Dysregulated invertebrate tropomyosin-dectin-1 interaction confers susceptibility to allergic diseases

Abstract

The key factors underlying the development of allergic diseases-the propensity for a minority of individuals to develop dysfunctional responses to harmless environmental molecules-remain undefined. We report a pathway of immune counter-regulation that suppresses the development of aeroallergy and shrimp-induced anaphylaxis. In mice, signaling through epithelially expressed dectin-1 suppresses the development of type 2 immune responses through inhibition of interleukin-33 (IL-33) secretion and the subsequent recruitment of IL-13-producing innate lymphoid cells. Although this homeostatic pathway is functional in respiratory epithelial cells from healthy humans, it is dramatically impaired in epithelial cells from asthmatic and chronic rhinosinusitis patients, resulting in elevated IL-33 production. Moreover, we identify an association between a single-nucleotide polymorphism (SNP) in the dectin-1 gene loci and reduced pulmonary function in two cohorts of asthmatics. This intronic SNP is a predicted eQTL (expression quantitative trait locus) that is associated with reduced dectin-1 expression in human tissue. We identify invertebrate tropomyosin, a ubiquitous arthropod-derived molecule, as an immunobiologically relevant dectin-1 ligand that normally serves to restrain IL-33 release and dampen type 2 immunity in healthy individuals. However, invertebrate tropomyosin presented in the context of impaired dectin-1 function, as observed in allergic individuals, leads to unrestrained IL-33 secretion and skewing of immune responses toward type 2 immunity. Collectively, we uncover a previously unrecognized mechanism of protection against allergy to a conserved recognition element omnipresent in our environment.

Fig. 1. Dectin-1 inhibits HDM-mediated allergic asthma

Asthmatic phenotype in (A) Clec7a^+/+ and Clec7a^−/− mice sensitized with PBS or HDM [10 µg, intraperitoneally (i.p.); days 0 and 7] and challenged with PBS or HDM [100 µg, intratracheally (i.t.)] on days 14 and 21. Seventy-two hours after the last challenge, (B) AHR as measured by airway pressure time index (APTI), (C) BAL eosinophils, (D) BAL neutrophils, (E and F) mucus (PAS staining), and (G) serum total IgE were assessed. (H) Clec7a^+/+ and Clec7a^−/− mice were sensitized and challenged with PBS or HDM through the airways [intratracheally on days 0, 7, and 14 and intranasally (i.n.) on days 16 to 20]. (I) Forty-eight hours after the last challenge, AHR to nebulized methacholine (Mch) was determined. (J) Male BALB/c mice were sensitized and challenged with PBS or 50 µg of HDM in combination with either 30 µg of isotype control or anti–dectin-1–blocking mAbs (intratracheally on days 0 and 14). Forty-eight hours after the last challenge, (K) AHR was determined (nebulized methacholine, 10 mg/ml), and BAL was collected for evaluation of (L) total cells and (M) eosinophils. Data are representative of two to three independent experiments each containing n = 3 to 8 animals per group (B to G) or from n = 7 to 14 mice per group, pooled from two independent experiments (I), or from n = 6 to 10 mice per group, pooled from two independent experiments (K and L), or are representative of two independent experiments (M). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, as determined by one-way ANOVA followed by post hoc (Newman-Keuls) analysis. In (I), *P < 0.05 indicates comparison with Clec7a^+/+ HDM (FDR).

Fig. 2. Dectin-1 regulates innate IL-13

Clec7a^+/+ and Clec7a^−/− mice were sensitized and challenged with PBS or HDM. Seventy-two hours after the last challenge, lungs were harvested for determination of (A) levels of Il17A mRNA, (B) Il4 mRNA, (C) Il5 mRNA, (D) Il13 mRNA, (E) IL-13⁺CD4⁺ cells, and (F) FoxP3⁺CD4⁺ T_regs. (G) Numbers of IL-13⁺ ILCs in mice 24 hours after single PBS or HDM (100 µg) intratracheal inhalation. Rag1^−/− or Rag1^−/−Clec7a^−/− mice were exposed to HDM as in Materials and Methods, and (H) AHR and (I) IL-13⁺ ILCs were determined. Data are means + SEM and are pooled from two independent experiments with n = 10 to 15 mice per group (A to E), representative of two experiments with n = 4 to 5 mice per group, or n = 6 to 13 animals per group (G to I). ^†P < 0.0001, as compared with PBS. *P < 0.05, **P < 0.01, as determined by one-way ANOVA followed by post hoc (Newman-Keuls) analysis.

Fig. 3. Dectin-1 regulates epithelial IL-33

BAL levels of (A) IL-33 and (B) IL-25 4 hours after a single PBS or HDM (100 µg) intratracheal exposure. n.s., not significant. (C) AHR in mice receiving HDM and blocking anti-ST2 or control antibodies. (D) AHR was determined in PBS- or HDM-treated Clec7a chimeric mice. WT, wild type; KO, knockout. (E) IL-33 levels in 16HBE cells treated with HDM in combination with isotype or neutralizing anti–hDectin-1 antibodies. IL-33 (F) and IL-6 (G) levels 4 hours after HDM treatment of 16HBE cells overexpressing human dectin-1 or empty vector. Data are means + SEM pooled from two to three independent experiments with n = 7 to 12 mice per group (A and B), n = 5 to 11 mice per group (C), or n = 4 to 13 mice per group (D) or are representative of two to three independent experiments with four replicate wells per condition (E to G). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, as determined by one-way ANOVA followed by post hoc (Newman-Keuls and Tukey’s) analysis.

Fig. 4. β-Glucans do not modulate epithelial IL-33 or AHR

(A) Supernatant IL-33 levels measured from 16HBE cells exposed to media or HDM, which was preincu-bated with PBS, vehicle, or β-glucanase. (B) AHR in A/J mice receiving PBS, HDM, β-glucanase–treated HDM, or HDM exposed to control conditions (veh + 56°C; see Materials and Methods). AHR in C57BL/6 mice exposed to either (C) PBS, HDM, or HDM + curdlan or (D) PBS, OVA, or OVA + curdlan (details in Materials and Methods). Data are means + SEM pooled from two independent experiment (A) or are representative of two independent experiments (B to D) with n = 3 to 8 mice per group. ****P < 0.0001, as determined by one-way ANOVA followed by post hoc analysis.

Fig. 5. Identification of invertebrate tropomyosin as a ligand for dectin-1

(A) Silver stain of a pull-down from rhDectin-1–Fc alone, protein G beads, or rhDectin-1–Fc incubated with HDM extract (see arrow). (B) Peptides identified by MS. (C) Coimmunoprecipitation (IP) of rDectin-1–Fc incubated with alder, peanut, shrimp, and HDM extracts and immunoblotted using anti–invertebrate tropomyosin (anti–Der p 10) or isotype control. WB, Western blot. (D) AHR, (E) BAL eosinophils, (F) lung PAS⁺ epithelial cells, and (G) BAL neutrophils from male BALB/c mice receiving PBS, HDM (50 µg), or HDM + 10 µg of recombinant Der p 10 intratracheally on days 0 and 5 and harvested on day 7 for analysis. (H) Four hours later, BAL IL-33 levels in male C57BL/6 mice were given PBS or HDM with isotype or anti–Der p 10 antibodies intratracheally. (I) Supernatant IL-33 from 16HBE cells treated for 2 hours with media or HDM in combination with isotype or anti–Der p 10 antibodies. (J) Change in body temperature in Clec7a^+/+ (WT) and Clec7a^−/− (KO) mice sensitized to shrimp extract. “†” represents death. (K) 16HBE human bronchial epithelial cells treated for 2 hours with PBS or shrimp extract with isotype control or neutralizing anti–hDectin-1 antibodies. Data are representative of two to three independent experiments (A and C) or are means + SEM of two to three independent experiments (I and K) with four replicate wells per condition or pooled from two to three independent experiments (D to G and H and J) and each containing n = 4 to 7 (D to G), n = 3 to 9 (H), or n = 9 to 11 (J) animals per group. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by one- or two-way (I) ANOVA followed by post hoc (Newman-Keuls, Dunnett’s, and Bonferroni) analysis.

Fig. 6. Dectin-1 is repressed in allergic individuals

CLEC7A expression in (A) bronchial epithelial cells (BEC) from controls and asthmatics and (B) NECs from control and CRS patients. (C) IL-33 levels in control and CRS NECs stimulated with media or HDM for 2 hours. (A to C) Data are means + SEM from n = 6 to 19 patients per group (A and B) or from a minimum of 7 patients (C). *P < 0.05, as determined by Student’s t test with Welch’s correction or one-way ANOVA followed by post hoc (Newman-Keuls) analysis.

Fig. 7. rs58677678 genomic location, allelic variation, and association with lung function

(A and B) Baseline FEV₁ by rs58677678 genotype for GALA II and SAGE. FEV₁ is strongly influenced by age, sex, height, and ethnicity; therefore, baseline FEV₁ is shown as the percentage of the predicted normal value achieved for each participant (y axis). Predicted normal values were derived using the Hankinson reference equation. (C) Forest plot of association analysis results. The size of the square represents the magnitude of the effect size, and the lines indicate the 95% confidence intervals in each study. β is the change in FEV₁ per addition of one G allele (risk allele) of rs58677678. (D) Schematic of CLEC7A indicating the genomic position of rs5867768. K, kilobases; E, exon. Intronic regions are indicated as blue horizontal lines between exons. (E) LocusZoom plot of meta-analysis for association between CLEC7A variants and FEV₁ across two independent studies (GALA II and SAGE). The top asthma-associated variant is highlighted in purple (rs58677678; P = 2.23 × 10⁻⁴, β = 0.100). The solid red line indicates the P value threshold for Bonferroni adjustment for 143 SNPs (3.50 × 10⁻⁴). Measurements of linkage disequilibrium with rs58677678 (r²) are from the 1000 Genome AMR (Ad Mixed American) population. Values of pairwise r² between rs58677678 and each of the other 142 SNPs in the region shown above are displayed using the color scheme shown in the top left corner of the figure. (F) Expression of CLEC7A in human lung tissue by rs58677678 genotype. The x axis indicates genotype of rs58677678 (n is the number of individuals with the associated genotype). The y axis represents rank-normalized CLEC7A expression in the lungs. Data used to calculate the correlation between rs58677678 genotype and expression of CLEC7A were ascertained from the GTEx public database portal. P values indicate the significance of the correlation between rs58677678 genotype and CLEC7A expression for the specified tissue.

Fig. 8. Invertebrate tropomyosin–dectin-1 interaction in mediating protection against allergic diseases

In healthy individuals, invertebrate tropomyosin binding of dectin-1 at mucosal surfaces results in suppression of IL-33–driven inflammation. In contrast, in allergic individuals, decreased levels of epithelial dectin-1 result in aberrant secretion of IL-33, which in turn drives IL-13 production from ILC2 and conditions local DCs to promote type 2 immune responses.