Distinct roles for LTalpha3 and LTalpha1beta2 produced by B cells contribute to their multi-faceted impact on ileitis

Abstract

B lymphocytes may facilitate chronic inflammation through antibody production or secretion of cytokines, including lymphotoxin (LT)-a₁b₂ associated with development of lymphoid tissue. Tertiary lymphoid structures (TLS) characterize human and murine ileitis by suppressing outflow from the ileum. Here, we show that B cell-derived secretory IgA protected against ileal inflammation, whereas B cell-derived LTa guarded against ileitis-associated loss of body mass. We initially hypothesized this protection resulted from formation of TLS that suppressed lymphatic outflow and thereby restrained systemic spread of inflammatory signals, but B cell-selective deletion of LTb did not exacerbate weight loss, despite eliminating TLS. Instead, weight loss driven by the cachectic cytokine TNF was exacerbated when LTa₃, another ligand for TNF receptors, was selectively neutralized. Thus, B cells' multi-faceted impact on ileitis includes generating secretory IgA, expressing LTa₁b₂ to drive formation of TLS, and producing LTa₃ for protecting against weight loss in the presence of TNF.

Figure 1

B cells are enriched in the inflamed mesentery of Crohn’s disease patients. a: Quantification of the number of CD19⁺ B cells in surgically resected sections of ileal-draining mesentery normalized to the weight of tissue (Control (n = 20), CD non-inflamed (n = 16), CD inflamed (n = 15)). b: A subset of the Fig. 1a data to show only CD patients that had matched mesentery draining from non-inflamed ileum and from inflamed ileum, lines connect paired samples from the same patient (n = 13). c–f: Single cell RNA sequencing (scRNA-seq) of sorted live, CD45⁺ mesenteric cells from 3 CD patients with matched non-inflamed and inflamed draining mesenteric samples. Uniform Manifold Approximation and Projection [UMAP] plot of mesenteric immune cells (c), the shift in proportion of cells in each cluster comparing non-inflamed and inflamed ileal draining mesentery (d), UMAP plots of a subset of genes used to identify clusters (e), and a dot plot showing average expression of RNA of genes in the TNF/LT family along with their receptors. g–h: Single cell BCR sequencing of 3 CD patients and 2 surgical controls of cells from the ileum and mesentery. The proportion of sequenced BCRs in the noninflamed ileum, inflamed ileum, non-inflamed draining mesentery, and inflamed mesentery that are clonal vs. non-clonal (g) and what isotype they are (h). Data in a and b represent mean ± SEM. * p < 0.05, **** p < 0.0001. Kruskal-Wallis test (a), Paired t test (b).

Figure 2. TNF^ΔARE/+ mice without B cells develop ileitis.

WT, TNF^ΔARE/+, and μMT-TNF^ΔARE/+ BM recipients 16–17 weeks post-transplant a: Schema to describe the bone marrow chimera groups. b: Semi-quantitative histological scoring on the distal ileum of BM chimera recipients (Three independent experiments (WT (n=9), TNF^ΔARE/+ and μMT-TNF^ΔARE/+ (n = 14)). c: Representative H&E pictures of the distal ileum of mice given WT, TNF^ΔARE/+, and B cell deficient μMTTNF^ΔARE/+ BM. Scale bars = 150 μm. d–e: Flow cytometry on the ileum for neutrophil (d) and T cells (e) numbers (Three independent experiments, WT (n = 15), TNF^ΔARE/+ and μMT-TNF^ΔARE/+ (n = 14)). f: Fold change in the amount of lipocalin-2 in the stool of mice normalized to the average amount per WT group per experiment (Three independent experiments, WT (n = 14), TNF^ΔARE/+ and μMT-TNF^ΔARE/+ (n = 13)). All data represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. One-way ANOVA with post-hoc Tukey test (b), Kruskal-Wallis test (d–f).

Figure 3. B cells are required for lymphatic remodeling in mice receiving TNF^ΔARE/+ BM.

WT, TNF^ΔARE/+, and μMT-TNF^ΔARE/+ BM recipients 16–17 weeks post-transplant. a-c: Representative whole mount images (LYVE-1 red, CSF1R green, B220 blue) staining mice that received WT (a), TNF^ΔARE/+ (b) and μMT-TNF^ΔARE/+ (c) BM. a–c, scale bars = 500μm. d: The number of neutrophils in the mesentery of mice in mice given TNF^ΔARE/+ and μMT-TNF^ΔARE/+ BM. (Three independent experiments, WT and TNF^ΔARE/+ (n = 14) and μMT-TNF^ΔARE/+ (n = 13)). e-g: Representative stereoscope images (left: brightfield and FITC channels, right: FITC channel alone) of the mesentery of anesthetized mice after FITC-dextran injection of 1 to 1.5 μl into the most distal Peyer’s patch, scale bars = 1.5 mm. Mice given WT (e), TNF^ΔARE/+ (f), and μMT-TNF^ΔARE/+ (g) BM. h: Quantification of time to MLN in tracer experiments. (Three independent experiments, WT (n = 4), TNF^ΔARE/+ (n = 6) and μMT-TNF^ΔARE/+ (n = 4)). All data represent mean ± SEM. ** p < 0.01, *** p < 0.001. Kruskal-Wallis test (d).

Figure 4. B cells sustain retention of lean mass in mice given TNF^ΔARE/+ BM.

a: Weekly percent weight change in mice given WT, μMT, TNF^ΔARE/+, or μMT-TNF^ΔARE/+ BM normalized to weight 2 weeks post-transplant (Six independent experiments, WT (n = 24), μMT (n = 9), TNF^ΔARE/+ (n = 22), μMT-TNF^ΔARE/+ (n = 23)). b–f: Mice given WT, TNF^ΔARE/+, and μMT-TNF^ΔARE/+ BM 14 weeks post-transplant. b: EchoMRI evaluation of the body composition of mice given WT, TNF^ΔARE/+, or μMTTNF^ΔARE/+ BM. c: Core body temperature of mice given WT, TNF^ΔARE/+, or μMT-TNF^ΔARE/+ BM. d-f: Mice were housed in metabolic cages for 24 hours, and activity (d), heat (e), and food intake (f) were measured. g: Weight of the gastrocnemius muscle in BM recipients. h: micro-CT analysis of paws of the bone mineral density. (Three independent experiments WT and TNF^ΔARE/+ (n = 14), μMT-TNF^ΔARE/+ (n = 13)). All data represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. One-way ANOVA with post-hoc Tukey test (c, g, h), Two-way ANOVA (to test if light vs. dark is different in addition to if there are group differences) (d–f).

Figure 5. Secretory antibody partially protects against ileal inflammation in TNF^ΔARE/+ BM recipients, but this protection is limited only to the ileum.

Lethally irradiated pIgR^−/− mice or pIgR^+/+ mice given TNF^ΔARE/+ bone marrow 16 weeks post-transplant. a, μg of IgA per gram of stool in pIgR^−/− vs. pIgR^+/+ recipients given TNF^ΔARE/+ BM. b, Fecal lipocalin-2 levels normalized to the average fecal lipocalin-2 amount in the pIgR^+/+ TNF^ΔARE/+ BM controls per experiment. (A–B, Two independent experiments, pIgR^+/+ (n = 7), pIgR^+/+ (n = 9). c–d, Flow cytometry on the ileum shows numbers of neutrophils (c) and T cells (d). Three independent experiments, pIgR^+/+ (n = 8), pIgR^−/− (n = 12)). e, Total body weight (Four independent experiments, pIgR^+/+ (n = 12), pIgR^−/− (n = 15)). f, Number of monocytes in the blood. (Three independent experiments, pIgR^+/+ (n = 8), pIgR^−/− (n = 12)). g, Number of TLS along the most distal branch of the mesentery (One experiment, pIgR^+/+ (n = 3), pIgR^−/− (n = 6)). All data represent mean ± SEM. * p < 0.05, ** p < 0.01. Mann-Whitney test for all.

Figure 6. B cell production of LTa₁b₂ is required for robust mesenteric TLS in ileitis.

a: Quantification of the number of TLS in the branch of the mesentery that drains the distal ileum. b,c: Representative whole mount images (LYVE-1 red, CSFR1 green, B220 blue) of lymphoid aggregates in the ileal associated mesentery of mice given WT/ μMT-TNF^ΔARE/+ (b) or LTb^−/−/μMT-TNF^ΔARE/+ (c) BM. Scale bars, 500 μm. d,e: Flow cytometry on the distal ileum of mice that received WT/μMT-TNF^ΔARE/+ or LTb^−/−/ μMT-TNF^ΔARE/+ BM to quantify neutrophils (d) and T cells (e). f,g: Flow cytometry on the blood of mice given WT/μMT-TNF^ΔARE/+ and LTb^−/−/μMT-TNF^ΔARE/+ BM to quantify neutrophils (f) and T cells (g). Two independent experiments were done, comparing WT/μMT-TNF^ΔARE/+ to LTb^−/−/μMT-TNF^ΔARE/+ BM recipients, WT/μMT-TNF^ΔARE/+ (n = 9), LTb^−/−/μMT-TNF^ΔARE/+ (n = 10). h,i: 8 weeks after TNF^ΔARE/+ BM transplant, mice were dosed weekly with isotype control or LTbR-Fc for an additional eight weeks in one experiment, isotype and LTbR-fc (n = 3 mice in each cohort). h: TLS were quantified in TNF^ΔARE/+ BM recipients given LTbR-Fc in the distal mesentery or isotype control. i: Flow cytometry on the ileum of mice given TNF^ΔARE/+ BM and treated with LTbR-fc or isotype control to quantify neutrophils. j,k: Representative stereoscope images of a CD19^cre/+Td^tomato/+/μMT-TNF^ΔARE/+ (j) and CD19^cre/+Td^tomato/+TNF^fi/fi/μMT-TNF^ΔARE/+ (k) BM recipient with Td^tomato signal overlaid onto a brightfield image. The length of the scale bars represents 1.5 mm. Graphic plots depict mean ± SEM. * p < 0.05, ** p < 0.01. Mann-Whitney tests were used in all graphs of this figure.

Figure 7. B cell production of LTa₃ protects against weight loss in ileal inflammation.

A: Quantification of the number of TLS in mice given LTa^−/−/μMT-TNF^ΔARE/+ BM compared to mice that received WT/ μMT-TNF^ΔARE/+ BM. b: Body weight of WT/ μMT-TNF^ΔARE/+, LTa^−/−/μMT-TNF^ΔARE/+ BM recipient mice and LTb^−/−/μMT-TNF^ΔARE/+ BM recipient mice at 30 weeks post-BM transplant. c: Percent weight change of mice comparing day 0 to day 2 in mice transplanted with WT or TNF^ΔARE/+ bone marrow 9 weeks post-transplant, then dosed with anti-LTa3 antibody or only isotype. d: Percent weight change of mice comparing day 0 to day 2 in mice transplanted with WT or TNF^ΔARE/+ bone marrow 9 weeks post-transplant, then dosed with anti-LTa3 antibody, anti-TNF antibody, both, or only isotype. e: MRI measurements of the body fat composition on day 3. f: Weights of the gastrocnemius muscle on day 5. g–i, Hydration ratio, activity, and food intake at day 3. All plots mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. One symbol represents data from one individual mouse.