DHEA induces autophagy in THP1-derived macrophages infected with Mycobacterium tuberculosis
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1
Instituto de Inmunología Clínica y Experimental de Rosario (IDICER-CONICET), Universidad Nacional de Rosario, Argentina
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2
Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Departamento de Patología, Mexico
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3
Instituto Nacional de Cardiología Ignacio Chávez, Departamento de Inmunología, Mexico
Tuberculosis (TB) constitutes an important health problem today. In 2014, an estimated 8.6 million people developed TB and 1.3 million died from the disease (including 320 000 deaths among HIV-positive people). The number of TB deaths is unacceptably large given that most of them are preventable. The disease is caused by Mycobacterium tuberculosis (Mtb), a facultative intracellular bacterium that is capable of surviving and persisting within host mononuclear cells.
In most cases, the immune response against Mtb is adequate and avoids the development of active disease. However, complete clearance of the pathogen is frequently not achieved. Mtb ensures its survival within host macrophages by arresting the maturation pathway that leads to phagosome–lysosome fusion, thus avoiding the phagolysosome that is rich in acid hydrolases capable of microbicidal degradation, and creating a suitable environment for bacillary survival and replication. As yet, while Mtb can block phagosome maturation, the induction of autophagy facilitates phagosome-lysosome fusion and bacilli clearance.
Among modulators of the immune response hormones like adrenal and gonadal steroids, and neurotransmitters are known to play an influential role in this regard. Given our earlier studies in which adrenal steroids were found to modify the cellular immune responses from TB patients, it was sensible to analyze the immunomodulatory capability of cortisol and DHEA on macrophages infected with Mtb.
In addressing this issue we have employed a model able to mimic the macrophage-Mtb interaction. Hence, we infected the human macrophage-like THP-1 cells with Mtb strain H37Rv and treated with Cortisol and DHEA at different doses. We monitored, cytokines gene expression and production, phagocytosis, intracellular-bacterial growth and autophagosoma formation. This approach provided new insight on the steroid hormones effects on the intracellular fate of Mtb. Cells were incubated in the presence or absence of Cortisol (10-6 M) and/or DHEA (10-6 M or 10-7 M) before Mtb (MOI 5:1).
Mtb-infected cultures had increased amounts of TNF-α, sTNFR (soluble TNF receptor), IL1-β and IL10 respect the control group. Except for sTNFR levels, which were significantly higher in Cortisol-treated cultures, this hormone decreased TNF-α, IL1-β and IL-10 levels compared to infected cultures left without hormones (p<0.001 vs Mtb alone). Values in DHEA-treated cultures remained within the range seen in cells exposed to Mtb. However, cultures exposed to Mtb treated with cortisol+DHEA continued to show lesser amounts of TNF-α, IL1-β and IL10 (p<0.001 vs Mtb+DHEA) together with higher quantities of sTNFR. In cultures left uninfected and treated with hormones (cortisol and/or DHEA), cytokines were undetectable.
Mtb induced the expression of TGF-β and IL23 compared to the control group (p<0.01 and p<0.05, respectively), with cortisol inhibiting the expression of the IL-23 but not TGF-β. DHEA failed to reverse such inhibitory effect. The pattern of IL23 expression in DHEA-treated cultures was similar to the one seen in M. tuberculosis-infected cultures. Infection with Mtb also led to a significant expression of TGF-β, although in this case treatment with Cortisol and/or DHEA resulted in no gross changes when compared with infected cultures left without hormones.
On the other hand, cortisol decreased the phagocytosis of Mtb (p<0.05) while the phagocytic capacity of macrophages was increased by DHEA without reaching statistical significance. The combination of Cortisol 10-6M and DHEA at 10-6M significantly reverted the Cortisol inhibitory effect (p<0.05).
Considering culture timing, macrophages exposed to Mtb alone showed an increase in the colony formation unit (CFU) numbers from day 1 to day 4 (p<0.01). By opposite, in cultures exposed to DHEA at 10-7 a trend to a decreased bacterial load as time elapsed was found, but differences remained statistically insignificant. Assessment at day 4 revealed that cultures treated with Gc+DHEA had significantly reduced CFU numbers with reference to their untreated counterparts.
In addition, infected macrophages showed numerous cytoplasmic vacuoles, like primary lysosomes, phagosomes and autophagosomes. The total number of lysosomes, phagosomes and autophagosomes were assessed in 30 cells by experimental condition and the number of autophagosomes was compared with the total number of cytoplasmic vacuoles. Control infected macrophages showed a mean of 13 ± 2 autophagosomes. A similar number was found when analyzing infected macrophages incubated with cortisol, whereas DHEA-treated macrophages displayed an increased autophagosome numbers, in 10-6 M and 10-7 M concentrations, respectively, p<0.01. Some of these cells showed bigger phagosomes forming large conglomerates. Respect to cells infected and treated with both hormones (Gc+DHEA), the Gc 10-6 M+DHEA 10-6 M combination displayed values similar to infected cultures without hormones. Unlike this, Gc 10-6 M+DHEA 10-7 M had a higher amounts of autophagosomes, some of them larger than those exhibited by infected macrophages without steroid hormones in the culture medium. Uninfected cells revealed no autophagosomes.
Interestingly the intra-macrophagic bacterial burden was found reduced by DHEA treatment. While this effect was not related to a different cytokine pattern, in terms of their production or mRNA expression, DHEA treatment did promote autophagy in Mtb-infected macrophages, irrespective of Cortisol presence.
Upon infection of macrophages, Mtb block phagosome maturation in order to survive but induction of autophagy facilitates phagosome-lysosome fusion and degradation of pathogen. In our hands, a lower dose of DHEA promoted autophagy in Mtb-infected macrophages, regardless of cortisol, suggesting that the CFU decrease from 1-4 days may be achieved through this mechanism.
In conclusion, DHEA in physiological conditions (that is in presence of cortisol) would leads to an autophagy induction and hence a better Mtb control. These results sound interesting since autophagy is not only important for bacterial clearance but also for the prevention of tissue damage. Last but not least, present results add support to the use of DHEA as an adjuvant therapy for TB.
Keywords:
Mycobacterium tuberculosis,
cortisol,
DHEA,
Macrophages,
autophagosome
Conference:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.
Presentation Type:
Oral Presentation
Topic:
Infectious and parasitic diseases
Citation:
Bongiovanni
B,
Mata-Espinosa
D,
D'Attilio
L,
León-Contreras
JC,
Marquez-Velasco
R,
Hernandez-Pando
R,
Bottasso
O and
Bay
ML
(2015). DHEA induces autophagy in THP1-derived macrophages infected with Mycobacterium tuberculosis.
Front. Immunol.
Conference Abstract:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología.
doi: 10.3389/conf.fimmu.2015.05.00299
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Received:
15 May 2015;
Published Online:
15 Sep 2015.
*
Correspondence:
PhD. Bettina Bongiovanni, Instituto de Inmunología Clínica y Experimental de Rosario (IDICER-CONICET), Universidad Nacional de Rosario, Rosario, Santa Fe, 2000, Argentina, bongiovanni@idicer-conicet.gob.ar