Infection with Mycobacterium avium subsp. paratuberculosis Results in
Rapid Interleukin-1? Release and Macrophage Transepithelial
Elise A. Lamont,aScott M. O’Grady,cWilliam C. Davis,dTorsten Eckstein,eand Srinand Sreevatsana,b
Department of Veterinary Population Medicine,aDepartment of Veterinary Biomedical Science,band Department of Animal Science,cUniversity of Minnesota, St. Paul,
Minnesota, USA; Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, USAd; and Department of Microbiology,
Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USAe
Pathogen processing by the intestinal epithelium involves a dynamic innate immune response initiated by pathogen-
epithelial cell cross talk. Interactions between epithelium and Mycobacterium avium subsp. paratuberculosis have not
been intensively studied, and it is currently unknown how the bacterium-epithelial cell cross talk contributes to the course
of infection. We hypothesized that M. avium subsp. paratuberculosis harnesses host responses to recruit macrophages to
the site of infection to ensure its survival and dissemination. We investigated macrophage recruitment in response to M.
avium subsp. paratuberculosis using a MAC-T bovine macrophage coculture system. We show that M. avium subsp. para-
tuberculosis infection led to phagosome acidification within bovine epithelial (MAC-T) cells as early as 10 min, which re-
sulted in upregulation of interleukin-1? (IL-1?) at transcript and protein levels. Within 10 min of infection, macrophages
were recruited to the apical side of MAC-T cells. Inhibition of phagosome acidification or IL-1? abrogated this response,
while MCP-1/CCL-2 blocking had no effect. IL-1? processing was dependent upon Ca2?uptake from the extracellular me-
dium and intracellular Ca2?oscillations, as determined by EGTA and BAPTA-AM [1,2-bis(2-aminophenoxy) ethane-
N,N,N=,N=-tetraacetic acid tetrakis (acetoxymethyl ester)] treatments. Thus, M. avium subsp. paratuberculosis is an oppor-
tunist that takes advantage of extracellular Ca2?-dependent phagosome acidification and IL-1? processing in order to
efficiently transverse the epithelium and enter its niche—the macrophage.
sal microorganisms that function together as a unique but non-
separable part of the host (22). Intestinal pathogens must over-
come several mechanisms employed by the epithelium, such as
the glycocalyx, to establish and promote their survival within the
host (14, 15, 53). Therefore, intestinal pathogens have developed
several strategies to circumvent, stun, and even manipulate the
The initial interaction between intestinal pathogens and the
epithelia sets the stage for ensuing infections and may determine
success, as defined by establishment, survival, and dissemination.
actions with Mycobacterium avium subsp. paratuberculosis, the
cobacterium avium subsp. paratuberculosis is thought to mainly
interface with macrophages despite the fact that the natural route
of infection occurs via the intestinal tract (10). Like other intra-
var Typhimurium, and Shigella spp., M. avium subsp. paratuber-
culosis infects M cells due to their lack of hydrolytic enzymes and
ever, further investigations have revealed that M. avium subsp.
goes epithelium processing and subsequent infiltration into the
Peyer’s patches (7, 43, 45). Studies by Patel et al. have shown that
processing of M. avium subsp. paratuberculosis by bovine mam-
mary epithelial cells (MAC-T cells) results in enhanced phagocy-
he intestinal epithelium is the largest surface area on humans
and animals and acts as a primary barrier against pathogens
surrogate for intestinal epithelia, increases expression of an oxi-
doreductase gene (MAP3464) to regulate the Cdc42 pathway (2).
The Cdc42 pathway is also initiated by other intracellular patho-
gens to form filopodia and consequent cytoskeleton rearrange-
ment (26, 33, 58). Research utilizing the closely related bacterium
Mycobacterium avium subsp. avium indicates that intestinal my-
cobacteria have developed several mechanisms, such as upregula-
to invade epithelium cells by a proposed regulatory role in GTP
binding and modulation of bacterial cell wall structure (32). We
have recently shown that M. avium subsp. paratuberculosis tran-
naturally infected cattle have distinct profiles in comparison to
those obtained from bovine macrophage infection (20). This di-
chotomy may be due to the various cell types present within the
ileum and mesenteric lymph node, which further highlights the
Received 16 December 2011 Returned for modification 1 February 2012
Accepted 27 June 2012
Published ahead of print 9 July 2012
Editor: J. L. Flynn
Address correspondence to Srinand Sreevatsan, email@example.com.
Supplemental material for this article may be found at http://iai.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
The authors have paid a fee to allow immediate free access to this article.
September 2012 Volume 80 Number 9 Infection and Immunityp. 3225–3235iai.asm.org
importance of cell-cell cross talk during natural infection. It is
likely that M. avium subsp. paratuberculosis encountered by lam-
ina propria macrophages is significantly altered from its original
state due to epithelium processing. Therefore, current research
initiatives solely utilizing M. avium subsp. paratuberculosis from
broth culture to infect bovine macrophages may not capture the
full complexity of host-pathogen interactions and may therefore
have limited utility in deciphering mycobacterial pathogenesis.
In the present study, we developed a MAC-T–bovine mono-
cyte-derived macrophage (MDM) coculture model to investigate
innate immune responses to M. avium subsp. paratuberculosis in-
leukin-1? (IL-1?). IL-1? production was determined to be criti-
cal for recruitment of MDMs to the site of M. avium subsp.
paratuberculosis infection. Phagosome acidification and IL-1?
transcript and protein levels were dependent upon an extracellu-
losis takes advantage of phagosome acidification enlistment of
IL-1? at the site of infection in order to efficiently transverse the
epithelium and arrive at its final niche—the macrophage.
MATERIALS AND METHODS
Ethics statement. All animal work was conducted in accordance with the
recommendations in the institutional guidelines and approved animal
care and use committee (IACUC) protocols at the University of Minne-
sota (approval number 1106A01161). All other experiments were carried
out in accordance with the University of Minnesota’s Institutional Bio-
safety Committee (IBC) approved protocol number 0806H36901.
Bacterial culture. M. avium subsp. paratuberculosis strains K-10 and
K-10(pWes4) expressing green fluorescent protein (GFP) were main-
tained in Middlebrook (MB) 7H9 medium containing 10% glycerol, 1%
(optical density at 600 nm [OD600] ? 1.0).
Mammalian cell culture. Monocyte-derived macrophages (MDMs)
from two JD-free dairy cows (253 and 2170) were elutriated and matured
as described previously (13, 19). Briefly, blood was collected from the
jugular vein into a gas-sterilized vacuum container (Paragon Medical,
Pierceton, IN) containing an equal volume of acid-citrate dextrose to
inhibit coagulation. Blood was divided into 40-ml aliquots and centri-
fuged for 20 min at 2,200 rpm at room temperature. Buffy coats were
collected, resuspended in 1? Dulbecco’s phosphate-buffered saline (D-
PBS), and layered on a 58% Percoll gradient (Sigma-Aldrich, St. Louis,
serum at 37°C in a humidified chamber (5% CO2) for 4 days. Cells were
later seeded for coculture experiments. Bovine mammary epithelial cells
(MAC-T) were maintained in Dulbecco’s modified Eagle medium
(DMEM) containing 10% fetal bovine serum (FBS) at 37°C in a humidi-
fied chamber (5% CO2).
Conjugation of mycobacterial cell wall lipoglycans to fluorescent
polystyrene beads. M. avium subsp. paratuberculosis-specific mannosy-
lated lipoarabinomannan (ManLAM) was obtained from Eckstein (Col-
orado State University) (5). The following reagents were obtained
Repository, NIAID, NIH: Mycobacterium tuberculosis strain H37Rv puri-
fied lipomannan (LM) (NR-14850) and Mycobacterium smegmatis puri-
fied non-mannose-capped lipoarabinomannan (AraLAM) (NR-14849).
Lipoglycans were purified using a previously reported method (5). Ap-
with 50 mM 2-(N-morpholino) ethanesulfonic acid hydrate, 4-morpho-
linoethanesulfonic acid (MES hydrate) buffer (pH 6.0) (Sigma-Aldrich,
St. Louis, MO), and 200 ?l of an aqueous suspension of fluorescein iso-
thiocyanate (FITC)-labeled 0.1-?m carboxylate-modified microspheres
(Sigma-Aldrich, St. Louis, MO) and incubated for 15 min at room tem-
perature. Forty mg of 1-ethyl-3-(3-dimethylaminopropyl carbodiimide)
(EADC) (Invitrogen, Carlsbad, CA) was added to the microsphere sus-
pension, and the pH was adjusted to 6.5 using 0.1 N sodium hydroxide
(NaOH). The reaction mixture was placed on a rocker and incubated
overnight at room temperature. Lipoglycan-microsphere suspensions
were quenched with 100 mM glycine, incubated for 30 min at room tem-
bound microspheres were resuspended in 5.0 ml of 50 mM PBS contain-
ing 1% BSA and 2 mM sodium azide and stored at 4°C until use in inva-
by heating microspheres to 95°C for 10 min and subjecting the superna-
tants to Western blot analysis using a rabbit polyclonal antibody against
whole-cell lysates of M. avium subsp. paratuberculosis (data not shown).
MAC-T–macrophage coculture and M. avium subsp. paratubercu-
losis invasion assay. Approximately 2.0 ? 104MAC-T cells were seeded
onto the apical side of a 3.0-?m-pore-size Snapwell insert (Transwell
permeable support; Corning, Lowell, MA) and incubated for 4 days in
5% CO2. Once semiconfluence was reached, the Snapwell insert was in-
inverted again to its original orientation, such that MAC-T cells and
MDMs were located on the apical and basolateral sides of the Snapwell
insert, respectively. Transwells were examined by phase-contrast micros-
copy to confirm macrophage adherence.
Subcultured M. avium subsp. paratuberculosis (either K-10 or GFP-
expressing K-10) was grown to an OD600of 0.5 (equivalent to 1.0 ? 106
cells/ml) and assessed for the number of live and dead cells using a
Baclight kit (Invitrogen, Carlsbad, CA) based on the manufacturer’s in-
culture was pelleted at 3,000 rpm for 10 min and washed three times in
sterile PBS. The pellet was resuspended, vortexed for 5 min, and repeat-
edly drawn through a sterile 21-gauge needle in DMEM containing 10%
FBS such that a 10:1 multiplicity of infection (MOI) was achieved. The
10:1 MOI reflected over 90% live M. avium subsp. paratuberculosis cells.
room temperature to sediment any bacterial clumps, and the upper two-
thirds of the resuspended culture was used for the invasion assay. M.
avium subsp. paratuberculosis was applied to the apical chamber and al-
ber containing 5% CO2followed by three washings with 1? D-PBS to
120 min postinfection (p.i.). The coculture was later washed using 1?
D-PBS and further processed for RNA extraction, confocal imaging, or
Blocking assays. The following reagents were purchased for use in
blocking assays: bafilomycin A1 (Sigma-Aldrich, St. Louis, MO), bovine 9.1
IL-1? blocking antibody (provided by William C. Davis), recombinant bo-
vine IL-1? (Thermo Scientific, Rockford, IL), human monocytic chemoat-
tractant protein-1/CCL-2 (MCP-1) (EMD Chemicals, Gibbstown, NJ), and
MCP-1/CCL-2blockingantibody(NovusBiologicals,Littleton,CO). In or-
der to access the role of phagosome acidification and IL-1? production,
the coculture infection assay was conducted as previously stipulated with
the exception of a preincubation period with either bafilomycin A1 (25
nM), 9.1 IL-1? blocking antibody (250 ng/ml or 500 ng/ml), or MCP-1/
CCL-2-blocking antibody (250 ng/ml or 500 ng/ml) for 1 h at 37°C in a
humidified chamber containing 5% CO2followed by three 1? D-PBS
(LPS; 1.0 ?g/ml) (Sigma-Aldrich, St. Louis, MO) control was included
Lamont et al.
iai.asm.orgInfection and Immunity
each coculture well in a separate bafilomycin A1 experiment to rescue
MDM recruitment. MCP-1/CCL-2 (5.0 ng/ml) was added to uninfected
cocultures to serve as a positive control and comparison for M. avium
subsp. paratuberculosis invasion and blocking assays. All blocking assays
were conducted three separate times, and the assay at each p.i. time point
was performed in triplicate.
Calcium signaling assays. The following reagents were purchased for
calcium signaling assays: 1,2-bis(2-aminophenoxy)ethane-N,N,N=,N=-
tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM) (Sigma-
Aldrich, St. Louis, MO), ethyleneglycol bis (aminoethylether) tetraacetic
acid (EGTA) (MP Biomedicals, Solon, OH), calcium-free DMEM (Invit-
rogen, Carlsbad, CA), and UTP (Sigma-Aldrich, St. Louis, MO). The
MAC-T–macrophage coculture invasion assay was conducted as stated
above, with the addition of either DMEM containing BAPTA-AM (25
nM) or EGTA (4 mM)-treated calcium-free DMEM alone or with UTP
(25 ?M) at p.i. time points. All calcium signaling assays were conducted
RNA extraction and quantitative real-time PCR. Upon completion
of p.i. time points, MAC-T cells were washed three times in 1? D-PBS,
incubated for 5 min at room temperature to ensure successful lysing. All
RNA work was conducted on RNase Away (Molecular Bioproducts, San
Diego, CA)-treated work surfaces. RNA was extracted per the manufac-
turer’s instructions (Invitrogen, Carlsbad, CA) and was later treated with
Turbo DNase (Ambion, Austin, TX) at 37°C for 30 min. The RNA reac-
tion was inactivated using phenol-chloroform for 2 min at room temper-
ature. RNA purity was assessed by measuring the 260/280 ratio obtained
by NanoDrop ND-1000 (NanoDrop Products, Wilmington, DE) and the
absence of amplification of the ?-actin gene with direct PCR. RNA used
tifast SYBR green one-step qRT-PCR kit (Qiagen, Valencia, CA) and a
Roche Light cycler 480II (Roche NimbleGen Inc., Madison, WI) with
corresponding software. The following program was used: 50°C for 10
were designed using Primer 3 (http://frodo.wi.mit.edu/primer3/). Fold
change was calculated using the ??CTmethod and the value for the
MAC-T cells. Products were examined on a 2% agarose gel. All samples
culosis invasion assays were collected at 10 and 30 min p.i., filtered with a
0.2-?m Millex syringe-driven filter unit (Millipore, Billerica, MA), and
concentrated using a Speedvac. The following controls were included in
Western blot analysis: human pro-IL-1? (Sino Biological Inc., Beijing,
China), mouse IL-1? (Abcam, Cambridge, MA), and bovine IL-1?
with 5.0 ?l of Laemmli buffer (Bio-Rad, Hercules, CA), denatured at
95°C, and loaded onto a Precise 4-to-20% Tris-HEPES gradient precast
trophoretically transferred onto a 0.2-?m-pore-size nitrocellulose mem-
brane (Bio-Rad, Hercules, CA) for 2 h (60 V) at 4°C. The membrane was
blocked overnight in 5% nonfat dried milk in 1? Tris-Tween 20 (Tris-
T20; 0.1% Tween 20 [vol/vol]) buffer at 4°C. Next, the membrane was
with a 1:1,000 dilution of anti-rabbit IL-1? polyclonal antibody (Abcam,
Cambridge, MA) with shaking at room temperature and further washed
as previously described. The membrane was incubated with a 1:10,000
dilution of horseradish peroxidase (HRP; R&D Systems, Minneapolis,
ning Ultra kit (PerkinElmer, Waltham, MA) per the manufacturer’s in-
sition from LabWorks 4.6 software (LabWorks Inc., Costa Mesa, CA).
Enzyme-linked immunosorbent assay (ELISA). Cell supernatants
from both M. avium subsp. paratuberculosis invasion assays and LPS (1.0
?g/ml; control) stimulation at 10 and 30 min p.i. were analyzed for pro-
IL-1? levels using a mouse IL-1? proform ELISA Ready-SET-Go! kit
dard curve was included using 2-fold dilutions of mouse pro-IL-1?. The
optical density was read at 450 nm with a wavelength correction of 570
nm. All samples were loaded into triplicate wells. ELISA was repeated
sis invasion and blocking assays with and without calcium were saved in
1.0-ml aliquots, filtered with 0.2-?m Millex syringe-driven filter units
(Millipore, Billerica, MA), dried via Speedvac, and resuspended in 250 ?l
of 1? D-PBS without calcium and magnesium. One ?l of each superna-
tant was spotted in duplicate technical replicates on a 0.45-?m-pore-size
nitrocellulose membrane using the Minifold I dot blot apparatus (GE
Healthcare, North America) per the manufacturer’s instructions. The ni-
trocellulose membrane was blocked in 5% nonfat dried milk in 1? PBS-
Tween 20 (PBS-T20; 0.1% T20 [vol/vol]) for 2 h at room temperature
with subtle shaking, washed five times in 1? PBS-T20 at 5-min intervals,
and incubated with rabbit anti-bovine IL-1? polyclonal antibody (AbD
Serotec, Raleigh, NC) for 1 h at room temperature. Next, the membrane
The nitrocellulose membrane was developed using a Western Lightning
Ultra kit (PerkinElmer, Waltham, MA) per the manufacturer’s instruc-
tions and imaged under Simple_Biochemi_Acquisition from LabWorks
4.6 software (LabWorks Inc., Costa Mesa, CA). Raw density values were
collected and converted to ng/ml. Concentrations were calculated based
on the concentration curve for recombinant bovine IL-1? protein
(Thermo Scientific, Rockford, IL).
Lactate dehydrogenase cytotoxicity assay. A lactate dehydrogenase
avium subsp. paratuberculosis as stipulated by the manufacturer (Clon-
tech, Mountain View, CA). Briefly, 100 ?l containing 4.0 ? 105MAC-T
microtiter plate and allowed to adhere overnight at 37°C in a humidified
chamber containing 5% CO2. Medium was removed, and all cells were
washed three times with 1? D-PBS to remove spontaneously released
LDH. M. avium subsp. paratuberculosis invasion of MAC-T cells was per-
formed as described above with the exception of 1% BSA in placement of
10% FBS. The following controls were included: background (medium
of LDH), high LDH release (2% Triton X-100), M. avium subsp. paratu-
nm with an applied correction at 600 nm. Optical density readings were
converted to LDH microunits based on a generated standard curve. As-
says at all time points were conducted in triplicate. The LDH assay was
repeated a total of three times.
Cell staining and confocal microscopy. All cells were allowed to ad-
here to glass coverslips (no. 1.5 thickness) in 24 well plates. Phagosome
acidification staining was based in part on a previously reported protocol
(25). Upon the final 30 min after infection of MAC-T cells, 25 nM Lyso-
TABLE 1 Primers used in this study
Gene product and direction Sequence (5=–3=)
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Tracker blue (Invitrogen, Carlsbad, CA) was added to culture medium
and incubated at 37°C in a humidified chamber with 5% CO2. After the
designated p.i. time point was completed, culture medium was decanted
in prewarmed (37°C) Deep Red CellMask plasma membrane stain (2.5
?g/ml) (Invitrogen, Carlsbad, CA) for 5 min and washed three times in
absolute methanol for 5 min at ?20°C followed by two washes with ice-
cold 1? D-PBS. In a separate experiment examining Rab7 expression,
in PBS containing 0.01% bovine serum albumin (BSA), permeabilized in
ice cold methanol at ?20°C for 5 min, and blocked with PBS containing
with 1:500 anti-mouse Rab7 monoclonal antibody (Abcam, Cambridge,
MA) for 1 h at 37°C in a humidified chamber containing 5% CO2, rinsed
three times in PBS, and stained with 1:1,000 Alexa Fluor 405 goat anti-
(Invitrogen, Carlsbad, CA) and sealed with nail polish. All slides were
stored at 4°C until confocal imaging.
Coculture Transwells were preprocessed by a method similar to the
Rab7 staining in MAC-T cells. Transwells were incubated with a 1:500
dilution of rabbit anti-bovine CD11b (Abcam, Cambridge, MA) and
mouse anti-bovine cytokeratin (Abcam, Cambridge, MA) for 2 h at 37°C
in a humidified chamber (5% CO2). Subsequently, cells were washed
three times with 1? D-PBS, incubated with 1:1,000 dilutions of Alexa
Fluor 405 goat anti-mouse IgG and Alexa Fluor 680 donkey anti-rabbit
IgG (Invitrogen, Carlsbad, CA) for 1 h at room temperature in the dark,
the support circumference with an 18.5-gauge needle, mounted on glass
coverslips, and stored as described above.
macrophages. Macrophages were incubated in RPMI 1640 containing
humidified chamber (5% CO2) to remove extracellular bacteria and sub-
sequently stained and fixed using the CellMask protocol described above.
The following lasers were used to visualize cells: Alexa Fluor 405, FITC,
Cy5, and/or DAPI (4=,6-diamidino-2-phenylindole). A Z series for each
slide was collected in 1.0-?m steps and stacked to render a complete
image. Three fields per slide were visualized.
and washed three times using sterile PBS. The pelleted cells were resus-
pended in 500 ?l of PBS, blocked using 1? PBS containing 1% BSA for 1
h on ice, and immediately washed three times with PBS. Cells were incu-
bated separately with 1:500 dilutions of phycoerythrin (PE)-conjugated
anti-human CD14 (R&D Systems, Minneapolis, MN) and anti-bovine
CD11b-FITC (Raybiotech Inc., Norcross, GA) primary antibodies or ap-
propriate isotype controls (R&D Systems, Minneapolis, MN) for 1 h on
ice and washed three times with PBS between stainings. Flow-cytometric
analysis was conducted using a FACSCanto equipped with FACSDiva
software (BD Biosciences, San Jose, CA). Bovine MDMs were defined as
CD11bhi(FITChi) and CD14hi(PEhi). Cell population was recorded at
10,000 events. Experiments were conducted three times using triplicate
biological samples (per p.i. time point).
Graphs and statistical analyses. Percent colocalization of Lyso-
Tracker staining and FITC was examined and calculated based on 20
randomly selected fields using the overlapping coefficient in Fluoview
graphs were generated using GraphPad Prism software (GraphPad Soft-
ware, La Jolla, CA). Means and standard errors of the means (SEM) were
calculated. qRT-PCR and dot blot results were analyzed using a two-way
than 0.01 were considered statistically significant.
M. avium subsp. paratuberculosis phagosome acidification
leads to interleukin-1? secretion at the epithelial interface not
epithelium-M. avium subsp. paratuberculosis interaction, we in-
fected MAC-T cells with a GFP-expressing strain of M. avium
subsp. paratuberculosis K-10 and imaged the cells using confocal
microscopy. M. avium subsp. paratuberculosis K-10 infection re-
sulted in early phagosome acidification within 10 min p.i. and
reached peak LysoTracker fluorescence intensity by 30 min
(Fig. 1A). This is in stark contrast to ManLAM, a mycobacterial
cell wall lipoglycan responsible for phagosome maturation arrest,
which did not display LysoTracker staining throughout the infec-
tion (see Fig. S2 in the supplemental material) (9). Phagosome
acidification was sustained in AraLAM-, LM-, and carboxylate-
culosis-guided phagosome acidification dissipated by 1 h p.i. (see
Fig. S2A and B). Phagosome acidification was validated by stain-
ing for the late endosomal marker Rab7 during the infection pro-
bacteria have developed several strategies to shut off the
acidification process (9, 21, 47, 54, 55, 56). It is important to note
that these previous studies were conducted with macrophages;
therefore, a different cell type, like the epithelium, may function
differently in response to mycobacteria. We found that IL-1?
pared to uninfected controls by 10 min p.i., which corresponded
to M. avium subsp. paratuberculosis-induced phagosome acidifi-
cation (Fig. 1C). IL-1? transcript levels were validated for activa-
tion by Western blot analysis and the absence of pro-IL-1? (see
munoblotting. MAC-T cells show maximum IL-1? production
(?200 ng/ml) by 30 min p.i. that also coincides with peak Lyso-
Tracker intensity (Fig. 1D). Although IL-1? has been reported to
be an integral component for host defense, it may also serve as a
chemoattractant for macrophages and has previously been iden-
tified to be an integral cytokine in granuloma formation during
mycobacteriosis (29, 35, 57). Therefore, M. avium subsp. paratu-
FIG1 M. avium subsp. paratuberculosis (MAP) induces phagosome acidification and IL-1? processing at the epithelium interface. (A) Confocal microscopy of
phagosome acidification in MAC-T cells. MAC-T cells were assessed for phagosome acidification at 10, 30, and 60 min after infection with M. avium subsp.
paratuberculosis using LysoTracker blue. Approximately 60% of MAC-T cells contained M. avium subsp. paratuberculosis. M. avium subsp. paratuberculosis
positive for Rab7. (C) qRT-PCR of uninfected and infected MAC-T cells at 10 min postinfection. In stark contrast to uninfected cells, M. avium subsp.
cells reached peak IL-1? expression at 30 min postinfection. **, P ? 0.01; ***, P ? 0.001.
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recruit macrophages to the site of infection to ensure its survival
Phagosome acidification and IL-1? production are neces-
sary for macrophage transepithelial migration and M. avium
subsp. paratuberculosis escape from the epithelium. We next
investigated macrophage recruitment during M. avium subsp.
paratuberculosis infection in a MAC-T–bovine MDM coculture
system. MAC-T cells and MDMs adhered to the apical and baso-
lateral sides of Transwells, respectively. If MDM recruitment to
the site of infection was induced by M. avium subsp. paratubercu-
losis, MDMs should permeate the Transwell pores and reside
within the apical chamber of the coculture system (Fig. 2). MDM
recruitment was assessed by flow cytometry of apical-chamber
supernatants. M. avium subsp. paratuberculosis infection in
MAC-T cells led to recruitment of MDMs (5,000 cells/10,000 re-
corded events) into the apical chamber within 10 min (Fig. 2A, B,
cal chamber by 120 min and was comparable to extravasation in
the MCP-1 control treatment (Fig. 2A). Confocal microscopy
confirmed M. avium subsp. paratuberculosis escape from MAC-T
cells and phagocytosis into MDMs during the migration process
(Fig. 2B and C). Next, we sought to determine the role of phago-
some acidification in the M. avium subsp. paratuberculosis pro-
moted macrophage transepithelial migration. We pretreated
MAC-T cells prior to M. avium subsp. paratuberculosis infection
with bafilomycin A1, an established inhibitor of vacuolar
A1 treatment resulted in a complete abrogation of MDM recruit-
ment (Fig. 2A). Furthermore, bafilomycin A1 treatment pre-
vented the production of IL-1? and was comparable to transcript
levels in uninfected MAC-T cells (Fig. 2D). Since the production
of IL-1? may be critical to macrophage transepithelial migration,
we supplemented the bafilomycin A1 treatment with the exact
concentration of IL-1? secreted during normal infection. Addi-
tion of IL-1? restored MDM migration to the apical chamber in
cell numbers comparable to those seen with M. avium subsp.
paratuberculosis infection alone (Fig. 2A). Furthermore, inhibi-
tion of IL-1? by a blocking antibody abrogated macrophage re-
cruitment (Fig. 2A and E). Despite the apparent macrophage re-
cruitment reliance on IL-1?, an alternative may be that another
chemoattractant induced IL-1? production and is actually re-
sponsible for transepithelial migration. For example, Gavrilin et
al. have shown that MCP-1 upregulates IL-1? expression in
monocytes (17). Therefore, we included an MCP-1-blocking an-
in the coculture system. MCP-1 did not affect macrophage re-
cruitment or IL-1? transcript and protein levels (Fig. 2A, E, And
effect. In other words, the potential remained that IL-1? was a
result of M. avium subsp. paratuberculosis-induced cytotoxicity,
which may have led to the destruction of the MAC-T monolayer
integrity and removed a barrier for the macrophages to cross. In
subsp. paratuberculosis infection, we analyzed cell culture super-
natants 10 and 30 min p.i. In contrast to the results obtained with
treatment with 2% Triton X-100, lactate dehydrogenase (LDH)
release in M. avium subsp. paratuberculosis-infected cells was not
elevated compared to the uninfected control (see Fig. S4B in the
supplemental material). LDH data determined that IL-1? pres-
ence in cell culture supernatants was not a result of cell content
release due to necrosis but is indicative of secretion from viable
MAC-T cells. Taken together, these data indicate that phagosome
acidification promotes IL-1? expression, which is the critical
component leading to macrophage transepithelial migration.
Phagosome acidification and IL-1? expression are depen-
into the lysosome requires calcium oscillations; therefore, we
migration is also dependent upon calcium signaling (11). We
intracellular and extracellular sources, during M. avium subsp.
paratuberculosis infection. BAPTA-AM treatment abolished both
phagosome acidification and upregulation of IL-1? (Fig. 3A and
B). Removal of calcium from the extracellular environment using
EGTA-treated calcium-free medium also blocked phagosome
acidification and IL-1? expression (Fig. 3B, C, And D). Further-
more, UTP supplementation restored phagosome acidification in
the calcium-free EGTA medium treatment, which indicates that
intracellular stores were still functional (Fig. 3E). Thus, M. avium
subsp. paratuberculosis preferentially utilizes an extracellular cal-
The intestinal epithelium serves as a gatekeeper that allows for
nutrient absorption and luminal sampling but blocks microbial
(both commensal and pathogen) passage (40). Therefore, intra-
have developed exquisite and complex mechanisms to overcome
this barrier (32, 48). The defining features of M. avium subsp.
paratuberculosis infection in ruminants are lesions and noncase-
ating granulomas present in the intestinal wall; however, there
remains a paucity of studies investigating how M. avium subsp.
FIG 2 M. avium subsp. paratuberculosis (MAP) enlistment of IL-1?-recruited macrophages to the initial site of infection. (A) FACS analysis of macrophage
infection readily recruits macrophages to the apical chamber. Macrophage recruitment was abolished when phagosome acidification was blocked with bafilo-
mycin A1 or IL-1? expression was prevented. Recruitment was rescued in bafilomycin A1 treatment with the addition of recombinant IL-1? protein. MCP-1
blocking did not impact macrophage recruitment. (B and C) Confocal microscopy of a coculture infection with M. avium subsp. paratuberculosis. (B) Macro-
phages are readily recruited to the apical chamber, as the Transwell contained only MAC-T cells. (C) Macrophages were found only in the apical chamber.
Recruited macrophages contained M. avium subsp. paratuberculosis. (D) Bafilomycin A1 treatment abrogated phagosome acidification and IL-1? transcription
blocking antibody. MCP-1 blocking does not impact IL-1? expression. (F) IL-1? dot blot. Cell supernatants were collected during M. avium subsp. paratuber-
culosis infection of MAC-T cells. The addition of the MCP-1 blocking antibody did not influence IL-1? protein levels. ***, P ? 0.001.
M. avium Transepithelial Migration
September 2012 Volume 80 Number 9 iai.asm.org 3231
paratuberculosis interacts with the epithelium. Given that the in-
testinal epithelium is the first host tissue that M. avium subsp.
paratuberculosis comes into contact with, it is likely that the intes-
tion (10, 18). For example, in a 1996 study, Alzuherri et al. re-
ported monocyte/macrophage infiltration into the intestinal
mucosa of M. avium subsp. paratuberculosis-infected Scottish
Blackface ewes as well as elevated levels of IL-1? (3). To our
knowledge, this is the first study to elucidate the mechanism be-
hind Alzuherri and colleagues’ observations utilizing a novel
MAC-T–bovine macrophage coculture system during M. avium
subsp. paratuberculosis infection. This is also the first report to
define M. avium subsp. paratuberculosis-directed macrophage re-
FIG 3 M. avium subsp. paratuberculosis-induced phagosome acidification and IL-1? processing is dependent upon an extracellular calcium flux. (A) Confocal
microscopy of BAPTA-AM-treated MAC-T cells during M. avium subsp. paratuberculosis infection. BAPTA-AM abolished LysoTracker staining. (B) qRT-PCR
of infected MAC-T cells treated with BAPTA-AM and calcium-free EGTA medium. BAPTA-AM and calcium-free medium prevented IL-1? transcription. (C)
Confocal microscopy of infected MAC-T cells treated with calcium-free EGTA medium 10 and 30 min postinfection. Calcium-free EGTA medium abrogated
LysoTracker staining. (D) IL-1? dot blot of infected MAC-T cells treated with calcium-free EGTA medium. The addition of calcium-free EGTA medium
prevented IL-1? expression at all postinfection time points. (E) Confocal microscopy of combined UTP and calcium-free treatment in infected MAC-T cells 10
min and 30 min p.i. The addition of UTP to calcium-free treatment restored phagosome acidification. Therefore, intracellular calcium stores remained
functional. ***, P ? 0.001.
Lamont et al.
iai.asm.orgInfection and Immunity
IL-1? production. The MAC-T–bovine macrophage coculture
system not only takes advantage of epithelium processing of M.
avium subsp. paratuberculosis but also takes into account the nu-
ances of cell-to-cell cross talk that takes place during infection.
This novel mechanism of host establishment may also explain
transepithelial migration observed in other phagosomal bacteria,
such as Brucella spp. For instance, Ackermann et al. noted trans-
rophages in a calf ligated ileal loop model (1). This dynamic
process at the epithelium interface may ultimately determine
Although epithelium-M. avium subsp. paratuberculosis in-
teractions may seem a bedlam of host and pathogen signals, the
results of the present study suggest that these signals are or-
gen establishment and survival. Previous studies have shown
that M. avium subsp. paratuberculosis exposure to a hyperos-
molar environment, like that found within epithelial cells, en-
hances phagocytosis during secondary infection (34). Further-
more, M. avium subsp. paratuberculosis entrance into the
epithelium mirrors that of other intestinal pathogens in that
there appears to be an upregulation of pathways involved in
cytoskeleton rearrangement (2). The closely related pathogen
Mycobacterium avium subsp. avium has been reported to up-
regulate a number of genes related to fatty acid and membrane
protein synthesis in HEp-2 cells (32). Therefore, it is likely that
M. avium subsp paratuberculosis has developed certain signal-
show that infection with M. avium subsp. paratuberculosis in
MAC-T cells leads to phagosome acidification and rapid IL-1?
expression from viable cells. Phagosome acidification may
seem to be a contradiction to M. avium subsp. paratuberculosis’
major goal of survival, as it is well documented that pathogenic
mycobacteria employ numerous mechanisms to inhibit the
acidification process (37, 50, 52, 56). However, a recent report
by Koo et al. suggests that phagosome acidification may actu-
ally serve as an effective survival and dissemination strategy
(24). Koo et al. have shown that phagosome acidification aids
more, IL-1? may serve as a chemoattractant for macrophages
and is also reported to be a necessary cytokine in granuloma
formation and maintenance (29, 35, 36, 57). We propose that
the intestinal epithelium represents a transition cell for M.
avium subsp. paratuberculosis based on data indicating that M.
avium subsp. paratuberculosis replicates inefficiently within
this tissue (7, 34). M. avium subsp. paratuberculosis temporar-
ily resides within the epithelium while macrophage recruit-
ment is elicited by phagosome acidification and IL-1? expres-
sion and release by the epithelium. Once macrophages arrive at
the site of infection, M. avium subsp. paratuberculosis enters
the cells and begins proliferation and dissemination to other
locations within the host. We show that M. avium subsp. para-
tuberculosis infection causes MDM recruitment to the primary
site of infection (MAC-T cells) in a coculture system. Macro-
phage transepithelial migration requires phagosome acidifica-
tion and IL-1? expression, and these two events are not
mutually exclusive. IL-1? is the true chemoattractant agent
responsible for this mechanism, since blocking of a previously
reported linked chemoattractant, MCP-1/CCL-2, failed to ab-
rogate macrophage recruitment and IL-1? processing.
Transepithelial migration may also be an important survival
and establishment strategy in other pathogenic mycobacteria.
Bermudez et al. developed a two-layer Transwell system contain-
ing A549 alveolar epithelial cells and endothelial cells to investi-
H37Rv, M. avium 101, and Mycobacterium bovis BCG strain Pas-
teur (8, 34). Passage through alveolar epithelial cells increased M.
tuberculosis translocation across the bilayer compared to direct
endothelial infection, which indicates that exposure to alveolar
epithelial cells allowed the emergence of an M. tuberculosis inva-
sive phenotype (8). Mycobacterial invasion in alveolar epithelial
cells also enhanced monocyte migration across the bilayer (8).
One briefly mentioned observation was the decrease in trans-
membrane resistance (TER), which may have influenced mono-
layer integrity. Interestingly, reduction in the TER has been ob-
served in intestinal epithelium cells infected with Salmonella
enterica serovar Typhimurium and Shigella flexneri (23, 39). The
radation of zona occludens 1, which promoted the migration of
neutrophils to the apical side of the epithelium. It is possible that
the TER reduction observed by Bermudez et al. may be due to a
similar mechanism of tight junction protein degradation. One
diffuse tight junctions. These tight junctions presumably offered
less of an impediment for macrophages to transverse the mono-
layer into apical chamber. Ongoing studies in our laboratory are
using a bovine intestinal epithelial (BIE) cell line, which forms
higher-resistance junctions, to determine if the reduction in TER
noted by Bermudez et al. also occurs with M. avium subsp. para-
tuberculosis infection (8).
Phagosome acidification and IL-1? expression were found to
be dependent on an extracellular calcium influx. Calcium plays a
dual role as it aids in phagolysosome fusion and is necessary for
the development of pro-IL-1? into its mature form (16, 38). Ad-
ditionally, calcium fluxes are necessary for leukocyte and macro-
of the transport mechanism responsible for calcium influx by an
appropriate blocker may provide a novel prophylactic for Johne’s
disease. It is likely that functional disruption of this transport
pathway will prevent IL-1? expression by the host during M.
avium subsp. paratuberculosis infection and consequently macro-
phage recruitment to the epithelium. For example, P2X receptors
have been linked to calcium influx from the extracellular milieu
and are currently under consideration as potential therapeutic
targets for inflammation-based neurodegenerative disorders (46,
49). Also, the connection between extracellular calcium, phago-
some maturation, IL-1?, and macrophage recruitment may be
exploited in M. avium subsp. paratuberculosis vaccine candidate
screening. Attenuation may be detected and candidates worthy of
future experimentation identified by the abrogation of calcium
influx and phagosome acidification at the epithelium interface.
More importantly, our MAC-T–MDM coculture model provides
a useful system to investigate M. avium subsp. paratuberculosis
In conclusion, we demonstrate that M. avium subsp. paratu-
berculosis infection promotes a cooperative self-destruction state
in the host epithelium, which entails maturation of the phago-
some and IL-1? production that is dependent upon extracellular
M. avium Transepithelial Migration
September 2012 Volume 80 Number 9iai.asm.org 3233
calcium. IL-1? in turn is available to act as a chemoattractant for
macrophages, which supply an escape route for M. avium subsp.
paratuberculosis. Although these results were identified in an in
vitro system, they may also be applied to natural infection. Ongo-
paratuberculosis effector molecules may play a role in phagosome
acidification and escape from the epithelium. Thus, IL-1?, which
is most widely known as a critical cytokine for control of patho-
genic mycobacteria, may paradoxically promote pathogen estab-
lishment and dissemination within the host.
We thank Raul G. Barletta (University of Nebraska-Lincoln) and Luiz E.
Bermudez (Oregon State University) for their generous gifts of GFP-ex-
pressing M. avium subsp. paratuberculosis strain K-10 and MAC-T cells,
respectively. We thank John P. Bannantine (U.S. Department of Agricul-
ture) for providing the rabbit polyclonal antibody against M. avium
subsp. paratuberculosis. We acknowledge the help of the University of
Minnesota’s bovine blood collection service.
This study was supported by USDA-CRIS (MIN-62-027), USDA-
CSREES NRI (2005-35204-16106), and University of Minnesota College
of Veterinary Medicine Agriculture Research Station (1802-11646-
AES0062027) grants awarded to S.S.
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