Blood First Edition Paper, prepublished online February 24, 2004; DOI 10.1182/blood-2003-11-4036
Thymosin D 1 activates dendritic cells for antifungal Th1
resistance through Toll-like receptor signaling
Luigina Romani1, Francesco Bistoni1, Roberta Gaziano, Silvia Bozza1, Claudia Montagnoli1,
Katia Perruccio2, Lucia Pitzurra1, Silvia Bellocchio1, Andrea Velardi2, Guido Rasi3, Paolo di
Francesco4 and Enrico Garaci4
From the 1Microbiology Section, Department of Experimental Medicine and Biochemical
Sciences, University of Perugia, Perugia, Italy;
2
Division of Hematology, Clinical
Immunology, Department of Clinical and Experimental Medicine, University of Perugia,
Perugia, Italy; 3Institute of Neurobiology and Molecular Medicine, CNR, Rome, Italy;
4
Microbiology Section, Department of Experimental Medicine and Biochemical Sciences,
University Tor Vergata, Rome, Italy.
This study was supported by the National Research Project on AIDS, contract n. 50D.27 and
the Project n. 1AF/F, both from the ISS, by the Basic Research Funds n.01p4B5-006 and the
Project n. 120.5/RF00.121, both from MIUR, Italy.
Corresponding author: Luigina Romani, M.D., Ph.D. Department of Experimental
Medicine and Biochemical Sciences - Microbiology Section, University of Perugia, Via del
Giochetto, 06122 Perugia, Italy
Telephone and Fax: 039-075-585-7411
E-mail: lromani@unipg.it
Running title: Thymosin and Dendritic Cells in Aspergillosis
Word count: 5048
Copyright (c) 2004 American Society of Hematology
Abstract
Dendritic cells (DCs) show a remarkable functional plasticity in the recognition of Aspergillus
fumigatus and orchestrate the antifungal immune resistance in the lungs. Here we show that
thymosin α 1, a naturally occurring thymic peptide, induces functional maturation and
interleukin-12 production by fungus-pulsed DCs through the p38 mitogen-activated protein
kinase/NF-KB-dependent pathway. This occurs by signaling through the myeloid
differentiation factor 88-dependent pathway involving distinct Toll-like receptors. In vivo, the
synthetic peptide activates T-helper (Th) cell 1-dependent antifungal immunity, accelerates
myeloid cell recovery and protects highly susceptible hematopoietic transplanted mice from
aspergillosis. By revealing the unexpected activity of an old molecule, our finding provide the
rationale for its therapeutic prescriptions and qualify the synthetic peptide as a candidate
adjuvant promoting the coordinated activation of the innate and adaptive Th immunity to the
fungus.
Introduction
Invasive aspergillosis (IA), characterized by hyphal invasion, destruction of pulmonary tissue
and dissemination to other organs, is the leading cause of both nosocomial pneumonia and
death in allogeneic bone marrow transplantation (BMT) with an estimated infection rate of 5
to 10% and an associated mortality rate of 90 to 100%.1-3 The most important risk factor for
IA has historically been neutropenia, such that reconstitution with myeloid progenitors
offered protection against IA in a murine model of allogeneic BMT.4 However, recent studies
on the epidemiology of IA in BMT recipients indicated a reduced neutropenia-related
2
infection and an increase "late-onset" infection, in concomitance with the occurrence of graftversus-host disease.3 These findings, together with the occurrence in non-neutropenic
patients,1 attest to the importance of specific defects in both the innate and adaptive immune
effector mechanisms in the pathogenesis of the disease.3,5
Clinical and experimental evidence point to the crucial role of a Th1 reactivity in the
control of IA.5,6-8 DCs instruct Th1 priming to the fungus in vivo and in vitro.9-11 Evidence
indicate that the ability of pulmonary DCs to instruct the appropriate T cell responses to
fungal antigens may be affected by local immuno-regulatory events, including signaling
through Toll-like receptors (TLRs).10-13 This is consistent with a certain degree of flexibility
of the immune recognition pathways to Aspergillus antigens and allergens, such that an
allergen could be converted to a potential protective antigen, provided CpG as adjuvant.11 The
model has brought DCs to center stage as promising targets for intervention for
immunotherapy and vaccine development14 and has shifted the emphasis from the "antigen"
towards the "adjuvant".15,16 Thus, the promise of a fungal vaccine will demand for an adjuvant
capable of both stimulating the appropriate type of response best tailored to combacting the
infection and being effective in conditions of immunosuppression.
Thymosin α 1 is a naturally occurring thymic peptide first described and characterized
by A. Goldstein.17 In the form of a synthetic 28-amino acid peptide, thymosin α 1 is in
clinical trials worldwide for the treatment of some viral infections, either as monotherapy or
in combination with interferon α.18,19 Additional indications are some immunodeficiencies20,
malignancies21 and HIV/AIDS.18 The mechanism of action of the synthetic polypeptide is not
completely understood but is thought to be related to its immuno-modulating activities,
centered primarily on the augmentation of T-cell function. Because of its immunomodulatory
function on cells on the innate immune system,22 including the ability to activate mitogenactivated protein kinases (MAPKs)23 and gene expression24 on macrophages, we hypothesized
3
that thymosin α 1 could act as an adjuvant capable of activating DCs for Th1 priming to
Aspergillus. In this article, we present evidence that thymosin α 1 activates DCs for antifungal
Th1 priming in vitro and in vivo by signaling through distinct TLRs and the MyD88 adaptor
protein.
Materials and methods
Animals
Female, 8- to 10-weeks old, BALB/c and C57BL6 mice were from Charles River (Calco,
Italy). NOD/SCID were from The Jackson. Breeding pairs of homozygous TLR2-, TLR9- and
MyD88-deficient mice, raised on C57BL6 background, and of homozygous IFN- - and IL-4deficient mice, raised on BALB/c background, were bred under specific-pathogen free
conditions at the breeding facilities of the University of Perugia, Perugia, Italy.
Microorganism, culture conditions, infection and treatments
The strain of A. fumigatus and the culture conditions were as described elsewhere.7,10 For
infection, mice were intranasally injected for 3 consecutive days with a suspension of 2 x 107
conidia/20 µl saline. For the quantification of fungal growth in the lungs, the chitin assay was
used as described.7,10 The chitin content was expressed as micrograms of glucosamine per
organ. The glucosamine content of lungs from uninfected mice was used as a negative control
(range, 0.80 and 2.25 µg glucosamine/organ). For histological analysis, lungs were excised
and immediately fixed in formalin. Sections (3 to 4 µm) of paraffin-embedded tissues were
stained with the periodic acid-Schiff procedure as described.7 Thymosin α 1 and the
scrambled polypeptide (both from SciClone Pharmaceuticals, Inc. San Mateo, California)
were supplied as purified (the endotoxin levels were less than 0.03 pg/ml, by a standard
limulus lysate assay) sterile lyophilized acetylated polypeptides. The sequences were as
4
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 (Thymosin α 1) and Ac-Ala-Lys-Ser-Asp-ValLys-Ala-Glu-Thr-Ser-Ser-Glu-Ile-Asp-Thr-Thr-Glu-Leu-Asp-Glu-Lys-Val-Glu-Val-Lys-AlaAsn-Glu-OH (sThymosin α 1). The lyophilized powders were reconstituted in sterile water.
Treatments were as follows: in BMT-mice, thymosin α 1, at different doses/intraperitoneally,
sthymosin α 1, 400 µg/kg/intraperitoneally and human recombinant G-CSF (Granocyte 34;
Aventis Pharma S.p.A. Antony Cedex, France), 250 µg/kg/intravenously, were given daily
beginning the day of the BM infusion, in concomitance with the infection and continuing for
additional 3 days; amphotericin B (A-2942, Sigma) was given daily for 3 days in
concomitance with the infection, at the dose of 4000 µg/kg/intraperitoneally. In preliminary
experiments, this dose was found to cure IA in cyclophosphamide-treated mice (data not
shown). Cyclophosphamide (Sigma), 150 mg/kg/intraperitoneally, was given a day before the
infection. In cyclophosphamide-treated mice, 400 µg/kg thymosin α 1 was given
intraperitoneally for 5 consecutive days beginning the day of the infection; neutrophil
depletion was obtained by treatment with 1 mg of Gr-1-neutralizing RB6-8C5 antibody
intravenously, a day before and after the infection. In preliminary experiments, lower amounts
of antibodies did not completely ablate the highly represented population of Gr1+F480neutrophils in the lungs of NOD/SCID mice (twice as much that present in wild type mice,
i.e., from 7 to 15%) or in the peripheral blood (a mean of 24.8 x 105 cells /ml, that is >70% of
white blood cells, the remaining being represented by monocytes (about 27%) and <2% by
lymphocytes). The treatment dramatically reduced the number of lung neutrophils (see
footnote of Table 1) but not that of DCs (between 5 to 6 x 105 CD11c+, MHC Class II+, F480cells before and after treatment). Control mice received an equivalent amount of purified rat
IgG2b (Cat. 02-9288, Zymed Laboratories Inc. South San Francisco, CA). FACS analysis of
lung cells a day after treatment with cyclophosphamide revealed a profound and long-lasting
5
(for up to 5 days) leukopenia (from 35% of CD4+ T cells in controls to <5 % in treated mice;
from 20 to 4 % of CD8+ T cells; from 19 to 7% of B220+ cells and from 7 to <2% of Gr1+
F480- cells. The percentages of F480+ cells (about 20%) and that of CD11c+, MHC Class II+,
F480- DCs (<3%) were unaffected by treatment.
TLR ligands
Zymosan from Saccharomyces cerevisiae, lipoteichoic acid (LTA) from Staphylococcus
aureus, lipopolysaccharide (LPS) from Salmonella minnesota Re 595 and Poly(I:C) were
purchased from Sigma. CpG oligonucleotides 1826 and 2006 of proven immunostimulatory
sequences were obtained as described.11
Generation of BMT mice
C57BL6 mice were exposed to a lethal dose of 9 Gy and infused with T cell-depleted donor
cells from BALB/c mice as described.25 More than 95% of the mice survived showing stable,
donor type hematopoietic chimerism, as revealed by donor type MHC class I antigen
expression on cells from spleens.25
DC isolation and culture
Blood CD1c+ myeloid DCs (MDCs) were generated from CD14+ mononuclear cells by
magnetic cell sorting (MiniMACs, Miltenyi Biotec S.R.L, Bologna, Italy) and cultured for 5
days in Iscove’s modified medium, containing 10% fetal bovine serum, 50
M 2-
mercaptoethanol, sodium pyruvate (1 mM), 2 mM L-glutamine, HEPES (10 mM), and 50
g/ml gentamycin in the presence of 50 ng/ml rHuman GM-CSF (Schering-Plough, Milano,
Italy) and 200 U/ml rHuman IL-4 (Peprotech, Inalco, Milano, Italy)26. Immature MDCs were
cultured for 24 hours with 1000 ng/ml trimeric human CD40 ligand-leucine-zipper fusion
6
protein (Immunex, Seattle, Washington State) to obtain mature DCs. CD123+ plasmacytoid
DCs (PDCs) were isolated using the BDCA-4 isolation kit (Miltenyi Biotec). Purity of
CD123+ cells was > 96%. For mature PDCs, immature DCs were cultured with the trimeric
human CD40 ligand as above and 10 ng/ml IL-3 (R&D System, Space Import-Export s.r.l.
Milano Italy). FACS analysis revealed that PDCs were CD123bright, CD4+, CD45RA+ and
CD11c- as opposed to MDCs characterized as being CD1a+, CD11c+, CD11b+, CD4+,
CD14low and CD8-. The expression of HLA Class II, CD80 and CD86 was high in both
immature and mature DCs. Murine lung CD11c+ DCs (between 5 to 7% positive for CD8α
and between 30 to 35% positive for Gr-1) were isolated by magnetic cell sorting.10 For
phagocytosis, DCs were pre-exposed to 100 ng/ml thymosin α 1 for 60 minutes and
subsequently incubated at 37°C with Aspergillus conidia for additional 60 minutes.10
Percentage of internalization was calculated as described.10 Photographs were taken using a
high Resolution Microscopy Colour Camera AxioCam, using the AxioVision Software Rel.
3.1 (Carl Zeiss S.p.A., Milano, Italy). For functional maturation and cytokine determination,
purified DCs were resuspended in Iscove's medium (with no serum but with polymixin B, to
avoid non-specific activation by serum components and endotoxin) and pulsed with 100
ng/ml thymosin α 1 for 24 hours either alone or together with TLR ligands or unopsonized
Aspergillus conidia.
Phenotypic analysis
Cell surface phenotype was assessed by reacting samples with FITC- or PE-conjugated rat
anti-mouse antibodies from PharMingen (San Diego, CA). Unrelated hisotype matched
antibodies were used as control. Analysis was performed on a FACScan (Becton Dickinson,
Mountain View, CA).
7
Antifungal effector activity
For phagocytosis, bronchoalveolar macrophages and peripheral neutrophils were pre-exposed
to 100 ng/ml thymosin α 1 for 60 minutes and incubated at 37°C with unopsonized
Aspergillus conidia for 60 minutes.10 For the conidiocidal activity, the number of colony
forming units and the percentage of colony forming units inhibition (mean ± SE), referred to
as conidiocidal activity, were determined as described.10
Assay with HEK293 transfected cells.
The human embryonic kidney cell line HEK293, wild type or stably transfected with human
TLR2, TLR9 and TLR4/CD1427 were cultured in low glucose Dulbecco’s modified Eagle’s
medium supplemented with 10% FCS, HEPES (10nM), L-glutamine (2 µg/ml), gentamycin
(50 µg/ml) (Invitrogen S.r.l. Life Technologies, Milano, Italy). Transfectants were
additionally supplemented with puromycin (100 µg/ml, Sigma). For stimulation experiments,
cells were cultured at a density of 3 to 5 x 105 cells/wells in 12-well tissue culture plates
overnight. Cells were washed and stimulated with 100 ng/ml thymosin α 1 either alone or
together with TLR ligands for 5 h before the assessment of IL-8 production in the
supernatants.
Cytokine and spot enzyme-linked immunosorbent (ELISPOT) assay
The levels of TNF-α, IL-10, IL-12 p70, IFN-α and IL-8 in culture supernatants were
determined by Kit ELISAs (Endogen Human Elisa Kits, S.r.l., R&D Systems and Euroclone,
Ltd, Milan, Italy). The detection limits (pg/ml) of the assays were <3 (human) and <32
(murine) for TNF-α, <12 (murine) and <5 (human) for IL-10, <16 (murine) and < 3 (human)
for IL-12 p70 and <25 (human) IL-8. For human IFN-α, <3 ng/ml. For enumeration of
8
cytokine-producing cells, an ELISPOT assay was used on purified CD4+ T cells and DCs
from lungs, as described.10
Proliferation assay by flow cytometric analysis
Proliferation of lung CD4+ T lymphocytes stimulated with 10 µg/ml Con A or heatinactivated conidia in the presence of lung DCs, was assessed by labeling with CFSE 5(6)carboxyfluorescein diacetate succinimidyl ester (Molecular Probes, Eugene, OR), as
described.11
Reverse transcriptase (RT)-PCR
Total RNA was extracted (TRIZOL Invitrogen) from immature DCs pre-treated with 100
ng/ml thymosin α 1 for 60 minutes followed by the exposure to unosponized Aspergillus
conidia for 60 minutes, as suggested by initial experiments. Synthesis and PCR of cDNA
were done as described.10 The forward and reverse PCR primers and the cycles used for
murine and human TLRs and HPRT were as described.28 The synthesized PCR products were
separated by electrophoresis on 2% agarose gel and visualized by ethidium bromide staining.
Analysis of p38 and NF-KB activation
The activation of p38 and NF-KB on lung DCs exposed for 20 minutes (as suggested by initial
experiments) at 37°C to Aspergillus conidia and/or 100 ng/ml thymosin α 1 was done as
described.29 Blots of cell lysates were incubated with rabbit polyclonal Abs recognizing either
the unphosphorylated form of p38 MAPK, or the double-phosphorylated (Thr-180/Tyr-182)
p38 MAPK (Cell Signaling Technology, Celbio S.r.l., Milano, Italy), or Abs specific for the
Rel A, 65 kDa DNA binding subunit of human NF-KB (Zymed Laboratories Inc.), followed
by horseradisch peroxidase-conjugated goat anti-rabbit IgG (Cell Signaling Technology), as
9
per manufacturer’ instructions. Blots were developed with the Enhanced Chemiluminescence
detection kit (Amersham Pharmacia Biothech, Cologno Monzese, Milano, Italy). Bands were
visualized after exposure the blots to a Kodak RX film. To ensure similar protein loading in
each lane, the phospho blots were stripped and the membranes were reprobed with Abs
against p38 and NF-KB.
Statistical analysis
Survival data were analyzed using the Mann-Whitney U test. Student’s t test was used to
determine the significance of values in experimental group (significance was defined as P <
0.05). In vivo groups consisted of 6 animals.
Results
Thymosin D 1 activates DCs
Previous studies have shown that murine DCs phagocytose Aspergillus in vitro and at the site
of infection.10 We show here that thymosin α 1, but not the scrambled peptide (sThymosin α
1) activates lung DCs for phagocytosis of unopsonized conidia (more than hyphae),
costimulatory antigen expression and cytokine production (Figure 1A). In line with previous
findings,10 Aspergillus conidia alone did not represent a sufficient stimulus to induce the
activation of DCs, but the combined exposure to thymosin α 1 remarkably increased the
expression of MHC Class II antigens, CD86 and CD40 molecules (Figure 1B) and the
frequency of IL-12 p70-producing DCs. Interestingly, IL-12 p70-producing DCs were also
increased by thymosin alone (Figure 1C).
10
11
The assessment of the actual cytokine production in supernatants of DCs confirmed the higher
production of IL-12 p70 along with TNF-α in response to conidia and thymosin α 1 as
compared to conidia or thymosin α 1 alone [from 0,75 ± 0,15 (conidia alone) or 1.4 ± 0,37
(thymosin alone) to 2.6 ± 0,51 (in combination) ng/ml of IL-12 p70 and from 147 ± 32
(conidia alone) or 289 ± 60 (thymosin alone) to 681 ± 97 (in combination) pg/ml of TNF-α].
The scrambled peptide failed to up-regulate Class II antigens and costimulatory molecule
expression and to induce cytokine production by DCs in response to conidia (data not shown).
Thymosin α 1 also activated human MDC and PDC subsets. Both immature and
mature DC subsets phagocytose conidia. Thymosin α 1 increased the phagocytic activity of
immature DCs, affected the DC morphology, as more cytoplasmic projections were detected
in immature MDCs (Figure 2A) and up-regulated the HLA Class II antigens and
costimulatory molecule expression in response to conidia (data not shown). In terms of
cytokine production, thymosin α 1 significantly increased the release of IL-12 p70 in response
to conidia (Fig. 2B) and to zymosan (data not shown) by immature MDCs and that of IL-10 in
response to conidia by immature PDCs (Fig. 2B). The production IFN-α , known to be
produced by immature PDCs,30 was also assessed and found not to be released by either DC
subset in response to conidia and/or thymosin α 1. However, it was produced by PDCs in
response to the TLR9 ligand CpG and significantly potentiated by thymosin α 1 (Figure 2B).
Together, these data point to a novel, previously undefined, immuno-regulatory role for
thymosin α 1 in the activation and functioning of DCs.
12
Thymosin D 1 activates the MyD88-dependent pathway through TLR signaling
TLRs signaling occurs in response to Aspergillus and mediates functional responses to the
fungus.11-13 Thymosin α 1 strongly activated the expression of TLR2, TLR5, and TLR9 on
13
murine DCs, as opposed to conidia that poorly or none activated TLR expression, with the
exception of TLR4. TLR2 and TLR9 were still activated upon the combined exposure to
conidia and thymosin α 1, whereas this was not the case for TLR5 whose expression was
instead inhibited. Importantly, the scrambled peptide failed to activate TLR2 and TLR9
expression in response to conidia (Figure 3A) or alone (data not shown). The pattern of TLR
expression was not different with and without polymixin B (data not shown). Similar to
murine DCs, conidia induced the expression of TLR4 on human MDCs and thymosin α 1,
alone or together with conidia, induced TLR2 expression on both DC subsets and TLR9 on
PDCs (Figure 3B). No TLRs expression was induced upon incubation of cells with the diluent
alone (Figures 3A and B).
To get further insights into the ability of thymosin α 1 to activate TLR-dependent signaling,
HEK293 cells transfected with TLR2, TLR9 and TLR4/CD14 were assessed for IL-8
14
production in response to thymosin α 1 alone or together with the relevant TLR ligands. The
results show that thymosin α 1 significantly increased the production of IL-8 by TLR9transfected cells either alone or in response to CpG; it did not stimulate the production of IL-8
by TLR2-transfected cells alone but slightly increased the production in response to zymosan
and it did not induce IL-8 by TLR4/CD14-transfected cells either alone or in response to LPS
(Fig. 3C). Therefore, thymosin α 1 appears to be able to signal directly through TLR9 and to
potentiate TLR2 signaling by the relevant ligand.
As NF-KB and p38 MAPK activation are early events in triggering TLR-induced gene
expression,31,32 and thymosin α 1 activates MAPK-transduction pathways,23 the nuclear
translocation of NF-KB as well as p38 phosphorylation in murine DCs was also assessed.
Conidia alone failed to induce NF-KB activation and p38 phosphorylation that were instead
induced upon treatment with thymosin α 1, but not with the scrambled peptide, either alone or
combined with conidia (Figure 3D).
15
The SN50 (an inhibitor of NF-KB nuclear translocation)33 or SB202190 (an inhibitor
of p38 MAPK)34 inhibitors ablated the effect of thymosin α 1 on DCs in terms of IL-12 p70
production [from 1.6 ± 0.62 ng/ml of the control to undetectable levels (<16 pg/ml) in the
presence of both inhibitors].
Further studies showed that thymosin α 1 affected the ability of murine DCs to
produce IL-12 p70 and IL-10 in response to known microbial TLR ligands.31,32,35 Thymosin
α 1 did not affect the cytokine production in response to Poly(I:C) and LPS. However, it
significantly increased (at about four folds) the production of IL-12 p70 and decreased that of
IL-10 to stimulation with the TLR2 ligands, such as zymosan and LTA. The production of IL12 p70 was also increased to stimulation with the TLR9 ligand CpG, although to a lesser
extent (Figure 4A). Among others,31,32 the myeloid differentiation factor 88 (MyD88) is an
adaptor protein essential for the activation of NF-KB and MAPK and the production of IL-12
16
p70 upon signaling by TLRs. We found here that the IL-12 p70 production was dramatically
ablated in MyD88-deficient mice in response to conidia and/or thymosin α 1. Interestingly,
IL-10 production was observed in response to conidia in MyD88-deficient mice but was not
modified by thymosin α 1 (Figure 4B). Therefore, it appears that the MyD88-dependent
pathway plays an essential role in the ability of thymosin α 1 to modulate IL-12 p70
production by DCs.
To assess whether the MyD88-dependent pathway could play an essential role also in
vivo, we resorted to an experimental model of IA, in which wild type, TLR2-, TRL9- and
MyD88-deficient mice were intranasally infected with Aspergillus conidia, treated with
thymosin
1 and assessed for the local fungal growth. The fungal growth in TLR2- and
TLR9-deficient mice was comparable to that of wild type mice; it was similarly impaired
upon thymosin treatment in wild type, TLR9- and, to a lesser extent, TLR2-deficient mice;
however, it was not impaired in MyD88-deficient mice and could not be restored by thymosin
α 1 (Figure 4C). Together, these data provide evidence that, despite a degree of redundancy in
the TLR usage, the MyD88-dependent signaling pathway is essential in the activity of
thymosin
1 in vitro and in vivo.
17
18
Thymosin D 1 protects BMT-mice from IA
Based on the above findings, we assess whether treatment with thymosin α 1 would increase
antifungal resistance in BMT mice with IA.25 Treatment with thymosin α 1, but not with the
scrambled peptide, cured the mice from the infection, as revealed by the increased survival
after the infection that paralleled the reduced fungal growth in the lungs. The effect on
protection was dose-dependent, full protection (>60 d survival) was achieved in mice treated
with 200 and 400 µg/kg thymosin α 1 and was superior to that of amphotericin B.
Interestingly, thymosin α 1 appeared to increase the therapeutic efficacy of amphotericin B,
as indicated by the increased survival and decreased fungal burden of mice treated with both
agents (Figure 5A). Thymosin α 1 also decreased lung pathology. Lung sections from
infected mice showed the presence of numerous Aspergillus hyphae infiltrating the lung
parenchyma, with severe signs of bronchial wall damage and necrosis and scarce
inflammatory cell recruitment (Figure 5B). These features were not observed in thymosintreated mice, whose lungs were characterized by healing infiltrates of inflammatory cells with
no evidence of fungal growth and bronchial wall destruction (Figure 5C). These data point to
the therapeutic efficacy of thymosin α 1 in IA and suggest the benefit of the association with
antifungals known to have a reduced activity in BMT settings.3
19
Thymosin D 1 accelerates myeloid and Th1 cell recovery in mice with IA
To evaluate whether thymosin α 1 would activate protective FN-γ-producing Th1 cells in
BMT-mice with IA,7,8,10 we assessed cell recovery by FACS analysis together with the
antigen-specific lymphoproliferation, pattern of local cytokine production and antifungal
activity of effector phagocytes. Although the absolute number of circulating lymphocytes and
neutrophils significantly increased after thymosin treatment (data not shown), a
cytofluorimetric analysis was performed on lung cells, as blood neutrophil levels do not
predict susceptibility to aspergillosis.4 The numbers of CD4+, CD8+ cells and neutrophils were
20
significantly increased in BMT mice upon treatment with thymosin α 1 (Figure 6A).
Recovered lung CD4+ T lymphocytes were functionally active as indicated by the antigenspecific proliferation (Figure 6B) and the IFN-γ production. The frequency of IFN-γproducing cells was higher and that producing IL-4 lower in mice treated with thymosin α 1
as compared to untreated mice (Figure 6C). On assessing the level of antifungal activity of
effector phagocytes it was found that the conidiocidal activity of both macrophages and
neutrophils was higher in thymosin-treated than untreated mice (data not shown). The finding
that the phagocytosis and killing of conidia by alveolar macrophages and circulating
neutrophils from uninfected mice were also significantly potentiated in vitro in the presence
of thymosin α 1 (Figure 6D) suggests that thymosin α 1 not only promotes DC maturation but
will also activate local effector cells for prompt phagocytosis and killing of the fungus.
21
However, recovery from neutropenia alone, as by treatment with a dose of G-CSF known to
accelerate neutrophil recovery in mice,4 was not sufficient to mediate a degree of antifungal
resistance comparable to that obtained with thymosin α 1, as BMT mice were unable to
survive the infection (Table 1). Similarly, despite a significant neutrophil recovery and the
ablation of resistance upon neutrophil depletion, thymosin α 1 had a limited therapeutic
efficacy in mice devoid of T cells or IFN- -producing Th1 cells. In contrast, the optimal
therapeutic efficacy of thymosin α 1 was achieved in the presence of an hightened antifungal
Th1 reactivity, such as that occurring in IL-4-deficient mice (Table 1). Therefore, although
neutrophils play an essential role in mediating antifungal resistance in the absence of an
adaptive Th1-dependent immunity, the achievement of a state of full protection to the fungus,
1, appears to rely on the coordinated action
as that obtained by treatment with thymosin
between innate effector phagocytes and protective Th1 cells.
Discussion
The results of our study identify a novel immuno-regulatory activity of thymosin α 1 that may
have important theoretical and therapeutic implications. Thymosin α 1 promoted the
production of IL-12 p70 in DCs through the MyD88-dependent pathway, which is known to
lead to production of Th1-promoting cytokines. The production of IL-12 p70 occurred to a
different degree between the different DC subsets, a finding in line with the observation that,
unlike murine PDCs that produce IL-12 p70 in response to TLR9 stimulation,36 human PDCs
are known to produce little IL-12 p70.26 However, human PDCs produced IL-10 more than
IL-12 p70 in response to conidia alone or together with thymosin α 1 and produced IFN-α in
response to CpG and, more, in response to CpG and thymosin α 1. In TLR-transfected cells,
thymosin α 1 directly activated TLR9 but not TLR2 signaling, the last being potentiated in
response to the relevant ligand. Preliminary experiments seem to indicate a role for MD2,
known to potentiate TLR2 signaling,37 in the TLR2 activating ability of thymosin α 1 (data
22
not shown). It appears, therefore, that thymosin α 1 activates TLR signaling either directly or
indirectly. However, whether TLR9 activation occurs in the endoplasmic reticulum of PDCs,
as recently reported28 and whether intermediate molecules are licensed by thymosin α 1 in
vivo to increase functional expression of TLRs are as yet unsolved issues. In this regard, it is
worth mentioning that the 28-residue peptide thymosin α 1 may undergo conformational
changes depending upon the environment.38 Whatever the case will be, it is conceivable that
thymosin α 1 may exploit the TLR2-dependent pathway on MDCs for IL-12 p70 production
and the TLR9-dependent on PDCs for IFN-α production. However, the production of IL-10
by PDCs in response to thymosin α 1 is also of interest. Much evidence indicates that the
TLR signaling pathways differ from one other and elicit different biological responses.30,31
Our data would suggest that the production of IL-10 in response to distinct TLR ligands and
to conidia mainly occurs in a MyD88-independent manner. Thymosin α 1 did not modify the
production of IL-10 in MyD88-deficient mice, a finding confirming the essential role of the
MyD88-dependent pathway in the regulation of IL-10 production by thymosin α 1. However,
an activity of thymosin α 1 on additional signaling pathways cannot be excluded. Regardless,
as IL-10 production by DCs is an essential component of memory protective antifungal
immunity, 39 balancing the IL-12/IL-10 production on DCs and/or different DC subsets may
represent the very essence of adjuvanticity of thymosin α 1 in aspergillosis.
The effects of thymosin α 1 on DCs are consistent with its anti-apoptotic activity40 as
well as with its ability to induce prostaglandins41 and to share structural homology with
human IFN-α,42 both agents known to induce DCs maturation.15 Given that DCs are central in
the balancing act between immunopathology, immunity and autoimmunity,43 and that PDCs
signaling through TLR9 are present in the thymus,44 the ability to modulate DC functioning
qualifies thymosin α 1 as a novel endogenous regulator of the innate and adaptive immune
systems acting through TLR exploitation. From a conceptual point of view, this may provide
23
an explanation for the, as yet elusive, function of naturally occurring thymosin α 1 that is
produced in vivo by lysosomal asparaginyl endopeptidase cleavage of prothymosin α in
diverse mammalian tissues45 and also the rationale for the therapeutic prescription of
thymosin α 1 in some viral infections, where PDCs producing IFN-α, for which TLR9 is
essentially required,46 are considered to play a central role.47,48 It is interesting that PDCs also
participate in immune responses after hematopoietic cell transplantation,49 a finding that may
explain, among others,50 the beneficial effect of thymosin α 1 in the immuno-reconstitution in
BMT mice.
TLRs activate the innate immune system not only to assist the adaptive immune
system but also for direct antimicrobial effector activity.31,32 The fact that thymosin α 1 not
only activated murine and human DCs for Th1 priming to A. fumigatus, but also effector
neutrophils to an antifungal state, further points to the beneficial effect of thymosin α 1 in
fungal infections.22 However, in line with the notion that the recovery from neutropenia is not
sufficient to confer protection against IA in BMT mice4 an action on neutrophils alone is not
sufficient to mediate the curative effect of thymosin α 1.
In conclusion, the unique nature of Aspergillus, a saprophytic fungus colonizing
immunocompromised hosts, demands for a better understanding of immunological
mechanisms required to both oppose fungal infectivity and optimize the efficacy of antifungal
therapy. This study shows that the deliberate targeting of cells and pathways of cell-mediated
immunity may increase resistance to Aspergillus and qualifies thymosin α 1 as a promising
candidate adjuvant programming the appropriate Th reactivity to the fungus through TLR
exploitation.
24
Table 1. Neutrophils and Th1 cells are required for the therapeutic efficacy of thymosin α 1
Group Micea
Treatmentb
Neutrophils/lung Chitin contentc
3 c
(days)c
(x 10 )
a
MST
1
BMT
None
71±8
44±6
4.5
2
BMT
G-CSF
544±28*
25±4*
10*
3
BALB/c
None
54±4
38±4
5.5
4
BALB/c
Thymosin α 1
714±12*
18±2
>60*
5
NOD/SCID
None
194±19
68±12
6
6
NOD/SCID
Thymosin α 1
491±22*
31±7*
15*
7
NOD/SCID
Thymosin α 1
+
neutrophil-depletion
69±14 *
59±10
5.5
8
IFN-γ-deficient
None
84±10
49±5
7.5
9
IFN-γ-deficient
Thymosin α 1
681±44*
25±4*
18*
10
IL-4-deficient
None
88±8
32±5
22
11
IL-4-deficient
Thymosin α 1
704±51*
0.9±0.3*
>60*
BMT (groups 1 and 2) or cyclophosphamide-treated (groups 3-11) mice were infected with 2
x 107 Aspergillus conidia intranasally for 3 consecutives days.
b
In BMT mice, G-CSF (250 µg/kg/i.v.) was given i.v. daily beginning the day of the BM
infusion, in concomitance with the infection and continuing for additional 3 days. In
cyclophosphamide-treated mice, thymosin α 1 (400 µg/kg/i.p.) was given for 5 consecutive
days beginning the day of the infection; neutrophil depletion was obtained by treatment with 1
mg of RB6-8C5 antibody i.v., a day before and after the infection.
c
Lungs were assessed for the presence of fungal cells (chitin content, expressed as µg
glucosamine/lung) and neutrophils (FACS analysis) a day (untreated mice, None) or 3 days
(treated mice) after the last Aspergillus inoculation. Values are the mean ± SE. MST, median
survival time (days). *P < 0.05, treated vs untreated mice. Each type of intact mice survived
the infection and efficiently controlled the fungal growth.
25
Acknowledgments
We thank Dr. Lara Bellocchio for superb editorial assistance, Dr. Paolo Mosci for the
histology, Drs. Giorgio Trinchieri and Elisabeth Beth from Schering-Plough, Dardilly,
France, for providing us with breeding pairs of TLR9-, TLR2- and MyD88-deficient mice,
Dr. Manfred Kopf from Swiss Federal Institute of Technology, Zurich, Switzerland, for
providing us with breeding pairs of IFN-γ- and IL-4-deficient mice and Dr. Terje Espevik
from the Norwegian University of Science and Technology, Trondheim, Norway for the
generous supply of transfected HEK cell lines through the courtesy of Prof. Giò Teti
(University of Messina, Italy).
26
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Figure legends
Figure 1. Thymosin D 1 activates murine DCs upon exposure to Aspergillus fumigatus.
Murine lung CD11c+ DCs were pre-treated with thymosin α 1 or the scrambled peptide
(sThymosin α1) before the exposure to conidia or hyphae for the assessment of (A)
phagocytosis (the data are the means ± SE of three independent experiments and expressed as
% internalization (numbers within figures). *P < 0.05, thymosin-exposed vs unexposed cells);
(B) costimulatory molecule expression by FACS analysis (data from one representative
experiment out of three. Black histograms represent cells stained with an irrelevant antibody.
The numbers refer to the median fluorescence intensity) and (C) frequency of IL-12 p70producing cells (the values are the mean ± SE per 105 cells of samples from 3-5 experiments,
calculated by ELISPOT assay *P < 0.05, exposed vs unexposed (None) or conidia-exposed
cells.
Figure 2. Thymosin D 1 activates human DCs upon exposure to Aspergillus fumigatus.
Immature and mature myeloid (MDCs) and plasmacytoid (PDCs) DCs were obtained from
blood mononuclear cells. For phagocytosis (A), see legend to Figure 1. The data are the
means ± SE of three independent experiments and expressed as % internalization (numbers
within figures). *P < 0.05, thymosin-exposed vs unexposed cells. (B) Cytokines were
determined in 24 h-culture supernatants of DCs stimulated (+) or not (-) with thymosin α 1 in
the presence of conidia or CpG 2006 (3 µg/ml). Bars indicate the standard errors. *P < 0.05,
cytokine production in thymosin-exposed vs unexposed cells. **P < 0.05, thymosin+CpG
versus CpG alone.
33
Figure 3. Thymosin D 1 activates TLR-dependent signaling.
Murine lung DCs (A) and human DC subsets (B), pretreated with thymosin α 1 or the
scrambled peptide (sThymosin α 1), were exposed to Aspergillus conidia and assessed for
TLR expression by RT-PCR. cDNA levels were normalized against the HPRT gene. None,
cells exposed to the diluent alone. (C) Production of IL-8 by wild type or TLR-transfected
HEK293 cells stimulated with thymosin α 1 and/or zymosan (10 µg/ml), CpG 2006 (3 µg/ml
) and LPS (1 mg/ml). *P < 0.05, stimulated versus unstimulated (None) cells. **P < 0.05,
CpG or zymosan+thymosin versus each single agent alone. (D) Activation of NF-KB and p38
on murine lung DCs exposed to Aspergillus conidia, thymosin α 1 or the scrambled peptide,
either alone or in combination. The assay was done by probing the cell lysates with specific
anti-phospho-38 and anti-NF-KB antibodies. Shown are the data from one representative
experiment out of three.
Figure 4. Thymosin D 1 activates the MyD88-dependent pathway of TLR signaling.
(A) Production of IL-12 p70 and IL-10 by murine lung DCs upon exposure to TLR ligands
and /or thymosin α 1. Zym (zymosan, 10 µg/ml), LTA (lipoteichoic acid, 1 µg/ml), Poly(I:C)
(polyinosine-polycytidylic acid, 10 µg/ml), LPS (lipopolysaccharide, 1 mg/ml) and
unmethylated CpG 1826 (CpG, 2 µM). *P < 0.05, cytokine production in the presence (+) or
not (-) of thymosin α 1. (B) Production of IL-12 p70 and IL-10 from lung DCs from wild type
or MyD88-deficient mice, upon exposure to conidia and/or thymosin α 1. Bars indicate the
standard errors. *P < 0.01, cytokine production in MyD88-deficient versus wild type mice.
(C) Mice were intranasally infected with Aspergillus conidia and treated with thymosin α 1
for 5 consecutive days, beginning the day of the infection. The fungal growth (chitin content
of the lungs) was assessed a day after treatment. Bars indicate the standard errors. *P < 0.05,
treated vs untreated mice. **P < 0.05, MyD88-deficient versus wild type mice.
34
Figure 5. Thymosin D 1 protects BMT mice from invasive aspergillosis.
BMT mice were intranasally infected with Aspergillus conidia a week after the BM infusion.
Thymosin α 1 or the scrambled peptide (group 7) was given intraperitoneally daily beginning
the day of the BM infusion, in concomitance with the infection and continuing for additional
3 days. Amphotericin B was given intraperitoneally for 3 days, in concomitance with the
infection. (A) MST, median survival times (days), calculated from the beginning of the
infection. The numbers refer to died animals over total injected. The fungal growth was
assessed at 1 (control) or 4 days after the last conidia inoculation. Bars indicate the standard
errors. *P < 0.05, treated vs untreated mice. **P < 0.05, combined treatment versus each
single treatment. (B and C) Periodic acid-Schiff-stained sections from lungs of infected BMT
mice either untreated (B at 1 day post-infection) or treated with thymosin α 1 (C at 4 days
post-infection). Numerous Aspergillus hyphae (arrows) infiltrating the lung parenchyma, with
severe signs of bronchial wall damage and necrosis and scarce inflammatory cell recruitment
are observed in the lungs of untreated mice, as opposed to what observed in thymosin-treated
mice, whose lungs were characterized by healing infiltrates of inflammatory cells with no
evidence of bronchial wall damage and fungal growth. Magnification x 400 in both panels.
Figure 6. Thymosin D 1 accelerates functional Th1 cell recovery in BMT-mice with IA.
BMT mice were infected and treated as in legend to figure 5. (A) The numbers refer to %
positive cells in the lungs on FACS analysis. (B) Lung CD4+ T lymphocytes were CFSEstained (M1) and stimulated with Con A (M4) or heat-inactivated Aspergillus (M5) in the
presence of lung DCs for 5 days before FACS analysis. M2 and M3, CD4+ T cells without
and with DCs alone, respectively. Shown are the results from one representative experiment
out of three. The numbers refer to the median fluorescence intensity, MFI. (C) Lung CD4+ T
cells producing cytokines were enumerated by ELISPOT assays. *P < 0.05, thymosin-treated
35
vs untreated (none) mice. In A, B and C, the assays were done 4 days after the last conidia
inoculation. (D) Bronchoalveolar macrophages and peripheral neutrophils from uninfected
mice were pre-exposed to thymosin α 1 and then to conidia before the assessment of the
phagocytic and conidiocidal activity. Values are the mean ± SE of samples taken from 3-5
experiments. *P < 0.05, thymosin-exposed vs unexposed (None) cells.
36