Crohn's Disease Fibroblasts Overproduce the Novel Protein KIAA1199 to Create Proinflammatory Hyaluronan Fragments

Abstract

Background & aims: Crohn's Disease (CD) is a chronic inflammatory disease of the gastrointestinal tract. Fibrosis, a serious complication of CD, occurs when activated intestinal fibroblasts deposit excessive amounts of extracellular matrix (ECM) in affected areas. A major component of the ECM is high-molecular-weight hyaluronan (HA) that, when depolymerized to low-molecular-weight fragments, becomes proinflammatory and profibrotic. Mechanisms for HA degradation are incompletely understood, but the novel protein KIAA1199 recently was discovered to degrade HA. We hypothesized that KIAA1199 protein is increased in CD colon fibroblasts and generates HA fragments that foster inflammation and fibrosis.

Methods: Fibroblasts were isolated from explants of surgically resected colon tissue from CD and non-inflammatory bowel disease control (ND) patients. Protein levels and tissue distribution of KIAA1199 were assessed by immunoblot and immunostaining, and functional HA degradation was measured biochemically.

Results: Increased levels of KIAA1199 protein were produced and deposited in the ECM by cultured CD fibroblasts compared with controls. Treatment of fibroblasts with the proinflammatory cytokine interleukin (IL) 6 increased deposition of KIAA1199 in the ECM. CD fibroblasts also produce significantly higher levels of IL6 compared with controls, and antibody blockade of IL6 receptors in CD colon fibroblasts decreased the level of KIAA1199 protein in the ECM. Colon fibroblasts degrade HA, however, small interfering RNA silencing of KIAA1199 abrogated that ability.

Conclusions: CD fibroblasts produce increased levels of KIAA1199 primarily through an IL6-driven autocrine mechanism. This leads to excessive degradation of HA and the generation of proinflammatory HA fragments, which contributes to maintenance of gut inflammation and fibrosis.

Keywords: CD, Crohn’s disease; Crohn’s Disease; DAMP, damage-associated molecular pattern; ECM, extracellular matrix; FBS, fetal bovine serum; Fibrosis; HA, hyaluronan; HBSS, Hank's balanced salt solution; HIF, human intestinal fibroblasts; HYAL, hyaluronidase; Hyaluronan; IBD, inflammatory bowel disease; IL, interleukin; IL6R, interleukin 6 receptor; KIAA1199; LC-MS, liquid chromatography–mass spectrometry; ND, non–inflammatory bowel disease control; NF-κB, nuclear factor-κB; PAGE, polyacrylamide gel electrophoresis; PBST, phosphate-buffered saline with 0.1% Tween-20; SDS, sodium dodecyl sulfate; TGF, transforming growth factor; TLR, Toll-like receptor; TNF, tumor necrosis factor; siRNA, small interfering RNA.

Figure 1

KIAA1199 is increased in the submucosal region of fibrotic CD colons. (A) A confocal image of a human colon tissue section from a non-IBD patient immunostained for KIAA1199 protein (red) and HA (green). (B) A confocal image of a human colon tissue section from a fibrotic CD patient immunostained for KIAA1199 protein (red) and HA (green). (C) Morphometric analyses of KIAA1199 protein in 10 CD and 10 control patient colon sections. Data points are the percentage of muscularis mucosae regions positive for KIAA1199 staining divided by the total muscularis mucosae areas analyzed. All data points have been normalized to the identical area on the nonspecifically stained serial section. All images were taken with a Leica TCS SP5 II Confocal/Multi-Photon high-speed upright microscope with Acousto-Optical Beam Splitter at 40× magnification, and acquisition was achieved with Leica confocal software. Scale bars: 100 μm. Insets are secondary antibody only (control) stained sections (*P < .05).

Figure 2

KIAA1199 is increased in fibroblasts derived from CD colons compared with fibroblasts from ND colons. (A) Fluorescent images of ND and CD colon fibroblasts grown for 13 days and immunostained for KIAA1199 (red) and 4′,6-diamidino-2-phenylindole (blue). Insets are secondary antibody (control) stained cells. Scale bars: 50 μm. Images were captured on a Leica DM upright microscope at 20× magnification with Image ProPlus acquisition software. (B) Average fold-change between densitometric measurements of immunoblots measuring KIAA1199 protein in CD and ND whole-cell lysate normalized to glyceraldehyde-3-phosphate dehydrogenase. Six independent experiments using 10 ND cell lines and 5 CD cell lines are shown. (C) Average fold-change between densitometric measurements from immunoblots of KIAA1199 protein in conditioned media from CD and ND fibroblasts grown for 13 days. Five independent experiments using 10 ND cell lines and 6 CD cell lines are shown. (D) Average fold-change of densitometric measurements from immunoblots comparing KIAA1199 protein in CD and ND ECM. Four independent experiments using 11 ND cell lines and 5 CD cell lines are shown. (E) Spectral counts from mass spectrometry measuring KIAA1199 protein in the ECM of CD and ND fibroblasts grown for 13 days. Two independent experiments using 2 ND lines and 3 CD lines are shown (*P < .05).

Figure 3

IL6 up-regulates KIAA1199 deposition into the ECM of colon fibroblasts. (A) Average fold-change between densitometric measurements of immunoblots measuring KIAA1199 protein in the ECM of cytokine-stimulated and unstimulated ND colon fibroblasts. Five independent experiments using 5 cell lines are shown. (B) Average fold-change between densitometric measurements of immunoblots measuring KIAA1199 protein in the ECM of IL6-stimulated and unstimulated CD colon fibroblasts. Four independent experiments using 4 cell lines are shown. (C) Comparison of IL6 protein levels in the conditioned media of CD and ND colon fibroblasts after 13 days of growth. Enzyme-linked immunosorbent assay determination of supernatants from 5 ND and 4 CD cell lines is shown. (D) Average densitometric measurements of immunoblots measuring KIAA1199 protein in the ECM of IL6R-blocking antibody–treated, mouse IgG-treated, and untreated CD colon fibroblasts. Five independent experiments using 5 cell lines are shown (*P < .05, **P < .01). Ab, antibody.

Figure 4

Colon fibroblasts cannot degrade exogenous HA in the absence of KIAA1199. (A) Representative carbohydrate-sizing electrophoresis gel showing hyaluronan after incubation with media only, mock siRNA-treated colon fibroblasts, and KIAA1199-knockdown colon fibroblasts. (B) Immunoblot image detecting KIAA1199 protein in mock siRNA and KIAA1199-knockdown colon fibroblasts. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) band included as loading control. (C) Average fold-change between densitometric measurements of carbohydrate-sizing electrophoresis gels measuring hyaluronan after incubation with media only, mock siRNA-treated colon fibroblasts, and KIAA1199-knockdown colon fibroblasts. Nine experiments using 9 cell lines are shown.