Archives of Virology

, Volume 158, Issue 8, pp 1755–1764

Progesterone suppresses interferon signaling by repressing TLR-7 and MxA expression in peripheral blood mononuclear cells of patients infected with hepatitis C virus

Authors

  • Sara S. Tayel
    • The Molecular Pathology Research Group, Department of Pharmacology and Toxicology, Faculty of Pharmacy and BiotechnologyGerman University in Cairo
  • Amal A. Helmy
    • Department of Endemic Medicine and HepatologyCairo University
  • Rasha Ahmed
    • Department of Endemic Medicine and HepatologyCairo University
  • Gamal Esmat
    • Department of Endemic Medicine and HepatologyCairo University
  • Nabila Hamdi
    • The Molecular Pathology Research Group, Department of Pharmacology and Toxicology, Faculty of Pharmacy and BiotechnologyGerman University in Cairo
    • The Molecular Pathology Research Group, Department of Pharmacology and Toxicology, Faculty of Pharmacy and BiotechnologyGerman University in Cairo
Original Article

DOI: 10.1007/s00705-013-1673-z

Cite this article as:
Tayel, S.S., Helmy, A.A., Ahmed, R. et al. Arch Virol (2013) 158: 1755. doi:10.1007/s00705-013-1673-z

Abstract

This study aimed at investigating the effect of progesterone on interferon signaling pathways in peripheral blood mononuclear cells (PBMCs) of patients infected with hepatitis C virus (HCV). PBMCs were isolated from peripheral blood of 38 treatment-naïve HCV-infected patients, pooled, and stimulated with progesterone in the presence and absence of its receptor antagonist, mifepristone, along with interferon alpha (IFN-α) or imiquimod. Toll-like receptor (TLR) 7 and myxovirus resistance protein A (MxA) were quantified in PBMCs using RT-qPCR. Imiquimod alone or combined with progesterone did not change MxA expression in HCV-infected PBMCs. Progesterone decreased the inducing effect of IFN-α on TLR-7 expression in both males and females. Moreover, progesterone stimulation prior to IFN-α treatment attenuated the Jak/STAT pathway, which was reflected by decreased expression of MxA in females. Progesterone showed a negative impact on the IFN signaling pathway in HCV-infected PBMCs as it decreased the expression of TLR-7 in both genders, while MxA expression was decreased only in females.

Introduction

Toll-like receptors (TLRs) are a family of receptors that are known to act early in innate immunity [1]. There are 11 known TLRs, of which TLR 3, 7, 8, and 9 recognize viral genomes, boosting an antiviral immune response through increased production of interferon (IFN) [2]. Specifically, TLR-7 recognizes RNA viruses and thus plays a major role in the innate immune response to hepatitis C virus (HCV) [3]. Following the activation of TLR-7, type I interferon (IFN1) is released from the cells, leading to the activation of the Jak/STAT pathway through binding of IFN-α to IFN-α receptor 1. The IFN-α receptor 1 subunits are dimerized with the activation of a series of kinases that phosphorylate IFN regulatory factor 7, which eventually leads to the transcription of interferon-stimulated genes (ISGs) [4, 5]. Myxovirus resistance protein A (MxA) is a powerful ISG that has a strong antiviral response even in the absence of other ISGs [6]. However, progression to a chronic HCV status is associated with numerous mechanisms by which the virus escapes the immune response [7]. Interference of HCV with different components of the Jak/STAT pathway has also been reported [8].

TLRs appear to show a gender-specific functional variation. The activation of TLR-7 with synthetic agonists in healthy peripheral blood cells has been shown to lead to higher interferon alpha (IFN-α) production in females compared to age-matched males [9]. This observation raises the question whether this functional variation is hormone dependent. Previous studies have revealed the capability of estrogen to affect the innate immunity of the host by suppressing the maturation of dendritic cells and significantly repressing the release of IFN during infection with Newcastle disease virus [10]. In contrast, 17-beta estradiol has been shown to enhance the response of healthy murine plasmacytoid dendritic cells to CpG, a TLR-9 agonist, causing an increased secretion of IFN-α [11]. Sex hormones have also shown their effects on the activation of the Jak/STAT pathway, where estrogen has been shown to be capable of attenuating the Jak/STAT pathway, as reflected by decreased production of ISGs, and this effect was reversed by treatment with both ICI 182/780 and Tamoxifen [12, 13].

Similarly, there is growing evidence that progesterone also affects the immune response in mammals [14]. Progesterone is capable of inhibiting the release of IFN-α in healthy plasmacytoid dendritic cells following stimulation with TLR-9 agonists [15]. Furthermore, progesterone fluctuation has been reported to cause a change in the status of autoimmune diseases and in the response to infection [16, 17]. However, the impact of progesterone on TLR-7 and the Jak/STAT pathway activation in HCV-infected peripheral blood mononuclear cells (PBMCs) requires further clarification. Our study aimed at investigating the effect of progesterone on the expression and activation of TLR-7 and subsequent release of IFN-α, as well as its impact on the downstream activation of the Jak/STAT pathway in HCV-infected PBMCs of males and females.

Materials and methods

Subjects

A total of 38 patients (22 males and 16 premenopausal females) chronically infected with HCV and 20 healthy volunteers (10 males and 10 premenopausal females) were included in this study. The mean age of male patients was 36, ranging from 20 to 59 years, and the mean age of females was 38, ranging from 33 to 45 years. HCV infection was confirmed by the presence of anti-HCV antibodies and HCV RNA in the serum. All patients were treatment-naïve but were eligible for peg-interferon/ribavirin combination therapy. All patients were negative for hepatitis B surface antigen, and fibrosis was assessed by Metavir score. Neither the premenopausal patients nor the healthy controls were on oral contraceptives or hormone replacement therapy, and all subjects gave their written informed consent. All experiments were performed in compliance with the guidelines of the institutional review board (IRB) of Kasr El Aini Medical School at Cairo University and were in accordance with the ethical standards of the Declaration of Helsinki. Table 1 summarizes the clinical features of the patients included in this study.
Table 1

Clinical data for the HCV-infected patients included in the study

Patient

Gender

Age

Year of HCV positivity

HCV RNA (IU/ml)

Fibrosis

ALT (IU/ml)

AST (IU/ml)

Genotype

1

Male

42

2005

9800

F1

60

38

4

2

Male

25

2010

3.913 × 10^5

F2

36

31

4

3

Male

20

2010

24700

F0

19

29

4

4

Male

52

2009

2.85 × 10^6

F4

64

95

4

5

Male

50

2009

7.487 × 10^5

F4

75

89

4

6

Female

35

2010

1.63 × 10^6

F1

47

52

4

7

Male

34

2009

3220

F2

65

32

4

8

Female

30

2010

7.675 × 10^4

F1-2

93

125

4

9

Male

37

2011

71000

F4

61

73

4

10

Male

29

2009

6.48 × 10^5

F2

62

73

4

11

Male

34

2010

3.06 × 10^6

F6

17

21

4

12

Female

42

2010

6.48 × 10^5

F1

35

37

4

13

Female

46

2010

1.14 × 10^6

F1

42

26

4

14

Male

45

2000

5.47 × 10^6

F3

149

73

4

15

Female

33

2010

1.99 × 10^5

F3

126

158

Mixed (1 + 4)

16

Male

26

2005

910 × 10^3

F2

97

105

4

17

Male

30

2010

1.77 × 10^6

F4

23

21

Mixed (1 + 4)

18

Male

54

2011

129 × 10^3

F3

90

119

4

19

Female

40

2010

2.32 × 10^5

F2

19

26

4

20

Female

45

2006

3.00 × 10^3

F4

54

62

4

21

Male

33

2003

1.24 × 10^5

F1

36

26

4

22

Female

29

2009

4.097 × 10^5

F4

13

12

4

23

Female

37

2010

1.73 × 10^6

F1

18

17

4

24

Male

37

2010

621800

F1-2

41

23

4

25

Female

33

2010

3.42 × 10^4

F2

28

26

4

26

Female

36

2010

1.25 × 10^5

F2

28

20

4

27

Male

21

2010

3.14 × 10^5

F1

28

45

4

28

Female

43

2010

2.62 × 10^4

F3

59

60

4

29

Female

39

2003

1.16 × 10^4

F1-2

42

37

4

30

Male

58

1995

1.38 × 10^5

F1-2

35

58

4

31

Male

44

2009

34381

F1-2

65

54

4

32

Male

34

2009

2.58 × 10^4

F1-2

25

20

4

33

Male

59

2010

4.28 × 10^3

F4

152

116

4

34

Male

50

2008

1.68 × 10^5

F4

115

70

4

35

Male

45

2006

3.49 × 10^5

F3-4

50

43

4

36

Female

36

2008

9.675 × 10^4

F1

34

12

4

37

Female

41

2010

6.57 × 10^4

F3

41

46

4

38

Female

39

2005

2.50 × 10^5

F1

42

35

4

Collection of blood samples and isolation of PBMCs

Four milliliters of peripheral venous blood was collected in the presence of an anticoagulant (EDTA) from healthy controls and patients for isolation of peripheral blood mononuclear cells (PBMCs). All samples were processed on the same day, within a few hours after collection. PBMCs were isolated using the Ficoll density gradient centrifugation method. Cells were then cryopreserved in 1 ml freeze mix (wash mix, 30 % FBS, 10 % DMSO) until use.

Cell culture and stimulation

PBMCs from males and females were checked for viability using trypan blue exclusion and then pooled according to gender. PBMCs were seeded in 24-well plates at a concentration of 1 × 106 cells/ml in each well. Cells were incubated at 37 °C in a 5 % CO2 atmosphere and 100 % humidity for 24 h prior to stimulation. All experiments were done three times, and stimulations were performed in duplicates. According to the experiment, different sets of PBMCs were subjected to one of the following conditions: (1) cells were either unstimulated for 30 hours then stimulated with IFN-α (Rhein, MinaPharm, Egypt) at a concentration of 10,000 units/ml for 24 hours. (2) Cells were left unstimulated for 6 hours and then stimulated with progesterone (Sigma, Aldrich) at a concentration of 10−6 mol/L for 24 hours, followed by treatment with interferon for 24 hours. (3) Cells were stimulated with mifepristone (RU486, Enzo Life sciences, Germany) at a concentration of 10−6 mol/L for 6 hours, followed by treatment with progesterone for 24 hours and interferon for 24 hours. (4) Cells were left unstimulated for 30 hours and then stimulated with the TLR-7 agonist imiquimod (Enzo Life sciences, Germany) at a concentration of 1 μg/ml for 24 hours. (5) Cells were left unstimulated for 6 hours and then stimulated with progesterone for 24 hours followed by imiquimod for 24 hours. (6) Cells were stimulated with mifepristone for 6 hours, followed by treatment with progesterone for 24 hours and imiquimod for 24 hours. In parallel, as controls, sets of PBMCs were left unstimulated during the entire course of the study. The concentrations used were described previously to be optimal for ex vivo stimulation studies [9, 18, 19].

Total RNA extraction and reverse transcription

Total RNA extraction was performed using a QIAamp RNA Mini Kit (QIAGEN, Germany), and total RNA was eluted in a final volume of 30 μl. Total RNA was reverse transcribed into single-stranded complementary DNA (cDNA) using a high-capacity cDNA reverse transcription kit (QIAGEN, Germany).

Quantitation of gene expression

The relative mRNA expression levels of the studied genes, MxA (Hs00182073_m1, Applied Biosystems, USA) and TLR7 (Hs00152971_m1, Applied Biosystems, USA), were quantified using a TaqMan real-time quantitative polymerase chain reaction (RT-qPCR) assay. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Applied Biosystems, USA) was used as a housekeeping gene for normalization of the amount of mRNA expression of the gene of interest. The quantification of gene expression was performed using an ABI 7000 real-time PCR instrument and system software (Applied Biosystems, USA).

Statistical analysis

Gene expression was expressed in relative units (RQ = 2−∆∆CT), and the data were expressed as mean ± standard error of the mean (SEM). Student’s t-test was used for statistical analysis of gene expression, where a P-value less than 0.05 was considered statistically significant. Calculations were performed using GraphPad Prism 5.00 software (GraphPad Software Inc., La Jolla, CA, USA).

Results

Effect of progesterone on MxA expression following TLR-7 stimulation

As shown in Fig. 1, the effects of progesterone on the activation of TLR-7 were assessed by stimulating TLR-7 with its agonist imiquimod in the presence or absence of progesterone, and the relative expression of MxA was then quantified using RT-qPCR.
https://static-content.springer.com/image/art%3A10.1007%2Fs00705-013-1673-z/MediaObjects/705_2013_1673_Fig1_HTML.gif
Fig. 1

Effect of progesterone on MxA expression following TLR-7 stimulation. a HCV-infected males. The baseline MxA expression did not differ significantly as compared to the baseline expression of healthy controls (P = 0.4649). Imiquimod stimulation did not result in any change in MxA expression when compared to baseline MxA expression (P = 0.1832). Stimulation with progesterone prior to imiquimod treatment did not cause any change in MxA expression when compared to stimulation with imiquimod alone (P = 0.6769). Stimulation with mifepristone prior to progesterone and imiquimod treatment did not alter MxA expression when compared to cells treated with imiquimod alone. b HCV-infected females. The same pattern of expression was observed as in HCV-infected males. c Healthy males. Imiquimod stimulation did not lead to any change in MxA expression compared to baseline, (P = 0.9939). d Healthy females. Imiquimod stimulation led to a significant increase in MxA expression compared to baseline, (P = 0.0196*). Stimulation with progesterone prior to imiquimod treatment caused a significant decrease in MxA expression when compared to stimulation with imiquimod alone (P = 0.0415*). Stimulation with mifepristone prior to progesterone and imiquimod treatment caused a significant decrease in MxA expression when compared to cells treated with imiquimod alone (P = 0.0023**). imi, imiquimod; Pg, progesterone; RU, mifepristone. The amount of MxA mRNA expression was calculated relative to the amount of mRNA expression of the housekeeping gene GAPDH in the same sample. Gene expression is expressed in relative quantitation (RQ = 2−∆∆CT). Results are expressed as mean ± standard error of the mean (SEM). Level of significance is indicated by stars (**)

In HCV-infected males, the baseline MxA expression did not differ significantly when compared to the baseline expression in healthy controls (1.293 ± 0.2917 [N = 22] and 1.039 ± 0.1771 [N = 10], respectively, P = 0.4649). Imiquimod stimulation did not result in any change in MxA expression when compared to baseline MxA expression (0.9353 ± 0.1020 [N = 22] and 1.293 ± 0.2917 [N = 22], respectively, P = 0.1832). Pretreatment with progesterone prior to imiquimod stimulation did not cause any change in MxA expression when compared to stimulation with imiquimod alone (0.9493 ± 0.4128 [N = 22] and 0.9353 ± 0.1020 [N = 22], respectively, P = 0.9602). Also, stimulation with mifepristone prior to progesterone and imiquimod treatment did not alter MxA expression when compared to cells treated with imiquimod alone (1.127 ± 0.1603 [N = 22] and 0.9353 ± 0.1020 [N = 22], respectively, P = 0.3760) (Fig. 1a).

Similarly, when the same experiments were performed using PBMCs from HCV-infected females, the baseline MxA expression did not differ significantly compared to the baseline expression of healthy controls (0.8411 ± 0.2406 [N = 16] and 1.447 ± 0.7631 [N = 10], respectively, P = 0.4255). Treatment with imiquimod did not cause any change in MxA expression when compared to baseline MxA expression (1.058 ± 0.1013 [N = 16] and 0.8411 ± 0.2406 [N = 16], respectively, P = 0.4384). Stimulation with progesterone prior to imiquimod treatment did not affect MxA expression when compared to stimulation with imiquimod alone (1.033 ± 0.03970 [N = 16] and 1.058 ± 0.1013 [N = 16], respectively, P = 0.8781). Additionally, stimulation with mifepristone prior to progesterone and imiquimod treatment did not alter MxA expression when compared to cells treated with imiquimod alone (1.114 ± 0.005405 [N = 16] and 1.058 ± 0.1013 [N = 16], respectively, P = 0.7303) (Fig. 1b).

In healthy males, imiquimod stimulation did not lead to any change in MxA expression as compared to baseline (1.041 ± 0.2449 [N = 10] and 1.039 ± 0.1771 [N = 10], respectively, P = 0.9939). In healthy females, imiquimod stimulation led to a significant increase in MxA expression as compared to baseline (4.674 ± 0.3890 [N = 10] and 1.477 ± 0.7631 [N = 10], respectively, P = 0.0196*) (Fig. 1c). Also in healthy females, stimulation with progesterone prior to imiquimod treatment caused a significant decrease in MxA expression when compared to stimulation with imiquimod alone (1.729 ± 0.9337 [N = 10] and 4.674 ± 0.3890 [N = 10], respectively, P = 0.0415*). Furthermore, stimulation with mifepristone prior to progesterone and imiquimod treatment caused a significant decrease in MxA expression when compared to cells treated with imiquimod alone (1.502 ± 0.2461 [N = 10] and 4.674 ± 0.3890 [N = 10], respectively, P = 0.0023**) (Fig. 1d).

Effect of progesterone on TLR-7 expression

Figure 2 shows the relative expression of TLR-7, quantified using RT-qPCR, upon stimulation with IFN in the absence or presence of progesterone, where PBMCs of HCV-infected males showed no significant difference in TLR-7 expression when compared to those of healthy controls (4.308 ± 0.8559 [N = 22] and 1.213 ± 0.6873 [N = 10], respectively, P = 0.1062). Stimulating the HCV-infected PBMCs with interferon significantly increased TLR-7 expression when compared to untreated PBMCs (91.11 ± 3.409 [N = 22] and 4.308 ± 0.8559 [N = 22], respectively, P = 0.0016**). Stimulation with progesterone prior to interferon treatment led to a significant decrease in TLR-7 expression when compared to treatment with interferon alone (22.50 ± 9.842 [N = 22] and 91.11 ± 3.409 [N = 22], respectively, P = 0.0223*). Also, stimulation with mifepristone prior to progesterone and interferon treatment led to a further decrease in TLR-7 expression when compared to treatment with interferon alone (19.11 ± 4.603 [N = 22] and 91.11 ± 3.409 [N = 22], respectively, P = 0.0063**) (Fig. 2a).
https://static-content.springer.com/image/art%3A10.1007%2Fs00705-013-1673-z/MediaObjects/705_2013_1673_Fig2_HTML.gif
Fig. 2

Effect of progesterone on TLR-7 expression. a HCV-infected males. HCV-infected PBMCs showed no significant difference in TLR-7 expression when compared to healthy controls (P = 0.1062). Interferon stimulation caused a significant increase in TLR-7 expression when compared to untreated PBMCs (P = 0.0016**). Stimulation with progesterone prior to interferon treatment led to a significant decrease in TLR-7 expression when compared to treatment with interferon alone (P = 0.0223*). Stimulation with mifepristone prior to progesterone and interferon treatment led to a further decrease in TLR-7 expression when compared to treatment with interferon alone (P = 0.0063**). b HCV-infected females. HCV-infected PBMCs showed a detectable level of TLR-7 expression compared to the undetectable TLR-7 expression in healthy PBMCs. Interferon stimulation caused a significant increase in TLR-7 expression when compared to untreated PBMCs (P = 0.0011**). Stimulation with progesterone prior to interferon treatment led to a significant decrease in TLR-7 expression when compared to treatment with interferon alone (P = 0.0055**). Stimulation with mifepristone prior to progesterone and interferon caused a decrease in TLR-7 expression to undetectable levels. c Healthy PBMCs. Interferon treatment in males caused a significant increase in TLR-7 expression when compared to that of unstimulated cells (P = 0.0243*). Healthy females. Interferon treatment led to increased expression of TLR-7 as well when compared to the undetectable levels in unstimulated cells. The increase in the TLR-7 induction was found to be the highest in HCV-infected male PBMCs. The increase in TLR-7 induction was generally higher in HCV-infected PBMCs when compared to the healthy PBMCs for both groups. IFN, interferon; Pg, progesterone; RU, mifepristone. The amount of TLR7 mRNA was calculated relative to the amount of mRNA expression of the housekeeping gene GAPDH in the same sample. Gene expression is expressed in relative units (RQ = 2−∆∆CT). Results are expressed as mean ± standard error of the mean (SEM). The level of significance is indicated by stars (**)

PBMCs from HCV-infected females showed detectable levels of TLR-7 when compared to the healthy controls. Stimulating the HCV-infected PBMCs with interferon significantly increased TLR-7 expression when compared to untreated cells (10.75 ± 0.3502 [N = 16] and 0.9424 ± 0.5795 [N = 16], respectively, P = 0.0011**). Treatment of PBMCs with progesterone prior to stimulation with interferon led to a significant decrease in TLR-7 expression when compared to treatment with interferon alone (2.416 ± 0.5121 [N = 16] and 10.75 ± 0.3502 [N = 16], respectively, P = 0.0055**). Also, stimulation of cells with mifepristone before treatment with progesterone and interferon led to a significant decrease in TLR-7 expression, rendering it undetectable again (Fig. 2b).

Treatment of healthy PBMCs from both males and premenopausal females with interferon resulted in increased expression of TLR-7 when compared to their respective healthy baselines. In healthy males, expression of TLR-7 in interferon-treated cells was significantly higher when compared to that of unstimulated cells (8.507 ± 0.9309 [N = 10] and 1.213 ± 0.6873 [N = 10], respectively, P = 0.0243*). In premenopausal females, interferon treatment also led to an increased expression of TLR-7 (2.546 ± 0.9263, N = 10) compared to the undetectable levels in unstimulated cells (Fig. 2c).

Effect of progesterone on MxA expression following exogenous interferon stimulation

Next, we investigated the effects of progesterone on the activation of the Jak/STAT pathway. This was evaluated by quantifying MxA expression by RT-qPCR after stimulation with IFN in the presence or absence of progesterone. In the case of HCV-infected males, interferon stimulation of PBMCs resulted in a significant upregulation of MxA expression when compared to untreated cells (2542 ± 547.7 [N = 22] and 5.231 ± 0.4810 [N = 22], respectively, P = 0.0112*). Stimulation with progesterone prior to interferon did not affect MxA expression when compared to stimulation with interferon alone (3104 ± 364.1 [N = 22] and 2542 ± 547.7 [N = 22], respectively, P = 0.5454). Also, stimulation with mifepristone prior to progesterone and interferon treatment did not lead to any change in MxA expression when compared to treatment with interferon alone (3882 ± 2716 [N = 22] and 2542 ± 547.7 [N = 22], respectively, P = 0.5102) (Fig. 3a).
https://static-content.springer.com/image/art%3A10.1007%2Fs00705-013-1673-z/MediaObjects/705_2013_1673_Fig3_HTML.gif
Fig. 3

Effect of progesterone on MxA expression following exogenous interferon stimulation. a HCV-infected males. Interferon stimulation resulted in a significant upregulation of MxA expression when compared to untreated cells (P = 0.0112*). Progesterone stimulation prior to interferon did not affect MxA expression when compared to stimulation with interferon alone (P = 0.5454). Stimulation with mifepristone prior to progesterone and interferon did not lead to any change in MxA expression when compared to treatment with interferon alone (P = 0.5102). b HCV-infected females. Interferon stimulation resulted in a significant upregulation of MxA expression when compared to untreated cells (P = 0.0071**). Progesterone stimulation prior to interferon treatment led to a significant decrease in MxA expression when compared to stimulation with interferon alone (P = 0.0377*). Stimulation with mifepristone prior to progesterone and interferon treatment led to a significant decrease in MxA expression when compared to treatment with interferon alone (P = 0.0230*). c Stimulation of PBMCs from healthy males showed effects comparable to those observed for the HCV-infected male PBMCs. d Stimulation of PBMCs from healthy females showed effects comparable to those observed in their HCV-infected counterpart. IFN, interferon; Pg, progesterone; RU, mifepristone. The amount of MxA and TLR7 mRNA expression was calculated relative to the amount of mRNA expression of the housekeeping gene GAPDH in the same sample. Gene expression is expressed in relative quantitation (RQ = 2∆∆CT). Results are expressed as mean ± standard error of the mean (SEM). The level of significance is indicated by stars (**)

In the case of HCV-infected females, treatment of PBMCs with interferon also led to a significant upregulation of MxA expression when compared to untreated cells (86135 ± 23469 [N = 16] and 0.8411 ± 0.2406 [N = 16], respectively, P = 0.0071**). However, stimulation with progesterone prior to interferon treatment led to a significant decrease in MxA expression when compared to stimulation with interferon alone (10891 ± 7392 [N = 22] and 86135 ± 23469 [N = 22], respectively, P = 0.0377*). Stimulation with mifepristone prior to progesterone and interferon treatment also led to a significant decrease in MxA expression when compared to treatment with interferon alone (1916 ± 314.9 [N = 16] and 86135 ± 23469 [N = 16], respectively, P = 0.0230*) (Fig. 3b).

In healthy males, stimulation of PBMCs with interferon showed a significant upregulation in MxA expression when compared to untreated cells (1681 ± 311.7 [N = 22] and 1.039 ± 0.1771 [N = 22], respectively, P = 0.0017**), and stimulation with progesterone prior to interferon treatment did not affect MxA expression when compared to stimulation with interferon alone (1519 ± 355.3 [N = 22] and 1681 ± 311.7 [N = 22], respectively, P = 0.7451). Treatment with mifepristone prior to progesterone and interferon stimulation did not lead to any change in MxA expression when compared to treatment with interferon alone (2242 ± 643.1 [N = 22] and 1681 ± 311.7 [N = 22], respectively, P = 0.4290) (Fig. 3c).

In healthy females, stimulation with interferon caused a significant increase in MxA expression when compared to untreated cells (95232 ± 3530 [N = 10] and 1.447 ± 0.7631 [N = 10], respectively, P < 0.0001***). Stimulation with progesterone prior to interferon treatment led to a significant decrease in MxA expression when compared to stimulation with interferon alone (8763 ± 242.9 [N = 10] and 95232 ± 3530 [N = 10], respectively, P = 0.0017**). Stimulation with mifepristone prior to progesterone and interferon treatment led to a further significant decrease in MxA expression (939.3 ± 485.6, N = 10) when compared to prestimulation with progesterone prior to interferon (8763 ± 242.9, N = 10, P = 0.0048**) and when compared to interferon treatment alone (95232 ± 3530, N = 10, P = 0.0014**) (Fig. 3d).

Discussion

Gender differences in response to IFN-based treatment of chronic HCV infection have been reported previously, with females showing higher rates of sustained virological response [20]. This exemplifies the importance of studying the impact of sex hormones on the IFN signaling pathway. Recent research has investigated the impact of estrogen on IFN signaling in HCV infection [13]. Therefore, the current study focused on the impact of progesterone on the IFN signaling pathway in PBMCs of HCV-infected patients, which has not been investigated previously. TLR-7 plays a major role in the innate immune response to HCV by recognizing the viral RNA and causing the subsequent release of IFN1, which is followed by the downstream activation of the Jak/STAT pathway, resulting in the transcription of ISGs with powerful antiviral effects [4]. MxA expression was used for the assessment of IFN1 release and the activation of the Jak/STAT pathway, as its expression is tightly regulated by IFN1 [21]. Since PBMCs are natural reservoirs for the virus as well as strong producers of interferon during viral infections, and since recent work in our lab has confirmed the susceptibility of PBMCs to viral infection as well as the ability of the virus to replicate inside these cells, PBMCs were selected as a suitable ex vivo model in our study [2224]. Given the number of stimulations performed in the current study, the most convenient way to achieve this was to pool PBMCs separately from females and males. In support of this, previous work in our lab showed no difference in mRNA expression between individual PBMCs and pooled PBMCs from the same patients [13]. In addition, previous studies have examined the difference between stimulation of pooled PBMCs and stimulation of the corresponding individual samples and found no difference between both groups [25, 26].

Initially, baseline MxA expression was compared between HCV-infected PBMCs and healthy controls in both males and females. The results showed no significant difference in MxA levels between healthy and infected cells (Fig. 1a, b). These results highlight an impaired IFN signaling in HCV-infected PBMCs and are in contradiction to the expected increase in ISGs during viral infection [27]. TLR-7 of healthy males and females was stimulated by its agonist, imiquimod, and the downstream release of IFN was evaluated by assessment of MxA expression. Only PBMCs from females showed a significant increase in MxA expression when compared to the untreated cells (Fig. 1c). This is consistent with previous results describing a significantly higher activation of TLRs in females compared to males [9]. Contrariwise, HCV-infected cells did not show the expected increase in MxA expression in either female or male patients (Fig. 1a, b). These results could be explained by an impaired expression and/or function of TLR-7 in HCV infection, as recently demonstrated in HCV-infected human hepatoma cells [28].

To assess whether the defect is in TLR-7 expression, TLR-7 baseline expression was quantified in HCV-infected patients compared to healthy controls. Interestingly, TLR-7 expression did not differ significantly between healthy and HCV-infected PBMCs (Fig. 2a, b), which refutes that TLR-7 expression is the main cause of decreased IFN release in our patients. These results, along with those shown in Fig. 1, show that the lack of an upregulation of MxA following imiquimod stimulation is due to an impaired function of TLR-7 in HCV-infected patients. Our results highlight a further mechanism by which the virus can evade the immune system by blunting TLR-7 function and the subsequent production of interferon and ISGs. Another major finding of our study is the significant increase in TLR-7 expression that occurs upon stimulation of PBMCs with IFN-α in both healthy and HCV-infected patients (Fig. 2). The inducing effect of IFN on TLR-7 was observed in both HCV-infected and healthy PBMCs; however, it was less pronounced in females than in males (Fig. 2). This induction could be due to either a possible positive feedback loop of IFN-α on TLR-7 or the antiviral effect of IFN-α suppressing viral interference with TLR-7 [28, 29]. Due to the low baseline expression of TLR-7 in HCV-infected PBMCs and the absence of visible effects of TLR-7 stimulation with imiquimod and progesterone, the impact of progesterone on TLR-7 was assessed after inducing TLR-7 expression by treatment with IFN-α. Our results revealed, for the first time, a significant suppressive effect of progesterone on the induction of TLR-7 in both males and females (Fig. 2a, b). Thus, it could be inferred that a higher physiological concentration of progesterone in women is the reason behind the lower baseline expression of TLR-7 in females (Fig. 2b) which, in turn, results in a lower induction by IFN. Blocking progesterone receptors with mifepristone did not reverse the effects of progesterone but resulted in a further decrease of TLR-7 expression. Previous research has reported the failure of mifepristone to reverse the action of progesterone on membrane progesterone receptors (mPRs) [30]. These receptors have been characterized in different types of immune cells, including peripheral blood leukocytes, and are thought to be the cause for the abrogated immune response during the luteal phase of the menstrual cycle [14]. Accordingly, it could be suggested that the suppressive actions of progesterone on IFN signaling are mediated through the newly identified mPRs and that the blockade of progesterone nuclear receptors in PBMCs by mifepristone has potentiated the action of progesterone on membrane receptors, exacerbating its suppressive effects. This explains the more pronounced downregulation of TLR-7 expression upon co-stimulation with mifepristone and progesterone when compared to progesterone alone (Fig. 2a, b). To test our hypothesis, we stimulated the female HCV-infected PBMCs with mifepristone prior to IFN without progesterone and compared the expression levels of TLR-7 to the PBMCs stimulated with IFN only. Results showed that TLR-7 induction was not affected by pre-stimulation with mifepristone (11.35 ± 0.2813, N = 16) when compared to IFN alone (10.75 ± 0.3502, N = 16, P = 0.3144), which argues against a mifepristone-related suppression of IFN signaling and additionally supports our explanation.

It has been shown previously that estradiol has an inhibitory effect on the Jak/STAT pathway in PBMCs of HCV-infected patients of both genders [13]. In the current study, the impact of progesterone on Jak/STAT pathway was investigated by comparing MxA gene expression after stimulation with IFN-α alone versus its expression upon stimulation with both progesterone and IFN-α. As expected, IFN-α activated the Jak/STAT pathway in both males and females, which was reflected in a significant induction of MxA gene expression with a higher induction of MxA expression in females compared to males (Fig. 3). This highlights gender-specific differences in the host immune response, showing a better response of female patients to IFN-α-based therapy (Fig. 3a, b), and appears to be in accordance with a previous study showing a positive correlation between the increase in ISG expression following IFN-α treatment and response to therapy [31]. On the other hand, progesterone stimulation prior to interferon led to a significant decrease in MxA expression only in female PBMCs (Fig. 3b). This might indicate that progesterone interferes with the Jak/STAT pathway only in females. It has been shown previously that progesterone cannot directly affect the level of MxA transcription [32]. Accordingly, it could be suggested that the suppressive actions of progesterone on MxA are mediated earlier in the Jak/STAT pathway and that it is likely to affect all ISGs similarly. The stimulation of PBMCs with mifepristone prior to progesterone and interferon resulted in a significant decrease in MxA expression only in female PBMCs and, again, mifepristone did not reverse the effects of progesterone (Fig. 3b). Consistent with this, PBMCs of healthy females showed MxA expression patterns that were similar to those of HCV-infected females, confirming that those effects are not due to any viral interference (Fig. 3d).

In continuation of previous work by our group on the impact of sex hormones on IFN signaling in HCV, this study illustrated a significant interference by progesterone with different components of the IFN signaling pathway in HCV-infected PBMCs. Previously, it was shown that estrogen causes attenuation of the Jak/STAT pathway, as reflected in the decreased levels of ISGs in an ex vivo stimulation study. In the current study, we extended our exploration to include the effects of progesterone on the expression of TLR-7, a key receptor for early viral recognition. We showed that progesterone inhibited the induction of TLR-7 by IFN in HCV-infected males and females. Moreover, progesterone interfered with the activation of the Jak/STAT pathway, as was reflected by decreased MxA expression following interferon treatment, but only in healthy and HCV-infected females. Consequently, we conclude that there is a suppressive effect of progesterone on IFN signaling in HCV-infected patients.

Acknowledgment

This work was funded by the German University in Cairo postgraduate fund.

Conflict of interest

The authors declare that they have no conflict of interest.

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© Springer-Verlag Wien 2013