Bovine serum albumin (BSA), phosphate buffered saline (PBS), phenylmethylsulfonyl fluoride (PMSF), Roswell Park Memorial Institute 1640 medium (RPMI), Dulbecco’s Modified Eagle’s Medium (DMEM), phorbol 12-myristate 13-acetate (PMA), and Triton-X-100 were purchased from Sigma-Aldrich Canada (Oakville, ON). Fetal bovine serum (FBS), 10,000 U/mL penicillin/10,000 $\mu$g/mL streptomycin solution, and 1M 4-(2-hydroyethyl)-1piperazineethanesulfonic acid (HEPES) buffer solution were obtained from Invitrogen Canada (Oakville, ON). Sodium dodecyl sulfate (SDS), Tris base, and Tween-20 were acquired from Bio-Shop Canada Inc. (Burlington, ON). Ethylene diamine tetraacetic acid (EDTA) was purchased from EM 46 Industries Inc. (Hawthorne, NY). Luminata${}^{TM}$ Forte Western HRP substrate were purchased from EMD Millipore (Etobicoke, ON). Bio-Rad Protein Assay Dye Reagent was obtained from Bio-Rad Laboratories Inc. (Mississauga, ON). Lactoferricin peptides (HCl salt) (Table 1, Ref. [22]) in a linear configuration were synthesized and purchased from American Peptide (Sunnyvale, CA).

Rabbit (Rb) anti-p65, Rb anti-phospho p44/42 MAPK (ERK1/2) (pTpY202/204), Rb anti-p44/42 MAPK (ERK1/2), Rb anti-phospho-I$\kappa$B$\alpha$ (Ser32), Rb anti-I$\kappa$B$\alpha$, Rb anti-phospho-c-Jun (Ser63), Rb anti-c-Jun and Rb anti-$\beta$-actin (horse radish peroxidase (HRP) conjugate) antibodies were all purchased from Cell Signaling Technology (Beverly, MA). HRP-conjugated donkey anti-rabbit secondary antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Goat-anti-rabbit secondary antibody conjugated to Alexa Fluor ® 488 was obtained from Invitrogen. TNF-$\alpha$, and IL-6 capture antibodies and biotin-conjugated detection antibodies for ELISA were purchased from eBioscience (San Diego, CA).

RAW 264.7 mouse macrophage-like cells were purchased from ATTC ® (Manassas, VA). THP-1 human monocytic-like cells were kindly provided by Dr. Brent Johnston (Dalhousie University, Halifax, NS).

THP-1 and RAW 264.7 cell lines were maintained at 37 ${}^{\circ }$C in a humidified 5% CO${}_2$ incubator and cultured in complete RPMI (cRPMI) 1640 medium. Cells were cultured in 75 mm${}^2$ tissue culture flasks (Fisher Scientific, Waltham, MA) and passaged at 80–90% confluency. RAW 264.7 cells were passaged using a 25 cm long cell scraper (VWR, Mississauga, ON).

C57BL/6 female mice, purchased from Charles River Laboratories (Wilmington, MA), were euthanized at 8-10 weeks of age, and bone marrow was collected from the tibias by flushing with PBS (pH 7.2) making a single cell suspension. Red blood cells were lysed by hypo-osmotic shock. The remaining bone marrow cells were differentiated over 7 d by culturing in cRPMI containing 15% (v/v) L929-conditioned DMEM as a source of M-CSF. After 3 d of culture, the cells were fed with fresh medium. At 6 d, culture medium and non-adherent cells were removed, and the remaining cells fed with fresh medium. At 7 d the resulting macrophages were used in experiments.

Both cell lines and primary cells were seeded 1 d prior to treatment to allow for adherence to plastic. For all cell types, cells were cultured in cRPMI. THP-1 monocytic like cells were treated with 200 ng/mL PMA in order to cause cells to differentiate into macrophages and adhere to the plate overnight prior to any additional treatment. For ELISA experiments THP-1 cells were seeded in 24-well plates at 2.5 $\times$ 10${}^5$ cells/well in 1 mL of medium. Cells were plated at 6 $\times$ 10${}^5$ cells/well in a 6-well plate for quantitative real-time polymerase chain reaction (q-PCR) experiments. For immunofluorescent assays, cells were seeded on 10 mm glass coverslips placed in a 12-well plate at 1.5 $\times$ 10${}^5$ cells/well in 1 mL medium. Once adhered, cells were cultured overnight in serum-free medium to allow for cell growth arrest. For western blot experiments, cells were seeded at 1 $\times$ 10${}^6$ cells in 75 mm${}^2$ tissue culture flasks and left for 36–48 h until 90% confluency was reached, then cells were treated with 200 ng/mL PMA overnight and serum-starved an additional night prior to treatment. For ELISA experiments, RAW 264.7 cells were seeded in 24-well plates at 1.5 $\times$ 10${}^5$ cells/well in 1 mL of medium. Cells were plated at 2.5 $\times$ 10${}^5$ cells/well in a 6-well plate for q-PCR. For western blot experiments cells were seeded at 1 $\times$ 10${}^6$ cells/well in 10 cm${}^2$ cell culture plates (Thermo Fisher) and left for 36–48 h until 90% confluency was reached, then they were serum-starved overnight prior to treatment. For immunofluorescent assays, cells were seeded on 10 mm glass coverslips placed in a 12-well at 1 $\times$ 10${}^5$ cells/well in 1 mL medium. BMDMs were seeded in 24-well plates at 2.5 $\times$ 10${}^5$ cells/well in 1 mL of medium. Cells were plated at 6 $\times$ 10${}^5$ cells/well in a 6-well plate for q-PCR experiments.

Supernatants were collected from THP-1, RAW 264.7, and BMDM cell cultures for detection of TNF-$\alpha$, and IL-6 using a sandwich enzyme-linked immunosorbent assay (ELISA) Ready-SET-Go!® ELISA kit from eBioscience (San Diego, CA) according to the manufacturer’s instructions. Cells were treated with medium alone, 100 ng/mL LPS alone, or with LPS in combination with 5 $\mu$M lactoferricin peptides (bovine, mouse, human). A subset of experiments used cadmium chloride (CdCl${}_2$), purchased from Sigma Aldrich. For these experiments, cells were treated with medium alone, 10 $\mu$M CdCl${}_2$ alone, or with CdCl${}_2$ in combination with 5 $\mu$M lactoferricin peptides. Supernatants being analyzed for TNF-$\alpha$ were collected after 6 h, and all other supernatants were collected after 24 h of treatment. Previous experiments indicated peak cytokine expression at these time-points after LPS-stimulation of macrophages. The absorbance values at 450 nm were determined using a ELx800 UV universal microplate reader (BioTek Instruments, Winooski, VT), Digiread software, and SOFTmax® PRO software (version 4.3; Molecular Devices Corp., Sunnyvale, CA).

Supernatants were collected from RAW 264.7 cells and BMDMs for detection of nitrite in solution as an indicator of NO production using Griess reagent purchased from Sigma Aldrich, as per the manufacturer’s instructions. Cells were seeded in a 24-well plate and left overnight. Cells were then treated with 500 ng/mL of LPS alone or with LPS in combination with 5 $\mu$M lactoferricin (bovine, mouse, or human) for 24 h. Supernatants were then collected and added to an equal volume of Griess reagent in a 96-well plate. Sodium nitrite purchased from Sigma Aldrich was used to make a standard curve. Plates were then incubated at room temperature in the absence of light. Absorbance values at 570 nm were determined using a ELx800 UV universal microplate reader (BioTek Instruments, Winooski, VT), Digiread software, an d SOFTmax® PRO software (version 4.3; Molecular Devices Corp., Sunnyvale, CA).

Previous experiments established that peak cytokine mRNA expression occurred 4 h after LPS-stimulation of macrophages. RNA was harvested using a RNeasy Mini Kit purchased from Qiagen (Valencia, CA) and stored at –80 ${}^{\circ }$C for the purpose of cDNA synthesis. Approximately 500 ng RNA was then reverse transcribed using an iScript${}^{TM}$ cDNA synthesis kit (Bio-Rad Laboratories; Hercules, CA) according to the manufacturer’s instructions. The iScript reaction mix (2 $\mu$L) and iScript reverse transcriptase (0.5 $\mu$L) was added to RNA template and nuclease-free water to a final volume of 10 $\mu$L and final concentration of RNA template 50 ng/$\mu$L. The reaction was incubated in a Bio-Rad T100${}^{TM}$ Thermocycler using the following reaction protocol: 5 min at 25 ${}^{\circ }$C, 30 min at 42 ${}^{\circ }$C, and 5 min at 85 ${}^{\circ }$C. Once synthesized, the cDNA was stored at –20 ${}^{\circ }$C for future use.

SsoFast EvaGreen${}^{TM}$ Supermix® (Bio-Rad Laboratories) was used for the q-PCR. The cDNA samples were diluted 1:4 in pyrogen-free water. Primers were optimized (data not shown) before primer mixes of 100 nM for glyceraldehyde-3phosphate dehydrogenase (GAPDH), TNF-$\alpha$, and iNOS (Table 2) (Integrated DNA Technologies; Skokie, IL) were made from 10 $\mu$L of both the forward and reverse primers added to 80 $\mu$L of water. A 1 $\mu$L sample of diluted cDNA was then added to a master mix solution containing 5 $\mu$L EvaGreen Supermix, 3 $\mu$L pyrogen-free water, and 1 $\mu$L primer mix in a final volume of 10 $\mu$L. Negative controls did not contain any cDNA. Reactions were conducted in triplicate using a Stratagene Mx3005p q-pcr system (Agilent Technologies, Santa Clara, CA) and a Rotor-Gene 6000 q-PCR machine (Qiagen, Valencia, CA). Cycling conditions consisted of a 30 s activation step at 95 ${}^{\circ }$C, followed by 40 amplification cycles for 5 s at 95 ${}^{\circ }$C and 30 s at an annealing temperature specific to each primer set used (refer to Table 2). To confirm that the PCR reaction had produced the specific and intended products, a melt curve analysis was conducted using MxPro q-PCR Software (Agilent Technologies, Santa Clara, CA) cycle threshold (CT) values, indicating the number of cycles it takes for the fluorescent signal to surpass the background fluorescence. The relative amounts of amplicons were determined by normalizing the CT values of the target gene to the endogenous control, GAPDH. These values were then normalized to the untreated control values, giving the expression fold values.

THP-1 cells, RAW 264.7 cells, and BMDMs treated with medium only, bovine, mouse, or human lactoferricin alone [5 $\mu$M], LPS [100 ng/mL], or a combination of lactoferricin peptides and LPS were lifted from tissue culture flasks using 5 mL of 10 mM EDTA and a cell scraper and collected in tubes that were centrifuged at 500 $\times$ g for 5 min. Cells were lysed in ice-cold RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 50 mM Na${}_2$HPO${}_4$, 0.25% sodium deoxycholate [w/v], 0.1% Nonidet P-40 [v/v], 5 mM ethylenediaminetetraacetic acid, and 5 mM ethyleneglycoltetraacetic acid) supplemented with protease and phosphatase inhibitors (1 mM Na${}_3$VO${}_4$, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 $\mu$g/mL aprotinin, 1 $\mu$g/mL leupeptin, and 1 $\mu$g/mL pepstatin) on ice for 15 min. Samples were centrifuged at 14,000 $\times$g for 10 min to remove cell debris. Supernatants containing protein were then collected and stored at –80 ${}^{\circ }$C for future use. A Bradford assay (Bio-Rad, Hercules, CA, United States) was used to quantify total protein concentrations from each sample. The proteins in each sample were then denatured by the addition of sample buffer (200 mM Tris-HCl [pH 6.8], 30% glycerol [v/v], 6% sodium dodecyl sulfate [w/v], 15% $\beta$-mercaptoethanol [v/v], and 0.001% bromophenol [w/v]) and placed in a heating block at 95 ${}^{\circ }$C for 5 min. If not used immediately, samples were stored at –80 ${}^{\circ }$C until future use. Equal amounts of protein sample (10 $\mu$g) were loaded onto Tris-HCl acrylamide resolving gels and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to a nitrocellulose membrane using the iBlot® dry blotting system (Life Technologies, Burlington, ON). Membranes were washed with Tris-buffered saline (TBS)-Tween-20 (TBST; 20 mM Tris-HCl [pH 7.6], 200 mM NaCl, 0.05% Tween-20 [v/v]) and blocked in TBST containing 5% fat-free milk powder [w/v] for 1 h at room temperature or overnight at 4 ${}^{\circ }$C with gentle rocking.

Membranes were washed and exposed to the primary antibody for 1 h at room temperature or overnight at 4 ${}^{\circ }$C with gentle rocking. Membranes were washed and then exposed to the HRP-conjugated secondary antibody (1:1000 in TBST blocking solution) for 1 h at room temperature with gentle rocking. Membranes were washed and reacted with Luminata${}^{TM}$ Forte Western HRP Substrate (Millipore; Taunton, MA), exposed to X-ray film (Sci-Med Inc.; Truro, NS), and then developed using a Kodak X-OMAT 1000A automated X-ray developer. In order to account for any variation of loading between protein samples, membranes were re-probed for $\beta$-actin. Image Studio${}^{TM}$ Software (LI-COR®; Guelph, ON) was used to determine the relative intensity of each band through densitometry. The ratio of actin normalized to phospho-protein was compared to that of total protein normalized to phospho-protein and subsequently normalized to the medium control.

Imaging of cells stained with fluorescent antibodies was used to determine nuclear localization of the inflammation-associated transcription factor NF-$\kappa$B. Cells were grown on coverslips that were placed in a 12-well plate, left for 24 h, serum-starved overnight, and treated with peptide and LPS for 4 h. Medium was removed and cells were fixed with 4% paraformaldehyde. Coverslips were washed in PBS and allowed to dry overnight. Slides were blocked and incubated with anti-p65 antibody diluted 1:100 in antibody dilution buffer. From this point on, all incubations were performed in the absence of light. Cells were washed in PBS, incubated in goat-anti-rabbit secondary antibody conjugated to Alexa Fluor ® 488 diluted 1:500 in antibody dilution buffer, washed again, and incubated in 30 $\mu$M 4’,6-diamidino-2-phenylindole (DAPI) diluted 1:100 in 1xPBS. Coverslips were lifted from the plate. Dako Fluorescent Mounting Medium was applied, and each coverslip was mounted to individual Fisherbrand Superfrost® Plus slides. Visualization of slides was done using a Zeiss Axioplan II Motorized Microscope (Zeiss Canada, North York, ON) and AxioVision 4.8 Microscopy Software.

A one-way analysis of variance (ANOVA) with a Dunnett’s post-test was conducted using GraphPad Prism Software (GraphPad Software Inc.; La Jolla, CA). Data were considered significantly different when the p value was less than 0.05 (indicated by *); when the p value was greater than 0.05, data were considered to not be statistically significant.

The ability of bovine, mouse and human lactoferricin peptides to downregulate pro-inflammatory cytokine mRNA expression by LPS-stimulated macrophages was investigated in a series of q-PCR experiments. A 5 $\mu$M concentration of each lactoferricin peptide dampened LPS-induced TNF-$\alpha$ mRNA expression in RAW 264.7 macrophages (Fig. 1A; p 0.01) and THP-1 macrophages (Fig. 1B; p 0.05). However, in BMDMs, only bovine lactoferricin downregulated LPS-induced TNF-$\alpha$ mRNA expression (Fig. 1C; p 0.05).

Fig. 1.

Species-specific lactoferricin peptides decrease LPS-induced TNF-$\alpha$ and IL-6 cytokine production in macrophages. (A, D, and G) RAW 264.7 cells, (B, E, and H) THP-1 cells, and (C, F and I) BMDMs were treated with 100 ng/mL LPS alone or in combination with 5 $\mu$M bovine, mouse, or human lactoferricin. RNA was isolated for cDNA synthesis and then qPCR was performed to determine expression levels of TNF-$\alpha$, 4 h after treatment, relative to untreated control treatments (A–C) (calculated by dividing the expression value for the lactoferricin treated cultures by the untreated control and multiplied by 100). Supernatants were collected and used in ELISA assays to detect TNF-$\alpha$ (D–F) 6 h after treatment and IL-6 (G–I) 24 h after treatment. Levels of TNF-$\alpha$ production are relative to untreated control treatments (calculated by dividing the expression value for the lactoferricin treated cultures by the untreated control and multiplied by 100). Data are the mean of 3–4 independent experiments $\pm$ SEM. * denotes p 0.05 compared to LPS positive control as determined by ANOVA with Dunnett’s multiple comparisons post-test. a.u. denotes arbitrary units.

In line with the mRNA expression findings, ELISA analysis of 24 h culture supernatants demonstrated that 5 $\mu$M bovine lactoferricin significantly reduced the production of TNF-$\alpha$ following LPS stimulation of RAW 264.7 cells, THP-1 cells, and BMDMs comparison to LPS treatment alone (Fig. 1D–F). Mouse and human lactoferricin also decreased LPS-induced TNF-$\alpha$ production by THP-1 cells (Fig. 1E), but did not affect TNF-$\alpha$ production by LPS-stimulated RAW 264.7 cells or BMDMs (Fig. 1D–F). Representative TNF-$\alpha$ concentrations from RAW 264.7 cells are as follows: no treatment, 63 pg/mL; LPS alone, 3120 pg/mL; bovine lactorferricin plus LPS, 1853 pg/mL.

We next asked whether lactoferricin peptides affected LPS-stimulated IL-6 production by RAW 264.7 cells, THP-1 cells, and BMDMs. ELISA analysis of 24 h culture supernatants from LPS-stimulated RAW 264.7 cells, THP-1 cells, and BMDMs that were treated with bovine lactoferricin showed a significant decrease in IL-6 production in comparison to the untreated control (Fig. 1G–I, p 0.05). Neither human nor mouse lactoferricin affected IL-6 production by LPS-stimulated RAW 264.7 cells or BMDMs. All three lactoferricin peptides decreased LPS-induced IL-6 production by THP-1 cells (Fig. 1H, p 0.05). Lactoferricin peptides had a similar effect on IL-6 mRNA expression by LPS-stimulated macrophages (data not shown). Lactoferricin treatment without LPS stimulation did not influence the production of TNF-$\alpha$ or IL-6 by RAW 264.7 cells, THP-1 cells, or BMDMs (data not shown).

To determine if lactoferricin peptides inhibited the LPS-stimulated production of NO, RAW 264.7 cells and BMDMs were stimulated with 500 ng/mL LPS in the absence or presence of different lactoferricin peptides for 24 h. Culture supernatants were tested for the presence of NO using Griess reagent. A significant decrease in LPS-induced NO production was observed in RAW 264.7 cells treated with either bovine or human lactoferricin (Fig. 2A, p 0.01). A decrease in NO production upon LPS-stimulation was observed with the addition of bovine, mouse, or human lactoferricin in BMDMs (Fig. 2B, p 0.05).

Fig. 2.

Lactoferricin peptides decrease nitric oxide production in LPS-stimulated macrophages. (A) RAW 264.7 cells and (B) BMDMs were treated with 500 ng/mL of LPS alone or in combination with 5 $\mu$M bovine, mouse, or human lactoferricin for 24 h. Supernatants were collected and used in a Griess assay to measure nitric oxide production. Data are the mean of 4 independent experiments $\pm$ SEM; * denotes p 0.05 and ** p 0.01 compared to LPS treatment as determined by ANOVA with Dunnett’s multiple comparisons post-test. BDL, below detectable levels.

To determine whether lactoferricin-mediated inhibition of NO was associated with decreased iNOS expression, RAW 264.7 and BMDM cells were treated with 500 ng/mL LPS alone or in combination with 5 $\mu$M bovine, mouse, or human lactoferricin for 4 h prior to RNA isolation. Levels of iNOS expression were normalized to the LPS control since iNOS mRNA was below the level of detection in untreated cells. A significant decrease in LPS-induced iNOS mRNA expression was seen in RAW 264.7 cells treated with either bovine or human lactoferricin (Fig. 3A, p 0.05). When LPS-stimulated BMDMs were treated with either mouse or human lactoferricin there was a significant reduction in iNOS expression in comparison to LPS treatment alone (Fig. 3B, p 0.05).

Fig. 3.

Lactoferricin peptides decrease inducible nitric oxide synthase expression in LPS-stimulated macrophages. (A) RAW 264.7 cells and (B) BMDMs were treated with 500 ng/mL of LPS in combination with 5 $\mu$M bovine, mouse, or human lactoferricin for 4 h. RNA was isolated for cDNA synthesis, and qPCR was performed to determine levels of iNOS expression. Data are expressed as a relative value of LPS induced iNOS expression (iNOS in lactoferricin-treated cells divided by iNOS in cells treated with LPS alone). Data are the mean of 3 independent experiments $\pm$ SEM; * denotes p 0.05 and ** p 0.01 compared to LPS positive control as determined by ANOVA with Dunnett’s multiple comparisons post-test. a.u. denotes arbitrary units.

To determine whether lactoferricin peptides influenced LPS-induced pro-inflammatory signaling cascades, RAW 264.7 and THP-1 cells were treated with LPS in the absence or presence of bovine, mouse, or human lactoferricin, and western blot analysis of cell lysates for phospho-I$\kappa$B$\alpha$ expression was conducted. Bovine lactoferricin reduced LPS-induced phospho-I$\kappa$B$\alpha$ expression in RAW 264.7 cells at 1 h but not at 30 min or 4 h (Fig. 4A). Subsequent western blot analysis was conducted after 1 h incubation with peptide, LPS, or LPS plus peptide. Treatment of RAW 264.7 cells with the combination of LPS and lactoferricin peptides led to a decrease in phospho-I$\kappa$B$\alpha$ expression in comparison to LPS treatment alone, whereas treatment with peptide alone had no significant effect on phospho-I$\kappa$B$\alpha$ expression (Fig. 4B–C p 0.01). In a similar manner to RAW 264.7 cells, there was a significant decrease in THP-1 cell expression of LPS-induced phospho-I$\kappa$B$\alpha$ following treatment with bovine, mouse, or human lactoferricin (Fig. 4D–E, p 0.01).

Fig. 4.

Lactoferricin peptides reduce expression of phosphorylated I$\kappa$B$\alpha$ in LPS-stimulated macrophages. (A) RAW 264.7 cells were treated with the indicated concentrations of bovine lactoferricin and 100 ng/mL LPS for the indicated times. Cell lysates were collected and protein expression was determined by western blotting. Nitrocellulose membranes were probed with the indicated antibodies and corresponding secondary antibodies. Data shown are representative of two independent time-course experiments. (B), (C) RAW 264.7 and (D), (E) THP-1 cells were incubated with 100 ng/mL LPS, 5 $\mu$M bovine, mouse, or human lactoferricin, or a combination of LPS and one species-specific lactoferricin peptide as indicated. Cell lysates were collected after 1 h and used in western blotting as described in (A). Data shown are one representative blot. (B), (D) One representative western blot and (C), (E) the mean density of phosphorylated I$\kappa$B$\alpha$ normalized to untreated controls and to total I$\kappa$B$\alpha$ and $\beta$-actin from 4 (RAW 264.7 cells) or 3 (THP-1 cells) independent experiments $\pm$ SEM; ** denotes p 0.01 and *** denotes p 0.001 compared to LPS alone treatments as determined by ANOVA with Dunnett’s multiple comparisons post-test.

To determine whether lactoferricin peptides inhibited the nuclear translocation of NF-$\kappa$B, RAW 264.7 and THP-1 cells were treated with LPS in the absence or presence of bovine, mouse, or human lactoferricin for 1 h and stained with anti-p65 (a subunit of NF-$\kappa$B) antibody and the nuclear stain DAPI. Cells were visualized using fluorescent microscopy. LPS stimulation of RAW 264.7 and THP-1 macrophages resulted in a prominent nuclear translocation of p65 (Fig. 5A–B). RAW 264.7 cells stimulated with LPS in the presence of bovine, mouse, or human lactoferricin showed reduced nuclear-localized p65 in comparison to the LPS-only control (Fig. 5A). Nuclear translocation of p65 in THP-1 cells was also inhibited by lactoferricin treatment (Fig. 5B). Species-specific lactoferricin treatments therefore inhibited the LPS-induced translocation of NF-$\kappa$B into the nucleus of mouse and human macrophages. Western blot analysis showed that lactoferricin peptides alone did not affect cytosolic p65 levels in LPS-stimulated RAW 264.7 and THP-1 macrophages in comparison to cells treated with LPS alone (data not shown).

Fig. 5.

Lactoferricin peptides inhibit the nuclear translocation of NF-$\kappa$B in LPS-stimulated macrophages. (A) RAW 264.7 cells and (B) THP-1 cells were treated with 100 ng/mL LPS alone or in combination with 5 $\mu$M bovine, mouse, or human lactoferricin for 1 h. Cells were fixed and incubated with nuclear stain, DAPI (30 $\mu$M), and rabbit anti-p65 antibody with secondary goat-anti-rabbit Alexa Fluor 488 ® conjugate, then imaged using fluorescent microscopy. Images are representative of two independent experiments. Scale bar represents 40 $\mu$m.

To determine if lactoferricin peptides target MAPK signaling pathways, western blot analysis was conducted using lysates from RAW 264.7 and THP-1 cells treated with lactoferricin alone, LPS alone, or LPS plus lactoferricin. RAW 264.7 cells treated with either bovine or mouse lactoferricin showed a significant reduction in LPS-induced phospho-ERK 1/2 expression in comparison to LPS treatment alone (Fig. 6A–B, p 0.05). THP-1 cells showed a similar trend of reduced phospho-ERK 1/2, demonstrating a decrease in LPS-induced ERK 1/2 expression with peptide treatment (Fig. 6C–D, p 0.05).

Fig. 6.

Lactoferricin peptides inhibit ERK phosphorylation in LPS-stimulated macrophages. (A), (B) RAW 264.7 and (C), (D) THP-1 cells were incubated with 100 ng/mL LPS, 5 $\mu$M bovine, mouse, or human lactoferricin, or a combination of LPS and one species-specific lactoferricin as indicated. Cell lysates were collected and protein expression was determined using western blotting. Nitrocellulose membranes were probed with the indicated antibodies and the appropriate secondary antibodies. (A), (C) Data shown are from one representative western blot. (B), (D) The mean density of phosphorylated ERK 1/2 normalized to untreated controls and to total ERK 1/2 and $\beta$-actin from 4 (RAW 264.7 cells) or 3 (THP-1 cells) independent experiments $\pm$ SEM.

Western blot analysis of phospho-c-Jun expression in lactoferricin-treated, LPS-stimulated RAW 264.7 cells demonstrated a significant reduction in phospho-c-Jun in comparison to cells treated with LPS alone (Fig. 7A–B, p 0.05). However, LPS-stimulated THP-1 macrophages treated in a similar manner did not show a significant decrease in phospho-c-Jun expression following lactoferricin treatment (Fig. 7C–D).

Fig. 7.

Lactoferricin peptides inhibit c-Jun phosphorylation in LPS-stimulated mouse macrophages. (A), (B) RAW 264.7 and (C), (D) THP-1 cells were incubated with 100 ng/mL LPS, 5 $\mu$M bovine, mouse, or human lactoferricin, or a combination of LPS and one species-specific lactoferricin peptide, as indicated. Cell lysates were collected and protein expression was determined using western blotting. Nitrocellulose membranes were probed with the indicated antibodies and the appropriate secondary antibodies. (A), (C) Data shown are from one representative western blot. (B), (D) The mean density of phosphorylated c-Jun normalized to untreated controls and to total c-Jun and $\beta$-actin from 4 (RAW 264.7 cells) or 3 (THP-1 cells) independent experiments $\pm$ SEM.

To determine whether species-specific lactoferricin peptides were able to downregulate a pro-inflammatory response induced by a stimulus other than LPS, RAW 264.7 and THP-1 cells were treated with CdCl${}_2$ alone or in the presence of bovine, mouse, or human lactoferricin. After 24 h, culture supernatants were collected, and ELISA was used to measure TNF-$\alpha$. As with LPS stimulation, there was a significant decrease in CdCl${}_2$-induced TNF-$\alpha$ production by THP-1 (Fig. 8A, p 0.05) and RAW 264.7 (Fig. 8B–C, p 0.05) cells treated with bovine, mouse, or human lactoferricin. Lactoferricin peptides therefore downregulated pro-inflammatory TNF-$\alpha$ induced by nonmicrobial activation with CdCl${}_2$.

Fig. 8.

Lactoferricin peptides decrease cadmium-induced TNF-$\alpha$ production in macrophages. (A) THP-1 cells and (B) RAW 264.7 cells were treated with 10 $\mu$M CdCl${}_2$ alone or in combination with 5 $\mu$M bovine, mouse, or human lactoferricin, for 24 h. Supernatants were collected and ELISA was used to determine relative levels of TNF-$\alpha$ production. (C) RNA was isolated from RAW 264.7 cells treated with 10 $\mu$M CdCl${}_2$ alone or in combination with 5 $\mu$M bovine, mouse, or human lactoferricin, for 4 h, cDNA was synthesized and q-PCR was performed to determine relative levels of TNF-$\alpha$ expression normalized to untreated control. Data show are the mean of 3-5 independent experiments $\pm$ SEM. ELISA data was normalized to untreated controls: 108 pg/mL and 118 pg/mL, respectively; * denotes p 0.05 compared to LPS control as determined by ANOVA with the Dunnett’s multiple comparisons post test; a.u. denotes arbitrary units.