The aryl hydrocarbon receptor instructs the immunomodulatory profile of a subset of Clec4a4+ eosinophils unique to the small intestine

Abstract

C-type lectin domain family 4, member a4 (Clec4a4) is a C-type lectin inhibitory receptor specific for glycans thought to be exclusively expressed on murine CD8α− conventional dendritic cells. Using newly generated Clec4a4-mCherry knock-in mice, we identify a subset of Clec4a4-expressing eosinophils uniquely localized in the small intestine lamina propria. Clec4a4+ eosinophils evinced an immunomodulatory signature, whereas Clec4a4− eosinophils manifested a proinflammatory profile. Clec4a4+ eosinophils expressed high levels of aryl hydrocarbon receptor (Ahr), which drove the expression of Clec4a4 as well as other immunomodulatory features, such as PD-L1. The abundance of Clec4a4+ eosinophils was dependent on dietary AHR ligands, increased with aging, and declined in inflammatory conditions. Mice lacking AHR in eosinophils expanded innate lymphoid cells of type 2 and cleared Nippostrongylus brasiliensis infection more effectively than did wild-type mice. These results highlight the heterogeneity of eosinophils in response to tissue cues and identify a unique AHR-dependent subset of eosinophils in the small intestine with an immunomodulatory profile.

Keywords: allergy; aryl hydrocarbon receptor; eosinophil; helminth; intestine.

Fig. 1.

A heterogeneous population of Clec4a4⁺ and Clec4a4⁻ eosinophils resides in the small intestine. (A) Targeting strategy to generate Clec4a4^mCherry/+ mice. CDS, coding sequence; H2B, human histone H2B; T2A, T2A self-cleaving peptide; UTR, untranslated region. (B) Expression of Clec4a4 and mCherry in splenic CD11b⁺ cDCs. (C) Frequency of mCherry⁺ cells among CD45⁺ cells (gated on live cells) in the indicated tissues (n = 3 to 7). DLN, skin-draining lymph node; LP, lamina propria; MLN, mesenteric lymph node. (D) mCherry expression on CD11c^high and CD11c^low CD45⁺ cells derived from siLP (gated on live cells). (E) Expression of CD11b, CD103, Siglec-F, and MHC class II on mCherry⁺ CD11c^high CD45⁺ cells and mCherry⁺ CD11c^low CD45⁺ cells derived from siLP (gated on live cells). (F) mCherry expression in Siglec-F⁺ eosinophils derived from siLP (gated on SSC^high MHC class II⁻ CD11b⁺ cells). (G) Representative pictures of FACS-sorted mCherry⁺ and mCherry⁻ eosinophils from siLP. (H) CCR3 expression in mCherry⁺ and mCherry⁻ eosinophils in siLP. (I) Representative image showing localization of Siglec-F⁺mCherry⁺ eosinophils in siLP villi of Clec4a4^mCherry/+ mice. Inset is an enlargement of I. DAPI, 4′,6-diamidino-2-phenylindole. (J) mCherry expression in eosinophils in the indicated tissues. (K) Parabiosis of CD45.1 WT and CD45.2 Clec4a4^mCherry/+ mice indicates that eosinophils recirculate in siLP, blood, and lung but much less so in BM; Clec4A4 mCherry is only expressed by eosinophils in the siLP. (L) Frequency of parabiont-derived eosinophils in the indicated tissues. (M) Frequency of parabiont-derived mCherry⁺ eosinophils in the indicated tissues. Results are shown as mean ± SEM. P values were calculated using one-way ANOVA and the Tukey's multiple comparisons test or the two-tailed Student’s t test. ns, not significant. ***P < 0.001; ****P < 0.0001.

Fig. 2.

Transcriptomic profiling and characterization of Clec4a4⁺ and Clec4a4⁻ eosinophil subsets. (A) Volcano plot of differentially expressed genes in mCherry⁺ and mCherry⁻ eosinophils (n = 5 replicates in each group). Transcripts with FC (fold change) > 2 and P < 0.05 in mCherry⁺ and mCherry⁻ eosinophils are depicted in red and blue, respectively. (B) Heat map of selected genes specific to Clec4a4⁺ and Clec4a4⁻ eosinophils. (C) Differential expression of cell surface (CD22, PECAM1, PD-L1, and LPAM1) and intracellular markers (CD68) by Clec4a4⁺ and Clec4a4⁻ siLP eosinophils. (D) Expression of the markers indicated in C by eosinophils from the indicated tissues. LP, lamina propria. (E, Left) Expression of Clec4a4 and PD-L1 in eosinophils derived from siLP. (E, Right) Frequency of Clec4a4⁺PD-L1⁺ and Clec4a4⁻PD-L1^lo eosinophils in the siLP. (F) Enrichment plots demonstrating up-regulated cholesterol homeostasis and TNFα signaling via NF-κB in mCherry⁺ and mCherry⁻ eosinophils, respectively. NES, normalized enrichment score; FDR, false discovery rate. (G) Biochemical pathways for de novo cholesterol synthesis. CoA, coenzyme A; HMG, hydroxymethylglutaryl. (H) Heat map of genes associated with cholesterol synthesis pathways in mCherry⁺ and mCherry⁻ eosinophils. Results are shown as mean ± SEM. P values were calculated using the two-tailed Student’s t test. **P < 0.01.

Fig. 3.

Development of the Clec4a4⁺ eosinophil subset is dependent on aging and on the AHR pathway. (A) Heat map of Ahr and Cyp1b1 in mCherry⁺ and mCherry⁻ eosinophils. (B) qRT-PCR of Ahr and Cyp1b1 transcripts in Clec4a4⁺ and Clec4a4⁻ eosinophils (n = 4 replicates). (C) Clec4a4 expression in splenic CD11b⁺ cDCs and siLP eosinophils from WT and Ahr^−/− mice. (D) Absolute numbers of Clec4a4⁺ and Clec4a4⁻ eosinophils in WT and Ahr^−/− mice (n = 3 in each group). (E) Experimental strategy for mixed BM chimeras. (F) Clec4a4 expression in splenic CD11b⁺ cDCs and siLP eosinophils derived from WT and Ahr^−/− BM cells after reconstitution. (G) Frequency of Clec4a4⁺ and Clec4a4⁻ splenic CD11b⁺ cDCs and siLP eosinophils in BM chimeras. (H) Frequency of Clec4a4⁺ and Clec4a4⁻ siLP eosinophils in WT mice at 1, 2, and 10 to 12 wk of age (n = 3, 6, and 7 replicates, respectively) and in GF mice (n = 5). (I) Experimental time line for feeding mice with a purified diet free of AHR ligands or a purified diet containing the AHR ligand I3C. (J) Representative Clec4a4 expression in siLP eosinophils derived from mice fed with a purified diet free of AHR ligands or a purified diet containing AHR ligands. (K) Frequency of Clec4a4⁺ and Clec4a4⁻ siLP eosinophils in mice fed with a purified diet free of AHR ligands (n = 4) or a purified diet containing AHR ligands (n = 5). Results are shown as mean ± SEM. P values were calculated using the two-tailed Student’s t test. NS, not significant. *P < 0.05; ***P < 0.001.

Fig. 4.

Clec4a4⁺ eosinophils modulate type 2 immunity during helminth infection. (A) Egg counts in feces of WT and Clec4a4-deficient (Clec4a4^{mCherry/mCherry}) mice (n = 13 in each group) at the indicated time points during H. polygyrus infection. (B) Expression of Clec4a4 and PD-L1 in siLP eosinophils derived from Ahr^fl/+ and Eo^CreAhr^fl/fl mice. (C) Frequency of Clec4a4⁺PD-L1⁺ and Clec4a4⁻PD-L1^lo siLP eosinophils in Ahr^fl/+ and Eo^CreAhr^fl/fl mice (n = 3 each group). (D) Worm counts in Ahr^fl/fl (n = 12) and Eo^CreAhr^fl/fl (n = 15) mice at day 6 postinfection with N. brasiliensis. (E) Representative percentages of KLRG1⁺ ILC2s among CD45⁺ cells in mesenteric lymph nodes (MLNs) of Ahr^fl/fl and Eo^CreAhr^fl/fl mice. A gate was applied on lineage-negative cells. Lineage markers included CD3, CD4, CD8, CD19, and NK1.1. (F) Frequency of ILC2s in MLNs from Ahr^fl/fl (n = 12) and Eo^CreAhr^fl/fl (n = 14) mice at day 6 postinfection with N. brasiliensis. Results are shown as mean ± SEM. P values were calculated using the two-tailed Student’s t test or one-way ANOVA and the Tukey's multiple comparisons test. NS, not significant. *P < 0.05; ***P < 0.001.