11β-Hydroxysteroid Dehydrogenases Are Regulated during the Pulmonary Granulomatous Response to the Mycobacterial Glycolipid Trehalose-6,6′-Dimycolate

Abstract

Objective: Tuberculosis has a staggering influence on world health, resulting in nearly 2 million deaths per year. The influence of glucocorticoids during Mycobacterium tuberculosis infection has been under investigation for decades; however, the identity of mycobacterial factors and the mechanism by which glucocorticoids are tissue specifically regulated to influence immune function during acute granuloma formation are unknown. Methods: One factor implicated in initiating immunopathology during M. tuberculosis infection is trehalose-6,6′-dimycolate (TDM), a glycolipid component of the mycobacterial cell wall. Intravenous administration of TDM causes inflammatory responses in lungs of mice similar to M. tuberculosis infection and has been used as a successful model to examine proinflammatory regulation and early events involved in the manifestation of pathology. Results and Conclusion: IL-6, IL-1α and TNF-α mRNA and protein peaked during the initiation of granuloma formation. Pulmonary corticosterone levels were elevated when the proinflammatory response was greatest, dropping to half of that upon the establishment of granuloma pathology on day 7. It is hypothesized that once corticosterone reaches the site of inflammation, the enzymes 11β-hydroxysteroid dehydrogenases (11βHSDs) can influence bioavailability by interconverting corticosterone and the inert metabolite 11-dehydrocorticosterone. RT-PCR demonstrated that pulmonary 11βHSD type 1 mRNA decreased 4-fold and 11βHSD type 2 (11βHSD2) mRNA expression increased 2.5-fold on day 3 after injection, suggesting that corticosterone regulation in the lung, specifically the reduction of active corticosterone by 11βHSD2, may influence the progression of granuloma formation in response to the mycobacterial glycolipid.

IL-6 Mediates 11βHSD Type 2 to Effect Progression of the Mycobacterial Cord Factor Trehalose 6,6′-Dimycolate-Induced Granulomatous Response

“…In vitro studies show that BMM produce IL-6, TNF-␣ and IL-1 ␤ following TDM treatment [25] . These findings emulate the response seen in vivo, where pro-inflammatory cytokine production is strongly upregulated as early as one day after intravenous administration of TDM [16,33] . Relevant to this study, Welsh et al [16] demonstrated that cohesiveness of the granulomatous structure is lost when mice are unable to produce IL-6, even when TNF-␣ levels remain sufficiently elevated.…”

“…Two micrograms of RNA was reverse transcribed and amplification of cDNA was assessed by the Taqman assay, using an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif., USA) per previously published methods and sequences [16,33] . Changes in gene expression for 11 ␤ HSD1 and 11 ␤ HSD2 were analyzed by using the -2 ⌬ ⌬ C T method as described [34] , with ␤ -actin used for normalization of samples.…”

“…Levels of IL-1 ␤ , IL-6, TNF-␣ and IL-12p40 were measured by a sandwich ELISA according to the manufacturer's instructions (R&D Systems), as previously described [16,30,35] . Briefly, capture antibody-coated Costar 96-well plates (Corning, Corning, N.Y., USA) were washed (0.5% Tween-20 in PBS) and blocked (1% BSA, 5% sucrose, 0.05% NaN 3 in PBS).…”

“…Specifically, the physiological role of 11 ␤ HSD type 2 (11 ␤ HSD2) is to convert active corticosterone (cortisol) into inert 11-dehydrocorticosterone (cortisone); whereas the inverse reaction is carried out by 11 ␤ HSD type 1 (11 ␤ HSD1). More recent findings indicate that in response to TDM, pulmonary corticosterone levels are altered in a pattern that is independent of serum concentrations [30] , suggesting that modulation of active GC moieties occurs directly within the lung itself. We hypothesize that induction of 11 ␤ HSD2 during granuloma development would convert corticosterone to the inert derivative, thus limiting corticosterone availability to GC receptors.…”

Influence of oral lactoferrin on Mycobacterium tuberculosis induced immunopathology

“…Total lung RNA was isolated by homogenizing lung tissue in 1 ml of RNA-Bee (Tel-Test, Friendswood, TX), followed by addition of 0.1 ml of chloroform (Sigma) 28–30 . The samples were mixed, incubated on ice for 15 min, and centrifuged for 15 min at 13,000 rpm.…”