Immunomodulatory Activity of Melaleuca Alternifolia Concentrate (MAC): Inhibition of LPS-induced NF-kB activation and cytokine production in myeloid cell lines

By:

Pauline Low -National Defence Center, Taipei

Amanda M. Clark – School of Medical Sciences and Griffith Health Research Institute, Gold Coast campus, Southport, Queensland 4222, Australia

Maxwell Reynolds – 98 Alive Pty Ltd, PO Box 82, Underwood, QLD 4119, Australia

Stephen J. Ralph – School of Medical Sciences and Griffith Health Research Institute, Gold Coast campus, Southport, Queensland 4222, Australia

 Abbreviations:

 

  1. MAC: Melaleuca Alternifolia Concentrate
  2. LPS: lipopolysaccharide
  3. TTO: tea tree oil
  4. iNOS: inducible nitric oxide synthase
  5. NO: nitric oxide
  6. HO-1: haeme oxygenase-1
  7. COX-2: cyclooxygenase

Abstract

Melaleuca Alternifolia concentrate (MAC) is a mixture predominantly composed of monoterpenoids and sequiterpenes, refined from the essential oil of the tea tree by removing up to 99% of the more toxic, hydrophobic monoterpenes.

MAC was examined here for its Immunomodulatory effects on human THP1 and murine RAW264.7 myloid leukemic cell lines as models for macrophage-like cells. Firstly, MAC levels were determined that did not affect either the survival or proliferation of these cell lines in vitro. Next, the levels of lipopolysaccharide (LPS)-induced production of cytokines (IL-6, TNFcx, IL-10, GM-CSF, INFy and IL-3) were examined from the myeloid cell lines using multiplex assays.

Many of the LPS-inducible cytokines produced by either cell lines could be significantly inhibited by MAC. Closer examination of the mechanism of action of MAC showed that it inhibited the LPS-induced activation of IkB phosphorylation and nuclear factor (NF)-k signalling and translocation, inhibiting iNOS protein expression and NO production.

These results demonstrate that MAC exerts its Immunomodulatory effects by inhibiting NF-kB signalling activation levels of cytokine production by macrophage-like cell lines.

Introduction

Natural products represent a source of clinically approved drugs that have contributed extensively to pharmaceutical drug development. Of note are essential oils, which are volatile, natural complex mixtures characterised by a strong odour and are formed by aromatic plants as secondary metabolites.

Common examples of essential oils are tea tree, eucalyptus, lavender and peppermint oils. They exhibit a vast array of activities, including antimicrobial, sedative, anti-inflammatory, skin penetration enhancement and spasmolytic properties. The largest class of secondary metabolites present in essential oils are the organic compounds terpenes, derived biosynthetically from the basic unit of isoprene (C5H8) as chains or ring structures. Terpenes that undergo chemical modification, such as by oxidation or rearrangement of the carbon skeleton, adding oxygen-containing groups are called terpenoids or isoprenoids. Together, terpenes and terpenoids are the major constituents of the essential oils of many plants and flowers.

Endemic Australian plants produce a wide range of essential oils. One of which, tea tree (TTO), is commonly available and from this, a new product has been derived. This refined material, obtained by further extraction, is known as Melaleuca Alternifolia Concentrate (MAC) and exhibits promising pharmacological activities.

Similar TTO, monoterpenoids and sequiterpenes comprise the main subtypes of terpenes present in MAC and terpinene-4-ol remains as the major constituent. However, the difference between TTO and MAC is in the proportion of monoterpenes that are present. In the process of generating MAC, approximately 90-99% of the monoterpenes are removed, including terpinolene, cx-pinene, cx-thujene, B-pinene, sabinene, cx-phellandrene, cx-terpinene, limonene, y-terpinene, and B-phellandrene. The international and Australian Standards stipulate a range of monoterpene content in TTO from 18-59.5%. Thus, as the monoterpene content within MAC falls outside the standard chemical range, it should not be considered as having the same chemical profile as conventional Melaleuca TTO and could subsequently be classed as a separate entity.

Melaleuca oils have been well established for their potent antibacterial, antifungal and antiviral properties. Toxicity from intake of large amounts of TTO has been reported in humans, rats, dogs, and cats, presenting with clinical signs of central nervous system depression. The toxicity of TTO has been largely attributed to the monoterpenes, which are predominantly removed from MAC because they show dermal toxicity, with the potential to induce strong allergic reactions. For example, limonene and cx-terpinene were identified as TTO reactants causing allergic contact eczema and cx-terpinene and cx-terpinolene were sensitizing agents for contact dermatitis, cx-Pinene and cx-terpinene, unlike the terpenoids, 1,8 cineole and cx-terpineol, were irritants in the rat popliteal lymph node assay and cx-terpinene acts as a pro-hapten, thereby inducing contact allergic reactions. Removing most of the monoterpene hydrocarbons from TTO during the process of producing MAC largely eliminates the toxicity inherent to TTO, which would otherwise result from the haptenating activity of the monoterpenes.

In contrast to the immune sensitizing activity of the monoterpenes, analysis of the monoterpene alcohol, terpinene-4-ol (the main component of TTO and MAC) has shown that it suppresses inflammatory cytokine production and superoxide levels by stimulating monocytes and neutrophils. In particular, the water soluble monoterpenoids portion of TTO, containing mostly monoterpene alcohols, terpinene-4-ol (42%), cx-terpineol (3%) and 1,8-ceneole (2%) was shown to significantly inhibit the production of cytokines TNF-cx, interleukin (IL)-1B and IL-10by activated human monocytes. In addition, terpinene-4-ol was found to be the only pure component which alone could account for inhibiting the activation of human monocytes. In a subsequent study, the same group reported that the water-soluble fraction of TTO significantly and dose-dependently suppressed agonist-stimulated superoxide production by human monocytes, and the suppression was not due to cell death. Terpinen-4-ol or cx-terpineol alone was also shown to significantly supress superoxide production in the monocytes.

In the present study, the Immunomodulatory action of MAC was investigated in vitro by examiningits effects on cytokine production and protein expression in murine or humane macrophage-like myeloid leukemic cell lines. The two cell lines, murine RAW264.7 and human THP1 represent commonly used and accepted model systems for studying the mechanisms of LPS and nuclear factor (NF)-kB dependent gene expression. LPS, a recognized potent activator of NF-kB signalling in myeloid cells and pro-inflammatory cytokine production was used as a critical activator of the immune responses during infection and inflammation.

 

Results

 

  1. MAC inhibits LPS-induced cytokine production from myeloid cell lines in in vitro.
  2. MAC suppresses LPS-induced IkB phosphorylation in the RAW264.7 cell line.
  3. MAC suppresses levels of LPS-induced Nitric Oxide production.
  4. MAC suppresses LPS-induced NF-kB nuclear translocation.

 

 

Discussion

Cytokines are regulatory immune signals involved in numerous steps of the inflammatory response and can be classified as either pro-inflammatory (e.g. IL-1B, GM-CSF, IFN-y and TNF-cx) or anti-inflammatory (e.g. IL-4, IL-10, and IL-13). Macrophages are of particular importance in immunity serving as major contributors to the production of the pro-inflammatory cytokines. Thus, the ability to modulate the inflammatory response by specifically inhibiting macrophage activation and subsequent cytokine production could provide an effective approach for immune modulating therapy. At the low levels of MAC used in this study (0.016%v/v), negligible cytoxicity was exhibited over the 24h time period towards the myeloid macrophage-like cell lines. In addition, MAC alone applied in these concentrations showed no effect on the basal levels of cytokine production by these myeloid cells.

However, the same low MAC levels exhibited potent anti-inflammatory effects in that dose-dependently inhibited several of the LPS-induced cytokines produced from the murine and human macrophage-like myeloid leukemic cell lines.

Several of the LPS-induced cytokines produced by the RAW264.7 cell line were inhibited by MAC, with substantial reductions in the levels of IL-3, IL-10, GM-CSF and IFN-y, whereas with the THP1 cells, the LPS-induced IL-1B, IL-6 and IL-10 levels were noticeably inhibited by MAC co-treatment. The RAW264.7 cells were very responsive to LPS for two of the cytokines, with high levels of IL-6 (15 ng/ml) and TNF-cx (6 ng/ml) detected.

Thus, these two cytokine genes in the RAW264.7 cell line were highly sensitive to even the lowest levels of LPS used (15.6 ng/ml), already sufficient to induce maximal levels of IL-6 and TNF-cx from this cell line. This result is consistent with the known LPS-inducible sensitivity of the promoters for these two cytokine genes which both contain proximal NF-kB binding sites. These results are also consistent with the sensitivity of the RAW264.7 cell line to iNOS induction and NO production reported previously for agents such as LPS and IFNy. This heightened responsiveness probably masked our ability to detect the actions of MAC affecting the levels of IL-6 or TNF-cx induced LPS in this cell line. As additional support for this observation, a marked difference was detected in responsiveness between the RAW264.7 and THP1 cell lines. Both IL-6 and TNF-cx levels produced from the THP1 pro-monocytic cell line were significantly inhibited by MAC co-treatment with LPS. It is likely that THP1 is less responsive to LPS because it represents a more undifferentiated type of pro-monocytic cell line, which has been previously reported to require priming or differentiation to enhance its LPS responsiveness. Similarly, very low levels of the cytokine, GM-CSF (<5 pg/ml) were produced by the THP1 cells in the present study, even when they were treated with the highest (0.5ug/ml) LPS level tested, such that no effects of MAC on GM-CSF production could be detected from these cells.

However, taken together, the data presented here are consistent with a significant anti-inflammatory activity of MAC capable of inhibiting the LPS-induced pro-inflammatory cytokine responses of macrophage-like myeloid leukemic cell lines.

Western immunoblotting analysis of intracellular signalling events indicated a mechanism for the MAC based suppression of the LPS-induced pro-inflammatory activity.

Activity of signalling events showed MAC inhibits IkB phosphorylation (IkB-p), normally required for the activation of the transcription factor, NF-kB and activation of downstream target genes, including cytokines.

This is the first report of such an effect being shown for TTO based products, and is consistent with previous reports establishing that other natural product-derived agents, such as triterpenoids and simploactone can decrease pro-inflammatory cytokine expression by inhibiting NF-kB activity. Activation and nuclear translocation of NF-kB is a critical step in the transcription of cytokine genes involved in the development of immune and inflammatory responses. NF-kB activity is inhibited when bound by IkB and phosphorylation of IkB results in the release and subsequent activation of NF-kB.

In the RAW264.7 cells, MAC inhibited LPS-induced phosphorylation of IkB (IkB –p), preventing the subsequent activation of NF-kB, whereas the anti-inflammatory agents astragaloside IV or dexamethasone were not effective.

The MAC treatment also inhibited LPS-induced expression of the iNOS protein. This is consistent with the induction of the iNOS gene expression requiring NF-kB activation. At the same concentration, terpinene-4-ol was not as effective an inhibitor as MAC.

Dexamethasone has a more complex mode of action and after binding to the glucocorticoid receptor (GR, the GR complex can act either in cis or trans to modulate NF-kB regulated genes. This could explain that Dex did not significantly inhibit lkB-p but did partially reduce the levels of iNOS protein levels. Alternately, it could also be explained by the direct action of GR binding to the iNOS mRNA to decrease its stability, as has been previously shown.

In support of MAC inhibition of iNOS, NO production induced by LPS was also reduced in both myeloid cell lines, even at the lowest concentration of MAC (0.004%v/v) tested.

Although significant reductions were not obtained in the RAW264.7 cells, a consistent trend of MAC inhibition was obtained at all concentrations. Nevertheless, MAC treatment resulted in very significant inhibition of the NO production by the THP1 cell line. Hence, pathways other than signalling via NF-kB may be operating in the RAW264.7 cell line, independently able to promote iNOS expression and NO production.

The anti-inflammatory activity of MAC in vitro may also partly involve the endogenous HO-1 cytoprotective pathway because MAC treatment was associated with elevated expression of HO-1. Several other studies of the LPS-induced activation of RAW264.7 cells or macrophages have previously reported similar results to ours in that co-treatment with other anti-inflammatory agents, including curcumin, chalcones or hydrogen sulphide stimulate HO-1 expression, reducing iNOS levels to inhibit NO production. Thus, HO-1 and its product carbon monoxide reduce the production of cytokines such as IL-6 from myeloid cells. In this manner, HO-1 is a potent immunosuppressive enzyme that protects tissues against inflammatory conditions and autoimmunity.

The in vitro studies presented here demonstrate that MAC exhibited anti-inflammatory effects by inhibiting the LPS activation of the NF-kB pathway. The net effect is to inhibit the cytokine production induced by stimulants, such as LPS, that would otherwise activate immune cells to enhance the immune response against bacterial pathogens.

The MAC mediated inhibition of the LPS activated NF-kB pathway was also supported by the studies applying immunofluorescence staining to examine changes in the intracellular localization of NF-kB in the myeloid cells. Thus, LPS and other factors are known to induce nuclear staining of NF-kB. However, the significant LPS-induced nuclear translocation of NF-kB was extensively prevented by the co-treatment with MAC, inhibiting NF-kB nuclear translocation, even at the lowest (0.004%) levels tested. Again, these results are entirely consistent with MAC inhibiting the LPS activated NF-kB signalling pathway in the myeloid cells.

 

Conclusion

Overall this study has used a number of independent analyses and different approaches to examine the effects of MAC on the pro-inflammatory responses induced by LPS in myeloid derived cell lines as model systems.

The data from these independent approaches, including:

·            Studies of cytokine production levels

·            iNOS

·            NO release

·            IkB signalling

·            NF-kB intercellular staining

The data is entirely consistent; it provides novel insights into some of the active agents within TTO and establishes that MAC inhibits the production of pro-inflammatory cytokines by macrophage-like immune cells in vitro by inhibiting the IKK mediated activation of the NF-kB signalling pathway in these cells.