International Immunology, Vol. 19, No. 5, pp. 645–655
ª The Japanese Society for Immunology. 2007. All rights reserved.
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Influenza A virus elevates active cathepsin B
in primary murine DC
Timo Burster1, Thierry Giffon1, Martin E. Dahl1,6, Pia Bjo ¨rck2, Matthew Bogyo2,3,
Ekkehard Weber4, Kutubuddin Mahmood5,7, David B. Lewis1and Elizabeth D. Mellins1
1Department of Pediatrics,2Department of Pathology and3Department of Microbiology and Immunology,
Stanford University School of Medicine, Stanford, CA 94305, USA
4Institute of Physiological Chemistry, Martin Luther University Halle-Wittenberg, Halle, Germany
5MedImmune Vaccines, Mountain View, CA 94043, USA
6Present address: Roche Palo Alto LLC, Palo Alto, CA 94304, USA
7Present address: Novavax, Inc. Malvern, PA 19355, USA
Keywords: Cathepsin B, dendritic cells, influenza A
Dendritic cells (DCs) act as a first-line recognition system for invading pathogens, such as
influenza A. The interaction of DC with influenza A virus results in DC activation via endosomal
Toll-like receptors and also leads to presentation of viral peptides on MHC class II molecules. Prior
work demonstrated that influenza A virus (A/HK331; H3N2) infection of BALB/c mice activates lung
DCs for antigen presentation, and that the enhanced function of these cells persists long after viral
clearance and resolution of the virus-induced inflammatory response. Whether influenza A virus has
acute or longer-lasting effects on the endo/lysosomal antigen-processing machinery of DCs has not
been studied. Here, we show that antigen presentation from intact protein antigen, but not peptide
presentation, results in increased T cell stimulation by influenza-exposed lung DCs, suggesting
increased antigen processing/loading in these DCs. We find that cathepsin (Cat) B levels and activity
are substantially up-regulated in murine lung DCs, harvested 30 days after A/HK331 infection. CatB
levels and activity are also increased in murine splenic and bone marrow-derived DCs, following
short-term in vitro exposure to UV-inactivated influenza A virus. Modest effects on CatX are also seen
during in vivo and in vitro exposure to influenza A virus. Using a cell permeable Cat inhibitor, we show
Cats in influenza-exposed DCs to be functional and required for generation of a T cell epitope from
intact ovalbumin. Our findings indicate that influenza A virus affects the MHC class II antigen-
processing pathway, an essential pathway for CD41T cell activation.
Influenza is a single-stranded RNA (ssRNA) enveloped virus
that expresses surface glycoproteins hemagglutinin (HA),
neuraminidase (NA) and matrix protein as well as numerous
internal proteins, including nucleoprotein, polymerase pro-
teins and non-structural proteins (1). Influenza virus strains
A, B and C are distinguished by differences in nucleoprotein
and matrix proteins. Influenza A virus is further subdivided
into subtypes based on variations in HA and NA, for exam-
Influenza A virus infects the mucosal epithelial cells in the
respiratory track of the host and causes acute respiratory dis-
ease. Influenza virus HA binds to sialated receptors on the
cell surface of host cells, including respiratory track epithelial
cells and dendritic cells (DCs), and is taken up into endoso-
mal compartments. The acidic environment of endosomes
induces a conformational change in HA, allowing the virus to
fuse with the endosomal membrane and enter the cytoplasm
(2). Thus, influenza antigens enter both the endosomal and
the cytoplasmic compartments, accessing the MHC class II
and MHC class I antigen presentation pathways, respectively.
Influenza A virus, having ssRNA, also activates DCs via
Toll-like receptor (TLR)-7. This endosomal TLR is expressed
by murine plasmacytoid dendritic cells (pDCs) and myeloid
dendritic cells (mDCs). ssRNA recognition does not require
active viral replication, and it is thought that viral ssRNA is
delivered to the endo/lysosomal compartments after viral
Received 29 July 2006, accepted 23 February 2007
Correspondence to: T. Burster; E-mail: firstname.lastname@example.org or E. Mellins; E-mail: email@example.com
Transmitting editor: R. MedzhitovAdvance Access publication 19 April 2007
by guest on June 6, 2013
internalization (3). TLR7 ligation appears to be crucial for the
murine DC response to live and inactivated influenza A (4).
We (M.E.D. and D.B.L.) demonstrated that thirty days after
influenza A virus infection in a murine model, lung DCs
showed increased stimulatory activity for CD4+T cells (5).
Cell-surface levels of MHC class II and co-stimulatory mole-
cules, such as CD40, CD80 and CD86, were increased on
lung DCs at this time point, suggesting one explanation for
their enhanced antigen-presenting cell (APC) function. We
were interested in whether influenza A virus has additional
effects on MHC class II antigen-presenting pathway that
might contribute to their increased stimulatory capacity.
MHC class II molecules are expressed in the endoplasmic
reticulum, where they interact with a trimer of invariant chain
(Ii) and the resulting nonomer traffics to endosomal compart-
ments. Upon arrival in endosomes, Ii is degraded to a nested
set of class II-associated invariant chain peptide (CLIP) frag-
ments. CLIP is exchanged for other endosomal peptides by
the action of H2-DM (reviewed by Busch et al.) (6).
Antigens access endosomes through internalization from
the extracellular environment or targeted delivery from other
intracellular compartments. In endosomes, antigen under-
goes several processing steps until it is suitable for loading
on MHC class II molecules (7). These processing steps in-
clude unfolding, disulfide reduction (for antigens with internal
disulfide bonds) and proteolysis. The most widely studied
proteases in this machinery are the cathepsins (Cats), and
they are classified into three different types according to the
amino acid in their active site: the aspartate (CatD and CatE)
(8–10), cysteine [C1: CatB, H, X and S and C13: asparagine-
specific endoprotease (AEP)] (11–15) and serine proteases
(CatG) (16, 17). After exposure to maturation signals, for ex-
ample LPS, DC induce and convert several proteases such
as CatL, CatS and AEP to their active form (17–20).
In this report, we show that influenza A virus infection has
effects on components of the processing machinery in lung
DCs. We also demonstrate that other DCs show similar
changes after exposure to influenza A virus and confirm that
the affected proteins are functionally important for presenta-
tion of an exogenous antigen.
BALB/cJ mice were purchased from Jackson laboratories.
Thy1.1 BALB/c mice were purchased from MMRRC and
bred in our facility. DO11.10 ovalbumin (OVA)-specific ab
TCR-transgenic mice were bred on a BALB/c background in
our facility. All animal experiments were approved by institu-
tional protocols of Stanford University.
DC purification from influenza A-infected or control mice
DCs were isolated from the spleens of uninfected BALB/c
mice or lung tissue of mice 30 days after intra-nasal inocula-
tion with 240 HA units of HK331 influenza A virus in allantoic
fluid from chicken eggs diluted with PBS or, as a control,
with virus-free normal allantoic fluid (NAF), as described (5).
DCs were isolated from splenic or lung tissue of mice by
mincing tissue followed by treatment with 300 U ml?1type I
collagenase (Worthington Biochemical, Lakewood, NJ, USA)
and 100 U ml?1DNase I (Sigma–Aldrich, St Louis, MO,
USA) for 90 min in RPMI 1640 medium without serum. After
filtration through nylon gauze to remove debris, the cells were
re-suspended in RPMI 1640 medium with 10% FCS and incu-
bated with CD11c mAb-coated paramagnetic microbeads
and applied to an AutoMacs according to the manufacturer’s
instructions (Miltenyi Biotec, Auburn, CA, USA). The A/HK3
31 influenza A virus is a recombinant between A/PR8/8/34
(H1N1) and A/Aichi/2/68 with the surface H3N2 glycoprotein
of A/Aichi and the internal components of A/PR8 (21).
DC-mediated T cell proliferation assay
OVA-specific CD4+T cells were purified from the spleen and
peripheral lymph nodes of DO11.10 Rag2?/? mice by mag-
netic cell sorting (MACS) with CD4 mAb-coated microbeads
(Miltenyi Biotec). Purified CD4+T cells (1 3 105) were then
cultured for 72 h in 96-well round-bottomed plates with 2 3
104MACS-purified CD11c+lung DCs from previously (30d)
infected (HK331) or mock-infected mice and OVA (100 lg
ml?1) or OVA323–339 peptide (5 lg ml?1) in a volume of
200 ll. Cell proliferation was measured by pulsing cell cul-
tures with [3H] thymidine ([3H]Tdr) (1 lCi per well) and de-
termining incorporation during the last 18 h of incubation
using a microbeta reader (PerkinElmer, Shelton, CT, USA).
In vitro exposure to influenza strains
Influenza virus A and B strains [cold-adapted (c.a.) A/Sydney/
5/97 and c.a. B/Harbin/7/94], provided as a kind gift by MedI-
mmune Vaccines, Inc. (Mountain View, CA, USA), were used
for in vitro experiments. These strains were 6:2 genetic reas-
sortants between (c.a.) master donor strains [c.a. A/Ann Arbor
6/60 (H2N2) or c.a. B/Ann Arbor 1/66] and strains that do-
nated the HA and NA gene segments: A/Sydney/5/97 (H3N2)
and B/Harbin/7/94-like virus, respectively (22). The reassortant
strains were plaque titered on MDCK cells, with titers
recorded as plaque-forming unit (pfu) per milliliter. For inacti-
vation, the c.a. viruses were exposed to a UV source light for
30 min and titered to confirm complete inactivation. Spleen
cells or DCs from BALB/c mice were treated (1 pfu per
cell) with UV-inactivated c.a. influenza A/Sydney/05/97 or UV-
inactivated c.a. influenza B/Harbin/7/94-like strain for 24 h in
RPMI 1640 complete medium (GIBCO Life Technologies,
Grand Island, NY, USA) and harvested after washing with PBS.
Determination of CatB activity
Hydrolysis of CatB activity was determined using a fluorogenic
substrate, as previously described (23). Briefly, 100 lM of the
fluorogenic substrate Z-FR-AMC (R&D Systems, Minneapo-
lis, MN, USA) in buffer [0.1 M citrate, pH 5.0, 4 mM dithio-
threitol (DTT), 4 mM aprotinin and 4 mM EDTA] and whole-
cell lysate (lysis buffer: 10 mM Tris, pH 7.5, 150 mM NaCl
and 0.5% NP-40) from uninfected or influenza A virus-infected
DCs were mixed on ice. Liberated fluorescence (excitation
380 nm, emission 460 nm) was measured every 5 min with
a Gemini XS multiwell fluorometer (Molecular Devices, Sunny-
vale, CA, USA). The fraction of the emitted fluorescence that
could be inhibited by addition of 10 lM CatB-specific inhibi-
tor CA074 [N-(L-3 trans-propylcarbamoyloxirane-2-carbonyl)-
L-isoleucyl-L-proline] (Caltag, Burlingame, CA, USA) was
considered CatB activity.
646Influenza A up-regulates cathepsin B
by guest on June 6, 2013
Activity-based probes to visualize active cysteine proteases
Cells were lysed in lysis buffer (10 mM Tris, pH 7.5, 150 mM
NaCl and 0.5% NP-40) and 10 lg of cell lysates were incu-
bated with reaction buffer (50 mM citrate, pH 5.0 and
50 mM DTT) in the presence of biotinylated DCG-04 (24) for
1 h at room temperature. Reactions were terminated by the
addition of SDS-reducing sample buffer and immediate boil-
ing. Samples were resolved by 12% SDS–PAGE gel, and
then transferred to a polyvinylidene difluoride-membrane
and visualized using streptavidin HRP and the enhanced
chemiluminescence-detection kit (Amersham Biosciences,
Pittsburgh, PA, USA).
Anti-Cat antiserum was generated against affinity-purified hu-
man CatB (rabbit anti-mouse CatB, E. Weber University
Halle-Wittenberg, Halle, Germany) and purified CatB from hu-
man liver was purchased from Calbiochem (Calbiochem, San
Diego, CA, USA). The monoclonal mouse anti-b-actin anti-
body was obtained commercially (Sigma–Aldrich). Cells were
lysed in lysis buffer (10 mM Tris, pH 7.5, 150 mM NaCl and
0.5% NP-40), adjusted for equal total protein quantified by
Bradford, resolved by SDS–PAGE and immunoblotted using
published conditions (17).
Flow cytometric staining
Spleen cells or bone marrow (BM)-derived mDCs from BALB/
c mice were incubated with blocking buffer (10% human AB
serum, Caltag) for 10 min at room temperature and then incu-
bated for 15 min with CD11c–FITC, CD45R–TC (Caltag) or
MHC class II (clone 2G9, which recognizes I-A and I-E, BD
PharMingen, San Diego, CA, USA) in blocking buffer, washed
with PBS (1% BSA, Sigma–Aldrich). Cells were incubated for
20 min with BD Fix/Perm (Becton Dickinson, San Jose, CA,
USA) and washed twice with BD Perm solution. Incubating
the cells for 10 min with 10% human AB serum diluted in BD
Perm solution blocked non-specific binding. Cells were then
stained with rabbit anti-mouse CatB, rabbit anti-mouse CatD
(E. Weber University Halle-Wittenberg) or the isotype controls
for 20 min, washed twice with BD Perm solution and incu-
bated for 10 min with the second antibody (goat anti-rabbit
PE, Caltag). Cells were analyzed using a FACScan flow
cytometer (Becton Dickinson). A total of 50 000 cells were
collected using CellQuest Software (Becton Dickinson) and
analyzed by FlowJo (Tree Star, Inc., Ashland, OR, USA).
Generation of BM-derived mDCs
Mouse DCs were prepared as previously described (25). In
brief, total BM cells from BALB/c mice were obtained by
flushing the femurs and tibia with PBS using a 23-gage nee-
dle. After washing and lysis of red cells, total leukocytes
were re-suspended in complete RPMI 1640 (GIBCO Life
Technologies) supplemented with 5% fetal bovine serum
(Cambrex, Walkersville, MD, USA), 10 mM HEPES, 2 mM
L-glutamine, antibiotics, 1 mM Na pyruvate, 0.1 mM non-
essential amino acids, 5 3 10?5M 2-mercaptoethanol (all
from GIBCO) and 100 ng ml?1rFlt3-L (PeproTech, Rocky
Hill, NJ, USA). Cells were cultured at 1 3 106cells ml?1in
six-well plates (Falcon, Franklin Lakes, NJ, USA) for 10 days.
Cells that detached from the plates were gently collected
and washed once in PBS. Cells were further incubated to-
gether with FcR-blocking antibodies (CD16/CD32, clone
2.4G2, BD PharMingen at 1 lg/1 3 106cells) for 30 min on
ice. After washing, cells were stained with antibodies spe-
cific for mDCs (CD11b–allophycocyanin–Cy7, PharMingen)
and pDCs and B220+DCs (B220–allophycocyanin, PharMin-
gen) were excluded. Propidium iodide was included to gate
out dead cells. Cells were sorted using a FACSVantage
(Becton Dickinson). Gates were set to exclude dead cells
and debris. Purity was usually ¥96% (data not shown). Post-
sort FACS analysis using directly fluorochrome-conjugated
antibodies showed that all cells expressed the common DC
marker CD11c and were immature based on low expression
of I-Adand CD86 (data not shown).
APC functional analysis in vitro
APCs were purified from spleen of Thy1.1 BALB/c mice (I-Ad)
and OVA-specific CD4+T cells (Thy1.2) were purified from
lymph nodes and spleen of DO11.10 Rag2?/? mice. APCs
(5 3 104) and CD4+T cells (1 3 105) were cultured with
OVA (100 lg ml?1) or OVA323–339peptide (5 lg ml?1), with
or without CA074-methyl ester (OMe) (10 lM, Caltag),
CA074 (10 lM, Caltag) or CatG-specific inhibitor (200 nM,
Calbiochem) (26), respectively, for 3–5 days at 37?C in a vol-
ume of 200 ll. Some of the cultured cells were exposed to
LPS (1 lg ml?1) or UV-inactivated c.a. A/Sydney influenza A
virus (1 pfu per cell). Cells were re-stimulated with phorbol
myristate acetate (20 ng ml?1) and ionomycin (0.2 ng ml?1)
for 6 h at 37?C, with 10 lg ml?1Brefeldin-A added for the
last 4 h. Anti-Thy1.2 mAb (clone 5a-8, Caltag) and anti-CD4
mAb were used for detection of the DO11-10 cells, and in-
tracellular IFN-c was detected with a PE-conjugated mAb
(clone XMG1.2, eBiosciences) using the Cytofix/Cytoperm
kit (BD Biosciences).
T lymphocyte proliferation was determined using carboxy-
fluoresceine diacetate succinimidyl ester (CFSE) labeling.
Purified CD4+T from DO11-10 Rag2?/? cells were washed
twice in serum-free medium (RPMI 1640) and incubated
with 0.5 lM CFSE for 15 min, washed twice and re-suspended
in complete RPMI 1640. CFSE-labeled cells (1 3 105) were
then cultured with 5 3 104APCs and OVA (100 lg ml?1)
for 3–5 days at 37?C. The DO11-10 TCR was detected with
an allophycocyanin-conjugated KJ1-26 clonotypic mAb and
CD69 with a PE-conjugated mAb (clone H1.2F3), all from
Caltag Laboratories. DO11-10 cells were analyzed for their
surface expression of CD69 versus CFSE on a FACSCalibur
flow cytometer (BD Biosciences). Data were analyzed using
FlowJo software (Tree Star, Inc.).
Lung DCs have enhanced presentation of intact protein
antigen but not peptide antigen after influenza A virus
We (M.E.D. and D.B.L.) have shown that lung DCs isolated
from mice that were previously (30d) inoculated with
influenza A virus have increased stimulatory capacity for
antigen-specific CD4+T cells when provided with intact pro-
tein as antigen (5). To assess the contribution of antigen
Influenza A up-regulates cathepsin B647
by guest on June 6, 2013
processing/peptide loading to this enhanced APC function,
we chose to compare T cell stimulation after incubation of
influenza-exposed DCs with intact OVA with stimulation after
incubation with OVA peptide (OVA323–339). To obtain influenza-
exposed DCs, we used our previous approach. We admin-
istered influenza A virus strain HK331 (H3N2) in allantoic
fluid intra-nasally to BALB/c mice; control mice received
an equivalent volume of identically diluted virus-free NAF.
Lung DCs were harvested 30 days after viral or NAF adminis-
tration and MACS purified, based on CD11c expression.
Purified splenic OVA-specific CD4+T cells from DO11.10
(OVA-specific TCR) Rag2?/? transgenic mice were cultured
for 72 h together with lung DCs, OVA (100 lg ml?1) or OVA
peptide (5 lg ml?1) and T cell proliferation was assessed by
the incorporation of [3H]Tdr. As shown in Fig. 1, lung DCs
from influenza-infected mice pulsed with OVA protein-stimu-
lated enhanced OVA-specific T cell proliferation compared
with control lung DCs from uninfected mice. In contrast, after
pulsing with OVA peptide, lung DCs from previously infected
mice did not mediate enhanced T cell proliferation, despite
having increased levels of surface class II and co-stimulatory
molecules, as previously shown (5). These findings imply
that differences that increase the generation of OVA peptide–
MHC complexes from intact antigen contribute to the en-
hanced T cell proliferation mediated by lung DCs from mice
previously infected with influenza A virus. Notably, when exog-
enous OVA peptide is the source of antigen, the T cell stimula-
tion is higher for both influenza-exposed and control DCs. This
finding indicates that naturally processed OVA peptide is limit-
ing for peptide–MHC complex generation when intact OVA
protein is the source of antigen, and thus argues that changes
in generation or loading of OVA peptide are critical to the in-
creased CD4+T cell stimulation observed with the DCs from
influenza A-infected mice. Our data also show that peptide
presentation by DCs isolated from uninfected control mice is
more effective, which likely reflects their increased capacity
for peptide exchange at the cell surface, as predicted by in-
creased H2-DM activity in DCs after microbial stimuli (27).
CatB is up-regulated in lung DCs from influenza A virus-
To pursue the implications of our findings on peptide versus
protein presentation of OVA by lung DCs from influenza-
infected mice, we chose to investigate Cat expression in lung
DCs after influenza A HK331 virus infection. We again admin-
istered virus or control NAF intra-nasally to BALB/c mice and
30 days after infection, lung DCs were isolated and purified
from both infected and control mice. Cell lysates of lung DCs
from both control and HK331-infected mice were incubated
with the biotinylated activity-based probe DCG-04 that labels
cysteine Cats through an activity-dependent reaction with the
active site thiol of these proteases. Thus, active-site labeling
provides an indirect, but quantitative readout of protease ac-
tivity. The lysates were loaded on a SDS–PAGE and visualized
by streptavidin HRP blot (Fig. 2A). In lung DCs from both con-
trol and HK331-infected mice, CatX was detected at a mo-
lecular weight of 35 kDa, CatB at 30 kDa, CatS at 25 kDa
and CatL at 20 kDa in agreement with published data from
BM-derived DCs (28). In lung DCs from HK331-infected
mice, there was a striking increase in levels of active CatB.
Modest changes in CatX levels suggested an increase in
this Cat as well. We also consistently noted an increase in
an unknown (presumed) cysteine protease of apparent mo-
lecular weight around 40 kDa. However, no significant in-
crease in levels of active forms of CatH, CatS and CatL was
found in lung DCs from HK331-infected mice compared
with control-treated mice. b-Actin levels were measured to
confirm that the same amount of protein was loaded on
SDS–PAGE from experimental and control DC lysates.
Next, we measured the turn over of the fluorogenic sub-
strate Z-FR-AMC in lung DCs from both control and HK331-
infected mice; substrate Z-FR-AMC detects CatB and CatL
activity at pH 5.0. The substrate turn over was higher
when we used lung DC lysate from HK331-infected mice
compared with lung DC lysate from control-treated mice
(Fig. 2B), consistent with an increase in CatB activity in
these cells. The relatively modest extent of the difference in
cells from infected versus uninfected animals likely reflects
the fact that CatL activity is also measured by this substrate,
Fig. 1. Enhanced presentation of OVA protein but not peptide by lung
DCs from influenza A virus-infected BALB/c mice. Lung DCs were
purified from BALB/c mice previously (30d) infected with influenza A
(HK331) or treated with NAF as a control. Lung DCs were used as
APCs to present either OVA protein or OVA peptide to OVA-specific
CD4+T cells from DO11.10 Rag2?/? mice. T cell proliferation was
assessed by the incorporation of [3H]Tdr. One of two independent
experiments with similar results is shown. *P < 0.05.
648 Influenza A up-regulates cathepsin B
by guest on June 6, 2013
and this activity is present in cells from both control and
We also investigated the changes in total CatB levels by
western blot, using an antibody that recognizes both proform
(inactive) of CatB and the mature, active form. The same
amount of protein from lung DCs from control and HK331-
infected mice was analyzed by SDS–PAGE, and CatB protein
was detected (Fig. 2C). We observed striking differences in
the relative levels of the mature and proforms of CatB. Mature
CatB protein was predominant in lung DCs from HK331-
infected mice, while the proform of CatB was predominant in
lung DCs from control-treated mice. We conclude that there
is an increase in the amount of active CatB in lung DCs
30 days after HK331 infection, and that this is likely due to
the turn over of inactive proform CatB to active CatB.
Both splenic and BM-derived murine DCs increase levels
of active CatB after exposure to UV-inactivated influenza A
virus in vitro
We were interested to determine whether in vitro exposure to
influenza virus affects CatB levels and activity in DCs acutely
or whether this effect requires longer term exposure of DCs
to the environment of the infected lung. To investigate this,
we exposed freshly isolated spleen cells or purified CD11c+
splenic DCs from BALB/c mice to influenza A virus. To avoid
cell lysis, which arises during influenza infection, we used
UV-inactivated influenza A or B viruses. Splenic DCs from
BALB/c mice were incubated with a UV-inactivated influenza
A virus strain that, like A/HK331, expresses H3N2 [c.a. A/
Sydney (H3N2); see Methods] at 1 pfu per cell or with UV-
inactivated influenza B virus (c.a. B/Harbin-like) at 1 pfu per
cell for 24 h in vitro. The DCs were harvested, and cell
lysates were incubated with the activity-based label DCG-04
before analysis by SDS–PAGE. The active polypeptide CatB
was visualized ;30 kDa and was dramatically increased
in influenza A virus-exposed spleen DCs, while influenza B
virus had only limited effects on CatB expression (Fig. 3A).
CatH, CatS and CatL were hardly affected by influenza A vi-
rus, but CatX was modestly increased. Thus, we observed
similar effects on these Cats in splenic DCs after acute ex-
posure to UV-inactivated influenza A virus to those seen after
influenza A infection in the whole animal, with up-regulation
Fig. 2. CatB is up-regulated in purified lung DCs derived from influenza A (HK331)-infected mice compared with control mice. (A) Cell lysates
from purified lung DCs of virus-free NAF control or influenza A virus (HK331)-inoculated mice were normalized for total protein content, labeled
with the biotinylated activity-based probe DCG-04, resolved by SDS–PAGE and papain-like cysteine proteases were visualized by streptavidin
HRP blot. b-Actin immunoblot was performed as a loading control. Data are representative of two experiments. (B) CatB/L activity was
determined at pH 5.0 by measuring the turn over of the fluorogenic substrate Z-FR-AMC in cell lysates from purified lung DCs of control and
influenza A virus (HK331)-inoculated mice. (C) CatB protein levels in cell lysates from purified DCs from indicated mice were assessed by
western blot with CatB-specific antibody. Purified recombinant CatB is used as a positive control.
Influenza A up-regulates cathepsin B 649
by guest on June 6, 2013
Fig. 3. CatB is up-regulated in both spleen and BM-derived DCs from BALB/c mice after exposure to UV-inactivated influenza A virus in vitro. (A)
Active papain family cysteine proteases were measured in cell lysates from purified splenic DCs exposed for 24 h to UV-inactivated influenza A
virus or mock-exposed cells, as a control. Cell lysates were incubated with activity-based cysteine protease probe, DCG-04, and comparable
650 Influenza A up-regulates cathepsin B
by guest on June 6, 2013
of CatB being especially notable. Interestingly, however,
when we exposed splenocytes to UV-inactivated influenza A
and stained with MHC class II-specific antibody, recognizing
I-A and I-E, and analyzed the MHC class II cell-surface ex-
pression of CD11c+gated cells by FACS, both influenza A-
exposed DCs and control DCs had comparable MHC class
II cell-surface expression (Fig. 3B), unlike DCs isolated from
control versus influenza A-exposed animals at 30d. This re-
sult suggests that influenza-related effects on class II ex-
pression in DCs require different kinetics or mediators than
effects on Cats.
DCs are divided into two subsets, mDC and pDC, and
can be distinguished by the co-expression of distinct cell-
surface markers.Murine mDC
CD11b molecules, whereas murine pDC expresses CD11c
and B220/CD45R. Next, we used FACS analysis to measure
CatB and CatD levels in both mDC and pDC. Splenic DCs
from BALB/c mice were exposed to UV-inactivated influenza
A for 24 h, stained with CD11c–FITC and CD45–TC and, af-
ter permeabilization, stained intracellularly with anti-CatB or
anti-CatD antibodies, which recognize both proform and ac-
tive form. B220/CD45R+and CD11clowcells represent the
pDCs, while CD45R?and CD11b/CD11chighcells are mDCs.
Figure 3(C) shows the histograms for CatB or CatD protein,
gated for pDCs and mDCs, as indicated. The solid histo-
gram shows the isotype control, the tinted histogram indi-
cates CatB or CatD levels in control splenic DCs and the
unfilled histogram denotes splenic DCs exposed to influenza
A. Control pDCs had less total CatB and CatD than influenza
A virus-exposed pDCs, and the control mDCs had less total
CatB and CatD than influenza A virus-exposed mDCs. The
extent of the increase in total CatD was substantially greater
than for CatB in both cell types. Thus, both mDCs and pDCs
appear to be comparably affected, and total CatB and CatD
levels are increased in both populations.
We also investigated the impact of exposure to UV-
inactivated influenza A virus on CatB levels and activity in
BM-derived DCs. BM cells from BALB/c mice were cultured
in the presence of rFlt3-L for 10 days. Sorted mDCs were ex-
posed to influenza A for 24 h. In Fig. 3(D), the left panel
shows labeling by the activity-based probe. Active CatB
was increased in influenza A virus-exposed cells and CatX
was possibly increased to a minor extent, but changes in
other Cats were not detected. The b-actin blot was used as a
loading control. We also detected an increase in CatB when
we performed intracellular staining with a CatB-specific anti-
body of control and influenza A virus-exposed mDCs and then
analyzed by FACS. Increased CatB protein was detected in
mDCs after influenza A virus exposure (unfilled histogram,
median 20.7) compared with control mDCs (tinted histogram,
median 11.7). The solid histogram represents the isotype
control (Fig. 3C, lower panel).
To quantify the increase in CatB activity in BM-derived
DCs after influenza A virus exposure, we used the fluoro-
genic substrate Z-FR-AMC (Fig. 3C, right panel). In addition,
we included the CatB-specific inhibitor CA074 (end concen-
tration 10 lM) in a separate reaction to assess the compo-
nent of the total enzyme activity attributable to CatB.
Increased substrate turn over was detected in cell lysates of
mDCs after influenza A virus exposure, reflecting increased
CatB activity. No substrate turn over was detected after ad-
dition of the CatB-specific CA074 inhibitor. These results
demonstrate that primary as well as in vitro generated
DCs can up-regulate CatB after exposure to UV-inactivated
influenza A virus.
The inhibitor, CA074-OMe, reduces T cell proliferation
Active-site labeling of CatB indicated active protease in influ-
enza A-exposed splenic DCs, but is an indirect measure of
functional activity. To corroborate those results with a func-
tional assay, we investigated processing and presentation of
OVA peptide (OVA323–339) by influenza-exposed and control
DCs and the generation of OVA323–339is known to require
CatB for processing from intact protein (29). APCs (T-
depleted spleen cells) from BALB/c mice were treated
in vitro with UV-inactivated influenza A or mock treated
for 24 h and then were incubated for 5 days with native
OVA as antigen. As another microbial stimulus, we also pre-
incubated some APCs with LPS. The response of I-Ad-
restricted, OVA-specific T cells purified from spleens of
DO11.10 Rag2?/? OVA-specific mice was measured in a T
cell proliferation assay. To directly test the role of CatB in
our assay, some APC/T cell/OVA protein cultures were simul-
taneously incubated with a cell permeable inhibitor CA074-
OMe, a non-cell permeable CatB-specific inhibitor (CA074)
or a CatG inhibitor as another control. CA074 [N-(L-3 trans-
an epoxy–peptide-based inhibitor that specifically inacti-
vates CatB, but cannot penetrate the cell membrane due
to its negatively charged carboxylate group. In contrast,
the methylated version, CA074-OMe, is inactive, but can
traverse the cell membrane. In the cytosol, CA074-OMe is
amounts of total protein from each were analyzed by SDS–PAGE. b-Actin immunoblot was performed as a loading control. (B) Splenocytes were
surface stained with antibody to MHC class II molecules that recognizes both I-A and I-E molecules and isotype control, as well as with anti-
CD11c antibody to detect DCs. Stained cells were analyzed by FACS. (C) Whole splenocytes were exposed to UV-inactivated influenza A virus
for 24 h, stained with CD45R–TC- and CD11c–FITC-conjugated antibodies, permeabilized and stained with anti-CatB–PE or anti-CatD–PE.
Stained cells were analyzed by FACS, with gating for pDCs (CD45R+and CD11c+) or mDCs (CD11c+high). Splenocytes exposed to UV-
inactivated influenza A virus are indicated by the unfilled histogram, control cells by the tinted histogram and the isotype control by the black
histogram. Data are representative of three independent experiments. (D) Upper left panel: BM-derived DCs were obtained from total BM from
BALB/c mice, and cultured with rFlt3-L for 10 days; cells were purified and exposed to UV-inactivated influenza A virus for 24 h. Control and BM-
derived DCs exposed to UV-inactivated influenza A virus were pre-incubated with the activity-based cysteine protease probe, DCG-04.Upper
right panel: equal amounts of cell lysate from control and UV-inactivated influenza A virus-exposed BM-derived DCs, as indicated, were
compared for their ability to turn over the fluorogenic substrate Z-FR-AMC. The CatB-specific inhibitor CA074 (10 lM) was used, as indicated, to
determine the component of enzyme activity attributable to CatB. Lower left panel: FACS analysis was performed after intracellular staining with
CatB-specific antibody. The unfilled histogram indicates BM-derived DCs exposed to UV-inactivated influenza A virus and tinted histogram
indicates control BM-derived DCs. The solid black histogram represents the isotype control.
Influenza A up-regulates cathepsin B651
by guest on June 6, 2013
CA074. However, because the intracellular conversion of
CA074-OMe to the CatB-specific inhibitor CA074 is slow, we
cannot rule out that other papain family cysteine proteases,
such as CatL, may also be inhibited by the OMe. OVA-
specific T cells were analyzed for their production of IFN-c
by intracellular staining followed by FACS analysis (Fig. 4A).
Consistent with prior results, an increased proportion of
T cells produced IFN-c production in the presence of DCs
treated with microbial stimuli and this increase was greater
for influenza A virus-treated compared with LPS-treated
DCs. In contrast, in presence of the cell permeable inhibitor
CA074-OMe, IFN-c production was reduced to background
level, indicating that functional CatB was expressed by
these cells and had access to the endocytosed OVA. Nei-
to generatethe CatB-specific inhibitor
ther the non-cell permeable CatB inhibitor CA074 nor a CatG
inhibitor affected T cell proliferation. In addition, Fig. 4(B)
(upper panel) shows that, while IFN-c production was de-
creased to background level using CA074-OMe and OVA
protein, IFN-c production was not decreased by using OVA
peptide (OVA323–339) and CA074-OMe. These results indi-
cate that CA074-OMe was not cytotoxic for T cells, but was
blocking an intracellular activity that is required for T cell
As a second assay to evaluate T cell responses to OVA
protein after exposure to influenza A, we evaluated Tcell pro-
liferation by dilution of CFSE label. Influenza A virus-treated
APCs were more effective in inducing proliferation of OVA-
specific T cells compared with untreated APCs (Fig. 4C). In
contrast, in the presence of CA074-OMe inhibitor, activation
Fig. 4. The cell permeable Cat inhibitor CA074-OMe blocks presentation of OVA protein by influenza A virus-exposed APCs. (A) Whole
splenocytes from BALB/c mice were exposed to UV-inactivated influenza A virus treated with LPS for 24 h or left untreated and then incubated
with OVA protein, with and without the cell permeable (CA074-OMe, 10 lM), the non-cell permeable (CA074, 10 lM) CatB-specific inhibitor or
a CatG-specific inhibitor for 5 days. IFN-c production of T cells (Thy1.2) from DO11.10 Rag2?/? OVA-specific mice was determined by
intracellular staining with IFN-c-specific antibody and FACS analysis. Cells were cultured without OVA as a control. (B) Splenocytes from BALB/c
mice were cultured with OVA peptide or OVA protein in the presence or absence of the inhibitor CA074-OMe (10 lM) (upper panel). IFN-c
production of T cells (Thy1.2) from DO11.10 Rag2?/? OVA-specific mice was determined by intracellular staining with IFN-c-specific antibody
and FACS analysis. (C) Tcell proliferation was determined by reduction of CFSE label. Data are representative of three independent experiments.
652 Influenza A up-regulates cathepsin B
by guest on June 6, 2013
of these cells was greatly decreased. Taken together, our
results show that papain family cysteine proteases (most
likely CatB) are functional in these cells, and indeed, are crit-
ical for the generation of the OVA323–339 epitope, as has
been observed for other APCs (29, 30).
DCs are critical APCs that bridge innate and adaptive im-
mune responses and uniquely prime naive T cells. In the
present study, we found the capacity to stimulate T cells by
lung DCs from influenza-infected mice was enhanced when
antigen was provided as intact protein but not peptide,
arguing for an effect of influenza infection on the antigen-
processing/presentation machinery of lung DCs. We therefore
investigated the influence of influenza A virus on Cat expres-
sion and activity in primary DCs. We found that (i) active CatB
was up-regulated in lung DCs of previously influenza A virus-
infected mice compared with lung DCs from uninfected
control mice and (ii) freshly isolated as well as in BM-derived,
cultured murine DCs also up-regulated active CatB after influ-
enza A virus exposure. Using a CatB inhibitor, we were able
to show that the CatB in influenza-exposed DCs was able
to access endocytosed OVA protein and was required for its
natural processing into the OVA323–339 peptide. Overall,
our functional studies of DCs exposed to influenza in vivo
(Fig. 1) and in vitro (Fig. 4) argue that increased active CatB
plays a role in the enhanced APC function of these cells.
However, our studies cannot definitively isolate the contribu-
tion of the increased amount of active CatB in the influenza-
treated APCs to their increased ability to stimulate T cells,
because multiple changes occur in these cells.
We also observed that the increased CatB activity is asso-
ciated with an increased conversion from proform to active
CatB, which is likely triggered by other proteases. However,
there are conflicting data in the literature regarding the pro-
cessing of the proform to the active form of CatB. In one re-
port, this processing event was inhibited when an aspartate
protease inhibitor pepstatin A was used, suggesting that the
aspartate protease CatD is a candidate for this function
(31). Different results were obtained by Hara et al. (32), who
used a metalloprotease inhibitor and proposed that a metal-
loprotease converted proform CatB to the active form. Yet
a third study used diverse inhibitors, such as inhibitors of
serine proteases (PMSF), aspartate proteases (pepstatin A)
and cysteine proteases (E64-d); only E64-d treatment had
an effect on the conversion from proform to active CatB
(33). We found here that the active form of CatB was sub-
stantially increased in influenza A virus-exposed DCs, CatX
was modestly increased and the level of several other active
forms of cysteine proteases were unchanged. In addition,
we observed increased total intracellular CatD expression
by FACS in splenic DCs after influenza A virus exposure. It
is possible that induction of CatD is necessary to convert
the proform to the active form of CatB. Alternatively, access
of CatD or other proteases, including those whose levels do
not change, to the proform of CatB is changed in affected
DCs, resulting in generation of mature, active CatB.
The carboxypeptidase CatX was previously determined to
be present in APCs, such as macrophages and DCs, and is
therefore hypothesized to be involved in antigen processing
(28). Recent findings also suggest that CatX plays a role in
T cell activation via regulation of the b2-integrin leukocyte
function-associated antigen-1 receptor (28, 34). We found
that CatX activity was modestly increased in several types
of DCs exposed to influenza A virus (Figs. 2A, 3A and C).
Kos et al. (35) found that the total level of CatX was un-
changed after maturation of human monocyte-derived DCs
stimulated with tumor necrosis factor-a, but did not measure
levels of active enzyme. In addition to this difference, both
the target DC subtype and the maturation stimuli are differ-
ent in the system of Kos et al. versus ours; a critical variable
may be that influenza A virus stimulates a cytokine milieu
that includes high levels of IFN-a. Thus, along with its func-
tion, the regulation of CatX upon DC maturation will be of in-
terest to study further in various DC subsets.
It is interesting that influenza-mediated changes in the cys-
teine Cats are relatively selective for CatB. Active CatB can
mediate destruction of perforin (36). After stimulation by anti-
genic peptide presented on MHC class I molecules, cytotoxic
T cells up-regulate surface CatB, which has been proposed
as a mechanism of protection against self-destruction by per-
forin (36). Peptide antigen-loaded DCs have been shown to
be subject to CTL-mediated elimination after they have initi-
ated an immune response in a murine model system (37).
We reasoned that virus-exposed DCs might up-regulate sur-
face CatB to bypass destruction by CTL. We looked at the
cell-surface expression of CatB in control and influenza A
virus-exposed splenic DCs, and detected CatB on the cell
surface of pDCs as well as mDCs by FACS analysis. How-
ever, we did not observe any difference in cell-surface CatB
expression after influenza A virus exposure on either pDCs
or mDCs (data not shown). We also failed to detect active
cysteine proteases in the supernatants from co-cultures of
influenza A virus-exposed DCs with antigen and T cells (data
not shown), suggesting that CatB was not secreted.
In addition, it was previously reported that CatB plays
a crucial role in processing of the hepatitis B core antigen
(38). Thus, we also considered the possibility that CatB is in-
duced in order to degrade a critical influenza antigen, such
as HA, one of the immunodominant influenza proteins. We
exposed purified HA to B lymphoblastoid cell-derived lyso-
somal proteases and isolated human CatB in vitro and found
that while lysosomal proteases can degrade HA, isolated hu-
man CatB alone is not sufficient (data not shown). The possi-
bility that increased CatB improves the efficiency of HA
processing remains to be explored.
The TLRs expressed by DCs allow for recognition of for-
eign viral and bacterial antigens (39, 40). Blander et al. (41)
show that TLR signaling might regulate Cat activity, as in-
creased Ii (p31) processing takes place in DC phagosomes
exposed to microbial TLR ligands, but not phagosomes en-
countering antigen without TLR ligands. We observe that
CatB activity is up-regulated after influenza A virus infection,
and this change in CatB activity is also induced by UV-
inactivated influenza A virus. This finding suggests the pos-
sible involvement of TLR signaling in our results. A candi-
date pathway is TLR7 recognition of ssRNA (3). An
alternative, but not mutually exclusive, possibility is that HA
protein may activate DCs, as we noted a difference in the
Influenza A up-regulates cathepsin B653
by guest on June 6, 2013
effects of influenza A and B strains in vitro, and there is a re-
cent evidence for HA-mediated initiation of innate activation
in B cells via a MyD88-dependent pathway that appears to
use a novel receptor (42). Another viral protein, human pap-
illoma virus type 16 E7, has been shown to increase CatB
activity, resulting in induction of apoptosis (43).
In the setting of in vivo infection with influenza A virus, an-
other possible contributor to the alterations in CatB is IFN-c.
It is known that lung infection with influenza A results in high
local IFN-c produced by CD4+T cells (5), and, more acutely,
IFN-c is produced by NK cells (44). In our in vivo system,
using IFN-c-deficient mice or IFN-c neutralization, we were
unable to observe influenza A virus-enhanced DC function,
thus demonstrating an important role for IFN-c in the cyto-
kine milieu that mediates the persistent effects on lung DCs
(5). It has been shown that IFN-c and LPS stimulate in-
creased CatB activity in human monocyte-derived DCs (17).
We found that the cell permeable inhibitor CA074-OMe re-
duced DO11.10 T cell proliferation. These results highlight
the importance of papain family of cysteine proteases, most
likely CatB, and not aspartate or serine proteases in the
generation of OVA323–339 peptide, the ligand required to
stimulate these T cells. In contrast, the non-cell permeable
CatB-specific inhibitor CA074 did not effect T cell stimulation
in our system. Mizuochi et al. (29) found that CA074 de-
creased IL-2 production by T cells after APCs were pulsed
with OVA protein, but not with OVA323–339peptide. However,
Mizuochi et al. (29) used a B cell lymphoma as APC,
whereas we have used primary DC, which may account for
this discrepancy. It has also been reported that the presence
of CA074 during stimulation with hepatitis B, Leishmania
major antigen or OVA induces a switch of cytokine produc-
tion from TH2 to TH1 type cytokines (30, 45, 46). This seems
unlikely to account for our results, however, as we observed
a striking block in proliferation of naive T cells by using
CA074-OMe. Last, CA074-OMe was shown to reduce cell
death in oral squamous carcinoma cells (47), consistent with
our finding that CA074-OMe is not directly toxic to cells.
In conclusion, our findings indicate that influenza A virus
increases CatB activity in freshly isolated DCs and suggest
that altering this antigen-processing machinery may be a key
effect of encounter with influenza A virus by these cells. As
this effect appears to be relatively long-lived in lung DCs, it
may contribute to enhanced processing and presentation of
other antigens, including allergens, after influenza infection.
This work was supported by the National Institutes of Health (NIH)
U54 AI057229 (to E.D.M.), the Deutsche Forschungsgemeinschaft
BU 1822/1-1 and BU1822/3-1 (to T.B.), the Stanford University
Immunology Training grant AI07290-19 (to M.E.D.), the American
Lung Association grant RT-017-N (to M.E.D.) and the NIH National
RR020843 (to M.B.). We thank P. Doherty (University of Melbourne,
Victoria, Australia) for providing HK331 influenza A virus and control
allantoic fluid and MedImmune Vaccines for the c.a. influenza strains
and control allantoic fluid.
carboxyfluoresceine diacetate succinimidyl ester
class II-associated invariant chain peptide
magnetic cell sorting
myeloid dendritic cell
normal allantoic fluid
National Institutes of Health
plasmacytoid dendritic cell
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