Burn Injury Induces Intestinal Inflammatory Response Mediated by Th17 in Burn-Primed Endotoxemic Mice

When faced with an infection, patients with severe injuries (e.g., burn and trauma) are susceptible to multiple organ dysfunction syndrome (MODS) following acute lung injury (ALI).¹ To elucidate the pathologic and immunologic mechanisms underlying organ failure in critically injured patients, we developed a 2-hit animal model that was first subjected to burn, followed by infectious secondary insult with endotoxin injection on 11^th day after burn, resulting in lethal ALI and MODS.^2,3 Although several researchers have reported on burn-induced immunosuppression by T-helper (Th) cells and macrophages,^4–11 our analyses of this 2-hit model have shown that administering lipopolysaccharide (LPS) to burn-primed mice causes lethal ALI and MODS due to excessive inflammatory cytokine production in the plasma and lungs.² This excessive inflammatory cytokine response by macrophage/monocytes to LPS contributes to ALI after burn injury.³ These immunological pathways may be involved in the intestinal ischemia and/or gut barrier injury observed after burns or shock.^12–16 The reasons for the development of burn-induced immunosuppression and MODS following ALI need to be clarified.

Many clinical and experimental studies have been performed to study gut function and disorders such as inflammatory bowel disease (IBD).^17–19 Researchers have shown that a Th17 cell (a new subset of Th cells that produces IL-17A) and other inflammatory cytokines induced by Th17 cells play critical roles in the gut in IBD. Although IL-17 is responsible for promoting inflammation through the recruitment of neutrophils and monocytes, Th17 cells are responsible for the facilitation of intestinal barrier functions and preventing bacterial mucosal invasion.^17,20 As Th17 cells are majorly present in the inflamed intestines of IBD patients, Th17-induced proinflammatory cytokines including IL-17 trigger and amplify inflammation in IBD.¹⁸ In clinical studies and various animal models of IBD,^18,21 the efficacy of probiotics, including Lactobacillus and Bifidobacteria, has been widely reported via several mechanisms involved in the displacement and suppression of the pathogen growth,²² functional improvement of the epithelial barrier,¹⁹ and immunomodulation of Th1, Th2, Th17, and regulatory T cell production.²² The administration of certain Lactobacillus species suppressed the Th17-mediated secretion of IL-17 via the down-regulation of TGFβ1 and IL-23 expression and downstream p-STAT3 phosphorylation in experimental colitis;²³ therefore, probiotics directly improve Th17-induced overproduction of downstream inflammatory cytokines and related symptoms through an anti-inflammatory effect in the gut of IBD patients.^18,21,22,23

As Th17 response is caused by burn injury (directly in the burned skin and indirectly in the cardiac tissue),^20,24,25 we hypothesized that the Th17 response and its modulation with probiotic preparation in the gut contribute to the pathogenesis of ALI and MODS after burn injury. In this study, we analyzed the intestinal cytokine kinetics in the burn-primed endotoxemic 2-hit model to clarify the role of the Th17 response in the gut treated with or without Lactobacillus treatment.

Materials and Methods

To assess the effects of probiotic preparation on host survival, a burn-primed murine model with sequential endotoxemia was applied as per a previously used method.^2,3 Seven-week-old Balb/c male mice (23–28 g) from the Charles River Laboratories Japan, Inc, were purchased. All mice were caged and provided ad libitum with water and standard powder chow (CE-2, Nippon Kurea, Tokyo, Japan) for at least a week. The mice were then assigned into burn and sham groups. The mice dorsum was shaved using an electrical clipper with appropriate pentobarbital anesthesia, one day pre-burn injury. The shaved dorsum was exposed to a 5-second hot steam using ether anesthesia on day 1, to produce a third-degree burn over 15% of the total body surface area (TBSA). Immediately post-burn injury, 4 mL of sterile saline was intraperitoneally administered to the burned mice and then they were observed. The same protocols were used for the sham mice except the burn injury.

The mice were bred with the standard powder chow before burn injury. We sought to assess the survival after lethal endotoxin challenge in burn-primed mice fed with a probiotic preparation containing Lactobacillus casei (L. casei). To assess the effects of Lactobacillus treatment on the burn-primed endotoxemic model, some of the burned mice were bred with the standard powder chow with 1.2% Biolactis (Yakult Honsha Company, Limited, Tokyo), which contains 500 mg of L. casei (1.5 × 10⁹ – 2.1 × 10¹⁰ bacteria)/1 g powder, after burn injury. The remaining mice received the standard powder chow containing an amount of cornstarch equivalent to that in the Biolactis as a control.

For survival studies, 3 mg/kg lipopolysaccharide (LPS) (E. coli 0111:B4; Sigma-Aldrich) that is not lethal for sham mice was administered through the tail vein on 11^th day after burn injury, and cumulative survival was determined for 72 hours (up to 96 hours) after lethal endotoxemia. All of these procedures and care for mice were deliberated and approved by the Keio University Institutional Animal Care and Use Committee, Tokyo, Japan.

Our preliminary experiments showed that cytokine contents were significantly higher in the ileum than those in the jejunum or the colon in burn-primed endotoxemic mice, indicating that the ileum was the primary location of cytokine production (unpublished data). To investigate the immunologic effects of burn injury and Lactobacillus treatment on gut mucosal mononuclear cells, we measured cytokine production by the isolated mononuclear cells from lamina propria in the ileum on 11^th day after burn injury with or without Lactobacillus treatment in the burned mice. We resected the entire ileum from the burned and sham mice on 11^th day after burn injury under appropriate pentobarbital anesthesia to isolate the lamina propria mononuclear cells using an enzyme method described in a previous study.²⁶ Dissected mucosa from the ileum was washed thoroughly using Ca²⁺ and Mg²⁺-free Hank's buffered saline to remove the mucus, cut into small pieces (2-mm cubes), and incubated at 37°C for 45 minutes in the medium with 1 mmol/L dithiothreitol (Sigma–Aldrich) and 2.5% fetal calf serum (FCS) to promote the release of epithelial and intraepithelial mononuclear cells from the mucosal tissue into supernatant. After the supernatant was removed by pipetting, the mucosal tissue including lamina propria mononuclear cells were incubated and stirred in a medium that contained 0.03% collagenase A from Clostridium histolyticum (Sigma–Aldrich) at 37°C for 2 hours. Thereafter, the supernatant in which the lamina propria mononuclear cells were released from the tissue was collected, and the cell suspension was separated using a Percoll gradient. The cell fractions were suspended in a 40% isotonic Percoll solution and then centrifuged using a Ficoll–Hypaque density gradient. After separation, the lamina propria mononuclear cells were resuspended and adjusted to 1.0 × 10⁶ cells/mL in RPMI-1640 with 5% FCS. The lamina propria mononuclear cells rich in T-lymphocytes and macrophages/monocytes (as determined using flow cytometry) were then immediately used for further experiments.

To evaluate the phenotypic changes of T-lymphocytes and macrophages/monocytes in the ileum mucosa after burn injury and Lactobacillus treatment, in vitro cytokine productions by the lamina propria mononuclear cells were examined in reaction to anti-CD3 antibody and LPS. As previously described,³ we used LPS for a specific macrophage/monocyte stimulation and anti-CD3 antibody (145-2C11, BD Biosciences) for a T-lymphocyte stimulation. Through preliminary experiments, we determined that 24-hour incubation with LPS (1 μg/mL) and anti-CD3 antibody (10 μg/mL) resulted in optimal cytokine production. To evaluate macrophage/monocyte and T-lymphocyte functions in the ileum mucosa, lamina propria mononuclear cells were adjusted to 1.0 × 10⁶ cells/mL in RPMI-1640 containing 5% FCS and incubated with anti-CD3 antibody (10 μg/mL) or LPS (1 μg/mL) for 24 hours at 37°C. After incubation, the supernatants of the cell suspensions were centrifuged and stored until analysis at −80°C.

For the analysis of cytokines in the supernatant from the gut tissue or a cell suspension of lamina propria mononuclear cells, we used the Cytometric Bead Array Mouse Th1/Th2/Th17 Cytokine Kit (BD biosciences) that allows researchers to determine the values of cytokines such as IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10, and IL-17.

We used Fisher's protected least significant difference (PLSD) comparison test for the observed values of cytokines and the Kaplan-Meier test (log-rank) for the 72-hour cumulative survival rates. Data are presented as mean ± SEM, and a P value of < 0.05 was considered significant.

Results

In the burn-primed murine model, only 15% TBSA burn was nonlethal after injury for any mice. Figure 1 demonstrates the 72-hour survival rate after lethal endotoxemia on 11^th day after burn injury for all groups. The survival rate was significantly lower in burned mice fed with standard powder chow as controls (Burn C group: n = 14, 21.4%) than that in burned mice fed with special powder chow containing 1.2% L. casei powder (Burn L group: n = 14, 64.3%) (P = 0.03, log-rank test), indicating that Lactobacillus treatment directly improved survival after lethal endotoxemia in burn-primed mice.

View Figure

• Download as Powerpoint
• Download Figure

In the in vitro studies, as shown in Fig. 2, the production of proinflammatory cytokines, such as TNF-α, IL-6, and IL-17 in reaction to anti-CD3 antibody significantly increased in the burn control (Burn C) group compared with that in the sham control (Sham C) group, demonstrating that burn injury exacerbated the inflammatory response by T-lymphocytes in the gut mucosa. The values of all the cytokines observed, except IL-4, significantly decreased in the burn with Lactobacillus treatment (Burn L) group compared with those in the Burn C group. In contrast, the values of IL-4 and IFN-γ significantly decreased in the sham mice that received Lactobacillus treatment (Sham L group) compared with those in the mice of the sham C group.

View Figure

• Download as Powerpoint
• Download Figure

Regarding the macrophage/monocyte functions in reaction to LPS, the production of all cytokines significantly increased in the Burn C group compared with that in the Sham C group, except TNF-α, and the cytokines were similarly suppressed in the Burn L and Sham L groups (Fig. 3). This indicated that exaggerated inflammatory and compensatory anti-inflammatory responses on the part of macrophages/monocytes were induced by burn injury. With the Lactobacillus treatment, the baseline cytokine levels measured in the gut mucosa were remarkably suppressed to the levels observed in the sham C group or even lower levels.

View Figure

• Download as Powerpoint
• Download Figure

Discussion

This study showed that burn injury increases the intestinal immune response, such as the up-regulation of cytokine productions by T cells and macrophages/monocytes in the ileum of burn-primed mice. It is noteworthy that the ileum cytokine kinetics by the T cells is contrary to past reports that Th1-cytokine production by peripheral monocytes and spleen cells is markedly suppressed, while Th2-cytokine responses became dominant after burn injury.^5,6,27 In contrast, those by macrophages/monocytes are similar to our previous observation of excessive cytokine production in response to LPS by splenic macrophages/monocytes in burn-primed mice.^2,3 The current findings suggest that the Th1-dominant in vivo reaction in the ileum causes exaggerated cytokine response and hypercytokinemia after burn injury.

Among CD3-induced productions of Th1 and inflammatory cytokines, the productions of TNF-α, IL-6, and IL-17 by lamina propria T-lymphocytes were significantly increased, whereas CD3-induced Th2 cytokines such as IL-4 and IL-10 were not altered as a result of burn priming. Additionally, LPS-induced cytokine response mainly from lamina propria macrophages/monocytes was significantly increased, except TNF-α, because of burn priming. We found that post-burn IL-17 production was enhanced from lamina propria lymphocyte and macrophage/monocyte, whereas our result that the other proinflammatory cytokine productions such as TNF-α and IL-6 were almost completely enhanced in the ileum after burn injury is consistent with the past several reports.^13,14,28 As Th17 cells are identified as the primary lymphocyte producer of IL-17, our results suggest that Th17 cells, or possibly other IL-17-producing cells (e.g., CD8 T cells, γδ T cells), should be differentiated and induced in the mucosa of the ileum after burn priming. Furthermore, as Th17 cells directly secrete several proinflammatory cytokines including TNF-α, IL-1β, IL-6, IL-12, IL-23, and TGF-β,^18,21 the post-burn overproduction of these inflammatory cytokines may be responsible for burn-primed induction of Th17 cells in lamina propria. Notably, this study showed that IFN-γ productions from lamina propria mononuclear cells were not only suppressed in T-lymphocytes at all but were also enhanced in macrophages/monocytes; major burns and trauma have been demonstrated to attenuate Th1 response associated with immunosuppression and susceptibility to sepsis.^4–7,11,27 Since some of the IL-17 producing T cells that also produce IFN-γ (termed as Th1/Th17 cells) increased in the gut mucosa from Crohn's disease patients,^29,30 the burn-induced Th17 cells in the gut might resemble those in Crohn's disease. It remains unclear why Th17 cells were rich in the gut after burn injury, as in Crohn's disease. Th17 cells are differentiated from naïve T-helper cells by IL-1β, IL-6, and TGF-β^18,30 and the plasma levels of proinflammatory cytokines, such as IL-1β and IL-6, are elevated after burn injury;^31,32 thus, these predisposing high systemic inflammatory cytokine levels might induce Th17 cells in the gut and other sites, such as the skin and cardiac tissue, after burn injury, as reported in previous studies.^8,24,25

This study also showed that probiotic treatment exerted a beneficial effect that burn-induced cytokine overproductions were suppressed through anti-inflammatory response, and resulted in an improved survival rate from lethal ALI in burn-primed endotoxemic mice. These anti-inflammatory effects exerted by probiotics against Th17-mediated hypercytokinemia may contribute to the attenuation of local and systemic inflammation, preventing distant lung injury and improving survival after lethal endotoxemia in our burn-primed model. Although the mechanism of the beneficial effects of Lactobacillus treatment is not fully elucidated in this study, probiotics prophylaxis may be a cheap and effective treatment for preventing septic ALI and MODS after burn injury.

This study has certain limitations; we did not perform investigation regarding the cross-reaction of cytokine production between T-lymphocytes and monocyte/macrophages and the direct flowcytometric analysis of Th17 cells using specific cell surface markers. Further studies are awaited regarding the beneficial mechanisms of probiotics in burn-primed mice and the clinical application of Lactobacillus treatment for burned patients.

Conclusions

Burn injury induced intestinal Th17 and Th17-mediated inflammatory response, which was associated with the survival of lethal endotoxemia after burn injury. Lactobacillus treatment after burn injury decreased intestinal Th17 and their inflammatory response and the mortality of lethal ALI in burn-primed endotoxemic mice.