Comparative proteomic analysis of growth hormone secretagogue A233 treatment of murine macrophage cells J774A.2 indicates it has a role in antiviral innate response

Background

Growth hormone secretagogues (GHS), among other factors, regulate the release of GH. The biological activity of the secretagogue peptide A233 as a promoter of growth and innate immunity in teleost fish has previously been demonstrated, but its role in the immune system of mammals is not well understood.

Methods

The effect of the peptide was investigated in J774A.2 macrophage cells using a comparative proteomics approach after 6 and 12 h of peptide stimulation.

Results

The functional analysis of differentially modulated proteins showed that A233 peptide treatment appears to promote activation and ROS-dependent cytotoxic functions in macrophages and enhanced expression of antiviral protein complexes such as MAVS. In accordance with this hypothesis, we found that A233 treatment enhanced superoxide anion production and the IFN-γ level in J774A.2 cells and mouse splenocytes, respectively, and reduced viral load in a dengue virus mouse model of infection.

Conclusions

The growth hormone secretagogue A233 peptide promotes activation of ROS-dependent cytotoxic functions and exerts immunomodulatory effects that enable an antiviral state in a dengue virus mouse model.

General Significance

The increase of IFN-γ level and the differential modulation of antiviral proteins by the A233 peptide suggest that the molecule could activate an innate immune response with a possible further impact in the treatment of acute and chronic diseases.

Abbreviations: DENV, dengue virus; GHRP-6, growth hormone releasing peptide-6; [d-Lys3]-GHRP-6t, ghrelin receptor antagonist; GHS, growth hormone secretagogue; IFN-γ, interferon gamma; LPS, lipopolysaccharide; O₂^•−, superoxide anion; RNS, reactive nitrogen species; ROS, reactive oxygen species

Keywords: Comparative proteomics, Growth Hormone Secretagogue, ROS production, Antiviral activity

2.1. Peptide synthesis

The decapeptide A233 (GKFDLSPEHQ), with a lactam bond between side chain functional groups of Lys² and Asp⁴, was manually synthesized on Fmoc-AM-PEG-PS resin (substitution level: 0.18 mmol/g) using the Fmoc/tBu strategy. All side-chains were protected with TFA-labile groups and the side-chain protections used for Lys and Asp were allyloxycarbonyl (Alloc) and allyl (All), respectively. Removal of the Fmoc group was carried out with 20% piperidine in DMF and the Fmoc-amino acids were coupled using DIC/HOBt activation. Once the complete peptide sequence had been assembled, and maintaining the Fmoc-Nα-protection, the Alloc-All groups were removed by treatment with phenylsilane and tetrakistriphenylphosphine palladium (0). The lactam bond was obtained by treatment of the peptide-resin with PyBOP/DIEA in DMF. Side-chain deprotection and cleavage from the resin were carried out with TFA-H₂O-TIS (95:2.5:2.5 v/v). The peptide was purified by reversed-phase high performance liquid chromatography (RP-HPLC). The A233 peptide was obtained with purity higher than 95%, as estimated by analytical RP-HPLC. The identity of the A233 was verified by ESI-MS analysis. The experimental (1137.58 Da) and calculated (1137.56 Da) monoisotopic molecular mass agreed, considering the formation of the lactam bond previously described. The ESI-MS/MS spectrum of the doubly-charged ion of A233 peptide (m/z 569.79, 2+) was acquired to obtain structural evidence of the lactam bond formation between Lys² and Asp⁴ from the backbone and internal fragment ions. The positive control GHRP-6 (Lipotec, Barcelona, Spain) and its antagonist [d-Lys³]-GHRP-6 (Sigma), both lyophilized, were reconstituted in PBS for use in in vitro and in vivo assays.

2.2. Reverse phase chromatography.

A233 peptide was analyzed on an AKTA 100 (GE Healthcare, USA) HPLC system. Analytical analysis was accomplished in a reverse-phase (RP) C18 analytical column (4.6 × 150 mm, 5 μm) (Vydac, USA). To assess the peptide purity, the column was equilibrated in 95% solution A, then a linear gradient from 5% to 60% solution B, over 35 min, was used keeping a flow rate of 0.8 mL/min. The solutions A and B used in the purification were 0.1% of TFA in water, and 0.05% of TFA in pure acetonitrile, respectively. Chromatograms were acquired at 226 nm, using the UNICORN 4.11 (GE Healthcare, USA) software package for data processing of the RP-HPLC chromatograms. The semipreparative purification was performed on a LaChrom (Merck-Hitachi, Germany) HPLC system. Separation was achieved using an RP C18 preparative column (Vydac, 25 × 250 mm, 25 μm). A linear gradient from 15% to 45% of solvent B over 50 min and a flow rate of 5 mL/min were used. Solvents A and B are composed of 0.1% (v/v) of TFA in water and 0.05% (v/v) of TFA in acetonitrile, respectively. Detection was monitored at 226 nm.

2.3. Mass spectrometric analysis.

ESI-MS analysis was performed in a hybrid quadrupole-time-of-flight (QTOF-2™) tandem mass spectrometer (Waters, Milford, MA, USA) equipped with a Z-nanospray ion source. Capillary and cone voltages were set to 900 and 35 V, respectively. A 5 μL aliquot of the peptide purified by RP-HPLC was loaded into a glass capillary coated with conductive material (New Objective, Woburn, MA, USA). The tip of the capillary was gently broken, while slightly pressurized with nitrogen, in order to get a stable spray during the measurement. The TOF and MCP were operated at 2.2 kV and 9 kV, respectively. The source was heated at 90 °C. The doubly-charged ion of the A233 peptide (m/z 569.79, 2+) was selected in the first quadrupole and fragmented inside the collision cell operated at collision energy of 35 eV to obtain an MS/MS spectrum with structural information. Ultrapure Argon (Gases Industriales, Cuba) was used as a collision gas. Data acquisition and processing were performed with the MassLynx (v. 4.0) software package (Waters, USA).

2.4. Growth conditions for J774.2 cells

The J774A.2 cell line was obtained from ECACC (85011428 Sigma). The cells were cultured as monolayers in Dulbecco's Modified Essential Medium (DMEM) F12 supplemented with 20% fetal bovine serum (FBS), 100 mg/mL penicillin/streptomycin, and 2 mM l-glutamine (Mediatech, Inc., Herndon, VA) at 37 °C in a humidified atmosphere of 5% CO₂. The cultured cells were grown to 70–80% confluence in 75 cm² flasks for preparation of samples that were used in further analyses.

2.5. Superoxide production by J774.2 cells

Superoxide anion production was measured in J774.2 cells by the reduction of nitro blue tetrazolium (NBT; Sigma, USA) following the procedure described by Yada et al. [10].

2.6. IFN-γ production in mouse splenocytes

Mouse splenocytes were obtained in aseptic conditions, and erythrocytes were lysed by adding 0.83% NH₄Cl solution. Cells were washed twice with PBS containing 2% FBS (PAA Laboratories, ON, Canada) and resuspended at 2 × 10⁶ cells/mL in RPMI 1640 medium (Sigma Aldrich, USA) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco, UK), 2 mM glutamine (Gibco), 5 × 10⁻⁵ M 2-mercaptoethanol (Sigma, USA) and 5% FBS. Finally, 2.5 × 10⁵ cells per well were cultured in 96-well round-bottom plates with the peptides at a final concentration of 10 μg/mL. Concanavalin-A (Sigma) was used as a positive control. In all experiments, three wells were plated for each antigen. After 4 days of culture, culture supernatants were collected and stored at −20 °C. IFN-γ was determined by ELISA (as recommended by Mabtech) in duplicate samples from culture supernatants.

2.7. Reduction and S-alkylation

Tubes containing proteins from each of the three experimental conditions (control at 0 h, and 6 h or 12 h of A233 treatment) received 45 µL of 50 mM Hepes, 2 M GuCl, pH 8.5 and the mixtures were reduced by adding dithiothreitol (DTT) to a final concentration of 10 mM and incubating the tubes for 4 h at 37 °C. The free thiol groups were alkylated by adding iodoacetamide to a final concentration of 20 mM, and allowing the reactions to proceed (in darkness) for an additional 30 min at room temperature.

2.8. Proteolytic digest

Hepes, pH 8.5 (50 mM) was added to 100 µL final volume, and proteins were digested with Lysyl-endopeptidase (Wako, Japan) using an enzyme:substrate ratio of 1:100 for 4 h at 37 °C. After digestion, the peptide mixture was diluted with water up to 0.5 mol/L guanidium hydrochloride. Sequencing grade trypsin (Promega, USA) was added to a concentration of 1:100 (enzyme: substrate), and the reaction proceeded for 16 h at 37 °C.

2.9. Mouse experiments and viral load detection in brain

Female BALB/c (Bc, H-2^d) mice (aged 6–8 weeks) were purchased from CENPALAB (Havana, Cuba) and housed in appropriate animal care facilities during the experimental period. Mouse experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Center for Genetic Engineering and Biotechnology (CIGB), Cuba. The protocols were approved by the CIGB Committee on Ethics for Animal Experiments.

Two groups of eight mice were injected intraperitoneally on days 0, 1, 3, 4, 6 and 7 with 10 µg of peptide A233 per animal or PBS (control) without adjuvant in a final volume of 100 μL. A third group was injected with the [d-Lys³]-GHRP6 antagonist peptide using the same schedule. As a positive control for the challenge with dengue virus (DENV), a fourth group of animals was inoculated with 10⁵ plaque-forming units (pfu) of an infectious preparation of DENV-3 (H87 strain, 27 passages in mice) on day 0.

On day 8, animals were challenged with 50 LD₅₀ of neuroadapted DENV-3 H87 by intracranial injection. Seven days after challenge, mice were euthanized to measure the viral load in brain as previously reported [11]. Brains were harvested and aseptically homogenized with 1 mL of PBS supplemented with 2% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. The suspensions were centrifuged for 10 min at 8000g, and supernatants were collected and stored at −80 °C.

For viral load measurement, 5×10⁵ Vero cells/well were cultured for 24 h in six-well plates with MEM (Sigma Aldrich) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine, 1 mM sodium pyruvate (Gibco, UK), and 2% FBS. Cells were infected with different dilutions of the infected brain supernatants in MEM medium supplemented with 1% FBS. After infection (4 h at 37 °C and 5% CO₂), the overlay medium was added (3% carboxymethylcellulose), and the plates were incubated at 37 °C and 5% CO₂ for 4 days. Infected cells were visualized by immunofocusing. Briefly, the cells were fixed with 4% formalin for 1 h, washed twice with PBS, and then incubated in PBS-1% Triton X-100 for 15 min. After permeabilization, blocking buffer (PBS supplemented with 10% FBS) was added to each well for 30 min at room temperature. After blocking, the cells were incubated with monoclonal antibody 4G2 (which recognizes the envelope protein of DENV) at 1 µg/mL (in PBS and 1% FBS) for 1 h at room temperature. After two washes with PBS, polyclonal anti-mouse IgG-HRP (Sigma Aldrich, USA) was added at a dilution of 1:2000 (in PBS and 1% FBS) and incubated for 1 h at room temperature. The cells were then washed twice with 50 mM Tris, pH 7.4, and a substrate solution of 0.5 mg/mL diaminobenzidine, 0.2 mg/mL CaCl₂, and 9 mg/mL NaCl in 50 mM Tris, pH 7.4, was added to each well. The plates were left on a shaker for 30 min until clear foci were visible for subsequent counting. The detection limit was 10 pfu/mL.

2.10. Statistical analysis

For statistical analysis, GraphPad Prism version 5.0 for Windows (GraphPad Software, San Diego, CA) was used. The normal distribution of data was analyzed with D'Agostino Pearson's test and the variance homogeneity with Bartlett's test. When normal or transformed data showed a normal distribution, a parametric test was performed; otherwise data were analyzed with a non-parametric test. The parametric analysis of more than two groups was performed by a one-way analysis of variance, using Newman–Keuls post-test. The non-parametric analysis of more than two groups was performed using Kruskal–Wallis and Dunn multiple comparison test. In all cases *: p<0.05; **: p<0.01; ***: p<0.001.

2.11. Bioinformatics analysis

The functional analysis of the differentially modulated proteins was performed in the context of classical macrophage activation. First, the function of the differentially modulated proteins was extracted from UniProtKB database [12] and the scientific literature. Information from the literature concerning macrophage activation and its relation with the differentially modulated proteins was explored with the information retrieval and text mining tool GoPubMed [13]. Next, an enrichment analysis of the differentially modulated proteins, at both 6 and 12 h and at each time separately, was performed based on the following biological annotations: GO biological process, GO molecular function, KEGG pathway, transcription factor binding site (TFBS), and mammalian phenotype ontology (MPO) [14]. GeneCodis [15] was used for GO annotations and KEGG pathway enrichment analyses, InnateDB [16] for TFBS enrichment analysis, and NetVenn [17] for MPO enrichment analysis. Default parameters were kept and the significance level was set at p<0.05.

Table 1.

Differentially modulated proteins at 6 and/or 12 h A233 peptide treatment that might be relevant for the effect on macrophages.

3.1. A233 treatment increases superoxide anion levels in J774.2 cells

Peptide treatment increases O2•− (superoxide anion) levels in tilapia head–kidney leukocyte cultures [2]. Together, the GO biological process and the molecular function enrichment analyses indicate an impact on oxidation–reduction processes after 6 h of treatment, with the differential up-regulation of superoxide dismutase Sod1 and Prdx6 peroxidase (Fig. 1, Supplemental Table 2). Prdx6 is primarily a phospholipid peroxidase that protects membranes from oxidation and is considered to be a cytoprotective enzyme; it also contains phospholipase A2 activity required for the activation of NADPH oxidase NOX2, and thus for ROS generation [20], [21].

Activated M1 macrophages mediate their cytotoxic and pro-inflammatory effects by producing ROS and RNS such as O2•− and NO (nitric oxide), respectively [22]. ROS generation induces cell damage, for example, by reacting with NO to yield ONOO⁻ (peroxynitrite anion) [23]. Sod1 and Prdx6 up-regulation might then suggest an increase in ROS production, while avoiding associated oxidative stress damage. Correspondingly, the effect of the GHS A233 and GHRP-6 on O2•− production was determined, demonstrating a significant increase of O2•− levels compared with the control in both cases (Fig. 2). Therefore, A233 treatment might potentiate macrophage activation and cytotoxic functions, as ROS production has been reported to have role in macrophage differentiation [24].

3.2. A233 treatment differentially modulates DNA repair and apoptosis related proteins

Activated macrophages are protected against ROS induced oxidative damage by showing, for instance, an enhancement of DNA repair and resistance to apoptosis [25]. Apoptosis is an ordered cascade of enzymatic events that culminates in cell death and the cleavage of DNA into characteristic nucleosomal fragments. Since peptide treatment increases O2•− levels, we investigated the modulation of these events. The GO biological process enrichment analysis highlighted the differential up-regulation of DNA repair proteins at 6 and/or 12 h post-treatment (Fig. 1, Supplemental Table 2). For example, the differentially modulated protein Nabp1 is a component of the SOSS complex required for efficient homologous recombination-dependent repair of double-strand breaks and ATM-dependent signaling pathways [26], [27]. Therefore, these findings indicate that DNA repair enhancement and resistance to apoptosis in J774.2 cells might be potentiated by A233 peptide treatment, which might promote survival under conditions with high-ROS levels.

3.3. A233 treatment differentially modulates STAT1 target proteins

The activation of the transcription factors NF-κB, STAT1 and AP-1 by stimuli like LPS, and/or IFN-α/β/γ induces the expression of inflammatory and microbicidal M1 genes. To investigate their activation in the A233 peptide-stimulated cells, we conducted a TFBS enrichment analysis at 6 and 12 h post-treatment and at each time separately. Noticeably, we detected a significant enrichment of the STAT1 targets at 6 and/or 12 h post-treatment, including Eef1g, Fabp4, Hadhb, Hk3, yar, Mcm3, Phb, Ptcd3,and Rtn3 (Supplemental Table 2).

In particular, the differentially up-regulated protein Fabp4 (Fig. 1, Table 1) is a target gene of the STAT1 transcription factor (Supplemental Table 2). The expression of Fabp4 in macrophages is induced by a pro-inflammatory environment, and its decrease reduces NF-κB activation and the levels of the inflammatory cytokines Cox2 and iNOS [29], [30]. This protein also displays an antioxidant activity against H₂O₂ in vitro [31]. The increase in Fabp4 protein may thus link peptide treatment with the establishment of a pro-inflammatory phenotype by responding to STAT1 and inducing NF-κB activation.

3.4. A233 treatment increases IFN-γ levels and induces an antiviral response

As suggested by the TFBS enrichment analysis, the A233 peptide may activate the STAT1 signaling pathway. IFN-γ polarizes macrophages to an M1 state through STAT1 activation, while deletion of STAT1 compromises viral immunity [32]. Activated STAT1 is transported to the nucleus, where it induces the expression of ROS-generating enzymes, iNOS and pro-inflammatory cytokines [33]. Therefore, we measured IFN-γ levels in mouse splenocytes stimulated with the peptide. As suspected, A233 treatment significantly increased IFN-γ levels (5099.73±86.16 pg/mL) compared to the control cells (558.05±26.48 pg/mL). Also, the stimulation with the antagonist peptide [d-Lys³]-GHRP6 induced a low cytokine secretion (430.45±7.22 pg/mL) (Fig. 3).

Considering that this cytokine is a mediator of antiviral activity against DENV [34], we evaluated the antiviral capacity of A233 peptide in a mouse model of DENV infection. This model, based on the intracranial inoculation of high viral doses of mouse-adapted strains, has previously been used to evaluate the efficacy of antiviral drugs against DENV [35], [36]. Our results reveal a significant reduction in viral load in treated mice after intracranial challenge with DENV-3 (161.43±29.71 pfu/mL), compared with the control group (714.29±34.87 pfu/mL) (Fig. 4). A similar outcome was observed after challenge with the DENV-2 NGC strain (data not shown). The administration of an antagonist [d-Lys³]-GHRP-6 peptide did not reduce viral load after challenge (765.71±57.54 pfu/mL) (Fig. 4). Further experiments could be conducted in mice and non-human primates to evaluate the efficacy of the molecule against infectious and non-infectious diseases.

On the other hand, peptide treatment of J774.2 cells differentially up-regulates the peroxisomal and mitochondrial protein MAVS required for innate immune defense against viruses (Fig. 1, Table 1). The mitochondrial isoform of MAVS activates an IFN-dependent signaling pathway that amplifies and stabilizes the antiviral response through the expression of type I IFNs and of other antiviral cytokines [37]. The differential up-regulation of MAVS, the increase in IFN-γ levels, and the protection against DENV infection indicates an anti-viral effect of A233 peptide treatment. Other mouse model studies examining mechanisms of antiviral innate immunity against DENV reveal an essential role for MAVS [38] and IRF-3/7 [39].

Supplementary material

Supplementary material

Supplementary material

• 1.Unniappan S., Peter R.E. Structure, distribution and physiological functions of ghrelin in fish. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 2005;140:396–408. doi: 10.1016/j.cbpb.2005.02.011. [DOI] [PubMed] [Google Scholar]
• 2.Martinez R., Ubieta K., Herrera F., Forellat A., Morales R., de la Nuez A., Rodriguez R., Reyes O., Oliva A., Estrada M.P. A novel GH secretagogue, A233, exhibits enhanced growth activity and innate immune system stimulation in teleosts fish. J. Endocrinol. 2012;214:409–419. doi: 10.1530/JOE-11-0373. [DOI] [PubMed] [Google Scholar]
• 3.Dixit V.D., Schaffer E.M., Pyle R.S., Collins G.D., Sakthivel S.K., Palaniappan R., Lillard J.W., Taub D.D. Ghrelin inhibits leptin- and activation-induced proinflammatory cytokine expression by human monocytes and T cells. J. Clin. Invest. 2004;114:57–66. doi: 10.1172/JCI21134. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 4.Waseem T., Duxbury M., Ito H., Ashley S.W., Robinson M.K. Exogenous ghrelin modulates release of pro-inflammatory and anti-inflammatory cytokines in LPS-stimulated macrophages through distinct signaling pathways. Surgery. 2008;143:334–342. doi: 10.1016/j.surg.2007.09.039. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 5.Bujold K., Rhainds D., Jossart C., Febbraio M., Marleau S., Ong H. CD36-mediated cholesterol efflux is associated with PPARgamma activation via a MAPK-dependent COX-2 pathway in macrophages. Cardiovasc. Res. 2009;83:457–464. doi: 10.1093/cvr/cvp118. [DOI] [PubMed] [Google Scholar]
• 6.Demers A., Rodrigue-Way A., Tremblay A. Hexarelin Signaling to PPARgamma in Metabolic Diseases. PPAR Res. 2008;2008:364784. doi: 10.1155/2008/364784. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 7.Biswas S.K., Mantovani A. Orchestration of metabolism by macrophages. Cell. Metab. 2012;15:432–437. doi: 10.1016/j.cmet.2011.11.013. [DOI] [PubMed] [Google Scholar]
• 8.Mantovani A., Sica A., Sozzani S., Allavena P., Vecchi A., Locati M. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol. 2004;25:677–686. doi: 10.1016/j.it.2004.09.015. [DOI] [PubMed] [Google Scholar]
• 9.Martinez F.O., Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Rep. 2014;6:13. doi: 10.12703/P6-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 10.Yada T., Kaiya H., Mutoh K., Azuma T., Hyodo S., Kangawa K. Ghrelin stimulates phagocytosis and superoxide production in fish leukocytes. J. Endocrinol. 2006;189:57–65. doi: 10.1677/joe.1.06187. [DOI] [PubMed] [Google Scholar]
• 11.Gil L., Marcos E., Izquierdo A., Lazo L., Valdés I., Ambala P., Ochola L., Hitler R., Suzarte E., Álvarez M., Kimiti P., Ndung’u J., Kariuki T., Guzmán M.G., Guillén G., Hermida L. The protein DIIIC-2, aggregated with a specific oligodeoxynucleotide and adjuvanted in alum, protects mice and monkeys against DENV-2. Immunol. Cell. Biol. 2015;93:57–66. doi: 10.1038/icb.2014.63. [DOI] [PubMed] [Google Scholar]
• 12.Magrane M., Consortium U. UniProt Knowledgebase: a hub of integrated protein data. Database (Oxford). 2011;2011 doi: 10.1093/database/bar009. bar009. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 13.Doms A., Schroeder M. GoPubMed: exploring PubMed with the Gene Ontology. Nucleic Acids Res. 2005;33:W783–W786. doi: 10.1093/nar/gki470. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 14.C.L. Smith, J.T. Eppig, The mammalian phenotype ontology: enabling robust annotation and comparative analysis, Wiley Interdiscip. Rev. Syst. Biol. Med., 1 (n.d.), 2009, 390–399. [DOI] [PMC free article] [PubMed]
• 15.Tabas-Madrid D., Nogales-Cadenas R., Pascual-Montano A. GeneCodis3: a non-redundant and modular enrichment analysis tool for functional genomics. Nucleic Acids Res. 2012;40:W478–W483. doi: 10.1093/nar/gks402. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 16.Breuer K., Foroushani A.K., Laird M.R., Chen C., Sribnaia A., Lo R., Winsor G.L., Hancock R.E.W., Brinkman F.S.L., Lynn D.J. InnateDB: systems biology of innate immunity and beyond--recent updates and continuing curation. Nucleic Acids Res. 2013;41:D1228–D1233. doi: 10.1093/nar/gks1147. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 17.Wang Y., Thilmony R., Gu Y.Q. NetVenn: an integrated network analysis web platform for gene lists. Nucleic Acids Res. 2014;42:W161–W166. doi: 10.1093/nar/gku331. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 20.Fisher A.B. Peroxiredoxin 6: a bifunctional enzyme with glutathione peroxidase and phospholipase A2 activities. Antioxid. Redox Signal. 2011;15:831–844. doi: 10.1089/ars.2010.3412. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 21.Chatterjee S., Feinstein S.I., Dodia C., Sorokina E., Lien Y.-C., Nguyen S., Debolt K., Speicher D., Fisher A.B. Peroxiredoxin 6 phosphorylation and subsequent phospholipase A2 activity are required for agonist-mediated activation of NADPH oxidase in mouse pulmonary microvascular endothelium and alveolar macrophages. J. Biol. Chem. 2011;286:11696–11706. doi: 10.1074/jbc.M110.206623. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 22.Xia Y., Zweier J.L. Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages. Proc. Natl. Acad. Sci. USA. 1997;94:6954–6958. doi: 10.1073/pnas.94.13.6954. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 23.Szabó C., Zingarelli B., O’Connor M., Salzman A.L. DNA strand breakage, activation of poly (ADP-ribose) synthetase, and cellular energy depletion are involved in the cytotoxicity of macrophages and smooth muscle cells exposed to peroxynitrite. Proc. Natl. Acad. Sci. USA. 1996;93:1753–1758. doi: 10.1073/pnas.93.5.1753. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 24.Covarrubias A., Byles V., Horng T. ROS sets the stage for macrophage differentiation. Cell. Res. 2013;23:984–985. doi: 10.1038/cr.2013.88. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 25.Bauer M., Goldstein M., Christmann M., Becker H., Heylmann D., Kaina B. Human monocytes are severely impaired in base and DNA double-strand break repair that renders them vulnerable to oxidative stress. Proc. Natl. Acad. Sci. USA. 2011;108:21105–21110. doi: 10.1073/pnas.1111919109. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 26.Huang J., Gong Z., Ghosal G., Chen J. SOSS complexes participate in the maintenance of genomic stability. Mol. Cell. 2009;35:384–393. doi: 10.1016/j.molcel.2009.06.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 27.Nam E.A., Cortez D. SOSS1/2: sensors of single-stranded DNA at a break. Mol. Cell. 2009;35:258–259. doi: 10.1016/j.molcel.2009.07.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 29.Makowski L., Brittingham K.C., Reynolds J.M., Suttles J., Hotamisligil G.S. The fatty acid-binding protein, aP2, coordinates macrophage cholesterol trafficking and inflammatory activity Macrophage expression of aP2 impacts peroxisome proliferator-activated receptor gamma and IkappaB kinase activities. J. Biol. Chem. 2005;280:12888–12895. doi: 10.1074/jbc.M413788200. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 30.Kajimoto K., Minami Y., Harashima H. Cytoprotective role of the fatty acid binding protein 4 against oxidative and endoplasmic reticulum stress in 3T3-L1 adipocytes. FEBS Open. Bio. 2014;4:602–610. doi: 10.1016/j.fob.2014.06.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 31.Durbin J.E., Hackenmiller R., Simon M.C., Levy D.E. Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease. Cell. 1996;84:443–450. doi: 10.1016/s0092-8674(00)81289-1. [DOI] [PubMed] [Google Scholar]
• 32.Sica A., Mantovani A. Macrophage plasticity and polarization: in vivo veritas. J. Clin. Invest. 2012;122:787–795. doi: 10.1172/JCI59643. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 33.Shresta S., Kyle J.L., Snider H.M., Basavapatna M., Beatty P.R., Harris E. Interferon-dependent immunity is essential for resistance to primary dengue virus infection in mice, whereas T- and B-cell-dependent immunity are less critical. J. Virol. 2004;78:2701–2710. doi: 10.1128/JVI.78.6.2701-2710.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 34.Huang P.H., White F.M. Phosphoproteomics: unraveling the signaling web. Mol. Cell. 2008;31:777–781. doi: 10.1016/j.molcel.2008.09.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 35.Lee J.-C., Won S.-J., Chao C.-L., Wu F.-L., Liu H.-S., Ling P., Lin C.-N., Su C.-L. Morusin induces apoptosis and suppresses NF-kappaB activity in human colorectal cancer HT-29 cells. Biochem. Biophys. Res. Commun. 2008;372:236–242. doi: 10.1016/j.bbrc.2008.05.023. [DOI] [PubMed] [Google Scholar]
• 36.Ohman T., Rintahaka J., Kalkkinen N., Matikainen S., Nyman T.A. Actin and RIG-I/MAVS signaling components translocate to mitochondria upon influenza A virus infection of human primary macrophages. J. Immunol. 2009;182:5682–5692. doi: 10.4049/jimmunol.0803093. [DOI] [PubMed] [Google Scholar]
• 37.Perry S.T., Prestwood T.R., Lada S.M., Benedict C.A., Shresta S. Cardif-mediated signaling controls the initial innate response to dengue virus in vivo. J. Virol. 2009;83:8276–8281. doi: 10.1128/JVI.00365-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 38.Chen H.W., King K., Tu J., Sanchez M., Luster A.D., Shresta S. The roles of IRF-3 and IRF-7 in innate antiviral immunity against dengue virus. J. Immunol. 2013;191:4194–4201. doi: 10.4049/jimmunol.1300799. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 39.Augustyniak D., Nowak J., Lundy.2012 F.T. Direct and indirect activities of neuropeptides and their therapeutic potential. Curr. Protein Pept. Sci. 2012;13:723–738. doi: 10.2174/138920312804871139. [DOI] [PMC free article] [PubMed] [Google Scholar]

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