Epithelial endoplasmic reticulum stress orchestrates a protective IgA response

Abstract

Immunoglobulin A (IgA) is the major secretory immunoglobulin isotype found at mucosal surfaces, where it regulates microbial commensalism and excludes luminal factors from contacting intestinal epithelial cells (IECs). IgA is induced by both T cell-dependent and -independent (TI) pathways. However, little is known about TI regulation. We report that IEC endoplasmic reticulum (ER) stress induces a polyreactive IgA response, which is protective against enteric inflammation. IEC ER stress causes TI and microbiota-independent expansion and activation of peritoneal B1b cells, which culminates in increased lamina propria and luminal IgA. Increased numbers of IgA-producing plasma cells were observed in healthy humans with defective autophagy, who are known to exhibit IEC ER stress. Upon ER stress, IECs communicate signals to the peritoneum that induce a barrier-protective TI IgA response.

Fig. 1.. Intestinal epithelial ER stress induces a protective IgA response.

(A) Absolute counts of SI LP IgA⁺ plasma cells in Xbp1^ΔlEC mice and Xbp1^fl/fl controls at 10 weeks of age (n = 7 or 8). (B) Ileal tissue IgA normalized by total soluble tissue protein (n = 8 to 10). (C) IgA concentration in SI washes (n = 6 to 10). (D) Circulating IgA concentration (n = 6 to 10 for each age). (E and F) Representative IHC images (E) and quantification (F) of LP IgA⁺ cells (brown) along ≥50 ileal crypt-villus axes (n = 6 or 7). Magnified area in (E) depicts basal plasmacytosis. (G) Representative IHC images and quantification of SI LP IgA⁺ cells (red) in Grp78^T-ΔIEC mice and Grp78^fl/fl controls after 3 days of tamoxifen treatment (n = 4). (H and I) Absolute counts of SI LP IgA⁺ plasma cells (H) and circulating IgA concentrations (I) of the indicated genotypes, treated with either TUDCA (2 mg/ml) in the drinking water or plain water (control) for two weeks (n = 7 or 8). (J and K) Enteritis scores of ileal (J) and jejunal (K) sections of indicated genotypes (n = 4 to 26). (L) Representative plots, frequencies, and absolute counts of SI LP IgM⁺ plasma cells (gated on CD45⁺CD3^– lymphocytes) of the indicated genotypes (n = 3 to 14). (M to O) Absolute flow cytometric counts of SI LP IgA⁺ plasma cells (M), representative IHC images and quantification of IgA⁺ cells in ileal sections (N), and enteritis scores (O) of Pigr^–/–Xbp1^ΔIEC mice and Pigr^–/–Xbp1^fl/fl controls (n = 9 to 18). (P) Frequencies of IgA-coated fecal bacteria from the indicated genotypes, as determined by flow cytometry (n = 2 to 20). B6 indicates a C57BL/6J background. Scale bars indicate 100 μm (low magnification) or 20 μmm [magnified view in (E)]. Symbols represent individual animals. Bars represent arithmetic means [(B), (D), (F), (G), (N), and (P)], medians [(J), (K), and (O)], or geometric means [(A), (C), (H), (L), and (M)]. Error bars indicate SEM. Data are representative of three [(A) and (B)] independent experiments or were compiled from two (M) or three [(L) and (P)] experiments. P values were calculated by unpaired Student’s t test [(A) to (D), (F), (G), (L) to (N), and (P)], Kruskal-Wallis test with Dunn’s post-test [(J) and (K)], Mann-Whitney U rank sum test (O), or two-way analysis of variance (ANOVA) with Fisher’s least-significant difference (LSD) method and two-stage step-up method of Benjamini, Krieger, and Yekutieli to control the false discovery rate [(H) and (I)]. *P<0.05; **P <0.01; ***P <0.001; ns, not significant.

Fig. 2.. ER stress–induced IgA is PP- and T cell–independent and involves recruitment of peritoneal B1b cells by a transmissible factor.

(A) Representative plots and percentages of germinal center (GC) B cells (gated on CD19⁺ lymphocytes) in MLN and PP of Xbp1^ΔIEC mice and Xbp1^fl/fl controls (n = 4 to 7). (B) Representative plots and percentages of MLN and PP T_FH cells (gated on CD3⁺CD4⁺ lymphocytes, n = 4 to 6). (C) Absolute counts of SI LP IgA⁺ plasma cells (PCs) in TCRβ^–/–Xbp1^ΔIEC mice and TCRβ^–/–Xbp1^fl/fl controls (n = 8 or 9). (D) Absolute counts of SI LP IgA⁺ plasma cells in PP-deficient Xbp1^ΔIEC mice and Xbp1^fl/fl controls (n = 6 to 8). (E and F) Representative plots, percentages, and absolute counts of peritoneal B1a and B1b cells in Xbp1^ΔIEC mice and Xbp1^fl/fl controls (n = 5 to 7). FSC, forward scatter. (G) Schematic representation of the parabiosis experiment (n = 7 or 8 pairs per genotype). (H) Frequencies of CD45.1⁺ circulating lymphocytes and CD45.1⁺ peritoneal B1 cells 3 weeks after parabiotic surgery. The dotted line indicates 50% chimerism. (I) Absolute numbers of CD45.1⁺ B1b cells in peritoneal cavities of CD45.1 animals conjoined with either Xbp1^fl/fl or Xbp1^ΔIEC mice. (J and K) Absolute numbers of SI LP CD45.1⁺IgA+ plasma cells in parabiotic Xbp1^fl/fl and Xbp1^ΔIEC mice (J) and in CD45.1 parabionts conjoined with either Xbp1^fl/fl or Xbp1^ΔIEC mice (K). Symbols represent individual animals. Bars represent arithmetic means [(A), (B), (E), and (H)] or geometric means [(C), (D), (F), and (I) to (K)]. Data are representative of three experiments [(E) and (F)] or were pooled from two experiments [(C), (D), and (G) to (K)]. P values were calculated by unpaired Student’s t test. *P <0.05; **P <0.01; ***P <0.001; ns, not significant.

Fig. 3.. Epithelial ER stress–derived IgA is microbiota- and inflammationindependent and polyreactive in nature.

(A) Absolute counts of SI LP IgA⁺ plasma cells in GF Xbp1^ΔIEC mice and Xbp1^fl/fl controls (n = 7 or 8). (B) Representative immunofluorescence images and quantification of LP IgA⁺ cells (green) along ≥50 ileal crypt-villus axes (n = 3 to 6). Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Arrows indicate basal plasmacytosis. Scale bar, 100 μm. (C and D) Frequencies (percentage of CD19⁺CD23^–CD43⁺ cells) (C) and absolute flow cytometric counts of peritoneal B1a and B1b cells (D) (n = 7 or 8). (E) Representative plots (gated on CD5^–CD19⁺CD43⁺ lymphocytes) and frequencies of IgA⁺ B1b-derived cells in SI LP of GF Xbp1^ΔIEC mice and GF Xbp1^fl/fl controls (n = 7 or 8). (F) t-Distributed stochastic neighbor embedding (t-SNE) plot depicting unsupervised clustering of single-cell transcriptomes (n = 11,104 cells) from peritoneal lavages of Xbp1^ΔIEC mice and Xbp1^fl/fl controls (aligned datasets). Numbers and colors indicate clusters. (G) Expression levels of canonical markers for macrophages (Csf1r), B cells (Cd79a), T cells (Cd3e), and peritoneal dendritic cells (Cd209a) in t-SNE plot. (H)t-SNE plot as in (F) with cells colored by genotype. Bar graph depicts the number of cells within each cluster by genotype. (I) Volcano plot showing log₂-transformed fold-change (log2FC) of gene expression in B1b cells from GF Xbp1^ΔIEC mice compared with that in B1b cells from GF Xbp1^fl/fl controls (n = 5 to 7). Differentially expressed genes [log2FC ≥ 1 or ≤ –1; false discovery rate (FDR) < 0.05] are highlighted in blue. FDR values that are <10^–5 are plotted at 10^–5 (triangles). (J) GSEA enrichment plots for selected gene sets. GO, gene ontology gene sets; HM, hallmark gene sets; OXPHOS, oxidative phosphorylation; NES, normalized enrichment score. (K) Circulating IgA concentrations in GF Xbp1^ΔIEC mice and Xbp1^fl/fl controls (n = 12 or 13). (L) Representative plots (gated on SYBR^hi events) and frequencies of IgA coating on fecal bacteria from μMT mice that were incubated with sera from GF Xbp1^ΔIEC mice or Xbp1^fl/fl controls (n = 4 or 5). (M) Polyreactivity enzyme-linked immune-sorbent assay optical density at 650 nm (OD₆₅₀) values of serum IgA from GF Xbp1^ΔIEC mice or Xbp1^fl/fl controls (n = 5 or 6) against the indicated antigens. LPS, lipopolysaccharide; KLH, keyhole limpet hemocyanin; dsDNA, double-stranded DNA. Symbols or lines represent individual animals. Bars represent arithmetic means [(B), (C), and (E)] or geometric means [(A), (D), and (K)]. Data are representative of at least two independent experiments [(B) to (D), (L), and (M)] or were pooled from two experiments [(A), (E), and (K)]. P values were calculated by unpaired Student’s t test. *P <0.05; **P < 0.01; ***P <0.001; ns, not significant.

Fig. 4.. Defective ATG16L1-dependent autophagy results in a peritoneal B1b response in mice and IgA induction in both mice and humans.

(A) Representative IHC images and quantification of LP IgA⁺ cells (brown) along ≥50 ileal crypt-villus axes of Atg16l1^ΔIEC mice and Atg16l1^fl/fl controls (n = 8). (B) Absolute counts of peritoneal B1a and B1b cells in Atg16l1^ΔIEC mice and Atg16l1^fl/fl controls (n = 7 to 12). (C) Representative IHC images and quantification of IgA⁺ cells (brown) in ileal biopsies of healthy human subjects, shown by ATG16L1 genotype as indicated by AA, AG, and GG (n = 8 to 16). Scale bars, 100 μm. HPF, high-power field. Symbols represent individual animals or human subjects. Bars represent arithmetic means [(A) and (C)] or geometric means (B). Data in (B) were pooled from two experiments. P values were calculated by unpaired Student’s t test [(A) and (B)] or one-way ANOVA with Holm-Šídák test (C). *P < 0.05; ns, not significant.