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Article history:
Received 31 March 2016
Received in revised form
18 July 2016
Accepted 20 July 2016
Available online 21 July 2016
Pineal melatonin is known for its immunomodulatory and anti-stress properties. It modulates stress
condition by regulating antioxidant responses and apoptosis in the immune cells. Stress causes increased
glucocorticoid level that acts through glucocorticoid receptor (GR) and is translocated into nucleus under
regulation of HSP90 based chaperone machinery. Melatonin inuences glucocorticoid and GR mediated
stress condition in rodents, but till date there are no reports which could suggest the effect of melatonin
treatment on GR mediated apoptosis and inhibition of Nrf-2/hemeoxygenase-1 (HO-1) induced antioxidant status in immunocompetent cells (peripheral blood mononuclear cells; PBMCs). Therefore, in the
present study, we considered GR mediated inhibition of Nrf2 and HO-1 along with anti-apoptotic Bcl-2
expression in PBMCs. The PBMCs were treated with synthetic glucocorticoid; dexamethasone (Dex) and
melatonin (Mel), to explore the effect of melatonin treatment in regulation of GR mediated apoptosis and
inhibition of antioxidant status in immune cells. It was noted that melatonin treatment retained GR into
cytoplasm by inhibiting the dissociation of HSP90 from GR-HSP90 complex and enhanced expression of
Nrf2/HO-1 and Bcl-2 expression. This led to increased HO-1 expression and elevated Bcl-2 led to
increased Bcl-2/Bax ratio that might ultimately enhanced the cellular antioxidant response and survival
under glucocorticoid mediated stress condition. Our observations suggest that the declined GR nuclear
translocation upon melatonin treatment might be responsible for the up-regulation of Nrf2 mediated
HO-1 activity and increased Bcl-2/Bax ratio in PBMCs to maintain the immune homeostasis under stress
condition.
2016 Published by Elsevier Ireland Ltd.
Keywords:
Glucocorticoid receptor
Melatonin
Nrf2
HO-1
PBMCs
Dexamethasone
1. Introduction
Stress is a constellation of events that acts as stimulus (stressor)
to activate stress response in the physiological system (Dhabhar
and McEwen, 2001). Stress has been reported to suppress or dysregulate immune function (Glaser and Kiecolt-Glaser, 2005) and
increase susceptibility to various infections (Cohen et al., 1991). The
detrimental effect of stress causes imbalance of the physiological
homeostasis by declining the immune function (such as the
reduction of immune cells activity, lymphocyte population, proliferation and NK cell activity) (Webster Marketon and Glaser, 2008)
along with decreased antioxidant response that leads to
* Corresponding author.
E-mail addresses: amareshsingh86@gmail.com (A.K. Singh), chaldar2001@
yahoo.com (C. Haldar).
http://dx.doi.org/10.1016/j.mce.2016.07.024
0303-7207/ 2016 Published by Elsevier Ireland Ltd.
60
A.K. Singh, C. Haldar / Molecular and Cellular Endocrinology 436 (2016) 59e67
(PPR). The goats were treated with antihelmintics twice per year
and 0.5% solution of malathion (acaricidal baths) was sprayed
externally at 7e20 days as described by Chowdhury et al. (2002).
2.2. Experimental plan
The blood was collected from jugular vein to isolate PBMCs from
all the goats. The PBMCs were cultured in four groups, i.e. 1) control,
2) melatonin (Mel), 3) Dexamethasone (Dex), and 4) Dexamethasone (Dex) along with melatonin (Mel). The dexamethasone, a
synthetic glucocorticoid was used to mimic the glucocorticoid
induced immunosuppression under in vitro condition. Immunosuppressive dose of dexamethasone (2 mM) and an immunoproliferative dose of melatonin (500 pg/ml) were used for in vitro
treatment to PBMCs. To check the proliferative response and
apoptotic markers cells were incubated for 48 h, for GR nuclear
translocation experiments cells were treated for 4 h at 37 C in CO2
incubator. The proliferative response of PBMCs, IL-2 secretion,
expression of GR, HSP90, Nrf2, HO-1 and apoptotic marker proteins
(Bcl-2, Bax, Caspase-3) were checked after the treatments.(Schematic representation).
A.K. Singh, C. Haldar / Molecular and Cellular Endocrinology 436 (2016) 59e67
61
2.4.2. Coimmunoprecipitation
The PBMCs (2 106) were incubated with and without melatonin in presence or absence of dexamethaonsone at 37 C in CO2
incubator. After 4 h of the hormonal treatment, cells were centrifuged at 1600 rpm for 3 mins and washed twice with cold PBS. The
coimmunoprecipitation was performed with commercially available Co-IP kit (Cat. No. 26,149; Thermon scientic, USA) following
Optical density of Challenged Con A manufacturer's protocol. Briey, Cells were lysed by adding lysis
% Stimulation ratio %SR
Optical density of Basal
buffer to the pellet and incubated for 5 min and then centrifuged at
100
13000 g for 10 min at 4 C to collect supernatant and quantication of protein was done by Bradford's method (Bradford, 1976).
Four microliters of anti-GR-antibody was immobilized to the resin
in column. 1 mg protein was added to the column and incubated
with gentle mixing or rocking for overnight at 4 C. At the end,
2.3.3. Estimation of Interleukin-2 in culture supernatants
elution was performed by adding elution buffer, incubating for
The cultured cells were centrifuged to collect medium at
10 min at room temperature. After elution the samples, the ex1600 rpm for 15 min. The quantication of IL-2 secretion was
pressions were checked by western blotting.
estimated with sandwich ELISA following manufacturer's protocol
(Immunotech, Marseille Cedex, France) published elsewhere
2.5. Apoptotic index of PBMCs
(Kaushalendra, 2012). Intra-assay variation was between 3.3% and
7.2%, interassay variation was between 6.2% and 8.2%, sensitivity
Apoptotic cells were analyzed microscopically following Acriwas 5 pg/ml, and recovery was between 80% and 132%.
dine OrangeeEthidium Bromide (AOeEtBr) double staining
method as described elsewhere (Sharma and Haldar, 2009).
2.4. Western blot analyses
AOeEtBr dye of volume 0.01 ml (1) was admixed gently with
0.2 ml of the diluted sample (1 106 cells/ml in PBS). A drop of this
2.4.1. Expression of GR, HSP90, Nrf2, HO-1, Bax, Bcl-2 and Caspasemixture was placed underneath cover-slip on a clean slide and cells
3
were observed immediately under uorescence microscope (Leitz
For the expression pattern of receptor and proteins, cultured
MPV3, Wetzlar, Hesse, Germany) at 440e520 nm. A minimum of
PBMCs were lysed with lysis buffer and quantied by the Bradford's
200 cells was counted in every sample to observed cell death.
method (Bradford, 1976). Four hours after the treatment PBMCs
were isolated by centrifugation of the complete medium at
No: of apoptotic cells early late apoptotic
100
1600 rpm for 15 min. The pellets were isolated was washed twice
Total no: of cells counted
with PBS (pH 7.4). The pellets were homogenized with buffer
containing 10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl2, 1 mM
DTT, 0.1 mM EDTA, and 0.1 mM phenylmethylsulfonyluoride
3. Statistical analyses
(PMSF) and incubated on ice for 15 min then centrifuged at 14000g
for 15 min for cytoplasmic extract. Further for nuclear proteins 10%
All statistical analyses were performed using SPSS version 17.0.
Nonidet P-40 (NP-40) solution was added and incubated for
The data were analyzed by performing one-way ANOVA followed
15e30 min and then centrifuged at 14000g for 15 min. Further,
by multiple comparisons by Tukey's HSD (Honest Signicant Difaliquots containing 50 mg protein were resolve on 10% SDS-PAGE gel
ference) multiple range tests. The values are expressed as
for GR, HSP90, and 12% SDS-PAGE for Nrf2, HO-1, Bax, Bcl-2 and
mean SE. A P-value of 0.05 was considered statistically
Caspase-3 respectively, followed by electro-transfer to nitrocellusignicant.
lose membrane (Bioscience, Keene NH, USA). Immunodetection
was carried out by using Glucocorticoid receptor (GR (H-300): sc8992, Santa Cruz Biotech, USA, diluted 1:250), HSP90 (#4874; cell
signaling, 1:500), Nrf2 (ab31163, Abcam 1:500), HO-1 (Thermo,
USA, 1:500), Caspase-3 (Caspase-3; H-277; sc-7148, Santa Cruz
Biotech, USA, diluted 1:200), Bax (N-20; sc-493, Santa Cruz Biotech,
USA, diluted 1:250) Bcl-2 (N-19; sc-492 Santa Cruz Biotech, USA,
1:200), b-actin antibody (A-2228, Sigma-Aldrich Chemicals, St.
Louis, USA, diluted 1:1000) and Lamin B1 antibody (ab-97775,
Abcam 1:500). All antibodies were diluted in phosphate buffer
saline (PBS; 0.1 M NaH2PO4, Na2HPO4, NaCl; pH 7.4) containing 5%
4. Results
4.1. Proliferative response of PBMCs
The proliferative response of PBMCs in terms of %SR was noted
towards a T-cell specic mitogen Concanavalin-A. % SR signicantly
(p < 0.01) increased upon melatonin treatment while a signicant
(p < 0.01) decline in %SR was noted upon dexamethasone treatment when compared to the control. Co-treatment of melatonin
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A.K. Singh, C. Haldar / Molecular and Cellular Endocrinology 436 (2016) 59e67
Fig. 1. Effect of in vitro melatonin and dexamethasone supplementation on (a) Blastogenic response (%SR) of peripheral blood mononuclear cells (PBMCs) stimulated with Con A and
(b) IL-2 concentration in the culture supernatant of PBMCs after in vitro supplementation with Mel, Dex, and Dex Mel. Values are expressed as mean, vertical bar on histograms
represents SEM, n 7. Signicance of difference; *p < 0.01, Con vs. all experimental groups and #p < 0.01, Dex vs. Dex Mel.
4.4. Coimmunoprecipitation
The melatonin treatment inhibited the dissociation of GR-HSP90
complex and a high expression of GR and HSP90 was noted in
melatonin treated group compared to control and dexamethasone
treatment (Fig. 3a).
A.K. Singh, C. Haldar / Molecular and Cellular Endocrinology 436 (2016) 59e67
63
Fig. 2. Western blot analysis showing the effect of in vitro melatonin and dexamethasone supplementation on expression of (a) complete GR and differential GR expression (b) in
cytoplasmic fraction and (c) nuclear fraction of PBMCs. The data are expressed as % band intensity of GR expression in PBMCs. Left side of the panel shows western blots and right
side shows the representative histograms. Values are expressed as mean, vertical bar on histograms represents SEM, n 7. Signicance of difference; *p < 0.01, Con vs. all
experimental groups and #p < 0.01, Dex vs. Dex Mel.
Fig. 3. Co-IP analysis of (a) GR-HSP90 interaction in PBMCs following Mel, Dex, and Dex Mel treatment. Western blot analysis showing the effect of in vitro melatonin and
dexamethasone supplementation on the (b) expression of nuclear HSP90 following Mel, Dex, and Dex Mel treatment. Values are expressed as mean, vertical bar on histograms
represents SEM, n 7. Signicance of difference; *p < 0.01, Con vs. all experimental groups and #p < 0.01, Dex vs. Dex Mel.
molecules (Julia, 2006) that modulate various physiological functions including immune system (Saplosky et al., 2000). Glucocorticoid exhibits biological actions via its nuclear receptors;
glucocorticoid receptor (GR) which is a low afnity receptor
(Saplosky et al., 2000; De Kloet et al., 1998). Glucocorticoids are
clinically relevant and widely used as anti-inammatory and
immunosuppressive agent for numerous conditions associated
with the host defense (Fauci and Dale, 1974). Dexamethasone, a
synthetic glucocorticoid, is used to treat various clinical conditions
like chronic asthma (Barnes, 1995), rheumatoid arthritis (Buttgereit
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A.K. Singh, C. Haldar / Molecular and Cellular Endocrinology 436 (2016) 59e67
Fig. 4. Western blot analysis showing the effect of in vitro melatonin and dexamethasone supplementation on the expression of (a) Nrf2 (b) HO-1 in PBMCs following Mel, Dex, and
Dex Mel treatment. b-actin expression was used as loading control. Left side of the panel shows western blots and right side shows the representative histograms. Values are
expressed as mean, vertical bar on histograms represents SEM, n 7. Signicance of difference; *p < 0.01, Con vs. all experimental groups and #p < 0.01, Dex vs. Dex Mel.
Fig. 5. Western blot analysis showing the effect of in vitro melatonin and dexamethasone supplementation on the expression of Bax, Bcl-2 and Caspase-3 in PBMCs. (a) Histogram
representing % Bcl-2/Bax ratio and (b) Histogram showing % band intensity of active Caspase-3. b-actin expression was used as loading control. Values are expressed as mean,
vertical bar on histograms represents SEM, n 7. Signicance of difference; *p < 0.01, Con vs. all experimental groups and #p < 0.01, Dex vs. Dex Mel.
and also in combination with dexamethasone. The declined proliferative response upon dexamethasone treatment could be due its
inhibitory effect on IL-2 secretion from immune cells. It has previously been reported that glucocorticoid treatment inhibits the
proliferation of immunocompetent cells (Ashwell et al., 2000) by
inhibiting IL-2 transcription and also affecting IL-2 mRNA stability
(McKay and Cidlowski, 1999). It compromises the activation of T
cells by down-regulating IL-2 R expression (Northrop et al., 1992).
On the contrary, melatonin due to its immunomodulatory property
(Conti et al., 2000) increases production of IL-2 from human PBMCs
at physiological dose (Garcia-Mauriiio et al., 1997). Furthermore,
melatonin avoids stress-induced immunosuppression by
A.K. Singh, C. Haldar / Molecular and Cellular Endocrinology 436 (2016) 59e67
65
Fig. 6. (a): Morphological changes observed in germ cells from (A) control, (B) Melatonin, (C) Dex and (D) Dex Melatonin-treated PBMCs after staining with acridine orangeeethidium bromide (AOeEtBr) dye under uorescence microscope. Arrow indicates normal, dollar ($) indicates early apoptotic cells, # indicates late apoptotic cells and dead cells.
(b): Effect of in vitro melatonin and dexamethasone supplementation on apoptotic index of PBMCs of goat Capra hircus. Values are expressed as mean SEM, n 7. Signicance of
difference: *p < 0.01, Con vs. all experimental groups and #p < 0.01, Dex vs. Mel Dex. (For interpretation of the references to colour in this gure legend, the reader is referred to
the web version of this article.)
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A.K. Singh, C. Haldar / Molecular and Cellular Endocrinology 436 (2016) 59e67
6. Conclusion
It might be suggested that use of melatonin with synthetic
glucocorticoid (like dexamethasone) under clinical condition as
well as in regulation of GR mediated stress condition could be
benecial in reducing the side effects of glucocorticoids and GR
mediated immunosuppression, declined cellular antioxidant
response and apoptosis.
Acknowledgement
Authors thank to Department of Biotechnology (Grant No. BT/
PR14505/AAQ/01/445/2010) and UGC-CAS (R/Dev/CAS-JRF/Zool/
03848) for nancial support to Mr. Amaresh Kumar Singh and Instrument gift from Alexandar Von Humboldt foundation, Bonn,
Germany is gratefully acknowledged. Authors wish to thank Dr.
Sameer Gupta, UGC-Research fellow, for various help during
manuscript preparation.
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