Published online before print October 3, 2007
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* Departments of Veterinary and Biomedical Science and
College of Biological Sciences, University of Minnesota, St. Paul, Minnesota, USA
1 Correspondence: Departments of Veterinary and Biomedical Science, University of Minnesota, 1971 Commonwealth Ave., St. Paul, MN 55108, USA. E-mail: weiss005{at}umn.edu
ABSTRACT
Pathogenic mycobacterial organisms have the capacity to inhibit macrophage activation and phagosome maturation. Although the mechanism is complex, several studies have incriminated signaling through TLR2 receptors with subsequent activation of the MAPK pathway p38 (MAPKp38) and overproduction of IL-10 in the survival of pathogenic mycobacterial organisms. In the present study, we compared the response of bovine monocytes with infection by Mycobacterium avium subspecies paratuberculosis (MAP), the cause of paratuberculosis in ruminants, with the closely related organism M. avium subspecies avium (Maa), which usually does not cause disease in ruminants. Both MAP and Maa induced phosphorylation of MAPKp38 by bovine monocytes; however, addition of a blocking anti-TLR2 antibody partially prevented MAPKp38 phosphorylation of MAP-infected monocytes but not Maa-infected monocytes. Addition of anti-TLR2 antibody enhanced phagosome acidification and phagosome-lysosome fusion in MAP-containing phagosomes and enabled monocytes to kill MAP organisms. These changes were not observed in Maa-infected monocytes. The effect on phagosome maturation appears to occur independently from the previously described inhibitory effects of IL-10 on phagosome acidification and organism killing, as IL-10 production was not affected by addition of anti-TLR2 antibody to monocyte cultures. Therefore, signaling through the TLR2 receptor appears to play a role in phagosome trafficking and antimicrobial responses in MAP-infected bovine mononuclear phagocytes.
Key Words: phagosome • MAPK • cytokines • bacterial killing • innate immunity
INTRODUCTION
The immune response to mycobacterial infection involves phagocytosis of organisms by mononuclear phagocytes and sequestration within phagosomes [1 2 3 ]. The capacity of the organism to prevent macrophage activation and phagosome maturation and to attenuate induction of a Th1 immune response appears to largely determine its pathogenicity [3 , 4 ]. Pathogenic mycobacteria block phagosome acidification and limit fusion between the phagosome and early and late endosomes [2 , 5 ]. The mechanism by which mycobacteria inhibit phagosome maturation appears to be complex. Factors incriminated include the receptors involved in phagocytosis, altered calcium signaling, iron acquisition, and receptor-organism interactions within the phagosome [6 7 8 9 10 ].
Recent studies have focused on the role of the TLR family of cell membrane receptors in initiating cell signaling associated with mycobacterial infections [10 11 12 13 14 15 16 17 ]. Mycobacteria interact with several TLR receptors including TLR2, TLR4, and TLR9 [11 , 12 ]. Several studies indicate that pathogenic mycobacteria signal through TLR2 to suppress macrophage antimicrobial responses and antigen presentation [18 , 19 ]. However, the role of TLR receptors in host defense and control of phagosome maturation and organism killing remains controversial [14 , 17 , 20 ].
We have studied the interaction of Mycobacterium avium subsp. paratuberculosis (MAP) with bovine monocytes [21 22 23 ]. MAP is the causative agent of Johne’s disease, a chronic, progressive enteritis in ruminants [24 ]. In vitro, MAP-infected bovine monocytes phosphorylate MAPK pathway p38 (MAPKp38) rapidly, express large amounts of IL-10, and fail to acidify phagosomes or kill MAP organisms [22 , 23 ]. Addition of a neutralizing anti-IL-10 antibody or pharmacologically blocking the MAPKp38 pathway increases phagosome acidification and enabled bovine monocytes to kill MAP organisms [21 , 23 ]. These data suggest that MAP-induced activation of MAPKp38 is a major mechanism involved in suppression of antimicrobial responses within bovine mononuclear phagocytes. As TLRs are a major mediator of MAPK activation, we hypothesized that TLR signaling regulates MAP survival within bovine mononuclear phagocytes [11 12 13 ]. The results of the present study provide evidence that addition of neutralizing anti-TLR2 antibody to bovine monocytes before addition of MAP facilitated phagosome maturation and organism killing. Alternatively, this effect could not be demonstrated with the less-pathogenic organism Mycobacterium avium subsp. avium (Maa).
MATERIALS AND METHODS
Bacterial strain and culture conditions
MAP was isolated from a naturally infected cow evaluated at the Minnesota Animal Health Diagnostic Laboratory (University of Minnesota, St. Paul, MN, USA). The organisms were determined to be MAP based on detection of species-specific DNA sequences by use of a standard PCR and culture characteristics [25
]. Maa strain 35716 was obtained from the American Type Culture Collection (Manassas, VA, USA). This organism had been isolated from a naturally infected cow. Organisms were grown to a concentration of 
108/mL, washed, and resuspended in broth media (OADC, Difco Labs, Detroit, MI, USA), Tween 80 (Sigma Chemical Co., St. Louis, MO, USA), mycobactin J (Allied Monitor Inc., Fayette, MO, USA), and 5% FBS (Allied Monitor Inc.). Viability of the organisms was determined by propidium iodide exclusion (Calbiochem, La Jolla, CA, USA).
Monocyte isolation and culture
Blood was obtained from three healthy adult Holstein cows, which tested negative for paratuberculosis, as determined by culture of fecal samples, PCR assay of fecal samples, and serum ELISA for paratuberculosis [25
]. Mononuclear cells were isolated by use of Percoll (Sigma Chemical Co.) density gradient centrifugation, as described [26
]. Isolated cells were washed in Dulbecco’s PBS solution and resuspended at 1 x 107 mononuclear cells/ml in RPMI-1640 medium containing 10% FBS. For isolation of monocytes, 3 x 107 mononuclear cells were incubated on 22 x 22 mm glass coverslips or in 60 x 15 mm tissue-culture plates for 90 min at 37°C to allow cells to adhere. Nonadherent cells were removed by repeated washing with RPMI-1640 medium warmed to 37°C. Adherent cells were cultured overnight at 37°C with 5% CO2 in RPMI-1640 medium supplemented with 10% FBS. Cell viability was monitored by use of trypan blue exclusion. Organisms were added to culture of bovine monocytes [multiplicity of infection (MOI): 10 bacilli/monocyte], and incubation was continued at 37°C and 5% CO2. In some experiments, the mRNA was harvested from plates using a commercial kit (RNeasy Kit, Qiagen, Valencia, CA, USA), following the manufacturer’s instructions. Integrity of RNA preparations was assessed by use of RNA agarose gel electrophoresis.
Chemical inhibitors and antibodies
Mouse anti-human TLR2 (Clone TL2.1, BioLegend Inc., San Diego, CA, USA) was added to monocyte cultures. Preliminary studies were performed to confirm that anti-human TLR2 bound to bovine monocytes. Cultured bovine monocytes were harvested using a cell scraper. The cells were resuspended in Dulbecco’s PBS containing 2% sheep serum, 5 mM sodium azide, and 0.1% glycine and were incubated with 2 µg/ml anti-TLR2 at 25°C for 30 min. After washing, the cells were incubated with 10 µl of a 1:30 dilution of FITC-labeled anti-mouse IgG (AbD Serotec, Oxford, UK). The cells were washed and evaluated using a flow cytometer (BD FACS Canto, Becton Dickinson, Rutherford, NJ, USA). A gate was set to identify the monocyte population (Fig. 1A
), and the percentage TLR2-positive cells was determined. Greater than 90% of bovine monocytes had evidence of labeling with anti-TLR2 (Fig. 1B)
. Dose-response studies were performed to determine the antibody concentration, which achieved maximal inhibition of MAP-induced phosphorylation of MAPKp38 (data not shown). This concentration was determined to be 10 µg/106 cells. Monocytes were washed with PBS and pretreated with anti-TLR2 or isotype-matched irrelevant mAb (clone MCA 1209EL, AbD Serotec) in RPMI-1640 medium for 30 min before infection with MAP organisms. Assessment by trypan blue exclusion indicated that monocyte viability was not affected by the presence of anti-TLR2 (data not shown).
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Figure 1. Binding of anti-TLR2 antibody (clone TL 2.1) to bovine monocytes as detected by flow cytometry. (A) Forward-angle versus side-angle light-scatter plot with the gate set to identify the monocyte population. (B) Forward-angle light-scatter versus log fluorescence intensity of the monocyte population gated in A. Cells in quadrant Q2 represent the cells with positive TLR2 labeling.
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comparative threshold cycle method [27
]. GAPDH expression was used to normalize the results, which showed no variation in the expression of GAPDH in MAP-infected monocytes treated with anti-TLR2 or AG18 compared with untreated, MAP-infected monocytes (data not shown).
IL-10 ELISA
Organisms were added to cultures of bovine monocytes (MOI 10:1), and supernatants were harvested at 6 h or 24 h. Cytokine sandwich ELISA was used to detect IL-10 from bovine monocyte culture supernatants [29
]. Briefly, 96-well microtiter plates (NUNC Inc., Rochester, NY, USA) were coated with mouse anti-bovine IL-10 (clone CC320, Serotec, Raleigh, NC, USA) overnight at 4°C. Plates were washed with PBS Tween 20 (0.05%) and blocked with PBS/BSA (0.5%) for 1 h at 25°C. Culture supernatants were added to the plates. A bovine IL-10 protein standard (gift from Dr. Chris Howard, Institute of Animal Health, Berkshire, UK) was serially diluted and added to the corner wells. Plates were incubated at 4°C overnight. After washing with PBS Tween 20 (0.05%), the plates were incubated with mouse anti-bovine IL-10 labeled with biotin (clone CC318, Serotec) at 37°C for 3 h. Plates were washed, and streptavidin HRP (Serotec) was added and incubated for 1 h at room temperature. A color-developing solution (BioRad Labs, Hercules, CA, USA) was added subsequently, and plates and were read at 413 nm using an ELISA plate reader.
Western blots
After incubation with organisms (MOI 10:1) for 30 min, monocytes were washed with ice-cold Dulbecco’s PBS, and cellular extracts were harvested using a lysis buffer containing 50 mM Tris-base, Dulbecco’s PBS, 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 1 mM sodium fluoride, 5 mg/ml leupeptin, 5 mg/ml aprotinin, and 2 mM sodium orthovanadate (Cell Signaling Technology, Beverly, MA, USA). Monocytes were incubated on ice for 5 min, scraped, and transferred to a 1.5-ml centrifuge tube. Lysates were centrifuged at 14,000 g for 10 min at room temperature, and supernatants were collected. Samples were loaded onto 4–20% polyacrylamide gels and electrophoresed at 110 V for 45 min. Proteins were transferred to polyvinyldifluoride membranes (Pierce, Rockford, IL, USA) by wet-blotting at 15 mA for 1.5 h. After blocking with 5% (wt/vol) nonfat dry milk in Tris-buffered saline solution containing 0.1% Tween 20, membranes were incubated overnight at 4°C with anti-mouse total MAPKp38 (clone 4377, Cell Signaling Technology) or anti-mouse phospho-MAPKp38 (clone 9102, Cell Signaling Technology) as described [23
]. Blots were washed three times with Tween-Tris-buffered saline [0.1% Tween 20 in 100 mM Tris-HCL, 0.9% NaCl (pH 7.5)] and were incubated for 1 h with HRP-conjugated goat anti-rabbit Ig (Pierce). Membranes were developed using a chemiluminescence assay and subsequent exposure to X-ray film for detection of phosphorylation (Pierce). To eliminate possible endotoxin contamination, MAP organisms were resuspended in endotoxin-free PBS. In addition, some organisms were incubated with polymyxin B (20 µg/ml, Sigma Chemical Co.) before addition to monocyte cultures.
Phagosome acidification
Organisms were labeled with FITC (Sigma Chemical Co.) before they were added to monocyte cultures. Bovine monocytes, grown on 22 x 22 mm coverslips, were incubated with fluorescein-labeled mycobacteria (MOI 10:1) for 18 h. LysoTracker Red (Invitrogen Inc., Carlsbad, CA, USA), at a final concentration of 50 nM, was added during the last 30 min of incubation. This stain is taken up by acidified phagosomes, where it is modified to become fluorescent. After incubation, the coverslips were inverted onto glass slides and evaluated immediately by use of a BioRad 1024 laser-scanning confocal microscope using the 488-nm and 567 laser (Nikon USA, Melville, NY, USA). Z-series of optical section images were collected using a 60x 1.4 n.a. Plan Apo objective. Images of green and red fluorescence were recorded sequentially at increments of 3 µM throughout the depth of the cell. Sequential images were merged, and intensity of green and red fluorescence of at least 100 phagosomes containing mycobacteria was quantified, and results were reported as a LysoTracker Red/FITC colocalization coefficient using ImagePro Plus, Version 6.0 (Media Cybernetics, Silver Spring, MD, USA). The colocalization coefficient was defined as the density of red fluorescence divided by the density of green fluorescence as described [21
22
23
]. The colocalization coefficients for at least 100 phagosomes in each of three separate experiments were used to calculate results. In addition, the percentage of green (i.e., nonacidified) and yellow (i.e., acidified) phagosomes was determined by counting at least 100 phagosomes in each sample. Controls consisted of monocytes incubated with unlabeled organisms, with or without Lysotracker Red, monocytes incubated with labeled organisms without anti-TLR2, and monocytes incubated with labeled organisms without addition of Lysotracker Red.
Phagosome-lysosome fusion
Lysosomal-associated membrane protein 3 (CD63) on the organism-containing phagosomes was quantified as an indication of phagosome-lysosome fusion. Organisms were labeled with FITC before they were added to monocyte cultures, which were grown on coverslips. Processing of cells for immunofluorescence was done using a Pelco model 3450 microwave oven equipped with a temperature-controlled ColdSpot load cooler (Ted Pella, Redding, CA, USA) as described [29
]. Monocytes, plated on coverslips, were fixed in 3% paraformaldehyde in PBS at pH 7.2 for a total of 40 s in the presence of microwaves at a set point temperature of 24°C at 175 W. The coverslips were rinsed in three changes of PBS and then incubated in 5% normal goat serum, 5% glycerol, and 1% cold water fish gelatin in PBS for 6 min (2 min microwave power at 175 W, 2 min no power, and 2 min microwave power at 175 W) at 24°C. Blocked cells were then treated with mouse anti-bovine CD63 antibody (clone CC25, Serotec), diluted 1–25, for a total of 6 min (2 min microwave power at 175 W, 2 min no power, and 2 min microwave power at 175 W). Specimens were washed in three changes of PBS and subsequently treated with Alexa 488-conjugated secondary antibody (Invitrogen, Eugene, OR, USA), diluted 1:400 for a total of 6 min (2 min microwave power at 175 W, 2 min no power, and 2 min microwave power at 175 W). The labeled coverslips were then mounted in 20% glycerol and 1% n-propyl gallate in PBS (pH 7.8) containing 2 µM 4',6-diamidino-2-phenylindole (Invitrogen, Eugene, OR, USA). Preparations were viewed using a Nikon C1si laser-scanning confocal microscope using the 488-nm line of the 40-mW argon laser and an 18-mW 561-nm diode laser (Nikon USA). Z-series optical section images were collected using a 100x 1.4 n.a. Plan Apo VC objective. Projections were made using ImagePro Plus, Version 6.0 software (Media Cybernetics). The intensity of green and red fluorescence of at least 20 phagosomes containing mycobacteria was quantified, and results were reported as a red/green colocalization coefficient, which was defined as the density of red fluorescence (CD63) divided by the density of green fluorescence (organisms). Controls included monocytes incubated with organisms and isotype-matched control antibody, monocytes incubated with organisms without addition of anti-TLR2, and monocytes incubated with organisms without addition of anti-CD63.
Phagocytosis and intracellular survival of mycobacterial organisms
Monocytes were incubated with MAP or Maa organisms (MOI 10:1) for 1 h before staining with the Ziehl-Neelsen carbolfuchsin stain (Sigma Chemical Co.). The percentages of monocytes containing organisms were determined by counting a minimum of 200 cells by use of light microscopy. Killing of organisms was assessed by use of a live-dead stain (BackLight kit, Invitrogen, Carlsbad, CA, USA), as described for mycobacteria [23
]. This technique was chosen rather than the standard serial dilution and colony-counting assay, as the particular strain of MAP used in this study grew poorly on solid media and as clumping of organisms after incubation with monocytes made quantitation by the colony-counting method problematic. After incubation with organisms for 72 h, monocytes were washed twice in Dulbecco’s PBS solution and lysed by incubation with 0.1% deoxycholate for 5 min. The lysate was incubated with a 1:1 mixture of a green fluorescent stain and propidium iodine stain (Calbiochem). Cells were placed on a microscope slide and examined on a Nikon E800 microscope (Nikon USA) at 1500x magnification using a dual-band filter set, which detected fluorescence in the green and red emission spectra. For this method, live organisms had green fluorescence, and dead organisms had red fluorescence. At least 200 organisms were counted.
Statistical analysis
All tests were done in duplicate or triplicate, and results of at least three separate experiments were evaluated. Results were expressed as mean ± SD. Differences between cell cultures incubated with and without addition of inhibitors were analyzed by use of the paired Student’s t-test. P < 0.05 was considered statistically significant.
RESULTS
MAP but not Maa activates MAPKp38 through TLR2
Bovine monocytes were incubated with MAP or Maa organisms (MOI 10:1), and MAPKp38 phosphorylation was determined by Western blotting (Fig. 2
). Addition of both organisms resulted in phosphorylation of MAPKp38 within 30 min. To determine cell membrane receptors involved in signaling, monocytes were preincubated with anti-TLR2. Addition of anti-TLR2 reduced MAP-induced MAPKp38 phosphorylation, suggesting a role of TLR2 in MAP-induced activation of the MAPKp38 pathway. Alternatively, anti-TLR2 had no effect on Maa-induced MAPKp38 phosphorylation.
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Figure 2. Effect of anti-TLR2 on phosphorylation of MAPKp38 associated with MAP or Maa infection of bovine monocytes, which were pretreated with or without anti-TLR2 (10 µg/ml), 30 min before infection (MOI 10:1) for 30 min. Cells were then lysed, and aliquots of total cell lysates were separated by SDS-PAGE and immunoblotted. Blots were incubated with specific anti-phospho-MAPKp38 (p-p38) or anti-total MAPKp38, followed by appropriate peroxidase-coupled secondary reagents and were visualized by X-ray.
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Figure 3. Effect of blocking TLR2 on IL-10 mRNA expression by bovine monocytes 6 h after addition of MAP or Maa organisms. Bovine monocytes were pretreated with or without anti-TLR2 (10 µg/ml), 30 min before infection with MAP or Maa. The incubation was continued for 6 h, and IL-10 mRNA was evaluated by RT-PCR. GAPDH was used to normalize the results. *, Statistically significant difference from the value for monocytes incubated with MAP organisms alone (P<0.05).
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Figure 4. Effect of blocking TLR2 on IL-10 protein production by bovine monocytes 6 h after addition of MAP or Maa organisms. Bovine monocytes were incubated with or without anti-TLR2 (10 µg/ml), 30 min before addition of MAP or Maa. Supernatants were collected after 6 h or 24 h of incubation, and the concentration of IL-10 was determined by ELISA. *, Statistically significant differences when compared with monocytes incubated with MAP alone (P<0.05).
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Figure 5. Effect of blocking TLR2 on phagosome acidification by bovine monocytes 18 h after addition of MAP or Maa organisms. Cultures of bovine monocytes were pretreated with or without anti-TLR2 (10 µg/ml), 30 min before infection with fluorescein-labeled MAP or Maa organisms (MOI 10:1). The intensity of red and green fluorescence for at least 100 phagosomes containing mycobacteria was quantified, and results were reported as a red/green colocalization coefficient. Increased levels of the coefficient indicate greater acidification of phagosomes. *, Statistically significant differences when compared with monocytes incubated with MAP alone (P<0.05).
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Figure 6. Effect of blocking TLR2 on phagosome maturation by bovine monocytes 4 h after addition of MAP or Maa organisms. Cultures of bovine monocytes were pretreated with or without anti-TLR2 (10 µg/ml), 30 min before infection with fluorescein-labeled MAP or Maa organisms (MOI 10:1). Thereafter, cells were fixed and permeabilized and incubated with anti-bovine CD63. The intensity of red and green fluorescence was quantified, and results were reported as a red/green colocalization coefficient. Increased levels of the coefficient mean increased association of CD63 with the phagosome. *, Statistically significant differences when compared with monocytes incubated with MAP alone (P<0.05).
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Results of our previous studies have established that the number of viable MAP organisms stays the same or increases slightly over time when incubated with bovine monocytes, whereas the number of viable Maa organisms decreases [22 ]. Consistent with our previous studies, bovine monocytes were unable to kill MAP organisms but were able to kill approximately half of the Maa organisms within 72 h of coincubation (Fig. 7 ). Preincubation of MAP-infected monocytes with anti-TLR2 resulted in a substantial increase in killing of MAP organisms. Alternatively, killing of Maa organisms was not affected by addition of anti-TLR2.
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Figure 7. Effect of blocking TLR2 on killing of mycobacterial organisms by bovine monocytes 72 h after addition of MAP or Maa organisms. Cultures of bovine monocytes were pretreated with or without anti-TLR2 (10 µg/ml), 30 min before infection with MAP or Maa organisms (MOI 10:1). After incubation with MAP or Maa organisms for 72 h, monocytes were lysed by incubation with 0.1% deoxycholate for 5 min. The lysate was incubated with a BackLight live-dead stain. Cells were placed on a slide and examined using a confocal microscope. Live organisms had green fluorescence, and dead organisms had red fluorescence. *, Statistically significant differences when compared with monocytes incubated with MAP alone (P<0.05).
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Pathogenic mycobacteria have developed highly specialized mechanisms for survival within mononuclear phagocytes. These mechanisms tend to be species-specific in that an organism, which is pathogenic in one species, is less pathogenic or nonpathogenic in another species. For example, MAP is highly pathogenic in ruminants, causing a severe enteritis, which leads to a chronic wasting disease and ultimately, to death [24 ]. Alternatively, Mycobacterium tuberculosis and Mycobacterium leprae, the causes of tuberculoid pneumonia and leprosy, respectively, in humans, have not been documented to be pathogens in ruminants. Maa is an opportunistic pathogen in humans and several other species but has rarely been isolated from ruminants [30 ]. Our laboratory has focused on identifying differences in how bovine monocytes respond to MAP and Maa with the intent of gaining insight into unique host-pathogen interactions, which determine pathogenicity [21 22 23 , 26 ]. In our previous studies, we determined that bovine monocytes or monocyte-derived macrophages incubated with MAP phosphorylated MAPKp38 rapidly, expressed large amounts of IL-10, and failed to acidify phagosomes or kill the organisms [22 , 23 ]. Alternatively, Maa-infected monocytes expressed less IL-10, partially acidified phagosomes, and killed approximately half of the organisms within 96 h [22 ]. Addition of a neutralizing anti-IL-10 antibody to bovine monocytes before addition of MAP organisms facilitated phagosome acidification and organism killing [21 ]. When MAPKp38 was inhibited, monocytes produced less IL-10 and phagosome acidification, and organism killing was enhanced [23 ]. These data implicate IL-10 as a mediator of MAP survival within bovine mononuclear phagocytes.
In the present study, we hypothesized that MAP-induced IL-10 transcription within bovine mononuclear phagocytes was mediated by TLR2 signaling. Through use of a blocking mAb, we showed that TLR2 was involved in MAP-induced MAPKp38 activation but not in Maa-induced MAPKp38 activation. This is interesting, as Maa has been reported to signal through TLR2 in mouse macrophages [31 ]. Although addition of anti-TLR2 reduced IL-10 expression, it did not reduce IL-10 protein production. The failure to reduce IL-10 protein production significantly may be the result of incomplete suppression of MAPKp38 by anti-TLR2. Even slight phosphorylation of MAPKp38 appears to be sufficient to initiate signaling throughout this pathway [32 ]. This is in part as a result of signal amplification within the cascade and signal integration between signaling cascades [32 ].
Unlike Maa-infected monocytes, addition of anti-TLR2 antibodies to MAP-infected monocytes promoted phagosome maturation and organism killing. These data suggest that MAP expresses TLR2 ligands and that the resultant signaling suppresses monocyte antimicrobial activity. We cannot eliminate the possibility that binding of anti-TLR2 antibody to the TLR2 receptor induced an agonist effect, which resulted in antimycobacterial activity. However, we consider this to be unlikely in that TLR2 is now a trigger activation of the MAPKp38, MAPK-ERK, MAPK-JNK, PI-3K, and NF-
B signaling pathways. In the present study, we demonstrated that anti-TLR2 blocked MAPKp38 activation. In recent studies, we have also shown that anti-TLR2 blocks activation of MAPK-ERK and NF-B (unpublished observations). Therefore, it is unlikely that the antibody is acting as an agonist to induce cell activation.
Several recent studies support TLR2 signaling in inhibition of macrophage antimicrobial responses [33
, 34
]. TLR2-deficient (–/–) mice had a normal Th1 immune response to M. tuberculosis infection and had a more extensive pathologic change compared with wild-type mice [36
]. In vitro studies indicated that engagement of TLR2 with the 19-kDa M. tuberculosis ligand decreased macrophage responsiveness to IFN-
and inhibited IFN--induced killing of M. tuberculosis organisms [33
, 36
]. TLR2–/– mouse macrophages were reported to have a greater capacity to kill Staphylococcus aureus organisms but not Escherichia coli organisms compared with wild-type macrophages [34
]. Taken together, these studies indicate that TLR2 receptor-associated signaling may be used by MAP and M. tuberculosis and perhaps by other pathogenic organisms to attenuate macrophage antimicrobial activity and to blunt the Th1 immune response.
Results of our previous studies lead us to conclude that IL-10 was the major mediator of MAP-induced suppression of bovine mononuclear phagocytes [21 22 23 ]. However, in the present study, addition of anti-TLR2 induced phagosome maturation and killing of MAP without altering IL-10 protein secretion significantly. There is some evidence to suggest that the IL-10-independent activity of TLR2 is mediated by signaling across the phagosome membrane. Previous studies have shown that mouse macrophage phagosomes containing M. tuberculosis fail to mature, as indicated by reduced recruitment of the phagosomal membrane-tethering molecule early endosomal autoantigen 1 (EEA1) [10 ]. Addition of a chemical inhibitor of MAPKp38 to mouse macrophages induced phagosome acidification and markers of phagosome maturation including acquisition of EEA1, lysosome-associated membrane protein-3, and lysobisphosphatidic acid [10 ]. In the present study, we were able to reproduce some of these changes by adding anti-TLR2 to bovine monocytes infected with MAP.
Generalization from studies of the maturation of mycobacteria-containing phagosomes is complicated by studies of phagosome maturation using inert particles and other organisms [17 , 20 ]. When compared with wild-type mice, MyD88–/– and TLR2–/–x 4–/– mice infected with E. coli had decreased phagosome acidification and arrested lysosomal fusion with phagosomes [17 , 36 ]. Pharmacologic inhibition of MAPKp38 in wild-type mice blocked phagosome maturation [17 ]. Understanding of the role of TLR2 in phagosome maturation is complicated further by studies of silica beads coated with TLR ligands [20 ]. When phagosomes from TLR2–/– or TLR4–/– mouse macrophages were compared with wild-type macrophages, no differences in phagosome acidification, phagosome-lysosome fusion, or proteinase activity in phagosomes was detected [20 ].
Based on these conflicting reports and our previous studies, we hypothesize that organisms have a complex interaction with the phagosome membrane, which determines phagosome maturation and its ultimate fate. In another study, we have shown that preincubating bovine monocytes with anti-CD11b or a receptor tyrosine kinase inhibitor increased acidification of phagosomes containing MAP organisms (unpublished observations). This suggests that the capacity of MAP to block phagosome maturation is dependent on a complex receptor-ligand relationship between the organism and the phagosome [37 ]. Although TLR2-dependent MAPKp38 signaling appears to be one of the pathways affecting phagosome maturation, other pathways remain to be elucidated. The complexity of this process is highlighted by proteomic analysis of phagosomes, which revealed several hundred distinct proteins associated with the phagosome [38 ].
In conclusion, unlike the less-pathogenic mycobacterial organism Maa, the pathogenic mycobacteria MAP interacts with TLR2 to activate MAPKp38, initiate IL-10 transcription, and inhibit phagosome maturation and organism killing. The effect on phagosome maturation appears to be partially independent from the previously described inhibitory effect of IL-10 on phagosome acidification and organism killing. Therefore, TLR2 ligation appears to play a role in suppressing antimicrobial responses in MAP-infected bovine mononuclear phagocytes.
ACKNOWLEDGEMENTS
This research was supported in part through U.S. Department of Agriculture grants CSREES-NRI 2004-35605-14243 and CSREES-NRI 2005-35204-16198. C. D. S. is a Research Fellow from Coordenacao de Aperfeicoamento de Pessoal de Nível Superior (CAPES; Brazil).
Received July 25, 2007; revised August 21, 2007; accepted September 8, 2007.
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