Although MIP-1α is an important chemokine in the recruitment of inflammatory cells, it remains unknown whether MIP-1α plays any role in the development of systemic inflammatory response following trauma-hemorrhage (T-H). C57BL/6J wild type (WT) and MIP-1α-deficient (KO) mice were used either as control, subjected to sham operation (cannulation or laparotomy only or cannulation plus laparotomy) or T-H (midline laparotomy, mean blood pressure 35 ± 5 mmHg for 90 min, followed by resuscitation) and sacrificed 2 h thereafter. A marked increase in serum α-glutathione transferase, TNF-α, IL-6, IL-10, MCP-1, and MIP-1α and Kupffer cell cytokine production was observed in WT T-H mice compared with shams or control. In addition lung and liver tissue edema and neutrophil infiltration (myeloperoxidase (MPO) content) was also increased following T-H in WT animals. These inflammatory markers were markedly attenuated in the MIP-1α KO mice following T-H. Furthermore, compared with 2 h, MPO activities at 24 and 48 h after T-H declined steadily in both WT and KO mice. However, normalization of MPO activities to sham levels within 24 h was seen in KO mice but not in WT mice. Thus, MIP-1α plays an important role in mediating the acute inflammatory response following T-H. In the absence of MIP-1α, acute inflammatory responses were attenuated; rapidly recovered and less remote organ injury was noted following T-H. Thus, interventions that reduce MIP-1α levels following T-H should be useful in decreasing the deleterious inflammatory consequence of trauma.
Despite numerous advances in intensive care medicine, sepsis and organ dysfunction, leading to multiple organ failure, remain a major cause of death in trauma patients as well as in patients following major surgery (1, 2, 3, 4, 5). Our previous studies have shown that trauma-hemorrhage (T-H)5 leads to activation of resident immunocompetent cells in different organs (6), resulting in an increased release of proinflammatory cytokines such as TNF-α and IL-6 (7). Increased levels of proinflammatory cytokines were associated with tissue damage caused by neutrophil infiltration (8). Besides chemokines, their receptors also play a pivotal role in mediating leukocyte transmigration from blood vessels to the inflamed tissue, which are released by both immunocompetent and intrinsic cells (9). Through this inflammatory reaction, infiltrated neutrophils can release cytokines, enzymes, and oxygen radicals that result in tissue damage and lead to organ dysfunction and eventual failure (10).
MIP-1α is a member of the CC subfamily of chemokines that is produced by a variety of immune cells such as macrophages, lymphocytes, neutrophils, and dendritic cells. MIP-1α orchestrates acute and chronic inflammatory host responses at the site of injury or infection mainly by recruiting proinflammatory cells (11). Standiford et al. (12) reported that MIP-1α expression in the lung tissue of mice increased after systemic LPS challenge in a time-dependent fashion, and this was accompanied by increased macrophage and neutrophil infiltration in the lungs. Pretreatment of anti-MIP-1α Ab decreased the influx of macrophages and neutrophils and reduced the LPS-induced lung permeability. Ajuebor et al. (13) also demonstrated that MIP-1α is proinflammatory in murine T cell-mediated hepatitis by recruiting CCR1-expression CD4+ T cells to the liver, and such hepatic injury is significantly attenuated in the MIP-1α-deficient mice. Since T-H activates immunocompetent cells and produces organ damage (14), we hypothesized that MIP-1α plays an important role in mediating organ injury following T-H by influencing cytokine/chemokine production by activated immunocompetent cells, and by recruiting polymorphonuclear leukocytes into the affected organs. To test this hypothesis, we compared the difference between tissue damage, neutrophil infiltration, serum cytokine/chemokine levels and Kupffer cell cytokine/chemokine productive capacity following T-H in MIP-1α-deficient knock out (KO) and wild type (WT) mice.
Materials and Methods
Animals and experimental groups
All animal studies were conducted in accordance with the guidelines of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham. Male C57BL/6J WT and CCL3/MIP-1α KO mice (on a C57BL/6J background) 8–10 wk old and weighing 18–22 g were obtained from The Jackson Laboratories. Both WT and KO mice were divided into five groups (control, cannulation only, laparotomy only, sham-operated, and T-H) and each group was subjected to a procedure described below. There were five to six animals in each group.
T-H shock model
Mice were fasted overnight but allowed water ad lib. They were anesthetized with isoflurane (Minrad) and restrained in a supine position. A midline laparotomy (2 cm) was performed, which was then closed in two layers with sutures (Ethilon 6/0, Ethicon). Both femoral arteries and the right femoral vein were cannulated with polyethylene-10 tubing (BD Biosciences). Blood pressure was measured via one of the arterial catheters using a blood pressure analyzer (MicroMed). Upon awakening, the mice were bled rapidly through the other arterial catheter to a mean arterial blood pressure of 35 ± 5 mmHg within 10 min, which was then maintained for 90 min. At the end of that interval, the animals were resuscitated via the venous line with four times the shed blood volume using Ringer’s lactate. After ligating the blood vessels, catheters were removed; the incisions were flushed with lidocaine and closed with sutures. Animals in the control group were not subjected to any surgical procedure. However, mice in cannulation only and laparotomy only groups had cannulation of femoral vessels or midline laparotomy performed, respectively. Sham-operated animals underwent the same surgical procedures as T-H groups, but were neither hemorrhaged nor resuscitated. The animals were sacrificed at 2 h after resuscitation or sham operation. An additional study examined myeloperoxidase (MPO) activity in the lung and liver at 24 and 48 h following T-H. For those measurements, two more groups of animals were sacrificed at 24 and 48 h following T-H.
The animals were anesthetized with isoflurane at 2 h following sham operation or resuscitation in the T-H groups and blood was obtained via cardiac puncture. Blood was centrifuged (2,500 × g, 10 min, 4°C) and the serum was collected and stored at −80°C until analyzed.
Lung and liver were removed aseptically, frozen in liquid nitrogen, and stored at −80°C. The frozen tissue samples were thawed and suspended in 1% proteinase inhibitor mixture (Sigma-Aldrich). The samples were sonicated on ice (Sonic Dismembrator, Fisher Scientific) and were then centrifuged at 12,000 × g for 10 min at 4°C. The supernatants were frozen and stored at −80°C until analyzed. Aliquots were used to determine protein concentration (Bio-Rad DC Protein Assay, Bio-Rad).
Preparation of Kupffer cells
Kupffer cells were isolated as previously described (15). In brief, the portal vein was catheterized with a 27-gauge needle, and the liver was perfused with 20 ml of HBSS (Invitrogen Life Technologies) at 37°C, which was immediately followed by perfusion with 15 ml of 0.05% collagenase IV (Worthington Biochemical) in HBSS with 0.5 mM CaCl2 (Sigma-Aldrich) at 37°C. The liver was then removed and transferred to a petri dish containing the above-mentioned collagenase IV solution. The liver was minced, incubated for 15 min at 37°C, and passed through a sterile mesh stainless steel screen into a beaker containing 10 ml of cold HBSS with 10% FBS. The hepatocytes were removed by centrifugation at 50 × g for 3 min. The residual cell suspension was washed twice by centrifugation at 800 × g for 10 min at 4°C in HBSS. The cells were then resuspended in complete RPMI 1640 medium containing 10% FBS and antibiotics (50 U/ml penicillin, 50 μg/ml streptomycin, and 20 μg/ml gentamicin, all from Invitrogen) and layered 16% Histodenz (Sigma-Aldrich) in RPMI 1640 medium and centrifuged at 3,000 × g for 45 min at 4°C. After removing the nonparenchymal cells from the interface, the cells were washed twice by centrifugation (800 × g, 10 min, 4°C) in complete RPMI 1640 medium. The cells were then resuspended in complete RPMI 1640 medium and plated in a 96-well plate at a cell density of 5 × 106 cells/ml. After 2 h of incubation (37°C, 95% humidity, and 5% CO2), nonadherent cells were removed by washing with RPMI 1640 medium. We compared the number of adherent cells at the end of 2 h and found no significant difference in the number of adherent cells from sham and T-H mice. The cells were then cultured under the above-mentioned conditions for 24 h with or without 1 μg/ml LPS (Sigma-Aldrich). The cell-free supernatants were harvested and stored at −80°C until assayed.
Flow cytometric analysis of cytokine/chemokine concentration
TNF-α, IL-6, IL-10, MCP-1, and MIP-1α concentrations in the serum and Kupffer cell supernatants were determined with cytokine bead array inflammatory kits using flow cytometry according to the manufacturer’s instructions (BD Pharmingen), as described previously (16). In brief, 50 μl of mixed capture beads were incubated with 50 μl samples for 1 h at 25°C, and then 50 μl of mixed PE detection reagent was added. After incubation for 1 h at 25°C in the dark, the complexes were washed twice and analyzed using the LSRII flow cytometer (BD Biosciences). Data analysis was conducted using the accompanying FACSDiva and FCAP Array software (BD Biosciences).
The accumulation of neutrophils in the lung and liver tissue was assessed by determination of the MPO activity as previous described (17). Frozen tissue samples were thawed and suspended in 10% phosphate buffer (pH 6.0) containing 1% hexadecyltrimethylammonium bromide (Sigma-Aldrich). The samples were sonicated on ice, centrifuged at 12,000 × g for 15 min at 4° C, and an aliquot (30 μl) was transferred into 180 μl of phosphate buffer (pH 6.0) containing 0.167 mg/ml o-dianisidine dihydrochloride and 0.0005% hydrogen peroxide (Sigma-Aldrich). The change in absorbance at 460 nm was measured spectrophotometrically for 10 min. MPO activity was calculated using a standard curve that was generated using human MPO (Sigma), and values were normalized to protein concentration.
Measurement of α-glutathione transferase (α-GST)
Hepatic injury was determined by measuring serum levels of α-GST using a commercially available enzyme immunoassay kit according to the manufacturer’s instructions (Biotin International). In brief, 100 μl of each sample or standard was added to a well of an Ab-precoated microassay plate which was provided with the kit; they were incubated for 60 min at room temperature with uniform shaking. After washing, 100 μl of conjugate was added to each well and was incubated for another 60 min at room temperature with uniform shaking. Again, after the washing procedure, 100 μl of substrate was added to each well and incubated at room temperature for exactly 15 min. One hundred microliters of stop solution was then added to each well and the color intensity of each well was read immediately at 450 nm by a spectrophotometer.
Determination of tissue edema formation
Water content of lungs and livers was used as a measure of tissue edema. Tissue samples were weighed immediately after removal (wet weight) and then subjected to desiccation in an oven at 80°C (Blue M) until a stable dry weight was achieved after 48 h. The difference between wet weight and dry weight is water weight, and is calculated as percentage of wet tissue weight.
Statistical analysis was performed using Sigma-Stat computer software (SPSS). Statistical significance was assumed where probability values of less than 0.05 were obtained. Comparisons between groups were performed using one-way ANOVA followed by the Fisher least significant difference test. Results are expressed as means ± SE.
Edema formation in lung and liver
There was significant edema formation following T-H and resuscitation in the lung tissue for both WT and KO mice compared with their respective sham-operated groups (Fig. 1,A) (p < 0.05). However, the lungs of the WT mice were more edematous than those of KO mice following T-H (p < 0.05). There was no difference between WT and KO sham-operated groups (Fig. 1,A). In contrast, while liver tissue became more edematous after T-H when compared with the sham-operated animal, the severity of edema was similar between WT and KO mice following T-H or sham operation (Fig. 1,B). Additionally, no significant difference was noted among control, cannulation only, laparotomy only, and sham-operated groups (Fig. 1).
Tissue neutrophil infiltration determined by MPO activity
Two hours following T-H, the MPO activity in lung tissue was not influenced by the presence or absence of MIP-1α in sham-operated animals. However, T-H induced a significant increase in MPO activity in both WT and KO animals but the MPO levels were significantly higher in WT animals compared with corresponding KO animals (p < 0.05) (Fig. 2,A). The results of MPO activity determined in the liver tissue revealed the same pattern as that observed in the lungs (Fig. 2,B). The MPO activities of the liver and lung among control, cannulation only, laparotomy only, and sham-operated groups remained similar (Fig. 2).
Twenty-four hours following T-H, the MPO activities in the lung and liver were significantly decreased compared with those at 2 h in both WT and KO animals. However, those MPO activities in WT T-H animals were still significantly higher than in the corresponding sham and KO animals. In contrast, MPO activity in the lung and liver in T-H KO mice had returned to the sham levels at 24 h. Moreover, 48 h following T-H, MPO activity in the lung from WT mice remained significantly higher than sham. In contrast, liver MPO activity in WT mice returned to sham levels at 48 h following T-H (Fig. 3).
Liver injury determined by serum α-GST
The serum α-GST levels were similar in WT and KO mice following sham operation. T-H induced a significant increase of α-GST level in both WT and KO compared with their respective shams (Fig. 4) (p < 0.05). The α-GST levels were also significantly higher in WT mice than in the KO after T-H (p < 0.05) (Fig. 4). There was no significant difference in the α-GST levels between control, cannulation only, laparotomy only, and sham-operated groups (Fig. 4).
Serum cytokine levels
There was no difference in serum TNF-α, IL-6, IL-10, and MCP-1 in WT and KO sham animals (Fig. 5, A–D). However, serum levels of TNF-α were increased following T-H in WT animals compared with the corresponding shams, and they were also significantly higher than those of T-H KO mice (p < 0.05). In contrast, TNF-α levels in the KO mice showed no significant difference between T-H and sham-operated animals (Fig. 5,A). On the other hand, serum levels of IL-6 were significantly increased following T-H in both WT and KO mice compared with their respective sham-operated animals (p < 0.05). However, compared with KO mice, IL-6 levels were significantly higher in WT mice following T-H (p < 0.05) (Fig. 5,B). The results of serum IL-10 and MCP-1 levels revealed a similar trend as those of IL-6 (Fig. 5, C and D). Serum levels of MIP-1α were significantly elevated in WT mice following T-H (p < 0.05), and as can be expected, MIP-1α was not detected in the serum of KO mice (Fig. 5,E). The serum cytokine levels from sham-operated groups did not show any difference between themselves or between themselves, control, cannulation only, and laparotomy only groups (Fig. 5).
Kupffer cell cytokine production capacities
T-H induced an increase in Kupffer cell cytokine productive capacity in both the WT and KO mice for all the cytokines measured (i.e., TNF-α, IL-6, IL-10, and MCP-1). Furthermore, Kupffer cell cytokine productive capacity was significantly higher in WT mice than in KO mice following T-H. However, the cytokine productive capacity of Kupffer cells in sham-operated animals remained similar between WT and KO groups (Fig. 6, A–D). MIP-1α was only measurable in WT animals (Fig. 6,E) and it was increased following T-H (p < 0.05). Kupffer cell cytokine production capacities of sham-operated group were similar to those of control, cannulation only, and laparotomy only groups (Fig. 6).
MIP-1 was initially described in 1988 as a purified 8-kd protein from the conditioned medium of endotoxin-stimulated macrophages (18). It was later shown to be comprised of two proteins termed MIP-1α and MIP-1β (19). In addition to its proinflammatory activities, MIP-1α is also known to inhibit proliferation of hematopoietic stem cells (20). However, studies have revealed that MIP-1α-null mice have no overt abnormalities in peripheral blood or bone marrow cells (21, 22), thus making it feasible to study the proinflammatory activities of MIP-1α following T-H using MIP-1α KO mice.
MIP-1α has a wide range of proinflammatory activities, e.g., Fahey et al. (23) demonstrated that purified recombinant MIP-1α alone was able to stimulate the secretion of TNF, IL-1, and IL-6 by peritoneal macrophages and acted as an autocrine modulator of its cells of origin. In this regard, studies have suggested that MIP-1α can play a role in the regulation of T cell differentiation since the addition of MIP-1α to Ag-activated T cells promoted the development of IFN-γ-producing TH1 cells (24). Furthermore, it has been shown that macrophage-derived dendritic cells produce high amounts of MIP-1α and promote development of IFN-γ-producing TH1 cells via a MIP-1α receptor (CCR5)-dependent pathway (25). Speyer et al. (26) reported that in a cecal ligation and puncture (CLP)-induced sepsis, blood neutrophils were found to express mRNA for several C-C chemokine receptors including CCR5 and were responsive chemotactically to MIP-1α. The MPO activity of the lung in CLP mice corresponded with an increase in lung MIP-1α and MCP-1 content, suggesting that MIP-1α and MCP-1 play an important role in accumulation of neutrophils in the lungs during sepsis. Thus, MIP-1α is a proinflammatory chemokine that has the potential to regulate a wide range of immune cells, i.e., macrophages, dendritic cells, lymphocytes, neutrophils, etc.
Our data indicate that in sham-operated or other controls groups (i.e., control, cannulation only, and laparotomy only groups), there was only minimal injury to the lung and liver and there were no differences in organ edema formation, MPO activity, and serum α-GST levels between WT and MIP-1α-deficient animals. T-H, on the other hand, induced significant organ injury in both WT and KO mice compared with their respective sham-operated and control groups. This was evidenced by significant edema formation in lung and liver as well as higher MPO activity and α-GST levels. Furthermore, T-H caused more severe lung edema formation and lung MPO activity in WT mice compared with KO mice. It also resulted in higher serum α-GST levels and higher liver MPO content in WT mice indicating that MIP-1α plays an important role in regulating neutrophil infiltration and mediating acute inflammatory injuries following T-H. This suggestion is consistent with the results reported by Krishnadasan et al. (27), who studied β-chemokine functions in experimental ischemic-reperfusion injury. They found that following normothermic ischemic reperfusion of the lung, those animals that received anti-MIP-1α Abs before reperfusion had significantly less inflammatory change in the lung, as demonstrated by reduced vascular permeability and reduced lung neutrophil accumulation. Similarly, in a model of T cell-mediated hepatitis induced by Con A, hepatic levels of MIP-1α increased in a time-dependent manner in the first 8 h after injection of Con A and were associated with the marked increase in plasma alanine transaminase levels. Furthermore, by using MIP-1α KO mice, they found that the MIP-1α gene deletion impaired the development of Con A-induced hepatitis and reduced the recruitment of T cells to liver (13). These results, together with our results, suggest that MIP-1α mediates proinflammatory response and induces organ injury during the acute phase of inflammation.
To further clarify the role of MIP-1α in regulating infiltration of neutrophils to vital organs following T-H, we measured the MPO activity at two additional time points, i.e., 24 and 48 h following T-H. Our data indicates that although MPO activity in both WT and KO mice declined significantly over time, the levels remained higher in WT animals compared with sham. Furthermore, lung MPO activity remained elevated in WT animals at 48 h following T-H. These findings indicate that MIP-1α plays a major role in mediating neutrophil infiltration during acute inflammation following the insult of T-H.
The serum cytokine and chemokine levels revealed a significant hypoinflammatory change in MIP-1α-deficient mice following T-H compared with that seen in the WT mice. However, with the exception of the serum TNF-α levels, the IL-6, IL-10, and MCP-1 levels in KO mice following T-H were still higher than those of sham-operated KO mice. Since these cytokines and chemokines can be secreted by various types of cells and they interact closely by feedback or autocrine mechanisms, a deficiency of one may cause subsequent hypoproduction of others, but still result in some inflammation (28). In an article that reported that MIP-1α mediates peritoneal neutrophil migration in immune inflammation via sequential release of TNF-α and LTB4 (leukotriene B4), the authors found that after i.p. challenge with OVA in OVA-sensitized mice, peritoneal neutrophil migration and TNF-α production were prominent in MIP-α WT mice, but not in KO mice. The TNF-α levels of peritoneal exudates of OVA-challenged KO mice were found to be the same as the control group, indicating that TNF-α production was severely suppressed due to the MIP-1α deficiency (29). This may, in part, explain our result that the serum TNF-α levels in MIP-1α T-H KO mice were similar to those observed in the sham group.
Due to the complexity of cytokine and chemokine interactions, these molecules can often be seen regulating each other during various adverse conditions such as tumor, infection, inflammation, or autoimmune diseases. For example, studies have shown that MIP-1α production was increased in multiple myeloma patients and its level correlated with the severity of the disease (30). MIP-1α also enhanced the expression of IL-6, which further increased bone destruction and tumor burden in these patients (30). In MIP-1α-deficient mice infected with Schistosoma mansoni, the size of the granuloma and the liver eosinophil peroxidase activity were diminished compared with WT mice, and the in vitro response of their lymphatic cells to Ag was characterized by lower levels of IL-10 (31). In a model of experimental acute pancreatitis, MIP-1α and MCP-1 were elevated simultaneously, and they were also inhibited at the same time following antagonist treatment (32). Goser et al. (33) also demonstrated the critical role of MIP-1α and MCP-1 in the induction of autoimmune myocarditis and blockade of MCP-1 or MIP-1α significantly reduced the severity of the disease. Overall, these findings support our results that MIP-1α plays an important role in regulating inflammatory conditions including those observed T-H. Our results have also showed that deficiency of MIP-1α attenuates inflammatory response following T-H either directly by influencing inflammatory cell activity or indirectly by the lower production of other proinflammatory cytokines.
In this study, we examined the cytokine production capacity of the Kupffer cells. Previous data from our laboratory showed that among lung, liver, and splenic macrophages, no significant MCP-1 production was observed by alveolar and splenic macrophages following T-H, and although IL-6 production by alveolar macrophages was increased, it was decreased in splenic macrophages. On the other hand, Kupffer cells are the only macrophages that produce significant amounts of MCP-1 and IL-6 simultaneously following T-H, and these have turned out to be the major players in producing remote organ dysfunction following T-H (14). Another study demonstrated that the expression of MIP-1α mRNA and protein in Kupffer cells were increased following ischemic reperfusion injury of liver in a rat model, and this was accompanied by a rapid increase of TNF-α and IL-1β release by Kupffer cells (34). Since Kupffer cells are both a major source and target of cytokines and chemokines, the cytokine production by Kupffer cells may reflect the severity of inflammatory response and organ damage. Our data revealed that in the absence of MIP-1α, cytokine production by Kupffer cells was less pronounced. Compared with WT mice, cytokine production (TNF-α, IL-6, IL-10, and MCP-1) by Kupffer cells following T-H was significantly decreased in KO mice. The decreased production of all the cytokines may thus have prevented remote organs such as liver and lung from severe inflammation and dysfunction.
It could be argued that since the sham-operated animals underwent midline laparotomy and cannulation of femoral vessels, there may be some significant inflammatory response in those animals compared with normal mice because of the above-mentioned surgical procedures. The major difference between the procedure of T-H and sham operation is that animals in the T-H group sustained hemorrhagic shock followed by fluid resuscitation. Although this is a well-established model in our laboratory and we have repeatedly shown significantly different inflammatory response between T-H and sham-operated mice (6, 7, 14, 15, 16, 17, 35), we again examined whether simple laparotomy and cannulation induce any inflammatory response. Our results indicate that inflammatory responses in sham-operated animals are similar to those of the control (true sham), cannulation only, and laparotomy only groups, and this is true for both WT and KO mice. In view of this, it can be concluded that MIP-1α plays an important role in the development of systemic inflammatory response following T-H.
Based on the above findings, one may consider the feasibility of modulating MIP-1α as a therapeutic approach to attenuate organ injury induced by acute inflammation following ischemia-reperfusion or T-H and resuscitation. In a model of experimental colitis, Ajuebor et al. (36) reported that colonic MIP-1α was elevated within 24 h of colitis and promoted colonic neutrophil accumulation; however, pretreatment with MIP-1α Ab significantly reduced neutrophil accumulation during the early phase (24 h) but not the late phase of colitis. On the other hand, some articles have addressed the importance of MIP-1α on innate immunity against infectious complications. Kobayashi et al. (37) found that PBMC from ∼90% of severely burned patients failed to produce MIP-1α in cultures. They also found that SCID mice chimeras that were inoculated with burn patient PBMC showed no resistance to sepsis as produced by CLP, while SCID chimeras inoculated with healthy human PBMC showed resistance to CLP (37). It has also been reported that MIP-1α KO mice were more susceptible to CLP-induced sepsis, and MIP-1α KO mice supplemented with recombinant MIP-1α were resistant (38). These data, along with our findings, indicate the importance of MIP-1α in mediating inflammation and host defense. While short-term manipulation of MIP-1α following T-H might be advantageous for diminishing the inflammatory response and reducing vital organ dysfunction, the increased risk of late infection by long-term suppression of MIP-1α activity should be carefully considered.
We thank Bobbi Smith for assistance with manuscript preparation.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by National Institutes of Health Grant R01 GM37127.
Abbreviations used in this paper: T-H, trauma-hemorrhage; KO, knock out; WT, wild type; MPO, myeloperoxidase; α-GST, α-glutathione transferase; CLP, cecal ligation and puncture.