Perillyl alcohol has antibacterial effects and reduces ROS production in macrophages

Abstract Natural products have emerged as a rich source of bioactive compounds for adjunctive treatments of many infectious and inflammatory conditions, including periodontitis. Among the monoterpenes with significant biological properties, there is the perillyl alcohol (POH), which can be found in several essential oils and has shown immunomodulatory properties in recent studies, which may be interesting in the treatment of non-neoplastic inflammatory disorders. Objective To determine the antibacterial and immune modulatory activities of the POH. Methodology The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the POH for two significant Gram-negative periodontal pathogens were determined by macrodilution and subculture, respectively. Cell proliferation and cytotoxicity in RAW 264.7 macrophages were determined by Trypan Blue and mitochondrial enzymatic activity assay. The modulation of reactive oxygen species (ROS) was analyzed by flow cytometry and expression of TNF and arginase-1 by real-time PCR. Results The POH was effective against P. gingivalis (ATCC 33277) and F. nucleatum (ATCC 25586) with MIC= MBC=1600 μM. No cytotoxicity up to 100 µM was observed on macrophages. The cell proliferation was inhibited from 48 hours at 100 μM (p<0.05) and 250 μM (p<0.01). The POH increased ROS production at both 10 μM and 100 μM (p<0.05) in unstimulated cells. The PMA-induced ROS production was not affected by POH, whereas 100 μM significantly reduced lipopolysaccharide-induced (LPS-induced) ROS. The expression of TNF was not affected by POH in unstimulated cells or in cells polarized to M1 phenotype, whereas both concentrations of POH reduced (p<0.05) the expression of arginase-1 in M2-polarized macrophages. Conclusion The POH has antibacterial activity against periodontal pathogens and reduced proliferation of murine macrophages without significant cytotoxicity at concentrations up to 100 μM. In addition, the POH reduced the LPS-induced ROS and the expression of arginase-1 in M2-polarized macrophages.


Introduction
In the last decades, advances in periodontal studies have increased the knowledge about the pathogenesis and its immunological mechanisms that modulate the host response to microbial challenge.
Thus, although the onset of periodontitis is dependent on the maturation of a complex microbial biofilm (essentially composed of Gram-negative anaerobes), the progression, the extent, and the severity of the disease are dependent on the balance between proinflammatory and protective mediators, which involves complex host-microbial interactions. 1 In this context, some cell types are particularly significant for tissue homeostasis in the periodontal microenvironment, such as macrophages. The macrophages are highly prevalent in diseased periodontal sites, and as the prototypical antigenpresenting cell, are directly involved in the response to the microbial challenge. 2 Activated macrophages also secrete important mediators in extracellular matrix degradation and resorption of alveolar bone, and can vary into bone-resorbing osteoclasts. [3][4][5] Depending on the external cues present in inflammatory microenvironment, the macrophages can assume different phenotypes in a spectrum between the extremes of the classically activated or pro-inflammatory (M1), and alternatively activated or reparative (M2) macrophages, according to the major cytokines secreted. The characterization of macrophage response and phenotype associated with inflammatory conditions, including periodontal diseases, may be useful in developing new treatment strategies. 6 There has been a growing interest in natural products with antimicrobial and anti-inflammatory properties as adjunct treatments for many infectious and inflammatory conditions, including periodontitis. [7][8][9] These products include essential oils, which are volatile aromatic liquids extracted from plants and are active ingredients of phytotherapy. 9 Monoterpenes are the main constituents of essential oils, and most studies of their effects on human health have been performed with limonene, carvone, camphor, and perillyl alcohol (POH). The POH can be found in several essential oils, [10][11][12] and medical interest in this compound was initially based on its anti-tumoral activity. [13][14][15][16][17] Recent studies have shown immunomodulatory properties of the POH, which may be of interest in the treatment of non-neoplastic inflammatory disorders.
D 'Alessio, et al. 18 (2014) reported that the POH promotes wound healing, likely by decreasing the pro-inflammatory cytokines IL-6 and TNF. The POH reduced oxidative activity, TNF production, and NF-kB activation in a rat model of ethanol-induced acute hepatic injury. 19 In addition, the antinociceptive activity

Statistical analysis
Before the analysis, the data normality was verified with the Shapiro-Wilk test. For comparisons between the experimental groups, the ANOVA followed by the Tukey's post hoc test were used. Data are expressed as the mean ± standard deviation, and the analysis were performed in the GraphPad version 7.0 with the significance level set at 95% (p<0.05).

Results
Antimicrobial activity of the POH against periodontal pathogens The POH had an antimicrobial activity against P. gingivalis and F. nucleatum at the same concentration (MIC 1600μM), and the MBC was equal to the MIC for both microorganisms (Table 1). However, it did not affect the PMA-induced production of ROS at either concentration (10 µM and 100 µM) ( Figure 2A). Conversely, the POH had a concentrationdependent effect on the LPS-induced ROS production.

Cytotoxicity and cell proliferation assays
At 10 µM, the POH had a significant (p<0.01) synergistic effect with the LPS on ROS induction, whereas at 100 µM there was a significant reduction in the LPS-induced ROS production (p<0.05) ( Figure 2B).

Gene expression of TNF and Arginase 1
The POH alone (10 µM and 100 µM) did not affect expression of TNF or Arginase-1 by macrophages.
The TNF was increased by stimulation with LPS and IFN-gamma, and pre-treatment with POH (10 and 100 µM) had no effect. The POH partially inhibited IL-4induced expression of Arginase-1 POH at both 10 and 100 µM (Figure 3).

Discussion
POH is a natural monoterpene found in several essential oils, and its antitumor activity has been well established in the literature and supported by in vitro and in vivo studies, and Phase II clinical trials in cancer patients. [10][11][12]15,27,28 Recent studies have suggested that the POH also plays a role in the immunoinflammatory response. 18,19 Periodontitis is a chronic inflammatory condition associated with a dysbiotic microbial biofilm that may  the ROS production to compare with macrophages.
In this study, the POH alone at 100 µM induced a statistically significant increase in the ROS production and did not affect ROS production induced by PMA.
Interestingly, the POH effect on the LPS-induced ROS was concentration-dependent: 10 µM further increased and 100 µM reduced LPS-induced ROS. This suggests that the POH modulates ROS production induced via activation of TLR4, 36 but not via the protein kinase Cα (PKCα) pathway activated by PMA. 37 An important characteristic of macrophages is their phenotypic plasticity in response to the microenvironmental cues. This study assessed the expression of TNF and Arginase-1 as genes representative of the M1/pro-inflammatory and M2/ reparative phenotypes, respectively. The POH alone had no effect on the expression of TNF or Arginase-1.
Moreover, the TNF expression induced by M1-polarizing stimuli was also not affected by POH. Interestingly, the POH at 10 and 100 µM reduced the expression of Arginase-1 induced by M2-polarizing stimulus. These results suggest that POH may favor the M1 phenotype, although it is important to note the limitation of assessing gene expression and not protein production.
Macrophage polarization is a complex process finely controlled by intracellular signaling pathways activated by the external stimuli. The M1-inducing stimuli used in this study activates predominantly STAT1 (IFNγ) and MAPKs and NF-kB. 38 The IL-4 used as an M2-polarizing stimulus activates primarily STAT6. 39 The reduction of IL-4-induced Arginase-1 by POH suggests an inhibition of STAT6, as inhibition of STAT6 by siRNA or the biochemical inhibitor AS1517499 inhibits expression of Arginase-1 and Arginase activity induced by  It is intriguing that the POH modulates the TLR-induced ROS, which also involves MAPK and NF-kB as major downstream signaling pathways, but not the TNF expression. These possibilities should be assessed in future experiments, which should also assess the POH effect on periodontitis and the associated inflammation in vivo.

Conclusion
POH has a strong antibacterial effect against P.
gingivalis. and F. nucleatum. Up to 100 µM, the POH was not cytotoxic to murine macrophages, but reduced proliferation, LPS-induced ROS production, and IL-4induced expression of Arginase-1.