 4

(a) HDM-exposed male (♂) and female (♀) mice had more CD68-positive cells in lung tissue as compared to control, but no differences were found between the HDM protocols. Combining all models, HDM exposure induced higher numbers of CD68-positive cells in females than in males (P < 0.05). (b) HDM-exposed male and female mice had more IRF5-positive cells in lung tissue as compared to control, but no differences were found between males and females when combining all models. The 14-day HDM protocol induced higher numbers of IRF5-positive cells as compared to the 24-day HDM protocol. (c) HDM-exposed male and female mice had more YM1-positive cells in lung tissue as compared to control, with higher numbers of YM1-positive cells in females than in males (P < 0.01). The 24-day HDM protocol induced higher numbers of YM1-positive cells as compared to the 14-day HDM protocol. (d) HDM-exposed male and female mice had lower numbers of IL-10-positive cells in lung tissue as compared to control, but no differences were found between the HDM protocols and between the sexes. The middle and right-hand panels are representative photos of control and HDM-exposed mice for all four stainings (magnification 200x). ^# P < 0.05, ^## P < 0.01 and ^### P < 0.001 compared to control. *P < 0.05.

Figure 5

HDM exposure induced higher levels ofYM1 in bronchoalveolar lavage fluid (BALF) of male (♂) and female (♀) mice as compared to control, but no differences were found between the HDM protocols. Combining all models, HDM exposure induced higher levels of YM1 in BALF in females than in males (P < 0.05). ^# P = 0.05 and ^### P < 0.001 compared to control.

Figure 6

YM1-positive cell numbers (cells/mm² lung tissue) correlated with percentage of eosinophils in BALF of male and female mice exposed to HDM according to three different protocols. No differences were found between males and females when combining all models.

Figure 7

(a) YM1-positive cell numbers correlated with IRF5-positive cell numbers in lung tissue of male and female mice exposed to HDM according to 3 different protocols. (b) YM1-positive cells numbers correlated with IL-10-positive cell numbers in lung tissue of male and female mice exposed to HDM according to three different protocols. No differences were found between males and females when combining all models.