Heparan sulfate and syndecan-1 are essential in maintaining murine and human intestinal epithelial barrier function

Abstract

Patients with protein-losing enteropathy (PLE) fail to maintain intestinal epithelial barrier function and develop an excessive and potentially fatal efflux of plasma proteins. PLE occurs in ostensibly unrelated diseases, but emerging commonalities in clinical observations recently led us to identify key players in PLE pathogenesis. These include elevated IFN-gamma, TNF-alpha, venous hypertension, and the specific loss of heparan sulfate proteoglycans from the basolateral surface of intestinal epithelial cells during PLE episodes. Here we show that heparan sulfate and syndecan-1, the predominant intestinal epithelial heparan sulfate proteoglycan, are essential in maintaining intestinal epithelial barrier function. Heparan sulfate- or syndecan-1-deficient mice and mice with intestinal-specific loss of heparan sulfate had increased basal protein leakage and were far more susceptible to protein loss induced by combinations of IFN-gamma, TNF-alpha, and increased venous pressure. Similarly, knockdown of syndecan-1 in human epithelial cells resulted in increased basal and cytokine-induced protein leakage. Clinical application of heparin has been known to alleviate PLE in some patients but its unknown mechanism and severe side effects due to its anticoagulant activity limit its usefulness. We demonstrate here that non-anticoagulant 2,3-de-O-sulfated heparin could prevent intestinal protein leakage in syndecan-deficient mice, suggesting that this may be a safe and effective therapy for PLE patients.

Figure 1. Sdc1 knockdown in HT29 cells causes protein leakage.

(A–E) Sdc1-targeting siRNAs (siRNA1 and siRNA2) reduce Sdc1 mRNA level (A), reduce cell-associated GAGs (B), increase relative albumin leakage (C), reduce TER (D), and amplify TNF-α–induced protein leakage (E), which can be reversed with heparin. Compared with effects of HS loss after exposure to HSase or scrambled siRNA (scr. siRNA) as negative control. White bars represent basal levels without intervention. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2. Loss of Sdc1 or HS causes increased intestinal protein leakage in mice (A) Sulfated GAG staining with colloidal gold (left and middle columns) and HS staining with bFGF-biotin probe (right column) in small intestinal sections from wild-type (top row), Sdc1^–/– (middle row), and Ext1^Δ/Δ mice (bottom row).

The middle column is a magnification of the left column. Original magnification, ×100 (left column); ×400 (middle column); ×200 (right column). (B) HS staining intensity on basolateral surface of IEC in Sdc1^–/– and Ext1^Δ/Δ mice relative to wild-type controls. (C) Intestinal protein leakage (⁵¹Cr or AAT) in Sdc1^–/–, Ext1^Δ/Δ, and HPA-Tg mice relative to respective littermate controls. All data represent assessment in a minimum of n = 5 mice (mean ± SD). **P < 0.01, ***P < 0.001.

Figure 3. Sdc1^–/– mice are more susceptible to cytokine-induced intestinal protein leakage.

(A and B) Intestinal protein leakage (⁵¹Cr) in Sdc1^+/+ and Sdc1^–/– mice in response to single (A) or multiple (B) i.v. injections of TNF-α (arrows) at 0.1 or 0.25 mg/kg. Line without symbols in A represents predicted leakage in Sdc1^–/– mice if effects of Sdc1 loss and TNF-α exposure were additive. (C) Intestinal protein leakage (AAT or ⁵¹Cr) in Sdc1^+/+ and Sdc1^–/– mice 48 h after exposure to TNF-α (i.v. 0.1 mg/kg), IFN-γ (i.v. 0.2 mg/kg), or a combination of both, relative to basal leakage in Sdc1^+/+ mice. Dashed lines represent predicted leakage in Sdc1^–/– if effects of Sdc1 loss, TNF-α, and/or IFN-γ were additive. (D) FACS analysis (median fluorescent activity ± SD) of TNFR1 expression in SGLT1-positive IEC from Sdc1^+/+ or Sdc1^–/– mice in response to IFN-γ exposure relative to basal expression in Sdc1^+/+ (which was set at 1.0; data not shown) mice. All data represent assessment in a minimum of n = 3 mice. **P < 0.01, ***P < 0.001.

Figure 4. Sdc1 loss and Ext1 or Ext2 haploinsufficiency increase protein leakage ex vivo.

Albumin leakage (mean ± SD) through stripped mouse mucosal explants mounted in Ussing chambers. Significances calculated compared with wild-type mice with the same interventions. All data represent assessment in a minimum of n = 6 mice. *P < 0.05, **P < 0.01, ***P < 0.001. n.d., not determined.

Figure 5. Sdc1 loss alone does not increase paracellular leakage.

Electron micrographs of mouse intestinal epithelium bathed in lanthanum. Lateral intercellular spaces were void of lanthanum phosphate precipitates (arrows) in wild-type (A) and Sdc1^–/– mice (C) without cytokine exposure but filled with lanthanum phosphate precipitates in wild-type (B), Sdc1^–/– (D), and Ext1^Δ/Δ mice (E) after IFN-γ/TNF-α exposure. Right column is a magnification of boxed areas in the left column. Scale bars: 5 μm.

Figure 6. Heparin and 2/3-DS-H alleviate protein leakage in vitro and in mice.

(A) Albumin leakage (mean ± SD) through HT29 monolayers relative to untreated cells. Maximum leakage (white bar) was induced by incubating cells with heparinase (HS loss), IFN-γ (10 ng/ml, 24 h), and TNF-α (2 ng/ml, 12 h). Cytokines were coincubated with heparin-like compounds or other GAG derivatives at 2.5 μg/ml or 25.0 μg/ml. Heparin (lane 2) and 2/3-DS-H (lane 9) were most effective in alleviating cytokine-induced protein leakage (arrows). Lane 1, HS; lane 2, high-molecular-weight heparin (unfractionated); lane 3, low-molecular-weight heparin; lane 4, sized heparin, dp 2; lane 5, sized heparin, dp 8; lane 6, sized heparin, dp 14; lane 7, sized heparin, dp 20; lane 8, 2,6-de-O-sulfated heparin; lane 9, 2/3-DS-H; lane 10, 6-O-desulfated heparin (chemical desulfation); lane 11, 6-O-desulfated heparin (enzymatic desulfation with endosulfatase [HSulf2]); lane 12, carboxyl-reduced heparin; lane 13, fully N-acetylated heparin; lane 14, fully O-sulfated N-acetylated heparin; lane 15, chondroitin sulfate; lane 16, fully O-sulfated chondroitin sulfate; lane 17, dermatan sulfate; lane 18, fully O-sulfated dermatan sulfate; lane 19, fully O-sulfated hyaluronic acid; lane 20, archaran sulfate; lane 21, sulfated cyclodextran; lane 22, sucrose octasulfate. (B–G) Intestinal protein leakage in Sdc1^+/+ (B, C, E, and F) and Sdc1^–/– mice (D and G) assessed by in vivo ⁵¹Cr labeling. Mice were injected daily with low (100 U/kg, 0.7 mg/kg) (B and E) or high doses (500 U/kg, 3.5 mg/kg) of heparin (C and F), 2/3-DS-H (C, D, F, and G), or PBS as control. Three days following the first injections (t = 0), intestinal protein leakage was induced by injection of either TNF-α (i.v., 0.1 mg/kg) (B–D) or IFN-γ (i.v., 0.2 mg/kg) and TNF-α (i.v., 0.1 mg/kg, 12 h after IFN-γ) (E–G). PBS was used as a control. All data represent assessment in a minimum of n = 4 mice (mean ± SD).