Stress granule assembly impairs macrophage efferocytosis to aggravate allergic rhinitis in mice

Abstract

Cytoplasmic stress granules (SG) assemble in response to stress-induced translational arrest and are key signaling hubs orchestrating cell fate and regulating various physiological and pathological processes. However, the role of SG formation in the progression of allergic diseases is incompletely understood. Here, by analyzing the nasal tissues of allergic rhinitis (AR) mouse models and AR patients, we find that SGs assemble specifically in the macrophages within the nasal mucosa and promote AR progression by restraining the efferocytotic ability of macrophages, ultimately resulting in reduced Mres generation and IL-10 production. Mechanistically, intracellular m⁷G-modified Lrp1 mRNA, encoding for a typical efferocytosis receptor, is transported by the m⁷G reader QKI7 into stress-induced SGs, where Lrp1 mRNA is sequestered away from the translation machinery, ultimately resulting in reduced macrophage efferocytosis. Therefore, SG assembly impairs macrophage efferocytosis and aggravates AR, and the inhibition of SGs bears considerable potential in the targeted therapy.

Fig. 1. SGs are assembled in the macrophages of nasal mucosa during AR.

a Immunofluorescence imaging of G3BP1 in the nasal mucosa from control and OVA-induced AR mice. Yellow arrows indicate the assembled SGs. b Immunofluorescence imaging of G3BP1, and RNA-scope staining of Krt5 and Cd45 mRNAs in the nasal mucosa from control and OVA-induced AR mice. Yellow arrows indicate the SG and Cd45 mRNA positive cells. c Immunofluorescence imaging of G3BP1 and CD11b in nasal mucosa from control and OVA-induced AR mice. d UMAP visualization of CD45⁺ cells in the nasal mucosa of control and OVA-induced AR mice. Each dot corresponds to one single cell colored according to the cell cluster. e Heatmap of gene expression analyzed by scRNA-seq displaying major markers for the main cell types. f UMAP visualization of G3bp1⁺ cells in the nasal mucosa of control and OVA-induced AR mice. g Shown is violin plot of G3bp1 expression level in neutrophils, T cells, macrophages, and plasma cells. h G3bp1 mRNA expression was detected by qRT-PCR analysis in PMs, BMDMs, BMNs, BMDEs, RAW 264.7 and iBMDM cells (n = 3, biological replicates). i G3BP1 was detected by Western blot in PMs, BMDMs, BMNs, BMDEs, RAW 264.7 and iBMDM cells. j G3BP1 was detected by Western blot in NMMs, NMNs and NMTs. Data are shown as mean ± s.d. or photographs from one representative of three independent experiments.

Fig. 2. SG assembly in macrophages promotes AR progression.

G3bp1^f/f and G3bp1^mac-/- mice were administrated with OVA or HDM to induce AR symptoms as depicted in Supplementary Fig. 2e. a Times of sneezes and scratches in each mouse were counted in 15 min after last i.n. challenge from control, OVA and HDM group (n = 5, biological replicates). (Tukey’s HSD). b, c H&E and PAS staining of the nasal mucosa and lung tissues from AR mice. d Flow cytometry of eosinophils and T cells in the nasal mucosa of AR mice (n = 3, biological replicates). (Tukey’s HSD). e Flow cytometry of eosinophils and neutrophils in the NLF obtained from control and AR mice (n = 3, biological replicates). (Tukey’s HSD). f Flow cytometry of Mres in the nasal mucosa of control and AR mice (n = 3, biological replicates). (Tukey’s HSD). g mRNA expressions of indicated cytokines, Prg2 (proteoglycan 2, pro-eosinophil major basic protein), Epx (eosinophil peroxidase), and Mpo (myeloperoxidase) were detected by qRT-PCR in the nasal mucosa of AR mice (n = 3, biological replicates). (two-tailed unpaired Student’s t-test). h ELISA assay of IL-4, IL-5, and IL-13 in the NLF obtained from control and AR mice (n = 3, biological replicates). (Tukey’s HSD). i mRNA expressions of Il-10 were detected by qRT-PCR in NMMs of AR mice (n = 3, biological replicates). (Tukey’s HSD). j Serum OVA-specific IgE level in the control and AR mice (n = 3, biological replicates). (Tukey’s HSD). Data are shown as mean ± s.d. or photographs from one representative of three independent experiments.

Fig. 3. SG assembly in macrophages impairs efferocytosis.

a, Pathway enrichment analysis of the differentially expressed genes according to KEGG database in macrophages from G3bp1^f/f and G3bp1^mac-/- mice. b The enrichment plot of the GSEA analysis for RNA-seq data on endocytosis-related gene sets (KEGG). c, d HDM (100 µg/well) or NaAsO₂ (50 µM)-inhibited in vitro macrophage efferocytosis of apoptotic cells (labeled with PKH26) was measured by flow cytometry (n = 3, biological replicates). (Dunnett’s multiple comparisons test). e Representative immunofluorescence assay showing efferocytosis of apoptotic cells (labeled with Claret) by macrophages (labeled with F4/80). Yellow arrows indicate phagocytotic macrophages. Magenta arrows indicate macrophages with SG assembly. f HDM (100 µg/well) or NaAsO₂ (50 µM)-inhibited in vivo efferocytosis of apoptotic Jurkat cells (labeled with PKH26) by PMs was measured by Flow cytometry (n = 3, biological replicates). (Dunnett’s multiple comparisons test). g In vitro efferocytosis assay of PKH26-labeled apoptotic Jurkat cells engulfed by G3bp1^f/f and G3bp1^mac-/- macrophages was measured by flow cytometry (n = 3, biological replicates). (Tukey’s HSD). h Representative immunofluorescence assay showing efferocytosis of apoptotic cells (labeled with Claret) by macrophages (G3bp1^f/f macrophages labeled with PKH26 red and G3bp1^mac-/- macrophages labeled with PKH67 green). i, Living cell imaging showing efferocytosis of apoptotic cells (labeled with Claret) by macrophages (G3bp1^f/f macrophages labeled with PKH26 red and G3bp1^mac-/- macrophages labeled with PKH67 green). j In vivo efferocytosis assay of PKH26-labeled apoptotic Jurkat cells engulfed by G3bp1^f/f and G3bp1^mac-/- macrophages was measured by flow cytometry (n = 3, biological replicates). (two-tailed unpaired Student’s t-test). Data are shown as mean ± s.d. or photographs from one representative of three independent experiments.

Fig. 4. SG assembly in macrophages sequesters Lrp1 mRNA to suppress its translation.

a Venn diagram indicating overlap of mRNAs with SG-enrichment and increased G3BP1-binding under stress conditions (using the OmicStudio tools at https://www.omicstudio.cn/tool). b The association between G3BP1 and Lrp1 mRNA in primary macrophages was determined by RIP-qRT-PCR (n = 3, biological replicates). (Tukey’s HSD). c The association between the NTF2 domain of G3BP1 and Lrp1 mRNA in RAW 264.7 cells was determined by RIP-qRT-PCR (n = 3, biological replicates). (Tukey’s HSD). d Immunofluorescence of G3BP1 and RNA-scope imaging of Lrp1 mRNA in PMs. e The association between G3BP1 and Lrp1 mRNA in PMs was determined by RNA pull-down assay. f G3BP1 and LRP1 protein levels in primary macrophages stimulated with HDM (100 µg/well) or NaAsO₂ (50 µM) were examined by Western blot. g LRP1 protein level on the surface of PMs stimulated with HDM (100 µg/well) or NaAsO₂ (50 µM) were examined by flow cytometry (n = 3, biological replicates). (Dunnett’s multiple comparisons test). h G3BP1 and LRP1 protein levels in primary macrophages of G3bp1^f/f and G3bp1^mac-/- mice stimulated with HDM (100 µg/well) were examined by Western blot. i LRP1 protein level on the surface of BMDMs of G3bp1^f/f and G3bp1^mac-/- mice stimulated with HDM (100 µg/well) were examined by flow cytometry (n = 3, biological replicates). (Tukey’s HSD). j LRP1 protein level on the surface of PMs of G3bp1^f/f and G3bp1^mac-/- mice were examined by flow cytometry (n = 3, biological replicates). (two-tailed unpaired Student’s t-test). Data are shown as mean ± s.d. or photographs from one representative of three independent experiments.

Fig. 5. Internal m⁷G-modified Lrp1 mRNA is shuttled by QKI7 into SGs thus repressing its translation.

a BMDMs were administrated with HDM (200 µg/well), and relative Lrp1 mRNA distribution in each ribosome fractions was analyzed by qRT-PCR (n = 3, biological replicates). (Tukey’s HSD). b In BMDMs from G3bp1^f/f and G3bp1^mac-/- mice, relative Lrp1 mRNA distribution in each ribosome fractions was analyzed by qRT-PCR (n = 3, biological replicates). (Tukey’s HSD). c Sequencing read clusters from m⁷G MeRIP-seq analysis of Lrp1 mRNA in PMs and top consensus motif identified by HOMER with MeRIP-seq peaks. d m⁷G modification of Lrp1 mRNA in primary macrophages was examined by m⁷G-RIP-qRT-PCR (n = 3, biological replicates). (two-tailed unpaired Student’s t-test with Welch’s correction). e Immunofluorescence of G3BP1 and m⁷G-modified mRNA, and RNA-scope imaging of Lrp1 mRNA in PMs. f Immunofluorescence of G3BP1 and QKI7, and RNA-scope imaging of Lrp1 mRNA in PMs. g The association between QKI7 and Lrp1 mRNA in primary macrophages was determined by RIP-qRT-PCR (n = 3, biological replicates). (two-tailed unpaired Student’s t-test). h The association between the KH domain of QKI7 and Lrp1 mRNA in RAW 264.7 cells was determined by RIP-qRT-PCR (n = 3, biological replicates). (Tukey’s HSD). i The association between QKI7 and Lrp1 mRNA in PMs was determined by RNA pull-down assay. j In QKI7-overexpressed RAW 264.7 cells stimulated by HDM (200 µg/well), relative Lrp1 mRNA distribution in each ribosome fractions was analyzed by qRT-PCR (n = 3, biological replicates). (Tukey’s HSD). Data are shown as mean ± s.d. or photographs from one representative of three independent experiments. Δ not significant.

Fig. 6. The SG-promoted AR depends on the inhibition of efferocytosis receptor LRP1.

a–d Lrp1^f/f and Lrp1^mac-/- mice were administrated with OVA to induce AR symptoms as depicted in Supplementary Fig. 2e. a Times of sneezes and scratches in each mouse was counted in 15 min after last i.n. challenge (n = 5, biological replicates). (Tukey’s HSD). b H&E staining of the nasal mucosa and lung tissues from AR mice. c Flow cytometry of eosinophils, neutrophils and T cells in the nasal mucosa of control and AR mice (n = 3, biological replicates). (Tukey’s HSD). d ELISA assay of IL-4, IL-5, and IL-13 in the NLF obtained from control and AR mice (n = 3, biological replicates). (Tukey’s HSD). e In vitro efferocytosis assay of PKH26-labeled apoptotic Jurkat cells engulfed by Lrp1^f/f and Lrp1^mac-/- BMDMs was measured by flow cytometry (n = 3, biological replicates). (Tukey’s HSD). f Representative immunofluorescence assay showing efferocytosis of apoptotic cells (labeled with Claret) by Lrp1^f/f and Lrp1^mac-/- macrophages (labeled with F4/80). g In vivo efferocytosis assay of PKH26-labeled apoptotic Jurkat cells engulfed by Lrp1^f/f and Lrp1^mac-/- macrophages was measured by flow cytometry (n = 3, biological replicates). (two-tailed unpaired Student’s test). h–j G3bp1^f/fLrp1^f/f, G3bp1^mac-/-, Lrp1^mac-/-, and G3bp1^mac-/-Lrp1^mac-/- mice were administrated with OVA to induce AR symptoms as depicted in Supplementary Fig. 2e. h Times of sneezes and scratches in each mouse was counted in 15 min after last i.n. challenge (n = 5, biological replicates). (Dunnett’s multiple comparisons test). i, H&E staining of the nasal mucosa and lung tissues from AR mice. j Flow cytometry of eosinophils and neutrophils in the nasal mucosa of control and AR mice (n = 3, biological replicates). (Dunnett’s multiple comparisons test). k In vivo efferocytosis assay of PKH26-labeled apoptotic Jurkat cells engulfed by G3bp1^f/fLrp1^f/f, G3bp1^mac-/-, Lrp1^mac-/-, and G3bp1^mac-/-Lrp1^mac-/- macrophages was measured by flow cytometry (n = 3, biological replicates). (Dunnett’s multiple comparisons test). Data are shown as mean ± s.d. or photographs from one representative of three independent experiments.

Fig. 7. SG assembly inhibitor alleviates AR symptoms, and SGs are assembled in nasal macrophages of AR patients.

a–f Mice were administrated by OVA and treated with PBS, NaAsO₂ (0.015 mg/kg), or G3Ia (0.05 mg/kg) as depicted in Supplementary Fig. 7b. a Times of sneezes and scratches in each mouse was counted in 15 min after last i.n. challenge (n = 5, biological replicates). (Dunnett’s multiple comparisons test). b H&E staining of the nasal mucosa and lung tissues from AR mice. c Flow cytometry of eosinophils, neutrophils and T cells in the nasal mucosa of AR mice (n = 3, biological replicates). (Dunnett’s multiple comparisons test). d ELISA assay of IL-4, IL-5, and IL-13 in the NLF obtained from AR mice (n = 3, biological replicates). (Dunnett’s multiple comparisons test). e mRNA expressions of cytokines, Prg2, Epx, and Mpo were detected by qRT-PCR in the nasal mucosa of AR mice (n = 3, biological replicates). (Dunnett’s multiple comparisons test). f Serum OVA-specific IgE level in the AR mice (n = 3, biological replicates). (Dunnett’s multiple comparisons test). g Representative immunofluorescence imaging showing the SG assembly in macrophages (CD68) in the nasal mucosa from healthy controls and AR patients. h Working model for SG assembly-inhibited macrophage efferocytosis through Lrp1 mRNA translational arrest to aggravate AR progression, created by Figdraw (export ID: IRIRWb0667). Data are shown as mean ± s.d. or photographs from one representative of three independent experiments.