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.