Heparan sulfate and syndecan-1 are essential in maintaining murine and human intestinal epithelial barrier function (original) (raw)
Sdc1 knockdown causes protein leakage in vitro. First we assessed whether Sdc1 loss causes protein leakage in vitro. Sdc1 siRNA knockdown in HT29 cells reduced Sdc1 mRNA levels by 87% ± 3% (Figure 1A) and decreased the amount of cell-associated glycosaminoglycans (GAGs) by 48% ± 7% (Figure 1B). In parallel, paracellular protein leakage increased 1.43 ± 0.26–fold and transepithelial electrical resistance (TER) decreased by 17.8% ± 6.9% (Figure 1, B–D). Neither cell growth nor apoptosis were affected (data not shown). Incubating HT29 cells with HS-cleaving heparinase III (HSase) reduced cell-associated GAGs by 81% ± 3%, increased protein leakage 1.62 ± 0.19–fold, and decreased TER by 23.4% ± 6.5%. The latter results are in accordance with our previous studies (9, 11) and also indicate that loss of HS increases protein leakage more than just the loss of the predominant HSPG, Sdc1.
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.
Sdc1 knockdown amplifies TNF-α–induced protein leakage in vitro. In the presence of HSPGs, TNF-α (20 ng/ml) increased protein leakage 4.11 ± 0.30–fold (Figure 1E). In the absence of cell-associated HS after exposure to HSase, TNF-α increased protein leakage 7.00 ± 0.29–fold, showing that HS loss and TNF-α synergize. Incubating Sdc1 knockdown cells with TNF-α increased protein leakage 5.55 ± 0.32–fold over baseline, indicating that the effects of Sdc1 loss and TNF-α also synergize. Again, loss of HS was more effective than just the loss of the major HSPG Sdc1. Coincubating TNF-α with soluble heparin (25.0 μg/ml) alleviated TNF-α–induced protein leakage.
Intestinal protein leakage is increased in Sdc1–/– or intestinal HS–deficient mice. To study whether HS loss also causes intestinal protein leakage in vivo, we established 2 independent methods to assess enteric protein loss in mice, one determining fecal loss of 51Cr-labeled albumin, the other measuring fecal levels of α1-antitrypsin (AAT) (10). The 2 methods gave comparable results, varying less than 10%. We applied them to mice deficient in Sdc1 or all HS. In _Sdc1_–/– mice HS expression on the basolateral surface of IEC was significantly reduced (Figure 2, A and B) and basal intestinal protein leakage was significantly increased compared to wild-type littermates (Figure 2C).
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.
Since treatment of cultured mucosal cells with HSase increased protein leakage to a greater extent than deletion of Sdc1, we next addressed whether general loss of intestinal epithelial HS further exacerbates protein leakage. Ext1 encodes a subunit of the copolymerase involved in HS formation (35). Since systemic knockout of Ext1 is embryonic lethal (36), we inactivated the gene selectively in IEC (_Ext1_Δ/Δ). HS expression on the basolateral surface of IEC was further reduced compared with _Sdc1_–/– mice (Figure 2, A and B), and basal fecal AAT levels were increased (Figure 2C). _Sdc1_–/– and _Ext1_Δ/Δ mice did not exhibit proteinuria or hypoalbuminemia. Gross intestinal phenotype and histology appeared normal in both lines.
Mice (_HPA_-Tg) overexpressing human heparanase, which selectively cleaves HS and causes a significant shortening of HS chains in organs (37), present with proteinuria (38), and we found they also show a 1.67 ± 0.12–fold increase in fecal AAT levels compared with wild-type littermates (Figure 2C). Notably, protein leakage was significantly higher in _Ext1_Δ/Δ and _HPA-_Tg mice than in _Sdc1_–/– mice.
TNF-α induces intestinal protein leakage in vivo, which is further enhanced in Sdc1–/– mice. PLE onset in patients is associated with moderately increased TNF-α levels (14), and TNF-α induces protein leakage in vitro (9, 11). To address whether TNF-α causes intestinal protein leakage in mice, we injected wild-type mice with a single dose of recombinant human TNF-α (rhTNF-α; 0.1 mg/kg, i.v.). These low doses were shown not to cause inflammation or necrosis (39). Intestinal protein leakage increased within 24 hours, reached a maximum after 48 hours, and then slowly decreased, reaching baseline levels after 6–7 days (Figure 3A). Recombinant mouse TNF-α (rmTNF-α) gave similar effects as rhTNF-α (data not shown). rhTNF-α and recombinant mouse TNF-α (rmTNF-α) have a very similar affinity for TNFR1, but rhTNF-α does not bind to murine TNFR2 (40), indicating that protein leakage is mostly mediated through TNFR1 signaling.
_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.
TNF-α–induced leakage further increased in _Sdc1_–/– mice. The combined effects of Sdc1 loss and TNF-α injections were synergistic (Figure 3, A and C), consistent with our in vitro results. For example, in wild-type mice, protein leakage increased 3.25 ± 0.76–fold 48 hours after injection of rhTNF-α (Figure 3C). Theoretically, the individual effects of Sdc1 loss and TNF-α presence together would predict a 3.84-fold increase in protein leakage in Sdc1–/– mice, but we measured a 4.62 ± 0.29–fold increase, which indicated that these factors worked synergistically.
Exogenous rhTNF-α or rmTNF-α were cleared from mouse plasma by more than 99% within 8 hours following injection (data not shown; the reported half-life is <20 min; ref. 39), and protein leakage only increased for a few days without causing any clinical signs of PLE, i.e., hypoalbuminemia or edema. More sustained protein leakage was observed when we injected TNF-α every 48 hours at higher concentrations (0.25 mg/kg) (Figure 3B). Effects on protein leakage progressively decreased, which is in accordance with observations that rats develop a partial tolerance to daily rhTNF-α injections (39). Plasma albumin levels in the mice remained normal, and there were no signs of edema.
IFN-γ upregulates TNFR1 and amplifies TNF-α–induced leakage, which is further enhanced in Sdc1–/– mice. PLE onset in patients is often associated with increased IFN-γ levels (13). We previously showed in HT29 cells that 12 hours of exposure to IFN-γ upregulates TNFR1 expression and amplifies TNF-α–induced protein leakage (11). We now assessed the effects of IFN-γ on protein leakage in mice. Injecting rmIFN-γ was reported to have no apparent effect on mouse intestinal architecture and histology over a wide concentration range (0.005–5.0 mg/kg) (39). We injected rmIFN-γ (0.2 mg/kg, i.v.) and measured no increase in protein leakage. We then assessed whether IFN-γ amplified TNF-α–induced leakage by injecting rmIFN-γ (0.2 mg/kg, i.v.) 12 hours prior to injecting TNF-α (0.1 mg/kg, i.v.). Intestinal protein leakage increased 4.17 ± 0.14–fold 48 hours after TNF-α injection, which was significantly higher than after TNF-α exposure alone, without prior IFN-γ injection. Calculating the additive effects of Sdc1 loss, IFN-γ, and TNF-α, we predicted a 4.76-fold increase in protein leakage but actually measured a 6.80 ± 0.49–fold increase in protein leakage _Sdc1_–/– mice after IFN-γ and TNF-α injections, again showing synergism. FACS analysis of SGLT1-positive IEC revealed that IFN-γ exposure increased TNFR1 expression 2.4 ± 0.2–fold and 3.0 ± 0.4–fold in wild-type and _Sdc1_–/– mice (P < 0.01), respectively (Figure 3D), showing the importance of HS(PG) for regulating IFN-γ–induced TNFR1 expression.
Pressure induces protein leakage ex vivo and synergizes with the effects of Sdc1 loss and cytokines. Mesenteric hypertension is common in PLE patients (4–6, 18). Our in vitro data showed that increased hydrostatic pressure alone causes protein leakage, which is further amplified by HS loss, IFN-γ, and TNF-α (11). To address whether increased pressure causes protein leakage in mice, we mounted stripped mucosal explants (consisting of epithelial cells, lamina propria, and muscularis mucosa but not circular and longitudinal muscle layers and serosa) in Ussing chambers and applied a hydrostatic pressure difference to the serosal side. Pressure alone increased albumin flux 6.9 ± 1.0–fold and 10.9 ± 1.4–fold in wild-type and _Sdc1_–/– mice, respectively, indicating that Sdc1 loss increases the susceptibility to pressure-induced leakage (Figure 4). Injecting mice with TNF-α (0.1 mg/kg, i.v.) 24 hours prior to preparing explants increased flux 1.8 ± 0.4–fold and 3.8 ± 0.5–fold in wild-type and _Sdc1_–/– mice, respectively, confirming that Sdc1 loss amplifies TNF-α–induced leakage. IFN-γ alone had no effect on albumin flux but significantly increased TNF-α–induced leakage when administered 12 hours prior to TNF-α injection, which was further enhanced in _Sdc1_–/– mice, confirming previous results. TNF-α alone or in combination with IFN-γ exacerbated pressure-induced protein leakage. The combination of IFN-γ, TNF-α, and pressure increased albumin flux 15.2 ± 1.3–fold in wild-type mice but 25.3 ± 1.8–fold in _Sdc1_–/– mice, again demonstrating the central role of Sdc1 in PLE pathogenesis. A similar fold increase in enteric protein loss was measured in PLE patients. Mice systemically haploinsufficient for either Ext1 (Ext1+/–) or Ext2 (Ext2+/–) also were more susceptible to IFN-γ/TNF-α– and/or pressure-induced protein leakage, but the effects were not as prominent as in _Sdc1_–/– mice.
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.
This ex vivo system showed that weak HS(PG) expression and IFN-γ/TNF-α, both alone and in combination, make the intestinal epithelial barrier more susceptible to pressure-induced leakage. To study the long-term effects of increased pressure, mesenteric hypertension would need to be increased in vivo. Mice with induced portal vein stenosis showed a significant increase in fecal AAT approximately 10 days after surgery, and AAT remained elevated for at least 2 weeks (data not shown).
HS(PG) loss alone does not induce paracellular leakage. HS(PG) loss caused protein leakage and amplified IFN-γ/TNF-α–induced protein leakage in vitro and in mice. However, how HS(PG) loss contributes to protein leakage is unknown. To assess whether HS(PG) loss increases paracellular leakage, we harvested mouse intestines in lanthanum buffer followed by phosphate precipitation (41). Lanthanum did not pass the junctional complex, and the extracellular space between adjacent epithelial cells was void of lanthanum precipitates in wild-type mice (Figure 5A) but also in _Sdc1_–/– mice (Figure 5B). These results indicate that HS(PG) deficiency alone has no effect on paracellular leakage. Sections from mice exposed to IFN-γ/TNF-α showed lanthanum passing the junctional complex and appearing in the interepithelial space and all the way down to the basal surface (Figure 5, C–E). These results confirm that IFN-γ/TNF-α alter junctional complex integrity and impair intestinal epithelial barrier function.
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.
Heparin and 2/3-DS-H alleviate cytokine-induced protein leakage. Heparin injections (100–500 U/kg) mitigate PLE in some post-Fontan patients (25–27) but have side effects mostly resulting from anticoagulant activity (27, 30). We screened a library of non-anticoagulant heparin-like compounds and other GAG derivatives for their ability to reduce IFN-γ/TNF-α–induced leakage in vitro (Figure 6A). Inhibition depended on molecular size (Figure 6A, lanes 3–7). Shorter fragments (degree of polymerization 2 [dp 2], lane 4) had almost no effect, whereas longer fragments (dp 20, lane 7) reduced IFN-γ/TNF-α–induced leakage by more than 50%. Low-molecular-weight heparin (lane 3) was much less effective than high-molecular-weight heparin (lane 2), supporting clinical data that low-molecular-weight heparin does not mitigate PLE (26). Heparin lost some of its activity after removal of 2-, 3-, and/or, 6-_O_-sulfates (lanes 8–11), reduction of the carboxyl group (lane 12), or substitution of _N_-sulfates with _N_-acetyl group (lanes 13 and 14). Other GAGs such as chondroitin, dermatan, or acharan sulfate (lanes 15–20) also slightly reduced IFN-γ/TNF-α–induced leakage. Sulfated cyclodextran (lane 21) or sucrose octasulfate (lane 22) had no effect. Next to unfractionated, high-molecular-weight heparin, non-anticoagulant 2/3-DS-H (lane 9) was most efficient in reducing IFN-γ/TNF-α–induced leakage.
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).
We then assessed whether heparin or 2/3-DS-H also reduce IFN-γ/TNF-α–induced protein leakage in mice by giving daily i.v. injections of either heparin or 2/3-DS-H. Three days after the initial injection, we administered either TNF-α alone or a combination of IFN-γ and TNF-α, as described above. Daily injections of heparin or 2/3-DS-H continued for 7 days. Low doses of either heparin (100 U/kg, 0.7 mg/kg) or 2/3-DS-H (0.7 mg/kg) reduced cytokine-induced intestinal protein leakage by more than 50% (Figure 6, B and E). Heparin doses 5-fold higher (500 U/kg, 3.5 mg/kg) completely prevented leakage (Figure 6, C and F) in wild-type mice. In _Sdc1_–/– mice, higher 2/3-DS-H doses (3.5 mg/kg) completely prevented TNF-α–induced leakage (Figure 6D) and reduced IFN-γ/TNF-α–induced leakage by more than 85% (Figure 6G). Administration of either heparin or 2/3-DS-H alone for 10 consecutive days had no adverse effects on mice and did not alter basal intestinal protein loss.