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. 2023 Mar 24;16(6):682–689. doi: 10.2174/1874467215666221005115007

Immunomodulatory Activity of Diterpenes over Innate Immunity and Cytokine Production in a Human Alveolar Epithelial Cell Line Infected with Mycobacterium tuberculosis

Alejandro David Hernández-Herrera 1, Julieta Luna-Herrera 2, Marisela del Rocío González-Martínez 3, Adria I Prieto-Hinojosa 4,5, Ana Monica Turcios-Esquivel 5, Irais Castillo-Maldonado 1,*, Dealmy Delgadillo-Guzmán 6, Agustina Ramírez-Moreno 7, Celia Bustos-Brito 8, Baldomero Esquivel 8, María-del-Carmen Vega-Menchaca 9, David Pedroza-Escobar 1,*
PMCID: PMC10240657  PMID: 36200155

Abstract

Background

Mexico has the largest number of the genus salvia plant species, whose main chemical compounds of this genus are diterpenes, these chemical compounds have shown important biological activities such as: antimicrobial, anti-inflammatory and immunomodulatory.

Objective

This study aimed to evaluate the immunomodulatory activity of three diterpenes: 1) icetexone, 2) anastomosine and 3) 7,20-dihydroanastomosine, isolated from Salvia ballotiflora, over innate immunity and cytokine production in a human alveolar epithelial cell line infected with Mycobacterium tuberculosis.

Methods

The immunomodulatory activity of diterpenes over innate immunity included reactive oxygen and nitrogen species (ROS and RNS) induction in response to infection; cytokine production included TNF-α and TGF-β induction in response to infection.

Results

The diterpenes anastomosine and 7,20-dihydroanastomosine showed a statically significant (p < 0.01) increase of RNS after 36 h of infection and treatment of 2.0 µg/mL. Then, the ROS induction in response to infection showed a consistent statically significant (p < 0.01) increase after 12 h of diterpenes treatments. The cell cultures showed an anti-inflammatory effect, in the case of TGF-β induction, in response to infection when treated with the diterpenes. On the other hand, there was not any significant effect on TNF-α release.

Conclusion

The diterpenes anastomosine and 7,20-dihydroanastomosine increased the production of RNS after 36 h of infection and treatment. Besides, the three diterpenes increased the production of ROS after 12 h. This RNS and ROS modulation can be considered as an in vitro correlation of innate immunity in response to Mycobacterium tuberculosis infection; and an indicator of the damage of epithelial lung tissue. This study also showed an anti-inflammatory immune response by means of TGF-β modulation when compared with control group.

Keywords: Inmmunomodulatory activity; icetexone; anastomosine; 7,20-dihydroanastomosine; innate immunity; chemokine production; Mycobacterium tuberculosis; A549

1. INTRODUCTION

Tuberculosis (TB) is a reemerging, infectious and chronic disease caused by the bacillus Mycobacterium tuberculosis (M. tb) [1, 2]. According to the world health organization (WHO), it is estimated that each year 10 million people become infected with M. tb and 1.5 million people die from this disease [3]. The pathological process of TB depends on the immunological status of the patient as well as the virulence factors of the bacteria [4, 5].

This microorganism can infect several cell types, including neutrophils, macrophages, endothelial cells and type II lung epithelial cells [6]. In these permissible cells the bacillus enters into the cell through phagocytosis [7]. M. tb antigens are recognized by toll-like receptors (TLR) mainly TLR-2 and TLR-4 [8, 9]. The activation of TLR initiates an inflammatory immune response in which the synthesis of cytokines, such as TNF-α and TGF-β [10, 11]; interleukins, such as IL-6 and IL-8 [12, 13]; as well as, reactive oxygen (ROS) and nitrogen (RNS) species is affected [14, 15]. The excessive production of these inflammatory mediators may cause necrosis, cavitation formation, and lung tissue damage [16, 17]. On the other hand, Mexico has the largest number of the genus salvia plant species, whose main compounds of this genus are terpenes [18, 19], these compounds have shown important biological activities such as: antimicrobial, anti-inflammatory and immunomodulatory [20, 21]. Thus, the aim of this study was to evaluate the immunomodulatory activity of diterpenes (icetexone, anastomosine and 7,20-dihydroanastomosine) isolated from Salvia ballotiflora (S. ballotaeflora) over innate immunity and cytokine production in a human alveolar epithelial cell line (A549) infected with M.tb.

2. MATERIALS AND METHODS

2.1. Antimycobacterial Activity of Diterpenes

The antimycobacterial activity of the diterpenes icetexone, anastomosine and 7,20-dihydroanastomosine, isolated from S. ballotaeflora [20], was evaluated using the microplate In vitro Toxicology Assay Kit, Resazurin based (Cat. Tox8. Sigma-Aldrich St. Louis, MO, USA) according to the specifications of the manufacturer [22]. Briefly, the M. tb reference strain H37Rv was cultivated in Middlebrook 7H9 supplemented broth, with 10% OADC growth supplement (5% Bovine albumin fraction V, 2% dextrose, 0.004% catalase, 0.0005% oleic acid, 0.85% NaCl) and 0.2% glycerol. The culture was grown to mid-log phase at 37°C until optical density (OD), at 600 nm, was 0.5. Then, a bacterial stock of M.tb was prepared by diluting the culture up to 107 CFU/mL. Subsequently, an aliquot of 104 CFU, adjusted in 200 μL final volume of a two-fold standard curve (0.00, 3.12, 6.25, 12.50, 25.00, 50.00 and 100.00 μg/mL) of diterpenes in Middlebrook 7H9 supplemented broth, was seeded per well into a ninety-six-well microplate. Isoniazid was used as a positive control drug at 1.00 μg/mL and drug-free broth was used as a negative control. Microplates were incubated for 7 days at 37°C. Thereafter, 20 μL of resazurin dye solution was added into each well. The microplates were incubated for another 24 h at 37°C. The antimycobacterial activity was measured by recording the decrease in OD at 600 nm. The minimal-inhibitory-concentration (MIC) results were presented as mean value. The lowest concentration that resulted to 90% inhibition was defined as the MIC. A pink color in the well indicated mycobacterial growth; and, a blue color in the well indicated no mycobacterial growth.

2.2. Citotoxicity Assay with the Haemolytic Activity Model

Blood samples of 50 μL, obtained in microtainer EDTA tubes from specific-pathogen-free (SPF) Wistar rats, were washed three times with 950 μL of sterile saline (0.9% w/v NaCl, endotoxin free solution) by centrifugation at 9,500 g for 1 min. Pellet diluted up to original volume with modified Hanks’ balanced salt solution (HBSS, without phenol red, 5.5 mM glucose, 137 mM NaCl, 5.3 mM KCl, 1.25 mM CaCl2, 830 μM MgSO4•7H2O, 1mM MgCl2, 430 μM NaH2PO4•H2O, 430 μM Na2HPO4, 4.25 mM NaHCO3, endotoxin free solution, sterile-filtered) in each wash. For making the erythrocytes suspension, in the last wash, the pellet diluted up to 1 mL with HBSS. An aliquot of 50 μL of the erythrocytes’ suspension was mixed with 950 μL of a two-fold standard curve of diterpenes (0.00, 3.12, 6.25, 12.50, 25.00, 50.00, 100.00, 200.00 and 400.00 μg/mL) in HBSS. Cell suspensions, into the two-fold standard curve of diterpenes, incubated at 37°C in an atmosphere of 5% CO2 and 95% humidity for 30 min, and centrifuged at 9,500 g for 1 min. Free hemoglobin in the supernatants was measured by recording OD at 412 nm. Sterile saline and distilled water were used as negative-hemolytic (0%) and positive-hemolytic (100%) controls, respectively.

2.3. Cell Culture

The A549 cell line, of human type II alveolar epithelium, was grown at confluency in a 25 cm2 cell culture flask with DMEM supplemented medium (with 10% decomplemented fetal bovine serum (FBS), 100 units/mL penicillin and 100 μg/mL gentamicin). The parameters of incubation were 37°C in an atmosphere of 5% CO2 and 95% humidity.

2.4. Immunomodulatory Activity of Diterpenes Over Innate Immunity: Evaluation of RNS and ROS Induction in Response to M. tb Infection

The RNS induction in response to M.tb infection was measured by means of nitrite released from A549 cells treated with the diterpenes. Twenty-four-well tissue culture plates were seeded with 105 A549 cells per well in a final volume of 2 mL DMEM supplemented medium. The cultures were infected for 4 h with a mycobacteria suspension adjusted at a multiplicity of infection (MOI) of 5.0; then, the cultures were washed 3 times with phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4), followed by the addition of the diterpenes (0.5 and 2.0 μg/mL). The plates were incubated for 36 h at 37°C in an atmosphere of 5% CO2 and 95% humidity. A sample of 50 µL of the supernatant was taken at three different times (12, 24 and 36 h). The samples were mixed with 50 µL of 1% sulfanilamide (in 5% phosphoric acid) and 50 µL of 0.1% N- (1-naphthyl) ethylenediamine, and incubated for 20 minutes. Thereafter, the OD at 520 nm was recorded, and the nitrite concentrations were calculated against a standard nitrite reference curve.

The ROS induction in response to M. tb infection was measured through p-nitro-blue-tetrazolium intracellular reduction by O2.-. Ninety-six-well tissue culture plates were seeded with 104 A549 cells per well in a final volume of 200 μL DMEM supplemented medium. The cultures were infected for 4 h with a mycobacteria suspension adjusted at a MOI of 5.0; then, the cultures were washed 3 times with PBS, followed by the addition of the diterpenes (0.5 and 2.0 μg/mL). Three sets of plates were prepared and incubated for 12, 24 and 36 h (each set of plates, respectively) at 37°C in an atmosphere of 5% CO2 and 95% humidity. After each incubation (12, 24 and 36 h), the DMEM medium was removed and cultures were washed 3 times with PBS, later 20 µL of 0.1% p-nitro-blue-tetrazolium chloride solution were added into each well and incubated at 37°C for 20 minutes. Subsequently, the wells were washed 3 times with PBS and the formazan crystals formed inside the cell cultures were dissolved with 108 µL of a solution 2M KOH and 92 µL of dimethyl sulfoxide; finally, the OD at 620 nm was recorded.

2.5. Immunomodulatory Activity of Diterpenes Over Cytokine Production: Evaluation of TNF-α and TGF-β Induction in Response to M. tb Infection

Twenty-four-well tissue culture plates were seeded with 105 A549 cells per well in a final volume of 2 mL DMEM supplemented medium. The cultures were infected with M.tb at a MOI of 5.0; then, the cultures were treated with the diterpenes at a final concentration 2.0 μg/mL. The plates were incubated for 36 h at 37°C in an atmosphere of 5% CO2 and 95% humidity. A sample of 100 µL of the supernatant was taken after three different incubation times (12, 24 and 36 h). The concentration of the cytokines TNF-α and TGF-β was determined in the supernatants of cell cultures by the commercially available kits Human TNF alpha Uncoated ELISA and Human/Mouse TGF beta 1 Uncoated ELISA ®invitrogen. These experiments were performed according to the specifications of the fabricant.

2.6. Statistic analysis

The results were expressed as mean ± standard errors (SE) and examined for statistically significant differences with analysis of variance (ANOVA) and multiple comparisons by post-hoc Tukey test. *p value < 0.05 and **p value < 0.01 were considered to be significant. The statistical package Graph Pad Prism Version 9.3.1 was used.

3. RESULTS

The MICs were calculated as follows 25 µg/mL for the anastomosine treatment, and >100 µg/mL for both icetexone and 7,20-dihydroanastomosine treatments. These results indicated that only anastomosine had antimycobacterial activity. On the other hand, the cytotoxicity test showed lethal doses 50% (LD50) higher than 50 µg/mL. Considering these results, the diterpene concentrations to be tested for immunomodulatory activity were set lower than 10% of the lowest MIC and LD50 being selected 0.5 and 2.0 µg/mL. The immunomodulatory activity of diterpenes over innate immunity was evaluated, in cell cultures of A549 cell line, through the quantification of RNS and ROS. The cell cultures included the following groups: 1) Uninfected control, 2) M. tb infected control, 3) Uninfected and treatment (0.5 and 2.0 µg/mL), 4) M.tb infected and treatment 0.5 µg/mL, and 5) M. tb infected and treatment 2.0 µg/mL. In the case of the group 3, no statistically significant (p > 0.05) differences were found when the cells were treated with 0.5 or 2.0 µg/mL; consequently, only one bar is presented for this group in Figs. (1 and 2). The RNS concentration (µM) of the uninfected control and M.tb infected control were 71.09 ± 2.88 and 72.52 ± 7.83, respectively. In the case of cell cultures infected with M.tb and treated with 2.0 µg/mL anastomosine and 7,20-dihydroanastomosine the RNS concentrations were 115.61 ± 17.96 and 96.33 ± 6.07, respectively. These results showed a statically significant (p < 0.01) increase of RNS after 36 h of infection with M.tb and treatment with any of both diterpenes. Besides, the 7,20-dihydroanastomosine treatment (2.0 µg/mL) also showed this pattern after 24 h (p < 0.01) of infection and treatment. The icetexone treatment of 2.0 µg/ml showed the same statically significant (p < 0.05) RNS increase after 12 h against the M. tb infected control (86.09 ± 8.21 and 72.52 ± 2.18 µM, respectively) as shown in Fig. (1).

Fig. (1).

Fig. (1)

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Immunomodulatory activity of diterpenes over innate immunity as means of RNS induction in response to M. tuberculosis infection. Treatments were as follows: (A). Icetexone, (B). Anastomosine, (C). 7,20-dihydroanastomosine. Statistical significance was *p<0.05 and **p<0.01

Fig. (2).

Fig. (2)

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Immunomodulatory activity of diterpenes over innate immunity as means of ROS induction in response to M. tuberculosis infection. Treatments were as follows: (A). Icetexone, (B). Anastomosine, (C). 7,20-dihydroanastomosine. Statistical significance was *p < 0.05 and **p < 0.01.

In the evaluation of ROS induction in response to M. tb infection the OD of the uninfected control and M. tb infected control were 0.064 ± 0.014 and 0.070 ± 0.009, respectively. The OD of cell cultures infected with M. tb and treated with 2.0 µg/mL icetexone, anastomosine and 7,20-dihydroanastomosine were 0.253 ± 0.026, 0.144 ± 0.021 and 0.113 ± 0.005, respectively. These results showed a statically significant (p < 0.01) increase of ROS after 12 h when the cell cultures were infected with M.tb and treated with any of three diterpenes. But, only 2.0 µg/mL icetexone treatment kept the ROS increase after 36 h (p < 0.01) as shown in Fig. (2).

The immunomodulatory activity of diterpenes over cytokine production was evaluated, in cell cultures of A549 cell line infected with M. tb, through the quantification of TNF-α and TGF-β. The cell cultures included the following groups: 1) Control, 2) Icetexone, 3) Anastomosine, and 4) 7,20-dihydroanastomosine. The control group did not receive any treatment while the diterpene groups were treated at a concentration of 2.0 µg/mL. There was not any significant effect on TNF-α release as seen in Fig (3A). This experiment was repeated three times. The TGF-β concentration of the control group, after 36 h of infection, was 227.62 ±1.32 pg/mL. In the case of cell cultures infected and treated with icetexone, anastomosine and 7,20-dihydroanastomosine, the TGF-β concentrations were 117.62 ± 2.21, 114.50 ± 3.97 and 179.50 ± 2.22, respectively. These results showed a statically significant (p < 0.01) decrease of TGF-β concentration after 36 h of infection and treatment with any diterpene, as shown in Fig. (3B).

Fig. (3).

Fig. (3)

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Immunomodulatory activity of diterpenes over cytokine production as means of TNF-α and TGF-β induction in response to M. tuberculosis infection. Data sets were as follows: (A). TNF-α, (B). TGF-β. Statistical significance was *p < 0.05 and ** p< 0.01. There was not any significant effect on TNF-α release.

4. DISCUSSION

Innate immunity mediates acute inflammation and pathogen killing. Acute inflammation is an immediate response characterized by leukocyte recruitment to sites of inflammation and local edema [23]. Acute inflammation originates from harmful stimuli, such as pathogens, and may lead to tissue damage as a consequence of dysregulated production of soluble mediators like cytokines, acute phase proteins, and chemokines [23-25]. M. tb has the ability to hijack the host innate and adaptive immune systems in numerous ways; for instance, modulating cytokine production, mainly RNS, ROS, TNF-α and TGF-β [26-30]. The A549 cell line was used because it is a type II epithelial cell with immunological activity [31, 32]. This study aimed to determine if the diterpenes isolated from S. ballotaeflora were able of modulating the molecular markers RNS, ROS, TNF-α and TGF-β in the A549 cell line infected with M. tb. The results, obtained after infection and treatment with 2.0 µg/mL of diterpenes, indicated that the concentration of RNS increased as a consequence of diterpene treatment. The most effective results were observed with icetexone and 7,20-dihydroanastomosine after 12 h and 24 h of infection and treatment, respectively. After 36 h of treatment, anastomosine and 7,20-dihydroanastomosine consistently increased the RNS concentrations. On the other hand, similar results were observed for the ROS after 12 h of infection and diterpene treatment (icetexone, anastomosin and 7,20-dihydroanastomosine). These results indicated that at both short and long treatment times there is an innate immune activity trough ROS and RNS induction.

Several studies have demonstrated the antimycobacterial, immunomodulatory and adjuvant activities of terpenoids [33]. For instance, the monoterpene camphene and its derivatives exhibited low MIC values against M. tb clinical isolates, synergism with the antitubercular-drug pyrazinamide and low cytotoxicity [34]. The terpenes commiphoroid E and F, at 200 μM concentration, inhibited the growth of both sensitive and clinically multi-drug resistant (MDR) M. tb isolated strains. In addition, these terpenes were weakly toxic in human A549 cells as indicated by the cell viability >80% [35]. Carvacrol showed good antitubercular activity and synergism with rifampicin [36]. Tanshinones from Salvia miltiorrhiza inhibited M. tb via disruption of the cell envelope surface and oxidative stress [37]. Ursolic acid reduced M.tb-induced ntric oxide release in human alveolar A549 cells [38]. And, similar results have been reported for andrographolide, laurinterol, aplysin, macfarlandin D and rosane derivatives [39-42].

As previously stated, several authors have reported that terpenes (monoterpenes, diterpenes and triterpenes) have anti-inflammatory properties [43-45]. These reports are consistent with the results found in this study because diterpene treatment induced an inverse concentration of cytokines when compared with the controls. This effect may be attributed to an inhibition of the pro-inflammatory pathway of Nf-κβ [46-48]. The Nf-κβ pathway is associated to the modulation of cytokines TNF-α and TGF-β [49, 50]. As observed in this study, diterpene treatments reduced the concentration of TGF-β, which suggest an inhibition of the inflammatory NF-κB pathway. The transcription factor NF-κB regulates multiple aspects of innate and adaptive immune functions and serves as a pivotal mediator of inflammatory responses. NF-κB induces the expression of various pro-inflammatory genes, including those encoding cytokines and chemokines, and also participates in inflammasome regulation [15, 51, 52]. In addition, NF-κB plays a critical role in regulating the survival, activation and differentiation of innate immune cells and inflammatory T cells [53]. Consequently, modulating NF-κB activation affects to the pathogenic processes of various inflammatory diseases [54].

The TNF-α is synthesized as a membrane pro-peptide with an intracellular amino terminus and an extracellular carboxyl terminus [55, 56]. On the other hand, the TGF-β is synthesized as a pro-peptide which forms a Large Latent Complex (LLC) with a Latency Associated Peptide (LAP) and a Latent TGF-β-Binding Protein (LTBP) [57, 58]. The release of TNF-α and TGF-β is mediated by matrix metallo-proteinases [55-58]. Based on the previous results, we propose that the specific reduction in TGF-β and not TNF-α is a consequence of the ROS increase. The hydroxyl radicals from ROS are able of modifying the LAP structure which consequently destabilizes the LLC and this may lead to the release or arrest of TGF-β depending on ROS concentration [59]. The ROS-mediated processes of TGF-β activation have been previously reported in several diseases [60-62]; however, more studies need to be developed to unravel the exact molecular mechanism of this immune modulation.

CONCLUSION

Terpenes are the main constituents of plants and exhibit many biological activities for humans. Terpenes classify according to the number of carbon atoms within their chemical structure; for instance, monoterpenes have 10 carbon atoms while diterpenes have 20 and triterpenes 30. The most studied biomedical activities of terpenes are antibacterial, anti-inflammatory and immunomodulatory.

The A549 cells have served as model of alveolar type II pulmonary epithelium, finding utility in research examining the metabolic processing of lung tissue and possible mechanisms of drug delivery to the tissue. The A549 cell line have been widely used in M. tb research; as a consequence of its permissibility to infection, the production of chemokines and the ability of producing RNS and ROS in response to M. tb infection. In the present study we determined that the diterpenes anastomosine and 7,20-dihydroanastomosine increased the production of RNS after 36 h of infection and treatment. Besides, the three diterpenes increased the production of ROS after 12 h. This RNS and ROS modulation can be considered as an in vitro correlation of innate immunity in response to M. tb infection; and an indicator of the damage of epithelial lung tissue. This study also showed an anti-inflammatory immune response by means of TGF-β modulation when compared with control group.

ACKNOWLEDGEMENTS

To the ‘Direccion de Investigacion y Posgrado (Universidad Autonoma de Coahuila; Torreon, Mexico)’ for the support provided for the publication of this manuscript.

LIST OF ABBREVIATIONS

TB

Tuberculosis

WHO

World Health Organization

TLR

Toll-Like Receptors

SPF

Specific-Pathogen-Free

FBS

Fetal Bovine Serum

OD

Optical Density

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

This protocol was approved by the Ethics Committee of the Faculty of Medicine, Universidad Autonoma de Coahuila; Torreon, México (reference number C.B.07/09/19).

HUMAN AND ANIMAL RIGHTS

No animal were used in this study. All animals research procedures were followed in accordance with The US National Research Council's “Guide for the Care and Use of Laboratory Animals”.

CONSENT FOR PUBLICATION

Not applicable.

AVAILABILITY OF DATA AND MATERIALS

The authors confirm that the data supporting the findings of this research are available within the article.

FUNDING

This work was supported by the authors.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

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