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Published in final edited form as: J Neuroimmunol. 2010 Dec 21;233(1-2):37–45. doi: 10.1016/j.jneuroim.2010.11.009

Additive effects of Orexin B and vasoactive intestinal polypeptide on LL-37-mediated antimicrobial activities

Kouji Ohta a,b, Mikihiko Kajiya a,b, Tongbo Zhu a,b, Hiromi Nishi a, Hani Mawardi a,b, Jane Shin a,b, Leena Elbadawi a,c, Nobuyuki Kamata d, Hitoshi Komatsuzawa e, Toshihisa Kawai a,b,*
PMCID: PMC3115685  NIHMSID: NIHMS256423  PMID: 21176972

Abstract

The present study examined the bactericidal effects of Orexin B (ORXB) and vasoactive intestinal peptide (VIP) alone or combined with cationic antimicrobial peptides, such as LL-37, on Escherichia coli, Pseudomonas aeruginosa, Streptococcus mutans and Staphylococcus aureus. The bactericidal effect of ORXB or VIP alone was detected in low NaCl concentration, but attenuated in physiological NaCl concentration (150 mM). However, such attenuated bactericidal activities of ORXB and VIP in 150 mM NaCl were regained by adding LL-37. Therefore, our results indicate that VIP and ORXB appear to mediate bactericidal effects in concert with LL-37 in the physiological context of mucosal tissue.

Keywords: neuropeptide, orexin B, vasoactive intestinal polypeptide, LL-37, antimicrobial activity

1. Introduction

In general, neurons produce both a conventional chemical neurotransmitter and one or more neuropeptides. Neuropeptides function as neurotransmitters in the brain and the peripheral nervous system. However, they also play roles in regulating immune function and neurogenic inflammatory responses through vasodilatation, plasma extravasation, and recruitment of immunocompetent cells (Toriya et al., 1997 ; Jonsdottir et al., 2000). Furthermore, recent studies revealed that some neuropeptides released from the neuroendocrine system, such as Neurokinin-1 (NK-1) and neuropeptideY (NPY), have antimicrobial properties (Brodgen et al., 2005a,b). Vasoactive intestinal polypeptide (VIP) is widely expressed in the central nervous system, as well as peripheral tissues, including the lung, stomach, skin, and oral cavity, where it has been shown to have a multitude of biological functions (Dickinson and Fleetwood-Walker, 1999; Awawdeh et al., 2002). VIP was recently reported to have direct antimicrobial activities against both Gram-positive and Gram-negative bacteria (El Karim et al., 2008). It is, therefore, plausible that the nervous system may deploy some antibacterial function in the form of released neuropeptides, such as VIP, whose delivery to innervated peripheral sites is considered to protect the nervous system from infection by microorganisms.

Orexins are novel hypothalamic neuropeptides that have been implicated in the regulation of feeding, arousal, and energy homeostasis (Kirchgessner, 2002; Ehrström et al., 2005). They are cleaved from a common precursor molecule, preopro-orexin, forming orexin A and orexin B (de Lesea et al., 1998; Sakurai et al., 1998). Since neurons and endocrine cells in the gut were found to display orexin-like immunoreactivity, it is implicated that orexins modulate the electrical properties and synaptic inputs of secretomotor neurons in the intestinal system and stimulate colonic motility (Kirchgessner and Liu, 1999). Besides the roles of neuronal activity mediated by orexins, it is also reported that orexin B (ORXB) modulates the function of peritoneal macrophages through activation of calcium-dependent potassium channels and induces enhancement of phagocytosis in mouse peritoneal macrophages (Ichinose et al., 1998; Ichinose and Watanabe, 2004). Interestingly, the chemical properties of ORXB, i.e., amino acid composition (28 amino acids), net charge, and isoelectric point, are very similar to those of VIP (Table 1). Since antimicrobial action of a peptide can be predicted by its chemical properties, especially amphipathic nature and cationic charge (Brogden et al., 2005), it is plausible that ORXB may function as an anti-infective molecule. However, it has not been reported whether ORXB has antimicrobial properties.

Table 1.

Characteristics of VIP, ORXB and LL-37

Name Sequence AA* Charge** IP***
Vasoactive intestinal peptide (VIP) HSDAVFTDNYTRLRKQMAVKKYLNSILN 28 3.1 10.2
Orexin B (ORXB) RSGPPGLQGRLQRLLQASGNHAAGILTM 28 3.1 12.1
LL-37 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES 37 6.0 11.1
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*

Nunber of amino acids;

**

Net charge in pH 7.0;

***

Isoelectric point

Cationic antimicrobial peptides are evolutionarily conserved small proteins which play a critical role in the host innate immune defense system against microorganisms. Specifically, LL-37, the sole human cathelicidin, is widely expressed in a variety of bodily fluids and tissues, including key immune cell types, such as monocytes, neutrophils and lymphocytes, as well as epithelial cells on the mucosal surface (Durr et al., 2006; Hosokawa et al., 2006; Mookherjee et al., 2007). LL-37 facilitates a broad spectrum of antimicrobial activities against Gram-negative and Gram-positive bacteria, fungi and viruses. In addition, LL-37 has been reported to have synergistic or additive effects with other antibacterial agents (Chen et al., 2005). Especially, some large antimicrobial proteins, such as lysozyme and lactoferrin, appear to function in concert with LL-37 to kill E. coli (Singh et al., 2000). However, it is unclear if intestinally produced neuropeptides, namely, ORXB and VIP, can exert additive effects on LL-37-mediated antimicrobial function.

In the present study, we hypothesized that ORXB and VIP not only exert antimicrobial activity by themselves, but they also work in concert with other mucosal antimicrobial peptides, such as LL-37, to protect against infection in the context of intestinal tissue. To prove this hypothesis, the present study examined the antimicrobial activities of ORXB and VIP alone and combined with LL-37, HBD-1, and HNP-1 against Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, and Gram-positive bacteria, Staphylococcus aureus and Streptococcus mutans.

2. Materials and Methods

2.1. Microorganisms and growth conditions

E. coli ATCC 27325, S. aureus ATCC6538P, and P. aeruginosa ATCC 15692 were cultured in brain heart infusion broth (BHI broth; Difco Laboratory, Detroit, MI, USA) overnight at 37°C. S. mutans ATCC 25175 was grown anaerobically in BHI broth overnight at 37°C.

2.2. Peptides

Neuropeptides, VIP and ORXB, were obtained from a commercial vendor (Genscript, Piscataway, NJ, USA). Cationic antimicrobial peptides LL-37 and human neutrophil peptide 1 (HNP-1) were generated by custom-synthesis using a commercial service (Biomatik Corporation, Wilmington, DE, USA). Generation of human beta-defensin (HBD-1) was previously reported (Midorikawa et al., 2003).

2.3. Bactericidal assays

Bacterial cultures in broth at mid-log phase were harvested, then washed with phosphate-buffered saline (PBS) and suspended in 10 mM sodium phosphate buffer (NaPi; pH 6.8), following the protocol published previously (Midorikawa et al., 2003). Bacterial suspension in NaPi (105 cells/mL, in a total volume of 200 µl/well) was incubated in the presence or absence of various concentrations of single peptide alone or combination of two neuropeptides (ORXB and LL-37, or VIP and LL-37) for 2 hours at 37°C. An appropriate dilution of the bacteria-peptide reaction mixture was applied to Brain Heart Infusion (BHI) agar plate and incubated at 37°C overnight. Colony-forming units (CFU) were assessed by counting the number of bacterial colonies found in the agar plate. The bactericidal effect was calculated as the ratio of surviving cells to total cells (Midorikawa et al., 2003). In some experiments, to elucidate the effects of NaCl, a bacteria-peptide reaction solution consisting of various concentrations of NaCl prepared in 10 mM NaPi (pH 6.8) was tested in the same manner as the bactericidal assay described above.

2.4. Checkerboard titration to measure the fractional inhibitory concentration (FIC)

To determine the characteristics of effects of VIP or ORXB on the LL-37-mediated antimicrobial activity, a checkerboard titration method was employed using a 96-well titer plates (Nalge Nunc Rochester, NY, USA) following a method published by others (Oren et al., 1999; Midorikawa et al., 2003; Tin et al., 2009). Test strains were diluted to a concentration of 106 CFU/ml and applied to quarter-strength Lurie-Bertani culture medium containing two-fold serial dilutions of peptide alone or combination of two different peptides (VIP and LL-37, or ORXB and LL-37). After 24 hours of incubation at 37°C, MICs were calculated by determining the minimum concentration at which no growth was visible. The fractional inhibitory concentration (FIC) was calculated as follows: FIC = (MIC of neuropeptide in combination/MIC of neuropeptide alone) + (MIC of LL-37 in combination/MIC of LL-37 alone). The minimal FIC detected in the checkerboard titration was defined as the FIC index between two different peptides. Synergy is defined as an FIC index of ≤ 0.5; an additive effect is defined as 0.5 < FIC index≤1; indifference is defined as 1< FIC index ≤ 2; and antagonism is defined as an FIC index of >2.

2.5. Statistical analysis

Data were analyzed by Student’s t-test or one-way analysis of variance (ANOVA), and the results were presented as the mean ± standard deviation.

3. Results

3.1. Antimicrobial activities of VIP and ORXB

Previously reported antimicrobial effects mediated by VIP were based on monitoring minimal inhibitory concentrations (MICs) which evaluated the growth inhibition, but not the bactericidal effects, mediated by VIP (El Karim et al., 2008). It is therefore conceivable that VIP may only inhibit the growth of bacteria, but have no killing effect. Thus, in the present study, bactericidal effects mediated by VIP were examined using Gram-negative bacteria, E. coli, and P. aeruginosa, and Gram-positive bacteria, S. aureus and S. mutans (Fig. 1). VIP displayed bactericidal activity against E. coli (IC50= 17.5 µg/ml), P. aeruginosa (IC50= 8.5 µg/ml) and S. mutans (IC50= 36.87 µg/ml) in a dose-dependent manner. However, S. aureus showed resistance to VIP at concentrations as high as 50 µg/ml (Fig. 1).

Fig. 1. Bactericidal activities of VIP.

Fig. 1

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Bacterial cells were incubated with VIP for 2 hours at 37°C in 10 mM NaPi (pH 6.8). Then serial dilutions were plated on each agar, and colony counts were obtained after 24 hours of incubation at 37°C. Bacterial survival is expressed as a percentage (number of cells that survived in the presence of peptides compared to the number of cells that survived without a peptide). Data are shown as the mean±-standard deviation of three independent experiments. *Significantly different from non-treated bacterial cells (Student’s t-test: P < 0.05).

In contrast to a previous study reporting the antibacterial potential of VIP (El Karim et al., 2008), it is totally unclear whether ORXB kills bacteria or only inhibits bacterial growth. The same battery of bacteria used for VIP was employed to examine the bactericidal effects of ORXB (Fig. 2). ORXB demonstrated bactericidal activities against Gram-negative bacteria, E .coli (IC50= 21.6 µg/ml) and P. aeruginosa (IC50= 23.1 µg/ml) in a dose-dependent manner, but this peptide did not kill either of the two tested Gram-positive bacteria, S. aureus and S. mutans. While ORXB mediated bactericidal effects against Gram-negative bacteria, both Gram-positive bacteria, S. mutans and S. aureus, showed resistance to ORXB (Fig. 2). These results demonstrated that VIP and ORXB possess bactericidal effects preferentially against Gram-negative bacteria, while VIP, but not ORXB, showed bactericidal effects against the Gram-positive oral bacterium S. mutans.

Fig. 2. Bactericidal activities of ORXB.

Fig. 2

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Bacterial cells were incubated with ORXB for 2 hours at 37°C in 10 mM NaPi (pH 6.8). Then serial dilutions were plated on each agar, and colony counts were obtained after 24 hours of incubation at 37°C. Bacterial survival is expressed as a percentage, as noted in Fig. 1. Data are shown as the mean±-standard deviation of three independent experiments. *Significantly different from non-treated bacterial cells (Student’s t-test: P < 0.05).

3.2. Effects of VIP and ORXB on LL-37-mediated bactericidal activities

Next, the effects of VIP and ORXB on LL-37-mediated bactericidal activities against E. coli and P. aeruginosa were examined (Fig. 3). Both demonstrated additive effects on the bactericidal activity mediated by LL-37 in a dose-dependent manner. However, when LL-37 was combined with either VIP or ORXB and tested with Gram-positive bacteria, S. aureus and S. mutans, LL-37-mediated bactericidal effects on these bacteria were not affected (Fig. 4). These results indicated that VIP and ORXB possess additive effects on LL-37-mediated bactericidal activity, but only on E. coli and P. aeruginosa.

Fig. 3. Effects of VIP and ORXB on LL-37-mediated antibacterial activities against E. coli and P. aeruginosa.

Fig. 3

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Various concentrations of VIP and ORXB, together with LL-37, were incubated for 2 hours at 37°C in 10 mM NaPi (pH 6.8) containing bacterial cells. Then serial dilutions were plated on each agar, and colony counts were obtained after 24 hours of incubation at 37°C. Bacterial survival is expressed as a percentage, as noted in Figure 1. Data are shown as the mean±-standard deviation of three independent experiments. #Significantly different from non-treated cells and VIP or ORXB alone. *Significantly different from VIP or ORXB alone and these neuropeptides + LL-37. $Significantly different from LL-37 alone and these neuropeptides + LL-37 (Student’s t-test: P < 0.05).

Fig. 4. Effects of VIP and ORXB on LL-37-mediated antibacterial activities against S. aureus and S. mutans.

Fig. 4

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Various concentrations of VIP and ORXB with LL-37 (1 µg/ml) were incubated for 2 hours at 37°C in 10 mM NaPi (pH 6.8) containing bacterial cells. Then serial dilutions were plated on each agar, and colony counts were obtained after 24 hours of incubation at 37°C. Bacterial survival is expressed as a percentage, as noted in Figure 1. Data are shown as the mean±-standard deviation of three independent experiments. #Significantly different from non-treated cells and VIP or ORXB alone. *Significantly different from VIP or ORXB alone and these neuropeptides + LL-37 (Student’s t-test: P < 0.05).

To determine possible additive or synergistic effect of VIP and ORXB on antibacterial activity of LL-37 against E. coli and P. aeruginosa, we performed checkerboard titration method. The combination of these neuropeptides and LL-37 showed additive effects (0.5 < FIC index≤1) (Table. 2)

Table 2.

FIC indexes for combinations VIP or ORXB with LL-37

Organism MIC (µg/mL) FIC Index*
VIP ORXB LL-37 VIP ORXB
E. coli 500 250 8 0.66 0.75
P. aeruginosa 250 125 16 0.75 0.55
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*

The FIC index for combinations of respective peptide, either VIP or ORXB, with LL-37 was calculated as described in Materials and methods. FIC index ≤ 0.5 = synergic effect; 0.5 < FIC index≤1 = additive effect; 1 < FIC index ≤ 2 = no effect; and FIC index > 2 = antagonism.

3.3. Effects of NaCl on the bactericidal activities of VIP, ORXB, and LL-37 against E. coli and P. aeruginosa

The bactericidal activities of antimicrobial peptides are often lower at physiological NaCl concentration (150 mM) (Midorikawa et al., 2003). Therefore, the effects of NaCl on the antimicrobial activity of VIP and ORXB were investigated for E. coli and P. aeruginosa (Fig. 5 and 6). The bactericidal activities of ORXB and VIP against E. coli and P. aeruginosa were attenuated by the addition of different concentrations of NaCl in a dose-dependent manner (Fig.5 and Fig.6). For example, the bactericidal effects of VIP and ORXB against E. coli in the absence of NaCl (0 mM) were monitored at bacterial survival rates of 10% and 33% respectively, demonstrating strong bactericidal activities (Fig. 5). However, in the presence of physiological NaCl concentration (150 mM), the survival rate of E. coli increased up to 82% and 78% in response to VIP and ORXB (Fig. 5), indicating the loss of bactericidal effects of both VIP and ORXB in 150 mM NaCl. Similar loss of bactericidal effects of both VIP and ORXB was detected when P. aeruginosa was used as the target bacterium (Fig. 6). In contrast to such dramatic loss of bactericidal effects in 150 mM NaCl for each individual neuropeptide, the combination of neuropeptide, either VIP or ORXB, and LL-37 demonstrated remarkable bacterialcidal effects on both E. coli and P. aeruginosa, even in the presence of 150 mM NaCl (Fig. 5 and Fig. 6).

Fig. 5. Salt sensitivity of antimicrobial peptides against E. coli.

Fig. 5

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a) Various concentrations of NaCl (0 to 300 mM) were added to 10 mM NaPi (pH 6.8), along with VIP (30 µg/ml) or ORXB (50 µg/ml); b) either VIP (30 µg/ml) or ORXB (50 µg/ml) was incubated with LL-37 (1 µg/ml) in the presence of a 150 mM concentration of NaCl with 10 mM NaPi (pH 6.8), then reacted with E. coli using the method described in Materials and Methods. Data are shown as the mean±-standard deviation of three independent experiments. The surviving cells (%) among the various concentrations of NaCl tested (50–300 mM) showed statistical difference (ANOVA, P < 0.01). #Significantly different from VIP or ORXB without NaCl. *Significantly different from VIP, ORXB, or LL-37 alone and these neuropeptides + LL-37 (Student’s t-test, P < 0.01).

Fig. 6. Salt sensitivity of antimicrobial peptides against P. aeruginosa.

Fig. 6

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a) Various concentrations of NaCl (0 to 300 mM) were added to 10 mM NaPi (pH 6.8), along with VIP (15 µg/ml) or ORXB (25 µg/ml); b) either VIP (15 µg/ml) or ORXB (25 µg/ml) was incubated with LL-37 (4 µg/ml) in the presence of a 150 mM concentration of NaCl with 10 mM NaPi (pH 6.8), then reacted with P. aeruginosa using the method described in Materials and Methods. Data are shown as the mean±standard deviation of three independent experiments. The surviving cells (%) among the various concentrations of NaCl tested (50–300 mM) showed statistical difference (ANOVA, P < 0.01). #Significantly different from VIP or ORXB without NaCl. *Significantly different from VIP, ORXB, or LL-37 alone and these neuropeptides + LL-37 (Student’s t-test, P < 0.01).

3.4. Effects of VIP and ORXB on HNP-1 and HBD-1-mediated bactericidal activities

To investigate the effects of VIP and ORXB on other cationic host defense peptides, human alpha-defensin, HNP-1, and human beta-defensin, HBD-1, were examined using bactericidal assays with E. coli and P. aeruginosa (Tables 3 and 4). We found that the combination of both neuropeptides and HNP-1 exhibited significant killing of E. coli in comparison to addition with either alone (Table 3). However, in combination with HBD-1, only VIP, but not ORXB, resulted in an increased killing rate of E.coli (Table 3). On the other hand, both VIP and ORXB increased the HBD-1-mediated bactericidal activities against P. aeruginosa, whereas HNP-1 did not affect the bactericidal activity mediated by VIP or ORXB against P. aeruginosa (Table 4). These results indicated that VIP or ORXB enhance various ranges of bactericidal activity against heterologous bacteria depending on the cationic antimicrobial peptide used as a partner peptide.

Table 3.

Effects of VIP and ORXB on LL-37, HNP-1, and HBD-1 mediated antibacterial activities against E.coli

VIP (µg/ml) None LL-37 (1µg/ml) HNP-1 (20µg/ml) HBD-1(10µg/ml)
0 100$±3.9 77.7±4.5 77.5±1.3 72.0±5.5
5 92.6±4.0 72.3±2.9 75.2±3.6 71.9±1.8
10 78.1±2.4 47.8*±4.9 62.3*±3.3 44.0*±1.8
20 38.1±2.9 0.3*±0.1 17.8*±3.0 0.3*±0.1
ORXB (µg/ml) None LL-37 (1µg/ml) HNP-1 (20µg/ml) HBD-1(10µg/ml)
0 100±3.9 77.7±4.5 77.5±1.3 72.0±5.5
10 77.9±5.7 72.3±2.9 78.8±4.2 67.4±2.7
20 45.9±2.2 30.3*±3.2 42.5±3.9 47.8±5.8
40 34.3±1.6 9.9*±2.0 15.1*±4.4 37.7±4.6
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Various concentrations of VIP or ORXB were incubated with LL-37 (1µg/ml), HNP-1 (20µg/ml) or HBD-1 (10µg/ml) for 2 hours at 37°C in 200 ml of 10mMNaPi (pH 6.8) containing E.coli. Then serial dilutions were plated on each agar, and colony counts obtained after 24 hours of incubation at 37°C. Bacterial survival is expressed as a percentage, as noted in Fig. 1. Data are shown as the mean±-standard deviation of three independent experiments.

$

Surviving cells (%)

*

Significantly different between viability of each combination and lower viability of peptide alone (Student’s t-test; p < 0.05).

Table 4.

Effects of VIP and ORXB on LL-37, HNP-1, and HBD-1 mediated antibacterial activities against P.aeruginosa

VIP (µg/ml) None LL-37 (3µg/ml) HNP-1 (25µg/ml) HBD-1(8µg/ml)
0 100$±2.2 90.4±2.6 92.0±1.9 93.9±1.4
2.5 98.0±5.0 86.9±1.9 95.4±2.6 85.4*±1.8
5 62.3±3.0 43.8*±1.9 64.1±2.9 37.4*±2.1
10 43.0±3.9 0.1*±0.01 49.8±2.9 0.1*±0.01
ORXB (µg/ml) None LL-37 (3µg/ml) HNP-1 (25µg/ml) HBD-1(8µg/ml)
0 100±2.2 90.4±2.6 92.0±1.9 93.9±1.4
5 98.1±2.5 59.1*±4.8 91.9±1.8 50.2*±2.8
10 92.7±3.7 34.8*±3.4 92.1±1.3 16.1*±2.8
20 64.3±3.8 4.0*±0.5 94.4±1.2 0.4*±0.02
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Various concentrations of VIP or ORXB were incubated with LL-37 (3µg/ml), HNP-1 (25µg/ml) or HBD-1 (8µg/ml) for 2 hours at 37°C in 200 ml of 10mMNaPi (pH 6.8) containing P.aeruginosa. Then serial dilutions were plated on each agar, and colony counts obtained after 24 hours of incubation at 37°C. Bacterial survival is expressed as a percentage, as noted in Fig. 1. Data are shown as the mean±-standard deviation of three independent experiments.

$

Surviving cells (%)

*

Significantly different between viability of each combination and lower viability of peptide alone (Student’s t-test; p < 0.05)

Discussion

The present study examined 1) the bactericidal activities of ORXB and VIP against microorganisms and 2) the effects of ORXB or VIP on bactericidal activities mediated by other mucosal antimicrobial peptides. Both ORXB and VIP demonstrated dose-dependent bactericidal activities against Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa. On the other hand, VIP, but not ORXB, showed bactericidal effects on Gram-positive S. mutans, whereas neither ORXB nor VIP killed S. aureus. Both ORXB and VIP displayed additive effects on the LL-37-mediated bactericidal activities against Escherichia coli and Pseudomonas aeruginosa. While, the enhancement of bactericidal activities was observed when ORXB and VIP were used in combination with HBD-1 or HNP-1. As noted above, the bactericidal activities of antimicrobial peptides are often lower at physiological NaCl concentration (150 mM) (Midorikawa et al., 2003). Although the effect of ORXB and VIP on LL-37-mediated bactericidal activity against E. coli and Pseudomonas aeruginosa was maintained in the presence of 150 mM NaCl, ORXB and VIP alone both lost bactericidal effects in 150 mM NaCl. Overall, these results indicated that VIP and ORXB appear to mediate bactericidal effects in concert with another mucosal antimicrobial peptide, such as LL-37, in the context of mucosal tissue.

Several neuropeptides with neural or neuroendocrine signaling functions have been shown to possess antimicrobial properties. In a previous study, the antimicrobial activities of various neuropeptides were evaluated using an assay to monitor minimal inhibitory concentrations (MIC), and VIP was found to have direct antimicrobial activities against a range of pathogens (El Karim et al., 2005). Herein, we investigated the antimicrobial activities of VIP and ORXB against E. coli, P. aeruginosa, S. aureus, and S. mutans, but by using an analytical method different from MIC, i.e., a bactericidal assay. It is noteworthy that, while MIC assays monitor the inhibition of bacterial growth mediated by the reagents (antimicrobial peptides or neuropeptides), the bactericidal assay detects the ability of these reagents to kill the target bacterium. Therefore, the present study, for the first time, demonstrated that two neuropeptides, VIP and ORXB, have lethal potency against Gram negative bacteria

It is thought that the mechanism underlying the bactericidal activities of antimicrobial peptides is related to their strong positive charge, which binds to negatively charged cell walls of microorganisms. Both VIP and ORXB have an abundance of positively charged amino acids, including arginine, lysine and histidine (ORXB 14.3% and VIP 17.9% of the total peptide sequence of 28 amino acids). While total net charge of either ORXB or VIP is lower than that of LL-37, both neuropeptides are still positively charged, and their isoelectric points are very similar to the antimicrobial peptide LL-37 (Table 1). Thus, the molecular chemistry of ORXB and VIP supports our hypothesis that ORXB and VIP function as cationic antimicrobial neuropeptides in a manner similar to LL-37, HNP-1 and hBD-1 (Brogden, 2005a, b).

VIP is widely expressed in the central nervous system as well as peripheral tissues, including lung, skin and oral cavity, where it has been shown to have a multitude of biological functions (Dickinson and Fleetwood-Walker., 1999). LL-37 is produced by innate immune cells, such as neutrophils and epithelial cells, and plays an important role in the host innate immune defense system against infection of pathogenic bacteria in the oral and intestinal mucosal cavities (Durr et al., 2006; Mookherjee et al., 2007). In the present study, VIP showed a strong additive effect on the bactericidal activities of LL-37 against E. coli and P. aeruginosa. Therefore, based on the fact that both VIP and LL-37 are produced in oral and gastrointestinal mucosa, it is plausible that these two peptides facilitate efficient antimicrobial function in the mucosal environment by their synergism.

Orexins were originally isolated from brain tissue (Sakurai et al.,1998), and then later found in the mucosa of the antrum of the stomach (Kirchgessner and Liu, 1999). Gastrin-producing cells of the stomach, as well as numerous endocrine cells in the stomach and pancreas, display orexin-like immunoreactivity (Kirchgessner and Liu, 1999). Orexins have been shown to be involved in modulating metabolic rate via stimulation of the sympathetic nervous system (Antunes et al., 2001). In addition, Orexin participates in the regulation of immune cells. In particular, ORXB was shown to modulate functions of macrophages and to promote their phagocytosis (Ichinose et al., 1999; Ichinose and Watanabe, 2004). LL-37 is also known to act on macrophages to induce their production of chemokines, such as MCP-1 (Scot et al., 2002). The present study demonstrated that ORXB and LL-37, either together or individually, exhibited antimicrobial activities against E. coli and P. aeruginosa. Therefore, LL-37 and ORXB released in gastrointestinal mucosa appear to play an important role in the regulation of innate immune responses not only by modulating macrophage functions but also by facilitating bactericidal effects.

It is well known that different bacterial species show a wide variety of susceptibility to the antimicrobial effects mediated by antimicrobial peptides. For example, Gram-negative bacteria exhibit higher susceptibility to antimicrobial activities by hBD-1 and hBD-2 than Gram-positive bacteria (Harder et al., 1997). It is also reported that S. aureus shows little or no susceptibility to antimicrobial effects mediated by neuropeptides, including neuropeptide Y, substance P, neurokinin A and calcitonin gene-related peptide (Hansen et al., 2006; El Karim et al., 2008). Furthermore, S. mutans has a lower level of susceptibility to neuropeptides, such as VIP, neuropeptide Y and substance P, compared to E. coli or P. aeruginosa (El Karim et al., 2008; Hansen et al., 2006). ORXB did not have a bactericidal effect against S. aureus and S. mutans (Fig. 2), a finding which agrees with the studies described above. Furthermore, LL-37-mediated bactericidal effects against S.aureus and S.mutans were not affected by the addition of VIP and ORXB (Fig. 4). The ability of S. aureus to subvert the actions of cationic antimicrobial peptides is thought to be caused by alterations in the electrostatic properties of the cell surface, resulting in a reduction in its net negative charge which, in turn, attenuates the bacterial binding potential of the cationic peptide (Peschel et al., 1999). Therefore, similar to cationic antimicrobial peptides, differences in the charges of bacterial cell surface may determine the wide range of susceptibilities of various bacterial species to neuropeptides.

The bactericidal activity of cationic antimicrobial peptides is known to be salt-sensitive. More specifically, bactericidal activities of LL-37, HBD-1 and HBD-2 were shown to be attenuated by the presence of 100 mM NaCl (Singh et al., 1998; Zasloff., 2002, Midorikawa et al., 2003). Although the underlying mechanism for this salt-sensitive property of cationic antimicrobial peptides remains to be elucidated, it appears that charge competition derived from Na+ inhibits the initial interaction between a cationic peptide and the negatively charged bacterial membrane (Lehrer et al., 1993). If cationic antimicrobial peptides do indeed lose bactericidal activity in physiological salt concentration, the functional relevance of secreted cationic antimicrobial peptides in mucosal tissue must be questioned. Our results showed that the bactericidal activities of ORXB and VIP against E. coli and P. aeruginosa were attenuated by NaCl in a concentration-dependent manner. Very importantly, however, the effects of VIP and ORXB on the bactericidal activities of LL-37 were maintained in the presence of 150 mM NaCl. These findings, for the first time, suggest that multiple different antimicrobial agents work in concert to facilitate bactericidal effects under normal physiological salt concentration. Our results also showed that addition of ORXB and VIP promoted the bactericidal activities of HBD-1 and HNP-1, further supporting this premise, i.e., that multiple different antimicrobial peptides, including ORXB and VIP, work in concert to elicit the functionally efficient antimicrobial activities in the normal physiological context of mucosal organs.

In conclusion, we demonstrated that VIP and ORXB not only possess direct antimicrobial activities but also promote bactericidal effects by cationic peptides, including LL-37, HBD-1 and HNP-1, against E. coli and P. aeruginosa. Most importantly, VIP and ORXB could up-regulate the bactericidal effects of LL-37 in the presence of physiological concentration of NaCl (150 mM). These new findings may provide additional biological roles of neuropeptides in the context of innate immune responses.

Acknowledgements

This study was supported by NIH grant DE-018310 from the National Institute of Dental and Craniofacial Research and Pilot Grant from Harvard Catalyst.

Footnotes

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References

  1. Antunes VR, Brailoiu GC, Kwok EH, Scruggs P, Dun NJ. Orexins/hypocretins excite rat sympathetic preganglionic neurons in vivo and in vitro. Am J Physiol Regul Integr Comp Physiol. 2001;281:1801–1807. doi: 10.1152/ajpregu.2001.281.6.R1801. [DOI] [PubMed] [Google Scholar]
  2. Awawdeh L, Lundy FT, Shaw C, Lamey PJ, Linden GJ, Kennedy JG. Quantitative analysis of substance P, neurokinin A and calcitonin gene-related peptide in pulp tissue from painful and healthy human teeth. Int. Endod. J. 2002;35:30–36. doi: 10.1046/j.1365-2591.2002.00451.x. [DOI] [PubMed] [Google Scholar]
  3. Brogden KA, Guthmiller JM, Salzet M, Zasloff M. The nervous system and innate immunity : the neuropeptide connection. Nat. Immunol. 2005a;6:558–564. doi: 10.1038/ni1209. [DOI] [PubMed] [Google Scholar]
  4. Brogden KA. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol. 2005b;3:238–250. doi: 10.1038/nrmicro1098. [DOI] [PubMed] [Google Scholar]
  5. Chen X, Niyonsaba F, Ushio H, Okuda D, Nagaoka I, Ikeda S, Okumura K, Ogawa H. Synergistic effect of antibacterial agents human b-defensins, cathelicidin LL-37 and lysozyme against Staphylococcus aureus and Escherichia coli. J Dermatol Sci. 2005;40:123–132. doi: 10.1016/j.jdermsci.2005.03.014. [DOI] [PubMed] [Google Scholar]
  6. de Lecea L, Kilduff TS, Peyron, Gao X, Foye PE, Danielson PE, Fukuhara C, Battenberg EL, Gautvik VT, Bartlett FS, 2nd, Frankel WN, van den Pol AN, Bloom FE, Gautvik KM, Sutcliffe JG. The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity. PNAS. 1998;95:322–327. doi: 10.1073/pnas.95.1.322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dickinson T, Fleetwood-Walker SM. VIP and PACAP. very important in pain. Trends Pharmacol Sci. 1999;20:324–329. doi: 10.1016/s0165-6147(99)01340-1. [DOI] [PubMed] [Google Scholar]
  8. Durr UH, Sudheendra US, Ramamoorthy A. LL-37, the only human member of the cathelicidin family of antimicrobial peptides. Biochim. Biophys. Acta. 2006;1758:1408–1425. doi: 10.1016/j.bbamem.2006.03.030. [DOI] [PubMed] [Google Scholar]
  9. Ehrström M, Gustafsson T, Finn A, Kirchgessner A, Grybäck P, Jacobsson H, Hellström PM, Näslund E. Inhibitory Effect of Exogenous Orexin A on Gastric emptying, Plasma Leptin, and the Distribution of Orexin and Orexin Receptors in the Gut and Pancreas in Man. J Clin Endocrinol Metab. 2005;90:2370–2377. doi: 10.1210/jc.2004-1408. [DOI] [PubMed] [Google Scholar]
  10. El Karim IA, Linden GJ, Orr DF, Lundy FT. Antimicrobial activity of neuropeptides against a range of micro-organisms from skin, oral, respiratory and gastrointestinal tract sites. J Neuroimmunol. 2008;200:11–16. doi: 10.1016/j.jneuroim.2008.05.014. [DOI] [PubMed] [Google Scholar]
  11. Harder J, Bartels J, Christophers E, Schroder JM. A peptide antibiotic from human skin. Nature. 1997;387:861. doi: 10.1038/43088. [DOI] [PubMed] [Google Scholar]
  12. Hansen CJ, Burnell KK, Brogden KA. Antimicrobial activity of Substance P and Neuropeptide Y against laboratory strains of bacteria and oral microorganisms. J Neuroimmunol. 2006;177:215–218. doi: 10.1016/j.jneuroim.2006.05.011. [DOI] [PubMed] [Google Scholar]
  13. Hosokawa I, Hosokawa Y, Komatsuzawa H, Goncalves RB, Karimbux N, Napimoga MH, Seki M, Ouhara K, Sugai M, Taubman MA, Kawai T. Innate immune peptide LL-37 displays distinct expression pattern from beta-defensins in inflamed gingival tissue. Clin Exp Immunol. 2006;146:218–225. doi: 10.1111/j.1365-2249.2006.03200.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ichinose M, Asai M, Sawada M, Sasaki K, Oomura Induction of outward current by orexin B in mouse peritoneal macrophages. FEBS Lett. 1998;440:51–54. doi: 10.1016/s0014-5793(98)01432-x. [DOI] [PubMed] [Google Scholar]
  15. Ichinose M, Watanabe M. Stimulation of phagocytosis in mouse peritoneal macrophages by orexin-A and orexin B. Biomed Res. 2004;25:249–254. [Google Scholar]
  16. Jonsdottir IH. Neuropeptides and their interaction with exercise and immune Function. Immunol Cell Biol. 2000;78:562–570. doi: 10.1111/j.1440-1711.2000.t01-10-.x. [DOI] [PubMed] [Google Scholar]
  17. Kirchgessner AL, Liu MT. Orexin synthesis and response in the gut. Neuron. 1999;24:941–951. doi: 10.1016/s0896-6273(00)81041-7. [DOI] [PubMed] [Google Scholar]
  18. Kirchgessner AL. Orexin in the brain in the brain-gut axis. Endocr Rev. 2002;23:1–15. doi: 10.1210/edrv.23.1.0454. [DOI] [PubMed] [Google Scholar]
  19. Lehrer RI, Lichtenstein AK, Ganz T. Defensins : antimicrobial and cytotoxic peptides of mammalian cells. Annu Rev Immunol. 1993;11:105–128. doi: 10.1146/annurev.iy.11.040193.000541. [DOI] [PubMed] [Google Scholar]
  20. Midorikawa K, Ouhara K, Komatsuzawa H, Kawai T, Yamada S, Fujiwara T, Yamazaki K, Sayama K, Taubman MA, Kurihara H, Hashimoto K, Sugai M. Staphylococcus aureus Susceptibility to Innate Antimicrobial Peptides,-Defensins and CAP18, Expressed by Human Keratinocytes. Infect Immun. 2003;71:3730–3739. doi: 10.1128/IAI.71.7.3730-3739.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mookherjee N, Rehaume LM, Hancock RE. Cathelicidins and functional analogues as antisepsis molecules. Expert Opin. Ther. Targets. 2007;11:993–1004. doi: 10.1517/14728222.11.8.993. [DOI] [PubMed] [Google Scholar]
  22. Peschel A, Otto M, Jack RW, Kalbacher H, Jung G, Götz F. Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins, and other antimicrobial peptides. J Biol Chem. 1999;274:8405–8410. doi: 10.1074/jbc.274.13.8405. [DOI] [PubMed] [Google Scholar]
  23. Oren Z, Lerman JC, Gudmundsson GH, Agerberth B, Shai Y. Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the molecular basis for its non-cell-selective activity. Biochem J. 1999;341:501–513. [PMC free article] [PubMed] [Google Scholar]
  24. Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H, Williams SC, Richarson JA, Kozlowski GP, Wilson S, Arch JR, Buckingham RE, Haynes AC, Carr SA, Annan RS, McNulty DE, Liu WS, Terrett JA, Elshourbagy NA, Bergsma DJ, Yanagisawa M. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell. 1998;92:573–585. doi: 10.1016/s0092-8674(00)80949-6. [DOI] [PubMed] [Google Scholar]
  25. Scott MG, Davidson DJ, Gold MR, Bowdish D, Hancock RE. The Human Antimicrobial Peptide LL-37 is a Multifunctional Modulator of Innate Immune Responses. J Immunol. 2002;169:3883–3891. doi: 10.4049/jimmunol.169.7.3883. [DOI] [PubMed] [Google Scholar]
  26. Singh PK, Jia HP, Wiles K, Hesselberth J, Liu L, Conway BA, Greenberg EP, Valore EV, Welsh MJ, Ganz T, Tack BF, McCray PB., Jr Production of beta-defensins by human airway epithelia. Proc. Natl. Acad. Sci. USA. 1998;95:14961–14966. doi: 10.1073/pnas.95.25.14961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Singh PK, Tack BF, McCray PB, Jr, Welsh MJ. Synergistic and additive killing by antimicrobial factors found in human airway surface liquid. Am J Physiol Lung Cell Mol Physiol. 2000;279:799–805. doi: 10.1152/ajplung.2000.279.5.L799. [DOI] [PubMed] [Google Scholar]
  28. Tin S, Sakharkar KR, Lim CS, Sakharkar MK. Activity of Chitosans in combination with antibiotics in Pseudomonas aeruginosa. Int J Biol Sci. 2009;5:153–160. doi: 10.7150/ijbs.5.153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Toriya Y, Hashiguchi I, Maeda K. Immunohistochemical examination of the distribution of macrophages and CGRP-immunoreactive nerve fibers in induced rat periapical lesions. Endod. Dent. Traumatol. 1997;13:6–12. doi: 10.1111/j.1600-9657.1997.tb00002.x. [DOI] [PubMed] [Google Scholar]
  30. Valore EV, Park CH, Quayle AJ, Wiles KR, McCray PB, Jr, Ganz T. Human beta-defensin-1: an antimicrobial peptide of urogenital tissues. J Clin Invest. 1998;101:1633–1642. doi: 10.1172/JCI1861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Zasloff M. Antimicrobial peptides of multicellular organisms. Nature. 2002;415:389–395. doi: 10.1038/415389a. [DOI] [PubMed] [Google Scholar]

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