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. 2014 Jan;7(1):114-23.
doi: 10.1038/mi.2013.29. Epub 2013 May 22.

Contribution of epithelial innate immunity to systemic protection afforded by prolyl hydroxylase inhibition in murine colitis

S Keely  1 , E L Campbell  2 , A W Baird  3 , P M Hansbro  1 , R A Shalwitz  4 , A Kotsakis  4 , E N McNamee  2 , H K Eltzschig  2 , D J Kominsky  2 , S P Colgan  2
Affiliations

Contribution of epithelial innate immunity to systemic protection afforded by prolyl hydroxylase inhibition in murine colitis

S Keely et al. Mucosal Immunol. 2014 Jan.

Abstract

Pharmacological stabilization of hypoxia-inducible factor (HIF) through prolyl hydroxylase (PHD) inhibition limits mucosal damage associated with models of murine colitis. However, little is known about how PHD inhibitors (PHDi) influence systemic immune function during mucosal inflammation or the relative importance of immunological changes to mucosal protection. We hypothesized that PHDi enhances systemic innate immune responses to colitis-associated bacteremia. Mice with colitis induced by trinitrobenzene sulfonic acid were treated with AKB-4924, a new HIF-1 isoform-predominant PHDi, and clinical, immunological, and biochemical endpoints were assessed. Administration of AKB-4924 led to significantly reduced weight loss and disease activity compared with vehicle controls. Treated groups were pyrexic but did not become subsequently hypothermic. PHDi treatment augmented epithelial barrier function and led to an approximately 50-fold reduction in serum endotoxin during colitis. AKB-4924 also decreased cytokines involved in pyrogenesis and hypothermia, significantly reducing serum levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α while increasing IL-10. Treatment offered no protection against colitis in epithelial-specific HIF-1α-deficient mice, strongly implicating epithelial HIF-1α as the tissue _target for AKB-4924-mediated protection. Taken together, these results indicate that inhibition of prolyl hydroxylase with AKB-4924 enhances innate immunity and identifies that the epithelium is a central site of inflammatory protection afforded by PHDi in murine colitis.

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Conflict of interest statement

The authors declare no financial conflicts in the work submitted here.

Figures

Figure 1
Figure 1. Influence of AKB-4924 treatment on disease activity in TNBS colitis
AKB-4924 (0, 0.3, 1.0 or 5.0 mg/Kg in cyclodextrin vehicle) was administered subcutaneously on day -1 relative to TNBS or EtOH intrarectal lavage. (A) Weight was monitored daily as an indicator of disease severity and normalised relative to initial weight and control animals. On day 7 animals were sacrificed and colons were excised and measured to assess (B) colon length. Panel C represents H&E staining of tissue isolated from control animals (i), TNBS animals treated with vehicle (ii), and TNBS animal treated with AKB-4924 (iii). (D) Disease activity was as assessed as sum of scoring (0–3) for colon thickening (colon weight/length), occult blood, fecal pellet consistency and % weight loss. (E) Temperature was measured by infrared (IR) thermometer at t=0, 6, 12 and 24 hrs and then at 24 hr intervals until sacrifice. N=6, *p<0.05, **p<0.01, ANOVA (A, E), two-tailed, Students T-test (B, D).
Figure 2
Figure 2. Influence of AKB-4924 treatment on pyrogen levels in TNBS colitis
AKB-4924 (5.0 mg/Kg in cyclodextrin vehicle) or vehicle-treated animals were sacrificed on day 2, 3 or 7 relative to TNBS or EtOH rectal lavage. Blood was collected by cardiac puncture and serum assayed in triplicate for each animal by multiplex ELISA for (A) IL-1β, (B) IL-6, (C) TNF-α, (D) IL-10. On day 7, whole colon tissue was homogenised in lysis buffer and assayed by ELISA for (E) IL-1β, (F) IL-6, (G) TNF-α, (H) IL-10. N=6, *p<0.05, **p<0.01 two-tailed Students T-test.
Figure 3
Figure 3. Influence of AKB-4924 on intestinal epithelial barrier function and HIF _target gene expression
AKB-4924 (0 or 5.0 mg/Kg in cyclodextrin vehicle) was administered subcutaneously on day -1 relative to TNBS or EtOH intrarectal lavage. Animals were sacrificed on day 2, 3 or 7 and colons were excised and tied off by suture into intestinal sacs. Sacs were loaded with FITC-dextran 4400 (FD-4; 500 μg/mL) and (A) the apparent permeability (Papp) was assessed. Blood was collected by sterile cardiac puncture and (B) serum assayed for LPS content. Colon epithelial mRNA was screened by qPCR for induction of the HIF _target genes CD73 and ITF (C) or ITGB1 (D). Panel E represents western blot analysis for HIF-1α in nuclear isolates from intestinal epithelial cells (IEC) and lamina propria cells (LP) in control and TNBS colitis animals in the presence of AKB-4924. TATA binding protein was employed as a housekeeper. N=6, *, ^, # p<0.05, **, ^^, ## p<0.01, ANOVA (A, B, D), two-tailed, Students T-test (C).
Figure 4
Figure 4. Influence of AKB-4924 on systemic bacteremia in TNBS colitis
Panel A shows recovery of viable bacteria translocated to blood and extraintestinal organs (liver, kidney, spleen and MLN; mesenteric lymph nodes in TNBS colitis animals after treatment with vehicle or AKB-4924 (5 mg/Kg). Bacterial counts are expressed in colony forming units, per gram tissue (CFU/g), except for ^ (blood) where counts are expressed as CFU/mL. (B) Murine neutrophils and macrophages were isolated by peritoneal lavage and incubated with 10μM AKB-4924 (AKB) or incubated in hypoxia (Hx; pO2, 20 torr for 6h) or normoxia (Nx) and assessed for the ability to phagocytose FITC-labelled E. Coli. N=5, *p<0.05, **p<0.01 two-tailed, Students T-test.
Figure 5
Figure 5. Functional epithelial HIF-1α is critical for AKB-4924-induced mucosal protection
Panel A represents Western blot analysis for HIF-1α from epithelial scrapings from IEC HIF-1α+/+ and IEC HIF-1α−/− animals subjected to TNBS colitis in the presence and absence of AKB-4924 (0 or 5.0 mg/Kg), which was administered subcutaneously on day -1 relative to TNBS or EtOH by intrarectal lavage. Cyclodextrin was administered as a control. (B) Weight was measured daily as an indicator of disease severity. On day 7 animals were sacrificed and colons were excised and measured to assess (C) colon shortening. N=5, *p<0.05, **p<0.01, ANOVA (B), two-tailed, Students T-test (C).
Figure 6
Figure 6. Influence of AKB-4924 on animals with active TNBS colitis
AKB-4924 (0 or 5.0 mg/Kg) was administered subcutaneously on day of peak weight loss (day 2) relative to TNBS or EtOH intrarectal lavage. Cyclodextrin was administered as a control. (A) Weight was measured daily as an indicator of disease severity and normalized relative to initial weight and control animals. On day 7 animals were sacrificed and (B) colons were excised and assessed for shortening, and (C) total cell populations in mesenteric lymph nodes (MLN) were counted using a haemocytometer. (D) Disease activity was as assessed as sum of scoring (0–3) for colon thickening (colon weight/length), occult blood, faecal pellet consistency and % weight loss. (E) Temperature was measured by IR thermometer at 24hr intervals from first AKB-4924 administration until sacrifice. N=6, *p<0.05, **p<0.01, ANOVA (A, E), two-tailed, Students T-test (B, C, D).
Figure 7
Figure 7. Influence of AKB-4924 on TNF ARE ileitis
AKB-4924 (0 or 5.0 mg/Kg) was administered to 10-week old TNFΔARE mice every second day over a 10 day period. Animals were assessed (A) histologically, and scored for (B) acute inflammatory index, (C) chronic inflammatory index, (D) villus inflammatory index and (E) total inflammatory index. N=5, *p<0.05, two-tailed, Students T-test.
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