mRNA expression of alpha and beta isoforms of glucocorticoid receptor in peripheral blood mononuclear cells of patients with tuberculosis and its relation with components of the immunoendocrine response

Brain, Behavior, and Immunity 25 (2011) 461–467

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mRNA expression of alpha and beta isoforms of glucocorticoid receptor in peripheral blood mononuclear cells of patients with tuberculosis and its relation with components of the immunoendocrine response Luciano D’Attlio a, Ernesto Trini b, Bettina Bongiovanni a, Griselda Dídoli a, Walter Gardeñez c, Luis J. Nannini d, Adriana Giri e, Oscar A. Bottasso a, María Luisa Bay a,⇑ a

Instituto de Inmunología, Facultad de Ciencias Médicas, Universidad Nacional de Rosario, 2000 Rosario, Argentina Laboratorio de Estudios Moleculares y Genómica, 2000 Rosario, Argentina c Servicio de Neumonología, Hospital Provincial del Centenario, 2000 Rosario, Argentina d Servicio de Neumonología, Hospital Eva Perón, 2152 Granadero Baigorria, Argentina e Instituto de Biología Molecular y Celular de Rosario (IBR)-CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, 2000 Rosario, Argentina b

a r t i c l e

i n f o

Article history: Received 22 June 2010 Received in revised form 31 October 2010 Accepted 10 November 2010 Available online 17 November 2010 Keywords: Glucocorticoids Glucocorticoid receptor Cytokines Tuberculosis Hormones Immune–endocrine interactions

a b s t r a c t We have analyzed the expression of glucocorticoid receptor (GR) isoforms by real time RT-qPCR in PBMCs from 19 controls (HCo) and 28 TB patients (8 mild; 12 moderate; 8 severe), HIV(-) and similar sex and age distribution. mRNA hGRa/b ratios were found higher in TB patients respect to those in HCo. However, when analyzing for disease severity such overall trend was at the expense of mild and moderate patients, with severe cases showing a lower mRNA hGRa/b ratio with respect to the other patient groups. This suggested some degree of resistance to endogenous glucocorticoids in patients with severe TB, since hGRaa dimer mediates the biological functions of GC, with the GRb isoform acting as an inhibitor of GC activity. Levels of IL-6, IL-18, IFN-c and Cortisol were significantly increased in severe and moderate cases, whereas DHEA values were found decreased in them (p < 0.05 respect to HCo). Analysis on the relationship between plasma levels of these immuno-endocrine mediators with the mRNA expression of hGRa and hGRb showed that IL-6 was positively associated with hGRa in mild TB patients (p < 0.01), whereas a negative correlation between IFN-c and hGRb was observed in severe cases (p < 0.01). As regard to hormones, DHEA was positively associated with hGRa in moderate and severe cases (p < 0.01). This group also showed a negative correlation between hGRa and Cortisol/DHEA ratios (p < 0.05). Changes in the systemic levels of cytokine and adrenal hormones are likely to affect GR expression in a differential fashion and according to the amount of pulmonary involvement. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Tuberculosis (TB) constitutes an important health problem today considering that 8.8 million new TB cases emerge and 1.6 million people die of this disease every year (WHO, 2008). Individuals are infected by the inhalation of air-borne droplet nuclei containing Mycobacterium tuberculosis. Infection largely takes place in the lungs wherein the requirement of oxygen is assured, and begins as a non-specific inflammatory reaction in the alveolar space, that progresses to a typical delayed type granulomatous reaction. If the infected host is immunocompetent, the immune system would keep the infection in check using mechanisms that prevent further bacillary proliferation and limit the ⇑ Corresponding author. Address: Instituto de Inmunología, Facultad de Ciencias Médicas, Universidad Nacional de Rosario, Santa Fe 3100, 2000 Rosario, Argentina. Fax: +54 341 4804569. E-mail address: [email protected] (M.L. Bay). 0889-1591/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbi.2010.11.006

spreading of the organism. In 5–10% of latently infected cases granulomas may evolve unsatisfactorily with softening of the caseum and presence of very high numbers of bacilli and the subsequent initiation of secondary infection develops (Flynn, 2004; Russell et al., 2009). Postprimary TB can manifest with involvement ranging from few foci in the upper parts of the lungs to intense inflammation, tissue destruction, and caseous necrosis, which usually disintegrates forming cavities. Immune protection results from phagocytosis and killing of, or controlling the growth of mycobacteria by activated macrophages. This cellular response is exerted by antigen-specific T cells producing interferon c (IFN-c) and tumor necrosis factor alpha (TNF-a) (Arcila et al., 2007; Cooper, 2009; Flynn, 2004; Rook et al., 2004). Disease pathology in TB is also known to have an immunemediated component which is partly due to a disturbance in immune homeostasis. This encompasses alterations intrinsic or extrinsic to the immune system. Within this setting, an increasing body of evidence demonstrates that there is a bidirectional

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communication between the neuroendocrine and immune systems, in particular, the hypothalamus-pituitary-adrenal (HPA) axis which plays a critical role in maintaining immune homeostasis. Endogenous glucocorticoids (GC), acting as key molecule of the HPA axis, take part in coordinating immune response and subsequent modulation of immune responses against infectious agents and other insults (Besedovsky and del Rey, 1996; Rook et al., 2000; Turnbull and Rivier, 1999; Webster et al., 2002). GC modulate a wide variety of immune cell functions, like cytokine and adhesion molecule expression, immune cell trafficking, maturation and differentiation. GC are able to shift the immune responses from a pro-inflammatory to an anti-inflammatory cytokine pattern and also to facilitate Th2 activity partly by inhibiting Th1 cells (Besedovsky and del Rey, 1996; Rook et al., 2000; Turnbull and Rivier, 1999; Webster et al., 2002). In our studies, TB patients showed increased cortisol levels in circulation (del Rey et al., 2007) whereas treatment of peripheral blood mononuclear cells (PBMC) with physiological concentrations of cortisol inhibited mycobacterial antigen-driven lymphoproliferation and IFN-c production (Mahuad et al., 2004). GC interact with two main receptors: GC receptor (GR) and mineralocorticoid receptor. The primary receptor for GC in immune cells is GR which is located in the cytoplasm in an inactive multiprotein complex form containing chaperones and co-chaperones. Upon GC binding a molecular rearrangement of the GR heterocomplex ensues promoting nuclear localization, homodimerization, and DNA-binding (Barnes and Adcock, 2009; Grad and Picard, 2007; Lu and Cidlowski, 2006). DNA specific elements by which activated GR homodimer mediates its genomic effect are termed GC response elements (GRE). These are located within regulatory regions of GCresponsive genes and can mediate positive or negative effects depending on the nature of the GRE (Barnes and Adcock, 2009; Grad and Picard, 2007; Lu and Cidlowski, 2006). GRE-independent transcriptional influences have also been described, since GR interacts with transcription factors like nuclear factor kappa B (NF-jB), activator protein-1 (AP-1) and signal transducers and activators of transcription (STAT) (Barnes, 2006). In parallel, non-genomic GC actions are known. For instance, GC were found to stimulate the release of Src kinase from GR heterocomplexes, leading to lipocortin-1 activation and inhibition of arachidonic acid release (Croxtall et al., 2000). It follows that multiple genomic and non-genomic mechanisms take place in the GC influences on cellular processes (Bartholome et al., 2004; Stahn and Buttgereit, 2008). Cloning of the human GR (hGR) has identified two isoforms, termed hGRa and hGRb, which originate from alternative splicing of the hGR primary transcript (Hollenberg et al., 1985; Yudt and Cidlowski, 2002). The hGRb lacks the ability to bind GC and seems to function as an inhibitor of hGRa-mediated transcriptional activation through the formation of hGRa/hGRb heterodimers (Oakley et al., 1996; Oakley et al., 1999). Overexpression of hGRb in cells stimulated with inflammatory cytokines was shown to result in reduced effectiveness of GC action (Webster et al., 2001). In turn, excessive expression of the hGRb isoform in relation to hGRa is thought to play a role in the regulation of GC sensitivity in several inflammation-based pathological situations (Hamid et al., 1999; Honda et al., 2000; Orii et al., 2002). In the present study we have analyzed the expression of hGRa and hGRb isoforms of hGR in PBMC of TB patients with different degree of pulmonary involvement and its potential relation with the endogenous levels of inflammation-related cytokines and adrenal steroids. Our data demonstrate a reduced mRNA hGRa/b ratio in PBMC of severe patients. Correlation studies showed that hormone and cytokine environment was likely to influence the expression of hGR isoforms.

2. Materials and methods 2.1. Subjects Twenty-eight newly diagnosed HIV-seronegative patients without previous histories of TB were included. All patients had acid fast bacilli demonstrable in their sputum. The severity of the disease was classified according to the extent and type of X-ray findings into 3 groups: mild, patients with a single lobe involved, and without visible cavities; moderate, patients presenting unilateral involvement of 2 or more lobes with cavities, if present, reaching a total diameter no greater than 4 cm; advanced, bilateral disease with massive affectation and multiple cavities (Mahuad et al., 2004). Patients were divided into three subgroups: mild, moderate and severe. The mean age for patients was 37.7 ± 14.5 years (mean ± SD) and 5 were females. None of the patients were under treatment with anti-tuberculous therapy. Numbers of samples available for processing in the different assays were: mild 6–8, moderate 9–12, severe 7–8 and control 12–19. Nineteen healthy subjects (HCo) aged matched (38.7 ± 12.2 years, 3 females) and living in the same area but not known to be in contact with tuberculosis patients, were included as controls. For all participants exclusion criteria included: pathologies affecting the hypothalamus-pituitary-thyroid- or gonadal-axis, or with direct compromise of the adrenal grand, pregnancy, age under 18, as well as systemic or localized pathologies requiring treatment with corticosteroids or immunosuppressants. All subjects had the studies explained to them and agreed to participate and provided their informed consent. The study was approved by the Ethical Committee of the Facultad de Ciencias Médicas, Universidad Nacional de Rosario. 2.2. Sample collection Blood samples were obtained between 8:00–9:00 am, prior the initiation of the antibiotic treatment using EDTA as anticoagulant. Blood samples were immediately centrifuged and plasma was treated with aprotinin (100 U/ml plasma; TrasylolÒ, Bayer, Germany) shortly after the extraction and preserved at 20 °C. PBMC were isolated by gradient centrifugation using Ficoll-Paque plus (Amersham Biosciences Inc., Piscataway, NJ, USA) (Bozza et al., 2007). Immediately after, 5–8  106 cells per ml of TRIzol (Invitrogen, Carlsbad, USA) were stored at 80 °C until use. 2.3. Strategy for the quantification of hGRa and hGRb mRNA in PBMC Total RNA from PBMC was isolated by TRIzol and cDNA was synthesized from total RNA using reverse primers and M-MLV reverse transcriptase (USB Corporation, Cleveland, USA). qPCR was performed with SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK) and in all reactions the transcript of PPIA [peptidylprolyl isomerase A (Cycophilin A)] gene, was used as endogenous control in each PBMC sample, to normalize results (Bookout and Mangelsdorf, 2003; He et al., 2008). Serially diluted cDNA samples synthesized from Jurkat cell line, which expresses both hGRa and hGRb mRNA, were used as external standards in each run. By using automated programs of the ABI PRISMÒ 7500 Real Time PCR System (Applied Biosystems, Foster City, USA) the amounts of hGRa, hGRb, and PPIA templates in each sample were calculated to be equal to the dilution-fold of the standard cDNA of Jurkat cells. The actual values of hGRa and hGRb templates in each sample were normalized by the value of internal control PPIA template. All reagents and PCR conditions are described below. Simi-

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larity and homogeneity of PCR products from samples were confirmed by automated melting curve analysis (SDS 2.0 software, Applied Biosystems, Foster City, USA), which revealed Tm values of the PCR products. Primers were designed using the software Primer Express v2.0 (Applied Biosystems, Foster City, USA) based on available sequences at http://www.ncbi.nlm.nih.gov/nuccore/. Primers were designed targeting two different exons, with the aim of prevent genomic amplification. In order to differentiate hGRa and hGRb transcripts, oligonucleotids were designed using common forward primers in exon 8 and specific reverse primers targeting exon 9a and 9b, respectively. 2.4. RNA isolation, cDNA synthesis and qPCR Total RNA was isolated from cells using TRIzol (Invitrogen, Carlsbad, USA) according to the manufacture’s recommendations. RNA pellets were dissolved in DEPC sterile water and stored at 80 °C until analysis. RNA quantity was calculating by OD 260 spectrophotometry (NanoVue Plus Spectrophotometer, GE Healthcare, Little Chalfont, UK). The integrity of the purified RNA was determined by 2% agarose gel electrophoresis. cDNA was synthesized from 5 lg of total RNA using 200 U M-MLV reverse transcriptase (USB Corporation, Cleveland, USA) specifics reverse primer (Table 1). Briefly, 5x M-MLV Reaction Buffer, 0.4 mM dNTP (Amersham Biosciences Piscataway, USA); 21.5 U RNAasa Inhibitor (RNAguard, Amersham Biosciences, Piscataway, USA), 0.4 lM of each reverse primers; 2 mM MgCl2 (Invitrogen) and DEPC sterile water for 50 ll of final volume. Retrotranscription programmes consisted of 5 min at 65 °C, 1 h at 40 °C followed by enzyme inactivation at 95 °C for 3 min. cDNA was stored at 80 °C until use it. qPCR was performed with the ABI PRISMÒ 7500 Real Time PCR System (Applied Biosystems, Foster City, USA) using 10 ll dilution 1/200 of cDNA, 0.4 lM of each primer and 25 ll of SYBR Green PCR Master Mix 2X at a final volume of 50 ll. Thermal cycling conditions were: 2 min at 50 °C, 10 min at 95 °C followed by 45 PCR cycles of denaturing at 95 °C for 15 s, annealing and elongation at 60 °C for 1 min. Fluorescence readings were performed on annealing/elongation steps. Data were expressed as arbitrary units -AU-, where 1 AU equals to one microgram of standard mRNA. 2.5. Cytokines and hormones determinations Levels of IL-6, IL-18 and IFN-c; cortisol, DHEA and DHEA-sulfate (DHEAS) were measured in plasma with commercially available ELISA kits according to the instructions of the manufacturer (Amersham Biosciences UK Limited, Little Chalfont Buckinghamshire, UK; MBL, Naka-Ku Nagoya, Japan; set OptEIA BD Biosciences, San Diego, USA; DRG Instruments GmbH, Marburg, Germany; respectively). Detection limits were: 0.1 pg/ml for IL-6; 25.6 pg/ ml for IL-18; 4.7 pg/ml for IFN-c; 2.5 ng/ml for cortisol; 0.1 ng/ml for DHEA and 0.044 lg/ml for DHEAS.

Table 2 Expression of hGRa and hGRb mRNA in PBMC from TB patients and healthy controls (HCo). Transcripts

mRNA hGRaa mRNA hGRba mRNA hGRa/b

Study groups HCo (n = 15)

TB (n = 22)

p value

16.54 (10.89–46.83) 32.42 ± 30.44 7.90 (3.92–10.95) 12.03 ± 13.84 3.07 (1.79–4.28) 3.09 ± 1.14

16.61 (12.38–26.92) 29.31 ± 28.20 5.79 (2.64–10.57) 7.54 ± 6.42 4.34 (2.35–6.43) 4.74 ± 2.70

ns ns <0.05*

a Data are represented as median (25–75 percentiles) and means ± SD of arbitrary units -AU- where 1 AU equals to one microgram of standard mRNA. ns: not significant. * Mann–Whitney U test.

2.6. Statistical analysis Comparisons among groups were performed by non parametric methods, such as the Kruskall–Wallis analysis of variance and Mann–Whitney U test. Correlations between hormone and cytokine levels with mRNA expression of hGR isoforms were analysed by the Spearman’s rank test. Data were considered statistically significant if p < 0.05. 3. Results 3.1. Expression of hGRa and hGRb by real-time RT-PCR Studies on the expression of hGRa and hGRb mRNAs in PBMC revealed no significant differences between TB and HCo groups (Table 2), although the expression of hGRb mRNA was on average 40% less in TB patients than in HCo. This reflected in a higher mRNA hGRa/b ratio of TB patients when compared to HCo (p < 0.05, same Table). Analysis by separating TB patients according to the extent of their lung compromise showed no significant differences in the expression of both hGR mRNA isoforms when each patient subgroups was compared to HCo (Table 3). A trend to a lessened expression of hGRb mRNA was found in mild and moderate cases (54% and 48%, respectively). As such, the mRNA hGRa/b ratio in PBMC of mild and moderate patients situated significantly above those observed in severe cases and HCo (p < 0.05, Table 3). There was no significant difference between mRNA hGRa/b ratio of severe patients and HCo (same Table). 3.2. Plasma cytokine levels As shown in Fig. 1 (panel A) plasma levels of IFN-c in TB patients grew proportionally with the severity of the disease, yielding a significant difference when moderate and severe cases were compared to HCo (p < 0.01, both cases). TNF-a plasma concentra-

Table 1 Real-time nucleotide primer sequence.

CycA; PPIA GeneID: 5478, NM_0211303 hGRa; NR3C1, GeneID: 2908, NM_001018077.1 hGRb; NR3C1, GeneID: 2908, transcript variant 6 NM_001020825.1

Forward primer

Reverse primer

Size

CycA-F (location: 356–377) 50 -gca tac ggg tcc tgg catc ttg-30 hGR-F (location: 3107–3129) 50 -gaa gga aac tcc agc cag aac tg-30 hGR-F (location:2605–2627) 50 -gaa gga aac tcc agc cag aac tg-30

CycA-R (location: 465–434) 50 -tgc cat cca acc act cag tct tg-30 hGRa-R (location: 3265–3244) 50 -gat gat ttc agc taa cat ctc g-30 hGRb-R (location: 2748–2730) 50 -tga gcg cca aga ttg ttg g-30

101 bp 159 bp 144 bp

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Table 3 Expression of hGRa and hGRb mRNA in PBMCs from healthy controls (HCo) and TB patients separated according to disease severity. Transcripts

Study groups

mRNA hGRaa mRNA hGRb

a

mRNA hGRa/b

HCo (n = 15)

Mild (n = 6)

Moderate (n = 9)

Severe (n = 7)

p

16.54 (10.89–46.83) 32.42 ± 30.44 7.90 (3.91–10.95) 12.03 ± 13.84 3.07 (1.79–4.28) 3.09 ± 1.14

18.46 (14.42–26.92) 27.72 ± 23.29 5.22 (2.81–7.02) 5.52 ± 3.46 5.10 (3.83–6.55) 5.38 ± 2.42

15.64 (11.2–17.5) 25.53 ± 26.68 3.54 (2.11–10.56) 6.34 ± 6.11 4.41 (3.46–6.45) 5.25 ± 3.25

18.73 (12.8–38.3) 35.54 ± 36.25 8.33 (5.65–16.75) 10.81 ± 8.11 2.57 (1.93–5.86) 3.53 ± 1.99

ns ns <0.05

Mild and moderate patients, different from severe cases and HCo (Kruskall–Wallis analysis of variance and Mann–Whitney U test). ns: not significant. a Data are represented as median (25–75 percentiles) and means ± SD of arbitrary units -AU- where 1 AU equals to one microgram of standard mRNA.

tions either in TB patients or in HCo were near to the limit of detection of the assay showing no differences among them (data not shown). IL-18 values also increased with disease severity, particularly in moderate and severe patients that showed levels significantly augmented respect to the HCo group (p < 0.05 and p < 0.01, respectively, panel B). Among TB patients, IL-6 levels were found significantly higher than those seen in the HCo, being more evident in moderate and advanced cases (p < 0.01; Fig. 1 panel C) but also in the mild group (p < 0.05, same panel). 3.3. Hormone circulating levels We next proceeded to analyze levels of adrenal steroids in TB patients and HCo. As most of these hormones show physiological age variations, pair wise correlations between age and individual plasma concentrations were performed, showing no significant associations no matter the combinations under analysis. Data from cortisol measurements showed significantly elevated levels in moderate and severe cases, respect HCo (p < 0.01 and p < 0.05, respectively; Fig. 2, panel A) and mild patients (p < 0.01; Fig. 2,

IFN- γ pg/ml

200

A

panel A). Plasma from TB patients was characterized by remarkably decreased levels of DHEA compared to HCo (p < 0.05, respect mild and moderate cases; Fig. 2, panel B). The magnitude of decrease was even higher in severe cases (p < 0.01, Fig. 2, panel B). Plasmatic levels of DHEAS were also reduced in TB patients but this trend did not reach statistical significance when compared to HCo (Fig. 2, panel C). Since the balance between cortisol and DHEA may be more relevant than single hormones when attempting to reflect plasma adrenal steroid profile, their ratios were also calculated. Both cortisol/DHEA and cortisol/DHEAS ratios were increased in all groups of TB patients (Fig. 2, panel D and E, respectively). Differences were statistically significant when moderate and severe cases were compared to HCo, either for cortisol/DHEAS (p < 0.05, both groups) or cortisol/DHEA ratios (p < 0.01 and p < 0.05, severe and moderate cases, respectively). Analysis on the relation among cytokines and hormones revealed a positive correlation between IL-18 with IFN-c and IFN-c with cortisol in TB patients (r = 0.59, p < 0.01 and r = 0.48, p < 0.05, respectively). Other pair wise correlations revealed no statistical differences.

**

150

** 100 50 0

1500

Hco

Mild

(n=16)

(n=7)

Mod (n=9)

Sev (n=8)

60

B

**

C

**

500

IL-6 pg/ml

IL-18 ng/ml

* 1000

40 **

* 20

0

0 HCo (n=16)

Mild

Mod

Sev

HCo

(n=8)

(n=12)

(n=8)

(n=12)

Mild (n=8)

Mod (n=10)

Sev (n=8)

Fig. 1. Plasma levels of IFN-c, IL-18 and IL-6 in TB patients with different degree of pulmonary involvement. Patients were separated according to disease severity into mild, moderate and severe cases. The exact number of plasma samples studied in each case is provided between brackets. Box plots show 25–75 percentiles of data values in each group with maximum and minimum values. The line represents the median values. Significant differences in relation to healthy controls (HCo) are depicted by ‘⁄’ and ‘⁄⁄’, p < 0.05 and p < 0.01, respectively. Panel A, IFN-c; Panel B, IL-18; Panel C, IL-6. Overall comparisons were performed by the Kruskall–Wallis analysis of variance followed by the Mann–Whitney U test, when applicable, for comparisons between two groups.

L. D’Attlio et al. / Brain, Behavior, and Immunity 25 (2011) 461–467

465

A

B

D

C

E

Fig. 2. Differential plasma levels of Cortisol, DHEA and DHEAS in pulmonary TB patients with different disease severity. Patients were separated according to disease severity into mild, moderate and severe cases. The exact number of plasma samples studied in each case is provided between brackets. Box plots show 25–75 percentiles of data values in each group with maximum and minimum values. The line represents the median value. Cortisol levels in moderate and severe cases differed significantly from values of mild patients p < 0.01. Significant differences in relation to healthy controls (HCo) are depicted by ‘⁄’ and ‘⁄⁄’, p < 0.05 and p < 0.01, respectively. Panel A, cortisol; Panel B, DHEA; Panel C, DHEAS; Panel D, cortisol/DHEA ratio; Panel E, cortisol/DHEAS ratio. Overall comparisons were performed by the Kruskall–Wallis analysis of variance followed by the Mann–Whitney U test, when applicable, for comparisons between two groups.

3.4. Correlation studies between GR isoforms expression and circulating levels of immuno-endocrine compounds Because expression hGR mRNA isoforms may be associated with cytokine- or hormone-induced GC insensitivity we further Table 4 Correlation analysis of hormone and cytokine plasma levels with hGRa and hGRb mRNA and mRNA hGRa/b ratio in TB patients. Pair

r coefficient

p value

Mild (n = 6) IL-6 vs. mRNA hGRa

0.92

<0.01

Moderate (n = 9) DHEA vs. mRNA hGRa Cortisol/DHEA vs. mRNA hGRa/b Cortisol/DHEAS vs. mRNA hGRa/b

0.93 0.83 0.82

<0.01 <0.01 <0.01

Severe (n = 7) DHEA vs. mRNA hGRa Cortisol/DHEA vs. mRNA hGRa IFN-c vs. mRNA hGRb

0.90 0.85 0.99

<0.01 <0.05 <0.01

Spearman’s coefficient of correlation: r.

investigated whether hGRa and hGRb transcripts and mRNA hGRa/b ratio were related with cytokine or adrenal steroids levels in circulation. Except for an association of IL-18 with mRNA hGRa/ b in HCo (r = 0.87, p < 0.01), relevant relations were recorded among TB patients. Data summarized in Table 4 indicate highly correlations in most cases. There was a positive correlation between hGRa mRNA with IL-6 and DHEA levels in mild and moderate patients, respectively (p < 0.01). In the latter patient group the Cortisol/DHEA and Cortisol/DHEAS balance was positively correlated with the mRNA hGR a/b (p < 0.01, both cases). Within severe cases, a positive correlation between DHEA levels with hGRa transcripts was found, whereas the Cortisol/DHEA balance and IFN-c concentrations were negatively correlated with hGRa and hGRb mRNA, respectively (p < 0.05 and p < 0.01, Table 4). 4. Discussion It is becoming clear that the sensitivity of a target cell to GC not only depends on the concentrations of the active GC but also on the specific intracellular environment, in turn determined by numer-

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ous molecules implicated in or influencing the GC signaling cascade (Kino et al., 2003; Bauer, 2005). The GR is a major component in this regard being implied in many GC actions under normal or pathological states. In the present study, we have analyzed the expression of hGR isoforms in PBMC in TB patients by real time RT-qPCR. Our results show that the mRNA hGRa/b ratio in TB patients as a whole was higher than those in the control group. However, when analyzing these ratios according to disease severity, such overall trend was found to be at the expense of mild and moderate TB patients, with severe cases showing a lower mRNA hGRa/b ratio respect the other patient groups. Findings in severe cases are compatible with a certain degree of GC resistance, which may serve to explain the increased inflammation and tissue destruction occurring in this type of patients. In fact, GC are pleiotropic hormones with a wide spectrum of immunomodulatory and anti-inflammatory properties. Homeostatic effects of endogenous GC are primarily exerted by inhibiting the production of various pro-inflammatory mediators (Besedovsky and del Rey, 1996; Rook et al., 2000; Turnbull and Rivier, 1999; Webster et al., 2002). This inhibition was shown to largely depend on the ability of the activated GR to hinder the activity of transcription factors, such as activator protein-1, nuclear factor of activated T cells, NF-jB, and STATs. The mechanisms by which mRNA hGRa/b ratio vary according to disease severity may be of different nature. Since cytokine and hormones are likely to regulate GR expression (Webster et al., 2001), and TB patients present a distinct immuno-endocrine profile as disease progresses (del Rey et al., 2007) cytokine and hormonal changes may be playing a role in these transcript differences. As such, the relationship between serum levels of adrenal steroid levels and cytokines with the expression of hGR isoforms was worth exploring. Findings suggest that the pre-existing hormone and cytokine environment influences the expression of GR isoforms, in a differential way depending on the disease extent. In some circumstances associations may be the reflection of a mechanism addressed to cope with the accompanying inflammatory response, for example the positive correlation between IL-6 and hGRa of mild patients. In the case of severe cases the negative relation of IFN-c with hGRb may be also viewed as an attempt to favor a better GR-mediated effect of GC, although the mRNA hGRa/b ratio turned out to be inappropriate for achieving such effect. As regards hormones, levels of adrenal steroids were closely related with hGR isoforms expression. To the well known anti-inflammatory effects of DHEA ‘‘per se’’ (Dillon, 2005), this steroid may be also favoring a better GC signaling in view of the positive correlation between DHEA and hGRa mRNA levels seen in moderate and severe patients. Also, the balance between cortisol and DHEA or DHEAS was clearly relevant as to the way that hGR expression is modulated. In the case of moderate patients such positive association also suggests a facilitating effect of adrenal steroids balance for hGR-mediated activity of GC. Conversely, in severe patients Cortisol/DHEA was negatively correlated with mRNA hGRa implying a more adverse scenario for GC activity. Our results also highlight other aspects of the intricate relationship between components of the inflammatory response. For instance, the positive correlation IL-18 and mRNA hGR a/b seen in healthy individuals. Moving to moderate and severe patients despite their above described correlations, levels of pro-inflammatory compounds were high. This may be part of a defensive mechanism accompanying disease development, although in light of the chronic nature of TB and the protracted response mounted by the host a defective regulation of the inflammatory response may be present, as well. With reference to this, while IFN-c and cortisol levels were positively correlated, a relative deficit of the HPA axis

cannot be discarded considering the modest increase of cortisol concentrations displayed by TB patients. In relation to GC resistance, besides the reduced mRNA hGRa/b ratio seen in severe cases, other mechanisms may be also working in parallel, i.e., defective hGR binding and translocation, increased P-glycoprotein, macrophage migration inhibitory factor and abnormal histone acetylation, among others (Barnes and Adcock, 2009). Whatever the case, it is clear that the greater the severity the higher the levels of cytokines capable of exerting a role in TB immunopathology. As a pleiotropic cytokine, IL-18 possesses powerful inflammatory properties and widespread immune-augmenting functions (Gracie et al., 2003). For example, in the presence of IL-12, IL-18 drives the Th1 polarization and is protective during experimental TB (Schneider et al., 2010), whereas in the absence of IL-12, IL-18 promotes Th2 differentiation (Gracie et al., 2003). The positive association between IL-18 and IFN-c in TB patients is in line with this demonstration. Nevertheless, increased levels of both cytokines, particularly in TB patients with progressive disease, suggest that they are devoid of their protective activity being mostly proinflammatory. In relation to IFN-c, this cytokine is of central relevance for controlling M. tuberculosis infection (Cooper et al., 2002), but recent studies suggested that IFN-c production is somehow related to the bacterial load of TB patients (Lalvani, 2004), serving to explain the present results. Confirming our previous findings (del Rey et al., 2007), circulating levels of IL-6 increased as disease aggravated. Increased IL-6 production was found during the early stages of mycobacterial infection (Hoheisel et al., 1998). Studies in IL-6-deficient mice revealed an increased susceptibility to infection with M. tuberculosis (Ladel et al., 1997), probably linked to a deficient production of IFN-c in the initial phase of the infection (Saunders et al., 2000). Even when IL-6 is known to exert both pro- and anti-inflammatory effects (Van Heyningen et al., 1997), present findings seem more compatible with a phlogistic activity. As reported earlier, DHEA levels were decreased in TB patients (del Rey et al., 2007; Bozza et al., 2009). Reduced amounts of DHEA may be partly due to immune-derived products, considering the effects of cytokines on endocrine functions (Besedovsky and del Rey, 1996; Rook et al., 2000; Turnbull and Rivier, 1999; Webster et al., 2002) and our studies in which culture supernatants from antigen-stimulated PBMC of TB patients inhibited DHEA production by adrenal cells (del Rey et al., 2007). Concomitantly, bacterial products and pro-inflammatory cytokines can inhibit the activity of DHEAS sulfatase in macrophages reducing the transformation of the sulfated mediator into DHEA (Hennebold and Daynes, 1994). This phenomenon may also account for the reduced levels of DHEA detected in our series of TB patients. Upon an infectious insult, the host mounts a complex response essential for maintaining the physiological homeostasis and good health. A common feature of this response is the activation of the HPA axis, attempting not only to optimize this pro-homeostatic response but also protecting against potentially damaging effects of such response. In a situation in which the pathogen cannot be cleared, like human TB, the neuro-immuno-endocrine response may be detrimental. Present studies add new evidence as to the immuno-endocrine features of progressive disease, in which the lower mRNA hGRa/b ratio coexists with increased amounts of compounds with substantial proinflammatory effects, slightly augmented cortisol levels, and reduced DHEA concentrations. Results also suggest distinct immunoregulation patterns throughout the disease spectrum as judged by the severity-associated differential relation between expression of GR isoforms with cytokine and hormonal influences. While correlation does not imply causation, the closeness of associations reported here underscores the deep bidirectional

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