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The W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA
Correspondence
Sabra L. Klein
saklein{at}jhsph.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Supplementary tables are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Klein & Calisher, 2007). Hantaviruses and their specific reservoir hosts represent a highly co-evolved system in which both virus and host survive (Plyusnin & Morzunov, 2001).
Rodent reservoirs sustain a presumably lifelong infection with hantaviruses, but do not display overt signs of disease (Botten et al., 2003; Lee et al., 1981). In contrast to rodents, spillover of hantaviruses to humans can cause hantavirus cardiopulmonary syndrome (HCPS), haemorrhagic fever with renal syndrome (HFRS), or nephropathia endemica (NE). Symptoms of HFRS and HCPS in humans are mediated by excessive proinflammatory and CD8+ T-cell responses (Khaiboullina & St Jeor, 2002; Kilpatrick et al., 2004; Mori et al., 1999). Seoul virus (SEOV) is the species-specific hantavirus that infects Norway rats. SEOV persists in the lungs of male rats and during infection, localized antiviral defences, proinflammatory factors, chemokines and adhesion molecules generally are reduced in males (Easterbrook & Klein, 2008; Hannah et al., 2008; Klein et al., 2004). Regulatory responses, including forkhead box P3 (FoxP3) and transforming growth factor (TGF)-β, however, are elevated in the lungs of male rats during persistent SEOV infection (Easterbrook & Klein, 2008; Easterbrook et al., 2007). Regulatory T cells contribute to host homeostasis by suppressing proinflammatory and CD8+ T-cell responses to protect the host, but can also contribute to viral persistence by suppressing responses necessary for viral clearance (Belkaid, 2007). Regulatory T cells reduce expression of tumour necrosis factor (TNF)-
and contribute to hantavirus persistence in the lungs of male rats, but mechanisms of regulatory T-cell induction remain unknown (Easterbrook et al., 2007; Schountz et al., 2007).
In addition to regulatory T cells, glucocorticoids are anti-inflammatory steroid hormones that suppress potentially damaging proinflammatory and cellular responses that contribute to the maintenance of host homeostasis, but concurrently may also suppress responses necessary for resolving an infection (Webster et al., 2002). Exposure to a stressor, including viral infection, can activate the hypothalamic-pituitary-adrenal (HPA) axis, which promotes the release of glucocorticoids from the adrenal cortex (Bailey et al., 2003; Silverman et al., 2005). Glucocorticoids suppress proinflammatory, CD8+ T-cell and Th1-polarized responses indirectly through effects on transcription factors, including nuclear factor (NF)-
B and AP-1, or directly by binding to glucocorticoid response elements (GRE) on responsive host genes (Kassel & Herrlich, 2007). Removal of glucocorticoids by adrenalectomy can result in more efficient viral clearance, but can also cause mortality mediated by excessive proinflammatory responses to viral infection (Bailey et al., 2003; Ruzek et al., 1999). Corticosteroids have been successfully administered to patients with HFRS or HCPS to alleviate symptoms of disease caused by excessive proinflammatory and cellular responses (Dunst et al., 1998; Seitsonen et al., 2006), but whether suppression of such responses may contribute to a reduced ability to clear the virus has not been examined.
Among humans and rodent reservoirs, more males than females are infected with hantaviruses (Klein & Calisher, 2007). When inoculated with the same dose of SEOV, male rats shed virus longer, via more routes, and have more viral RNA present in target organs, such as the lungs, than females (Hannah et al., 2008; Klein et al., 2000, 2004). During SEOV infection, innate immune responses, including the expression of pattern recognition receptors [PRR; e.g. Toll-like receptor (Tlr)7 and retinoic acid inducible gene (Rig)I] and innate antiviral genes [e.g. interferon (Ifn)β and myxovirus resistance (Mx)2], as well as proinflammatory and chemokine responses, are higher in the lungs of female than male rats (Hannah et al., 2008; Klein et al., 2004). These sex differences may be dependent on oestradiol in females and testosterone in males, as gonadectomy reverses these differences (Hannah et al., 2008). Like sex steroids, glucocorticoids can be differentially regulated between the sexes (Kitay, 1961) and also may contribute to differential immune responses in males and females during SEOV infection. We hypothesized that elevated concentrations of glucocorticoids in male rats may suppress host proinflammatory and effector immune responses during SEOV infection to contribute to increased viral persistence in male compared with female rats.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Surgical procedure and glucocorticoid replacement.
Male and female rats (n=400) were anaesthetized with 80 mg ketamine kg–1 and 6 mg xylazine kg–1 (Phoenix Pharmaceutical) and were subjected to bilateral adrenalectomy (ADX) or received sham surgery. Exogenous corticosterone was replaced in 250 mg pellets containing 10 or 80 % corticosterone (MP Biomedicals), with the remainder of the pellet consisting of cholesterol (MP Biomedicals). Animals assigned to the sham surgery or ADX0 groups were implanted with pellets containing 100 % cholesterol. Pellets were implanted in the intrascapular region and all animals were allowed 2 weeks to recover from surgery.
Infection.
Male and female rats (n=8–12/sex/corticosterone treatment/time point) were inoculated intraperitoneally with 104 p.f.u. of SEOV (strain SR-11) or vehicle alone, as described previously (Klein et al., 2001, 2002). At days 0, 3, 15, 30 and 40 post-inoculation (p.i.), animals were anaesthetized with isoflurane (Abbot Animal Health) and blood was obtained from the retro-orbital sinus. To circumvent the rise in corticosterone concentrations following exposure to common laboratory handling procedures, blood samples were collected within 3 min of moving the cage (Place & Kenagy, 2000). Rats were euthanized with CO2 and lung samples were collected and stored at –80 °C until processed.
Corticosterone enzyme immunoassay (EIA).
Corticosterone was extracted from sera using diethyl ether. The ether layer was removed and evaporated and samples were reconstituted in assay buffer (Cayman Chemical). Corticosterone concentrations in serum (final dilution 1 : 100) were measured using a commercial EIA kit and the manufacturer's protocol (Cayman Chemicals).
RNA isolation.
RNA was isolated from the lungs using TRIzol LS (Invitrogen) and the manufacturer's protocol as described previously (Klein et al., 2000, 2001). Following 2-propanol precipitation and washing in 75 % ethanol, RNA pellets were briefly air-dried and resuspended in DEPC-treated water.
Real-time quantitative RT-PCR for SEOV detection.
First-strand synthesis of the S segment of SEOV cDNA was prepared as described previously using 0.1 µM gene-specific primers and the Invitrogen Superscript III First Strand Synthesis reagents (Easterbrook & Klein, 2008; Hannah et al., 2008). Negative- and positive-sense SEOV was measured by real-time RT-PCR as described elsewhere (Hannah et al., 2008).
Real-time quantitative RT-PCR for host gene expression.
First strand cDNA was prepared and gene expression was measured by real-time RT-PCR, as described previously (Easterbrook & Klein, 2008; Hannah et al., 2008). Custom primer and probe sets were generated using Primer Express 2.0 software (Applied Biosystems) and the NCBI sequences for genes in Rattus norvegicus, with the exception of murine Ror
t. Gene expression patterns from SEOV-infected animals were normalized to Gapdh expression and are presented as relative to the expression levels from uninfected rats (i.e. day 0 p.i.) in the appropriate corticosterone treatment group and are expressed as fold change.
Cytokine protein quantification by ELISA.
Lung tissue was homogenized in lysis buffer, as described previously (Easterbrook & Klein, 2008). Cytokine protein in the supernatant was measured using ELISA kits for rat TNF- and active mouse TGF-β1 (R&D Systems). Protein concentrations are expressed as fold change relative to protein concentrations in uninfected rats (i.e. day 0 p.i.).
FACS analyses.
Lung tissue was digested in collagenase (1 mg ml–1; Invitrogen) and DNase (3 µg µl–1; Roche) to produce a single cell suspension. Following red blood cell lysis, non-specific binding was minimized by incubation with anti-rat CD32 (FcII receptor). Cells were stained for viability using ethidium monoazide (EMA; Invitrogen) and the appropriate anti-rat monoclonal antibodies (BD Biosciences, unless otherwise specified) as follows:
CD4+ T cells: fluorescein isothiocyanate (FITC)-CD3 (clone G4.18), phycoerythrin (PE)-CD25 (clone OX-39) and allophycocyanin (APC)-CD4 (clone OX-35); CD8+ T cells: FITC-CD8 (clone OX-8), PE-CD3 (clone G4.18) and APC-CD4; B cells: FITC-CD45R (clone HIS24) and APC-CD4; NK cells: FITC-CD3, PE-CD161a (clone NKR-P1A) and APC-CD5 (clone HIS47; eBioscience); regulatory T cells: FITC CD4 (clone OX-35) and PE-CD25; and macrophages: PE-CD3. Following fixation and permeabilization, regulatory T cells and macrophages were further labelled with APC-FoxP3 (clone FJK-16a; eBioscience) and FITC-CD68 (ED-1; Serotec), respectively. Isotype controls for each fluorochrome were run using the same procedure as surface and intracellular labelling and were used to determine gating limits. Cells were identified using CellQuest Pro (BD Biosciences) and data were analysed using FlowJo software (Tree Star).
Statistical analyses.
Quantitative variables were analysed using ANOVAs or the non-parametric equivalent. Significant interactions were further analysed using the Tukey or Dunn method for pairwise multiple comparisons. Mean differences were considered statistically significant if P<0.05. Correlational analyses were performed using the Pearson product moment.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Kitay, 1961; You-Ten & Lapp, 1996). Removal of the adrenal glands and subsequent replacement with 0 (ADX0), 10 (ADX10) and 80 % (ADX80) corticosterone resulted in low (12.6±1.4 ng ml–1), medium (35.1±2.7 ng ml–1) and high (93.0±7.3 ng ml–1) concentrations of corticosterone in the serum of male and females rats throughout SEOV infection (Fig. 1a and b; P<0.001 in each case). In male rats that received sham surgeries and had intact adrenal glands (sham), concentrations of corticosterone were reduced throughout SEOV infection (Fig. 1a; P<0.001) and were similar to concentrations in ADX0 males. In sham females, corticosterone concentrations were not affected by SEOV infection (Fig. 1b).
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; P<0.001 in each case), but to a greater extent in males than females (P<0.01). At day 15 p.i., ADX0 and sham males had more SEOV RNA in the lungs than ADX10 or ADX80 males (Fig. 2a; P<0.001). Manipulation of corticosterone did not affect the amount of SEOV RNA in the lungs of female rats. Although the amount of SEOV RNA was reduced at day 40 p.i., it was still detectable in the lungs of male and female rats in all treatment groups, but to a greater extent in sham male (9/10) than sham female (6/10) rats.
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; Hannah et al., 2008). Positive-sense SEOV mRNA was detected in the lungs of males and females and paralleled the distribution of negative-sense SEOV RNA copies at day 15 p.i., but at an approximately 195-fold lower copy number (P<0.001). The copies of negative- and positive-sense SEOV RNA were positively correlated at all time points during infection (r=0.65, P<0.001).
Innate, proinflammatory, CD8+ and CD4+ T-cell responses do not mediate the glucocorticoid-dependent sex difference in SEOV load
To test a likely hypothesis that immunomodulation by glucocorticoids caused the increased amount of SEOV RNA in the lungs of male rats with low corticosterone, proportions of various immune cell types and genes that are indicative of cellular activity were examined. There was no significant effect of SEOV infection or corticosterone treatment on the percentage of macrophages in the lungs of female or male rats (Supplementary Table S1, available in JGV Online). Expression of several innate and proinflammatory mediators was elevated (e.g. Tnf and Ifnβ) or unchanged (i.e. Myd88 and Nos2) in the lungs of sham and ADX females with low corticosterone concentrations during SEOV infection (P<0.05 for both cases); the administration of a high dose of corticosterone suppressed or eliminated the elevated expression of these innate and proinflammatory mediators in females (Fig. 3a, c, d, and f and Supplementary Table S2; P<0.05 for each case). The expression of Ifnβ, Tnf, Myd88 and Nos2 was reduced in the lungs of male rats throughout SEOV infection, regardless of glucocorticoid manipulation (Fig. 3a, b, d, and e and Supplementary Table S2; P<0.05 for each case). The expression of Il6 was reduced or remained unchanged in the lungs of both males and females throughout SEOV infection, regardless of corticosterone treatment (Supplementary Table S2; P<0.05).
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t) and Il17, were not altered by SEOV infection or glucocorticoid manipulation in the lungs of male and female rats (Supplementary Tables S1–S3). The proportions of CD8+ T cells and expression of Ifn and granzyme B (Gzmb) genes were not affected by corticosterone manipulation in males or females; proportions of CD8+ T cells and expression of Ifn, however, were elevated in the lungs of sham females compared with sham males during persistent infection (Supplementary Tables S1 and S3; P<0.001 for each case).
Elevated regulatory T-cell activity in the lungs is associated with low circulating corticosterone in male, but not female, rats during SEOV infection
Previous data demonstrate that male rats have elevated proportions of regulatory T cells in the lungs during persistent SEOV infection (Easterbrook et al., 2007). The proportions of regulatory T cells and expression of Tgfβ and Foxp3 in the lungs were elevated in male rats with low corticosterone concentrations, but not in ADX80 males, 30 and 40 days p.i. (Fig. 4a and b and Supplementary Tables S1 and S2; P<0.05 in each case). There was no effect of SEOV infection or corticosterone treatment on either the proportion of regulatory T cells or the expression of Tgfβ and Foxp3 in the lungs of female rats (Fig. 4a and c and Supplementary Tables S1 and S2). Low concentrations of corticosterone correlated with increased regulatory T-cell activity in the lungs of males (Tgfβ: r=–0.27, P<0.001 and Foxp3: r=–0.17, P=0.04). Similar patterns of TGF-β protein production as Tgfβ gene expression in the lungs were observed for both males and females throughout infection (data not shown).
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; Lafrenie et al., 1996; Nilsson et al., 2007). Expression of Mmp9 was higher in the lungs of sham males than sham females during SEOV infection (Fig. 4d
; P<0.001). Mmp9 expression was elevated early during infection at day 3 p.i. in ADX0 and sham male rats as compared with ADX10 and ADX80 males (Fig. 4e; P<0.05 for each case). In the lungs of female rats, expression of Mmp9 was reduced during SEOV infection, regardless of glucocorticoid treatment (Fig. 4f; P=0.006). Tissue inhibitor of metalloproteinase (TIMP)-1 is a specific inhibitor of Mmp9 expression and the expression of Timp1 in the lungs did not differ among corticosterone treatment groups in either males or females (Supplementary Table S2). TGF-β causes increased expression of Mmp9 and our data consistently revealed a correlation between Tgfβ and Mmp9 expression across all time points during SEOV infection (r=0.23, P=0.009). Thus, the expression of Mmp9 correlated with SEOV load in males with low circulating corticosterone. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Silverman et al., 2005). Hantaviruses and their reservoir hosts, however, represent a highly co-evolved system in which viral persistence is maintained and the host does not display symptoms of disease. The role of glucocorticoids in maintaining the balance between survival of SEOV and survival of the rat host, a model system of host homeostasis, was examined. Circulating concentrations of corticosterone were reduced during SEOV infection in male, but not female, rats. Peak infection, as defined by the highest amount of SEOV RNA in the lungs, varies across studies, but consistently ranges between 15 and 30 days p.i., after which point viral load is reduced, but remains detectable presumably for the lifetime of the rat (Easterbrook & Klein, 2008; Easterbrook et al., 2007; Hannah et al., 2008; Klein et al., 2001, 2002). Male rats with the lowest concentrations of corticosterone had the highest amount of SEOV RNA in the lungs during peak infection (i.e. day 15 p.i.). No relationship between glucocorticoids and SEOV RNA was observed among females; thus, low corticosterone is associated with elevated SEOV replication or dissemination in the lungs of males only. These data suggest that, in addition to the known effects of sex steroid hormones (Hannah et al., 2008; Klein et al., 2002), glucocorticoids also contribute to different patterns of SEOV infection in male and female rats.
The lungs consistently are a site of elevated hantavirus replication and persistence (Botten et al., 2003; Easterbrook & Klein, 2008; Hutchinson et al., 1998; Lee et al., 1981). Because glucocorticoids have immunomodulatory properties, immune responses in the lungs were analysed to evaluate potential mediators of hantaviral persistence and replication at a primary site of infection. Glucocorticoids did not affect proportions of CD3+, non-regulatory CD4+ T cells, CD8+ T cells, B cells or macrophages in the lungs of male and female rats during SEOV infection. Every nucleated cell expresses glucocorticoid receptors which, when bound by glucocorticoids, translocate to the nucleus to alter transcription (Miller et al., 1998; Silverman et al., 2005). Thus, glucocorticoids were hypothesized to exert regulation of cell function at the transcriptional level rather than on the number or proportion of cells present in the lungs. The expression and production of proinflammatory, antiviral, as well as CD8+ T cell, Th1, Th2 and Th17 transcription factors and cytokines, however, did not correlate with elevated SEOV RNA load in the lungs of male rats with low concentrations of corticosterone.
Localized regulatory T cell responses (i.e. FoxP3 and TGF-β production) are elevated during persistent infection and contribute to hantavirus persistence in male rodents (Easterbrook et al., 2007; Schountz et al., 2007). This study shows that, during SEOV infection, proportions of regulatory T cells, as well as the expression and production of Foxp3 and TGF-β, are elevated in the lungs of male, but not female, rats with low circulating concentrations of corticosterone (i.e. sham and ADX0 males). Although glucocorticoids and regulatory T cells both contribute to host homeostasis, glucocorticoids negatively regulate regulatory T-cell responses through a negative glucocorticoid response element (GRE) on the TGF-β promoter (Parrelli et al., 1998). In males, reduced concentrations of corticosterone may contribute to subsequently elevated production of TGF-β and regulatory T-cell responses.
The immune mediators that were examined did not fully explain the association between reduced circulating corticosterone and elevated SEOV RNA during peak infection in the lungs of male rats. Glucocorticoids also regulate the transcription of non-immune genes, including metabolic factors (e.g. rat arginase and tyrosine aminotransferase), cell proliferation factors (e.g. cyclin D3), and enzymes (e.g. MMP-9) (Bishayi & Ghosh, 2003; Newton, 2000). MMP-9 is a glycogenase that disrupts the basement membrane and extracellular matrix to permit mobility of cells through tissues and can contribute to extravasation of HIV-infected monocytes (Lafrenie et al., 1996). Hantaviruses also infect monocytes and macrophages and could increase dissemination into tissues by upregulating MMP-9 to disturb the protective basement membrane barrier. The expression of Mmp9 is negatively regulated by glucocorticoids (Eberhardt et al., 2002). Male rats with low circulating corticosterone had elevated expression of Mmp9 in their lungs during the acute phase of SEOV infection as compared with males with higher corticosterone concentrations. Among several species, females express less Mmp9 than males, which is mediated by the suppression of Mmp9 expression by oestradiol (Ailawadi et al., 2004; Nilsson et al., 2007). In contrast to male rats, females had reduced expression of Mmp9 in the lungs during SEOV infection, suggesting that oestradiol may inhibit Mmp9 expression in females, regardless of glucocorticoid concentrations. Additionally, TGF-β induces the expression of Mmp9 in macrophages and endothelial cells (Puyraimond et al., 1999; Xie et al., 1994). Male rats with the highest expression of Tgfβ also had elevated expression of Mmp9, suggesting that production of TGF-β may contribute to regulatory T-cell activity and Mmp9 expression in male rats during early SEOV infection. Elevated production of MMP-9 during the acute phase of SEOV infection (e.g. day 3 p.i.) may contribute to more efficient viral dissemination and, subsequently, more SEOV RNA in the lungs of male than female rats during peak infection (e.g. day 15 p.i.). The initial dissemination of SEOV-infected cells may contribute to an increased viral load in lung tissue, but, once infection is established, extended production of MMP-9 may be unnecessary for maintenance of viral persistence. Immune mediators, including regulatory T cells, are more likely to be the predominant mediators of SEOV persistence in the lungs of rats. Whether SEOV-infected or bystander macrophages and endothelial cells contribute to elevated Mmp9 expression in the lungs of males requires further investigation. Additionally, the temporal dynamics of MMP-9 production at time points between days 3 and 15 p.i. and the role of MMP-9 in mediating SEOV dissemination should be further evaluated.
The mechanism by which SEOV infection causes a reduction of corticosterone concentrations in male rats remains unknown. SEOV RNA has been identified in the adrenal glands of male and female rats (Klein et al., 2002; Hinson et al., 2004), so whether SEOV may act directly on cellular production of corticosterone in the adrenal cortex or indirectly through other host immunological or endocrine factors to modulate corticosterone production requires further investigation. The role of immune and endocrine mediators in contributing to the maintenance of zoonotic viruses, including hantaviruses, in the environment should continue to be examined.
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ACKNOWLEDGEMENTS |
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Received 26 April 2008;
accepted 26 June 2008.
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