Sex-dependent influence of LMAN1 on allergen-induced airway hyperresponsiveness

Abstract

Allergic asthma is a chronic inflammatory disease of the airways characterized by a type 2-high adaptive immune response towards common aeroantigens such as dust mite, pollen, and animal dander. Despite the advances made toward translation of various biologics into the clinic, the limited efficacy of these therapies in certain populations, combined with the ineligibility of some patients for treatment (clinically or economically), have led to the continued need for the development of more widely effective allergic asthma therapies. Our lab previously identified lectin mannose-binding 1 (LMAN1) as a novel receptor for house dust mite (HDM) and showed that in vitro, LMAN1 downregulated inflammatory NF-κB signaling in DCs in response to HDM. In this follow-up work, we investigated the in vivo relevance of LMAN1 by subjecting LMAN1 knockout (KO) mice and wild type (WT) littermate controls to a model of HDM-induced allergic asthma. Surprisingly, we discovered that loss of LMAN1 led to opposing effects on airway hyperresponsiveness (AHR), which were dependent on the sex of the mice. HDM-treated female LMAN1 KO mice showed increased AHR, while HDM-treated male KO mice showed decreased AHR, compared with their WT counterparts. We further identified the features of HDM-induced asthma which may account for the gender-biased effects of LMAN1 on lung function. This work not only highlights the complexity of the loss of LMAN1 in vivo but also suggests that such sex-dependent responses should be taken into consideration when pursuing LMAN1 as a therapeutic target for treatment of allergic asthma.

Keywords: ERGIC-53; HDM; LMAN1; airway hyperresponsiveness; asthma.

Figure 1.

LMAN1 influences HDM-induced AHR in a sex-dependent manner. WT and LMAN1 KO mice were left untreated or were subjected to an HDM-induced asthma model. On day 14, mice were anesthetized, intratracheally cannulated, and connected to the FlexiVent ventilator. Increasing doses of methacholine were administered to the nebulizer, and measurements were obtained of (A) maximum respiratory system resistance (Rrs), (B) tissue damping (G), (C) respiratory system elastance (Ers), and (D) tissue elastance (H). Data represent mean ± SEM. A Student T-test was used to assess significance at each dose of methacholine. Aggregated data from multiple experiments is shown with n = 7-13 per group. *P < 0.05, **P < 0.01.

Figure 2.

Loss of LMAN1 in males reduces airway inflammation in an HDM asthma model. WT and LMAN1 KO mice were left untreated or were subjected to an HDM-induced asthma model. Mice were euthanized on day 14 and bronchoalveolar lavage (BAL) samples from (A) female mice and (B) male mice were analyzed via flow cytometry. Lines behind scatterplots represent means ± SEM. Results were analyzed using two-way ANOVA with multiple comparisons. Aggregated data from multiple experiments is shown with n = 6 to 13 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3.

Male LMAN1 KO mice exhibit decreased lung pathology and lumen narrowing. WT and LMAN1 KO mice were left untreated or were subjected to an HDM-induced asthma model. On day 14, mice were euthanized, and lungs were inflated and fixed in formalin. Lung samples were stained with H&E, Periodic Acid Schiff (PAS), or trichrome blue. (A) Representative 40x images of bronchioles in female mice (left) and male mice (right) are shown. (B) Lung inflammatory index was scored for a maximum score of 16, mucus production for a maximum of 4, and collagen deposition for a maximum of 6. Lumen area and epithelial thickness were also measured. Lines behind scatterplots represent means ± SEM. Results were analyzed by two-way ANOVA with multiple comparisons. Aggregated data from multiple experiments are shown with n = 7 to 14. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4.

Male LMAN1 KO mice show reduced Th2 polarization. WT and LMAN1 KO mice were left untreated or were subjected to an HDM-induced asthma model. On day 14, mice were euthanized for the collection of lungs and serum. Lungs were enzymatically digested and mechanically homogenized to obtain single-cell suspensions, and intracellular cytokine staining (ICS) was performed for females (A) and males (B). ELISA was used to measure HDM-specific IgG1 in serum obtained from female (C) and male (D) mice. Lines behind scatterplots represent means ± SEM. Results were analyzed by two-way ANOVA with multiple comparisons. Aggregated data from multiple experiments is shown with n = 3 to 15 mice per group for ICS and n = 6 to 12 mice per group for ELISA. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

LMAN1 does not influence lung ILC2 recruitment. WT and LMAN1 KO mice were left untreated or were subjected to an HDM-induced asthma model. On day 14, mice were euthanized and lungs were enzymatically digested and mechanically homogenized to obtain single- cell suspensions for flow cytometric identification of ILC2s. Results are shown for CD45⁺ Lin^-Thy1.2⁺ Sca-1⁺, CD45⁺ Lin^- Thy1.2⁺ ST2⁺, CD45⁺ Lin^- Thy1.2⁺ KLRG1⁺, and CD45⁺ Lin^- Thy1.2⁺ Sca-1⁺ ST2⁺ KLRG1⁺ ILC2 cells in female (A) and male (B) mice. Lines behind scatterplots represent means ± SEM. Results were analyzed by 2–-way ANOVA with multiple comparisons. Aggregated data from multiple experiments are shown with n = 4 to 14 per group *P < 0.05.

Figure 6.

Sex-specific differences in the early production of inflammatory cytokines in WT and LMAN1 KO HDM-treated mice. Male and female WT and LMAN1 KO mice were exposed to HDM intratracheally. On day 1 after exposure, mice were euthanized and lungs were homogenized in cell lysis buffer with protease inhibitors. Cell lysates were standardized and subjected to a multiplex bead-based assay (A) or ELISAs (B) to determine the production of proinflammatory cytokines. Lines behind scatterplots represent means ± SEM. Results were analyzed by 2-way ANOVA with multiple comparisons. Aggregated data from multiple experiments are shown with n = 5 to 6 per group *P < 0.05, **P < 0.01.