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* Novartis Horsham Research Center, Novartis Pharmaceutical Ltd., Horsham, United Kingdom;
SR Pharma, Center Point, London, United Kingdom; and
Department of Medical Microbiology, Windeyer Institute of Medical Sciences, Royal Free and University College Medical School, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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)
or total IgE levels were observed. Furthermore, transfer of splenocytes
from OVA-immunized and M. vaccae-treated mice into
recipient, OVA-immunized mice significantly reduced the
allergen-induced eosinophilia by an IFN--independent mechanism,
clearly indicating that the mechanism by which M. vaccae
induces its inhibitory effect is not due to a redirection from a
predominantly Th2 to a Th1-dominated immune response. The protective
effect of M. vaccae on the allergen-induced eosinophilia
lasted for at least 12 wk after its administration, and the treatment
was also effective in presensitized mice. Moreover, the allergen
specificity of the inhibitory effect could be demonstrated using a
double-immunization protocol, where M. vaccae treatment
before OVA immunization had no effect on the eosinophilic inflammation
induced by later immunization and challenge with cockroach extract Ag.
Taken together, these results clearly demonstrate that M.
vaccae is effective in blocking allergic inflammation by a
mechanism independent of IFN-, induces long term and Ag-specific
protection, and therefore has both prophylactic and therapeutic
potential for the treatment of allergic
diseases. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Epidemiological and clinical studies have provided compelling evidence
that suggest a link between the relative lack of infectious diseases
and the increase in allergic disorders. There are indeed many studies
suggesting that bacterial infections and/or bacterial products can
inhibit the development of allergic diseases (6). It has
been demonstrated that a positive tuberculin test result suggestive of
past infections with Mycobacterium tuberculosis was
inversely related to the subsequent development of atopy and asthma
(7). Moreover, the use of antibiotics during infancy
correlated with an increased risk of developing asthma, suggesting that
bacterial infections early in life may help to inhibit the development
of asthma (8). In animal models of allergic inflammation,
bacillus Calmette-Guérin (BCG) inoculation in mice delivered 14
days before allergen immunization reduced the formation of specific IgE
in response to allergen and the eosinophilic and AHR responses to
allergen with an increase in the production of IFN- (9, 10). Bacterial products such as oligonucleotides containing
unmethylated CpG motifs have been found to be potent stimuli of IL-12
and IFN- production, leading to a strong inhibition of allergic
airway inflammation (11). Furthermore, several studies
have demonstrated that heat-killed Listeria monocytogenes,
Lactobacillus plantarum, or Mycobacterium vaccae
could also suppress allergic responses in mice (12, 13, 14).
In particular, there is evidence that the fast-growing M.
vaccae, which induces a vigorous cell-mediated immune response and
shares several immunodominant epitopes with other mycobacteria, might
be a potential candidate for an anti-asthma vaccine. Preliminary
clinical studies in adults with grass pollen-induced asthma and
rhinitis found that treatment with heat-killed M. vaccae was
associated with fewer asthmatic symptoms and significantly less use of
bronchodilator relief medication during the pollen season
(15). More recently, it has been demonstrated that
intradermal administration of a killed M. vaccae suspension
was associated with an improvement in the severity of the atopic
dermatitis in children with moderate to severe disease
(16).
To further investigate the potential of mycobacteria to treat, prevent, or even cure atopic diseases, we tested whether heat-killed M. vaccae, which in other contexts has undergone extensive investigations in human cancer and tuberculosis patients, was able to alter the Ag-induced inflammation and AHR to inhaled methacholine in a murine model of pulmonary inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice (20–25 g, 5–6 wk old) were obtained from Harlan (Oxon, U.K.), All experimental protocols complied with the Home Office 1986 Animals Scientific Act and were approved by the Novartis Horsham Research Center animal welfare committee.
Ags and M. vaccae suspension
OVA was obtained from Sigma-Aldrich (grade V; Sigma-Aldrich, St. Louis, MO). Cockroach extract Ag was obtained from Hollister-Stier Laboratories (Spokane, WA). Sterile vials of M. vaccae (0.3 ml of a suspension containing 10 mg heat-killed M. vaccae/ml) were provided by SR Pharma (London, U.K.).
Experimental design
Short term immunization protocol.
BALB/c mice were immunized i.p. on days 0 and 14 with 10 µg OVA in
0.2 ml alum (Serva, Heidelberg, Germany) as described in Fig. 1
. On day 21 animals were exposed for 20
min to an aerosol of OVA in sterile PBS (50 mg/ml) or sterile PBS
alone.
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. On day 63
animals were exposed for 20 min to an aerosol of OVA in sterile PBS (50
mg/ml) or sterile PBS alone. Double immunization protocol. On days 0 and 14 mice received an i.p. injection of OVA in the presence of alum, and on days 42 and 56 of immunization animals were immunized with cockroach extract Ag (CEA; 10 µg in 0.2 ml alum) by i.p. and s.c. routes, respectively. On day 63 mice received an intranasal Ag challenge of 50 µl PBS containing 10 µg CEA. Control animals received 50 µl PBS.
Treatment with M. vaccae
Mice were treated, via the s.c. route, with 0.1 mg M.
vaccae (SR Pharma) in 200 µl saline. This dose was chosen based
on a previous report (14) and from pilot experiments
demonstrating an optimal response using these conditions (data not
shown). Control animals received an s.c. injection of 200 µl saline
alone, and treatment regimens are detailed in Fig. 1. Briefly, in the
short term immunization protocol mice were treated with M.
vaccae 21 days before the first OVA immunization. In the long term
immunization protocols M. vaccae was administered either
before the first or the third immunization to investigate whether the
compound could also interfere with an established allergic phenotype.
Finally, in the double-immunization protocol animals were treated with
M. vaccae 21 days before the first OVA immunization or 21
days before the first CEA immunization.
Assessment of inflammation
At the specified time point animals were anesthetized with
pentobarbitone sodium (60 mg/kg i.p.). The trachea was cannulated, and
bronchoalveolar lavage (BAL) was performed by injecting 0.4 ml PBS into
the lung via the trachea. The fluid was withdrawn and stored on ice.
This procedure was repeated three times. Total cell count was
determined, and cytospin preparation (Shandon Scientific, Cheshire,
U.K.) was performed. Cells were stained with DiffQuik (Baxter
Dade, Dudingen, Switzerland), and a differential count of 200 cells was
performed using standard morphological criteria. The remaining BAL
fluid was centrifuged (400 x g for 10 min), and
supernatant was collected and stored at -80°C for cytokine and
chemokine measurements. BAL cytokine (IL-5, IL-4, IL-13, and IFN-)
and chemokine (macrophage inflammatory protein-1
, RANTES, and
eotaxin) levels were measured using commercially available kits (R&D
Systems, Oxon, U.K.).
Morphometric analysis of eosinophils was accomplished by examining 10 high power fields (x1000 magnification) in histological sections from each lung. Blood smears were prepared by taking blood from the abdominal aorta. Peripheral eosinophils were identified and counted after staining with DiffQuik stain. Bone marrow cells were flushed from femurs with 1 ml RPMI 1640 medium supplemented with 10% FBS, and eosinophils were enumerated after cytocentrifugation and staining with O-phenylenediamine and counterstaining with thiazine.
Measurement of airway reactivity to inhaled methacholine
All mice were immunized i.p. with 10 µg OVA in alum (short
term immunization, Fig. 1). Twenty-one days after the beginning of the
immunization mice were challenged four times, each 1 day apart, with
either 50 mg/ml OVA or PBS for 20 min by aerosol. Twenty-four hours
after the last challenge mice were used to assess airway
hyperresponsiveness to inhaled methacholine by barometric
plethysmography (Buxco Electronics, Troy, NY) using whole body
plethysmography as described previously (17). To evaluate
airway responsiveness, conscious, spontaneously breathing mice were
initially placed in the main chamber of the plethysmograph. Following a
short settlement period, baseline airway function over 1 min was
recorded and averaged. Mice were then exposed for 1 min to nebulized
PBS, and recordings were taken. Following that, mice were exposed to
0.3 M methacholine (Aldrich, Madison, WI) for 1 min, and recordings
were taken for a total of 4 min, but were averaged for each minute. The
highest of the four 1 min averaged enhanced pause (Penh) values during
the 1 min after exposure to methacholine was expressed as a percentage
of the baseline Penh value obtained following PBS exposure.
Serum IgE and IgG2a levels
Twenty-four hours after Ag challenge animals were anesthetized with pentobarbitone sodium (60 mg/kg i.p.), a blood sample was taken from the abdominal aorta, serum was prepared, and total IgE and IgG2a Ab titers were determined by ELISAs as previously described (18). Briefly, total serum IgE and IgG2a were measured using sandwich ELISA, 4B3-39 (rat IgG anti-murine IgE mAb; Novartis, Basel, Switzerland) and goat IgG anti-mouse IgG2a were the capture reagent for IgE and IgG2a, respectively. Biotin-conjugated 3-11 (rat IgG anti-mouse IgE mAb) and biotin-conjugated RA33-18-12 (rat IgG anti-mouse IgG2a mAb) were used as detection reagents along with alkaline phosphatase-streptavidin. Affinity-purified monoclonal IgE anti-trinitrophenyl (A3B1; BD Biosciences, Oxford, U.K.) and monoclonal IgG2a anti-PC (Novartis) were used as standards.
Splenocyte activation
To assess the effect of M. vaccae on anti-CD3 or
OVA-driven IFN- release by splenocytes, mice were s.c. treated 3 wk
before the immunization with saline or 0.1 mg M. vaccae.
Briefly, spleens were collected on day 21, and single-cell suspensions
were prepared by passing the cells through a cell strainer. RBC were
removed by hypotonic lysis. After three washes in RPMI (Life
Technologies, Paisley, U.K.), 5 x 105
splenocytes/250 µl RPMI supplemented with 10% FBS (Life
Technologies), 2 mM L-glutamine (Life
Technologies), 100 µg/ml streptomycin (Life Technologies), and 100
U/ml penicillin (Life Technologies) were added to each well in
triplicate and then incubated for 72 h at 37°C in 5%
CO2 in the presence or the absence of 80 µg/ml
OVA. For anti-CD3 activation cells were cultured in 96-multiwell
plates coated with 4.5 µg/ml immobilized anti-CD3 mAb 145-2C11
(BD PharMingen, Oxford, U.K.) for 48 h (19). Plates
were then centrifuged (80 x g, 5 min, 4°C), and IL-4
and IFN- levels in the supernatants were determined by ELISA (R&D
Systems, Oxon, U.K.). The detection limit for both IL-4 and IFN- was
2 pg/ml.
Adoptive cell transfer and anti-IFN- treatment
Twenty-four hours after the OVA challenge (day 22), spleens from saline- or M. vaccae-treated mice were collected, and single-cell suspensions were prepared by passing the cells through a cell strainer. Splenocytes were then isolated by Ficoll-Hypaque density gradient centrifugation. Recipient immunized mice received on day 20 an i.v. injection of 50 µl PBS containing 2 x 106 splenocytes or PBS alone. On day 21 mice were challenged with either PBS or OVA, and BAL was obtained 48 h postchallenge.
In another set of experiments immunized mice (day 20) were injected
i.p. with 0.5 ml preimmune rabbit serum or an equivalent amount of
anti-IFN- immune serum (provided by Prof. S. Kunkel, Department
of Pathology, University of Michigan Medical School, Ann Arbor, MI) or
with saline 2 h before and 48 h after the i.v. injection of
2 x 106 splenocytes from M.
vaccae-treated and OVA-immunized and -challenged mice. Twenty-four
hours after the cell transfer mice were challenged with OVA, and BAL
was obtained 48 h postchallenge. This treatment regimen with the
anti-IFN- immune serum has been previously shown to
significantly attenuate systemic levels of IFN- in a mouse model of
inflammation (20).
Statistical analysis
Results are expressed as the mean ± SEM of the indicated number of animals. One-way ANOVA was used to determine significance among the groups. If a significant variance was found, an unpaired Student’s t test was used to assess comparability between means. A value of p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a first set of experiments the effect of M. vaccae
treatment on the eosinophilic inflammation of OVA-immunized and
-challenged mice using a short immunization protocol was analyzed. As
shown in Fig. 2, single OVA aerosol
challenge to immunized mice resulted in a significant increase in the
number of total cells, eosinophils, neutrophils, and macrophages in BAL
fluid compared with PBS challenged-animals. The increase in eosinophils
and macrophages peaked between 24 and 48 h after OVA challenge
(Fig. 2, A and C), whereas the highest number of
neutrophils was detected between 4 and 24 h (Fig. 2B).
To analyze the effect of M. vaccae, mice were treated by
s.c. injection with 0.1 mg M. vaccae 24 h before the
immunization, 4 h before allergen challenge, or 21 days before the
first immunization. No effect on eosinophilic inflammation was observed
in mice treated either 24 h before the immunization (0.71 ±
0.16 and 1.12 ± 0.37 x 105
eosinophils/ml for saline-treated and OVA-challenged mice and M.
vaccae-treated and OVA-challenged mice, respectively) or shortly
before the allergen challenge (1.35 ± 0.16 and 1.43 ±
0.13 x 105 eosinophils/ml for
saline-treated and OVA-challenged mice and M. vaccae-treated
and OVA-challenged mice, respectively). However, in mice treated 21
days before the first immunization, M. vaccae induced
a significant reduction of the allergen-induced inflammation, as
demonstrated by the significantly reduced numbers of eosinophils,
neutrophils, macrophages, and total cells present in the BAL fluid of
these animals (Fig. 2). Based on these results the treatment schedule
of 21 days before the immunization and the dose of 0.1 mg (see
Materials and Methods) was selected for all additional
experiments.
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To further investigate whether the reduction of inflammatory cells
in BAL fluid of M. vaccae-treated animals was due to the
retention of these cells within the bronchial tissue, reduced
infiltration from the circulation, or suppressed release from the bone
marrow, eosinophil accumulation following allergen challenge in these
compartments was analyzed. As shown in Fig. 3, a single aerosol Ag challenge on day
21 to immunized mice induced an increase in the number of eosinophils
in bone marrow, blood, and peribronchial wall at 24 h
postchallenge compared with PBS-challenged mice. The s.c.
administration of 0.1 mg M. vaccae 21 days before
immunization reduced the numbers of eosinophils in all these
compartments (Fig. 3), suggesting that the mechanism of how M.
vaccae inhibits the inflammatory cell infiltrate found in BAL
fluid is not due to a selective blockage of cell movement from these
compartments or retention of cells within the bronchial tissue.
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As shown in Fig. 4, the single Ag
challenge of OVA to immunized mice induced a characteristic increase
not only in the Th2 cell cytokines IL-4, IL-5, and IL-13, but also in
chemokines associated with eosinophilic inflammation, such as eotaxin,
macrophage inflammatory protein-1, and RANTES. In contrast, IFN-,
a cytokine normally associated with a Th1 cell response, was
significantly reduced in BAL fluid 24 h following the allergen
challenge. However, although pretreatment with M. vaccae
effectively blocked BAL and lung eosinophilia, no effect on Ag-induced
cytokine and chemokine release was observed 24 h after challenge;
in particular, no effect on BAL fluid IFN- levels was observed,
suggesting that the mechanism of action of the M.
vaccae-mediated anti-inflammatory effect does not involve a
strong Th1 cell cytokine production.
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The total IgE level in immunized animals was significantly higher
than that in naive mice (753 ± 25 ng/ml), indicating allergic
immunization of these animals. When M. vaccae at 0.1 mg was
administered to mice 21 days before the beginning of immunization, no
decrease in the serum levels of IgE was found compared with
saline-treated and OVA-immunized and -challenged mice (Fig. 4C). Specific OVA-IgE was also unaffected by M.
vaccae treatment (data not shown). Furthermore, M.
vaccae was unable to change IgG2a serum levels compared with
saline-treated and OVA-immunized and -challenged mice, suggesting again
that the main mechanism by which M. vaccae induces the
suppression of allergic inflammation does not involve the induction of
a Th1 cell response, expected to decrease IgE and increase IgG2a
levels.
Effect of M. vaccae on IL-4 and IFN- release by
splenocytes in response to in vitro anti-CD3 mAb and OVA
stimulation
To confirm that the effect of M. vaccae on Ag-induced
airway eosinophilia does not involve allergen-specific Th1 cells,
splenocytes from saline- or M. vaccae-treated and immunized
mice (short term immunization, day 21) were stimulated with immobilized
anti-CD3 mAb for 48 h. Challenge with anti-CD3 mAb induced
a marked IFN- release, which was unaffected by M. vaccae
treatment. In vitro anti-CD3 activation also triggered IL-4 release
by splenocytes, and this response was not modified by previous
treatment with M. vaccae (Table I).
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release regardless of the treated
group analyzed (Table I). Effect of adoptive cell transfer on Ag-induced airway eosinophilia
M. vaccae treatment is able to block allergic
eosinophilia, without in vitro and in vivo changes in IFN- levels.
To better understand the mechanism by which M. vaccae blocks
airway eosinophilia, splenocytes from M. vaccae-treated mice
were transferred into recipient immunized animals before the Ag
challenge. Indeed, the i.v. administration of 2 x
106 splenocytes obtained from M.
vaccae-treated and OVA-immunized and -challenged mice
significantly reduced OVA-induced airway eosinophilia in recipient mice
compared with PBS-treated and OVA-challenged mice (45% inhibition;
n = 6–8; p < 0.05; Fig. 5). In contrast, the transfer of
splenocytes from saline-treated and OVA-challenged as well as from
naive mice to recipient OVA-immunized mice failed to inhibit Ag-induced
airway eosinophilia (data not shown). Furthermore, treatment of
recipient mice with an Ab against IFN- before cell transfer failed
to reverse the inhibitory effect of M. vaccae-treated
splenocytes on airway eosinophilia (Fig. 5).
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Next, the effect of M. vaccae treatment on the
induction of AHR was analyzed using a multiple allergen challenge model
(see Materials and Methods). OVA immunization followed by
multiple challenges induced a marked increase in airway reactivity to
inhaled methacholine 24 h after the last OVA challenge compared
with control mice (Fig. 6). This
OVA-induced AHR to inhaled methacholine was reduced by 72% after
treatment with M. vaccae 21 days before the first
immunization (n = 8–10; p < 0.05;
Fig. 6). In a separate set of experiments the possibility that M.
vaccae treatment itself might interfere with the bronchial
response to methacholine was ruled out, since treatment of
OVA-immunized, PBS-challenged mice with M. vaccae failed to
modify the extent of methacholine-induced bronchoconstriction (increase
in Penh of 83 ± 18.6% and 126 ± 28.5% for PBS-challenged,
M. vaccae-untreated and -treated animals, respectively;
n = 5). AHR to inhaled methacholine was accompanied by
airway eosinophilia 24 h after the last OVA challenge. Treatment
of mice with M. vaccae, given s.c. 21 days before
immunization, resulted in 40% inhibition of eosinophil accumulation in
BAL fluid at 24 h compared with saline-treated and OVA-challenged
mice (saline-treated and OVA-challenged mice, 3.59 ± 0.75
eosinophils x 105/ml; M.
vaccae-treated and OVA-challenged mice, 2.11 ± 0.38
eosinophils x 105/ml; n =
7–10; p < 0.05), demonstrating the protective
anti-inflammatory effect of this treatment even after multiple
allergen challenges.
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The results obtained to date clearly demonstrate that pretreatment
with M. vaccae results in inhibition of the Ag-induced
allergic inflammation and airway hyperresponsiveness in a short term
immunization model. However, these experiments do not answer the
questions of whether this protective effect is long lasting, or whether
M. vaccae treatment is also effective in established
immunization states. To answer these questions, a long-term
immunization protocol was established, which allowed us to analyze the
long term and Ag-specific effect of M. vaccae treatment.
Mice were immunized on days 0, 14, 42, and 56 and were challenged with
an aerosol of OVA on day 63 (Fig. 1). As shown in Fig. 7, A and B, airway
eosinophilia induced by a single OVA challenge on day 65 was
significantly reduced by a single administration of 0.1 mg M.
vaccae either 3 wk before the first (day -21) or the third
immunization (day 21). When administered before the third immunization
(day 21), M. vaccae showed a more pronounced inhibitory
effect on airway eosinophilia (80% reduction; n =
8–10; p < 0.05) than when administered before the
first immunization (day -21; 42% reduction; n =
8–10; p < 0.05; Fig. 7B), suggesting that
M. vaccae treatment is able to interfere with an established
immunization state and therefore may have therapeutic potential.
Similar to the results obtained in the short immunization protocol
(Fig. 4), no change in total IgE and IgG2a levels was observed,
although treatment with M. vaccae before the second round of
immunization (day 21) slightly, but not significantly, reduced the
total serum IgE levels (saline-treated and OVA-challenged mice,
22,700 ± 3,133 ng/ml; M. vaccae-treated and
OVA-challenged mice, 15,363 ± 2,882 ng/ml). Moreover, M.
vaccae treatment failed to inhibit the allergen-induced increase
in cytokine or chemokine levels in BAL fluid of OVA-challenged mice
(IL-5 levels in saline-treated and OVA-challenged mice, 235
± 50 pg/ml compared with 263 ± 65 pg/ml in M.
vaccae-treated and OVA-challenged mice; IL-13, 160 ± 26 vs
210 ± 55 pg/ml; RANTES, 39 ± 7 vs 28 ± 7 pg/ml).
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In an attempt to verify the specificity of the M.
vaccae treatment mice were treated with 0.1 mg M.
vaccae 3 wk before the first immunization with OVA. On days 42 and
56 of immunization animals were immunized with CEA and were challenged
intranasally with 10 µg CEA on day 63 (Fig. 1). Intranasal CEA
challenge to these double-immunized mice resulted in an increase in the
number of eosinophils in the BAL fluid at 48 h postchallenge with
CEA. In contrast to the results obtained with OVA (Fig. 7A),
treatment with M. vaccae before the OVA immunization had no
effect on the eosinophilic inflammation induced by later immunization
and challenge with CEA, suggesting that the M.
vaccae-mediated inhibitory effect is Ag specific (Fig. 7C). This is further supported by experiments showing that
using this double-immunization, long term protocol, pretreatment with
0.1 mg M. vaccae 3 wk before CEA immunization (Fig. 1)
inhibited CEA-induced eosinophilic inflammation (saline-treated and
CEA-challenged mice, 0.46 ± 0.1 eosinophils x
105/ml; M. vaccae-treated and
CEA-challenged mice, 0.25 ± 0.1 eosinophils x
105/ml; n = 9–10;
p < 0.05).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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independent, long lasting, and Ag specific and was also effective in
previously immunized animals, indicating the therapeutic potential of
the treatment.
Pharmacological agents that selectively inhibit Th2 cell activation and
cytokine production, antagonize cytokines associated with allergic
immune responses, or reprogram the immune response toward a potentially
protective Th1 pathway are of considerable interest in the treatment of
allergic asthma (21). In this respect it is well
documented that many bacterial infections associated with suppression
of allergic inflammation may induce profound Th1 responses with high
levels of IL-12 and IFN- and therefore have the potential to
interfere with the mainly Th2-driven allergic response (6, 9, 10, 11, 12, 13, 14). In contrast to these studies, our results using s.c.
treatment with heat-killed M. vaccae had no effect on
IFN- production and did not modify the BAL fluid levels of IL-4,
IL-5, IL-13, or serum IgE. It is therefore very unlikely that a shift
from a Th2-dominated immune response to a Th1-dominated immune response
is the major mechanism by which M. vaccae exerts its
inhibitory effect. This conclusion is supported by the findings that 1)
no Th1-inducing, Th2-suppressing activity could be demonstrated; 2) the
suppressive effect of M. vaccae treatment on the allergic
pulmonary response was not restricted to eosinophils, but also extended
to other inflammatory cell types, such as neutrophils and macrophage;
3) splenocytes from immunized and M. vaccae treated-mice
when activated in vitro failed to release increased levels of IFN-;
4) adoptive transfer of splenocytes from in vivo M.
vaccae-treated mice into OVA-immunized mice inhibited the
allergen-induced eosinophilia, which was not reversible by blocking
IFN-; and 5) M. vaccae treatment had no effect on IgE or
IgG2a production. It is noteworthy that the inhibitory effect of
M. vaccae on the allergic eosinophilia was not observed when
the bacterial extract was administered 1 day before the immunization or
4 h before the challenge, suggesting that the timing of exposure
to heat-killed M. vaccae is crucial for the establishment of
an immunomodulatory cascade that needs maturation over a period of
several days before being able to induce a suppressive effect. This
observation is therefore not compatible with rapid and high level IL-12
or IFN- production as seen with other bacterial infections, which
subsequently inhibit the Th2-driven inflammatory response (6, 9, 10, 11, 12, 13, 14). Moreover, most of the latter studies were performed with
live bacteria (e.g., BCG), which may induce a different form of immune
response compared with heat-killed mycobacterial extracts and therefore
could explain at least some of the differences observed in our model
compared with other reported data (9). On the other hand,
very recent studies comparing the immunoregulatory effects of live BCG
and M. vaccae also demonstrated that although the overall
responses were qualitatively similar, M. vaccae was much
weaker in producing some of the key features associated with a Th1
response, such as IL-12 and IFN- production, suggesting differences
in the ability to induce a Th1 response in response to different
mycobacterial Ags (22).
Another often discussed possibility of how bacterial infection might
interfere with Th2-driven inflammation is the exposure to bacterial DNA
containing unmethylated CpG motifs. Indeed, oligonucleotides containing
unmethylated CpG motifs have been found to be potent stimuli of IL-12
and IFN- production, leading to a strong inhibition of allergic
airway inflammation (11). Multiple studies demonstrated
that such CpG oligonucleotides inhibit allergic inflammatory responses
when administered during the immunization period as well as shortly
before the challenge, change the cytokine pattern into a predominantly
Th1-like profile, and are potent IgE synthesis inhibitors (11, 23, 24). Although the preparation of M. vaccae used
in our study most likely contains CpG DNA, it is very unlikely that
this is a major mechanism of how this bacterial extract induces its
inhibitory effect, since no Th1-inducing activity was found, and the
treatment was not effective using treatment and Ag sensitization and
challenge protocols similar to those described for testing CpG
oligonucleotides.
Moreover, and in support of the failure of redirecting the Th2 to a Th1 immune response by M. vaccae are the data for serum IgE and IgG2a levels, which were not affected by the treatment. Similar conclusions were reached in a recent study in which intradermal administration of heat-killed M. vaccae induced a significant improvement in atopic dermatitis in children with moderate to severe disease (16). In this study the ability of M. vaccae to alter Th2 activity was also not convincing, as no effect on serum IgE or blood eosinophil numbers were found. Similar findings were reported by other investigators, demonstrating that a single injection of M. vaccae into OVA-preimmunized mice did not cause significant changes in serum IgE levels (25). In contrast, Wang and Rook (14) reported that M. vaccae injection to OVA-immunized mice significantly suppressed serum IgE (14). Therefore, it is likely that the discrepancy in effects on IgE levels observed with Mycobacterium treatment may be due to different protocols of immunization, which account for a difference in the kinetics of IgE production and turnover.
Besides its effect on the allergic inflammation and AHR in a short term
immunization protocol, the present study also demonstrates that
M. vaccae treatment confers long term and Ag-specific
protection and therefore has both prophylactic and therapeutic
potential for the treatment of allergic diseases. Of particular
importance in this context are the data showing that M.
vaccae treatment is still able to block eosinophilic inflammation
in previously immunized mice (Fig. 7B) and that the
protection against OVA did not confer protection against CEA-induced
eosinophilic inflammation in mice treated 21 days before the first OVA
immunization (Fig. 7C). One interpretation of these data
could be that the treatment with M. vaccae induces the
generation of specific regulatory cells that might be able to block the
allergic response in an Ag-specific way for a long duration of time.
The induction of such regulatory cells by M. vaccae would
also explain why there is a need for a time gap between treatment with
the bacteria and immunization with OVA. Supporting this hypothesis, we
show here that splenocytes from M. vaccae-treated and
OVA-immunized mice are able to prevent airway eosinophilia in recipient
immunized mice. This protective effect was not abrogated in recipient
mice treated with an anti-IFN- Ab, reinforcing the concept that
M. vaccae attenuates allergic inflammation by an
IFN--independent mechanism. The identification of the splenocyte
population responsible for this protective effect is currently under
investigation.
In conclusion, the ability of s.c. administration of M. vaccae to provide long term protection against allergic lung inflammation suggests that it may have therapeutic benefit in asthma and other allergic diseases. Indeed, preliminary clinical studies in adults with grass pollen-induced asthma and rhinitis found that treatment with M. vaccae was associated with fewer asthmatics symptoms and significantly less use of bronchodilator relief medication during the pollen season (13). Moreover, it has been demonstrated that intradermal administration of a killed M. vaccae suspension was associated with an improvement in the severity of the atopic dermatitis in children with moderate to severe disease (16). These studies clearly support our hypothesis that M. vaccae treatment may provide an alternative to the current practice of asthma therapy and also demonstrate that the inhibitory effects of M. vaccae noted in murine models of experimental allergic airway inflammation appear to translate well to human allergic diseases. However, the exact mechanism of how M. vaccae exerts its effect remains open and is the topic of future studies.
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Acknowledgments |
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2 Abbreviations used in this paper: AHR, airway hyperreactivity; BAL, bronchoalveolar lavage; BCG, bacillus Calmette-Guérin; CEA, cockroach extract Ag; Penh, enhanced pause.
Received for publication August 16, 2001. Accepted for publication May 17, 2002.
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) or with 0.1 mg M. vaccae
(
) and challenged with OVA. Control mice were treated with saline
and challenged with PBS (
). Data are the mean ± SEM of 8–10
mice for each group. *, p < 0.05 compared with
saline-treated and PBS-challenged mice. #, p <
0.05 compared with M. vaccae-treated and OVA-challenged
mice.
