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. 2017;9(2):162-180.
doi: 10.1159/000450955. Epub 2016 Nov 18.

Inflammatory Properties and Adjuvant Potential of Synthetic Glycolipids Homologous to Mycolate Esters of the Cell Wall of Mycobacterium tuberculosis

Hermann Giresse Tima  1 , Juma''a Raheem Al Dulayymi, Olivier Denis, Pauline Lehebel, Klarah Sherzad Baols, Mohsin Omar Mohammed, Laurent L'Homme, Mohaned Mohammed Sahb, Georges Potemberg, Sylvie Legrand, Roland Lang, Rudi Beyaert, Jacques Piette, Mark Stephen Baird, Kris Huygen, Marta Romano
Affiliations

Inflammatory Properties and Adjuvant Potential of Synthetic Glycolipids Homologous to Mycolate Esters of the Cell Wall of Mycobacterium tuberculosis

Hermann Giresse Tima et al. J Innate Immun. 2017.

Abstract

The cell wall of mycobacteria is characterised by glycolipids composed of different classes of mycolic acids (MAs; alpha-, keto-, and methoxy-) and sugars (trehalose, glucose, and arabinose). Studies using mutant Mtb strains have shown that the structure of MAs influences the inflammatory potential of these glycolipids. As mutant Mtb strains possess a complex mixture of glycolipids, we analysed the inflammatory potential of single classes of mycolate esters of the Mtb cell wall using 38 different synthetic analogues. Our results show that synthetic trehalose dimycolate (TDM) and trehalose, glucose, and arabinose monomycolates (TMM, GMM, and AraMM) activate bone marrow-derived dendritic cells in terms of the production of pro-inflammatory cytokines (IL-6 and TNF-α) and reactive oxygen species, upregulation of costimulatory molecules, and activation of NLRP3 inflammasome by a mechanism dependent on Mincle. These findings demonstrate that Mincle receptor can also recognise pentose esters and seem to contradict the hypothesis that production of GMM is an escape mechanism used by pathogenic mycobacteria to avoid recognition by the innate immune system. Finally, our experiments indicate that TMM and GMM, as well as TDM, can promote Th1 and Th17 responses in mice in an OVA immunisation model, and that further analysis of their potential as novel adjuvants for subunit vaccines is warranted.

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Figures

Fig. 1
Fig. 1
Synthetic TMM and TDM esters stimulate the in vitro production of TNF-α and IL-6 by BMDCs. a, b BMDCs derived from C57BL/6 mice were stimulated for 24 h in triplicate cultures with increasing concentrations of plate-coated synthetic cis-methoxy TMM-KB51, cis-methoxy TDM-KB52, or TDB. The supernatants were harvested from separate wells and the volume of pro-inflammatory cytokines (TNF-α and IL-6) was determined by sandwich ELISA. Results are expressed as the mean amount of cytokines ± SD of 3 independent experiments. c Separate BMDC cultures derived from C57BL/6 mice were stimulated for 24 h in triplicate with 9 pairs of synthetic TDM versus corresponding TMM esters at 0.1 µM, with evaporated ISO as a negative control and TDB or natural TDM at the same concentration as positive controls. The supernatants were harvested and the amount of TNF-α was determined by sandwich ELISA. Results are expressed as the mean amount of TNF-α ± SD and data of 4 representative experiments have been pooled. Statistical analysis of results obtained for the TDM/TMM pairs was performed by the Mann-Whitney test. * p < 0.05, ** p < 0.01, and *** p < 0.001. d Separate BMDC cultures derived from C57BL/6 mice were stimulated for 24 h in triplicate with single TDMs representative of the different classes and configurations of mycolates present in mycobacteria at 1 µM, with evaporated ISO as a negative control and TDB or natural TDM at the same concentration as positive controls. The supernatants were harvested and the amount of TNF-α was determined by sandwich ELISA. Results are expressed as the mean amount of TNF-α ± SD and data of 3 representative experiments have been pooled. Statistical analysis of the results of the different groups was performed by the Mann-Whitney test. ** p < 0.01 and *** p < 0.001.
Fig. 2
Fig. 2
Synthetic GMMs and AraMMs activate BMDCs. a–d BMDC cultures from C57BL/6 mice were stimulated for 24 h in triplicate with escalating concentrations of plate-coated synthetic mycolate esters composed of the same cis-methoxy mycolate bound twice to trehalose (TDM-KB52) or once to trehalose (TMM-KB51), glucose (GMM-SMP73), arabinose (AraMM-MOD16), or controls (natural TDM mix, TDB, or evaporated ISO). The supernatants were harvested and the volumes of pro-inflammatory cytokines (IL-6, a; IL-1β, b; TNF-α, c; IL-12p40, d) were determined by ELISA. Results are expressed as the mean amount of cytokines ± SD (n = 4 independent experiments). Statistical analysis on the results obtained for synthetic compounds and natural TDM mix compared to TDB was performed by the Mann-Whitney test. n.s., not significant. ** p < 0.01, and *** p < 0.001. e Upregulation of costimulatory molecules (CD86 and CD80) and MHC-II was evaluated by flow cytometric analysis after 48 h of stimulation with 10 µg/mL of synthetic mycolate esters composed of the same cis-methoxy mycolate bound twice to trehalose (TDM-KB52) or once to trehalose (TMM-KB51), glucose (GMM-SMP73), arabinose (AraMM-MOD16), or controls (unstimulated, LPS 100 ng/mL, or TDB 10 µg/mL). Results are the mean ± SD and representative of at least 3 independent experiments. Statistical analysis on results obtained for synthetic compounds, TDB, or LPS compared to unstimulated cells was performed by the Mann-Whitney test: n.s., not significant. * p < 0.05. f BMDCs were treated for 30 min with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) and stimulated for 4 h with 10 µg/mL of synthetic mycolate esters composed of the same cis-methoxy mycolate bound twice to trehalose (TDM-KB52) or once to trehalose (TMM-KB51), glucose (GMM-SMP73), arabinose (AraMM-MOD16), or controls (unstimulated, PMA 100 ng/mL, or TDB 10 µg/mL). The production of ROS by the tested compound (black line) was measured by flow cytometry and compared to unstimulated cells (grey shading).
Fig. 3
Fig. 3
Synthetic GMMs and AraMMs activate BMDCs by Mincle-dependent mechanisms. a–c BMDCs from C57BL/6 WT, MyD88−/−, FcRγ−/−, Mincle−/−, and MALT1−/− mice were stimulated in triplicate with 10 µg/mL of different synthetic mycolate esters, TDB, natural TDM mix, or negative control (evaporated ISO) for 24 h. The supernatants were analysed for their content in pro-inflammatory cytokines (TNF-α) by ELISA. Results are expressed as the mean volume of TNF-α ± SD and are representative of at least 4 independent experiments. d HEK293 reporter cells expressing murine Mincle were used to evaluate binding to the Mincle receptor. SEAP release after NF-κB activation was evaluated by colorimetric assay using Quantie-blue substrate and by measuring the OD at 655 nm.
Fig. 4
Fig. 4
Synthetic glucose and arabinose mycolate esters activate the NLRP3 inflammasome. a BMDCs prepared from C57BL/6 mice were primed for 3 h with 1 µg/mL of ultrapure LPS and then stimulated for 5 h with 10 µg/mL of synthetic mycolate esters, with evaporated ISO as a negative control and TDB or natural TDM mix at the same concentration as positive controls. The supernatants were harvested and the amount of IL-1β in the supernatants was determined by ELISA. b The secretion of active caspase-1 p20 was detected by Western blot in the supernatant after methanol-chloroform precipitation. NS, non-specific band. c Quantification of caspase-1 p20 in the supernatant. The relative intensity of caspase-1 p20 was determined relative to a non-specific band used as a loading control in the supernatant. d, e BMDCs from C57BL/6 mice were primed with 1 µg/mL of ultrapure LPS for 2 h, incubated with 10 µM of Z-VAD-FMK or control medium for 1 h and stimulated with 10 µg/mL of synthetic mycolate esters and controls for 5 h. Secretion of IL-1β and TNF-α was determined by ELISA in the supernatants. f, g BMDCs were prepared from WT, NLRP3−/−, and caspase-1/11−/− C57BL/6 mice and primed for 3 h with 1 µg/mL of ultrapure LPS before stimulation for 5 h with 10 µg/mL of synthetic mycolate esters and controls. The amount of IL-1β and TNF-α in the supernatants was determined by ELISA. Results represent the mean ± SD of triplicate cultures and are representative of at least 2 independent experiments.
Fig. 5
Fig. 5
In vivo inflammatory properties of synthetic mycolate esters. C57BL/6 mice were injected subcutaneously in the 2 hind footpads with w/o/w emulsions composed of 30% IFA, 10 µg/mouse of specified synthetic mycolate esters, and 50 µg/mouse of ovalbumin. In the emulsion of the vehicle control group no glycolipid was present. In the TDB control group 10 µg/mouse of TDB replaced the synthetic glycolipids. a Mean footpad size (mm) of 5 mice tested individually was measured with a caliper on the day of injection and 1, 3, and 7 days after injection. b The percentage of granulocytes (Ly6G+ and Ly6C+) was determined in cells isolated from the footpad by flow cytometry 7 days after administration. c–f The relative expressions of cytokines and chemokines were determined by qRT-PCR in the footpad cells. Fold changes were determined in comparison to vehicle. Footpad swelling and the percentage of granulocytes induced by synthetic mycolate esters were statistically analysed in comparison to TDB using the Mann-Whitney test. n.s., not significant. * p < 0.05, ** p < 0.01, and *** p < 0.001.
Fig. 6
Fig. 6
In vivo adjuvant potential of synthetic mycolate esters in an experimental OVA model. a C57BL/6 mice were injected subcutaneously in the 2 hind footpads with w/o/w emulsions composed of 30% IFA, 10 µg/mouse of specified synthetic mycolate esters, and 50 µg/mouse of ovalbumin. In the emulsion of the vehicle control group no glycolipid was present. In the TDB control group 10 µg/mouse of TDB replaced the synthetic glycolipids. Mice were sacrificed 7 days after immunisation and cells from pooled popliteal and inguinal lymph nodes were collected, counted (a) and stimulated with culture medium (RPMI) or 5 µg/mL of ovalbumin (OVA) for 24 or 72 h. Supernatants were harvested and the concentrations of IL-2 (b), IFN-γ (c), and IL-17A (d) were determined by ELISA. Results are means ± SD of 5 mice tested individually and representative of at least 4 independent experiments. n.s., not significant. * p < 0.05, and ** p < 0.01 as determined by the Mann-Whitney test.
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