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International Immunology Advance Access originally published online on September 5, 2007
International Immunology 2007 19(11):1261-1270; doi:10.1093/intimm/dxm097
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Thymosin {alpha}1 activates the TLR9/MyD88/IRF7-dependent murine cytomegalovirus sensing for induction of anti-viral responses in vivo

Silvia Bozza1,2, Roberta Gaziano1, Pierluigi Bonifazi1, Teresa Zelante1, Lucia Pitzurra1, Claudia Montagnoli1, Silvia Moretti1, Roberto Castronari1, Paola Sinibaldi3, Guido Rasi4, Enrico Garaci5, Francesco Bistoni1,2 and Luigina Romani1,2

1 Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Via del Giochetto, 06122 Perugia, Perugia, Italy
2 Fondazione "Istituto di Ricovero e Cura per le Biotecnologie Trapiantologiche" I.B.i.T., Perugia, Italy
3 Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Tor Vergata, Rome, Italy
4 Institute of Neurobiology and Molecular Medicine, Centro Nazionale Ricerche, Rome, Italy
5 National Institute of Health, Rome, Italy

Correspondence to: L. Romani; E-mail: lromani{at}unipg.it


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 Funding
 References
 
Reactivation of latent human cytomegalovirus following allogeneic transplantation is a major cause of morbidity and mortality and predisposes to severe complications. Thymosin {alpha}1 (T{alpha}1), a naturally occurring thymic peptide, is approved for treatment of some viral infections and as an immune adjuvant. T{alpha}1 successfully primed dendritic cells (DCs) for anti-microbial T helper type 1 resistance through Toll-like receptor (TLR) 9 signaling. We sought to determine here whether T{alpha}1 could play a role in murine cytomegalovirus infection (MCMV). To this purpose, susceptible, resistant and TLR-deficient mice were infected with MCMV, treated with T{alpha}1 and assessed for protection in term of microbiological and immunological parameters. T{alpha}1 protected susceptible and resistant mice from MCMV infection. The anti-viral effect of T{alpha}1 occurred through the activation of plasmacytoid DCs via the TLR9/myeloid differentiation primary response gene 88-dependent viral recognition sensing, leading to the activation of IFN regulatory factor 7 and the promotion of the IFN-{alpha}/IFN-{gamma}-dependent effector pathway.

Keywords: dendritic cells, immunomodulation, innate immunity, viral infections


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 References
 
Medical advances such as allogeneic transplantation can expose patients to periods of marked immunosuppression, during which human cytomegalovirus (HCMV) reactivation is a major cause of morbidity and mortality (1–3). HCMV, a member of the Herpesviridae family, is a ubiquitous opportunistic pathogen that has intimate lifelong relationship with its human host and establishes latency after clearance of primary infection (4). Reactivation of latent virus following allogeneic transplantation results in progressive tissue damage manifesting as overt HCMV disease that usually presents as pneumonia, colitis or hepatitis or complications of this infection, including acute and chronic graft rejection, graft-versus-host disease and superinfection by other viruses, bacteria and fungi, particularly Aspergillus spp (3). Currently available anti-viral pharmacotherapy is limited by toxicities and lack of efficacy in established HCMV disease. Efforts have therefore focused on the development of adoptive immunotherapeutic strategies to hasten host immune reconstruction (5–7). Control of infection will depend ultimately on the restoration of adequate anti-viral immunity, and cellular immunotherapy is an attractive approach to improving immune protection (6, 7).

The immune control of murine cytomegalovirus (MCMV) infection requires elements from both innate and adaptive immune systems (4, 8–10). Through the participation of member of the Toll-like receptors (TLRs) (11–13) and IFN regulatory factor (IRF) families (14–16), MCMV induces early dendritic cell (DC)-dependent type I IFN and IL-12 responses that are essential for mouse resistance to MCMV (17–21). The TLR9/myeloid differentiation primary response gene 88 (MyD88)-signaling pathway mediates anti-viral cytokine responses by plasmacytoid dendritic cell (pDC) that, through their unique capacity to secrete IFN-{alpha}, and to a lesser extent IL-12 and other innate cytokines, is a cornerstone in the initiation of both innate and adaptive immune responses to MCMV. However, conventional CD11b+DCs also produce IFN-{alpha} independently of TLR9 and MyD88 (11, 12, 22). In addition to directly interfering with viral replication through ubiquitous cellular mechanisms, IFN-{alpha} controls NK cell cytotoxic activity (18) and regulates T cell functions by activating classical DC to more efficiently present antigens (18). IL-12 and IL-18 secretion are instead required to prime a strong NK cell-dependent IFN-{gamma} response (21, 23, 24), a process that is essential to counteract MCMV infection in the liver, in contrast to a perforin-dependent mechanism in the spleen (25).

Thymosin {alpha}1 (T{alpha}1), a naturally occurring thymic peptide, is approved in 30 countries as a biological response modifier for treatment of some viral infections, either as monotherapy or in combination with IFN-{alpha}, and as an immune adjuvant (26). Additional indications are some immunodeficiencies, malignancies and acquired immune deficiency syndromes. It has recently been shown that T{alpha}1 modulated DC functioning through direct and indirect effects on TLR9 signaling, thus acting as an endogenous regulator of the innate and adaptive immune systems (27). Given the prominent role of TLR9 as a pattern recognition receptor for murine MCMV, we sought to determine whether T{alpha}1 could play a role in MCMV infection. To this purpose, susceptible, resistant and TLR-deficient mice were infected with MCMV, treated with T{alpha}1 and assessed for protection in term of microbiological and immune parameters. T{alpha}1 decreased the viral load in both susceptible and resistant mice. The anti-viral effect of T{alpha}1 occurred through the activation of pDC via the TLR9/MyD88-dependent viral recognition sensing leading to the activation of IRF7 and the promotion of the IFN-{alpha}/IFN-{gamma}-dependent effector pathway.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
Mice
Wild-type (WT) inbred C57BL6 and BALB/c mice, 8- to 12-week old, were purchased from Charles River Breeding Laboratories (Calco, Italy). Breeding pairs of homozygous TLR9–(Tlr9–/–)-, TLR4–(Tlr4–/–)- and MyD88–(Myd88–/–)-deficient mice (on the C57BL6 background) were bred under specific pathogen-free conditions at the breeding facilities of the University of Perugia, Perugia, Italy. Experiments were performed following protocols approved by the Institutional Animal Committee and in accordance with European Economic Community Council Directive as well as Institutional Animal Care and Use guidelines.

Virus, infection and treatments
Stocks of Smith strain MCMV salivary gland extracts were prepared from BALB/c mice and titered in a standard plaque assay on BALB/c murine embryonic fibroblast (MEF) cells (28). Mice were injected intra-peritoneally (i.p.) with 1 x 105 (BALB/c) or 5 x 105 (C57BL6) plaque-forming units (PFU) of MCMV. Virus titers were quantified on MEF cells by standard plaque assay on tissues removed at different times. Treatments were as follow: T{alpha}1 (>90% pure on HPLC, product number T3410, from Sigma–Aldrich, Milan, Italy) was supplied as purified (the endotoxin levels were <0.03 pg ml–1, by a standard limulus lysate assay) sterile, lyophilized polypeptide. The sequence was as follows: Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-IIe-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Glu-Glu-Ala-Glu-Asn-O. The lyophilized powder was reconstituted in sterile water and 200 µg kg–1 i.p. was given daily for 7 or 14 consecutive days beginning the day of the infection. Gancyclovir (GCV) (Cymevene, from Recordati, Milan, Italy) was administered at 40 mg kg–1 i.p., three times a week, every other day, beginning 6 h after infection. Controls received the scrambled peptide (27).

DC subset generation
DCs were obtained by culturing 106 per ml BALB/c bone marrow cells in Iscove's modified medium, containing 10% filtered bovine serum, 50 µM 2-mercaptoethanol, sodium pyruvate (1 mM), 2 mM L-glutamine, HEPES (10 mM) and 50 µg ml–1 gentamycin in the presence of 150 U ml–1 mouse rGM-CSF (Sigma) and 75 U ml–1 rIL-4 (R&D Systems) for 7 days to obtain CD11b+DC or 200 ng ml–1 FLT3L (Immunex Corporation, Seattle, WA, USA) for 9 days to obtain pDC. Final maturation was accomplished by the addition of 1 µg ml–1 LPS or 2 µg ml–1 cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG-B ODN 1668) (Coley Pharmaceutical Group, Wellesley, MA, USA) for additional 24 h to CD11b+DC or pDC, respectively (29). CD11b+DCs were discriminated on CD11chigh expression and were distinctly composed of CD8{alpha}+DCs and CD11b+DCs. pDCs were defined as CD11clow, Ly6C+ and CD8{alpha}+/– cells.

Flow cytometry analyses
After blocking of FcRs with the anti-CD16/32 (2.4G2) antibody, cells were analyzed for antigen expression with a FACScan flow cytofluorometer (Becton Dickinson, Mountain View, CA, USA) equipped with CELLQuestTM software. Control staining of cells with irrelevant antibody was used to obtain background fluorescence values. Antibodies were from BD Biosciences PharMingen (San Diego, CA, USA).

Plaque assay
Plaque assay was determined on cells grown to subconfluence and incubated with serially diluted virus samples for 2 h at 37°C (30). All organs from uninfected animals were negative viruses. Virus titers are expressed as log10 (mean ± SE) per gram of tissue.

Inhibition of viral replication
DCs (106 per well) were pre-incubated for 2 h at 37°C with 50 µg ml–1 T{alpha}1 diluted in serum-free DMEM and then added of 105 PFU MCMV. Infectivity was measured 48 h later.

NK cell cytotoxic activity
NK cells were purified from spleens by DX5 microbeads (Miltenyi Biotec). NK cytolytic activity was assessed against Cr-labeled YAC-1 lymphoma cells (7).

IRF7 analysis
DCs were exposed for 3 h to 50 µg ml–1 T{alpha}1 and/or 105 PFU MCMV. For immunoblot analysis, nuclear extracts were prepared with NE-PER nuclear and cytoplasmic extraction reagents (Pierce, Celbio S.r.l., Milan, Italy) and the protein concentration of the nuclear extracts was determined with a BCA-200 protein assay kit (Pierce) following the manufacture’s instructions. Nuclear content of IRF7 was determined by western blot using polyclonal rabbit anti-IRF7 (H-246) antibody and bovine anti-rabbit HRP (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Visualization was performed with the ECL Western blotting analysis system from Amersham and Kodak Biomax films. Intracellular flow for IRF7 was performed as described (31) using indirect cytoplasmic staining on 2 x 106 DCs exposed as above for 6 h. Briefly, after stimulation, DCs were stained with PE-labeled hamster anti-mouse CD11c (BD Biosciences PharMingen) and then fixed overnight at 4°C with 1% PFA (Fisher Scientific, Hampton, NH, USA) in PBS. After washing, the cells were permeabilized with 0.5% saponin (Sigma–Aldrich) for 15 min at room temperature (RT) and then incubated with polyclonal rabbit anti-IRF7 (400 ng) or normal rabbit IgG (as a control) (Santa Cruz Biotechnology) for 30 min at room temperature. The cells were washed twice with permeabilization buffer and incubated 30 min with goat anti-rabbit IgG FITC (Sigma–Aldrich). The cells were fixed and analyzed using FACScan flow cytofluorometer. Nuclear translocation by fluorescence microscopy was done on fixed cells, seeded on glass slides by cytospin and then permeabilized with 0.2% Triton X-100. Cells were blocked with wash buffer containing 3% BSA and 10% normal goat serum for 30 min and incubated with the primary antibody, anti-IRF7 at a 1/20 dilution or normal rabbit IgG, as an isotype control. After washing, cells were incubated with FITC-conjugated anti-rabbit IgG and 4',6'-diamidino-2-phenylindole at 50 ng ml–1. Following extensive washing of the slides, cover slips were placed with PBS. Slides were scanned with an Axioskop 2 plus (Carl Zeiss S.p.A., Milan, Italy) fluorescence microscope with digital image capture (Color Camera AxioCam, using the AxioVision Software Rel. 3.1).

Immunoblot analysis of IRF3 phosphorylation
DCs were exposed for 3 h to 50 µg ml–1 T{alpha}1 and/or 105 PFU MCMV. Immunoblot analysis was conducted by a standard procedure in conditions in which the phosphorylation status of proteins is maintained (32). The antibodies used were rabbit anti-IRF3 and bovine anti-rabbit HRP (Santa Cruz Biotechnology). This procedure allows the simultaneous detection of the two non-activated IRF3 forms (I and II) and the activated, C-terminally phosphorylated IRF3 (P). Visualization was performed with the ECL Western blotting analysis system from Amersham and Kodak Biomax films.

Quantification of MCMV mRNA
Reverse transcription–PCR assay was used for amplification of the 356-bp segment of MCMV glycoprotein B (gB) DNA from total cellular RNA (33). Synthetic DNA oligonucleotide primers were selected from the published sequence of the MCMV gB gene (11). The sense primers were based on the cDNA no. 2416–2443: 5'-AAG-CAG-CAC-ATC-CGC-ACC-CTG-AGC-GCC-3' and the anti-sense on no. 2745–2772: 5'-CCA-GGC-GCT-CCC-GGC-GGC-CCG-CTC-TCG-3'. Cycling conditions were initial denaturation for 3 min at 95°C, followed by cycles of 1 min at 95°C, 1 min at 50°C and 20 s at 72°C, and a final extension for 10 min at 72°C.

Quantification of cytokines by ELISA and ELISPOT assays
The levels of cytokines in the culture supernatants of mitogen-stimulated spleen cells (48 h stimulation with 10 µg ml–1 ConA) or MCMV-pulsed DCs (24 h) were determined by ELISA (R&D Systems and PBL, Biomedical Lab, Milan, Italy). The detection limits (pg ml–1) of the assays were <16 for IL-12p70, <10 for IFN-{gamma}, <3 for IL-10 and <10 for IFN-{alpha}. IFN-{gamma}-producing cells were enumerated by ELISPOT assay on purified cells (29). CD8+ and CD4+T cells were isolated from spleens by positive selection using magnetic-activated cell sorting enrichment kits (Miltenyi Biotec). The purity of enriched samples was >90%. Results are expressed as the mean number of cytokine-producing cells (±SE) per 105 cells, calculated using replicates of serial 2-fold dilutions of cells.

Statistical analyses
Student's t-test was used to determine the statistical significance of values in experimental groups. Significance was defined as P < 0.05. In vivo groups consisted of six animals. Unless otherwise indicated, data are mean ± SE.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
T{alpha}1 protects from MCMV infection
We assessed the effects of T{alpha}1 administration in the acute primary MCMV infection of either susceptible (BALB/c) or resistant (C57BL6) mice. Mice were infected with a sub-lethal dose of MCMV, treated with T{alpha}1 or GCV and the titer loads in lung, salivary gland, spleen and liver were determined at different weeks after the infection by standard plaque assay titration. MCMV replicated to higher titers in the visceral organs of susceptible (Fig. 1A) than resistant (Fig. 1B) mice, particularly in the early phase of the infection. T{alpha}1, given at 200 µg kg–1 for 7 days significantly decreased the viral load in different visceral organs, in both susceptible and resistant mice. The effect was similar to that of GCV and was higher with T{alpha}1 given for 14 days. In resistant mice, the effect of T{alpha}1 was particularly evident in the salivary gland and liver (Fig. 1B). T{alpha}1 also significantly decreased the viral load in BALB/c mice infected with a higher PFU (data not shown). These results suggest that T{alpha}1 may exert therapeutic effects in MCMV infection.


Figure 1
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  		Fig. 1. T{alpha}1 protects from primary MCMV infection in vivo. BALB/c (A) and C57BL6 (B) mice were infected with 105 or 5 x 105 PFU of MCMV, respectively. Virus titers were quantified on MEF cells by standard plaque assay on tissues removed at different times after infection and expressed as log10 (geometric means ± SDs). T{alpha}1 (200 µg kg–1 given for 7 or 14 days) and GCV (40 mg kg–1 given three times a week) were administered beginning the day of the infection. Controls received the scrambled peptide. Results are representative of four independent experiments. Bars indicate the standard deviations. *P < 0.05, treated versus untreated mice.

 
T{alpha}1 activates the TLR9/MyD88-dependent anti-viral pathway
Effective anti-MCMV immune surveillance, including early NK cell responses, required the TLR9/MyD88-signaling pathway, TLR3 and TLR2 whereas the TLR4 did not seem to play a significant role (11–13, 34, 35). To assess the involvement of TLR in the effect of T{alpha}1 in infection, TLR-deficient mice were challenged with MCMV, treated with T{alpha}1 and followed for viral replication, NK cell activity and cytokine production. Figure 2(A) shows that Tlr9–/– and Myd88–/– mice were more susceptible to infection than C57BL6 mice, while deficiency for TLR4 did not significantly affect mouse resistance. T{alpha}1 was effective in C57BL6 and in Tlr4–/– mice but completely ineffective in Tlr9–/– or Myd88–/– mice, a finding suggesting the involvement of the TLR9/MyD88-dependent signaling in the anti-viral activity of T{alpha}1. CD69+NK cells were expanded (Fig. 2B) and fully activated, in terms of cytolytic activity and IFN-{gamma} production (Fig. 2C and D), by T{alpha}1 in WT and Tlr4–/– but not Myd88–/– mice. In line with previous findings (11), CD69+NK cells were poorly expanded and activated, at least in term of IFN-{gamma} production, in Tlr9–/– mice. As early activation of NK cells in MCMV infection is mediated by IFN-{alpha}/ß which promotes cytotoxicity and proliferation of NK cells and IL-12 which induces IFN-{gamma} production (17–21), we measured IL-12p70, IFN-{alpha}, IFN-{gamma} and IL-10 production in culture supernatants of spleen cells from infected mice treated with T{alpha}1. The efficacy of T{alpha}1 directly correlated with the production of IFN-{alpha}, more than IL-12p70 and IFN-{gamma} whose levels were significantly increased in supernatants of splenocytes from C57BL6 and Tlr4–/– mice, but not Tlr9–/– or Myd88–/– mice (Fig. 2E).


Figure 2
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  		Fig. 2. T{alpha}1 activates the TLR9/MyD88-dependent pathway. C57BL6, Tlr4–/–, Tlr9–/– and Myd88–/– mice were infected with 5 x 105 PFU of MCMV and treated with 200 µg kg–1 T{alpha}1 for a week before the assessment of (A) viral load, (B) DX5+CD69+NK cell percentage, (C) cytotoxic activity and (D) number of IFN-{gamma}+NK-producing cells and (E) cytokine production. Virus titers, expressed as log10, were quantified on MEF cells by standard plaque assay on infected tissues removed a day after the last T{alpha}1 injection. *P < 0.05, Tlr9–/– and Myd88–/– versus C57BL6 mice. The number of splenic DX5+CD69+NK cells on total spleen cells was done by FACS analysis, the cytotoxic activity of purified splenic DX5+CD69+NK cells by standard 51Cr-release assay against YAC-1 targets and the frequency of IFN-{gamma}-producing NK cells by ELISPOT assay (mean ± SE, x105). Cytokine (pg ml–1) levels in culture supernatants of spleen cells were determined by ELISA assay. C, uninfected control mice. Symbols are as follows: filled triangles, uninfected mice; mice at 2 (filled circles) or 7 (filled squares) days after the infection; T{alpha}1-treated mice at 2 (open circles) or 7 (open squares) days after the infection. *P < 0.05, infected versus uninfected mice. **P < 0.05, T{alpha}1-treated versus untreated mice. Results are representative of three independent experiments.

 
T{alpha}1 promotes viral infection, IRF7 activation and cytokine production in pDC
As early activation of NK cells in MCMV infection is both TLR9 and DC dependent (12), we evaluated DC activation upon exposure to MCMV in the presence of T{alpha}1. Acute infection with MCMV induces a transient, but profound, immunosuppression in susceptible BALB/c mice, which can be linked to infection of CD11b+DCs (22). CD11b+DCs support productive infection of MCMV both in vitro and in vivo (22), whereas MCMV does not replicate in pDCs (13, 18, 22, 36). We resorted to bone marrow-derived CD11b+DC or pDC subsets from uninfected mice to allow us to directly assess the effect of T{alpha}1 on viral replication, activation and cytokine production by the different DC subsets from WT and Tlr9–/– mice. In line with previous studies (13, 18, 22, 36), MCMV replicated in CD11b+DC but not in pDC. Viral replication was not affected by T{alpha}1 in CD11b+DC but, interestingly, was actually promoted in pDC, as assessed by the levels of MCMV gB transcripts (Fig. 3A) paralleled by similar results obtained upon measuring infectious viral particles in culture supernatants (data not shown). Exposure to CpG-B ODN failed to induce viral replication in pDC, at least in the same conditions used for T{alpha}1 (data not shown). As pre-treatment of virus with T{alpha}1 prior to DC exposure also promoted, albeit partially, viral replication in pDC, this finding suggests a cell-independent effect T{alpha}1 (data not shown).


Figure 3
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  		Fig. 3. T{alpha}1 promotes viral replication, IRF7 activation and cytokine production in pDC. Bone marrow-derived CD11b+DCs or pDCs from uninfected mice were exposed in vitro to MCMV and/or T{alpha}1 and assessed for (A) viral replication by reverse transcription–PCR of MCMV gB transcripts expression, (B) IRF7 activation as intracellular IRF7 content by FACS analysis, (C) nuclear translocation detected by immunoblotting and (D) immunofluorescence with anti-IRF7; (E) cytokine production. In (A), DCs were infected with MCMV in the presence (+) or not (–) of 50 µg ml–1 T{alpha}1 for 3 h at 37°C. Dash indicates uninfected cells. The results shown represent one representative experiment out of three. (B) Intracellular content of IRF7 in uninfected (lane 1), MCMV-infected (lane 2), T{alpha}1-exposed (lane 3) and MCMV+T{alpha}1-treated DCs (lane 4). Cells were exposed for 6 h to 50 µg ml–1 T{alpha}1 and/or MCMV, before sequential incubation with anti-IRF7 or normal rabbit IgG (as a control) and goat anti-rabbit IgG FITC. Data are the mean fluorescence intensity minus means of isotype control ± SEs of three independent experiments. *P < 0.05, lanes 3 and 4 versus lane 2. (C) Immunoblotting of IRF7 (arrow indicates an inducible band) done after 3 h exposure to T{alpha}1 and/or MCMV. Immunoblotting of IRF3 was done in CD11b+ DCs or pDCs from uninfected C57BL6 mice exposed as above. The two non-activated IRF3 forms (I and II) and the activated, C-terminally phosphorylated IRF3 (P) are indicated. Nuclear fractionation was checked by blotting with anti-aldolase (as negative control) antibodies. Lanes are as in (B). (D) IRF7 nuclear translocation was assessed using anti-IRF7 or normal rabbit IgG, as an isotype control (IgG/MOCK), via a FITC-conjugated anti-rabbit IgG, and DAPI 4',6-diamidino-2-phenylindole staining to demonstrate the location of the nucleus. Slides were scanned with a fluorescence microscope with digital image capture. (E) Cytokine production (pg ml–1 at 24 h) by DC subsets treated as above. Bars indicate the standard errors. *P < 0.05, cytokine production in MCMV-exposed DC versus unexposed DC. **P < 0.05, T{alpha}1-treated versus untreated DC.

 
HCMV elicits a coordinated cellular anti-viral response for IFN production which includes the temporal activation of IRF3 and IRF7 that results in nuclear accumulation of these factors (16). As IRF7 is the master regulator of anti-viral type 1 IFN production in pDC (15, 31, 37), and the TLR9-signaling pathway has a crucial role for the IRF7-dependent anti-viral program (15), we examined IRF7 expression in DC subsets from both C57BL/6 and Tlr9–/– mice upon virus and/or T{alpha}1 exposure in vitro by intracellular staining, immunoblotting with anti-IRF7 antibody and nuclear translocation. IRF7 was present constitutively at high levels in pDC more than CD11b+DC from either WT or Tlr9–/– mice and was unmodified upon viral exposure (Fig. 3B). In contrast, T{alpha}1, either alone or combined with the virus, significantly increased the IRF7 intracellular content in a TLR9-dependent manner (Fig. 3B). Importantly, we noted translocation of IRF7 upon either virus or T{alpha}1 exposure, either alone or in combination, and this occurred in pDC from WT but not Tlr9–/– mice (Fig. 3C and D). Consistent with previous findings (31), a diffuse staining of IRF7 was seen in the cytosol of unstimulated pDC from both types of mice. Following virus or T{alpha}1 exposure alone, but particularly in combination, a very bright nuclear staining of IRF7 was observed. Nuclear translocation of IRF7 did not occur in CD11b+DC, whose cytosolic content was also low (Fig. 3D). In contrast to the IRF7 activation in pDC, IRF3 phosphorylation and translocation was observed in CD11b+DC from C57BL6 mice upon viral exposure alone or together with T{alpha}1 but not in pDC (Fig. 3C). As already reported (36), both DC subsets from WT mice produced IFN-{alpha} and IL-12p70 in response to the virus, although pDC more than CD11b+DC and WT DC more than Tlr9–/– DC. Pre-treatment with T{alpha}1 greatly increased the production of IFN-{alpha} by pDC and this was TLR9 dependent. Interestingly enough, T{alpha}1 slightly promoted IFN-{alpha} by CD11b+DC (Fig. 3E). These data suggest that T{alpha}1 promotes the TLR9/IRF7-dependent anti-viral program in pDC. However, the finding that significant IFN-{alpha} production was still observed in Tlr9–/– mice indicated that signaling through other TLR is also contributing to the production. As a matter of fact, contaminating CD11b+DC may likely contribute to cytokine production in Flt3-L-cultured DC.

T{alpha}1 recovers NK and CD4+T cell reactivity in susceptible mice with MCMV infection
Although Ly49H+ NK cells are pivotally involved in resistance to MCMV (38), NK cell proliferation and production of IFN-{gamma} is not dependent on Ly49H expression during early MCMV infection (39). We assessed therefore whether T{alpha}1 would affect the activation of Ly49H– NK cells in the spleen of MCMV-infected BALB/c mice at different times after the infection. Figure 4(A) shows that treatment with T{alpha}1 increased the number of fully activated, as revealed by the increased expression of the activation marker CD69, DX5+NK cells at 2 and 7 days after the infection. The cytotoxic activity (Fig. 4B) and the frequency of IFN-{gamma}-producing cells (Fig. 4C) of ex vivo purified splenic NK cells at both times after infection were also significantly up-regulated upon T{alpha}1 treatment. Concomitant depletion of NK cells greatly limited the beneficial effects of T{alpha}1 in infection (data not shown), thus suggesting that the effect on NK cells is instrumental for the anti-viral efficacy of T{alpha}1 in susceptible mice (data not shown).


Figure 4
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  		Fig. 4. T{alpha}1 promotes NK and T cell functions in susceptible mice with MCMV infection. Phenotypic analysis of NK (A) cells from spleens of MCMV-infected BALB/c mice untreated (–) or treated (+) with 200 µg kg–1 T{alpha}1 for a week. Dash indicates uninfected mice. Numbers refer to the percentage of positive cells by FACS analysis at different days after infection. Data are representative of one out of three independent experiments. (B) Cytotoxic activity (by standard 51Cr-release assay against YAC-1 targets) and (C) frequency of IFN-{gamma}-producing NK cells by ELISPOT assay from mice treated as above. Symbols are as follows: filled triangles, uninfected mice; mice at 2 (filled circles) or 7 (filled squares) days after the infection; T{alpha}1-treated mice at 2 (open circles) or 7 (open squares) days after the infection. (D) Frequency of IFN-{gamma}-producing splenic T cells purified from MCMV-infected mice, treated as above, a day after the last T{alpha}1 treatment. Bars indicate the standard errors. The results shown are from three independent experiments. (E) Cytokine production in mice during MCMV infection. Cytokine levels (pg ml–1) in culture supernatants of spleen cells from MCMV-infected mice, treated as above, a day after the last treatment. *P < 0.05, infected versus uninfected mice. **P < 0.05, T{alpha}1-treated versus untreated mice. ND, not done.

 
In primary BALB/c infected mice, CD8+T cells play a major and protective role by clearing productive infections in visceral organs, with the exception of the epithelial cells of the salivary glands whose persistent productive infection is controlled by CD4+T cells and IFN-{gamma} (40). To evaluate whether treatment with T{alpha}1 also affects CD8+ and CD4+T cell-dependent anti-viral activity, we assessed the IFN-{gamma}-producing activity of purified splenic CD8+ and CD4+T cells from infected mice after T{alpha}1 treatment. Figure 4(D) shows that T{alpha}1, while not affecting the expansion of CD4+ or CD8+ T cells (data not shown), increased the number of IFN-{gamma}-producing cells, particularly in the CD4+ compartment. Therefore, despite the fact that the absolute number was not increased, the effector anti-viral activity of both T cell subsets was increased by treatment with T{alpha}1. Together, these findings suggest an activity of T{alpha}1 on both the innate and adaptive anti-viral immune responses. On measuring IL-12p70, IFN-{alpha}, IFN-{gamma} and IL-10 production in culture supernatants of spleen cells from infected and T{alpha}1-treated BALB/c mice, we found that treatment with T{alpha}1 significantly increased IFN-{alpha}, IL-12p70 and IFN-{gamma} productions, particularly in the first week of the infection. These effects were comparable to those obtained with GCV. Interestingly, T{alpha}1 also dramatically increased IL-10 production (Fig. 4E).

T{alpha}1 is effective in combination with GCV
Finally, we assessed whether T{alpha}1 could work in combination with GCV. To this purpose, susceptible or resistant mice infected with MCMV were concomitantly treated for a week with GCV and/or T{alpha}1 and assessed for viral titers and patterns of cytokine production. The results (Fig. 5) confirmed that either agent alone similarly decreased the viral titers in the lung of either type of mice and promoted the production of IFN-{alpha} and IFN-{gamma}. The anti-viral effect of GCV was not modified when given in combination with T{alpha}1, at least at the optimal doses of either agent used.


Figure 5
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  		Fig. 5. T{alpha}1 is effective in combination with GCV. BALB/c or C57BL6 mice were infected with 105 or 5 x 105 PFU of MCMV, respectively. Virus titers, expressed as log10, were quantified on MEF cells by standard plaque assay on lung tissues removed 7 days after the infection. T{alpha}1 (200 µg kg–1 daily for a week) and/or GCV (40 mg kg–1, three times a week) were administered beginning the day of the infection. Cytokine (pg ml–1) levels in culture supernatants of mitogen-activated splenocytes (7 days) were determined by ELISA assay. *P < 0.05, treated versus untreated mice. Results are representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
T{alpha}1 has been shown to have an important role in the defense against different pathogens (26). This study broadens the spectrum of anti-microbial activity of T{alpha}1, which includes its ability to protect from MCMV infection and reactivation by inducing the production of IFN-{alpha} and IFN-{gamma} through the TLR9/MyD88/IRF7-dependent pathway.

Herpesviruses utilize a complex route of entry into cells that involves multiple interactions between several distinct viral envelope glycoproteins and cellular receptors (41). Large DNA viruses such as MCMV use an array of immune evasion strategies to ensure that viral replication proceeds successfully despite anti-viral host immune responses. It is therefore not unexpected that the host uses multiple TLR to recognize MCMV and to ensure that an appropriate immune response is mounted. TLR9 has been linked to the activation of immune responses during MCMV infection (11–13), although pathways independent of TLR9 are also involved in generating immune responses to MCMV. We found here that T{alpha}1 increased anti-viral immune resistance in both susceptible and resistant mice through the TLR9/MyD88-dependent pathway. Our findings are consistent with a role of T{alpha}1 as an important cellular immunomodulator and imply the existence of a cellular receptor. In this regard, the finding that T{alpha}1 affected the anti-viral cytokine production in DC is in line with the notion that T{alpha}1 promotes the maturation and cytokine production in human and murine pDC by signaling through TLR9 (27, 42). T{alpha}1 increased IFN-{alpha} and, to a lesser extent, IL-12p70 production of pDC more than CD11b+DC in response to the virus and this was TLR9 and MyD88 dependent. Interestingly, T{alpha}1 also promoted viral replication in pDC known to not support viral replication (13, 18, 22, 36). This finding is of relevance as viral replication in the cytosol may affect viral sensing by pDC (43). The mechanism behind this observation is at present unknown. We found that pre-treatment of virus with T{alpha}1 prior to exposure to DC also promoted, albeit partially, viral replication in pDC, a finding suggesting a possible direct effect of T{alpha}1 on the virus itself. However, how T{alpha}1 affects viral replication and whether it affects also viral routing inside pDC remains to be fully elucidated.

A well-characterized collaboration exists between IRF3 and IRF7 that serves to amplify the anti-viral response. Specifically, during viral infection, IRF3 has an early role in inducing transcription of IFN-{alpha} and IFN-ß that can then induce IRF7 expression. The newly synthesized IRF7 then promotes transcription of different IFN subunits, thereby creating a positive feedback mechanism that augments the host anti-viral response. T{alpha}1 did not modify IRF3 activation in CD11b+DC upon infection, but activated IRF7 in pDC, a finding in line with its TLR9-dependent activity as well as its ability to act as a unique activator of the tumor necrosis factor receptor-associated factor 6-signaling pathway (44).

The production of type I IFN early after MCMV infection by pDC is critical for the early control of the infection through the activation of NK cell cytotoxicity (36), although CD11b+DCs have recently been found to contribute to NK cell activation (36). T{alpha}1 increased recovery of splenic Ly49H+ NK cell number and functions (IFN-{gamma} production and cytotoxicity) in C57BL6 mice with MCMV infection through a TLR9/MyD88-dependent pathway. Moreover, T{alpha}1 also promoted NK cell activity in BALB/c mice, in which early NK cell responses are ineffective (45). These findings suggest that T{alpha}1, by promoting specific and non-specific NK cell activation during MCMV infection, may counteract viral evasion of NK cells in infection (46). However, given the ability of T{alpha}1 to increase the efficiency and maturation of cellular functions along the T cell pathway (47), an independent activity of T{alpha}1 on NK cells is also conceivable.

One interesting observation of the present study is the finding that IL-10 is also produced after treatment with T{alpha}1 in both susceptible and resistant mice. Adaptive immunity is required for the termination of the productive infection and the establishment of latency (45). Whether IL-10 can be considered an integral component of the anti-viral immune responses, as seen in other microbial infections, or play an autoregulatory role in response to anti-viral therapy with IFN-{alpha} (48) is presently unclear. Undoubtedly, the effects of T{alpha}1 goes beyond an activity on the innate immune system to include the activation of appropriate adaptive CD4+T cell responses, a finding consistent with the relevant anti-viral activity of T{alpha}1 in the salivary gland whose persistent productive infection is controlled by CD4+T cells and IFN-{gamma} (49, 50).

The results of this study expand upon the immunoregulatory activity of T{alpha}1 on pDC. T{alpha}1 has recently been shown to promote the differentiation and activation program of pDC through the activation of a TLR9-dependent immunosuppressive pathway of tryptophan catabolism (42). T{alpha}1-primed DC fulfilled multiple requirements, including the induction of priming and tolerance to a fungal pathogen and allo-antigen hematopoietic transplantation. We showed here that T{alpha}1 also promotes the anti-viral program of murine pDC and human pDC (BS (Bozza Silvia) unpublished observations), thus providing the rationale for the therapeutic prescription of T{alpha}1 in some viral infections, where pDCs producing IFN-{alpha} are considered to play a central role (51). Ultimately, the exploitation of TLR as adjuvant receptors may provide new options for optimizing anti-CMV therapeutic strategies, as recently reported (32). In this regard, the ability of T{alpha}1 to act in concert with GCV qualifies T{alpha}1 as a useful and safe immunomodulator to be used alone or in conjunction with chemotherapy against CMV infection in transplantation.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
National Research Project on AIDS (contract 30G.28), "Ricerca Clinica e Terapia della Malattia da HIV", Italy; Ministero Istruzione Università e Ricerca (Project FIRB to R.L.) R.L. Romani Luigina.


   Acknowledgements
 
We thank Cristina Massi Benedetti for dedicated editorial assistance and Santo Landolfo and Patrizia Caposio (University of Torino, Torino, Italy) for providing us with the MCMV. The authors have no conflicting financial interests.


   Abbreviations
 
CpG-B ODN, cytosine-phosphorothioate-guanine oligodeoxynucleotide
DC, dendritic cell
gB, glycoprotein B
GCV, gancyclovir
HCMV, human cytomegalovirus
IRF, IFN regulatory factor
i.p., intra-peritoneally
MCMV, murine cytomegalovirus
MEF, murine embryonic fibroblast
MyD88, myeloid differentiation primary response gene 88
pDC, plasmacytoid dendritic cell
PFU, plaque-forming units
RT, room temperature
TLR, Toll-like receptor
T{alpha}1, thymosin {alpha}1
WT, wild type

   Notes
 
Transmitting editor: S. Romagnani

Received 2 January 2007, accepted 7 August 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 References
 

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The Rapidly Expanding Therapeutic Role of Thymosin {alpha}-1 in the Management of Gastrointestinal and Systemic Infectious Disorders
J Intensive Care Med, May 1, 2009; 24(3): 210 - 211.
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