The Helminth-Derived Immunomodulator AvCystatin Reduces Virus Enhanced Inflammation by Induction of Regulatory IL-10+ T Cells
The Helminth-Derived Immunomodulator AvCystatin Reduces Virus Enhanced Inflammation by Induction of Regulatory + IL-10 T Cells
Editor: Jie Sun 0
Mayo Clinic Minnesota 0
UNITED STATES 0
Martijn J. Schuijs 0 1
Susanne Hartmann 0
Murray E. Selkirk 0
Luke B. Roberts 0
Peter J. M. Openshaw 0 1
Corinna Schnoeller 0 1
0 Current address: VIB Inflammation Research Center, laboratory of Immunoregulation and Mucosal Immunology, Ghent, Belgium and Department of Respiratory Medicine, Ghent University , Ghent , Belgium
1 Respiratory Science Division, National Heart and Lung Institute, Imperial College London , London , United Kingdom , 2 Department of Life Sciences, Imperial College London , London , United Kingdom , 3 Centre for Infection Medicine, Institute for Immunology, Freie Universität Berlin , Berlin , Germany
Respiratory Syncytial Virus (RSV) is a major pathogen causing low respiratory tract disease (bronchiolitis), primarily in infants. Helminthic infections may alter host immune responses to both helminths and to unrelated immune triggers. For example, we have previously shown that filarial cystatin (AvCystatin/Av17) ameliorates allergic airway inflammation. However, helminthic immunomodulators have so far not been tested in virus-induced disease. We now report that AvCystatin prevents Th2-based immunopathology in vaccineenhanced RSV lung inflammation, a murine model for bronchiolitis. AvCystatin ablated eosinophil influx, reducing both weight loss and neutrophil recruitment without impairing anti-viral immune responses. AvCystatin also protected mice from excessive inflammation following primary RSV infection, significantly reducing neutrophil influx and cytokine production in the airways. Interestingly, we found that AvCystatin induced an influx of CD4+ FoxP3+ interleukin-10-producing T cells in the airway and lungs, correlating with immunoprotection, and the corresponding cells could also be induced by adoptive transfer of AvCystatin-primed F4/80+ macrophages. Thus, AvCystatin ameliorates enhanced RSV pathology without increasing susceptibility to, or persistence of, viral infection and warrants further investigation as a possible therapy for virus-induced airway disease.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This study was supported by a Wellcome
Trust Program grant to PO (087805/Z/08/Z). MJS
was supported by an Erasmus Placement grant
(UUER/2010/010744) from the European Commission
Life-Long Learning programme. The funders had no
role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors of this manuscript
have the following competing interests: C.S. and S.H.
Respiratory Syncytial virus (RSV) is the commonest single cause of hospitalization during
infancy, resulting in approximately 160,000 deaths annually worldwide [
]. Infection with RSV
causes inflammatory cell recruitment to the lung leading to bronchiolar occlusion [
] and RSV
hold a patent for uses of Cystatin (2 056 867). M.J.S.,
M.E.S., L.B.R and P.J.M.O declare no relevant
conflict of interest. The stated conflicts of interest do
not alter our adherence to PLOS ONE policies on
sharing data and materials.
bronchiolitis may be associated with recurrent wheezing and asthma in later life [
the great need for a vaccine, none is yet available. Vaccination with alum-adjuvanted
formalininactivated RSV vaccine caused increased disease severity and deaths, probably associated with
eosinophilic disease [
]. Prophylactic administration of the humanised anti-RSV monoclonal
palivizumab (Synagis1) prevents infection in high-risk infants [
] and reduces the frequency
of subsequent wheezing [
], but is costly. The great burden of RSV disease and the lack of an
effective treatment underscores the importance of developing new intervention strategies.
Epidemiological studies suggest that chronic helminthic infection might protect humans
from allergic sensitization and reduce allergic and inflammatory responses [
]. In animal
models, helminthic infections prevent or cure inflammation of mucosal tissues such as lung
and gut [
]. However, the mechanisms underlying these effects remain largely obscure
and varies depending on species and disease [
]. Moreover, studies on co-infection of viruses
and helminths have indicated a general impairment of antiviral immunity leading to increased
virus persistence [
], but very little is known on the effect of specific, recombinant parasite
immunomodulators in viral disease.
We have previously shown that AvCystatin, a recombinant cysteine protease inhibitor
derived from the filarial nematode Acanthocheilonema viteae, abrogates Th2-related
inflammatory disease in in vivo models of OVA-induced allergic airway inflammation, Th1-related
inflammation in dextran sulphate sodium-induced colitis [
] and grass pollen-specific allergic
]. The anti-inflammatory activity of AvCystatin appears to be mediated by
enhancement of IL-10 production by host macrophages since protection is lost after
macrophage depletion or blocking of IL-10 signalling [
]. In addition, the increased number of
regulatory T cells (Tregs) observed in the peribronchial lymph nodes (PBLN) in the OVA allergy
model suggests a possible role for Tregs in cystatin-induced suppression of inflammatory
] as may be the case in other situations [
To test the ability of AvCystatin to modify virus-enhanced immunopathology we
investigated its effects in primary RSV infection and two models of virus-induced lung eosinophilia in
mice: RSV challenge of mice sensitised with recombinant vaccinia encoding the major surface
glycoprotein G (vvG/RSV) [
] and RSV challenge of formalin-RSV vaccinated mice (FI-RSV/
]. AvCystatin suppressed eosinophilic, as well as primary neutrophillic
immunopathology without impairing immune defence against viral infection without altering viral
clearance or virus load. Immunoprotection in either eosinophilic model correlated with induction
of IL-10 producing CD4+ T cells; adoptive transfer of AvCystatin treated macrophages
activated IL-10-secretion by CD4+ T cells. AvCystatin therefore selectively blocks viral
immunopathology without dampening anti-viral immunity, and might be used to counter virus-enhanced
immunopathology at mucosal sites.
AvCystatin treatment reduced RSV-induced immunopathology in
Th2-based viral lung eosinophilia
To analyse efficacy and safety of AvCystatin in virus-enhanced lung inflammation we first
compared its effect in a model reflecting RSV-induced bronchiolitis (Fig 1A). The vvG/RSV
model uses live attenuated vaccinia-virus as a vehicle for RSV-G protein instead of
denaturation and adjuvant (day -14), and live virus challenge (d0). Pathology in vvG/RSV mice causes
enhanced weight loss after RSV challenge (Fig 1B) accompanied by pulmonary inflammation
with both eosinophils and neutrophils (Fig 1C). AvCystatin treatment [
] was administered
during both the sensitization and challenge phases to the relevant sites (i.p. and i.n.) and time
points (d-14, d-7 and d-2, d-1) (Fig 1A). This combined treatment significantly reduced
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Fig 1. AvCystatin treatment reduced RSV-induced immunopathology in Th2-based models of viral lung eosinophilia. A) Schematic
of the vvG RSV model: e.d. epidermabrasive; i.p. intraperitoneal; i.n. intranasal application. B) Weight loss in vvG RSV over an eight day
period C) Eosinophil, neutrophil, and macrophage influx into BAL: total numbers in vvG RSV. D) RSV-specific serum immunoglobulin
levels. E) L gene copy numbers in lungs, d4 after RSV/mock challenge. Naïve (light grey bars): mock infected and mock treated with PBS;
vvG/RSV (black bars): scarified with vvG and challenged with RSV; vvG/AvCystatin/RSV (white bars): vvG/RSV plus intraperitoneal (d -14
and -7) and intranasal (d -2 and -1) injection of AvCystatin. Representative data from 2 independent experiments, 5 mice per group. Error
bars indicate SEM. P values reflect Mann-Whitney t-test: * p<0.05, **p<0.01.
vaccine-enhanced RSV immunopathology, reducing weight loss (Fig 1B), neutrophil and
eosinophil influx (Fig 1C)), and mucus production in the airways (S4A Fig). However, levels of
RSV-specific Immunoglobulin (Ig) G1 and IgG2a, measured 8 days post RSV infection, were
unchanged (Fig 1D). Furthermore, CD8+ T-cell responses, including IFNγ production, were
not affected by AvCystatin treatment (data not shown). Clinical immunosuppressants and
some live helminth infections induce generalised suppression of immune responses and might
thus result in enhanced virus load or extended virus persistence. However, AvCystatin did not
increase the load or prolong the persistence of RSV in the lung indicating that beneficial
antiviral immune responses were not suppressed (Fig 1E).
To assess the ability of AvCystatin to suppress enhanced Th2 pathology in an alternative
model, we induced Th2-based disease by vaccinating mice with FI-RSV (S1A Fig). This scenario
mimics the human Lot100 trails in the early 1960’s that enhanced disease severity in infants. In
this case, treatment with AvCystatin completely prevented weight loss (S1B Fig). FI-RSV-driven
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Fig 2. AvCystatin reduced chemokine and cytokine release in the airways. A) IL-4, B) IL-13, C) CCL11/Eotaxin, D) CCL5 / RANTES,
E) TNF- α, F) IFN-γ, G) CCL3 / MIP-1α, Detection limit: 0.3pg/ml; 7.8pg/ml; 2.2pg/ml; 5.2pg/ml; 3.4pg/ml; 1.8pg/ml; 12.5pg/ml, respectively).
Day 4 post RSV/mock challenge. Naïve (light grey bars): mock infected and mock treated with PBS; vvG/RSV (black bars): scarified with
vvG and challenged with RSV; FI-RSV/AvCystatin/RSV (white bars). Representative data from 2 experiments, 5 mice per group. Error bars
indicate SEM. P values reflect Mann-Whitney t-test: * p<0.05, **p<0.01, ***p<0.001.
eosinophil influx into the airways was also significantly reduced (S1C Fig) whilst macrophage
numbers were enhanced (S1D Fig). Again we observed no increased viral load or prolonged
persistence of RSV (S1E Fig). These findings again demonstrate that AvCystatin effectively reduces
RSV-enhanced Th2-immunopathology without impairing antiviral responses.
AvCystatin reduced chemokine and cytokine release in the airways
Since cytokine expression in the airways reflects and influences the outcome of enhanced RSV
disease, we analysed Bronchiolar Alveolar Lavage (BAL) fluid in the vvG/RSV model for the
presence of both Th1 or Th2 specific cytokines and chemokines. Treatment of mice sensitised
as described previously (Fig 1A) with AvCystatin significantly reduced the levels of IL-4, IL-13,
eotaxin (CCL11) and CCL5/RANTES (Fig 2A–2D). TNF-α, IFN-γ and MIP1α (CCL3) were
also suppressed (Fig 2E–2G), whereas IL-6 and IL-17 remained unaltered (data not shown).
Thus, AvCystatin down-regulates the expression of specific Th1 and Th2 cytokines as well as
chemokines implicated in RSV-induced airway pathology and eosinophilia, but does not cause
general cytokine and chemokine suppression or interfere with antiviral defence.
AvCystatin induces IL-10 production by CD4+ T cells in airways and
To investigate the mechanisms responsible for the observed protection, we analysed cellular
recruitment into airways, lungs and draining lymph nodes. Administration of AvCystatin
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Fig 3. AvCystatin induced IL-10 production by CD4+ T cells in airways and lungs. Total CD4+ T cell counts in lungs (A) and BAL (B).
CD4+ T cells were stimulated with PMA/ionomycin for 3h, IL-10+ and IFN-γ+ cells were gated in lung (B+C) and BAL (E+F). Ratios of IL-10+
versus IFN-γ+ CD4+ T cells in lung and BAL (G). Naïve (light grey): mock infected and mock treated with PBS; vvG/RSV (black bars):
scarified with vvG (d-14) and challenged with RSV (d0); vvG/Av17/RSV (white bars): model plus injection of AvCystatin (d-14 and -7 i.p., d-2
and -1 i.n.). (A) Combined data from 2 independent experiments, (B-G) Representative data from 2 independent experiments, 5 mice per
group. Error bars indicate SEM. P values reflect Mann-Whitney t-test: * p<0.05, **p<0.01.
induced an increase in the numbers of CD4+ T cells in the lungs at day 8 post RSV challenge
(Fig 3A), but not in the airways (Fig 3D). Notably, both the number and frequency of CD4+ T
cells producing IL-10 was increased in both lungs and airways (Fig 3B, 3E and S2A–S2E Fig)
whereas the number of those producing IFN-γ was decreased in BAL (Fig 3F) but not in the
lungs (Fig 3C). Notably, comparison of CD4+IL-10+IFN-γ- with CD4+IL-10-IFN-γ+
populations showed a shift from pro-inflammatory to immunosuppressive environment (Fig 3G).
Thus, the observed protective effects of AvCystatin were associated with the induction of IL-10
production by CD4+ T cells.
AvCystatin reduces airway inflammation in primary (Th1) RSV infection
We next investigated whether the protective effects of AvCystatin were limited to Th2-induced
pathologies or depended on the route of application. Primary RSV infection causes a
predominantly Th1 response, with an influx of neutrophils to the airways by day 4, and T
cell-dependent pulmonary immunopathology by day 8 post infection (Fig 4A). Either intranasal (i.n.) or
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Fig 4. AvCystatin treatment in primary RSV infection. A) Schematic of the primary RSV model with AvCystatin treatment regimen: i.p.
intraperitoneal; i.n. intranasal application. Neutrophil influx in the BAL was shown (B). RSV L gene copy numbers in the lungs (C) and viral
load (D) measured 4 days post RSV/mock challenge. RSV-specific IgG2a detected in serum 8 days post infection (E). IFN-γ, TNFα, IL-6,
CCL3, and CCL5 cytokine and chemokine production (F). Naïve (dark grey bars): mock infected and mock treated with PBS, RSV
challenged day 0 (black bars); AvCystatin/RSV; AvCystatin treatment i.n. or i.p. on day -1 (white bars or light grey bars, respectively).
Representative data of at least 2 independent experiments, 5 mice per group. Error bars indicate SEM. P values reflect Mann-Whitney
ttest: * p<0.05, **p<0.01.
intraperitoneal (i.p.) administration of AvCystatin significantly reduced influx of neutrophils
to the airways by day 4 (Fig 4B) without affecting viral load and CD8+ T-cell responses (Fig 4C
and 4D, and data not shown). Furthermore, intranasal administration significantly reduced
MUC5a expression in the lungs (S4B Fig). AvCystatin treatment did not alter serum IgG2a
levels measured 8 days post infection (Fig 4E). However, administration of AvCystatin via either
route suppressed expression of IFN-γ, TNF-α, IL-6, CCL3/MIP-1α and CCL5/RANTES in the
airways (Fig 4F). Mirroring the vaccine enhanced vvG/RSV model (Fig 1A), we observed an
increased influx of CD4+ T cells in the lungs and airways in AvCystatin treated mice
undergoing primary infection (Figs 4A, 5A and 5D). Moreover, the infiltrating CD4+ T cells revealed
an increased frequency of IL-10 producers after either i.n. or i.p. administration of AvCystatin
(Fig 5B and 5E) accompanied by an remarkable increase in CD4+CD25+Foxp3+ positive Treg
cells in the airways (Fig 5F), but not in the lungs (Fig 5C). These results indicate that
AvCystatin also selectively protect mice from excessive inflammation during primary RSV infection.
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Fig 5. IL-10+ CD4+ T cell induction by AvCystatin conditioned macrophages. Lung and BAL CD4+ T cells detected by flow cytometry
(A and D). Total number of IL-10 producing T cells determined by intracellular staining for IL-10 after restimulation with PMA/ionomycin for
3h in Lung and BAL (B and E). Number of CD4+ T cells positive for FoxP3 in lung (C) and BAL (F). Percentage of F4/80+CD11b+ cells
infiltrating the lungs 24h post RSV challenge (G) and total cell number (H). Total cell number of CD4+ IL-10+ T cells infiltrating the BAL, 8
days post PEC transfer (I). Naïve (dark grey bars): mock infected and mock treated with PBS, RSV challenged day 0 (black bars);
AvCystatin/RSV; AvCystatin treatment i.n. or i.p. on day -1 (white bars or light grey bars, respectively). Representative data of 2
experiments, 5 mice per group. Error bars indicate SEM. P values reflect Mann-Whitney t-test: * p<0.05, **p<0.01.
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Additionally, examination of cells from the regional nodes showed that intraperitoneal, but
not intranasal, AvCystatin induced systemic CD4+FoxP3+ cells; in contrast, only intranasal
treatment induced LN IL-10 production (S3A and S3B Fig). Thus, AvCystatin induced
regulatory T cells and especially CD4+IL-10+ T cells in primary RSV infection, limiting RSV induced
AvCystatin conditioned macrophages induce IL-10 production in airway
CD4+ T cells
Previous studies [
], indicate that macrophages may be involved in causing the
immunoprotection induced by AvCystatin. We therefore looked at the role and function of these cells
in primary RSV infection. We found that recruitment of F4/80+CD11b+ macrophages was
enhanced by AvCystatin treatment via airways 24h after RSV challenge (Fig 5G and 5H).
Interestingly, lung F4/80+ macrophages showed no increase in production of IL-10 at 4 and 8 days
post-RSV infection (data not shown) although peritoneal macrophages have been shown to
bind AvCystatin  and migrate to the spleen and mediastinal lymph nodes after adoptive
]. To determine whether AvCystatin primed macrophages might induce IL-10
production by CD4+ T cells, we primed peritoneal exudate cells (PEC; 70% macrophages and 30%
B cells) with AvCystatin and adoptively transferred these cells to naïve mice prior to RSV
infection. Transfer of AvCystatin-primed PEC resulted in elevated numbers of IL-10+ CD4 T cells
in the airways at 8 days post-RSV infection (Fig 5I). In contrast, PECs primed with
heat-inactivated AvCystatin prior to transfer had no effect. PECs therefore appear to be crucial in the
uptake and processing of active AvCystatin, leading to the induction of IL-10-producing CD4+
T cells which may mediate protective effects observed in the lung. This observation
complements previous research showing protection by AvCystatin treated macrophages, via the
induction of IL-10 producing CD4+ T-cells, in an experimental model of DSS colitis [
Testing the therapeutic potential of AvCystatin in viral lung disease, we show that it has
beneficial effects in both primary and in vaccine-enhanced lung pathology. It reduces
immune-mediated disease that is seen in RSV infection in its various forms without impairing antiviral
defences. It beneficial effects appear to be mediated via macrophages that promote regulatory
and IL-10 producing CD4+ T cells, and include a potent inhibition of eosinophilic lung disease.
These findings help to explain epidemiological findings that show an inverse relationship
between the prevalence of parasitic worm infection and that of allergy and autoimmunity [
]. This correlation is often explained in the context of the hygiene hypothesis, which suggests
that diseases characterized by an over-reactive immune response against harmless or
self-antigens are caused by a lack of immunological ‘training’, related to a reduced rate of infections
that were once common but are now mainly found in developing countries [
Epigenetic ‘imprinting’ has been recently implied by the observation of protective effects of
previous bacterial infections on RSV disease [
]. An effect of parasite colonisation on RSV
disease would seem to be via different mechanisms, since Th2-driven immunopathology (as
observed in allergic inflammation), shares many of the features of anti-helminth parasite
immune responses (e.g. eosinophilia, production of IgE, IL-13 and eotaxin). Parasites appear to
have evolved complex mechanisms whereby they inhibit Th2 immune responses, presumably to
survive better within their hosts. There is some evidence that these immunomodulatory
mechanisms may also suppress unrelated allergic and inflammatory responses, but the relationships
and mechanisms are complex and difficult to investigate in man. However, experiments in mice
show that helminth infections can abrogate allergic airway inflammation ,[
co8 / 14
infection studies indicate that helminths may enhance susceptibility to viruses. In Schistosoma
mansoni and lymphocytic choriomeningitis virus (LCMV) infection in mice, helminth-related
immunosuppression of antiviral type I IFN responses resulted in infiltration of high numbers of
LCMV-specific CD8 cells into the liver, leading to increased morbidity and hepatotoxicity [
Additionally, latent herpes infection could also be reactivated by helminthic worms in a STAT6
dependent manner, by the production of IL-4 [
]. Or in the case of Norovirus by inhibition of
T cell proliferation by alternatively activated macrophages developing in the presence of IL-4
]. The use of purified recombinant immunomodulators rather than live helminth infection
provides a controlled means by which to alleviate immunopathology [
In our present studies, AvCystatin suppressed Th2 cytokine production (especially IL-4),
minimising the recruitment of inflammatory cells into the airways. Furthermore, production of
chemokines linked to eosinophilia (RANTES and eotaxin) was strongly inhibited, and this was
reflected by the inhibition of virus-related eosinophilia. However, AvCystatin did not generally
suppress cytokine production, allowing for effective antiviral responses, as for example IL-6
expression was unaltered. IL-6 is considered a pro-inflammatory cytokine induced in RSV
] and known to be crucial for follicular helper cell and germinal centre
development in chronic virus infection [
] and acute antiviral responses [
]. AvCystatin thus
modulates rather than generally suppresses immune responses, a feature also seen in some long
persisting helminths (e.g. Heligmosomoides polygyrus [
Recent work confirms a crucial role for IL-10 in the outcome of RSV infection, with T cells
identified as the major source [
]. In the current study, we observed induced numbers of
F4/80+CD11b+ macrophages, identified as the major population of cells in peritoneal exudates
which ingest AvCystatin [
]. Previous in vitro and in vivo studies (OVA airway
hyperreactivity), indicate a key role for IL-10 and macrophages in the AvCystatin-induced amelioration of
]. Application of AvCystatin in vvG- RSV and primary RSV infection
models resulted in the induction of IL-10-producing CD4+ T cell populations. By adoptive
transfer of AvCystatin-primed peritoneal exudate cells we were able to induce IL-10 producing
CD4 T cells and ameliorate airway inflammation. This finding is in support of the
demonstration that adoptive transfer of AvCystatin-primed macrophages, but not B cells, mediated
protection in a model of allergic airway inflammation and colitis via induction of IL-10 producing
T cells . The current study shows that exposure to AvCystatin leads to enhanced
recruitment of IL-10-producing CD4+ T cells in viral infection; however, FoxP3 expression did not
coincide with IL-10 production in the regional lymph nodes. The source of IL-10 can therefore
differ depending on the infection, tissue environment and type of inflammatory insult.
However, we do not rule out a role for FoxP3+ Treg cells in the control of RSV pathology, as we
have observed an increase in Treg cells in the airways after AvCystatin treatment during
primary infection and previous work has implicated a role for Treg cells in suppressing RSV
In relation to human RSV-induced bronchiolitis, neutrophils appear to be the instigators of
pathology and dampening neutrophillic inflammation by AvCystatin or other neutrophil
suppressors, may well be a way forward for future patient care. However, due to the proposed
antiviral function of neutrophils, current practice suggests to target neutrophils under the umbrella
of antiviral treatment [
This is, to our knowledge, the first time that a recombinant parasite-derived
immunomodulator has been shown to down-regulate virally-enhanced inflammation without impairing
responses important for anti-viral immunity. We provide evidence that AvCystatin
immunomodulation may act through the production of CD4+ T cell derived IL-10. Collectively, our
findings support the testing of AvCystatin as a potential novel agent to counter virus-enhanced
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Materials and Methods
All mouse experiments were ethically approved by the Imperial College Central Biological
Services (CBS) ethics committee performed in accordance with approved UK Home Office
guidelines (Project License No. PPL 70/6785).
Mice were treated with 20 μg AvCystatin prepared as previously described [
] or PBS
intraperitoneally (i.p.) or intranasally (i.n.).
RSV (strain A2) and rVV-G (vaccinia virus expressing the RSV G-protein) were propagated in
Hep-2 cells (ATCC) as described previously [
]. FI RSV was obtained by formalin inactivation
of RSV A2 according to FI RSV Lot 100 protocols, with alum as adjuvant [
]. For FI-RSV
models, eight week old female BALB/c mice (Harlan, UK) were injected with FI-RSV
intramuscularly (day -14) and infected intranasally with 5 x 105 PFU RSV 2 weeks thereafter. For rvv-G
RSV models, mice were primed with vaccinia expressing RSV-G protein (3 x 106 PFU) by
scarification of the rump and infected intranasally with 5 x 105 PFU RSV 14 days later. RSV titres
were assessed by titration of lung homogenates on Hep-2 cell monolayers [
]. RSV L-gene
copy numbers were determined by TaqMan technology using the delta delta ct method, and
normalization with 18S as previously described [
Bronchoalveolar lavage (BAL) and cell isolation
BAL-accessible cells were obtained by repeated instillation of 1 ml of PBS/12 mM lidocaine via
the trachea in sacrificed animals. Cytospin preparations were stained with hematoxylin and
eosin and analysed by light microscopy (300 cells per slide). Lungs were homogenized and
digested with collagenase D (50ug/ml, Sigma, Gillingham, UK) for 30 min. Mediastinal
(peribronchial) lymph nodes were dissociated and filtered through a 100 μm cell strainer.
Flow cytometry was performed as previously described [
]. Briefly, cells were incubated with
LIVE/DEAD Fixable cell stain (Invitrogen), then stained for 20 min with: MHCII-PacificBlue,
F4/80-APC (Biolegend), CD4-AF700, CD8-PacificBlue, CD25-APC, CD86-APC,
CD49b-PeCy7 (eBioscience), CD11b-PE-Cy7, CD45R/B220-PerCP-Cy5.5 (BD-Pharmingen). For
intracellular staining, the samples were stimulated for 3 h (100 ng/ml phorbol 12-myristate
13-acetate, 1 μg/ml ionomycin; monensin added after 1 h). Cells were fixed and permeabilized with
Cytofix/Cytoperm (BD), then stained with IL-10-FITC, IFN-γ-PerCP-Cy5.5, IL-17-PE,
FoxP3-AF488 (BD-Pharmingen) antibodies before analysis on LSRII flow cytometer (BD),
collecting 100,000 live events. Data was analyzed using FlowJo (v7.6.5, Tree Star, Ashland, OR, USA).
Adoptive transfer of peritoneal exudate cells (PEC)
Donor BALB/c were injected i.p. with 20 μg active or heat-inactivated AvCystatin and
peritoneal exudate cells (PEC) recovered 20 h later in PBS/2 mM EDTA/0.2% BSA. Recipient mice
were injected intravenously with 3x106 PEC and infected with RSV i.n. 4 h later.
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Cytokine and Chemokine detection
Quantification of BAL chemokines and cytokines was performed using a 13-plex Luminex kit
(Millipore, Watford, UK). Data were acquired with a Luminex 100 (Applied Cytometry
systems, Sheffield, UK) and Starstation software.
GraphPad Prism software (La Jolla, CA, USA) was used to analyse data, generally shown as
mean ±SEM of 5 animals per group. Mann-Whitney t-test and 2 way ANOVA were used to
compare data. P values of <0.05 were considered significant.
S1 Fig. Reduced RSV-induced immunopathology by AvCystatin treatment in a model of
FI-RSV of viral lung eosinophilia. A) Schematic of the FI RSV model: i.m. intramuscular; i.p.
intraperitoneal; i.n. intranasal application. B) Weight loss in the FI-RSV model. Total cell
number of eosinophil (C) and macrophages in the BAL (D). Viral load in the lungs measured by
RSV L-gene copies (E). Representative data of 2 experiments, 5 mice per group. Error bars
indicate SEM. P values reflect Mann-Whitney t-test: p<0.05, p<0.01.
S2 Fig. Frequency of IL-10 producing T-cells in the lungs and airways after AvCystatin
treatment. Flowcytometric analysis of IL-10 intracellular cytokine content in RSV challenged
or AvCystatin/RSV challenged mice is shown (A). Graphical visualization of IFNγ and IL-10
production by CD4+ T cells in the BAL (D and E) and lungs (B and C). Representative data of
2 experiments, 5 mice per group. Error bars indicate SEM. P values reflect Mann-Whitney
ttest: p<0.05, p<0.01.
S3 Fig. AvCystatin induced FoxP3+ T cell induction in the mediastinal lymph node. Total
number of FoxP3+ CD4+ T cells (A) and the number of IL-10+ CD4+ T cells in the mLN (B)
after AvCystatin treatment and RSV challenge. Representative data of 2 experiments, 5 mice
per group. Error bars indicate SEM. P values reflect Mann-Whitney t-test: p<0.05, p<0.01.
S4 Fig. AvCystatin treatment reduces Muc5a production in lung. Relative expression of
MUC5a in mice lungs after the vvG RSV model (A) or primary RSV model (B). Error bars
indicate SEM. P values reflect Mann-Whitney t-test: p<0.05.
The authors thank Bettina Sonnenburg and Emilia Danilowicz-Luebert for AvCystatin
purifications, Jacky Flipse for data discussions and Michelle Goritzka for excellent technical help.
This study was supported by a Wellcome Trust Program grant to PO (087805/Z/08/Z). MJS
was supported by an Erasmus Placement grant (UU-ER/2010/010744) from the European
Commission Life-Long Learning program.
Conceptualization: MJS CS SH MES PJMO.
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Formal analysis: MJS CS.
Funding acquisition: MJS PJMO.
Investigation: MJS CS LBR.
Methodology: MJS CS PJMO.
Resources: SH MES PJMO.
Supervision: CS PJMO.
Visualization: MJS CS.
Writing – original draft: MJS CS MES PJMO.
Writing – review & editing: MJS CS MES PJMO.
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