Chaperone-dependent E3 ligase CHIP ubiquitinates and mediates proteasomal degradation of soluble guanylyl cyclase

The nitric oxide receptor soluble guanylyl cyclase (sGC) exists in multimeric protein complexes, including heat shock protein (HSP) 90 and endothelial nitric oxide synthase. Inhibition of HSP90 by geldanamycin causes proteasomal degradation of sGC protein. In this study, we have investigated whether COOH terminus of heat shock protein 70-interacting protein (CHIP), a co-chaperone molecule that is involved in protein folding but is also a chaperone-dependent ubiquitin E3 ligase, could play a role in the process of degradation of sGC. Transient overexpression of CHIP in COS-7 cells degraded heterologous sGC in a concentration-related manner; this downregulation of sGC was abrogated by the proteasome inhibitor MG-132. Transfection of tetratricopeptide repeats and U-box domain CHIP mutants attenuated sGC degradation, suggesting that both domains are indispensable for CHIP function. Results from immunoprecipitation and indirect immunofluorescent microscopy experiments demonstrated that CHIP is associated with sGC, HSP90, and HSP70 in COS-7 cells. Furthermore, CHIP increased the association of HSP70 with sGC. In in vitro ubiquitination assays using purified proteins and ubiquitin enzymes, E3 ligase CHIP directly ubiquitinated sGC; this ubiquitination was potentiated by geldanamycin in COS-7 cells, followed by proteasomal degradation. In rat aortic smooth muscle cells, endogenous sGC was also degraded by adenovirus-infected wild-type CHIP but not by the chaperone interaction-deficient K30A CHIP, whereas CHIP, but not K30A, attenuated sGC expression in, and nitric oxide donor-induced relaxation of, rat aortic rings, suggesting that CHIP plays a regulatory role under physiological conditions. This study reveals a new mechanism for the regulation of sGC, an important mediator of cellular and vascular function.     

AH23848 accelerates inducible nitric oxide synthase degradation through attenuation of cAMP signaling in glomerular mesangial cells.

Excessive release of nitric oxide (NO) by mesangial cells contributes to the pathogenesis of glomerulonephritis. Prostaglandin E(2) (PGE(2)) produced at inflammatory sites regulates the release of NO through its downstream signaling. In glomerular mesangial cells (MES-13 cells), PGE(2) modulated NO production mainly through EP4 receptor in a cAMP-dependent manner. Lipopolysaccharide and interferon-gamma (LPS+IFNgamma)-induced NO production, inducible nitric oxide synthase (iNOS) gene and protein expression were greatly inhibited by AH23848, an EP4 antagonist. Further investigation indicated that AH23848 attenuated endogenous cAMP accumulation in MES-13 cells and modulated NO production through declination of iNOS gene expression and acceleration of iNOS protein degradation. AH23848 downregulated the iNOS protein in MES-13 cells through protein kinase A (PKA) since KT5720, a PKA-specific inhibitor, reduced iNOS protein stability. A short exposure of activated MES-13 cells to okadaic acid augmented iNOS activity. AH23848 and KT5720 attenuated serine/threonine phosphorylation of iNOS protein in LPS + IFNgamma-stimulated MES-13 cells. The results of this study led us to speculate that cAMP might regulate iNOS-stimulated NO synthesis through posttranslational mechanisms. Attenuation of cAMP signaling and the phosphorylation status of the iNOS protein may account for the effect of AH23848 in accelerating iNOS protein degradation in MES-13 cells.     

Oxalomalate affects the inducible nitric oxide synthase expression and activity

Inducible nitric oxide synthase (iNOS) is an homodimeric enzyme which produces large amounts of nitric oxide (NO) in response to inflammatory stimuli. Several factors affect the synthesis and catalytic activity of iNOS. Particularly, dimerization of NOS monomers is promoted by heme, whereas an intracellular depletion of heme and/or L-arginine considerably decreases NOS resistance to proteolysis. In this study, we found that oxalomalate (OMA, oxalomalic acid, alpha-hydroxy-beta-oxalosuccinic acid), an inhibitor of both aconitase and NADP-dependent isocitrate dehydrogenase, inhibited nitrite production and iNOS protein expression in lipopolysaccharide (LPS)-activated J774 macrophages, without affecting iNOS mRNA content. Furthermore, injection of OMA precursors to LPS-stimulated rats also decreased nitrite production and iNOS expression in isolated peritoneal macrophages. Interestingly, alpha-ketoglutarate or succinyl-CoA administration reversed OMA effect on NO production, thus correlating NO biosynthesis with the anabolic capacity of Krebs cycle. When protein synthesis was blocked by cycloheximide in LPS-activated J774 cells treated with OMA, iNOS protein levels, evaluated by Western blot analysis and (35)S-metabolic labelling, were decreased, suggesting that OMA reduces iNOS biosynthesis and induces an increase in the degradation rate of iNOS protein. Moreover, we showed that OMA inhibits the activity of the iNOS from lung of LPS-treated rats by enzymatic assay. Our results, demonstrating that OMA acts regulating synthesis, catalytic activity and degradation of iNOS, suggest that this compound might have a potential role in reducing the NO overproduction occurring in some pathological conditions.     

Quercetin tetraacetyl derivative inhibits LPS-induced nitric oxide synthase (iNOS) expression in J774A.1 cells

In inflammation, nitric oxide (NO) acts as a pro-inflammatory mediator, which is synthesized by inducible nitric oxide synthase (iNOS) in response to pro-inflammatory agents such as lipopolysaccharide (LPS). Quercetin (Qt) has anti-inflammatory properties through its ability to inhibits nitric oxide production and iNOS expression in different cellular types. In the present study, we evaluated the effect of a semi-synthetic acetyl (quercetin-3,5,7,3′-tetraacetyl: TAQt) Qt derivative and two natural sulphated (quercetin-3-acetyl-7,3′,4′-trisulphate: ATS and quercetin-3,7,3′,4′-tetrasulphate: QTS) Qt derivatives on the LPS-induced NO production and iNOS expression in J774A.1 cells. Our results demonstrate that only TAQt inhibited the NO production by decreasing the iNOS mRNA and protein levels. In addition, we showed that TAQt blocked the LPS-induced nuclear NF-κB translocation by inhibiting the IκB-α degradation. Hence, as TAQt inhibited the LPS-induced iNOS expression and NO production, it could therefore be considered as a potential therapeutic agent for the treatment of inflammatory diseases related with the NO system.     

Inhibition of inducible nitric oxide synthesis by the herbal preparation Padma 28 in macrophage cell line

Padma 28 is a mixture of herbs used in traditional Tibetan medicine with anti-inflammatory activities. We investigated the effects of Padma 28 on nitric oxide (NO) production by the inducible nitric oxide synthase (iNOS) in lipopolysaccharide stimulated mouse macrophages (RAW 264.7). Padma 28 (0-900 microg/mL) induced a concentration dependent inhibition of inducible nitric oxide synthesis. iNOS protein expression showed a concentration dependent reduction as revealed by immunoblotting when cells were incubated with increasing amounts of Padma 28. Padma 28 decreased iNOS mRNA levels as shown by RT-PCR. Aqueous extracts from costi amari radix (costus root, the dried root of Saussurea lappa) and the outer cover of myrobalani fructus (the dried fruit of Terminalia chebula), constituents of the complex herb preparation Padma 28, were found to inhibit inducible nitric oxide synthesis by decreasing iNOS protein and iNOS mRNA levels. The inhibition of inducible nitric oxide synthesis might contribute to the anti-inflammatory activities of Padma 28.     

Salmonella typhimurium mutants that downregulate phagocyte nitric oxide production

To examine the potential and strategies of the facultative intracellular pathogen Salmonella typhimurium to increase its fitness in host cells, we applied a selection that enriches for mutants with increased bacterial growth yields in murine J774-A.1 macrophage-like cells. The selection, which was based on intracellular growth competition, rapidly yielded isolates that out-competed the wild-type strain during intracellular growth. J774-A.1 cells responded to challenge with S. typhimurium by mounting an inducible nitric oxide synthase (iNOS) mRNA and protein expression and a concomitant nitric oxide (NO) production. Inhibition of NO production with the use of the competitive inhibitor N-monomethyl-L-arginine (NMMA) resulted in a 20-fold increase in bacterial growth yield, suggesting that the NO response prevented bacterial intracellular growth. In accordance with this observation, five out of the nine growth advantage mutants isolated inhibited production of NO from J774-A.1 cells, despite an induction of iNOS mRNA and iNOS protein. Accompanying bacterial phenotypes included alterations in lipopolysaccharide structure and in the profiles of proteins secreted by invasion-competent bacteria. The results indicate that S. typhimurium has the ability to mutate in several different ways to increase its host fitness and that inhibition of iNOS activity may be a major adaptation.     

Interleukin-1beta-induced nitric oxide production in rat aortic endothelial cells: inhibition by estradiol in normal and high glucose cultures.

Expression of inducible nitric oxide synthase (iNOS) and the resultant increased nitric oxide (NO) production are associated with septic shock, atherosclerosis, and cytokine-induced vascular injury. Estrogen is known to impact vascular injury and vascular tone, in part through regulation of NO production. In the current study, we examined the effect of physiological concentrations of estradiol on interleukin-1beta (IL-1beta)-induced NO production in rat aortic endothelial cells (RAECs). 17Beta-estradiol significantly decreased IL-1beta-induced iNOS protein levels and reduced NO production in RAECs. High glucose (25 mM) elevated the increase in IL-1beta-induced iNOS protein and NO production. Nevertheless, estradiol still inhibited IL-1beta-induced iNOS and NO production even in the presence of high glucose. These data suggest that estradiol may exert its beneficial effects in part by inhibiting induction of endothelial iNOS, a possible mechanism for the protective effect of estradiol against diabetes-associated cardiovascular complications.     

8-Hydroxyquinoline inhibits iNOS expression and nitric oxide production by down-regulating LPS-induced activity of NF-kappaB and C/EBPbeta in Raw 264.7 cells

In activated macrophage, large amounts of nitric oxide (NO) are generated by inducible nitric oxide synthase (iNOS), resulting in acute or chronic inflammatory disorders. In Raw 264.7 cells stimulated with lipopolysaccharide (LPS) to mimic inflammation, 8-hydroxyquinoline (8HQ) inhibited the LPS-induced expression of both iNOS protein and mRNA in a parallel dose-dependent manner. 8HQ did not enhance the degradation of iNOS mRNA. To investigate the mechanism by which 8HQ inhibits iNOS gene expression, we examined the activation of MAP kinases in Raw 264.7 cells. We did not observe any significant change in the phosphorylation of MAPKs between LPS alone and LPS plus 8HQ-treated cells. Moreover, 8HQ significantly inhibited the DNA-binding activity of nuclear factor-kappaB (NF-kappaB) and CCAAT/enhancer-binding protein beta (C/EBPbeta), but not activator protein-1 and cAMP response element-binding protein. Taken together, these results suggest that 8HQ acts to inhibit inflammation through inhibition of NO production and iNOS expression through blockade of C/EBPbeta DNA-binding activity and NF-kappaB activation.     

P38 MAPK, but not p42/p44 MAPK mediated inducible nitric oxide synthase expression in C6 glioma cells

Recently mitogen-activated protein kinase (MAPK) has been reported to play an important role in phosphorylation cascades governing cell growth and protein expression in numerous cell types. In order to explore the signaling mechanism by which inducible nitric oxide synthase (iNOS) is regulated in C6 glioma cells, we investigated the role of MAPK in iNOS expression by using the specific MAPK inhibitors. First the induction of nitric oxide by lipopolysaccharide (LPS), tumor necrosis factor alpha (TNFalpha), interferon gamma (IFNgamma), alone or their combination, was studied in C6 glioma cells. Administration of LPS, TNFalpha, or IFNgamma alone had no detectable stimulatory effect on the production of nitric oxide (NO). However, combination of the three factors elicited a significant elevation of NO level in C6 cell culture medium. Subsequently pretreatment of C6 cells with a specific inhibitor of p38 MAPK, SB202190, resulted in a dose-dependent inhibition of NO production and iNOS expression, but PD98059, an inhibitor of p42/p44 MAPK activation, had no effect. These data suggest that p38 MAPK mediates iNOS expression in C6 glioma cells, but p42/p44 MAPK is not involved in this process.     

Penehyclidine hydrochloride attenuates LPS-induced iNOS production by inhibiting p38 MAPK activation in endothelial cells

The aim of this study was to investigate the inhibitory effect of penehyclidine hydrochloride (PHC) on lipopolysaccharide (LPS)-induced nitric oxide (NO) and inducible nitric oxide synthase (iNOS) production in human endothelial cell. Cultured endothelial cells were pretreated with PHC, followed by LPS treatment. NO activity were determined. iNOS expression and p38 mitogen-activated protein kinase (p38 MAPK) protein expression were measured by Western blot analysis. LPS treatment significantly induced p38 MAPK activation, iNOS expression, and NO production, which could be attenuated by 2 μg/ml PHC pretreatment. Furthermore, our study showed LPS-induced NO production and iNOS expression were suppressed by p38 MAPK inhibitor SB203580 pretreatment. We concluded that PHC attenuates NO production and iNOS expression by suppressing the activation of p38 MAPK pathway, thereby implicating a mechanism by which PHC may exert its protective effects against LPS-induced endothelial cell injury.     

Overexpression of the human inducible nitric oxide synthase gene enhances radiation-induced apoptosis in colorectal cancer cells via a caspase-dependent mechanism.

Nitric oxide (NO) has been reported to sensitize cancer cells to radiation. Since delivery of NO to tumors is limited in vivo by systemic toxicity of NO, we examined the potential of gene delivery of the human inducible nitric oxide synthase (iNOS) gene as a means of achieving high output NO production. We successfully transduced two colorectal cancer cell lines as evidenced by increased iNOS protein accumulation and nitrite production. We found that overexpression of iNOS enhanced the effects of radiation on apoptosis in both cell lines in a caspase-dependent fashion. Gene transfer of iNOS holds much promise as a potential radiosensitizer of cancer cells since it increases apoptosis in an additive manner with radiation.     

Lipid hydroperoxides inhibit nitric oxide production in RAW264.7 macrophages

The effects of oxidatively modified low density lipoprotein (oxLDL) on atherogenesis may be partly mediated by alterations in the production of nitric oxide (NO) by vascular cells. Lipid hydroperoxides (LOOH) and lysophosphatidylcholine (lysoPC) are the major primary products of LDL oxidation. The purpose of this study was to characterize the effects of oxLDL, LOOH and lysoPC on NO production and the expression of inducible nitric oxide synthase (iNOS) gene in lipopolysaccharide (LPS) stimulated macrophages. LDL was oxidized using an azo-initiator 2,2'-azobis (2-amidinopropane) HCl (ABAP) and octadecadienoic acid was oxidized by lipoxygenase to generate 13-hydroperoxyl octadecadienoic acid (13-HPODE). Our study showed that oxLDL markedly decreased the production of NO, the levels of iNOS protein and iNOS mRNA in LPS stimulated macrophages. The inhibition potential of oxLDL on NO production and iNOS gene expression depended on the levels of LOOH formed in oxLDL and was not due to oxLDL cytotoxicity. Furthermore, 13-HPODE markedly reduced NO production and iNOS protein levels, whereas lysoPC showed only slight reduction. The effects of 13-HPODE and lysoPC did not require an acetylated LDL carrier. Our results suggest that 13-HPODE is a much more potent inhibitor of NO production and iNOS gene expression than lysoPC in LPS stimulated RAW264.7 macrophages.     

The protective effect of aminoguanidine on cerebral ischemic damage in the rat brain

The NADPH-diaphorase (NADPH-d) histochemical technique is commonly used to localize the nitric oxide (NO) produced by the enzyme nitric oxide synthase (NOS) in neural tissue. The expression of inducible nitric oxide synthase (iNOS) is induced in the late stage of cerebral ischemia, and NO produced by iNOS contributes to the delay in recovery from brain neuronal damage. The present study was performed to investigate whether the increase in nitric oxide production via inducible nitric oxide synthase was suppressed by the administration of aminoguanidine, a selective iNOS inhibitor, as it follows a decrease of NADPH-diaphorase activity (a marker for NOS) after four-vessel occlusion used as an ischemic model. The administration of aminoguanidine (100 mg/kg i.p., twice per day up to 3 days immediately after the ischemic insult) reduced the number of NADPH-diaphorase positive cells to control levels. Our results indicated that aminoguanidine suppressed NADPH-diaphorase activity, and also decreased the number of NADPH-diaphorase positive cells in the CA1 region of the hippocampus following ischemic brain injury.     

High iNOS mRNA and protein localization during late pregnancy suggest a role for nitric oxide in mouse pubic symphysis relaxation

Remodeling and relaxation of the mouse pubic symphysis (PS) are central events in parturition. The mouse PS remodels in a hormone-controlled process that involves the modification of the fibrocartilage into an interpubic ligament (IpL), followed by its relaxation prior to parturition. It is recognized that nitric oxide synthase (NOS) and consequently nitric oxide (NO) generation play important roles in extracellular matrix modification, and may promote cytoskeleton changes that contribute to the remodeling of connective tissue, which precedes the onset of labor. To our knowledge, no studies thus far have investigated inducible nitric oxide synthase (iNOS) expression, protein localization, and NO generation in the mouse PS during pregnancy. In this work, we used a combination of the immunolocalization of iNOS, its relative mRNA expression, and NO production to examine the possible involvement of iNOS in remodeling and relaxation of the mouse IpL during late pregnancy. The presence of iNOS was observed in chondrocytes and fibroblast-like cells in the interpubic tissues. In addition, iNOS mRNA and NO production were higher during preterm labor on Day 19 of pregnancy (D19) than NO production on D18 or in virgin groups. The significant increase in iNOS mRNA expression and NO generation from the partially relaxed IpL at D18 to the completely relaxed IpL at D19 may indicate that NO plays an important role in late pregnancy during relaxation of the mouse IpL.     

Toxoplasma gondii infection of activated J774-A1 macrophages causes inducible nitric oxide synthase degradation by the proteasome pathway

Classically activated macrophages produce nitric oxide (NO), which is a potent microbicidal agent. NO production is catalyzed by inducible nitric oxide synthase (iNOS), which uses arginine as substrate producing NO and citruline. However, it has been demonstrated that NO production is inhibited after macrophage infection of Toxoplasma gondii, the agent of toxoplasmosis, due to iNOS degradation. Three possible iNOS degradation pathways have been described in activated macrophages: proteasome, calpain and lysosomal. To identify the iNOS degradation pathway after T. gondii infection, J774-A1 macrophage cell line was activated with lipopolysaccharide and interferon-gamma for 24 h, treated with the following inhibitors, lactacystin (proteasome), calpeptin (calpain), or concanamycin A (lysosomal), and infected with the parasite. NO production and iNOS expression were evaluated after 2 and 6 h of infection. iNOS was degraded in J774-A1 macrophages infected with T. gondii. However, treatment with lactacystin maintained iNOS expression in J774-A1 macrophages infected for 2 h by T. gondii, and after 6 h iNOS was localized in aggresomes. iNOS was degraded after parasite infection of J774-A1 macrophages treated with calpeptin or concanamycin A. NO production confirmed iNOS expression profiles. These results indicate that T. gondii infection of J774-A1 macrophages caused iNOS degradation by the proteasome pathway.     

Oltipraz inhibits inducible nitric oxide synthase in vitro and inhibits nitric oxide production in activated microglial cells

The clinically relevant drug oltipraz (OPZ) has previously been shown to inhibit cytochrome P450 enzymes [Chem. Res. Toxicol. 13 (2000) 245]. The current study reveals that OPZ is also able to inhibit *NO formation by purified inducible nitric oxide synthase (iNOS) but not by neuronal nitric oxide synthase in hemoglobin assays. The inhibition of iNOS by OPZ is reversible and competitive with an IC(50) of 5.9 microM and Ki of 0.6 microM. In murine BV-2 microglial cells, an immortalized cell line that produces *NO in response to lipopolysaccharide (LPS), OPZ is able to block the formation of nitrite in LPS treated cells. The inhibitory effect of OPZ on LPS treated cells is not due to cell toxicity. Finally, treatment of cells with OPZ does not induce or suppress expression of iNOS protein as shown by Western blot analysis.     

Inhibition of nitric oxide synthase inhibitors and lipopolysaccharide induced inducible NOS and cyclooxygenase-2 gene expressions by rutin, quercetin, and quercetin pentaacetate in RAW 264.7 macrophages

Several natural flavonoids have been demonstrated to perform some beneficial biological activities, however, higher-effective concentrations and poor-absorptive efficacy in body of flavonoids blocked their practical applications. In the present study, we provided evidences to demonstrate that flavonoids rutin, quercetin, and its acetylated product quercetin pentaacetate were able to be used with nitric oxide synthase (NOS) inhibitors (N-nitro-L-arginine (NLA) or N-nitro-L-arginine methyl ester (L-NAME)) in treatment of lipopolysaccharide (LPS) induced nitric oxide (NO) and prostaglandin E2 (PGE2) productions, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expressions in a mouse macrophage cell line (RAW 264.7). The results showed that rutin, quercetin, and quercetin pentaacetate-inhibited LPS-induced NO production in a concentration-dependent manner without obvious cytotoxic effect on cells by MTT assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide as an indicator. Decrease of NO production by flavonoids was consistent with the inhibition on LPS-induced iNOS gene expression by western blotting. However, these compounds were unable to block iNOS enzyme activity by direct and indirect measurement on iNOS enzyme activity. Quercetin pentaacetate showed the obvious inhibition on LPS-induced PGE2 production and COX-2 gene expression and the inhibition was not result of suppression on COX-2 enzyme activity. Previous study demonstrated that decrease of NO production by L-arginine analogs effectively stimulated LPS-induced iNOS gene expression, and proposed that stimulatory effects on iNOS protein by NOS inhibitors might be harmful in treating sepsis. In this study, NLA or L-NAME treatment stimulated significantly on LPS-induced iNOS (but not COX-2) protein in RAW 264.7 cells which was inhibited by these three compounds. Quercetin pentaacetate, but not quercetin and rutin, showed the strong inhibitory activity on PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS co-treated RAW 264.7 cells. These results indicated that combinatorial treatment of L-arginine analogs and flavonoid derivates, such as quercetin pentaacetate, effectively inhibited LPS-induced NO and PGE2 productions, at the same time, inhibited enhanced expressions of iNOS and COX-2 genes.     

A critical role for CHIP in the aggresome pathway

Recent evidence suggests that aggresome formation is a physiologic stress response not limited to misfolded proteins. That stress response, termed “physiologic aggresome,” is exemplified by aggresome formation of inducible nitric oxide synthase (iNOS), an important host defense protein. CHIP (carboxy terminus of Hsp70-interacting protein) is a highly conserved protein that has been shown to mediate substrate ubiquitination and degradation by the proteasome. In this study, we show that CHIP has a previously unexpected critical role in the aggresome pathway. CHIP interacts with iNOS and promotes its ubiquitination and degradation by the proteasome as well as its sequestration to the aggresome. CHIP-mediated iNOS targeting to the proteasome sequentially precedes CHIP-mediated iNOS sequestration to the aggresome. CHIP is required for iNOS preaggresome structures to form a mature aggresome. Furthermore, CHIP is required for targeting the mutant form of cystic fibrosis transconductance regulator (CFTRΔF508) to the aggresome. Importantly, the ubiquitin ligase function of CHIP is required in targeting preaggresomal structures to the aggresome by promoting an iNOS interaction with histone deacetylase 6, which serves as an adaptor between ubiquitinated proteins and the dynein motor. This study reveals a critical role for CHIP in the aggresome pathway.     

Antimicrobial peptide P18 inhibits inflammatory responses by LPS- but not by IFN-γ-stimulated macrophages

Antimicrobial peptide P18 markedly inhibited the expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells, whereas magainin 2 did not inhibit these activities. P18 dose-dependently reduced nitric oxide (NO) production by LPS-stimulated RAW 264.7 macrophage cells, with complete inhibition at 20 microg P18 ml(-1). In contrast, P18 had no effect on NO production and the expression of iNOS mRNA and iNOS protein by interferon-gamma (IFN-gamma)-stimulated RAW264.7 cells, suggesting P18 selectively inhibits LPS-stimulated inflammatory responses in macrophages. An LAL assay showed that P18 has strong LPS-neutralizing activity, indicating that P18 inhibits the inflammatory responses in LPS-stimulated macrophages by direct binding to LPS. Collectively, our results indicate that P18 has promising therapeutic potential as a novel anti-inflammatory as well as antimicrobial agent.