Carrageenan-induced Colonic Inflammation Is Reduced in Bcl10 Null Mice and Increased in IL-10-deficient Mice

Abstract

The common food additive carrageenan is a known activator of inflammation in mammalian tissues and stimulates both the canonical and noncanonical pathways of NF-κB activation. Exposure to low concentrations of carrageenan (10 μ g/mL in the water supply) has produced glucose intolerance, insulin resistance, and impaired insulin signaling in C57BL/6 mice. B-cell leukemia/lymphoma 10 (Bcl10) is a mediator of inflammatory signals from Toll-like receptor (TLR) 4 in myeloid and epithelial cells. Since the TLR4 signaling pathway is activated in diabetes and by carrageenan, we addressed systemic and intestinal inflammatory responses following carrageenan exposure in Bcl10 wild type, heterozygous, and null mice. Fecal calprotectin and circulating keratinocyte chemokine (KC), nuclear RelA and RelB, phospho(Thr559)-NF-κB-inducing kinase (NIK), and phospho(Ser36)-IκBα in the colonic epithelial cells were significantly less (P < 0.001) in the carrageenan-treated Bcl10 null mice than in controls. IL-10-deficient mice exposed to carrageenan in a germ-free environment showed an increase in activation of the canonical pathway of NF-κB (RelA) activation, but without increase in RelB or phospho-Bcl10, and exogenous IL-10 inhibited only the canonical pathway of NF- κ B activation in cultured colonic cells. These findings demonstrate a Bcl10 requirement for maximum development of carrageenan-induced inflammation and lack of complete suppression by IL-10 of carrageenan-induced inflammation.

Figure 1

Histopathology of intestinal tissue in Bcl10 wild type and null mice following carrageenan exposure. (a), (b) Bcl10 WT, heterozygous, and null mice were sacrificed after ingestion of carrageenan (10 μg/mL) added in their water for 30 days. Histopathology of H&E sections of cecum demonstrated disruption of the mucosa in a WT mouse, with no similar findings in the Bcl10 null mice, consistent with reduced inflammation in the absence of Bcl10 [WT: wild type]. (c) Throughout the mouse intestine, the extent of inflammatory infiltrate, including granulocytes, lymphocytes, and plasma cells, was greater in the Bcl10 WT mice, than in the Bcl10 null mice. The histopathology was scored for 3 WT, 3 heterozygous, and 3 null mice, and the mean scores for leukocyte infiltration for each site are compared. The scores for the WT mice are higher at each site than for the null mice, although the differences are not statistically significant. The extent of inflammatory infiltrate was significantly greater in the small intestine than in the colon and rectum for each of the groups (P < 0.01). Cecal inflammation, including leukocyte infiltration and edema, was greater in the WT mice than in the heterozygous or null mice. (d) The mouse weights at the onset of the carrageenan exposure and at termination are presented and indicate no significant differences in weight and slight weight gain in all groups. (n for no CGN control=3; n for WT with CGN = 6; n for het with CGN = 3; n for null with CGN = 8) (WT: wild type; het: heterozygous; CGN: carrageenan).

Figure 2

Carrageenan-induced increases in KC and calprotectin are less in Bcl10 null mice. (a) KC, the mouse homolog of IL-8, increased less in the Bcl10 null mice (n = 8) than in the wild type (n = 6) or heterozygous (n = 3) mice following carrageenan to ~77% of the higher value (142.2 ± 9.0 ng/L versus 188.5 ± 6.4 ng/L; P < 0.001). (b) Fecal calprotectin, an indicator of colonic inflammation, also increased less following carrageenan in the Bcl10 null mice than in the wild type or heterozygous mice, to 115.1 ± 8.0 μg/kg versus 182.7 ± 14.3 μg/kg in the wild type mice (P < 0.001). CGN: carrageenan; KC: keratinocyte chemokine; WT: wild type; het: heterozygous.

Figure 3

Serum IL-6 and MCP-1 increase post-carrageenan exposure, and increases are similar in Bcl10 WT, heterozygous, and null mice. Carrageenan exposure produced significant increases in serum levels of IL-6 and MCP-1 in the WT, heterozygous, and null mice (P < 0.001), compared to the unexposed controls. WT, heterozygotes, and null mice serum levels of MCP-1 and IL-6 both increased ~85%. Serum levels of TNF-α, IFNγ, IL-1β, IL-10, IL-12, and IL-23 did not change.

Figure 4

Carrageenan-induced increase in RelA is reduced and is absent in RelB in Bcl10 null mice. (a) Nuclear RelA, measured by oligonucleotide assay, increased by ~89% in the wild type and heterozygous mice following carrageenan but only by about 40% in the Bcl10 null mice (P < 0.001). (b) In contrast, the increase in RelB was completely inhibited in the Bcl10 null mice but increased by ~75% in the heterozygous and wild type mice following carrageenan (P < 0.001). CGN: carrageenan; WT: wild-type; Het: heterozygous; N.D.: no difference.

Figure 5

Phosphorylation of Bcl10 is absent and of IκBα is reduced in Bcl10 null mice following carrageenan. (a) Phospho(Ser138)-Bcl10 was increased to ~400% of the no carrageenan baseline when the Bcl10 wild type and heterozygous mice were exposed to carrageenan (P < 0.001) but not in the Bcl10 null mice. (b) The increase in phospho(Ser32)-IκBα is less in the Bcl10 null mice, compared to the wild type and heterozygous mice (increase of ~37% versus ~91%), but the increase from the baseline no carrageenan value is also significant (P < 0.001). CGN: carrageenan; WT: wild type; Het: heterozygous.

Figure 6

Different responses of phospho(Thr559)-NIK and of phospho(Thr184)-Tak1 following carrageenan in Bcl10 null mice. (a) The immunoblot shows that phospho-NIK does not increase following exposure to carrageenan in the Bcl10 null mice, in contrast to the finding in the wild type mice. (b) Densitometry confirms the increase in phospho-NIK in the Bcl10 WT mice treated with carrageenan compared to the Bcl10 null mice (P = 0.01, unpaired t-test, two-tailed). (c) In contrast, phospho-Tak1(Thr184) was somewhat increased in the Bcl10 null mice's intestinal tissue, although less than in the wild type mouse tissue. (d) Densitometry confirms the visual impression that the phospho-Tak1 is significantly reduced in the Bcl10 null versus Bcl10 WT mouse following CGN exposure (P < 0.05, unpaired t-test, two-tailed). CGN: carrageenan; WT: wild type; NIK: NF-κB inducing kinase; Tak: TGF-β activated kinase.

Figure 7

Effects of carrageenan exposure in IL-10-deficient mice compared to C57BL/6 control in germ-free environment and in standard housing. (a) KC was not different in the C57BL/6 germ-free mice versus mice with standard housing at baseline or following carrageenan exposure. In the IL-10-deficient mice, KC was greater at baseline (P < 0.05) and following carrageenan (P < 0.001) than in the control mice, and the increase in KC was greater in the IL-10-deficient mice than in the controls (~271 ng/L versus ~87 ng/L). (b) Fecal calprotectin was not different in the germ-free mice versus the standard housing control mice with or without carrageenan exposure. Fecal calprotectin was greater in the IL-10-deficient mice at baseline (P < 0.05) and following CGN (P < 0.001). (c) Bcl10 was similar in the C57BL/6 mice in the germ-free environment as in standard housing. Baseline Bcl10 was significantly greater in the IL-10 null mice than in the controls (P < 0.001), and increased more in the IL-10-deficient mice (~229% versus ~144%) following carrageenan (P < 0.001) than in the control mice. (d) The germ-free environment did not affect the RelA results in the C57BL/6 mice. Baseline RelA was significantly greater in the IL-10 null mice than in the control C57Bl/6 mice (P < 0.05) and increased more following carrageenan in the IL-10-deficient mice than in the controls (P < 0.001). (e) Carrageenan-induced increase in RelB was unaffected by IL-10 deficiency. (f) Carrageenan-induced increase in phospho(Ser138)-Bcl10 was unaffected by IL-10 deficiency or the germ-free environment. CGN: carrageenan; N.D.: no difference.

Figure 8

In NCM460 cells, exogenous IL-10 inhibits the canonical but not the non-canonical NF-κB pathway that is activated by carrageenan and mediated by Bcl10. (a) Increase in RelA following exposure to carrageenan was partially inhibited by exogenous IL-10 in the NCM460 cells. When Bcl10 was silenced by siRNA, the increase in RelA was reduced and completely inhibited by IL-10 (20 μg/L × 24 h). (b) The increase in phospho(Ser32)-IκBα was partially inhibited by exogenous IL-10 in the control and control siRNA cells. The increase was less when Bcl10 was silenced and was almost completely inhibited by exogenous IL-10. (c) In contrast to the above findings, the increase in RelB was unaffected by exogenous IL-10. (d) Phospho(Ser138)-Bcl10 increased to over four times the baseline in the control and control siRNA cells, and the increases were not inhibited by exogenous IL-10. ND: no difference; IL-10: interleukin-10; si: small interfering siRNA; CGN: carrageenan.