Formation of Foamy Macrophages by Tuberculous Pleural Effusions is triggered by the IL-10/STAT3 Axis through ACAT up-regulation

FRANCO MARIN, JOSÉ L.; MILILLO, M. AYELÉN; POGGI, SUSANA; GONZALEZ-DOMINGUEZ, E; REARTE, BÁRBARA; ASIS, AGOSTINA CROTTA; NEYROLLES, OLIVIER; HERNÁNDEZ-PANDO, R.; SASIAIN, MARÍA DEL CARMEN; GENOULA, MELANIE; KVIATCOVSKY, DENISE; MORAÑA, EDUARDO J; MATA-ESPINOZA, D.; BARRIONUEVO, PAULA; FONTANALS, ADRIANA; COUGOULE, CÉLINE; SANCHEZ-TORRES, C.; LUGO-VILLARINO, GEANNCARLO; DUPONT, MAEVA; SCHIERLOH, PABLO; PALMERO, DOMINGO; CONTRERAS, JUAN CARLOS LEÓN; CORDOBA MORENO, M.; GAGO, GABRIELA; MARIDONNEAU-PARINI, ISABELLE; VEROLLET, CHRISTEL; BALBOA, LUCIANA

Cancún

Congreso; XII Congress of the Latin American Association of Immunology & XXIII Congress of the Mexican Society of Immunology; 2018

ALAI

Tuberculosis (TB) is a highly contagious disease caused by Mycobacterium tuberculosis (Mtb) infection. The ability of Mtb to persist in its human host relies on numerous immune evasion strategies, such as the deregulation of the lipid metabolism leading to the formation of foamy macrophages (FM). The formation of FM is caused by infectious agents through deregulation in the balance between the influx and efflux of lipids. The free cholesterol released from the endocytosed lipoproteins moves from lysosomes to the endoplasmic reticulum, where it is either re-esterified to cholesteryl ester by the enzyme acylCoA:cholesterol acyltransferase (ACAT), which can then be stored in cytosolic droplets (lipid bodies) (1). Lipid body accumulation within leukocytes is a common feature in both clinical and experimental infections, especially in mycobacterial infections (2, 3). Mtb infection leads to the induction of FM, process which is promoted by several mycobacterial lipids (4, 5, 6, 7). This event enables the fusion between Mtb-containing phagosomes and lipid bodies resulting in an abundant supply of lipids for the pathogen (4) which enables Mtb to switch into a dormancy phenotype and to become tolerant to several front-line antibiotics (8). Besides, lipid bodies represent a secure niche in order to avoid bactericidal mechanisms such as respiratory burst (9). Concerning the impact of FM on the host immunity against Mtb, it was shown that human macrophages exposed to lipids prior to Mtb infection failed to produce TNF-α and to clear the infection (10, 11). Taking into account that FM generated prior to Mtb infection impair the host immune response, there is a keen interest to identify the host-derived cytokines released at the site of Mtb infection, and to understand how these signals contribute to FM differentiation and alter host defense against Mtb. In this regard, it is well known that different activation programs in macrophages driven by pro or anti-inflammatory cytokines are associated to changes in the lipid metabolism (12). Therefore, it is likely that host cytokines produced in response to Mtb infection contribute to lipids turnover promoting FM formation, and consequently lead to Mtb persistence. To model those soluble host-immune factors we employed a physiological relevant sample derived from active TB patients such as the acellular fraction of tuberculous pleural effusions (TB-PE). Then, we treated human M-CSF-driven macrophages with cell-free preparations of pleural effusions derived from patients with tuberculosis (TB-PE) in order to mimic a genuine microenvironment derived during Mtb infection. According to the pattern of staining of neutral lipids with Oil Red O (ORO), we observed that TB-PE treatment induced lipid bodies accumulation in macrophages to the same extent as Mtb infection or exposure to Mtb-derived lipids and that it was specific for TB-PE treatment in comparison to pleural effusions from patients with heart failure (HF-PE). This foamy phenotype induced by TB-PE was accompanied by higher abundance of cholesteryl esters and triacylglycerols and higher CD36 expression in comparison to HF-PE-treated macrophages. Besides, we showed that the foamy formation was dependent on ACAT activity, as judged both by the increase of ACAT expression after TB-PE treatment and by the prevention of lipid bodies accumulation in the presence of Sandoz, a specific inhibitor of ACAT. Noticeably, IL-10 depletion from TB-PE prevented the augmentation of all these parameters but not that of IL-1β, IL-6, IFN-γ, IL-4, or TNF-α. In line with it, the complementation of IL-10-depleted TB-PE with exogenous IL-10 restored the foamy phenotype in a dose-dependent manner. Additionally, macrophages treated with IL-10-depleted TB-PE showed smaller sized lipidic vacuoles than those exposed to non-depleted TB-PE when cells were observed by electron microscopy. Moreover, we found a positive correlation between the levels of IL-10 and the number of lipid-laden CD14+ cells among the pleural cells in TB patients, demonstrating that FM differentiation occurs within the pleural environment. Additionally, we observed that the acquisition of the foamy phenotype was associated with immunosuppressive properties such as a higher expression of anti-inflammatory markers, like CD163, mannose receptor (MRC1), and PD-L1, and a lower expression of HLA-DR, high IL-10 release, low TNF-α production, impaired Th1 activation, and high bacillary loads. To our concern, we provide evidence for the first time that FM display a reduced ability to activate a recall Th1 response of specific anti-mycobacterial T cell clones. Downstream of IL-10 signaling, we found that the transcription factor STAT3 was activated by TB-PE, detecting its phosphorylated form by western blot and immunofluorescence microscopy, and its chemical inhibition with Stattic prevented the accumulation of lipid bodies and ACAT expression in macrophages. Finally, we confirmed our results comparing whether BMDM derived from WT or IL-10-KO mice differed in their propensity to accumulate lipid bodies in response to Mtb-derived lipids. IL-10 deficiency partially prevented foamy phenotype induced by Mtb lipids, which in turn could be reverted by the addition of exogenous IL-10. Based on our findings, we propose a model for the modulation of FM in the context of a physiologically relevant microenvironment promoted by Mtb infection in which the axis IL-10/STAT3 promotes the accumulation of lipid bodies throughout ACAT up-regulation that is accompanied by an increase of CD36 and the acquisition of immunosuppressive properties such as a reduced induction of anti-mycobacterial Th1 clones, an enhanced production of IL-10 and a more permissive phenotype for bacillary growth.In conclusion, our results provide additional mechanisms by which the environment created by the infection process can drive the foamy differentiation even in the absence of the pathogen such as we observed that uninfected macrophages can be driven into FM in the presence of IL-10 and a source of lipids. These uninfected lipid-rich cells can abrogate the host innate and adaptive cellular defense mechanisms, and when these cells become infected, they may further favor pathogen persistence. Therefore, this knowledge may contribute to the identification of host molecular pathways that could be modulated to the benefit of the patient. In this regard, a better understanding of the molecular mechanisms underlying host-pathogen interactions could provide a rational basis for the development of effective anti-TB therapeutics.