Heme oxygenase-1–derived carbon monoxide enhances the host defense response to microbial sepsis in mice (original) (raw)
Enhanced susceptibility of HO-1 null mice to polymicrobial infection. We performed cecal ligation and puncture (CLP) (19 gauge, 1 hole unless otherwise stated) in WT (HO-1+/+), heterozygous (HO-1+/–), and HO-1 null (HO-1–/–) littermate mice on a pure BALB/c genetic background to induce polymicrobial peritonitis, bacteremia, and sepsis. HO-1–/– mice suffered markedly higher mortality rates compared with HO-1+/+ and HO-1+/– mice after CLP-induced sepsis (Figure 1A). While CLP led to an infiltration of inflammatory cells and edema in the villi of the ileum and the submucosal region of the colon in HO-1+/+ mice, HO-1–/– mice experienced complete destruction of ileal villi and the mucosal surface of the colon (Figure 1B). Blood was cultured from HO-1+/+ and HO-1–/– mice during the first 48 hours after CLP, and CFUs of bacteria were assessed to determine the level of bacteremia. Compared with HO-1+/+ mice, circulating levels of bacteria were significantly greater in HO-1–/– mice (Figure 1C). Analogous to cultures of blood, organ homogenates (lungs, livers, and spleens) from HO-1–/– mice showed significantly higher levels of bacteria than HO-1+/+ mice (Figure 1D). Thus, in the absence of HO-1, CLP-induced sepsis leads to a destructive process in the ileum and colon and increased bacteremia, resulting in end-organ seeding. These data demonstrate the importance of endogenous HO-1 in protecting mice against the lethal effects of polymicrobial sepsis.
Deficiency of endogenous HO-1 leads to reduced survival after CLP-induced polymicrobial sepsis. (A) Rates of survival were determined for HO-1+/+ (n = 7), HO-1+/– (n = 8), and HO-1–/– (n = 8) littermates after CLP surgery. P = 0.001. These data are the composite of 3 independent experiments. (B) H&E staining of representative paraffin-embedded ileum and colon sections of HO-1+/+ and HO-1–/– mice 48 hours after sham or CLP surgery. Original magnification, ×100. Arrows depict destruction of the ileal villi and colonic mucosa. During the first 48 hours after CLP, blood was collected from the right atrium of hearts (C), and organs (lungs, livers, and spleens) were homogenized with 1 ml PBS (D). Serial dilutions were made of blood and organ homogenates and then plated on LB agar plates. CFUs were determined after incubating at 37°C overnight. Blood: HO-1+/+, n = 14; HO-1–/–, n = 13. Organs: HO-1+/+, n = 9; HO-1–/–, n = 8. Horizontal bars represent mean values. These experiments were performed independently 3 times.
Overexpression of HO-1 has beneficial effects during polymicrobial sepsis. Due to the detrimental effects of HO-1 deficiency, we hypothesized that targeted overexpression of HO-1 would be beneficial during microbial-induced sepsis. Since the route of infectious dissemination in sepsis is the bloodstream, we overexpressed HO-1 in the vasculature. Tg mice on a pure C57BL/6 background were generated using the promoter of aortic carboxypeptidase-like protein (ACLP) (29) to target human HO-1 (hHO-1) in vascular SMCs. ACLP is able to target Tg expression not only in large-sized and medium-sized vessels but also in small vessels down to the arteriole (29). The Tg construct is shown in Figure 2A. The founder mice were identified by Southern blot analysis of genomic DNA (Figure 2B), and we obtained mice with either 1 (Tg1) or 2 (Tg2) copies of the Tg. To confirm mRNA expression of the Tg, total RNA was isolated from aortas of WT and Tg2 mice and RT-PCR performed using hHO-1–specific primers. The hHO-1 Tg was only present in the Tg mice (Figure 2C). Also, the level of HO-1 protein expression was detected in the aortas of Tg2 mice by immunostaining and compared with expression in WT mice (Figure 2D). We found that the expression of HO-1 protein was significantly increased, by 3.5-fold, in the vasculature of Tg2 mice compared with WT mice (n = 3 in each group; P = 0.0034) by colorimetric analysis as described previously (30, 31).
Generation and characterization of HO-1 Tg mice. (A) Schematic of the Tg construct. ACLP promoter indicates 2.5 kb of the ACLP promoter known to express in SMCs. hHO-1, 1-kb hHO-1 cDNA; pA, 300-bp bovine growth hormone polyadenylation sequences. The position of the Southern probe is shown below the construct. (B) Southern blot analysis of EcoRI-digested genomic DNA from WT and Tg1 and Tg2 Tg mice is shown. En, endogenous. (C) Total RNA was isolated from aortas of WT and Tg2 mice, and RT-PCR performed using hHO-1–specific primers. Mouse β-actin was used as an internal positive control for the PCR reaction, and “No RT” indicates the negative control. Sections from aortas (D) and villi and crypts of the ileum (E) from WT and Tg2 mice were stained with HO-1 antiserum. Sections from ileum were also stained with antiserum against smooth muscle (SM) α-actin. Brown staining indicates HO-1 expression (D, 4 left panels of E), and red staining indicates smooth muscle α-actin expression (4 right panels of E). Original magnification, ×100 (D and E). Arrows depict HO-1 and smooth muscle α-actin–expressing cells in villi and crypts of the ileum and colon, respectively.
HO-1–derived CO is known to exhibit biologic effects, including the modulation of vascular tone (32) and the inhibition of inflammatory signaling, by suppressing proinflammatory cytokines (TNF-α and IL-1β) and upregulating the antiinflammatory cytokine IL-10 (24). Since hypotension and the systemic inflammatory response are critical contributors to the morbidity and mortality of sepsis, we assessed baseline systolic blood pressure, as described previously (17), and levels of circulating cytokines. Tg2 mice showed comparable baseline blood pressures (Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI32730DS1) and no difference in circulating levels of proinflammatory (TNF-α, IL-1β, IL-6) and antiinflammatory (IL-10) cytokines compared with WT mice (Supplemental Figure 1B).
With evidence that baseline blood pressure and inflammatory cytokine production was comparable, we next wanted to determine whether vascular overexpression of HO-1 would improve outcome in polymicrobial sepsis. Thus, Tg mice (Tg1 and Tg2) and WT mice on a pure C57BL/6 background underwent CLP. Note that survival rate of WT mice after 19-gauge, 1-hole CLP is lower on a C57BL/6 compared with a BALB/c genetic background (see Methods). Both Tg1 and Tg2 mice demonstrated significantly improved survival after CLP compared with WT mice (Figure 3A). Bowel histopathology after CLP surgery showed similar tissue injury in the ileum of WT, Tg1, and Tg2 mice, with evidence of inflammation and villi shortening (Supplemental Figure 2). In the colon, a greater amount of submucosal edema was noted in the WT mice (Supplemental Figure 2) compared with Tg1 and Tg2 mice. However, in contrast to HO-1–/– mice, there was no evidence of gross tissue destruction in the ileum or colon of WT, Tg1, or Tg2 mice. To help elucidate why mortality was different among the groups, we performed blood cultures in WT, Tg1, and Tg2 mice. Tg1 mice had significantly lower bacterial counts in the circulating blood than WT mice, and the decrease in bacterial counts was even more pronounced in Tg2 mice compared with either WT or Tg1 mice (Figure 3B). Lower bacterial counts were also seen in end-organ cultures from lungs, livers, and spleens of Tg mice compared with WT mice (Figure 3C). Similar to the circulating blood, bacterial counts in the end organs decreased in a dose-dependent fashion depending on the copy number of HO-1 Tgs. These data suggest that, beyond protecting bowel integrity, vascular overexpression of HO-1 has a more direct antimicrobial effect during CLP-induced polymicrobial sepsis. Due to the similarity in survival response to CLP in Tg lines, we focused on the Tg2 mice for the remainder of the experiments.
Improved survival from polymicrobial sepsis in HO-1 Tg mice. (A) Survival was assessed in HO-1 Tg (Tg1, n = 9; Tg2, n = 10) and WT (n = 12) mice after CLP-induced polymicrobial sepsis. P = 0.016. These data are a composite from 2 independent experiments. Analogous to what was reported in Figure 1, blood was collected from the right atrium of hearts (B), and organs (lungs, livers, and spleens) were homogenized (C) from WT, Tg1, and Tg2 mice. Serial dilutions were made of blood and organ homogenates, and CFUs were determined after incubating at 37°C overnight on LB agar plates. Blood: WT, n = 12; Tg2, n = 8. Organs: WT, n = 8; Tg2, n = 6. Horizontal bars represent mean values. These experiments were performed independently at least 2 times.
Overexpression of HO-1 has beneficial effects against Gram-positive Enterococcus faecalis–induced sepsis. Because of the mixed bacterial flora of CLP-induced sepsis, we wanted to know whether overexpression of HO-1 would have a different effect on infection from Gram-positive versus Gram-negative organisms. Aerobic cultures of cecal contents determined that the most prevalent organisms in the bowel were Enterococcus faecalis (Gram-stain positive) and Escherichia coli (Gram-stain negative) and the bacterial flora did not differ between WT and Tg2 mice (data not shown). To allow a slow and progressive release of microorganisms, similar to the CLP model of polymicrobial sepsis, we used a fibrin clot model of single organism sepsis (33), with placement of the bacterial fibrin clot in the abdominal cavity. Tg2 mice experienced no protection from _E. coli_–induced mortality (Figure 4A); however, overexpression of HO-1 significantly improved survival from _E. faecalis_–induced sepsis in Tg2 mice compared with WT mice (Figure 4B).
Beneficial effects of HO-1 Tg on Gram-positive but not Gram-negative bacteria. Survival was assessed in Tg2 (n = 11) and WT (n = 11) mice after fibrin clot–induced microbial sepsis by E. coli (3 × 108) bacteria (A) and in mice (Tg2, n = 8; WT, n = 15) receiving E. faecalis (2 × 108) bacteria (B). P = 0.04 for E. faecalis and not significant for E. coli.
HO-1–derived CO increases phagocytic activity of E. faecalis in a NOD2-dependent fashion. With this differential survival in HO-1 Tg mice to E. faecalis but not E. coli infection, additional studies were performed to determine whether a difference was evident in the ability of inflammatory cells to phagocytize the bacteria. Nonlabeled or FITC-labeled E. coli and E. faecalis were injected into the peritoneum of WT or Tg2 mice. After 24 hours, a peritoneal lavage was performed and phagocytic rates were determined using flow cytometry as described in Methods. Phagocytosis was not different between WT and Tg2 mice exposed to E. coli (P = 0.462); however, exposure to E. faecalis led to a 48% increase (P = 0.001) in phagocytic rate in mice overexpressing HO-1 (Figure 5A). To determine whether there was a difference in the inflammatory response between WT and Tg2 mice, we measured total inflammatory cells/phagocytic cells 24 hours after injection of heat-killed E. faecalis into the peritoneum. Flow cytometry analysis revealed that cells were predominantly neutrophils and that the total neutrophil count was not different between WT and Tg2 mice (Figure 5B). Inflammatory cells were also analyzed from the peripheral blood after CLP surgery, and analogous to what is shown in Figure 5B, circulating levels of total inflammatory cells (leukocytes) were not different between WT and Tg2 mice (Supplemental Figure 3A). Moreover, circulating levels of TNF-α, IL-1β, IL-6, and IL-10 were not significantly different between WT and Tg2 mice at 48 hours, a point in time when mortality differences were starting to become apparent between groups (Supplemental Figure 3C). In addition, circulating levels of TNF-α and IL-6 were not different between WT and Tg2 mice 6 hours after CLP surgery (Supplemental Figure 3B). Thus, overexpression of hHO-1 did not change the overall inflammatory response, as judged by circulating inflammatory cells and the production of inflammatory cytokines 6 and 48 hours after CLP, but it did enhance the ability of neutrophils to phagocytize E. faecalis bacteria.
HO-1–derived CO enhances phagocytosis. (A) Tg2 (n = 6) and WT (n = 6) mice were injected with FITC-labeled E. coli or E. faecalis. After 24 hours, phagocytosis was analyzed. *P = 0.001 versus WT. (B) Total neutrophil counts were measured 24 hours after injection of nonlabeled E. faecalis into Tg2 (n = 6) and WT (n = 7) mice. (C) WT mice were injected with CO-RM (10 μM/kg, n = 5) or vehicle (12.5% DMSO, n = 5) 12 hours and 2 hours before injection of FITC-labeled E. faecalis. After 24 hours, phagocytosis was measured. †P = 0.004 versus V. (D) NOD2 and TLR4 mRNA levels were quantified by real-time PCR of total ileum RNA isolated at baseline (WT, n = 5; Tg2, n = 4) and 48 hours after CLP-induced sepsis (WT, n = 13; Tg2, n = 15). ‡P = 0.019 and 0.043 versus WT. (E) NOD2–/– mice were injected with CO-RM (n = 5) or vehicle (n = 6) 12 hours and 2 hours before injection of FITC-labeled E. faecalis. After 24 hours, phagocytosis was measured. Χ_P =_ 0.038 versus V. Data are presented as mean ± SEM.
ACLP targeting is able to increase hHO-1 expression in SMCs but not inflammatory cells. However, since CO — a product of heme catabolism by HO-1 — is able to diffuse across cell boundaries and exhibit biological functions on neighboring cells (34), we proposed that targeting HO-1 to vascular cells would allow biological actions on adjacent inflammatory cells infiltrating into critical organs during sepsis. To test this hypothesis, we investigated whether CO could enhance the phagocytic properties of inflammatory cells in vivo. CO-releasing molecule (CO-RM) or an equal volume of vehicle was injected into the peritoneum of WT C57BL/6 mice 12 hours and 2 hours before injection of nonlabeled or FITC-labeled E. faecalis. Administration of CO-RM to mice promoted a significant increase in the phagocytic rate (2.3-fold; P = 0.004) compared with vehicle-treated mice (Figure 5C). The importance of CO in regulating this response, in contrast to another metabolite of HO-1, was confirmed by experiments showing that administration of biliverdin did not enhance the phagocytic response during E. faecalis infection (data not shown).
Interestingly, hHO-1 Tg is also expressed in the ileum of Tg mice (Figure 2C) in cells adjacent to intestinal epithelial cells and Paneth cells of the villi and crypts (Figure 2E). These hHO-1–expressing cells also stain positive for smooth muscle α-actin (Figure 2E) and are consistent with myofibroblasts. To further investigate mechanisms by which HO-1 may promote antibacterial effects, we assessed the expression of TLR4 and NOD2 in tissue from the ileum of WT and HO-1 Tg mice. Studies using real-time PCR analyses revealed that baseline NOD2 expression was increased in Tg2 mice compared with WT mice (Figure 5D). Moreover, increased expression of NOD2 after CLP-induced polymicrobial sepsis was enhanced in Tg2 mice compared with WT mice. In contrast, mRNA levels of TLR4 were not detectable at baseline, and after CLP-induced sepsis, the levels of TLR4 were similar between WT and Tg2 mice (Figure 5D).
To elucidate whether enhanced phagocytic activity by CO is related to expression of NOD2, we performed phagocytic assays in NOD2–/– mice after administration of CO-RM or vehicle. Beyond ileal tissue, NOD2 was expressed in phagocytic cells from peritoneal lavage of WT mice exposed to heat-killed E. faecalis (data not shown). In contrast to the increased phagocytosis in WT mice, NOD2–/– mice exposed to CO-RM demonstrated no evidence of an increase and, in fact, exhibited a modest decrease in phagocytic activity (18.5%) compared with vehicle-treated mice (Figure 5E). These data suggest that expression of NOD2 is critical for enhancing phagocytic activity by CO in inflammatory cells.
CO-RM enhances bacterial clearance during CLP-induced polymicrobial sepsis. With evidence of enhanced phagocytosis by CO-RM, we wanted to determine whether this response resulted in decreased bacteremia. Thus, CO-RM or an equal volume of vehicle was administered intraperitoneally to WT C57BL/6 mice 12 hours and 2 hours prior to CLP surgery. The mice were then sacrificed 24 hours after CLP surgery and bacteremia quantitated. The mice receiving CO-RM had significantly lower circulating bacterial counts (CFUs) than vehicle-treated mice (Figure 6A). The lower bacterial counts in the CO-RM group were also seen in end organs, as shown in cultures from lungs, livers, and spleens (Figure 6B). These data are consistent with decreased bacteremia and end-organ infection in Tg2 mice (Figure 3, B and C).
CO-RM enhances bacterial clearance. WT mice were injected with CO-RM (n = 10) or vehicle (n = 10) 12 hours and 2 hours before CLP surgery. After 24 hours, CFUs were determined from blood (A) and organs (B). Horizontal bars represent mean values. These experiments were performed independently at least 2 times.
CO-RM has therapeutic effects and rescues HO-1–/– mice from the increased mortality of CLP-induced polymicrobial sepsis. Our data suggest that CO is a key product of HO-1–derived heme catabolism, contributing to the host defense response during polymicrobial sepsis. We next administered CO-RM or vehicle to HO-1–/– mice 12 hours and 2 hours prior to CLP surgery and every 24 hours after the initiation of sepsis. CO-RM administration to HO-1–/– mice resulted in significantly improved survival compared with vehicle-treated HO-1–/– mice (Figure 7A). These data demonstrated that CO can rescue HO-1–/– mice from the detrimental outcome of CLP-induced sepsis and verified the critical importance of CO in the microbial response to sepsis. To determine the therapeutic potential of CO, we administered CO-RM to WT BALB/c mice starting 6 hours after CLP surgery (19 gauge, 2 holes). At this point in time, bacteria have already begun to seed end organs (data not shown). As revealed in Figure 7B, CO-RM is able to significantly improve survival from CLP surgery, even when administered after the onset of sepsis.
CO-RM rescues mice from the mortality of CLP-induced polymicrobial sepsis. (A) Treatment (Tx) with CO-RM (10 μM/kg, n = 8) or an equal volume of vehicle (12.5% DMSO in PBS, n = 8) was administered intraperitoneally to HO-1–/– mice 12 hours and 2 hours before and every 24 hours after CLP surgery (19 gauge, 1 hole). Survival rates were then assessed over the subsequent 8 days. P = 0.03. The data are a composite of 3 independent experiments. (B) Treatment with CO-RM (10 μM/kg, n = 14) or an equal volume of vehicle (n = 14) was administered intraperitoneally to HO-1+/+ mice starting 6 hours after CLP surgery (19 gauge, 2 holes), after 12 hours, and then every 24 hours. Survival rates were assessed over the subsequent 8 days. P = 0.04. Data are a composite of 5 independent experiments. Short arrows depict the time of CLP surgery, and the longer arrows represent the initiation of treatment, either CO-RM or vehicle.