Nfkb2 deficiency and its impact on plasma cells and immunoglobulin expression in murine small intestinal mucosa

Abstract

The alternative (noncanonical) nuclear factor-κB (NF-κB) signaling pathway predominantly regulates the function of the p52/RelB heterodimer. Germline Nfkb2 deficiency in mice leads to loss of p100/p52 protein and offers protection against a variety of gastrointestinal conditions, including azoxymethane/dextran sulfate sodium (DSS)-induced colitis-associated cancer and lipopolysaccharide (LPS)-induced small intestinal epithelial apoptosis. However, the common underlying protective mechanisms have not yet been fully elucidated. We applied high-throughput RNA-Seq and proteomic analyses to characterize the transcriptional and protein signatures of the small intestinal mucosa of naïve adult Nfkb2^-/- mice. Those data were validated by immunohistochemistry and quantitative ELISA using both small intestinal tissue lysates and serum. We identified a B-lymphocyte defect as a major transcriptional signature in the small intestinal mucosa and immunoglobulin A as the most downregulated protein by proteomic analysis in Nfkb2^-/- mice. Small intestinal immunoglobulins were dramatically dysregulated, with undetectable levels of immunoglobulin A and greatly increased amounts of immunoglobulin M being detected. The numbers of IgA-producing, cluster of differentiation (CD)138-positive plasma cells were also reduced in the lamina propria of the small intestinal villi of Nfkb2^-/- mice. This phenotype was even more striking in the small intestinal mucosa of RelB^-/- mice, although these mice were equally sensitive to LPS-induced intestinal apoptosis as their RelB^+/+ wild-type counterparts. NF-κB2/p52 deficiency confers resistance to LPS-induced small intestinal apoptosis and also appears to regulate the plasma cell population and immunoglobulin levels within the gut.NEW & NOTEWORTHY Novel transcriptomic analysis of murine proximal intestinal mucosa revealed an unexpected B cell signature in Nfkb2^-/- mice. In-depth analysis revealed a defect in the CD38+ B cell population and a gut-specific dysregulation of immunoglobulin levels.

Keywords: NF-κB; Nfkb2; RelB; immunoglobulins; intestinal mucosa; plasma cells.

Graphical abstract

Figure 1.

B cell defect is a major transcriptional signature in Nfkb2^−/− small intestinal mucosa. Total RNA was isolated from proximal small intestine and used for RNA sequencing (n = 6). A: volcano graph of the differentially regulated genes that were further filtered, under high stringency, using a cut off of 1.5 log2-fold change for both downregulated genes (blue) and upregulated genes (red). B: top 10 downregulated genes in Nfkb2^−/− small intestinal tissue with genes that are expressed in B cells shown in red. C: validation of St6gal1, Igtp, Gbp2, and Gbp8 gene expression by real-time qPCR analysis in Nfkb2^+/+ (black circles) and Nfkb2^−/− (open circles) samples (n = 6–8/genotype, equal number of males and females). D: proximal small intestinal sections showing lamina propria staining for St6gal1 in C57BL/6, but not in Nfkb2^−/− mice (representative image of n = 4). Mann–Whitney test for pairwise comparisons was applied. Differences were considered statistically significant when P < 0.05.

Figure 2.

Proteomics analysis of Nfkb2^−/− small intestinal mucosa reveals immune system category and immunoglobulins as being the most affected. Total protein lysates from proximal small intestinal epithelium of both C57BL/6 wild-type and Nfkb2^−/− mice (n = 4) were used for proteomics analysis. A: volcano plot showing the differentially regulated proteins (Welch´s T test, FDR < 0.05) for both downregulated (blue) and upregulated proteins (red). B: pathway analysis with down-weighting of overlapping genes (PADOG) showing the most downregulated [blue, lowest value log2(fold change) = −3.31] and upregulated [yellow, highest value log2(fold change) = 1] enriched pathways affected by Nfkb2 deletion. Scale bar represents the log2 (fold change). C and D: top 10 most upregulated and downregulated proteins identified by proteomics analysis in proximal small intestine isolated from naïve Nfkb2^−/− mice.

Figure 3.

Severe dysregulation of immunoglobulins in Nfkb2^−/− mice. Small intestinal tissue lysates and sera from Nfkb2^+/+ (black circles), Nfkb2^+/− (black and white circles), and Nfkb2^−/− mice (open circles) (n = 5 or 6/genotype) were used for quantification of immunoglobulins. Total protein in tissue lysates was used for normalization purpose. Kruskal–Wallis multiple-comparison test was applied. *P < 0.05, **P < 0.01.

Figure 4.

Absence of CD138-positive (CD138+ve) plasma cells in Nfkb2^−/− and RelB^−/− intestinal lamina propria. Small intestine from C57BL/6 and Nfkb2^−/− mice (males and females) and RelB^−/− females (along with their wild-type and heterozygous littermates) were fixed and 4 µm sections were used for staining for CD138 (×40 magnification). A: representative images of stained villi of the four genotypes are presented. Tissues were counterstained with hematoxylin. B: comparison of CD138+ve plasma cells numbers between C57BL/6 (black diamonds) and the respective wild-type littermate controls [Nfkb2^+/+ (black circles) or RelB^+/+ (black squares)]. C: effect of Nfkb2 deficiency on lamina propria CD138+ve plasma cell numbers in Nfkb2^+/+ (black circles), Nfkb2^+/− (black and white circles), and Nfkb2^−/− (open circles) samples. D: effect of RelB deficiency on lamina propria CD138+ve plasma cell numbers in RelB^+/+ (black squares), RelB^+/− (black and white squares), and RelB^−/− (open squares) samples. Ten villi per mouse were counted (n = 3–7 mice/genotype). Kruskal–Wallis multiple-comparison test was applied. *P < 0.05. Scale bars represent 100 μm.

Figure 5.

RelB-deficient and Nfkb2 heterozygous mice are sensitive to LPS-induced small intestinal apoptosis in vivo. A: quantification of apoptotic and shedding intestinal epithelial cells (ICEs) in RelB^+/+ (black squares), RelB^+/− (black and white squares), and RelB^−/− (open squares), small intestinal sections labelled for active caspase-3, 1.5 h after 0.125 mg/kg LPS injection (n = 5 mice). B: quantification of apoptotic and shedding IECs in Nfkb2^+/+ (black circles), Nfkb2^+/− (black and white circles), and Nfkb2^−/− (open circles), small intestinal sections labelled for active caspase-3, 1.5 h after 0.125 mg/kg LPS injection (n = 4–6 mice). C: IHC (×40) for active caspase 3 in small intestinal sections from C57BL/6 mice without treatment (−) or 1.5 h post 0.125 mg/kg LPS injection (+). Kruskal–Wallis multiple-comparison test was applied. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars represent 100 μm. LPS, lipopolysaccharide.