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Naturally Occurring Antimicrobial Peptide OH-CATH30 Selectively Regulates the Innate Immune Response To Protect against Sepsis Sheng-An Li,† Yang Xiang,† Yan-Jie Wang,† Jie Liu,†,‡ Wen-Hui Lee,† and Yun Zhang*,† †

Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China ‡ University of the Chinese Academy of Science, Beijing 100049, China S Supporting Information *

ABSTRACT: Sepsis, which is a systemic inflammatory response that follows a bacterial infection, has a high mortality rate and limited therapeutic options. Here we show that the antimicrobial peptide OHCATH30, which naturally occurs in snake, selectively regulates the innate immune response to protect mice from lethal sepsis. The administration of OH-CATH30 significantly improves the survival rate of mice infected by antibiotic-susceptible and -resistant pathogens, including Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. OH-CATH30 selectively up-regulates the production of chemokines and cytokines without harmful immune response. Recruitment of monocytes, macrophages, and neutrophils to the infection site is pivotal to the protective capacity of OH-CATH30. Furthermore, the alternative activation of the innate immune response by OH-CATH30 depends on p38 mitogen-activated protein kinase signaling. Taken together, our study demonstrates that OH-CATH30, a naturally occurring antimicrobial peptide, selectively stimulates the innate immune response to protect against sepsis.

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INTRODUCTION The innate immune response plays a key role in host defense against infection.1 Moderate activation of the innate immune response is beneficial for eradicating invading pathogens. However, hyperactivation of the innate immune system may lead to the excessive production of pro-inflammatory cytokines (such as TNF-α and IL-6) and sepsis.2 Sepsis, which is a systemic inflammatory response that follows a severe bacterial infection, has a high mortality rate and limited therapeutic options.3 Current treatments for sepsis focus on the control of the infection, modulation of the immune response, hemodynamic support, and palliative care.4,5 Early initiation of an adequate antibiotic treatment is crucial for the outcome of patients who have been admitted to the intensive care unit for sepsis.6 However, antibiotic treatments have lost their efficacy in many cases because of both the emergence of antibioticresistant bacteria and the dramatic decline in the development of effective antibiotics.7−9 Emerging evidence suggests that modulation of the innate immune response is an effective strategy to prevent and treat bacterial infection.2,10−12 The nonspecific properties of the innate immune response allow its modulation to provide broad-spectrum protection against various bacterial pathogens, including antibiotic-resistant bacteria.10 However, successful application of an immunomodulatory therapy requires controlled stimulation of protective immunity that avoids an increase in systemic inflammatory responses.2,13 Naturally occurring antimicrobial peptides are a class of small cationic peptides with antimicrobial activity that have been © 2013 American Chemical Society

isolated from various organisms, particularly in nonmammalian species.14,15 Antimicrobial peptides have a variety of functions, including neutralization of LPS, chemoattraction of immune cells, release of histamine from mast cells, and induction of angiogenesis.16 Antimicrobial peptides are considered viable alternatives to conventional antibiotics to treat antibioticresistant pathogens, and several antimicrobial peptides are being tested in clinical trials.17,18 However, most naturally occurring antimicrobial peptides are abandoned during the development phase because of poor antimicrobial activity and high toxicity.18−20 Studies have investigated the use of synthetic antimicrobial peptides to decrease the toxicity of naturally occurring antimicrobial peptides while maintaining or enhancing their antimicrobial activity and immunomodulatory properties.10,21−23 However, the immunomodulatory properties of naturally occurring antimicrobial peptides, especially peptides that have been derived from nonmammalian species, have not been thoroughly investigated. In our previous studies, we identified a small antimicrobial peptide, OH-CATH30, from the king cobra that exhibited potent, broad-spectrum, salt-independent antimicrobial activity.24−26 Although OH-CATH30 was shown to lose its antibacterial activity after 3 h of incubation with 100% human serum, it was effective against drug-resistant bacteria in a mouse bacteremia model.26 Therefore, we hypothesize that OHCATH30 possesses immunomodulatory properties. In this Received: July 26, 2013 Published: October 23, 2013 9136

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Figure 1. OH-CATH30 prevents lethal sepsis in mice. (A) OH-CATH30 (10 mg/kg) was administered by ip injection at −24, −5, 0, +0.5, or +3 h after intraperitoneal inoculation of E. coli 25922 (n = 10). (B−D) Mice were intraperitoneally challenged with E. coli 25922 (n = 10), P. aeruginosa 27853 (n = 8), or S. aureus 25923 (n = 10). OH-CATH30 was intraperitoneally administered 5 h before infection. Survival curves were significantly different between OH-CATH30 and vehicle-treated mice (P < 0.05 by log-rank test). (E) Sepsis was induced using clinically derived drug-resistant bacterial strains, including E. coli (n = 12), P. aeruginosa (n = 10), and S. aureus (n = 15). OH-CATH30 (10 mg/kg) was intraperitoneally administered 5 h before infection. (F) The therapeutic potential of OH-CATH30 in a CLP sepsis model was evaluated (n = 10). (G) Viable bacteria in peritoneal lavage are shown. Mice were pretreated with OH-CATH30 (ip, 10 mg/kg) 5 h before challenge with E. coli 25922 (n = 7), P. aeruginosa 27853 (n = 6), and S. aureus 25923 (n = 8), respectively. The solid line represents the arithmetic mean for each group (*, P < 0.05; by unpaired t test).

manner from 1 to 10 mg/kg (Figure 1B). OH-CATH30 delivered by a single ip injection 5 h before Pseudomonas aeruginosa (>85% survival, P < 0.005) or Staphylococcu aureus (>60% survival, P < 0.05) infection also significantly enhanced mouse survival rates (Figure 1C,D). Pretreatment with OHCATH30 also exhibited significant protection to mice infected with clinical isolated drug-resistant E. coli (92.5% survival, P < 0.005), P. aeruginosa (95% survival, P < 0.005), and S. aureus (63% survival, P < 0.05), compared to animals treated with conventional antibiotic (CFP) (Figure 1E). Furthermore, to mimic clinical sepsis conditions, we also evaluated the efficacy of OH-CATH30 in a CLP-induced sepsis model. Administration of OH-CATH30 by intravenous (iv) injection 2 and 12 h after CLP resulted in a 40% survival rate, and 70% of mice were rescued when OH-CATH30 was administered in combination with CFP (Figure 1F). We further investigated whether OH-CATH30 had an ability to improve the bacterial clearance in vivo. Mice were infected with E. coli, P. aeruginosa, and S. aureus, respectively. The bacterial burden in the peritoneal lavage was significantly reduced by pretreatment with a single ip injection of OH-CATH30 (P < 0.05, Figure

study, we aim to investigate the immunomodulatory properties of OH-CATH30 using mouse models of sepsis and to explore the potential mechanism of action of this peptide. We show that OH-CATH30 acts directly on the host to selectively trigger the innate immune response to protect mice from lethal sepsis.

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RESULTS OH-CATH30 Protects Mice from Lethal Sepsis. OHCATH30 exhibited potent antibacterial activities against a wide range of bacteria in vitro.26 To exclude the direct bactericidal activity of OH-CATH30, OH-CATH30 was administered by intraperitoneal (ip) injection before inoculation. Antibacterial activity was not observed in peritoneal fluids collected 1 h after OH-CATH30 administration using a radial diffusion assay (data not shown). Mice infected with a lethal dose of Escherichia coli were rescued by administration of OHCATH30 before (−24 h) to after (+3 h) the bacterial challenge (Figure 1A), which suggests that the protective capacity of OH-CATH30 does not depend on its direct bactericidal activity. In addition, OH-CATH30 promoted the survival rate of mice infected with E. coli in a dose-dependent 9137

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Figure 2. OH-CATH30 selectively induces cytokine production in macrophage. (A) OH-CATH30-induced cytokine expression in BMDMs was determined by qRT-PCR. Expression of the target genes in the samples was normalized against the expression of GAPDH, and gene expression in untreated cells was normalized to 1. (B) OH-CATH30-induced cytokines in BMDM culture supernatant were determined by ELISA. BMDMs were stimulated with OH-CATH30 or LL37 at the indicated concentrations for 18 h. The results are reported as the mean ± SD from three independent experiments (*, P < 0.05; **, P < 0.01; by unpaired t test). (C) OH-CATH30-induced cytokines in human monocyte THP-1 cells. The results are reported as the mean ± SEM from two independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001; by unpaired t test).

1G). Because OH-CATH30 has a short half-life of less than 1 h (Supplementary Figure 1, Supporting Information), these results demonstrate that the protective capacity of OHCATH30 may depend on a direct effect on the host immune response. OH-CATH30 Selectively Induces the Production of Cytokines in Macrophages/Monocytes. Macrophages/ monocytes are the main cell types in the mouse peritoneal cavity. Therefore, we determined whether OH-CATH30 acted on macrophages/monocytes to trigger an immune response. The qRT-PCR analysis revealed that various cytokines were upregulated in BMDMs after stimulation with OH-CATH30 (Figure 2A). mRNA levels of CCL and CXCL chemokines, including CCL2, CCL3, CCL4, and CCL7 and CXCL1,

CXCL2, CXCL3, CXCL5, and CXCL11, were significantly increased (P < 0.05). mRNA levels of anti-inflammatory cytokines, including IL-10 (5-fold) and IL-1Ra (25-fold), were also increased. Interestingly, mRNA levels of IL-17α, which is a primary cytokine that initiates immune response, were increased 51-fold in cells treated with OH-CATH30 relative to nontreated cells. Although IL-1β and IL-6 mRNA levels also increased by 2.5- and 4.5-fold, respectively, upon OH-CATH30 treatment, these levels were much lower than the increases of 193-fold and 3000-fold, respectively, observed following LPS treatment (Figure 2A). We confirmed via enzyme-linked immunosorbent assay (ELISA) that OH-CATH30-induced protein levels of IL-10, IL-1Ra, CXCL1, CXCL2, and CCL2 were also significantly increased in BMDMs (P < 0.05, Figure 9138

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2B). However, the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were not significantly up-regulated in the BMDM culture supernatant (Figure 2B). In contrast, at the same concentration (10 μg/mL) of OH-CATH30, the cytokine levels (269, 1450, 232, and 156 pg/mL for CXCL1, CXCL2, IL1-Ra, and IL-10, respectively) were much higher than those induced by LL37. Consistent with the results in BMDMs, OHCATH30 also selectively induced the production of cytokines in PMA-differentiated THP-1 cells (Figure 2C) and in human peripheral blood mononuclear cells (PBMCs) (Supplementary Figure 2, Supporting Information). MTT and LDH assays revealed that the concentrations of OH-CATH30 (5−20 μg/ mL) that were used for cell stimulation were not toxic to the cells (Supplementary Figure 3, Supporting Information). To determine whether OH-CATH30 improves the bactericidal activity of the macrophages in addition to the regulation of cytokine production, we investigated the effects of OHCATH30 on the phagocytic activity and H2O2 production of macrophage. OH-CATH30 did not improve the phagocytosis of fluorescently labeled E. coli by mouse macrophage RAW 264.7 cells (Supplementary Figure 4A, Supporting Information). In addition, the H2O2 levels in the cell culture supernatant were also not elevated after OH-CATH30 stimulation (Supplementary Figure 4B), suggesting no direct stimulation of the bactericidal activity of macrophages by the antimicrobial peptide OH-CATH30. OH-CATH30-Induced Cytokine Production in Vivo. To confirm the results observed in cultured cells, we further investigated the effect of OH-CATH30 on cytokine production in the mouse peritoneum in the absence and presence of bacterial infection. The concentrations of IL-10, IL-1Ra, CXCL1, CXCL2, and CCL2 were significantly increased in the mouse peritoneal lavage 5 h after an ip injection of OHCATH30 (P < 0.05) (Figure 3A). In contrast, the proinflammatory cytokines TNF-α, IL-1β, and IL-6 were not significantly up-regulated. We also investigated whether pretreatment with OH-CATH30 affected cytokine production in the presence of a P. aeruginosa infection (Figure 3B). The concentrations of TNF-α and IL-6 in the group pretreated with OH-CATH30 were markedly lower than those in the vehicletreated group (P < 0.05). Interestingly, preadministration of OH-CATH30 did not affect the production of CCL2 and CXCL2 chemokines after infection. The Efficacy of OH-CATH30 Depends on Monocytes and Neutrophils. Because OH-CATH30 selectively induced the production of CCL and CXCL chemokines in the macrophages and mouse peritoneal lavage, we hypothesized that monocytes and neutrophils may play a pivotal role in the protective capacity of OH-CATH30. The total number of leukocytes in the mouse peritoneal lavage markedly increased by 4 × 106 cells/mouse after ip administration of OH-CATH30 compared to PBS treatment (P < 0.005, Figure 4A). Neutrophils, resident monocytes/macrophages, and inflammatory monocytes were gated as GR1+/F4/80−, GR1−/F4/80+, and GR1+/F4/80+ cells, respectively.27 The number of monocytes/macrophages was modestly increased (P < 0.05), and the number of neutrophils increased to a great extent (P < 0.01, Figure 4B). The dynamic appearance of monocytes/ macrophages and neutrophils is consistent with the dynamic production of CCL2 and CXCL2 in the mouse peritoneal cavity (Figure 4C). To investigate the role of neutrophils in the protective capacity of OH-CATH30, the efficacy of OHCATH30 was evaluated in neutropenic mice following a P.

Figure 3. Cytokines induced by OH-CATH30 in vivo. (A) Mice were intraperitoneally injected with OH-CATH30 (10 mg/kg), and the peritoneal lavage was collected in 2 mL of cold sterile saline 5 h after injection. Bars represent the mean ± SD from four mice (*, P < 0.05; **, P < 0.01; by unpaired t test). (B) OH-CATH30 decreased the production of TNF-α and IL-6 but not CCL2 and CXCL2 in a P. aeruginosa-induced sepsis model. Mice were pretreated with OHCATH30 (ip, 10 mg/kg) 5 h before the bacterial challenge. The serum was collected 5 h after inoculation, and TNF-α and IL-6 levels were determined by ELISA. The peritoneal lavage was collected 3 h after inoculation to determine the CCL2 and CXCL2 levels. Bars represent the mean ± SD from four mice (**, P < 0.01; by unpaired t test).

aeruginosa infection. No increase in the number of neutrophils or inflammatory monocytes was observed in the peritoneal lavage of neutropenic mice up to 24 h after OH-CATH30 administration (Figure 4D). However, OH-CATH30 administration increased the amount of resident monocytes/macrophages in neutropenic mice, which is similar to the effect of OH-CATH30 administration in normal mice. Furthermore, pretreatment with OH-CATH30 reduced the bacterial burden in the peritoneal cavity (Figure 4E) and rescued 40% of infected neutropenic mice (P = 0.0337, Figure 4F). We further investigated whether OH-CATH30 was efficacious after monocyte/macrophage depletion (Supplementary Figure 5, Supporting Information). The administration of clodronate liposomes led to a more than 80% decline in the number of monocytes/macrophages compared with the number in normal mice. The administration of OH-CATH30 did not significantly increase the production of CXCL2 and the recruitment of neutrophils. Meanwhile, the administration of OH-CATH30 failed to eradicate invading bacteria and rescue infected mice. OH-CATH30-Induced CXCL2 Is p38 Signaling Pathway Dependent. To explore the potential signaling pathway by which OH-CATH30 selectively induced the production of CXCL2, we investigated the effects of OH-CATH30 on the TLR-mediated immune response. TLR4 is the signaling receptor for LPS, and TLR2 is the signaling receptor for LTA and PGN.28 OH-CATH30 blocked the production of LPS-induced pro-inflammatory mediators, including IL-6, TNF-α, and NO, in RAW 264.7 cells (Figure 5A), but had no effect on LTA- or PGN-induced TNF-α (Figure 5B), suggesting that OH-CATH30 selectively inhibits the TLR4mediated inflammatory response. On the basis of the above 9139

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Figure 4. The efficacy of OH-CATH30 depends on monocytes and neutrophils. (A−C) Normal mice were intraperitoneally injected with OHCATH30 (10 mg/kg). (A) Total cells were collected from the peritoneal lavage with 2 mL of PBS for cell counting 5 h after OH-CATH30 administration. The results are reported as the mean ± SD from three mice (*, P < 0.05; **, P < 0.01; by unpaired t test). (B) Cell types in the peritoneal lavage were analyzed by flow cytometry. (C) Peritoneal lavage was collected at the indicated time to determine CCL2 and CXCL2 levels by ELISA. (D) Neutropenic mice were intraperitoneally injected with OH-CATH30 (10 mg/kg). Peritoneal cells were collected and analyzed by flow cytometry. (E, F) The efficacy of OH-CATH30 was determined in a sepsis model in neutropenic mice (n = 10) (*, P < 0.05; by unpaired t test).

observations, we hypothesized that TLR4 may be involved in OH-CATH30-induced CXCL2 production. Therefore, we first investigated whether OH-CATH30 had an effect on the TLR4/ MD2 complex in the plasma membrane of mouse macrophage. A 30 min pretreatment with OH-CATH30 allowed OHCATH30 to bind to macrophages (Figure 5C). Furthermore, the plasma membrane TLR4/MD2 complex was downregulated by 20% with OH-CATH30 treatment, which is

similar to that of LPS treatment (Figure 5D). We next evaluated the role of TLR4/MD2 in OH-CATH30-induced CXCL2 production using a monoclonal TLR4/MD2-neutralizing antibody. OH-CATH30-induced CXCL2 levels were significantly attenuated from 1250 ± 97 to 850 ± 48 pg/mL after the BMDMs were cultured with antimouse TLR4/MD2 Ab (clone MTS510, Biolegend) for 30 min (P < 0.05). A broad-spectrum G protein-coupled receptor inhibitor (pertussis 9140

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Figure 5. Receptors involved in the OH-CATH30-induced CXCL2 production. (A, B) Effects of OH-CATH30 on the TLR-mediated immune response. Detailed methods are provided in the Supporting Information. The results are reported as the mean ± SEM from three independent experiments (*, P < 0.05; by unpaired t test). (C) Binding of OH-CATH30 to mouse macrophages. (D) OH-CATH30 down-regulated the plasma membrane TLR4/MD2 complex of RAW 264.7 cells. (E) BMDMs were cultured with antimouse TLR4/MD2 Ab (10 μg/mL), CD14 Ab (10 μg/ mL), WRW4 (10 μg/mL), KN-62 (2 μM), and pertussis toxin (100 ng/mL) for 30 min, respectively, and stimulated with OH-CATH30 for 6 h. CXCL2 levels in the cell culture supernatant were analyzed by ELISA (*, P < 0.05; by unpaired t test).

Figure 6. The production of CXCL2 induced by OH-CATH30 partially depends on the alternative activation of macrophages. (A) Effects of specific signaling pathway inhibitors on OH-CATH30-induced CXCL2 in mouse BMDMs. The detailed method is provided in the Supporting Information. The results are reported as the mean ± SEM from three independent experiments (*, P < 0.05; by unpaired t test). (B) Effects of OH-CATH30 on the MAPK pathway. (C) Effects of OH-CATH30 on the NF-κβ pathway.

toxin) also significantly inhibited OH-CATH30-induced CXCL2 production (P < 0.01, Figure 5E). In contrast, antimouse CD14 Ab (clone Sa14-2, Biolegend), WRW4 (formyl peptide receptor-like 1 antagonist), and KN-62 (P2X7

inhibitor) did not significantly attenuate the OH-CATH30induced CXCL2 (Figure 5E). We further investigated the effects of the immune-related intracellular pathway on the OH-CATH30-induced CXCL2 9141

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primary chemokines that recruit monocytes and neutrophils, respectively.10,30 Therefore, monocytes and neutrophils may be pivotal for the protective capacity of OH-CATH30. An influx of macrophages, neutrophils, and inflammatory monocytes was detected in the peritoneal lavage following OH-CATH30 administration. Neutrophils and monocytes, which are the main phagocytes in the innate defense system, are pivotal for the clearance of invading pathogens.21,31 Bacterial elimination by phagocytes is nonspecific and effective across a broad spectrum of pathogens. Therefore, bacteria may have difficulty developing resistance to naturally occurring antimicrobial peptides in vivo because these peptides exert anti-infective properties via modulation of the innate immune response. Other cell types in addition to monocytes/macrophages and neutrophils were increased in the peritoneal lavage after OHCATH30 administration. This result is not surprising because OH-CATH30 is able to induce other chemokines in addition to CCL2 and CXCL2. Although these additional cell types were not characterized in the current study, they may also contribute to the protective capacity of OH-CATH30. Further characterization of these cell types may facilitate the development of OH-CATH30 as a potential immunotherapy agent. Previous studies have demonstrated that cationic antimicrobial peptides are able to block TLR-mediated inflammatory responses, including those mediated by TLR4, TLR2, and TLR9.32 In our study, OH-CATH30 selectively decreased the levels of LPS-induced inflammatory cytokines but not the levels of the mediators of the TLR2-induced production of TNF-α, namely, LTA and PGN. However, preadministration of OHCATH30 resulted in a ∼60% survival rate in antibioticsusceptible and nonsusceptible S. aureus-infected mice. There are two possible explanations for this result: (1) OH-CATH30 has the ability to selectively induce the production of CCL and CXCL chemokines, which recruit monocytes/macrophages and neutrophils, respectively, to the site of infection to eradicate invading pathogens; and (2) anti-inflammatory cytokines such as IL-10 and IL-1Ra that are induced by OH-CATH30 in vivo may also contribute to the efficacy of OH-CATH30 in S. aureus-induced sepsis. This is consistent with previous studies reported by Monisha and Anasstasia.10,21 In their studies, two representative immunomodulatory peptides (IDR-1 and IDR1002) have been well studied. Both synthetic cationic peptides enhance the levels of chemokines while reducing proinflammatory cytokine responses to provide protection in vivo. Activation of TLR4 regulates multiple downstream molecules, including NF-kB and MAPKs, to trigger immune response.33 In our study, pretreatment with OH-CATH30 modestly reduced the number of TLR4/MD2 complexes on the plasma membrane of murine macrophages, indicating that OH-CATH30 may improve the internalization of TLR4/MD2 to trigger immune response.33 Although preincubation with antimouse TLR4/MD2 Ab reduced the levels of OH-CATH30induced CXCL2 (P < 0.05), pertussis toxin also significantly inhibited OH-CATH30-induced CXCL2 (P < 0.01), indicating a Gi protein receptor may be involved in OH-CATH30induced CXCL2 production in addition to TLR4/MD2. The immunomodulatory properties of LL37 have been demonstrated to be mediated through various receptors such as P2X7 and FPRL-1.34,35 However, a pharmacological inhibitor of these receptors did not affect the OH-CATH30-induced CXCL2 levels. LL37 induces cytokine production though both the MAPK and NF-κβ pathways.36,37 In our study, a pharmacological inhibitor of p38 MAPK, but not NF-κβ, significantly

using specific pharmacological inhibitors, including PI3K, ERK, JNK, p38, and NF-κβ. OH-CATH30-induced CXCL2 (1275 ± 124 pg/mL) was markedly attenuated in the presence of SB 202190 (464.9 ± 18 pg/mL), a specific inhibitor of p38 MAPK (P < 0.05, Figure 6A). Although the inhibitor of JNK (SP600125), MEK1/2 (U0126), and NF-κβ (Bay11-7082) also slightly reduced the levels of OH-CATH30-induced CXCL2 (952.0 ± 31, 979.6 ± 170, and 1000 ± 141 pg/mL for SP600125, U0126, and Bay11-7082, respectively), the changes were not significant (P > 0.05, Figure 6A). Consistent with the inhibitory assay, OH-CATH30 (10 μg/mL) markedly activated p38 in a time-dependent manner and modestly activated ERK and JNK (Figure 6B). We also investigated the effects of OH-CATH30 on NF-κβ signaling. Activation of the NF-κβ pathway was determined by the phosphorylation of p65 and the degradation of Iκβα. No phosphorylation of p65 or degradation of Iκβα was observed with OH-CATH30 (Figure 6C). In contrast, slightly increasing phosphorylation of p65 was observed with the human cathelicidin peptide, LL37 (10 μg/ mL).

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DISCUSSION The current strategies to treat sepsis focus on the neutralization of one or more inflammatory mediators, such as the antiendotoxin monoclonal antibodies, IL-1 receptor antagonists, and anti-TNF-α antibodies; however, none of these strategies have been entirely effective in clinical trials.3 In addition, anti-inflammatory therapies used during the hypoimmune phase may increase secondary infection and mortality.3,29 Therefore, balancing rather than merely suppressing the immune response may be an advantageous strategy in addition to controlling the infection. In the present study, we show that the antimicrobial peptide OH-CATH30, which is derived from the king cobra, protects mice from lethal sepsis by activating macrophages via p38 MAPK signaling. Our study suggests that selective modulation of the innate immune response by naturally occurring antimicrobial peptides may be a potential therapy for bacterial infections. In animal models, pretreatment with OH-CATH30 5 h before a bacterial challenge significantly reduced the bacterial burden and provided protection from sepsis in various mouse models. OH-CATH30 substantially decreased TNF-α and IL-6 levels, but did not affect CCL2 and CXCL2 levels 5 h after P. aeruginosa infection. This result was consistent with the observation of OH-CATH30-induced cytokine production in macrophages. OH-CATH30 selectively induced chemokines and anti-inflammatory cytokines. However, the cytokine profile induced by OH-CATH30 was not similar to that induced by LPS. These results suggest that OH-CATH30 balances rather than simply suppresses the immune response during infection. Our study shows that the protection of OH-CATH30 administered postinfection in the mouse sepsis model is dependent on both its selective immunomodulatory properties and its direct bactericidal activity,26 indicating OH-CATH30 may be efficacious to patients who get to the emergency room already infected, especially those infected by antibiotic-resistant pathogens. In addition, OH-CATH30 may be administered prophylactically to patients who are at a high risk of infection in the hospital, such as immunocompromised patients, severely burned patients, and cancer patients. Following administration of an ip injection of OH-CATH30, levels of CCL2 and CXCL2 chemokines were largely increased in the mouse peritoneal lavage. CCL2 and CXCL2 are the 9142

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a 22 gauge needle. The animals received an iv injection of CFP (40 mg/kg), OH-CATH30 (10 mg/kg), or OH-CATH30 and CFP 2 and 12 h after the procedure. To assess the action of OH-CATH30 in immunocompromised mice, the mice were rendered neutropenic by an ip injection of 150 and 100 mg of cyclophosphamide/kg of body mass on days 0 and 3, respectively. Peptides were administered 5 h before the induction of infection on day 5. The viable bacteria in the peritoneal cavity were evaluated 8 h after the bacterial inoculation. Mortality in all experimental sepsis models was recorded for up to 7 days. Humane end points were used in our survival studies. The mice were monitored every 6 h, and if the animals were not able to roll over from side to chest, they were humanely euthanized by CO2 inhalation. Cytokine Determination. ELISA was used on the cell culture supernatant, peritoneal lavage, and serum to assay for cytokine and chemokines according to the manufacturer’s instructions. The following ELISA kits were used: CCL2, CXCL1, CXCL2, IL-10, IL1Ra, IL-6, TNF-α, and IL-1β (R&D Systems). The OH-CATH30induced cytokines in the THP-1 cells were assayed using a Proteome Profiler Array (R&D Systems) according to the manufacturer’s instructions (Supporting Information). qRT-PCR. BMDMs were treated with OH-CATH30 (20 μg/mL) and LPS (10 ng/mL) for 3 h, and then the RNA was isolated using an RNeasy kit (Takara). cDNA was synthesized from the total RNA (500 ng) using a single-strand cDNA synthesis kit (Takara) and assessed by qRT-PCR using the SYBR Premix Ex Taq II (Tli RNaseH Plus) twostep qRT-PCR kit (Takara) on a LightCycler real time PCR system (Roche Diagnostics). The cycle counts of the target genes in the samples were normalized to those of GAPDH, and the fold changes of the transcript levels of the target genes were analyzed by Pfaffl’s method.39 The primers that were used for this analysis are listed in Supplementary Table 1 (Supporting Information). Flow Cytometry Analysis. To determine the cell types in the peritoneal lavage, cells were collected at indicated time points and stained with antimouse F4/80-PE and GR1-FITC (BioLegend) for 30 min at room temperature. To evaluate the binding of OH-CATH30 to RAW 264.7 cells, the cells were incubated with FITC−OH-CATH30 or FITC−NC30 (10 μg/mL) for 30 min at 37 °C and washed two times with PBS. To determine the effect of OH-CATH30 on the plasma membrane TLR4/MD2 complex of RAW 264.7 cells, the cells were incubated with OH-CATH30 (20 μg/mL) and LPS (1 μg/mL) for 30 min. The cells were then washed with ice-cold PBS and stained with antimouse PE-TLR4/MD2 (2 μg/mL, BioLegend) for 30 min on ice. All samples were analyzed by flow cytometry (FACSVantage SE, BD Biosciences, San Jose, CA). Data were analyzed using FlowJo software 7.6.1 (Tree Star Inc.). Western Blotting. Western blotting was performed as previously described.40 Briefly, samples were separated by 10% SDS−PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked with 5% BSA and probed with the following specific primary antibodies: anti-IκBα, antip38, anti-ERK1/2, anti-JNK, antiphospho-p65, antiphospho-JNK, antiphospho-ERK1/2, and antiphospho-p38 (Cell Signaling Technology, Danvers, MA). Blots were then incubated with HRP-conjugated goat antirabbit IgG (Cell Signaling Technology) and developed using the SuperSignal WestPico chemiluminescence substrate (Pierce Chemical Co.). Data Analysis. The survival rates were compared between groups by the Kaplan−Meier log-rank test. The significance of the difference in viable bacterial counts and cytokine levels between groups was determined by unpaired t test. Differences with P < 0.05 were considered statistically significant. All statistical analyses were conducted using Prism 5 GraphPad software.

attenuated the OH-CATH30-induced production of CXCL2. In addition, OH-CATH30 activated p38 phosphorylation but did not activate NF-κβ, which indicates that OH-CATH30 selectively stimulates the innate immune response via an alternative activation of macrophage. A previous study shows that chemokine induced by IDR-1002 is mediated through a Gicoupled receptor and the PI3k, NF-κβ, and MAPK signaling pathways.21 However, the peptides OH-CATH30 and IDR1002 share no sequence similarity in addition to cationic properties, which may account for the difference in signaling pathways involved. Currently, we do not know other immuneassociated signaling pathways involved in the OH-CATH30induced CXCL2 in addition to the p38 MAPK pathway that need further investigation. In conclusion, our study demonstrates that OH-CATH30, a snake antimicrobial peptide, selectively triggers an innate immune response to protect against sepsis. In addition, our study provides further insight into the development of naturally occurring antimicrobial peptides, especially those derived from nonmammalian species, as immunotherapy agents against antibiotic-resistant pathogens.

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EXPERIMENTAL SECTION

Reagents. LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES), OH-CATH30 (KFFKKLKNSVKKRAKKFFKKPRVIGVSIPF), NC30 (PRKIKGKVSKPFIFFVKLKNAKKSVKRKFF), and FITC-labeled peptides were synthesized by GL Biochem (Shanghai, China). The identity and purity (≥96%) of each peptide was confirmed by matrix-assisted laser desorption ionization time-offlight (MALDI-TOF) mass spectrometry and high-performance liquid chromatography (HPLC) (Supplementary Figures 6 and 7, Supporting Information). LTA from S. aureus, PGN from S. aureus, LPS from E. coli 0111:B4 (Sigma-Aldrich), and the peptides were dissolved in endotoxin-free water. The peptides were tested by chromogenic limulus amebocyte lysate assay for the absence of endotoxin (LPS) according to the manufacturer’s instructions from the LAL assay kit (Xiamen Houshiji Ltd., China). The Griess reagent system (Promega) was used to measure nitric oxide (NO) according to the manufacturer’s instructions. Cell Culture. Human monocyte THP-1 cells were seeded at 5 × 105 cells/mL in RPMI 1640 supplemented with 10% fetal calf serum (FCS) under an atmosphere of 5% CO2 at 37 °C. THP-1 cells were differentiated into macrophages with 50 ng/mL PMA (Sigma-Aldrich) for 12 h and rested for 24 h before stimulation. Mouse peritoneal macrophages were isolated as described by Nijnik et al.21 Mouse peritoneal macrophages and murine macrophage RAW 264.7 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FCS, as described above. BMDMs were prepared by culturing mouse bone marrow cells in RPMI 1640 supplemented with 10% FCS and 20% L929-conditioned medium.21 Mouse Sepsis Models. Female Kunming mice (18−22 g) were provided by the Animal Center of Kunming Medical University. All procedures, care, and handling of the animals were approved by the Ethics Committee of the Kunming Institute of Zoology at the Chinese Academy of Sciences. To develop sepsis induced by bacteria, mice were challenged with E. coli 25922 (2.5 × 108 CFU/mice), P. aeruginosa 27853 (8 × 107 CFU/ mice), S. aureus 25923 (2 × 108 CFU/mice), or antibiotic-resistant pathogens by ip injection. OH-CATH30 was intraperitoneally administered in sterile saline at the indicated time points. Peritoneal lavage was collected in 2 mL of cold sterile saline. The viable bacteria in the peritoneal lavage were counted as described previously.26 The CLP-induced sepsis model was performed as described by Rittirsch et al.38 Briefly, mice were anesthetized with ketamine (100 mg/kg, intramuscularly (im)) and xylazine (10 mg/kg, im). Once exposed, the cecum was ligated at a level 5.0 mm from the cecal tip away from the ileocecal valve. Then the ligated cecal stump was punctured once with

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ASSOCIATED CONTENT

S Supporting Information *

Description of the pharmacokinetic study, biological activity of OH-CATH30 in PBMCs, biological properties of OHCATH30 in the monocyte depletion mouse model, primers used in qRT-PCR, toxicity of OH-CATH30 in BMDMs, and 9143

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effects of OH-CATH30 on the bactericidal properties of macrophage. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-871-65194279. Fax: +86-871-65191823. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the National Basic Research Program of China (973 Program, 2010CB529800), the National Natural Science Foundation of China (NSFCYunnan joint funding, U1132601, 31071926, 31270835, and 81302539), and the key research program of the Chinese Academy of Sciences (KJZD-EW-L03).

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ABBREVIATIONS USED TLR, Toll-like receptor; MAPK, mitogen-activated protein kinase; FITC, fluorescein isothiocyanate; LTA, lipoteichoic acid; PGN, peptidoglycan; LPS, lipopolysaccharide; PMA, phorbol 12-myristate 13-acetate; BMDMs, bone marrowderived macrophages; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; LDH, lactate dehydrogenase; qRT-PCR, quantitative real-time PCR; CFP, cefoperazone sodium; CCL, CC chemokine ligand; CXCL, CXC chemokine ligand; CLP, cecal ligation and puncture

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