Discussion
TB-T2D comorbidity is a new area of interest due to its recent increased incidence worldwide. T2D is a metabolic endocrine disease associated with abnormal secretion and action of insulin and chronic hyperglycemia affecting multiple organ systems. T2D compromises host immune responses and impairs host ability to control microbial infections. Indeed, T2D produced several abnormalities in diverse immune mechanisms against Mtb that worsens the course of the disease requiring longer courses of chemotherapy (7, 8). Our results confirm the more aggressive course of TB in an experimental model of T2D/TB; and for the first time demonstrates that one significant pathogenic factor is the higher expression of 11-βHSD1 and corticosterone production during late advanced disease in the lungs and liver. The treatment with BEA efficiently decreases the expression of 11-βHSD1 and antagonizes corticosterone producing significant reductions of bacillary burdens and hyperglycemia. These results suggest that this synthetic hormone can contribute to the treatment of this co-morbidity.
Mtb affects mainly the lungs producing chronic and excessive inflammation in which innate and adaptive immunity are deeply affected (2, 3). Infection starts by inhalation of saliva droplets with Mtb that are engulfed by alveolar macrophages, which are key cells in bacilli elimination and together with dendritic cells process Mtb antigens that are presented to T lymphocytes in regional lymph nodes. Lymphocytes migrate to the lung and with macrophages and other cells form containment structures known as granulomas which are the histopathological hallmark of TB (5). In early stages of active infection, Th1 cellular immune responses are protective, as IFN-γ, TNFα and other cytokines such as IL-12 induce macrophage activation, allowing bacterial growth control. Nevertheless, during late active disease, extensive inflammation produces a shift toward anti-inflammatory responses, such as Th-2 cytokine pattern in which IL-4, IL-13 and other anti-inflammatory cytokines such as IL-10 and transforming growth factor-β (TGF-β) induce a local immunosuppressive/anti-inflammatory milieu, resulting in poor containment of infection and progression of tissue damage. Mortality rates approximate 1%. (30). Interestingly, besides these immunologic responses, there is an intense neuroendocrine response that creates a complex network of cytokines, hormones, and neurotransmitters that influence the outcome of TB pathogenesis (8, 9, 31). These neuroendocrine responses include abnormal production of several hormones, that is partly mediated by the cytokines released during the immune response to Mtb (pro-inflammatory mediators), and also contribute to modify the central nervous system (CNS) response by activating the two major stress systems, the HPA axis (32) and the sympathetic nervous system (SNS) (7,33).
HPA and GCs have an important contribution to the physiopathology of TB (34, 35). It was reported in the experimental model of progressive TB in BALB/c mice that at the time of maximal protective activity mediated by IFN-γ, TNF-α, IL-1β and NO production (day 21 after infection), pro-inflammatory cytokines such as TNF-α and IL-1β strongly activate the HPA axis, producing high expression of CRF in the hypothalamus and adrenal hyperplasia with high serum concentrations of corticosterone (32). Then, during the chronic or late phase after 28 days of infection, there is progressive adrenal atrophy and a corticosterone decrease in sera co-existence with extensive pneumonia (32). Thus, there is high GCs adrenal production apparently with the aim to avoid tissue damage produced by excessive lung inflammation. During the late phase of the disease when adrenal glands are atrophic, it seems that there is another source of corticosterone available by production in peripheral macrophages.
Our results show, for the first time, that this extra-adrenal source of GCs are macrophages, particularly foamy cells which strongly express the enzyme 11-βHSD type 1 and serve as a new source of corticosterone in the inflamed tuberculous lung. This local source of corticosteroids acts in an autocrine and paracrine manner. However, this local excess of corticosterone also inhibits Th1 lymphocyte activity and induces differentiation of Th-2 lymphocytes that favors bacterial survival and proliferation causing animals death (14). Thus, this local anti-inflammatory activity mediated by high production of corticosterone in affected organs during active late TB have deleterious effect. Antagonistic hormones such as DHEA can restore balance to the system.
DHEA is the most abundant product of the human adrenal gland after puberty with maximum levels occurring in the late 20s. Its production then falls steadily with increasing age. DHEA has anti-glucocorticoid properties in several systems. For example, DHEA shows opposite effects to GCs on enzyme expression in the liver in obesity (1) and in the immune system (4). In a previous study, treatment with DHEA starting on day 60 after Mtb infection in BALB/c mice reduced the pulmonary bacillary burden and decreased tissue damage by reactivating the Th-1 cytokine response (36). In humans a deficit in DHEA relative to cortisol can be striking in severe human TB (8). However, DHEA is suboptimal for human use, partly because it is metabolized into sex steroids.
16α-Bromoepiandrosterone (BEA; 16α-bromo-5α-androstan-3β-ol-17-one) is a synthetic adrenal steroid derivative that does not enter sex steroid pathways and when tuberculous BALB/c mice were treated with BEA, every other day beginning on day 60, it produced a significant inhibition of bacterial proliferation and higher expression of protective cytokines (TNF-α, IFN-γ). Moreover, when given as an adjunct to conventional chemotherapy, BEA enhanced bacterial clearance (36). Interestingly, this synthetic hormone showed therapeutic benefit in patients with TB (2), malaria (37) and in the Acquired Immune Deficiency Syndrome (AIDS). In the HIV/AIDS trial BEA reduced the incidence of TB co-infection by 42·2% and the cumulative incidence of opportunistic infections (38).
In the present study a new water-miscible formulation of BEA was used. Its ability to form a stable suspension in water is derived from a new method of formulating the molecule. Water miscibility avoids the problems associated with the organic solvents required to administer the older, lipid soluble formulation. Mice treated with this new BEA formulation showed similar results as the old synthetic analog, producing significant increase of IFN-γ and TNF-α, in coexistence with pulmonary bacillary loads reduction, and now we demonstrated a new activity, which is a significant pulmonary decrease in the expression of 11-βHSD1 and corticosterone. 11-βHSD2 which shifts the equilibrium to inactive cortisone was overexpressed as well. Thus, BEA can induce CD4 Th1 cells and macrophages activation by direct activity, and as shown in the present work, by the suppression of the local production of corticosterone in the lungs.
Obesity is commonly associated with T2D and other manifestations of the metabolic syndrome [1, 2]. The metabolic syndrome manifests as obesity, hyperlipidemia and T2D, and is related to insulin resistance in adipose tissue, skeletal muscle and liver [1, 3]. In obesity and T2D, GCs are important pathogenic factors because they are antagonists of insulin action. GC impair glucose uptake, enhance lipolysis and hepatic gluconeogenesis and promote proteolysis. Moreover, GCs directly inhibit pancreatic secretion of insulin, enhance glucose secretion by inhibiting gluconeogenesis in the liver and oppose other metabolic actions, such as insulin signaling and glucose uptake by inhibiting the translocation of the glucose transporter GLUT4 to the plasma membrane (19).
Our results show that in comparison with healthy control animals, mice with T2D without Mtb infection exhibited higher 11-βHSD1 expression and strong corticosterone immunostaining in bronchial epithelial cells and in hepatocytes. BEA treatment decreased the expression of both enzyme and hormone in lung and liver and corrected glucose concentration in blood. Thus, BEA significantly improved metabolic abnormalities by down-regulating 11-βHSD1 gene expression as one of the mechanisms. These results are in agreement with previous publications, and contribute new information such as the increase of corticosterone production in the airways epithelium of T2D individuals which could be a factor for higher predisposition of respiratory infections. One of these infections is TB.
Our results demonstrated a higher pulmonary bacillary load in T2D/TB than in TB mice without T2D, as well as lower expression of IFN-γ and TNF-α, in co-existence with higher expression of 11-βHSD1 and strong immunostaining to corticosterone in the pulmonary inflammatory intra-alveolar infiltrate, particularly in foamy macrophages. Thus, it seems that the higher pulmonary production of corticosterone mediated by 11-βHSD1 is a factor that worsens the course of TB in T2D and this could be a new therapeutic target.
In this regard, our results suggest that BEA can block 11-βHSD1 in the lung and in the liver, becoming an effective treatment for this co-morbidity. Trials for the use of BEA in the treatment of human TB, T2D and the co-morbidity T2D/TB are now justified.