Ovocalyxin-36 is an effector protein modulating the production of proinflammatory mediators

Sepsis is a systemic inflammatory response syndrome during infection. Therapeutic agents are essential to protect the host from sepsis. Ovocalyxin-36 (OCX-36) is a chicken eggshell membrane protein and shares protein sequence and gene organization homology with bactericidal permeability-increasing protein (BPI), lipopolysaccharide-binding protein (LBP) and palate, lung and nasal epithelium clone (PLUNC) proteins that play a major role in innate immune protection. We recently reported that OCX-36 binds to both lipopolysaccharide (LPS) and lipoteichoic acid (LTA) (Cordeiro et al., 2013, PLoS ONE 8, e84112), which is an important activity to neutralize endotoxins and non-endotoxin pyrogens during an inflammatory response. Here we investigated the immune modulating effects of OCX-36 and enzymatically digested OCX-36 (dOCX-36) in vitro and in a mouse model of endotoxemia. OCX-36 alone dose-dependently induced both TNF-α and nitric oxide (NO) production by RAW 264.7 macrophage cells, and this immunostimulatory effect was reduced by enzymatic digestion. In the presence of LPS, dOCX-36 was more effective than intact OCX-36 at reducing LPS-induced secretion of TNF-α from RAW 264.7 cells, but did not reduce NO production. In contrast, OCX-36 increased LPS-induced NO production, both in the presence and absence of FBS, PCR array analysis confirmed that OCX-36 and dOCX-36 differentially regulated genes involved in innate immunity, and dOCX-36 down-regulated the expression of genes involved in LPS signaling and inflammatory responses. In vivo, dOCX-36 was more effective at reducing LPS-induced inflammatory symptoms and inhibiting the local production of pro-inflammatory mediators in the small intestine. These results suggest that OCX-36 and OCX-36 derived peptides may differentially modulate innate immune responses, and support our hypothesis that OCX-36 derived peptides have potential therapeutic applications in sepsis.

Abbreviations

ACE, angiotensin-I converting enzyme; BCA, bicinchoninic acid; BPI, bactericidal permeability-increasing protein; BW, body weight; CCL2, MCP-1, monocyte chemotactic protein-1; CXCR4, chemokine receptor 4; Cybβ, cytochrome b-245, beta polypeptide; DMEM, Dulbecco's modified Eagle's medium; dOCX-36, digested ovocalyxin-36; HRP, horseradish peroxidase; HTAB, hexadecyltrimethyl ammonium bromide; iNOS, inducible nitric oxide; LAL, Limulus Amebocyte Lysate; LBP, lipopolysaccharide-binding protein; LTA, lipoteichoic acid; MPO, myeloperoxidase; MWCO, molecular weight cut-off; MyD88, myeloid differentiation primary response gene 88; NO, nitric oxide; OCX-36, ovocalyxin-36; PAMPs, pathogen-associated molecular patterns associated molecular patterns; Pglyrp1, peptidoglycan recognition protein 1; PLUNC, palate, lung and nasal epithelium clone; PMSF, phenylmethanesulfonyl fluoride; PRR, pattern recognition molecule; Proc, protein C; TMB, tetramethylbenzidine; TLR, Toll-like receptor

Keywords: Lipopolysaccharide; Ovocalyxin-36; Gene expression; Innate immunity

Sepsis is a disease characterized by the invasion of bacterial pathogens into the bloodstream that activates an inflammatory response. The uncontrolled immuneresponse leads to septic shock that involves tissue damage and multiple organ dysfunction and failure (Opal, 2007).

LPS is the main component of the Gram-negative bacterial cell wall and the principal activator of the innateimmune system which promotes the production of pro-inflammatory mediators during infection (Beutler and Rietschel, 2013). LPS is one of several pathogen-associated molecular patterns (PAMPs) and is recognized by Toll-like receptor (TLR) 4 which is expressed on the surfaceof macrophages. This stimulates the host cells to secrete alarge amount of proinflammatory mediators and cytokinessuch as nitric oxide (NO), tumor necrosis factor (TNF)-α,, and interleukins (ILs) (Kumar et al., 2009). NO and TNF-α are associated with antimicrobial activity, the host innate immune response to pathogens and tumor cell killing (Bogdan, 2001).

The toxic effect of LPS is modulated by a large family of proteins such as the LBP/BPI/PLUNC protein family.These proteins bind LPS and mediate the LPS signal to innate immune receptors (Wiesner and Vilcinskas,2010). For example, BPI protein suppresses the delivery of LPS to immune receptors and promotes LPS uptake by macrophages via the macrophage phagocytic process (Iovine et al., 2002). On the other hand, low concentrations of LBP deliver LPS to CD14 molecules and then boost the inflammatory response induced by LPS; in contrast, high concentrations of LBP reduce LPS activation of macrophages (Lamping et al., 1998). Some studies have reported that LBP/BPI/PLUNC proteins inhibit proinflammatory activities of LPS in macrophages such as induction of cytokines secretion, stimulation of neutrophil oxidaseenzymes and NO formation (Schumann, 2001; Lukinskieneet al., 2011).

The current therapy for severe sepsis and septic shock includes treatment of circulatory failure, the administration of antibiotics and the use of activated protein C (Riverset al., 2001). Newer strategies are the identification and development of improved antimicrobial peptides that also neutralize the LPS functionality that leads to overproduction of proinflammatory mediators (Schuerholz et al.,2012).

OCX-36 is an avian protein enriched in the eggshellmembranes of chicken eggs. OCX-36 shares similarity inprotein sequence and gene structure with LBP, BPI andPLUNC proteins, which is the origin of our hypothesisthat OCX-36 participates in the innate immune protectionagainst pathogens (Gautron et al., 2007). We have recentlycharacterized the biological function of purified OCX-36extracted from eggshell membranes, demonstrating that itis a pattern recognition molecule (PRR) which has antimi-crobial activity against S. aureus and the ability to bind toEscherichia coli LPS and to S. aureus LTA (Cordeiro et al.,2013).

In order to evaluate the potential of OCX-36 for therapeutic and neutraceutical applications, we compared whole and enzymatically digested OCX-36 to determine their immune-stimulating and anti-endotoxin properties in vivo and in vitro. 

Dulbecco’s modified Eagle’s medium (DMEM), sodium pyruvate and penicillin–streptomycin were purchasedf rom Gibco. FBS was purchased from Cansera. 48-Well tissue culture plates and 96-well medium binding microplates were purchased from Corning Costar. Recombinant mouse TNF-α, IL-6, and IL-1β, anti-mouse TNF-α,IL-6, and IL-1α antibodies, biotinylated anti-mouse TNF-α, IL-6, and IL-1α antibodies, and avidin-conjugated HRP were purchased from BD Biosciences. Mouse TNF-α andIL-6 ELISA Ready-SET-Go®kits were purchased frome Bioscience. WST-1 Cell Proliferation Reagent was purchased from Roche Applied Science. Bicinchoninic acid (BCA) protein assay, bovine serum albumin (BSA), cellculture grade water (endotoxin-free, <0.005 EU/mL) andLimulus Amebocyte Lysate (LAL) Chromogenic Endotoxin Quantification Kit were purchased from Thermo Scientific. Pepsin from porcine gastric mucosa, LPS fromE. coli O111:B4, PMSF, aprotinin, leupeptin, pepstatinA, 3,3',5,5''-tetramethylbenzidine (TMB) and hexade-cyltrimethyl ammonium bromide (HTAB) were purchased rom Sigma–Aldrich. The Griess reagent system was purchased from Promega. AurumTMTotal RNA Mini Kit was purchased from Bio-Rad Laboratories. RT²First Strand cDNA Kit and Mouse Innate and Adaptive ImmuneResponse RT² Profiler PCR Array were purchased from SA Biosciences.

OCX-36 was extracted from eggshell membranes and purified as previously described (Cordeiro et al., 2013). Purified OCX-36 was dissolved in PBS buffer (10 mMsodium phosphate buffer, 0.154 M NaCl, pH 7.4) prepared with endotoxin-free water (<0.005 EU/mL) and the concentrations of OCX-36 for all assays were determined by theBCA protein assay using BSA as standard. Endotoxin levels in OCX-36 samples were measured by the LAL assay (Thermo Scientific).

To prepare pepsin-digested OCX-36, freeze-dried OCX-36 (2 mg/mL) was dissolved in 0.15 M HCl, and pepsin was added to the OCX-36 solution at an enzyme to substrateratio of 1:250 (w/w). Samples were incubated at 37ºC for 0, 30 s; 1.5, 5 and 30 min; and 1.5 h, 5 h and 10 h, followed by heating at 90ºC for 5 min to inactivate the enzyme.The digested samples were dialyzed against water (MWCO100 Da; Spectrum Laboratories, Inc.) and lyophilized forfurther use in cell culture and animal studies.

To prepare thermolysin-digested OCX-36, freeze dried OCX-36 was dissolved in endotoxin-free PBS buffer anddiluted in thermolysin digestion buffer (50 mM Tris–HCl,0.5 mM CaCl₂, pH 7.4). Thermolysin was dissolved in the same buffer and was added at an enzyme to substrate ratioof 1:100 (w/w). Samples were incubated at 65ºC for 15 min, 30 min; and 1 h, 1.5 h and 4 h. After digestion, the enzyme was inactivated by heating at 95ºC for 15 min and then cooled for 10 min at room temperature. Samples were centrifuged at 13,000 rpm (Biofuge Pico Heraeus Instruments) at 4ºC for 10 min.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as previously described (Hincke and Nairn,1992), followed by staining with Coomassie Blue and destaining.

RAW 264.7 cells (ATCC) were cultured in DMEM supplemented with 1 mM sodium pyruvate, 50 U/mL of penicillin–streptomycin and 10% FBS, and were grown at37?C in a 5% CO₂chamber. Cell passages 2–10 were used.Cells were seeded into 48-well tissue culture plates at a density of 5 × 10⁵cells/well and allowed to adhere for16–18 h before treatment. OCX-36 or dOCX-36 (with or without LPS) was either diluted into the culture medium(500 µL) from concentrated stocks (10 µL) (for measurements of NO secretion) or prediluted in medium and added to the wells (500 µL) after aspiration of the culture medium (TNF-α secretion), in order to obtain the final, stated, concentrations.

RAW 264.7 cells were treated with OCX-36 or enzymatically-digested OCX-36 (dOCX-36), in the presence or absence of E. coli O111:B4 LPS, in DMEM containing 10% FBS. To examine the effect on NO production,cells were incubated for 24 h in the presence of medium alone, 10 ng/mL LPS, OCX-36 or dOCX-36 alone, or LPS pre-incubated for 1 h with OCX-36 or dOCX-36 at the indicated concentrations. For TNF-α secretion and gene expression analysis by PCR array, cells were incubated for 6 h in the presence of medium alone, 100 ng/mL LPS, OCX-36 or dOCX-36 alone, or LPS pre-incubated with OCX-36 ordOCX-36 at the indicated concentrations.

To evaluate the potential role of LBP on the anti-endotoxin activity of OCX-36, cells were washed twice withPBS and treated as described above with OCX-36 in the presence or absence of LPS in serum-free DMEM. After incubation for 6 or 24 h, culture supernatants were collected for measurement of TNF-α and NO, respectively.

Balb/c mice (18–20 g, 6–8 mice/group; Charles River Laboratories, Inc.) were injected intraperitoneally (i.p.) with 2 or 10 mg/kg body weight (BW) OCX-36 or dOCX-36 combined with 25 µg E. coli O111:B4 LPS in sterile saline, in a total volume of 100 µL. Positive control mice received LPS alone, and negative control mice received saline only. Two control groups were given 10 mg/kg BW OCX-36 or dOCX-36 alone in sterile saline. Mice were monitored for clinical symptoms and weighed 24 h after LPS administration.

To further examine the acute local and systemic effects,mice were injected i.p. with 10 mg/kg BW OCX-36 or dOCX-36 combined with 25 µg E. coli O111:B4 LPS in sterile saline. Positive control mice received LPS alone, and negative control mice received saline. After 2 h, mice were humanely euthanized and blood, liver, and small intestine (ileum) samples were collected. Blood was processed for serum and stored at −20ºC, and liver and ileum samples were flash frozen and stored at −80ºC until further analysis.

All animal procedures were carried out in accordance with the Canadian Council of Animal Care Guide to the Care and Use of Experimental Animals and were approved by the University of Guelph Animal Care Committee (AUP#07R116).

Cell viability was measured using the WST-1 Cell Proliferation Reagent (Roche) according to the manufacturer’s instructions.

The NO concentration in culture supernatants wasdetermined using the Griess Reagent System (Promega) according to the manufacturer’s protocol.

Measurement of TNF-α concentration in culture supernatants was carried out by ELISA according to the manufacturer’s instructions (BD Biosciences), and results are expressed as percent TNF-α secretion relative to positive control (LPS) cells.

To measure TNF-α, IL-6 and IL-1 concentrations inliver and ileum samples, tissues were homogenized in 1 mLice-cold PBS containing 0.5% Triton X-100, 1 mM PMSF,10 µg/mL aprotinin, 10 µg/mL leupeptin, and 10 µg/mL pepstatin A using a Polytron® homogenizer (PT 1200;Kinematica, Inc., Switzerland). Homogenates were clarified by centrifugation at 12,000 rpm for 15 min at 4ºC (Biofuge Fresco, Heraeus Instruments). TNF-α, IL-6 andIL-1β concentrations were measured by ELISA according to the manufacturer’s instructions (BD Biosciences).TNF-α and IL-6 concentrations in serum were determined using mouse TNF-α and IL-6 ELISA Ready-SET-Go®kits(eBioscience) according to the manufacturer’s instructions.Cytokine concentrations are expressed as pg or ng cytokineper mL serum, or ng cytokine per g tissue.

Homogenate pellets from liver and ileum samples(Section 2.10) were re-homogenized in 1 mL of 50 mM potassium phosphate, pH 6.0 containing 0.5% (w/v) HTAB,and subjected to two freeze–thaw cycles. Samples were clarified by centrifugation at 12,000 rpm for 10 min at 4ºC (Biofuge Fresco, Heraeus Instruments), and supernatants were assayed for MPO activity. Briefly, samples were diluted in HTAB buffer and mixed with a 1.6 mM TMB solution in 0.3 mM H₂O₂. One unit (U) of enzyme activity was defined as the amount of MPO present that caused achange in absorbance of 1.0/min at 655 nm. MPO activities are expressed as mU MPO per g tissue.

Total RNA was extracted from RAW 264.7 cells using the Aurum^TM Total RNA Mini Kit (Bio-Rad Laboratories) according to the manufacturer’s instructions. One microgram of RNA was reverse transcribed using the RT² First Strand cDNA Kit (SA Biosciences), and the expression of 84 genes involved in the host responseto bacterial infection and sepsis were analyzed simultaneously using a Mouse Innate and Adaptive Immune Response RT² Profiler PCR Array (SA Biosciences) according to the manufacturer’s instructions. PCR was carried out using a MyiQ Single Color Real-Time PCR Detection System (Bio-Rad) and data were analyzed using the instructions and template provided by the manufacturer (http://www.sabiosciences.com/dataanalysis.php). Results are expressed as -fold change relative to untreatedcells. A gene was considered to be differentially expressedwhen it had a fold change of at least ±2.0.

All analyses were performed in triplicate unless specified otherwise. Statistical analyses were carried out using GraphPad Prism version 5.0 (GraphPad). Statistical significance was determined by Student’s t-test with p < 0.05 taken as significant. Results are reported as mean ± SEM. 

To investigate the effect of OCX-36 on TNF-α and NO production in murine macrophages, RAW 264.7 cells were incubated with varying concentrations of OCX-36 and in the presence or absence of E. coli LPS. Treatment with LPS significantly increased TNF-α production in RAW 264.7when compared to untreated control cells (p < 0.05), and this was decreased (p < 0.05) by OCX-36 at concentrations of 0.1, 1 and 10 µg/mL (Fig. 1A). In contrast, OCX-36 had no pronounced effect on LPS-induced NO production (Fig. 1B).Surprisingly, treatment of RAW 264.7 cells with OCX-36 alone, without LPS stimulation, significantly increased both TNF-α and NO production (p < 0.05) compared to untreated control cells, when added at concentrations greater than1 µg/mL (Fig. 1A and B).

This was not associated with endotoxin contamination in OCX-36 samples, since the endotoxin levels measured in OCX-36 samples using the LAL assay were lower than 1 ng/mL (0.11 ± 0.04 EU/µg ofOCX-36, 0.02 ± 0.01 ng/µg of OCX-36), and this level of endotoxin (LPS) was found not to induce NO productionin RAW 264.7 cells. 

Fig. 1. Effect of OCX-36 on TNF-α and NO secretion in LPS-stimulated RAW 264.7 cells. (A) Cells were treated with 0, 0.1, 1, 10 or 100 µg/mLOCX-36 in the presence or absence of 100 ng/mL E. coli LPS for 6 h. TNF-αconcentration was measured by ELISA, and is presented as percent TNF-α relative to positive control cells treated with LPS alone. (B) Cells were treated with 0, 1, 10 or 100 µg/mL OCX-36 in the presence or absence of 10 ng/mL E. coli LPS for 24 h. NO concentration was measured using Griess reagent and is presented as percent NO relative to positive control cells.Data represent means ± SEM, n = 3. Values without a common letter are significantly different at p < 0.05.

 

To further examine the effect of LBP, which is naturally present in FBS, on the anti-endotoxin activity of OCX-36, RAW 264.7 cells were treated with OCX-36 and LPS in serum-free medium. Similar to the results obtained in medium containing FBS, OCX-36 at concentrations of 0.1, 1 and 10 µg/mL significantly reduced TNF-α secretion (p < 0.05) (Fig. 2A). However, in the absence of serum, OCX-36 treatment at 1 and 10 µg/mL resulted in a >25%decrease in LPS-induced TNF-α secretion, compared to only a 12% reduction in the presence of FBS, at the same OCX-36 concentrations. OCX-36 did not reduce NO productionin LPS-activated RAW 264.7 cells in the absence of serum,but rather OCX-36 at 100 µg/mL significantly increased LPS-induced NO secretion when compared to cells treated with LPS alone (p < 0.5) (Fig. 2B). No effect of OCX-36 or LPS treatment was observed on cell viability (data not shown). 

Fig. 2. Effect of OCX-36 on TNF-α and NO production in LPS-stimulated RAW 264.7 cells in the absence of FBS. (A) Cells were treated with 0, 0.1,1, 10 or 100 µg/mL OCX-36 in the presence of 100 ng/mL E. coli LPS for 6 h in serum-free medium. TNF-α concentration was measured by ELISA,and is presented as percent TNF-α relative to positive control cells treated with LPS alone. (B) Cells were treated with 0, 0.1, 10 or 100 µg/mL OCX-36 in the presence of 10 ng/mL E. coli LPS for 24 h in serum-free medium,and NO concentration was measured using Griess reagent. Data represent means ± SEM, n = 3. Values without a common letter are significantly different at p < 0.05.

The effect of OCX-36-derived peptides (dOCX-36) on TNF-α and NO release by RAW 264.7 macrophages stimulated with E. coli LPS was also examined. OCX-36 was digested with pepsin for up to 10 h, and digestion was monitored at intervals by SDS-PAGE. OCX-36 appeared to be completely digested by pepsin after 1.5 h and by 10 h only peptide fragments were visible (Fig. 3A); thus this timepoint was chosen for OCX-36 digestion. OCX-36 was also digested with thermolysin for up to 4 h, and digestion was similarly monitored by SDS-PAGE (Fig. 3B). The peptides generated from digestion of OCX-36 with thermolysin were only tested for NO analysis in vitro.

dOCX-36 significantly reduced LPS-induced TNF-α secretion from RAW 264.7 cells (p < 0.05) in a dose-dependent manner, and an almost 50% reduction was observed at the highest dose tested (100 µg/mL) when compared to cells treated with LPS alone (Fig. 4A). Digestion with pepsin also appeared to abrogate the stimulatory effect of OCX-36 on RAW 264.7 cells, as dOCX-36 alone did not induce TNF-α secretion when compared to untreated cells. Peptides derived from OCX-36 digested with pepsin (Fig. 4B) and thermolysin (data not shown), however, did not show any effect on NO secretion in LPS-stimulated cells when compared to cells treated with LPS alone. As with TNF-α, dOCX-36 alone did not induce NO production when compared to untreated cells. Treatment with OCX-36 digested with pepsin or thermolysin did not affect cell viability (data not shown). 

Fig. 3. SDS-PAGE analysis of OCX-36 digested with pepsin and thermolysin. OCX-36 was incubated with (A) pepsin or (B) thermolysin for the times indicated. The position of molecular weight standards (kDa) is indicated and position of OCX-36 is indicated by the asterisk

 

Since both OCX-36 and dOCX-36 exerted differential effects on TNF-α and NO production in RAW 264.7 cells, we further examined the effects of OCX-36 and dOCX-36 at the transcriptional level. Cells were incubated for 6 h with medium alone, OCX-36 or dOCX-36 alone, LPS alone, or LPS pre-incubated with dOCX-36(LPS + dOCX-36), and relative gene expression was analyzed by PCR array. Differentially expressed genes are shown in Table 1. Notably, treatment of cells with dOCX-36 alone down-regulated expression of Cybβ (cytochromeb-245, beta polypeptide), IL-1f6 (IL-1 family, member 6),IL-1rn (IL-1 receptor antagonist), Pglyrp1 (peptidoglycan recognition protein 1) and up-regulated Proc (protein C)expression when compared to OCX-36. Treatment withOCX-36 alone down-regulated IL-1 when compared toLPS, and along with dOCX-36 down-regulated IL-6 expres-sion. Moreover, when combined with LPS (LPS + dOCX-36), dOCX up-regulated expression of CCL2 (MCP-1, monocyte chemotactic protein-1), and down-regulated expression of CXCR4 (chemokine receptor 4), MyD88 (myeloid differentiation primary response gene 88) and Prg2 (Proteoglycan2). 

Table 1 Differentially expressed genes in RAW 264.7 cells treated with OCX-36, dOCX-36, LPS, or LPS+dOCX-36.

 

To examine the anti-endotoxin effects in vivo, mice were injected with a sublethal dose of E. coli O111:B4LPS and monitored for clinical signs and body weights. Mice given LPS along with native OCX-36 (LPS + OCX-36) or pepsin-digested OCX-36 (LPS + dOCX-36) displayed slightly reduced clinical signs (ruffled fur, lethargy) than mice given LPS alone. While all mice lost weight 24 h after LPS administration, mice given the high dose (10 mg/kgBW) OCX-36 or dOCX-36 lost less weight than positive control (LPS) mice (p < 0.05) (Fig. 5). Therefore this dose was chosen for further study. There was no effect of OCX-36 or dOCX-36 administered alone.

To examine the effects of OCX-36 and dOCX-36 on acute LPS-induced endotoxemia, levels of pro-inflammatory mediators in the serum, liver and intestine (ileum) were measured 2 h after i.p. injection of LPS alone, or combined with OCX-36 or dOCX-36. Administration of LPS + dOCX-36 reduced serum IL-6 concentrations (p < 0.05) when compared to LPS-treated mice (Fig. 6A), but did not significantly affect serum TNF-α (Fig. 6A), or liver IL-6 and TNF-α(Fig. 6B). LPS + OCX-36 had no effect on serum or livercytokine concentrations when compared to mice treated with LPS alone. Levels of pro-inflammatory cytokines and myeloperoxidase (MPO) activity in the ileum were also measured to examine the effect of OCX-36 and dOCX-36 onlocal inflammation. IL-6, TNF-α, and IL-1β concentrations were elevated in the ileum of positive control (LPS) mice, but were significantly reduced in mice administered LPS + dOCX-36 (p < 0.05) (Fig. 7A). LPS + OCX-36 did not significantly affect cytokine levels in the ileum when compared to mice treated with LPS alone. The activity of MPO, an indicator of neutrophil infiltration into the intestinal mucosa, was significantly decreased by treatment with both LPS + OCX-36 and LPS + dOCX-36 when compared topositive control (LPS) mice (p < 0.05) (Fig. 7B). 

OCX-36 is a chicken eggshell protein specifically expressed in the chicken reproductive and digestive tracts. Based on similarities in the protein sequence and exon/intron gene organization with LBP, BPI and PLUNC family members, it has been proposed that OCX-36 plays a role in host defense (Gautron et al., 2007; Tian et al., 2010). LBP/BPI/PLUNC family proteins are capable of recognizing and neutralizing the effects of LPS (Wurfel et al., 1994; Wiesner and Vilcinskas, 2010). LBP also binds to other bacterial components such as LTA, peptidoglycan breakdown products and lipopeptides, and has been shown to modulate the effects of LTA in macrophages and monocytes(Schumann, 2011). We recently demonstrated that OCX-36 has antimicrobial activity against S. aureus and affinity for bacterial endotoxin (E. coli LPS) and non-endotoxinpyrogen (S. aureus LTA) (Cordeiro et al., 2013). OCX-36 can be readily extracted directly from eggshell membranes.This is a clear difference in comparison to the other innate immune proteins such as LBP, BPI and PLUNC proteins since most studies examining these proteins utilize recombinant proteins (Amura et al., 1998; Lamping et al., 1998; Chen et al., 2007). In the current study, we generated peptides by OCX-36 digestion and compared the effect of full-length OCX-36 and OCX-36-derived peptides on LPS-induced TNF-α and NO production in RAW 264.7 macrophage cellsin vitro, and in vivo using a mouse model of endotoxemia. 

Fig. 4. Effect of OCX-36-derived peptides (dOCX-36) on TNF-α and NO secretion in LPS-stimulated RAW 264.7 cells. (A) Cells were treated with 0, 0.1, 1,10 or 100 µg/mL dOCX-36 in the presence or absence of 100 ng/mL E. coli LPS for 6 h. TNF-α concentration was measured by ELISA, and is presented as percent TNF-α relative to positive control cells treated with LPS alone. (B) Cells were treated with 0 or 60 µg/mL of pepsin-digested OCX-36 in the presence or absence of 10 ng/mL E. coli LPS for 24 h, and NO concentration was measured using Griess reagent. Data represent means ± SEM, n = 3. Values without acommon letter are significantly different at p < 0.05.

Macrophages are phagocytic cells that attack infectious pathogens through the secretion of immune modulating mediators (Rosenberger and Finlay, 2003). Activation of macrophages in vitro by LPS induces production of pro-inflammatory mediators, including TNF-α, IL-6, IL-1 and NO (Zhu et al., 2013). Our in vitro experiments demonstrated that OCX-36 displayed a moderate inhibitory effect on LPS-induced TNF-α secretion and a stimulatory activity on NO production in RAW 264.7 cells. Previous studies have shown that human recombinant BPI and LBP inhibited the ability of LPS to stimulate TNF-α, but only BPI was able to suppress the production of TNF-α and NO on mouse macrophages (Amura et al., 1997, 1998). This is in line withour findings that OCX-36 exerted distinct effects on LPS-induced TNF-α and NO secretion from RAW 264.7 cells.The authors were unable to detect differences in TNF-α or inducible nitric oxide synthase (iNOS) gene expression, LPS-induced phosphorylation or activation of the NF-B transcription factor by either LBP or BPI, and suggested downstream regulation of LPS-mediated signaling events or the presence of two independent membrane bindingsites for LPS used by LBP and BPI protein to activate LPS stimulated murine macrophages (Amura et al., 1997, 1998). We also observed that at high doses, OCX-36 alone could significantly enhance the production of TNF-α and NO by mouse macrophages, indicating that OCX-36 can also exert immunostimulatory effects.

The inhibitory effect of LBP on TNF-α secretion inmurine macrophages stimulated with LPS was previously reported to occur in both the presence and absence of murine serum (Lamping et al., 1998). Here, OCX-36 likewise reduced TNF-α secretion by LPS-activated RAW 264.7 cells in both the absence and presence of 10% FBS. FBS is a source of LBP and a percentage of bioactive LBP in FBS is able to promote cell activation with murine and human TLRs (Meszaros et al., 1995). The reduction in TNF-α secretion from LPS-stimulated RAW 26.4 cells treated with OCX-36 in the absence of serum suggested that OCX-36 may compete with LBP for LPS and lead to an inhibition of TNF-α secretion. The observation that OCX-36 did not reduce TNF-α secretion when added to the cells at different time points independent of LPS and without pre-incubation (data not shown) further supports the role of the interaction of OCX-36 with LPS in anti-inflammatory activity. 

Fig. 5. Effect of OCX-36 and dOCX-36 on LPS-induced weight loss in mice.Mice were injected i.p. with 25 g LPS combined with 2 or 10 mg/kgBW OCX-36 or dOCX-36 in sterile saline. Positive control (LPS) mice received E. coli LPS alone, and negative control (Saline) mice received saline only. Mice were weighed before and 24 h after LPS injection.n = 5–10 mice/group. Each data point represents an individual animal, and horizontal lines indicate mean values. *p < 0.05.

 

We next examined the effect of OCX-36-derived peptides on TNF-α and NO secretion in RAW 264.7cells. Synthetic peptides derived from LBP and BPI have been shown to prevent LPS-induced TNF-α secretionby macrophages, and have been suggested as a potential adjunctive therapy to conventional sepsis treatments (Battafarano et al., 1995; Dankesreiter et al., 2000). More recently, a synthetic peptide (GL13NH2) from parotidsecretory protein, a PLUNC member protein, was similarly found to reduce the LPS-stimulated release of TNF-α from RAW 264.7 cells (Abdolhosseini et al., 2012). Here, OCX-36 was digested with pepsin, to mimic digestion in the gastrointestinal tract. Several studies have shown that peptides derived from chicken egg proteins digested with pepsin exhibit broad antimicrobial activity spectrum, anti-oxidant activity and reduced allergenicity (Kovacs-Nolanet al., 2000; Mine et al., 2004). OCX-36-derived peptides showed a different effect on TNF-α and NO secretion by RAW 264.7 cells when compared to full-length OCX-36. Pepsin-digested OCX-36 had enhanced anti-endotoxin effects and reduced LPS-induced TNF-α secretion by almost 50%. On the other hand, OCX-36 digested with pepsin orthermolysin did not show any inhibitory effect on NO release by LPS-stimulated murine macrophages. Moreover, in contrast to full-length OCX-36, OCX-36-derived peptides alone had no immunostimulatory activity and did not induce NO or TNF-α production.

The differential effects of OCX-36 and OCX-36-derived peptides on NO and TNF-α production in RAW 264.7macrophages, both alone and in the presence of LPS, were further examined by the expression profiling of severalgenes involved in innate immunity and inflammation. OCX-36 alone down-regulated the expression of pro-inflammatory cytokines IL-1β and IL-6, but up-regulated molecules involved in bacterial antigen recognition and presentation, such as CD1d and TLR8, which can lead to downstream activation of NF-kB (Joyce, 2001;Cervantes et al., 2012), further supporting its role inactivation of innate immunity. Likewise, dOCX-36 alonedown-regulated the expression of molecules involvedin inflammation, including the oxidative stress-related molecule cytochrome b-245, IL-1F6, a member of the IL-1 cytokine family that activates NF-B, and IL-1 receptor agonist, which regulates IL-1-mediated inflammation (Towneet al., 2004). In addition, dOCX-36 up-regulated protein C, which has been shown to exert anti-inflammatory effects in acute inflammation and sepsis (Frommhold et al., 2011). In the presence of LPS, dOCX-36 up-regulated CCL2(MCP-1), an important mediator of monocyte/macrophage recruitment, which has been shown to induce NO production in mouse macrophages in vitro (Biswas et al., 2001).dOCX-36 also down-regulated the expression of MyD88 and CXCR4 in LPS-activated RAW 264.7 cells. MyD88 isa TLR adapter protein important for TLR4 cell signaling pathways and is involved in systemic inflammation and mortality during sepsis. The CXCR4 receptor plays important functions, which, along with CD14, TLR4 and MD-2, may also play a role in LPS binding and signaling (Triantafilou and Triantafilou, 2002; Feng et al., 2011). Surprisingly, expression of the anti-inflammatory cytokineIL-10 was down-regulated in cells treated with dOCX-36,LPS, and to a lesser extent, LPS + dOCX-36. Decreased IL-10 production in LPS-stimulated macrophages has been described in response to anti-inflammatory treatments (Babcock et al., 2002), and Amura et al. (1998) found that BPI and LBP did not alter IL-10 expression in LPS-stimulated mouse macrophages despite a significant reduction in LPS-induced TNF-α secretion. While these results suggest that IL-10 may not be involved in the anti-endotoxin effects of dOCX-36 observed here, further gene expression analysis at different time points may be required to elucidate the role of IL-10 in response to OCX-36/dOCX-36 treatment.

The anti-endotoxin activity of pepsin-digested OCX-36(dOCX-36) in vitro motivated us to use a mouse model ofendotoxemia to examine the effects of dOCX-36 on the levels of pro-inflammatory cytokines in various organs.Our data showed that dOCX-36 significantly reduced IL-6,but not TNF-α levels in the serum of mice following i.p.treatment with LPS. While TNF-α is important in inflammation, IL-6 plays a key role in the acute phase response during sepsis and endotoxemia, and in fact circulating IL-6 levels have been found to be more closely correlated with disease severity and mortality than other inflammatory cytokines in patients with septic shock (Damas et al.,1992; Liaw et al., 1997). In addition, dOCX-36 was able to decrease the levels of IL-6, TNF-α and IL-1β, as well as MPO activity, in the ileum of mice administered LPS + dOCX-36.Both OCX-36 and dOCX-36 were able to decrease local LPS-induced MPO activity, suggesting the potential to reduce the recruitment of neutrophils in the ileum of septic mice. TNF-α, IL-6 and IL-1 are pro-inflammatory cytokines that play important roles in the intestinal mucosa during endotoxemia. Increased levels of these cytokines might affect the intestine as well as the function and integrity of remote organs and tissues (Meyer et al., 1995; Pritts et al., 2002). The intestinal anti-inflammatory activity of OCX-36 peptides suggested that dOCX-36 can modulate the intestinal mucosal immune response during endotoxemia. Both LBP and BPI are present in the intestinal epithelium, and overexpression of BPI was shown to attenuate bacteria-induced inflammation in intestinal epithelialcells, suggesting the potential for oral administration of OCX-36-derived peptides (Vreugdenhil et al., 2000; Cannyet al., 2006). There are a number of reports describing the anti-endotoxin activity of BPI and recombinant BPI peptides in vivo (Levy, 2002). Recombinant chimeric protein BPI₂₃-Fcy1 displayed anti-endotoxin and bactericidal activity and increased the survival rate of mice with sepsis caused by Gram-negative infection (Chen et al., 2007), and Jiang et al. (2004) found that a synthetic BPI peptide at a dose of 10 mg/kg could protect animals from lethalendotoxemia and reduce production of TNF-α and IL-6. While recombinant LPB at high doses has also been shown to protect mice against septic shock in vivo, it is highly dependent on LBP concentration, as lower doses have been found to pontentiate cell responses to LPS (Lamping et al.,1998), and as such LBP-derived peptides that can prevent LPS-induced TNF-α secretion in vitro and in vivo, independent of LBP concentration, have also been reported (Ara˜naet al., 2003). Likewise, the OCX-36-derived peptides tested here lacked the immunostimulatory activity of native OCX-36, and may therefore be beneficial for the treatmentof sepsis and inflammation. 

Fig. 6. Effect of OCX-36 and dOCX-36 on TNF-α and IL-6 concentrations in the (A) serum and (B) liver of LPS-treated mice. Mice were injected i.p. with 25 gE. coli LPS combined with 10 mg/kg BW OCX-36 or dOCX-36 in sterile saline. Positive control (LPS) mice received LPS alone. Blood and liver samples were collected 2 h after injection and cytokine levels in serum and liver homogenates measured by ELISA. Values shown are means ± SEM for n = 6–8 mice/group.*p < 0.05 compared to LPS.

Overall, in vitro studies revealed that purified OCX-36 reduces LPS-induced secretion of TNF-α from macrophagesand that OCX-36-derived peptides possess potent anti-endotoxin properties. The neutralizing activity of digested OCX-36 was confirmed by its capability to down-regulate the expression of genes involved in LPS signaling and inflammatory responses. OCX-36 might promote LPS activation in RAW 264.7 cells to augment some macrophage functions such as NO and TNF-α production. This suggests that OCX-36 is also a potential candidate as an immunostimulator of NO and TNF-α, which are important cytotoxic mediators contributing to the bactericidal activity of macrophages. Similar to our in vitro data, OCX-36-derived peptides were found to have an inhibitory effect on the production of LPS-induced proinflammatory mediators associated with endotoxemia in vivo. A future study to isolate and identify the effective anti-inflammatory OCX-36 peptides may lead to development of a novel endotoxin-neutralizing therapeutic agent or to delivery of OCX-36 as a nutraceutical by oral ingestion is conceivable. 

Fig. 7. (A) Cytokine and (B) MPO concentrations in the ileum of LPS-treated mice. Mice were injected i.p. with 25 µg E. coli LPS combined with 10 mg/kg BWOCX-36 or dOCX-36 in sterile saline. Positive control (LPS) mice received LPS alone. Ileum sections were collected 2 h after injection and cytokine levels and MPO activity in ileum homogenates were measured by ELISA and MPO assay, respectively. Values shown are means ± SEM for n = 6–8 mice/group. *p < 0.05compared to LPS.

These studies were supported by the Canadian NSERC Strategic grant program (STPGP 365046-08). CMMC isgrateful to Dr. Chantal Matar and her post-doctoral fellow, Dr. Tri Vuong for providing cell culture training andlab facilities. We would like to thank Dr. Yves Nys for valuable discussion and insight during the preparation ofthis manuscript, and are grateful to Hamed Esmaili, Megan Rose-Martel, Dr. Prithy Rupa and Hua Zhang for their valuable help. 

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