Iron dysregulation combined with aging prevents sepsis-induced apoptosis

Abstract

Background: Sepsis, iron loading, and aging cause independent increases in gut epithelial and splenic apoptosis. It is unknown how their combination will affect apoptosis and systemic cytokine levels.

Materials and methods: Hfe-/- mice (a murine homologue of hemochromatosis) abnormally accumulate iron in their tissues. Aged (24-26 months) or mature (16-18 months) Hfe-/- mice and wild type (WT) littermates were subjected to cecal ligation and puncture (CLP) or sham laparotomy. Intestine, spleen, and blood were harvested 24 h later and assessed for apoptosis and cytokine levels.

Results: Gut epithelial and splenic apoptosis were low in both aged septic and sham Hfe-/- mice, regardless of the amount of iron in their diet. Mature septic WT mice had increased apoptosis compared to age-matched sham WT mice. Mature septic Hfe-/- mice had similar levels of intestinal cell death to age-matched septic WT mice but higher levels of splenic apoptosis. Apoptosis was significantly lower in septic aged Hfe-/- mice than septic mature Hfe-/- animals. Interleukin-6 was elevated in septic aged Hfe-/- mice compared to sham mice.

Conclusions: Although sepsis, chronic iron dysregulation, and aging each increase gut and splenic apoptosis, their combination yields cell death levels similar to sham animals despite the fact that aged Hfe-/- mice are able to mount an inflammatory response following CLP and mature Hfe-/- mice have elevated sepsis-induced apoptosis. Combining sepsis with two risk factors that ordinarily increase cell death and increase mortality in CLP yields an apoptotic response that could not have been predicted based upon each element in isolation.

FIG. 1

Graph demonstrating all variables examined in overall study design including age, genotype, iron loading status, injury and number of animals examined.

FIG. 2

Number of gut epithelial apoptotic cells in 100 crypts in aged Hfe^−/− mice subjected to either sham laparotomy or CLP, fed either an iron deficient or high iron diet 2 weeks prior to surgical manipulation. Apoptosis is low in all groups.

FIG. 3

Comparison of gut epithelial apoptosis between (A) mature WT mice subjected to sham laparotomy or CLP, (B) mature WT and Hfe^−/− mice subjected to CLP, and (C) mature and aged Hfe^−/− mice subjected to CLP. Asterisks represent p values <0.05.

FIG. 4

Gut epithelial apoptosis in (A) mature WT mice subjected to sham laparotomy, (B) mature WT mice subjected to CLP, (C) mature Hfe^−/− mice subjected to CLP, and (D) aged Hfe^−/−mice subjected to CLP. Apoptotic cells are identified by arrows.

FIG. 5

Splenic apoptosis in 5 random high powered fields in aged Hfe^−/− mice subjected to either sham laparotomy or CLP, fed either an iron deficient or high iron diet 2 weeks prior to surgical manipulation. Apoptosis is low in all groups.

FIG. 6

Comparison of splenic apoptosis between (A) mature WT mice subjected to sham laparotomy or CLP, (B) mature WT and Hfe^−/− mice subjected to CLP, and (C) mature and aged Hfe^−/− mice subjected to CLP. All groups are statistically different.

FIG. 7

Systemic levels of inflammatory cytokines (A) IL-6, (B) TNFα, and (C) IL-10 in aged Hfe^−/− mice subjected to either sham laparotomy or CLP, fed either an iron deficient or high iron diet 2 weeks prior to surgical manipulation. Absolute levels of all mediators are generally higher in septic animals demonstrating CLP changes the inflammatory profile without altering gut and splenic apoptosis (compare to fig. 1, 4).