Suppression of inflammatory and infection responses in lung macrophages by eucalyptus oil and its constituent 1,8-cineole: Role of pattern recognition receptors TREM-1 and NLRP3, the MAP kinase regulator MKP-1, and NFκB

Abstract

Eucalyptus oil (EO) used in traditional medicine continues to prove useful for aroma therapy in respiratory ailments; however, there is a paucity of information on its mechanism of action and active components. In this direction, we investigated EO and its dominant constituent 1,8-cineole (eucalyptol) using the murine lung alveolar macrophage (AM) cell line MH-S. In an LPS-induced AM inflammation model, pre-treatment with EO significantly reduced (P ≤0.01or 0.05) the pro-inflammatory mediators TNF-α, IL-1 (α and β), and NO, albeit at a variable rate and extent; 1,8-cineole diminished IL-1 and IL-6. In a mycobacterial-infection AM model, EO pre-treatment or post-treatment significantly enhanced (P ≤0.01) the phagocytic activity and pathogen clearance. 1,8-cineole also significantly enhanced the pathogen clearance though the phagocytic activity was not significantly altered. EO or 1,8-cineole pre-treatment attenuated LPS-induced inflammatory signaling pathways at various levels accompanied by diminished inflammatory response. Among the pattern recognition receptors (PRRs) involved in LPS signaling, the TREM pathway surface receptor (TREM-1) was significantly downregulated. Importantly, the pre-treatments significantly downregulated (P ≤0.01) the intracellular PRR receptor NLRP3 of the inflammasome, which is consistent with the decrease in IL-1β secretion. Of the shared downstream signaling cascade for these PRR pathways, there was significant attenuation of phosphorylation of the transcription factor NF-κB and p38 (but increased phosphorylation of the other two MAP kinases, ERK1/2 and JNK1/2). 1,8-cineole showed a similar general trend except for an opposite effect on NF-κB and JNK1/2. In this context, either pre-treatment caused a significant downregulation of MKP-1 phosphatase, a negative regulator of MAPKs. Collectively, our results demonstrate that the anti-inflammatory activity of EO and 1,8-cineole is modulated via selective downregulation of the PRR pathways, including PRR receptors (TREM-1 and NLRP3) and common downstream signaling cascade partners (NF-κB, MAPKs, MKP-1). To our knowledge, this is the first report on the modulatory role of TREM-1 and NLRP3 inflammasome pathways and the MAPK negative regulator MKP-1 in context of the anti-inflammatory potential of EO and its constituent 1,8-cineole.

Fig 1. Selection of non-cytotoxic concentration of eucalyptus oil (EO) for treatment of alveolar macrophages.

Murine alveolar macrophage cell line MH-S (1×10⁶ cells/well) was cultured in presence of varying concentrations of EO (0.01–0.10%) for 24 hours and cell viability (%) was assessed using Trypan blue staining. Values represent mean ± SEM based on three independent experiments. Asterisks (**) indicate statistically significant (P ≤ 0.01, respectively) difference when compared to the vehicle control.

Fig 2. Modulation of LPS-induced pro-inflammatory response in alveolar macrophage cells by pre-treatment with eucalyptus oil (EO).

MH-S cells (1x10⁶ cells/ml culture/well) were treated with (i) Vehicle (0.1% MeOH), (ii) EO only (0.02% vol/vol), (iii) LPS only (2 μg/ml), and (iv) LPS (2 μg/ml) +EO (0.02%); EO was added 3hours before LPS addition (“pre–treatment”). NO and cytokines were periodically measured in the culture supernatant through 24 hour time-point. Values are presented as mean ± SEM based on three independent treatments. Asterisks (* and **) indicate statistically significant (P ≤ 0.05 and P ≤ 0.01, respectively) difference when compared to the positive control (LPS only).

Fig 3. Modulation of LPS-induced pro-inflammatory response in alveolar macrophage cells by pre-treatment with 1,8-cineole (Cin).

MH-S cells (1x10⁶ cells/ml culture/well) were treated with i) vehicle (0.1% MeOH), (ii) Cin only (0.02% vol/vol), (iii) LPS only (2 μg/ml), (iv) LPS (2 μg/ml) +Cin (0.02%); Cin was added 3hours before LPS addition (“pre-treatment”). NO and cytokines were periodically measured in the culture supernatant up to 24 hour time-point. Values are presented as mean ± SEM of based on three independent treatments. Asterisks (*) and (**) indicate statistically significant (P ≤ 0.05 and P ≤ 0.01, respectively) difference as compared to the positive control (LPS only).

Fig 4. Effect of eucalyptus oil (EO) and 1,8-cineole on phagocytosis of Mycobacterium smegmatis and its clearance during infection of alveolar macrophages.

Panels A and C: Effect of EO and cineole on phagocytosis activity (1h); Panels B and D: Effect of EO and cineole on bacterial clearance (24 h). MH-S cells (1x10^6 cells/ml culture/well) were treated with EO (0.02% v/v) or cineole either 3h before phagocytosis (“pre-treatment”) or right after phagocytosis (“post-treatment”) during infection with M. smegmatis. Bacterial counting (CFU analysis) was performed on macrophage cell lysates at 1hour (phagocytosis) or 24 hours (pathogen load) post-infection challenge. Values are presented as mean ± SEM based on three independent treatments. Asterisks (*) and (**) indicate statistically significant (P ≤ 0.05 and P ≤ 0.01, respectively) difference as compared to the vehicle control.

Fig 5. Modulation of LPS-activation of MAPKs in alveolar macrophages by pre-treatment with eucalyptus oil (EO) or 1,8-cineole (Cin).

(A-D) EO pre-treatment blots; (E-H) Cin pre-treatment blots for p38, SAPK/ JNK, ERK1/2, and NF-kB. MAPKs, respectively. Activation was assessed in terms of increase in both the total content and the phosphorylated form; NF-kB activation was assessed in terms of increase in its phosphorylated form. Densitometry analysis of Western blots was done using NIH software image J. Lanes1-4 represent vehicle control (VC), EO-only, LPS-only and EO+LPS for EO pre-treatment group or VC, Cin, LPS and Cin +LPS for Cin pre-treatment group, respectively. Details on the treatments and antibodies for total- and phospho- MAPKs and β-Actin are described in Materials and Methods section. Values represent means ± SEM based on three independent treatments. Asterisks (*) and (**) indicate statistically significant (P ≤ 0.05 and P ≤ 0.01, respectively) difference as compared to the LPS treatments while the number sign (#) indicates statistical significance as compared to the vehicle control. See S1 File for the original Western blot images.

Fig 6. Modulation of LPS-activation of NLRP3 inflammasome in alveolar macrophages by pre-treatment with eucalyptus oil (EO) or 1,8-cineole (Cin).

NLRP3 protein expression was assessed in cell lysates of EO- and Cin- pretreatment groups (3 hours pre-treatment with either EO or Cin at 0.02% concentration followed by induction with 2 μg/ml LPS for 4 hours). Cell lysates were analyzed by Western blotting using anti-NLRP3 antibodies. Densitometry analysis of Western blots was done using NIH software image J. Lanes1-6 represent VC, EO, Cin, LPS, and EO+LPS, Cin +LPS, respectively. Values represent means ± SEM based on three independent treatments. Asterisks (*) and (**) indicate statistically significant (P ≤ 0.05 and P ≤ 0.01, respectively) difference as compared to the LPS-only treatment while the number sign (#) indicates statistically significance as compared to the vehicle control. See S1 File for the original Western blot images.

Fig 7. Modulation of mRNA expression of key target genes of LPS-signaling pathway by pre-treatment with eucalyptus oil (EO) and 1,8-cineole.

(A-E) Target genes encoding MKP-1, TREM-1, LBP, TLR4 and CD14, respectively. mRNA expression was analyzed by quantitative RT-PCR using total RNA isolated from different treatment groups, as detailed in Fig 6 legend. The housekeeping gene GAPDH was used as internal control for normalizations and expression difference as fold-change was determined using the formula (2^-ΔΔCt) method. Values are presented as mean ± SEM based on three independent treatment groups. Asterisks (*) and (**) indicate statistically significant (P ≤ 0.05 and P ≤ 0.01, respectively) difference as compared to the LPS-only treatment.

Fig 8. Schematic representation of immune-modulatory mode of action of eucalyptus oil and its constituent 1, 8-cineole on different targets of LPS/infection-induced pathways in alveolar macrophage.

Abbreviations: EO, Eucalyptus oil; Cin, 1,8-Cineole; LPS, Lipopolysaccharide; TLR, Toll-like receptor; LBP, LPS-binding protein; CD14, Cluster of differentiation 14; TREM-1, Triggering receptor expressed on myeloid cells 1; IRAKs, IL-1 Receptor-Associated Kinases; TRAF6, TNF receptor-associated factor 6; MAPKs, Mitogen-activated protein kinases; ERK, Extracellular signal–regulated kinases; JNK: c-Jun N-terminal kinases; MKP-1, MAP kinase phosphatase-1; NF-κB, Nuclear factor kappa-light-chain-enhancer of activated B cells; NLRP3, Nod-like receptor family pyrin domain containing 3; NO, Nitric oxide: TF, Transcription factor.