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. 2023 Nov 2;29(11):1778-1792.
doi: 10.1093/ibd/izad092.

Deletion of Endogenous Neuregulin-4 Limits Adaptive Immunity During Interleukin-10 Receptor-Neutralizing Colitis

Jessica K Bernard  1   2 , Edie B Bucar  1 , Cambrian Y Liu  1   3 , Kay Katada  1 , Mary K Washington  4 , Michael A Schumacher  1   5 , Mark R Frey  1   5   6
Affiliations

Deletion of Endogenous Neuregulin-4 Limits Adaptive Immunity During Interleukin-10 Receptor-Neutralizing Colitis

Jessica K Bernard et al. Inflamm Bowel Dis. .

Abstract

Background: Growth factors are essential for maintenance of intestinal health. We previously showed that exogenous neuregulin-4 (NRG4) promotes colonocyte survival during cytokine challenge and is protective against acute models of intestinal inflammation. However, the function(s) of endogenous NRG4 are not well understood. Using NRG4-/- mice, we tested the role of endogenous NRG4 in models of colitis skewed toward either adaptive (interleukin-10 receptor [IL-10R] neutralization) or innate (dextran sulfate sodium [DSS]) immune responses.

Methods: NRG4-/- and wild-type cage mate mice were subjected to chronic IL-10R neutralization colitis and acute DSS colitis. Disease was assessed by histological examination, inflammatory cytokine levels, fecal lipocalin-2 levels, and single cell mass cytometry immune cell profiling. Homeostatic gene alterations were evaluated by RNA sequencing analysis from colonic homogenates, with real-time quantitative polymerase chain reaction confirmation in both tissue and isolated epithelium.

Results: During IL-10R neutralization colitis, NRG4-/- mice had reduced colonic inflammatory cytokine expression, histological damage, and colonic CD8+ T cell numbers vs wild-type cage mates. Conversely, in DSS colitis, NRG4-/- mice had elevated cytokine expression, fecal lipocalin-2 levels, and impaired weight recovery. RNA sequencing showed a loss of St3gal4, a sialyltransferase involved in immune cell trafficking, in NRG4-null colons, which was verified in both tissue and isolated epithelium. The regulation of St3gal4 by NRG4 was confirmed with ex vivo epithelial colon organoid cultures from NRG4-/- mice and by induction of St3gal4 in vivo following NRG4 treatment.

Conclusions: NRG4 regulates colonic epithelial ST3GAL4 and thus may allow for robust recruitment of CD8+ T cells during adaptive immune responses in colitis. On the other hand, NRG4 loss exacerbates injury driven by innate immune responses.

Keywords: ErbB receptor tyrosine kinases; adaptive immunity; experimental colitis; neuregulin growth factors.

Plain language summary

Neuregulin-4 (NRG4) is a growth factor that protects the epithelial cells lining the colon from injury and restrains innate (non-specific) immune responses. Here we show that NRG4’s role in inflammation is context-specific, and mice that lack NRG4 have impaired adaptive immunity in a model of chronic immune-mediated colitis.

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Conflict of interest statement

M.R.F. and M.A.S. are inventors on a patent held by Children’s Hospital Los Angeles on the possible therapeutic use of neuregulin-4 in intestinal inflammation. The other authors have no conflicts.

Figures

Figure 1.
Figure 1.
Whole body neuregulin-4 (NRG4) deletion leads to reduced interleukin-10 receptor (IL-10R) neutralization–induced inflammation in the colon. Five-week-old NRG-/- and wild-type (WT) cage mate mice were injected with IL-10R–neutralizing antibody (1 mg/kg) for 4 weeks. A, Hematoxylin and eosin–stained sections from paraffin-embedded colonic Swiss rolls collected on experimental week 5. B, Total colonic damage score; n = 7 WT and n = 12 NRG4-/-. Analyzed by Kruskal-Wallis test (*P < .05). C, Fecal lipocalin-2 enzyme-linked immunosorbent assay (****P < .0001, ** P < .001); n = 10 WT and n = 19 NRG4-/- analyzed by 2-way analysis of variance. D, Plasma myeloperoxidase (MPO) concentration levels by enzyme-linked immunosorbent assay (**P = .0005); n = 5 WT and n = 8 NRG4-/-. E, Total spleen weight (**P = .001). F, Weekly weights of mice (*P < .05). G, Colon length. H, Bulk RNA sequencing analysis of colonic homogenates. Data are presented as the mean ± SEM. Statistical testing by Student’s 2-sided t tests. TPM, transcripts per million.
Figure 2.
Figure 2.
Neuregulin-4 (NRG4) deletion reduces inflammatory cytokines and T cell infiltration in interleukin-10 receptor (IL-10R) neutralization colitis. A, Relative messenger RNA (mRNA) levels (quantitative polymerase chain reaction) of Tnf, Ifnγ, Il1b, and Il6 from full-thickness colonic homogenates after 5 weeks of IL-10R neutralization colitis, compared with baseline colon homogenates; n = 10 baseline WT, n = 10 baseline NRG4-/-, n = 8 colitic WT, and n = 12 colitic NRG4-/-, analyzed by 1-way analysis of variance. The following outliers were identified and removed by ROUT testing (Q = 1.0%): Tnf (1 from the WT control group), Ifnγ (1 from the NRG4-/- control group), Il1b (1 from the WT control and 2 from the NRG4-/- colitis groups), and Il6 (1 from the NRG4-/- control and 1 from NRG4-/- colitis groups). B, CyTOF analysis of colonic cells isolated after IL-10R–induced injury, expressed as % of total cellularity in the sample. C, Expression of homing genes were analyzed by quantitative polymerase chain reaction in colonic homogenates. Data are presented as the mean ± SEM. Statistical testing used Student’s 2-sided t tests. *P < .05; **P < .01; ***P < .001; ****P < .0001.
Figure 3.
Figure 3.
Neuregulin-4 (NRG4) deletion moderately limits splenic CD8+ T cells but does not alter CD8+ T cell proliferation. A, Colonic cells were isolated from 5-week-old NRG4-/- mice and wild-type (WT) cage mates and analyzed by flow cytometry; n = 4 WT and n = 4 NRG4-/-. B, Baseline RNA sequencing from colonic homogenates in adult NRG4-/- and WT cage mates was analyzed using CIBERSORT to predict changes in immune cell abundance between groups; n = 6 WT and n = 5 NRG4-/-. C, Splenocytes were isolated and analyzed by flow cytometry. D, Thymus and spleen collected from unchallenged mice were analyzed by quantitative polymerase chain reaction for expression of CD45 (Ptprc) and the interleukin-10 receptor (Il10ra, Il10rb). E, Isolated CD8+ T cells were analyzed for Nrg4 expression. F and G, Isolated T cells were stimulated and/or treated with tumor necrosis factor and analyzed for (F) interleukin-10 receptor or (G) Cyclin D1 and Ki67. Data are presented as the mean ± SEM. Analyzed by Student’s 2-sided t test. *P < .05. mRNA, messenger RNA.
Figure 4.
Figure 4.
Neuregulin-4–null (NRG4-/-) mice have impaired recovery in dextran sulfate sodium (DSS) colitis. NRG4-/- and wild-type (WT) littermates were subjected to a recovery model of DSS colitis (6 days with 3% DSS in drinking water followed by 6 days with no DSS). A, Daily weights of mice. B, Fecal lipocalin-2 levels. C, Colon lengths. D, Histological scores. E, Cytokine expression in colonic homogenates. Data are presented as the mean ± SEM. Analyzed by Student’s 2-sided t test. *P < .05; **P < .01. mRNA, messenger RNA.
Figure 5.
Figure 5.
RNA sequencing (RNA-seq) analysis indicates significant loss of St3gal4 in mice with neuregulin-4 (NRG4) deletion. RNA-seq of full-thickness colonic biopsies from n = 5 NRG4-/- and n = 6 wild-type (WT) cage mates 11 to 12 weeks of age. A, Heat map of expression levels of altered genes. Red indicates an increase in relative expression and blue indicates a decrease in relative expression, as shown in the figure legend. B, Volcano plot of altered genes identified by RNA-seq; St3gal4 P = .0008. C, Pathway analysis of downregulated genes that are above 2 -log10P value by BioPlanet_2019 in NRG4-/- mice compared with WT cage mates.
Figure 6.
Figure 6.
Neuregulin-4 (NRG4) deletion leads to the loss of colonic epithelial ST3GAL4. A,St3gal4 in situ hybridization from NRG4-/- and wild-type (WT) colonic Swiss roll sections at homeostasis. B, Relative St3gal4 messenger RNA (mRNA) expression from NRG4-/- and WT colonic organoids after starvation (***P = .0007), analyzed by Student’s 2-sided t test. C, Relative St3gal4 mRNA expression from full-thickness NRG4-/- and WT colonic homogenates at baseline and after interleukin-10 receptor neutralization colitis. Statistical analysis by 1-way analysis of variance, with 1 outlier from the NRG4-/- control group was identified and removed by ROUT testing (Q = 1.0%). D, NRG4-/- mice were intraperitonally injected with NRG4 (100 µg/kg) or phosphate-buffered saline for 24 hours. Relative St3gal4 mRNA expression from n = 4 NRG4-/- and n = 4 WT full-thickness colonic biopsies (**P = .0011). E, Thymus St3gal4 expression. F, Spleen St3gal4 expression. G, Mucosal thickness assessed by Alcian blue staining on distal colonic sections. Statistical testing used Student’s 2-sided t tests.
Figure 7.
Figure 7.
The loss of neuregulin-4 (NRG4) may shift microbial composition, promoting an increase in known protective bacterial species. Six wild-type (WT) mice (WT breeding cage) and 6 NRG4-/- mice (NRG4-/- breeding cage) were weaned together at approximately 3 weeks of age, in 3 different cages. Stool was collected at 5 and 9 weeks of age. Stool from WT and NRG4-/- dams were collected at litter weaning. Colonic scrapings were collected at 9 weeks. A, 16S analysis of microbial diversity for each age and genotype visualized from rarefaction. Statistical analysis using Student’s 2-sided t test; stool at 5 weeks (P = .09) and at 9 weeks (P = .06). B, Principal component (PC) analysis of microbial abundances for each age and genotype, as compared with each dam. C, Random forest techniques identify discriminating taxa as those whose elimination reduces the accuracy of the classifier for the tested NRG4-/- and WT genotypes. D, The abundance, as determined by the percentage of biomass, from discriminating species predicted by the random forest methods in WT and NRG4-/- cage mates. Statistical testing by Student’s 2-sided t-test.
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References

    1. Harari D, Tzahar E, Romano J, et al. . Neuregulin-4: a novel growth factor that acts through the ErbB-4 receptor tyrosine kinase. Oncogene. 1999;18:2681-2689. doi:10.1038/sj.onc.1202631 - DOI - PubMed
    1. Bernard JK, McCann SP, Bhardwaj V, Washington MK, Frey MR.. Neuregulin-4 is a survival factor for colon epithelial cells both in culture and in vivo. J Biol Chem. 2012;287:39850-39858. doi:10.1074/jbc.M112.400846 - DOI - PMC - PubMed
    1. Schumacher MA, Hedl M, Abraham C, et al. . ErbB4 signaling stimulates pro-inflammatory macrophage apoptosis and limits colonic inflammation. Cell Death Dis. 2017;8:e2622. doi:10.1038/cddis.2017.42 - DOI - PMC - PubMed
    1. Chen Z, Wang GX, Ma SL, et al. . Nrg4 promotes fuel oxidation and a healthy adipokine profile to ameliorate diet-induced metabolic disorders. Mol Metab. 2017;6:863-872. doi:10.1016/j.molmet.2017.03.016 - DOI - PMC - PubMed
    1. Schumacher MA, Dennis IC, Liu CY, et al. . NRG4-ErbB4 signaling represses proinflammatory macrophage activity. Am J Physiol Gastrointest Liver Physiol. 2021;320:G990-G1001. doi:10.1152/ajpgi.00296.2020 - DOI - PMC - PubMed

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