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,1
* Department of Pathology, and
Departments of Pediatrics and Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA;
Department of Biochemistry and Molecular Biology, University of North Dakota Medical School, Grand Forks, North Dakota, USA; and
Department of Traumatology, Hand- and Reconstructive Surgery, University of Ulm Medical School, Ulm, Germany
2Correspondence: Department of Pathology, The University of Michigan Medical School, 1301 Catherine Rd., Ann Arbor, MI 48109-0602, USA. E-mail: pward{at}umich.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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β T cells or neutrophils. In sharp contrast, 
T cell-knockout or T cell-depleted mice displayed baseline IL-17A plasma levels after CLP. Neutralization of IL-17A by two different antibodies improved sepsis (survival from 
10% to nearly 60%). Unexpectedly, antibody treatment was protective, even when administration of anti-IL-17A was delayed for up to 12 h after CLP. These protective effects of IL-17A blockade were associated with substantially reduced levels of bacteremia together with significant reductions of systemic proinflammatory cytokines and chemokines in plasma. In vitro incubation of mouse peritoneal macrophages with lipopolysaccharide (LPS) in the copresence of IL-17A substantially increased the production of TNF-, IL-1β, and IL-6 by these cells. These data suggest that, during experimental sepsis, T cell-derived IL-17A promotes high levels of proinflammatory mediators and bacteremia, resulting in enhanced lethality. IL-17A may be a potential therapeutic target in sepsis.—Flierl, M. A., Rittirsch, D., Gao, H., Hoesel, L. M., Nadeau, B. A., Day, D. E., Zetoune, F. S., Sarma, J. V., Huber-Lang, M. S., Ferrara, J. L. M., Ward, P. A. Adverse functions of IL-17A in experimental sepsis.
Key Words: • T cells • survival • colony-forming units • cytokines • chemokines
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and septicemia ranks now as the 10th leading cause of death in the United States, as opposed to 13th in 1990 (2)
. It is now becoming apparent that sepsis might not be a solitary, clearly defined disease, but rather a puzzling interplay of various biological systems and cascades with the immune system, resulting in a highly variable clinical picture. As a result, we are only able to use crude clinical parameters like body temperature, leukocyte count, heart rate and respiratory rate in an attempt to define sepsis. Thus, many clinicians refer to sepsis as a syndrome rather than a disease. In parallel with the difficulties encountered in the clinical setting, sepsis research has suffered from many setbacks. In many cases, promising experimental strategies have failed to be successful in the clinical setting (3
, 4)
. Therefore, there is a compelling need to broaden our comprehension of the pathophysiology of inflammation and its regulation. Recently, novel mediators of sepsis and inflammation have been discovered, such as HMGB-1 (5
, 6)
, putting new promising therapeutical targets on the murky horizon. Further, the discovery of the IL-17 cytokine family has provided a new pathway for crosstalk between adaptive and innate immunity. To date, several sources of IL-17 have been identified: Th17 cells, CD8 T cells, natural killer (NK) cells, β T cells, T cells, and neutrophils (7)
. It is now becoming clear that IL-17A (the first described member of the IL-17 family) is a mediator of neutrophil stimulation and mobilization by T lymphocytes (8)
. This presents IL-17A with a uniquely powerful position at the interface between adaptive and innate immunity. Thus, it is not surprising that the IL-17 family has been shown to be involved in various physiological and pathophysiological processes such as granulopoesis, defense against bacteria, rheumatoid arthritis, allograft rejection, tumor modulation, and asthma and allergic reactions (7
, 9
10
11
12
13
14
15
16)
.
In the current study, we sought to evaluate the role of IL-17A during experimental sepsis induced by CLP in mice. Given the proinflammatory effects of IL-17A in various pathophysiological settings, we hypothesized that elevated systemic levels of IL-17A during sepsis might be harmful, connecting the inflammatory arsenal of adaptive and innate immunity and ultimately resulting in the well-described inflammatory downward spiral occurring in sepsis. In the setting of experimental sepsis, we identify T cells, but not β T cells or neutrophils, as the source of elevated plasma IL-17A levels following CLP. Moreover, we describe protective effects of IL-17A blockade on sepsis mortality, effective even when carried out up to 12 h after CLP, associated with reduced blood-borne bacteria (colony forming units, CFUs) and decreased levels of proinflammatory cytokines and chemokines. Accordingly, IL-17A may be a target in human sepsis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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β T cell-knockout (B6.129S2-Tcratm1Mom/J) or T cell-knockout (B6.129P2-Tcrdtm1Mom/J) mice (9–10 wk of age, 22–25 g; Jackson Laboratories, Bar Harbor, ME, USA) were used. Both knockout mice are on a C57BL/6 background. Intraperitoneal ketamine (100 mg/kg body weight) (Fort Dodge Animal Health, Fort Dodge, IA, USA) was used for anesthesia and intraperitoneal xylazine (13 mg/kg body weight) (Bayer Corp. Shawnee Mission, KS, USA) for sedation. Experimental sepsis was induced by CLP, as described previously (17)
. In short, a 1-cm abdominal midline incision was made; the cecum was exposed, ligated, and punctured through and through with a 19-gauge needle; and a small portion of feces was pressed out to ensure persistence of the punctures. After repositioning of the bowel, the abdomen was closed in layers using 6-0 surgical sutures (Ethicon Inc., Somerville, NJ, USA) and metallic clips. Before and after the surgery, animals had unrestricted access to food and water.
Plasma collection
At given time points after CLP, whole blood was collected by cardiac puncture into syringes containing anticoagulant citrate dextrose (ACD; Baxter, Deerfield, IL, USA) in a 9:1 ratio. Samples were centrifuged (3000 rpm, 10 min, 4°C); plasma was obtained and immediately stored at –80°C until further analysis.
Cytokine/chemokine measurements
Plasma was harvested as described above. The following mouse ELISA kits were used: TNF-, IL-1β, IL-6, IL-10, MIP-2, MIP-1, MCP-1, IL-17, TGFβ (all R&D Systems, Minneapolis, MN, USA). For quantification of HMGB1, a commercially available ELISA kit (Shino-Test Corporation, Kanagawa, Japan) was used. All experiments were performed according to the manufacturer’s protocol.
T cell depletion
T cell depletion was induced using 500-µg hamster anti-mouse TCR monoclonal antibodies (Biolegend, San Diego, CA, USA) 5 days before CLP, as described previously (18
, 19)
. Control animals received an intraperitoneal injection of isotype-matched Armenian hamster IgG (500 µg; Biolegend, San Diego, CA, USA).
Anti-IL-17A antibodies
A specific neutralizing monoclonal anti-mouse IL-17A antibody was obtained from eBioscience (San Diego, CA, USA) and a polyclonal anti-mouse IL-17A antibody from R&D Systems. The monoclonal neutralizing antibody was used in all experiments, while the polyclonal R&D antibody was exclusively used to confirm the survival data (data not shown). Both antibodies have been characterized by the manufacturers to specifically neutralize mouse IL-17A and contain <0.001 endotoxin/µg protein. For in vivo neutralizing experiments, 45 µg of anti-IL-17A or control IgG (Jackson ImmunoResearch, West Grove, PA, USA) was administered intravenously in a total volume of 200 µl at the time of CLP. In some survival studies, infusions of blocking antibodies to IL-17A were delayed 6 or 12 h after CLP where indicated.
Determination of CFUs in blood
Blood was obtained by cardiac puncture and was drawn into syringes containing 10% citrate. Following appropriate dilution in 0.9% saline, 100-µl samples were plated in duplicates on 5% sheep blood agar (Remel, Lenexa, KS, USA) and incubated for 24 h at 37°C under aerobic conditions. CFUs were then determined and multiplied by the dilution factor.
Isolation and incubation of murine peritoneal macrophages
Mouse peritoneal macrophages were harvested 5 days after intraperitoneal injection of 1.5 ml thioglycolate (BD Biosciences, Sparks, MD, USA; 2.4 g/100 ml) by peritoneal lavage with PBS. After washing, the purity of cell suspension was >95% macrophages. Macrophages (5x106 per experimental condition) were allowed to adhere to tissue culture plates over 2 h and incubated in RPMI medium containing 0.5% BSA for 4 h at 37°C. Recombinant, endotoxin-free mouse IL-17A was obtained from R&D Systems, and LPS (Escherichia coli 0111:B4) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
Statistical analysis
All values are expressed as means ± SE. Data were analyzed with a one-way ANOVA, and individual group means were then compared with a Student-Newman-Keuls test. Differences were considered significant when P 
0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A).
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Systemic IL-17A requires T cells but not β T cells or neutrophils
Because IL-17A has been reported to be produced by β T cells, T cells, NK cells, CD4 T cells, and neutrophils (7)
, we sought to determine the cell population chiefly responsible for the elevated levels of IL-17A after CLP. Experimental sepsis was induced by CLP in wild-type, β T cell-knockout mice (B6.129S2-Tcratm1Mom/J) or T cell-knockout mice (B6.129P2-Tcrdtm1Mom/J), comparing plasma levels of IL-17A 12 h after CLP to wild-type littermates. As shown in Fig. 1B
, levels of IL-17A were not significantly altered in β T cell-knockout mice when compared to wild-type littermates. In sharp contrast, there was complete abolition of systemic IL-17A buildup in septic animals lacking T cells (Fig. 1C
), suggesting a key role for T cells in the sepsis-induced production of systemic IL-17A. These findings were confirmed in CLP mice depleted of T cells using a monoclonal hamster antibody when compared to isotype IgG-treated mice (Fig. 1D
). Neutrophils (PMNs) have been reported to contain intracellular IL-17A message and protein, but release of free, soluble IL-17A from PMN has not been reported (20
21
22)
. In further experiments, blood PMNs were depleted using a monoclonal antibody, Ly-6G. With blood PMNs falling to less than 5% of normal blood values, there was no significant reduction in plasma levels of IL-17A after CLP (data not shown), indicating that during CLP-induced sepsis, PMNs were not a significant source of IL-17A. Moreover, CD4 T cell-knockout mice (B6.129S6-Cd4) or animals that were T cell depleted with a monoclonal antibody targeting CD4 cells (GK1.5) (23)
showed no significant changes in IL-17A levels 12 h after CLP in comparison to CD4+/+ mice and isotype IgG-treated littermates, respectively (data not shown). These data indicate that T cells are the source of IL-17A in the CLP model of experimental sepsis.
Neutralization of IL-17A in CLP mice treated with monoclonal anti-IL-17A
To determine the in vivo efficacy of a neutralizing monoclonal rat IgG antibody to mouse IL-17A, levels of IL-17A were determined in plasma obtained from animals treated intravenously with either control IgG or neutralizing monoclonal anti-IL-17A 12 h after CLP. As determined by ELISA, mice treated with an isotype control IgG displayed similar levels as wild-type mice, while administration of 45 µg monoclonal anti-IL-17A reduced plasma levels of IL-17A to negative control levels (data not shown).
Improved survival in CLP after anti-IL-17A treatment
To investigate the role of IL-17A in CLP-induced sepsis, a neutralizing monoclonal antibody to IL-17A (as described above) was given intravenously at the time of CLP or at 6 or 12 h after CLP. Mice treated with an isotype control IgG, administered at the same time points, served as a control. As shown in Fig. 2
A, control IgG-treated mice displayed 8% survival by day 7 (bottom line). In contrast, when mice were administered anti-IL-17A at the time of CLP, survival was 58% at day 7 (top line, Fig. 2A
, P<0.05). To investigate whether delayed infusion of anti-IL-17A would still protect in the setting of CLP, intravenous infusion was delayed until 12 h after CLP, resulting in survivals of 33% (P<0.05), indicating that delayed infusion of anti-IL-17A was still significantly protecting against CLP-induced sepsis (middle line, Fig. 2A
). Delay of anti-IL-17A infusion until 6 h after CLP also resulted in an improved survival of 40% (P<0.05 vs. ctrl IgG-treated animals) (data not shown). When mice were given a different neutralizing antibody to IL-17A (polyclonal) at time 0, survival was 62% at day 5, resulting in a similar improvement of survival (data not shown).
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IL-17A blockade reduces bacteremia after CLP
To determine the effects of in vivo blockade of IL-17A after CLP might be linked to the level of systemic bacteremia, anticoagulated blood was collected 0–48 h after CLP by cardiac puncture, and aerobic blood cultures were obtained using sheep blood agar plates. After 24 h of incubation at 37°C, CFUs were quantified. As shown in Fig. 2B
, blood CFUs peaked at 24 and 48 h after CLP. Accordingly, CLP was induced in mice treated with either anti-IL-17A or control IgG, and blood samples were obtained 24 h after CLP and plated for aerobic CFUs. As shown in Fig. 2C
, anti-IL-17A-treated mice displayed a significant reduction in CFUs when compared to control IgG-treated littermates 24 h after CLP. These data suggest that, during CLP, IL-17A reduces the ability of mice to clear blood-borne bacteria and that with its blockade, the levels of bacteremia are reduced.
T cell-knockout mice display improved survival during experimental sepsis
Because we have identified T cells as the main source of IL-17A production during experimental sepsis (Fig. 1)
and because blockade of IL-17A was protective during CLP (Fig. 2A
), we hypothesized that T cell-knockout mice might display improved survival during CLP. As shown in Fig. 2D
, T cell–/– mice exhibited greatly improved survival during CLP-induced experimental sepsis when compared to C57/BL6 wild-type littermates.
IL-17A increases macrophage in vitro production of proinflammatory cytokines
To evaluate whether elevated levels of IL-17A during sepsis might also be related to elevated levels of proinflammatory cytokines, murine peritoneal macrophages were obtained and incubated with either LPS (50 ng/ml), IL-17A (10 pg/ml), or a combination of LPS (50 ng/ml) and increasing doses of IL-17A (1 pg/ml to 10 ng/ml). As shown in Fig. 3
, the addition of IL-17A greatly potentiated the LPS-induced production of TNF-, IL-1β, or IL-6 by 2- to 3-fold in a dose-dependent manner, whereas IL-17A alone (10 pg/ml) only slightly augmented the baseline proinflammatory mediator response. This effect was observed over a wide range of doses (1 pg to 10 ng). Interestingly, these effects were exclusively observed for proinflammatory cytokines but not the proinflammatory chemokines MCP-1, MIP-1, or MIP2 (data not shown). Collectively, these findings suggest that, in the copresence of LPS, IL-17A increases the in vitro production of proinflammatory cytokines by macrophages.
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Blockade of IL-17A reduces plasma levels of proinflammatory cytokines and chemokines after CLP
Given these in vitro observations, we sought to determine whether blockade of IL-17A in CLP mice affected the levels of inflammatory mediators in plasma. Mice were randomly assigned to three groups. The first group was given sham treatment, the second group was infused with 45 µg of IgG immediately after CLP (control IgG treatment), and a third group was intravenously administered 45 µg of monoclonal anti-IL-17A at the time of CLP. Plasma was obtained at time 0 (sham-treated mice) and 24 h after CLP (IgG- or anti-IL-17A-treated mice), since proinflammatory mediators peak in blood 24 h after CLP (unpublished data). There were very low or unmeasurable levels of mediators in plasma from all sham-treated mice (Fig. 4
). In animals treated with nonspecific IgG, there were sharp increments in the cytokines TNF-, IL-1β, IL-6, and in the chemokines MIP-1, MIP-2, and MCP-1 in plasma 24 h after CLP (Fig. 4A-C, F-H
). Levels of HMGB-1 were also increased after CLP (Fig. 4I
). When CLP mice were infused with anti-IL-17A at time 0, systemic levels of all proinflammatory mediators were substantially reduced, suggesting that IL-17A facilitates the systemic production of proinflammatory cytokines and chemokines and HMGB-1 after CLP. Interestingly, anti-IL-17A caused increases in levels of the anti-inflammatory cytokine IL-10 (Fig. 4D
) and TGFβ (Fig. 4E
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Lately, a distinct lineage of CD4 T cells, Th17 cells, has been identified as the main source of IL-17A and has clearly been the focus of recent research (16
, 24
25
26
27
28
29
30
31
32
33
34
35
36)
. However, besides Th17 cells, other sources of IL-17A have been identified: β T cells, T cells (37)
, NK cells, and neutrophils (7)
. In a recent study, patients suffering from trauma, systemic inflammatory response syndrome (SIRS), or sepsis syndrome were found to have a rapid and prolonged activation of T cells but not β T cells, suggesting a significant role for T cells as early responders during trauma, SIRS, and sepsis (38)
. It is now becoming evident that these cells are key players in bridging the innate and adaptive early immune responses. In line with the current study, in which we identified T cells as the predominant source of IL-17A during sepsis (Fig. 1C, D
), recent studies reported that resident T cells control neutrophil-mediated innate immune responses via the production of IL-17A (15
, 37)
. After intraperitoneal injection of E. coli, the rapid and transient production of IL-17A was found to mainly originate from T cells and assist in recruitment of neutrophils (15)
. Collectively, these data suggest that T cell-derived IL-17A enhances innate immunity via recruitment of neutrophils. This rapid recruitment of PMNs by IL-17A seems, in part, to be regulated by enhancement of chemokine gene expression (39)
. Interestingly, IL-17A and TNF- seemed to act synergistically to achieve this goal. Although TNF- initiated chemokine gene transcription through activation of NF-
B, IL-17A extended the half-life of mRNA via activator of NF-B1 protein (Act-1) -dependent mechanisms (39)
. In the present study, we have also found synergistic effects of IL-17A on LPS-induced in vitro activation of peritoneal macrophages (Fig. 3)
. The requirement of Act-1 for IL-17A and IL-17A receptor (IL-17RA) -dependent signaling during the immune response has been confirmed in other studies (40
, 41)
. Moreover, IL-17A has been reported to be a major orchestrator of sustained neutrophil mobilization and associated activity through induction of specific cytokines and colony-stimulating factors in resident lung cells (42)
.
By incubating cells with LPS in the copresence of various doses of IL-17A, we attempted to mimic septicemia and elevated IL-17A levels in vitro to investigate whether macrophage production of cytokines and chemokines can be altered by exposure to IL-17A and LPS. Interestingly, the copresence of IL-17A with LPS synergistically increased levels of the proinflammatory cytokines TNF-, IL-1β, and IL-6 (Fig. 3)
, whereas levels of MCP-1, MIP-1, and MIP-2 were not found to be increased under similar conditions. The exact molecular mechanism of this observation remains to be determined, but the data would suggest that IL-17A produced during sepsis can be implicated in the "cytokine storm," which connotes an unregulated development of inflammatory response.
IL-17A has been found to be elevated in a variety of inflammatory conditions, such as rheumatoid arthritis, pneumonia, systemic lupus erythematosus, allograft rejection, etc. (43
, 44)
Thus, it is not surprising that IL-17A acts on various cells, including neutrophils, fibroblasts, epithelial cells, and endothelial cells, inducing the expression of proinflammatory mediators such as IL-6, CXC chemokines, and matrix metalloproteinases (43)
. In the current study, we found reduced levels of proinflammatory cytokines and chemokines in septic animals treated with anti-IL-17A when compared to control IgG-treated littermates (Fig. 4)
and that T cells were the major source of sepsis-associated increased IL-17A levels (Fig. 1)
. Chung et al. (45)
recently reported decreased concentrations of TNF-, IL-6, and IL-12 in T cell-knockout animals during sepsis. It is tempting to speculate whether these decreased levels of proinflammatory mediators are in fact due to the absence of IL-17A in T cell-knockout mice or T cell-depleted animals during sepsis (Fig. 1C, D
), as IL-17A significantly potentiates the inflammatory mediator production in macrophages in vitro (Fig. 3)
. Collectively, the data in the current report suggest that CLP causes increased levels of IL-17A in plasma, which enhances production of proinflammatory cytokines. The mechanism by which IL-17A causes enhanced levels of CFUs in blood after CLP is unclear, but it may be that the bactericidal function of blood neutrophils is in some manner compromised under such conditions. An analogous situation in sepsis has been shown to be related to uncontrolled generation of C5a, which paralyzes MAPK signaling pathways in neutrophils (46)
.
The unique position of IL-17A at the interface between innate and acquired immunity puts this mediator in the lead as a crucial signal for the reinforcement and crosstalk of host defense systems. Thus, IL-17A might constitute a useful target for therapeutic intervention in sepsis and other inflammatory settings.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication December 31, 2007. Accepted for publication January 31, 2008.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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