Hemorrhage-induced intestinal damage is complement-independent in Helicobacter hepaticus-infected mice

Abstract

With more than half of the world population infected, Helicobacter infection is an important public health issue associated with gastrointestinal cancers and inflammatory bowel disease. Animal studies indicate that complement and oxidative stress play a role in Helicobacter infections. Hemorrhage (HS) induces tissue damage that is attenuated by blockade of either complement activation or oxidative stress products. Therefore, we hypothesized that chronic Helicobacter hepaticus infection would modulate HS-induced intestinal damage and inflammation. To test this hypothesis, we examined HS-induced jejunal damage and inflammation in uninfected and H. hepaticus-infected mice. Helicobacter hepaticus infection increased HS-induced midjejunal mucosal damage despite attenuating complement activation. In addition, infection alone increased chemokine secretion, changing the HS-induced neutrophil infiltration to a macrophage-mediated inflammatory response. The HS-induced macrophage infiltration correlated with increased secretion of tumor necrosis factor-α and nitric oxide in the infected mice. Together, these data indicate that Helicobacter infection modulates the mechanism of HS-induced intestinal damage and inflammation from a complement-mediated response to a macrophage response with elevated tumor necrosis factor-α and nitric oxide. These data indicate that chronic low-level infections change the response to trauma and should be considered when designing and administering therapeutics.

Figure 1. Helicobacter infection increased intestinal damage at 2 h post-hemorrhage

Mucosal injury (A) and villus height/crypt depth ratio (B) were determined from H&E stained jejunal sections from Helicobacter-infected (solid bars) and uninfected (open bars) mice after Sham or 1, 2 or 3 h post-hemorrhage (HS) treatment.. Each bar is the average ± SEM of 6–10 mice per group. (^∗) indicates p ≤ 0.05 compared to respective Sham treatment and (ϕ) indicates p ≤ 0.05 compared to similar treatment of uninfected animals. C–H) Representative photomicrographs of H&E stained intestinal sections from infected (F, G, H) and uninfected (C, D, E) Sham-treated (C, F), 2 h post-HS treated (D, G) or 3 h post-HS treated (E, H) mice. Original photomicrographs are 200X magnification.

Figure 2. Helicobacter infection induced intestinal chemotactic factors

Ex vivo jejunal chemokines CCL3 (A), CCL4 (B), CXCL10 (C), and KC (D) produced by intestinal tissue sections from infected (solid bars) and uninfected (open bars) mice subjected to Sham or hemorrhage (HS) treatment were determined by multiplex analysis. Chemokine concentrations were normalized to tissue protein content and expressed as pg per mg of intestinal tissue. Each bar represents the average ± SEM with 4–10 mice per group. (^∗) indicates p ≤ 0.05 compared to respective Sham treatment and (ϕ) indicates p ≤ 0.05 compared to similar treatment of uninfected animal.

Figure 3. Intestinal LTB₄ and neutrophil infiltration are induced by hemorrhage and Helicobacter infection

A) Ex vivo intestinal LTB₄ production by Helicobacter-infected (solid bars) and uninfected (open bars) mice subjected to Sham or hemorrhage (HS) treatment was measured. Each bar represents the average ± SEM with 5–10 animals per group. B) Neutrophils (PMN) present in villi of each treatment group were counted from formalin fixed H&E stained mid-jejunal intestinal sections at 1000X and are presented as PMN per high powered field. Each bar represents the average of 4–10 animals. (^∗) indicates p ≤ 0.05 compared to Sham treatment and (ϕ) indicates p ≤ 0.05 compared to similar treatment of uninfected animals.

Figure 4. Helicobacter infection decreased intestinal C3 deposition and increased DAF (CD55) expression following hemorrhage

Mid-jejunal sections from uninfected (open bars, C) and Helicobacter-infected (solid bars, D) C57Bl/6 mice after Sham or hemorrhage (HS) treatment were stained for C3 deposition (A) and DAF expression (C, D). Sera C5a was quantitated by ELISA (B). Each bar represents the average ± SEM of 8–10 mice per group. (^∗) indicates p ≤ 0.05 compared to the respective Sham treatment and (ϕ) indicates p ≤ 0.05 compared to similar treatment of uninfected animals. Original magnification of photomicrographs is 200X.

Figure 5. Cobra Venom Factor (CVF) depletion of C3 did not attenuate hemorrhage-induced jejunal tissue damage in Helicobacter-infected mice

After Helicobacter-infected (solid bars) and uninfected (open bars) mice were subjected to Sham, or Hemorrhage (HS) in the presence of CVF treatment, mucosal injury (A) or jejunal LTB₄ (B) production were quantitated at 2 hr. Each bar represents the average ± SEM of 6–10 mice per group. (∗) indicates p ≤ 0.05 compared to similar Sham treatment and (ϕ) indicates p ≤ 0.05 compared to respective uninfected treatment.

Figure 6. Hemorrhage and infection increased F4/80 macrophages in the intestinal villi

Mid-jejunal tissue sections from uninfected (A, C) and Helicobacter-infected (B, D) C57Bl/6 mice after Sham (A, B) or hemorrhage (HS) (C, D) treatment were stained for F4/80. Original magnification of photomicrographs (A–D) is 400X and the inset (D) is enlarged 3 fold. Data are representative of five individual experiments with 3–4 photos per treatment group in each experiment. Bar = 20 µm

Figure 7. Helicobacter infection increased jejunal TNF-α and NO production

Ex vivo generated mid-jejunal concentrations of TNF-α (A) and nitric oxide (NO) (B) from H. hepaticus infected (solid bars) and uninfected (open bars) mice were measured. Each bar represents the average ± SEM with 3–10 mice per group. (∗) indicates p ≤ 0.05 compared to similar Sham treatment and (ϕ) indicates significant difference (p≤ 0.05) compared to similarly treated uninfected mice. (C, D) After hemorrhage, F4/80 (red) co-localized (orange) with TNF-α (C) or iNOS (D) expression (green) in the mid-jejunum of Helicobacter-infected mice. Arrows indicate F4/80 positive cells expressing TNF or iNOS.