Hyaluronan 35 kDa enhances epithelial barrier function and protects against the development of murine necrotizing enterocolitis

Abstract

Background: Disruption of tight junctions (TJs) predisposes to bacterial translocation, intestinal inflammation, and necrotizing enterocolitis (NEC). Previously, studies showed that hyaluronan (HA), a glycosaminoglycan in human milk, maintains intestinal permeability, enhances intestinal immunity, and reduces intestinal infections. In this study, we investigated the effects of HA 35 kDa on a NEC-like murine model.

Methods: Pups were divided into Sham, NEC, NEC+HA 35, and HA 35. Severity of intestinal injury was compared using a modified macroscopic gut scoring and histologic injury grading. The effect of HA 35 on intestinal permeability was determined by measuring FITC dextran and bacterial translocation. RNA and protein expression of TJ proteins (claudin-2, -3, -4, occludin, and ZO-1) were compared between the groups.

Results: Pups in the NEC+HA 35 group had increased survival and lower intestinal injury compared to untreated NEC. In addition, HA 35 reduced intestinal permeability, bacterial translocation, and proinflammatory cytokine release. Ileal expression of claudin-2, -3, -4, occludin, and ZO-1 was upregulated in NEC+HA 35 and HA 35 compared to untreated NEC and shams.

Conclusion: These findings suggest that HA 35 protects against NEC partly by upregulating intestinal TJs and enhancing intestinal barrier function.

Figure-1:. HA 35 administration in Dithizone/Klebsiella murine NEC model.

Pups were randomized to one of the following groups: Sham; NEC; NEC + HA, and HA alone. HA 35 was given to pups in the NEC+ HA and HA group by gavage at a concentration of 15mg/kg or 30mg/kg body weight once daily for three days prior to induction of NEC and one hour prior to bacterial administration. NEC was induced using the Paneth cell disruption and Klebsiella infection NEC-like model. I.P. injection of dithizone (33 mg/kg) was given at P14–16, followed by gavage administration of 1×10⁸ CFU Klebsiella pneumoniae /kg. Pups were monitored for 10 hours after gavage for clinical illness and survival.

Figure 2.. Kaplan-Meier survival curve (A) showing increased survival in HA administered groups.

Survival in low dose HA (15 mg/kg) at 70 %, high dose HA (30mg/kg) at 90 % as compared to untreated NEC at 50% (p=0.05 & p=0.04 respectively). (B) Serum FITC levels were lower in NEC + HA 35 treatment compared to NEC with mean ± S.E.M. of 1,554 ± 250 ng/ml in NEC + HA 30 mg/kg, 2,456 ± 212 ng/ml in NEC + HA 15mg/kg, and 5,274 ± 389 ng/ml in untreated NEC. **** p<0.0001. (C) Scatter plot of bacterial colony counts from blood cultures being significantly lower in NEC + HA 30mg/kg compared to untreated NEC (mean ± S.E.M of 5,426 ± 378 CFU/ml versus 12,722 ± 1519 CFU/ml respectively. ***p<0.0001. One-way ANOVA with Turkey’s multi-comparison test.

Figure-3:. HA 35 reduces the severity of small intestinal injury in murine NEC.

Representative gross (A) and H&E images (B) of the small intestine from the groups. (C) HA 35 administration was associated with decreased gross appearance of injury with a mean ± S.E.M macroscopic score of 2.125 ± 0.31 compared 3.25 ± 1 in the NEC group. ** p <0.01. (D) Histological NEC severity scores of a mean ± S.E.M of 1.2 ± 0.25 in the NEC + HA 35 group versus 2.3± 0.15 in the untreated NEC group. ** p <0.01. Data are in mean ± S.E.M. Results are representative of at least ≥ six animals and at least two separate experiments. One-way ANOVA with Dunnet’s multi-comparison test.

Figure-4:. HA 35 administration decreases the systemic (plasma) pro-inflammatory cytokine release in NEC-like model.

Pro-inflammatory cytokine levels of TNF α (*p= 0.0483, **p=0.0018), Gro-α (*** p=0.0003, **** p<0.0001), IL-12ρ70 (*** p =0.0003), IL-6 (****p<0.0001) were decreased in NEC + HA 35 compared to untreated NEC. Data are in mean ± S.E.M. Results are representative of at least > six animals and at least two separate experiments. One-way ANOVA with Turkey’s multi-comparison test.

Figure-5:. Immunohistochemical staining images (10x and 40x inserts) for TJ proteins and the corresponding qPCR RNA analysis.

HA 35 treated pups showed increased intensity of staining in healthy and NEC challenged pups for all TJ proteins. Insert 40 x images showing increased cryptal intensity of staining in HA 35 treated healthy and NEC challenged pups for TJ occludin (A) and claudin-3(C). Claudin −4 (B) staining was increased in the lateral cytoplasmic membranes of the tips of villi. Insert 40x images showing cytoplasmic staining for ZO-1 (D) in tips of villi in all groups and increased cytoplasmic cryptal staining for claudin −2 (E) noted in the NEC group. RNA PCR results showed upregulated expression of occludin, claudin −4, claudin −3, ZO-1, and claudin −2 in HA 35 and NEC + HA 35 groups compared to untreated NEC (F-J). Claudin −3 RNA expression was downregulated in the NEC group compared to sham (H), while RNA expression for ZO-1 and Claudin −2 was upregulated in HA 35 and NEC + HA 35 group compared to untreated NEC (I, J). Data represent mean ± S.E.M. Analysis was done by two-way ANOVA with Turkey’s multi-comparison test.