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Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Munich, Germany
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Abstract |
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production in zymosan A-stimulated mouse
macrophages and the human monocytic Mono-Mac-6 cell line. The
underlying mechanism of TNF- suppression could be assigned to
LcrV-mediated IL (IL)-10 production, because 1) LcrV induces IL-10
release in macrophages, 2) anti-IL-10 Ab treatment completely
abrogated TNF- suppression, and 3) TNF- suppression was absent in
LcrV-treated macrophages of IL-10-deficient (IL-10-/-)
mice. The relevance of LcrV-mediated immunosuppression for the
pathogenicity of yersiniae became evident by experimental infection of
mice; in contrast to wild-type mice, IL-10-/- mice were
highly resistant against Yersinia infection, as shown by
lower bacterial load in spleen and liver, absent abscess formation in
these organs, and survival. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and mitogen-activated protein (MAP)
kinase activities (8, 9, 10).
Because these Yops are injected directly into the host cell cytoplasm
by a needle-like apparatus (11), they are not accessible
to the Ab immune response. However, protection in mice against
yersiniae can be achieved by treatment with Abs against YadA of
Y. enterocolitica (12) or against the V Ag
(LcrV) of Y. enterocolitica and Y. pestis,
respectively (13, 14, 15, 16). The V Ag is one of the
virulence-associated Ags of Y. pestis recognized as early as
50 years ago. LcrV is a secreted protein encoded in the
lcrGVHYopBD operon located on pYV (17, 18) and
thus belongs to the common virulence-associated Ags of the three
Yersinia spp. Although LcrV has been studied for many
decades, it remains one of the most enigmatic proteins involved in
Yersinia virulence. LcrV appears to be a multifunctional
protein in that 1) it is required for regulation of Yop production, 2)
it is required for translocation of Yops into host cells (together with
YopB and D), and 3) it is capable of forming channels in artificial
membranes (19, 20, 21). Moreover, it has been demonstrated
that treatment of mice with a staphylococcal protein A-Y.
pestis V Ag fusion peptide results in suppression of TNF- and
IFN-
and in amplification of IL-10 in spleen homogenates
(22, 23, 24). Evidence is accumulating that V Ag is on one
hand associated with the Yop translocation complex that connects the
target cytoplasm membrane with the needle apparatus of yersiniae and on
the other is released into the environment where it presumably affects
cells of the immune system (17, 18). Considering
translocated Yops and released V Ag, we would expect a two-process
strategy by which yersiniae suppress the immune response: 1)
suppression of contacted cells by injected Yops (short distance
effect); and 2) suppression of bystander cells by released V Ag (long
distance effect). This model would also explain why active or passive
immunization with V Ag or anti-V Abs, respectively, has such a high
protective effect.
Recently, it has been demonstrated that the opportunistic pathogen Pseudomonas aeruginosa is also equipped with a TTS apparatus for secretion/translocation of anti-host effector proteins (25). It is also striking that the TTS apparatus of P. aeruginosa is closely related to that of Yersinia spp. including a LcrV homolog, denoted PcrV. Moreover, it has been shown that active and passive immunization of mice with PcrV and anti-PcrV, respectively, protects against P. aeruginosa infection (26) and that PcrV has also the capacity to form channels (21). However, it is still unknown whether PcrV is also capable to suppress proinflammatory cytokine production or to induce IL-10 release in spleen tissue as it is known for LcrV.
For these reasons, the major aim of this study was to elucidate
comparatively the immunomodulatory function of Y.
enterocolitica O8 and P. aeruginosa V Ags in murine
peritoneal macrophages and human cells of the monocyte-macrophage line
Mono-Mac-6 by analyzing their role in TNF- suppression and IL-10
induction. When TNF- suppression by V Ag of Y.
enterocolitica was found to be dependent on IL-10 induction, we
were prompted to check whether IL-10-deficient mice are resistant to
Y. enterocolitica infection because of subversion of the
infection strategy of the pathogen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c and LPS nonresponder C3H/HeJ mice were purchased from Charles River Wiga (Sulzfeld, Germany). LPS nonresponder mice (C57BL/10ScCR and BALB/c LPSd) (27), a gift from M. A. Freudenberg and C. Galanos, were bred under specific-pathogen-free conditions at the Max Planck Institut für Immunbiologie (Freiburg, Germany). IL-10-deficient (IL-10-/-) mice on a C57BL/6 background and C57BL/6 mice, serving as wild-type control animals, were obtained from The Jackson Laboratory (Bar Harbor, ME). Female mice were used at 6–8 wk of age.
Preparation of recombinant proteins
Expression, production, and purification of recombinant V Ag (rLcrV) derived from Y. enterocolitica O8 strain WA-314 (28) was performed as described previously (16). For construction of the recombinant YopH (rH) and rPcrV, the P. aeruginosa strain PAO1 homolog of the Yersinia V Ag, the QIAexpress histidine-tagged protein expression and purification system (Qiagen, Hilden, Germany) was used as described previously for rLcrV (16). To amplify yopH and pcrV, the primer pairs yopH1 (CTC GGA TCC ATG AAC TTA TCA TTA AGC GAT) and yopH2 (CTC GAG CTC AGC AGA TAA ACC TCA ACT AAT) (29) as well as pcrV1 (ATA GGA TCC ATG GAA GTC AGA AAC C) and pcrV2 (ATA AAG CTT CTA GAT CGC GCT GAG AAT GTC) were used, respectively. Protein concentrations were measured by the bicinchoninic acid protein assay (Pierce, Freiburg, Germany). rLcrV, rH, and rPcrV were virtually LPS free as measured by the Limulus amebocyte assay (Pyroquant, Walldorf, Germany). The purity of the recombinant proteins was also checked by SDS-PAGE (data not shown). Aliquots of the preparations were stored at -80°C, thawed immediately before the experiments, and used only once to avoid repeated freezing and thawing cycles.
Preparation of Abs against rLcrV and rH
Antiserum against rLcrV (anti-rLcrV) was produced in rabbits as described previously (16). Polyclonal anti-rH serum was prepared similarly. Briefly, 2-month-old New Zealand rabbits were immunized at four different times with 150 µg rH in 1 ml PBS mixed with lyophilized adjuvant (ABM adjuvant; Sebak, Aidenbach, Germany). After the animals were sacrificed, blood was obtained by heart puncture, and serum was collected after clotting. The serum (anti-rH) was tested in immunoblots using different Yops and rH and reacted specifically with a 51-kDa protein at a serum dilution of 1/104. The Ig fraction was enriched by ammonium sulfate precipitation and subsequent extensive dialysis against PBS as described (16).
Preparation and stimulation of murine peritoneal macrophages in vitro
Proteose peptone-elicited peritoneal macrophages (PPMs) were prepared as follows. Peritoneal exudate cells were obtained from mice that had received an i.p. injection of 1 ml 10% proteose peptone (Difco Laboratories, Detroit, MI) 3 days before. The cells were washed three times and resuspended in ice-cold RPMI 1640 (Biochrom, Berlin, Germany) supplemented with 2 mM L-glutamine (Biochrom), 10 mM HEPES (Biochrom), 100 µg/ml streptomycin (Biochrom), 100 U/ml penicillin (Biochrom), and 10% heat-inactivated FCS (Life Technologies, Karlsruhe, Germany). Cells (1 x 106/ml) were plated in 24-well tissue culture plates (Nunc, Roskilde, Denmark). After the cells had been incubated at 37°C in a humidified 5% CO2 atmosphere for 2 h, nonadherent cells were removed by vigorous washing with sterile endotoxin-free PBS. Macrophage monolayers were pretreated for 3 h with rLcrV, rH, rPcrV, or LPS from Salmonella abortus equi (Sigma, Deisenhofen, Germany) as control. Denaturation and degradation of LcrV were achieved by 1 h boiling or digestion with 20 µg/ml proteinase K (Sigma) for 30 min, respectively. To remove rLcrV specifically, the rLcrV-containing solution was treated with purified antiserum against rLcrV, and the immune complexes were subsequently separated using protein A-Sepharose beads (Pharmacia, Upsala, Sweden). As control, solubilized rLcrV was incubated similarly with anti-rH.
After the 3-h pretreatment period of macrophages with the mentioned
proteins or LPS, cells were stimulated with 1 mg/ml zymosan A (Sigma)
for 18 h. Supernatants were collected, and levels of TNF- were
determined. For IL-10 measurements, macrophage supernatants were
collected after rLcrV treatment for 2 h and stored until
tested.
To analyze the influence of IL-10 on TNF- production, inhibiting rat
anti-murine IL-10 Abs (JES5-2A5) were purchased from BD PharMingen
(Hamburg, Germany). Inhibiting anti-TGF-
(R&D Systems,
Minneapolis, MN) and IL-4 (R&D Systems) Abs served as controls. Abs and
rLcrV were coincubated with PPMs for 3 h. Thereafter, PPMs were
stimulated with 1 mg/ml zymosan A for 18 h for TNF- production.
Stimulation of Mono-Mac-6 cells
Cells of the human monocytic cell line Mono-Mac-6
(31) grown in RPMI 1640 supplemented with 10% FCS for 3
days at a cell density of 2 x 105 cells/ml
were used for experiments testing responsiveness to yersiniae and
Pseudomonas V Ag. Cells (2 x
105/ml) were plated in 24-well tissue culture
plates and pretreated for 18 h with different amounts of rLcrV,
rLcrV boiled for 1 h, or rPcrV. Unpretreated cells served as
control. After pretreatment, supernatants were collected for
measurement of IL-10. For TNF- production cells pretreated as
indicated above were stimulated with 1 mg/ml zymosan A. After 6 h
incubation, supernatants were investigated for TNF- levels.
Assays for murine and human TNF- and IL-10
Murine TNF- levels were measured in Nunc-Immuno plates (Nunc)
by using a capture ELISA using rat anti-mouse TNF- mAb
(G281-2626) and biotin-labeled anti-TNF- mAb (MP6XT3) as
described previously (30). Murine IL-10 was determined by
a commercial ELISA (R&D Systems) according to the manufacturer’s
recommendations. Human TNF- levels were measured by a capture ELISA
using mouse anti-human TNF- mAb (mAb1) and biotin-labeled
anti-TNF- mAb (mAb11) as recommended by the manufacturer (BD
PharMingen). Similarly, human IL-10 was determined by a capture ELISA
based on the mAbs JES3-19F1 and JES3-12G8 (BD PharMingen).
Experimental infection of mice.
Y. enterocolitica O8 strain WA-314 carrying the virulence plasmid pYVO8 (28) was grown in Luria-Bertani medium at 27°C overnight, sedimented, resuspended in 20% glycerol, and frozen at -80°C. For infection of mice, aliquots of glycerol stock cultures were thawed, washed in sterile PBS (pH 7.4), and diluted to the appropriate bacterial concentrations (16). Mice were given i.p. injections of 0.2 ml bacterial solution containing 1 x 104 CFU. Survival was monitored for 14 days.
Determination of the number of yersiniae in spleen and liver
Mice were injected i.p. with 1 x 104 CFU Y. enterocolitica O8 strain WA-314. After 4 days, the mice were sacrificed. Spleens and livers were dissected and homogenized as described previously (16). Yersiniae CFU were determined by plating serial dilutions on Yersinia selective agar (CIN agar; BD Biosciences, Heidelberg, Germany) and counting the CFU after an incubation period of 40 h at 27°C.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production in murine peritoneal macrophages is suppressed
by rLcrV, but not by rPcrV, in a dose-dependent manner
TNF- plays an important role as a defense effector in innate
immunity (32). Therefore, it may be speculated that for
bacteria the suppression of macrophage-dependent TNF- production
might be important, especially in the initial phase of infection when
mainly macrophages build the first line of defense. A similar
phenomenon is seen in LPS tolerance. To differentiate V Ag-induced
immunomodulation from LPS tolerance, we chose LPS nonresponder mice for
our experiments. For testing of the TNF--suppressing capacity of
rLcrV, PPMs from the LPS nonresponder mouse strain C3H/HeJ were
pretreated for 3 h with rLcrV at different concentrations
(33). Macrophages were subsequently stimulated for 18
h with zymosan A, because zymosan A is known to be a potent TNF-
inducer in macrophages (34). TNF- in culture
supernatants was measured by ELISA. Pretreatment with rLcrV impaired
the ability of C3H/HeJ macrophages to produce TNF- in a
dose-dependent manner, whereas pretreatment with rH or with
Pseudomonas rPcrV did not result in TNF- suppression
(Fig. 1
). A similar dose-dependent
immunomodulating effect of rLcrV was also seen in PPMs of the LPS
nonresponder C57BL/10ScCR and BALB/c LPSd mice as well as in PPMs from
the LPS responder mouse strains BALB/c and C57/BL6 (data not
shown).
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suppression, two additional
control approaches were chosen besides the use of LPS nonresponder
macrophages: 1) boiling of our rLcrV preparation which was expected to
inactivate LcrV, but not LPS, abolished the TNF- suppression
completely in LPS nonresponder C3H/HeJ macrophages (Fig. 1
); and 2)
proteolytic degradation of rLcrV by preincubation with proteinase K
inhibited the TNF--suppressive effect of rLcrV in a similar manner
(Fig. 1).
Specific removal of rLcrV using anti-rLcrV abolishes TNF-
suppression
To show that the TNF- suppression was specifically attributable
to V Ag, rLcrV was removed from the preparation after incubation with
anti-rLcrV using protein A-Sepharose beads. Specific binding of
anti-rLcrV to rLcrV was shown previously (16). After
removal of rLcrV, no TNF- suppression could be observed in C3H/HeJ
peritoneal macrophages (Fig. 1). A similar treatment using anti-rH
Abs did not influence the immunosuppressive capacity of rLcrV (data not
shown).
TNF- suppression by rLcrV is abolished by anti-IL-10 Abs
To test whether the TNF- suppression could be attributed to
IL-10, C3H/HeJ peritoneal macrophages were pretreated with rLcrV and
anti-IL-10, anti-IL-4, or anti-TGF- Abs, respectively.
After zymosan A restimulation, no TNF- suppression was found in
anti-IL-10-treated macrophage cultures suggesting a role for IL-10
in the TNF--suppressive effect (Fig. 2). Anti-IL-4 and anti-TGF- Abs
were not able to abolish the rLcrV-induced TNF- suppression.
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suppression
Proteose-peptone elicited C3H/HeJ peritoneal macrophages were
stimulated with rLcrV; boiled rLcrV; rLcrV after incubation with
anti-rLcrV using protein A-Sepharose beads, rH, or rPcrV; or
remained untreated. After 2 h cultivation, macrophage supernatants
were collected for IL-10 measurement by ELISA. IL-10 was found in
supernatants of rLcrV-treated macrophages, whereas incubation with rH
or rPcrV did not result in IL-10 induction (Fig. 3). Furthermore, both boiling and removal
of rLcrV by the anti-rLcrV-protein A-Sepharose strategy impaired
the IL-10 induction (Fig. 3). To investigate whether IL-10 is
sufficient and essential for rLcrV-caused TNF-
suppression, PPMs from IL-10-/- mice were
pretreated for 3 h with rLcrV and subsequently stimulated with
zymosan A. No TNF- suppression upon zymosan A stimulation could be
observed in rLcrV-pretreated macrophages from IL-10-deficient mice when
compared with PBS-pretreated macrophages (Fig. 4). In contrast, rLcrV caused marked
TNF- suppression in macrophages derived from wild-type C57BL/6 mice
(Fig. 4).
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suppression and elicits
IL-10 production in human monocytic Mono-Mac-6 cells
To analyze whether the immunomodulatory capacity of
Yersinia V Ag not only is restricted to mouse macrophages
but can also be found in the human system, the human monocytic cell
line Mono-Mac-6 was used. Pretreatment of Mono-Mac-6 cells with rLcrV
resulted in TNF- suppression, whereas boiling of rLcrV completely
abolished the cytokine-suppressive effect (Fig. 5A). Accordingly, IL-10 could
be detected after 18 h pretreatment of Mono-Mac-6 cells with
rLcrV, whereas boiled rLcrV did not induce IL-10 production (Fig. 5B). The P. aeruginosa homolog rPcrV was not able
either to suppress TNF- production or to induce IL-10. Additionally,
these data indicate that TNF- suppression by rLcrV does not require
the participation of a cell type other than macrophages.
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To evaluate the in vivo role of IL-10 in Y.
enterocolitica infection, wild-type and
IL-10-/- mice were infected i.p. with 1 x
104 CFU Y. enterocolitica, and their
survival was monitored for 14 days. Although >80% of
IL-10-/- mice survived for 14 days, all
wild-type mice were dead on day 10 postinfection, with 50% of
wild-type mice succumbing to Y. enterocolitica within 7 days
(Fig. 6). The high resistance of
IL-10-/- mice to Y. enterocolitica
was paralleled by the absence of macroabscesses in spleen after 4 days
of infection, whereas the spleens of wild-type mice showed impressive
macroabscesses. Accordingly, bacterial numbers in spleens and livers of
IL-10-/- mice obtained 4 days after i.p.
infection with Y. enterocolitica were significantly lower
than in those from wild-type mice, consistent with low morbidity and
mortality of IL-10-/- mice after Y.
enterocolitica infection (Fig. 7).
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Discussion |
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is a proinflammatory cytokine which is primarily released
by activated macrophages and plays a crucial role in limiting the
severity of bacterial infections (32). To evade the host
immune defense, the inhibition of TNF- would be a powerful strategy
that a pathogen might develop. Two ways of TNF- suppression have
been described in Yersinia infection: one is dependent on
the injection of the effector proteins YopP in Y.
enterocolitica (8, 10) or YopJ in Y.
pseudotuberculosis into target cells (9); the other
has been attributed to secreted Yersinia V Ag and YopB.
Although the molecular mechanism of TNF- suppression by YopP/YopJ
could be assigned to inhibition of NF-B and of the extracellular
signal-regulated kinase 2, c-Jun N-terminal kinase, and p38 MAP kinase
activities (8, 9), the underlying mechanisms of YopB- and
V Ag-caused immunomodulation have not been studied yet.
A role for V Ag in TNF- and IFN- suppression was shown in spleens
of infected mice (22) and in mixed macrophage-T cell
cultures (33). Besides V Ag, YopB of Y.
enterocolitica has been claimed to suppress TNF- production in
a murine peritoneal macrophage model (34). In this study,
YopB purified from culture supernatants of induced pYV plasmid-bearing
Y. enterocolitica was used, but no recombinant YopB
(35). V Ag, however, has been shown to bind to YopB
(36). Therefore, it cannot be ruled out that the observed
TNF- suppression was due to V Ag possibly bound to YopB.
In our study using recombinant rLcrV, we could demonstrate V Ag-caused
TNF- suppression in a macrophage cell culture model. rLcrV was able
to reduce TNF- production induced by zymosan A in LPS nonresponder
peritoneal macrophages in a dose-dependent manner.
One aim of this study was to clearly establish TNF- suppression as a
rLcrV-specific effect and differentiate it from LPS tolerance.
Denaturation of the protein component in the rLcrV preparation by
boiling or proteinase K degradation and the use of PPMs of three LPS
nonresponder mouse strains allowed attribution of the TNF-
suppression to the protein component of rLcrV and exclusion of a
LPS-induced tolerance phenomenon due to contaminating LPS.
Additionally, the latter is corroborated by the use of zymosan A for
TNF- induction, because it had been shown that LPS pretreatment of
PPMs of LPS responder C3H/HeSlc mice did not affect zymosan-induced
TNF- production (34).
The TNF--suppressive effect observed by us could clearly be
attributed to rLcrV, because removal of rLcrV from the preparation
after incubation with anti-rLcrV using protein A-Sepharose beads
abolished the TNF- suppression completely in PPMs of C3H/HeJ mice.
Moreover, the capacity of V Ag to down-modulate a TNF- response is
not restricted to the murine host, because rLcrV was able to suppress
TNA- production in the human monocytic cell line Mono-Mac-6.
As control, we used similarly produced recombinant YopH which was nonactive in any of the murine and human experimental cell models presented here. Surprisingly, we were unable to detect any LcrV-like immunomodulating activity for the P. aeruginosa homolog PcrV. This lack of immunosuppressive effects of rPcrV in the macrophage models tested is especially striking, because other functions of rLcrV such as participation in translocation of type III effectors and channel formation in lipid bilayer membranes are shared by PcrV (26).
TNF- expression in macrophages can be inhibited by a number of
factors. Of TNF- down-regulating cytokines produced by macrophages,
the anti-inflammatory IL-10 has been shown to be increased in
spleens of BALB/c mice after injection of Y. pestis V
Ag-polyhistidine fusion peptide (Vh)
(24). Our data underline the decisive role of IL-10 in the
rLcrV-caused TNF- suppression, because anti-IL-10-Abs completely
abolished the cytokine-suppressive effect of rLcrV in murine PPMs.
Furthermore, it could be shown that rLcrV induces IL-10 directly in
murine PPMs and in human Mono-Mac-6 cells. The final confirmation
that IL-10 induction is the key mechanism by which rLcrV establishes
TNF- suppression could be achieved by showing that rLcrV-caused
TNF- suppression was absent in PPMs derived from
IL-10-/- mice.
Inhibition of TNF- production has been described in several
bacterial infections (37, 38, 39, 40, 41). In most of these cases,
however, the underlying cytokine-suppressive mechanism has not yet been
elucidated. IL-10 induction by a bacterial protein leading to TNF-
suppression has thus far been found only for V Ag of
Yersinia spp., as shown in the present study.
The cell type(s) involved in V Ag-caused TNF- suppression have not
yet completely elucidated. Schmidt et al. showed that activated T cells
are strictly required for V Ag-caused TNF- suppression upon
LPS-IFN- stimulation in a peritoneal exudate cell model
(33). Our findings using the Mono-Mac-6 cells, however,
suggest that a rather direct effect of V Ag on cells of the
macrophage-monocytic lineage seems to be sufficient to cause TNF-
suppression on zymosan A stimulation without involving other cell
types. Because cells of the macrophage-monocytic lineage are the main
or the only possible source of both IL-10 and TNF- in our two cell
models, it is tempting to speculate that rLcrV-induced IL-10 leads to
TNF- suppression by "silencing" macrophages in a paracrine or
autocrine loop.
The importance of IL-10 for sustaining a Y. enterocolitica
infection was highlighted by in vivo experiments showing that
IL-10-deficient mice were highly resistant to i.p. Y.
enterocolitica infection compared with wild-type mice. Because
rLcrV was shown in vitro to induce IL-10 leading to TNF-
suppression, it may be concluded that V-Ag released by yersiniae
supports evasion of the innate immunity effector TNF- in Y.
enterocolitica infection by stimulating IL-10 production, thus
exploiting the TNF- down-regulating capacity of endogenous host
IL-10. This conclusion is supported by the finding that i.p.
injection of Vh into BALB/c mice resulted in an
early increase of IL-10 in spleens (24).
The role of IL-10 in microbial infection has been investigated for
several bacterial and parasitic pathogens using IL-10-deficient mice
(for a review, see Ref. 42). After infection with
Toxoplasma gondii (43) or Trypanosoma
cruzi (44), IL-10-deficient mice died rapidly due to
overproduction of proinflammatory cytokines from
CD4+ T cells. Similarly, in an in vivo model of
acute endotoxic shock, the high mortality rate of LPS-challenged
IL-10-deficient mice was accompanied by an uncontrolled TNF-
production (45). In contrast, up-regulation of
proinflammatory type 1 cytokine responses resulted in increased innate
and acquired immunity in IL-10-deficient mice when challenged with
sublethal doses of Listeria monocytogenes (46).
In the early state of experimental i.p. Mycobacterium bovis
bacillus Calmette-Guérin infection (47),
IL-10-deficient mice exhibited a significantly lower bacteria burden in
spleen and liver, which was explained to be a macrophage- rather than a
T cell-dependent phenomenon. Only one study thus far using
IL-10-deficient mice allowed the attribution of an IL-10-dependent
pathomechanism to a "microbial" virulence factor; mice lacking
IL-10 showed decreased lesion development and reduced parasite burdens
after local Leishmania major infection which was linked to
the absence of IL-12- and TNF--down-regulating IL-10 that is
normally induced by host IgG bound to the Leishmania
amastigote surface in wild-type mice (48).
The striking similarity between V Ag-induced innate
immunity modulation and LPS tolerance might prompt one to assume that V
Ag and LPS share a common Toll-like receptor (TLR) and a corresponding
signal transduction pathway. However, from our study, it can be
concluded that TLR 4 which has been found to transmit LPS effects is
not involved in V Ag-induced cellular responses, because TNF-
suppression could be achieved in PPMs from mice with a missense
mutation in the Tlr4 gene (C3H/HeJ, BALB/c LPSd) and a null
mutation of Tlr4 (C57BL/10ScCr), respectively
(49). It is tempting to speculate that, in analogy to
other bacterial products, V Ag uses a different TLR to exhibit its
immunomodulating features on monocytic cells. Experiments to identify V
Ag-signaling receptors are currently undertaken in our laboratory.
In conclusion, we propose that yersiniae have established
two strategies for suppression of the proinflammatory cytokine TNF-:
1) direct suppression of TNF- via translocated YopP/YopJ (short
distance effect); and 1) indirect suppression of TNF- by released V
Ag via IL-10 induction in bystander macrophages (long distance effect).
The exploitation of endogenous IL-10 by microbes has thus far been
described only for intracellular pathogens (50); in the
present study, we show for the first time that also an extracellular
bacterium is able to use this immunomodulating strategy. Moreover, the
differences between LcrV and PcrV regarding their IL-10-inducing and
TNF--down-regulating capacity might explain why Yersinia
spp. act as pathogenic bacteria, whereas P. aeruginosa must
be considered an opportunistic microorganism.
The perturbation of cytokine networks is increasingly
recognized as a pathogenicity mechanism of bacteria (for a review, see
Ref. 51). For the bacterial proteins responsible for these
effects, the term bacteriokine has been coined. Besides its regulatory
and translocatory features, V Ag causes immunomodulation by interfering
with the host’s TNF- response. Therefore, V Ag may belong to this
newly recognized group of bacterial proteins.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jürgen Heesemann, Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Pettenkoferstrasse 9a, 80336 Munich, Germany. E-mail address: heesemann{at}m3401.mpk.med.uni-muenchen.de
3 Abbreviations used in this paper: TTS, type III protein secretion/translocation apparatus; Yop, Yersinia outer protein; rH, recombinant YopH; MAP, mitogen-activated protein; PPM, proteose peptone-elicited peritoneal macrophage; TLR, Toll-like receptor.
Received for publication September 7, 2001. Accepted for publication November 26, 2001.
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  R. Mittal and N. V. Prasadarao Outer Membrane Protein A Expression in Escherichia coli K1 Is Required to Prevent the Maturation of Myeloid Dendritic Cells and the Induction of IL-10 and TGF-{beta} J. Immunol., August 15, 2008; 181(4): 2672 - 2682. [Abstract] [Full Text] [PDF]
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 J. J. Lazarus, M. A. Kay, A. L. McCarter, and R. M. Wooten Viable Borrelia burgdorferi Enhances Interleukin-10 Production and Suppresses Activation of Murine Macrophages Infect. Immun., March 1, 2008; 76(3): 1153 - 1162. [Abstract] [Full Text] [PDF]
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 J. E. Broms, M. S. Francis, and A. Forsberg Diminished LcrV Secretion Attenuates Yersinia pseudotuberculosis Virulence J. Bacteriol., December 1, 2007; 189(23): 8417 - 8429. [Abstract] [Full Text] [PDF]
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V. Auerbuch and R. R. Isberg Growth of Yersinia pseudotuberculosis in Mice Occurs Independently of Toll-Like Receptor 2 Expression and Induction of Interleukin-10 Infect. Immun., July 1, 2007; 75(7): 3561 - 3570. [Abstract] [Full Text] [PDF]
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 A. McNally, T. Dalton, R. M. L. Ragione, K. Stapleton, G. Manning, and D. G. Newell Yersinia enterocolitica isolates of differing biotypes from humans and animals are adherent, invasive and persist in macrophages, but differ in cytokine secretion profiles in vitro. J. Med. Microbiol., December 1, 2006; 55(Pt 12): 1725 - 1734. [Abstract] [Full Text] [PDF]
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 A. Ben Nasr, J. Haithcoat, J. E. Masterson, J. S. Gunn, T. Eaves-Pyles, and G. R. Klimpel Critical role for serum opsonins and complement receptors CR3 (CD11b/CD18) and CR4 (CD11c/CD18) in phagocytosis of Francisella tularensis by human dendritic cells (DC): uptake of Francisella leads to activation of immature DC and intracellular survival of the bacteria J. Leukoc. Biol., October 1, 2006; 80(4): 774 - 786. [Abstract] [Full Text] [PDF]
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 H. BRAAT, M. P PEPPELENBOSCH, and D. W HOMMES Immunology of Crohn's disease. Ann. N.Y. Acad. Sci., August 1, 2006; 1072: 135 - 154. [Abstract] [Full Text] [PDF]
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 S. A. Handley, P. H. Dube, and V. L. Miller From the Cover: Histamine signaling through the H2 receptor in the Peyer's patch is important for controlling Yersinia enterocolitica infection PNAS, June 13, 2006; 103(24): 9268 - 9273. [Abstract] [Full Text] [PDF]
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R. A. Lukaszewski, D. J. Kenny, R. Taylor, D. G. C. Rees, M. G. Hartley, and P. C. F. Oyston Pathogenesis of Yersinia pestis Infection in BALB/c Mice: Effects on Host Macrophages and Neutrophils Infect. Immun., November 1, 2005; 73(11): 7142 - 7150. [Abstract] [Full Text] [PDF]
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A. Sing, D. Reithmeier-Rost, K. Granfors, J. Hill, A. Roggenkamp, and J. Heesemann A hypervariable N-terminal region of Yersinia LcrV determines Toll-like receptor 2-mediated IL-10 induction and mouse virulence PNAS, November 1, 2005; 102(44): 16049 - 16054. [Abstract] [Full Text] [PDF]
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 G. Maloney, M. Schroder, and A. G. Bowie Vaccinia Virus Protein A52R Activates p38 Mitogen-activated Protein Kinase and Potentiates Lipopolysaccharide-induced Interleukin-10 J. Biol. Chem., September 2, 2005; 280(35): 30838 - 30844. [Abstract] [Full Text] [PDF]
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C. Cowan, A. V. Philipovskiy, C. R. Wulff-Strobel, Z. Ye, and S. C. Straley Anti-LcrV Antibody Inhibits Delivery of Yops by Yersinia pestis KIM5 by Directly Promoting Phagocytosis Infect. Immun., September 1, 2005; 73(9): 6127 - 6137. [Abstract] [Full Text] [PDF]
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K. A. Overheim, R. W. DePaolo, K. L. DeBord, E. M. Morrin, D. M. Anderson, N. M. Green, R. R. Brubaker, B. Jabri, and O. Schneewind LcrV Plague Vaccine with Altered Immunomodulatory Properties Infect. Immun., August 1, 2005; 73(8): 5152 - 5159. [Abstract] [Full Text] |
) or PBS (
). TNF-
) mice after i.p.
infection of 1 x 104 CFU Y.
enterocolitica (WA-314).