Materials and Methods

Bacterial strain and culture—Mycobacterium avium subsp paratuberculosis (strain 166) was obtained from a cow with naturally acquired paratuberculosis that was evaluated at the Minnesota Animal Health Diagnostic Laboratory. The organisms were identified as MAP on the basis of their dependency on mycobactin J for growth and on detection of species-specific DNA sequences by use of a standard PCR assay.¹⁶ The organisms were grown to a concentration of approximately 10⁸/mL, washed, and resuspended in broth medium^a supplemented with Tween 80,^b mycobactin J,^c and 5% fetal bovine serum.^c Viability of the organisms varied between 85% and 94%, as determined by propidium iodide^d exclusion. Immediately before addition to monocyte cultures, organisms were washed and resuspended in culture medium.

NF-κβ pathway inhibitor—A specific inhibitor of NF-κβ activation, pyrrolidine dithiocarbamate,^d was used in the study.^17,18,19 Pyrrolidine dithiocarbamate blocks phosphorylation of Iκβ (an inhibitory protein of NF-κβ), thereby preventing degradation of Iκβ and subsequent release of NF-κβ.¹⁹ In preliminary experiments, we evaluated concentrations of pyrrolidine dithiocarbamate that ranged from 10μM to 50μM and determined that a concentration of 25μM resulted in optimal reduction in NF-κβ nuclear DNA binding (data not shown). This concentration was used for the remainder of the study. Monocyte viability, as determined by trypan blue exclusion, was not affected by addition of pyrrolidine dithiocarbamate alone to monocyte cultures (data not shown). At 60 minutes after exposure to the organisms, phagocytosis of MAP by monocytes was not affected by addition of pyrrolidine dithiocarbamate, as determined by light microscopy after staining of cells with acid-fast stain (percentage of phagocytic monocytes with and without addition of pyrrolidine dithiocarbamate was 80 ± 6% and 82 ± 5%, respectively).

Cell isolation and culture—Blood samples were collected from 3 healthy adult Holstein cows that belonged to the University of Minnesota dairy herd. The animals in this herd were regularly tested for paratuberculosis and were known to be negative for MAP infection on the basis of results of microbial culture of feces and a serum ELISA. Peripheral blood mononuclear cells were isolated by use of a density gradient centrifugation medium,^e as described.^5,7 Isolated cells were washed in Dulbecco PBS solution and resuspended (at a concentration of 1 × 10⁷ mononuclear cells/mL) in RPMI medium containing 10% fetal bovine serum. For isolation of monocytes, 3 × 10⁷ mononuclear cells were incubated in tissue culture plates (60 × 15 mm) for 90 minutes at 37°C to allow cells to adhere. Nonadherent cells were removed by repeated washing with RPMI medium that had been warmed to 37°C. Adherent cells were cultured overnight (approx 18 hours) at 37°C in RPMI medium supplemented with 10% fetal bovine serum and 5% carbon dioxide. Mycobacterium avium subsp paratuberculosis organisms (multiplicity of infection, 10 bacilli/monocyte) were added to monocytes, and incubation was continued at 37°C and 5% carbon dioxide_. The mRNA was harvested from plates at 2 or 6 hours by use of a commercial kit.^f Integrity of each RNA preparation was assessed by use of RNA agarose gel electrophoresis.

Determination of NF-κβ–DNA binding by electrophoretic mobility shift assay—After incubation with MAP organisms for 30 or 60 minutes, medium was decanted from the monocyte cultures, 100 μL of icecold cell lysis buffer^g was added, and cells were scraped into 1.5-mL microcentrifuge tubes kept on ice, as described.²⁰ After vortexing vigorously, tubes were centrifuged and pellets were washed in 1 mL of cold buffer solution. The resulting nuclear pellet was resuspended in 100 μL of ice-cold buffer (20μM HEPES [pH, 7.9], 0.4μM NaCl, 1μM EDTA, 1μM ethylene glycol tetraacetic acid, 1μM dithiothreitol, and 1μM phenylmethyl sulfonyl fluoride). After thorough mixing and centrifugation, the supernatant was harvested and frozen at −80°C until used.

The electrophoretic mobility shift assay was conducted by use of a commercially available kit with chemiluminescent detection.^h The nuclear protein extract and biotin end-labeled target DNA were loaded onto a 4% to 20% polyacrylamide gel and underwent electrophoresis at 100 V for 45 minutes. Proteins were transferred to a polyvinyldifluoride membraneⁱ by wet blotting at 380 mA for 30 minutes. The membrane was crosslinked by use of a UV lamp. Blocking steps and addition of streptavidin-horseradish peroxidase conjugate were performed according to the manufacturer's instructions. Membranes were placed in film cassettes and exposed to radiographic film for 2 to 5 minutes.

Determination of cytokine gene expression by real time-PCR assay—Commercially available kits^f,j were used to extract mRNA and remove genomic DNA according to the manufacturers' instructions.⁵ First-strand cDNA was synthesized by addition of random primers in 20 μL of reverse transcription mix (1X first-strand buffer containing dithiothreitol [10μM], dinitrophenyl [500nM], 20 units of RNase inhibitor, and 100 units of reverse transcriptase). Then, cDNA was diluted to 100 μL (total volume), and SYBR green master-mix^k was added. Samples were analyzed in triplicate in a 96-well optical reaction plate^k; each sample contained 5 μL of cDNA and 15 μL of SYBR green master mix. Primers were designed by use of a Web-based program (Appendix).^l Results were expressed as relative fold expression by use of the Delta cycle threshold method.²¹ Glyceraldehyde-3-phosphate dehydrogenase expression was used to adjust (normalize) the results. Preliminary results indicated no variation in the expression of glyceraldehyde-3-phosphate dehydrogenase in MAP-exposed monocytes treated with pyrrolidine dithiocarbamate, compared with untreated MAP-exposed monocytes.

Acidification of phagosomes—Prior to their addition to monocyte cultures, MAP organisms were labeled with fluorescein isothiocyanate.^5,6 Bovine monocytes that had been grown on cover slips (22 × 22 mm) were incubated with fluorescein-labeled mycobacteria (multiplicity of infection, 10 bacilli/monocyte) for 6 hours. During the last 30 minutes of incubation, a red-fluorescent dye^m that stains acidic compartments in live cells was added (final concentration, 50nM). This stain is modified to become fluorescent within acidified phagosomes. After incubation, the coverslips were inverted onto glass slides and evaluated by use of a confocal microscope.ⁿ Intensity of green and red fluorescence was sequentially recorded at increments of 3 μm throughout the depth of a cell; sequential images were merged, and the intensity of red and green fluorescence of phagosomes containing mycobacteria was quantified. Results were reported as a red-green colocalization coefficient. The colocalization coefficient was defined as the density of red fluorescence divided by the density of green fluorescence. The colocalization coefficients of at least 100 phagosomes within each experiment were determined, and colocalization coefficients for at least 3 separate experiments were used to calculate results. Control specimens consisted of monocytes incubated with unlabeled MAP organisms with or without the red fluorescent probe, monocytes incubated with labeled organisms without pyrrolidine dithiocarbamate, and monocytes incubated with labeled organisms without addition of the red fluorescent probe.

Phagocytosis and intracellular survival of MAP organisms—Monocytes were incubated with MAP for 1.5 hours before staining with Ziehl-Neelsen carbolfuchsin stain.^b The percentage of monocytes containing organisms was determined by counting a minimum of 200 cells via light microscopy. Killing of organisms was assessed by use of a live-dead stain^o as described.⁵ This technique was chosen rather than use of the more standard serial dilution and colony-counting assay because the particular organism used grew poorly on solid media and because clumping of organisms after incubation with monocytes made quantitation by the colony-counting method problematic. After incubation with MAP organisms for 72 hours, monocytes were washed twice in Dulbecco PBS solution and were lysed by incubation with 0.1% deoxycholate for 5 minutes. The lysate was incubated with a 1:1 mixture of a green fluorescent stain and propidium iodine stain.^o Samples were placed on a microscope slide and examined by use of a confocal microscope.ⁿ For this method, live organisms had green fluorescence and dead organisms had red fluorescence. At least 200 organisms were counted, and the percentage of live organisms was determined.

Apoptosis—Apoptosis of bovine monocytes was evaluated by use of a nuclear staining technique.^22,p Other methods to detect apoptosis were not used because of technical problems with the assays; bovine monocytes do not stain with annexin V, and use of the terminal deoxynucleotidyltransferase-mediated uridine triphosphate-biotin nick end-labeling assay on those cells has been reported to yield inconsistent results.²² Monocytes were incubated with and without MAP organisms (multiplicity of infection, 10 bacilli/monocyte) for 24 hours. Pyrrolidine dithiocarbamate or vehicle was added to some cultures 1 hour before incubation with MAP. Culture plates were then incubated with a fluorescent stainⁿ for 10 minutes.²² Cultures were examined by use of a fluorescence microscope^o; at least 200 cells were counted on each of 2 slides, and the percentage of fluorescent nuclei was determined.

Statistical analysis—All tests were done in duplicate or triplicate, and results of at least 3 separate experiments were evaluated. Results were expressed as mean ± SD. Differences in variables of interest between cell cultures incubated with and without addition of pyrrolidine dithiocarbamate were analyzed by use of the paired student t test. A value of P < 0.05 was considered significant.

Results

MAP-induced signaling through the NF-κβ pathway—Monocytes were incubated with MAP organisms for 30 or 60 minutes, and NF-κβ–DNA binding was determined by use of an electrophoretic mobility shift assay. In unexposed monocytes, NF-κβ–DNA binding was detectable (Figure 1); addition of pyrrolidine dithiocarbamate to unexposed monocytes did not alter NF-κβ–DNA binding (data not shown). At both 30 and 60 minutes after exposure to MAP, NF-κβ–DNA binding was markedly increased when compared to findings for monocytes that were not incubated with MAP. Incubation of monocytes with pyrrolidine dithiocarbamate before addition of MAP organisms reduced NF-κβ–DNA binding to levels similar to that detected in unexposed monocytes.

Nuclear factor-κβ–DNA binding in bovine monocytes alone and in bovine monocytes incubated with MAP organisms for 60 minutes with or without pyrrolidine dithiocarbamate (inhibitor of NF-κβ activation; 25μM) as detected by analysis of nuclear extracts via an electrophoretic mobility shift assay. Similar data were obtained in 3 independent experiments. Arrows indicate NF-κβ–bound biotin-labeled DNA probe (NF-κβ) and excess unlabeled biotin-labeled DNA probe (free probe). In some experiments, an excess of unlabeled DNA probe was added (free probe [competitor]; negative control treatment). – = Not present. + = Present.

Citation: American Journal of Veterinary Research 69, 6; 10.2460/ajvr.69.6.804

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Nuclear factor-κβ–DNA binding in bovine monocytes alone and in bovine monocytes incubated with MAP organisms for 60 minutes with or without pyrrolidine dithiocarbamate (inhibitor of NF-κβ activation; 25μM) as detected by analysis of nuclear extracts via an electrophoretic mobility shift assay. Similar data were obtained in 3 independent experiments. Arrows indicate NF-κβ–bound biotin-labeled DNA probe (NF-κβ) and excess unlabeled biotin-labeled DNA probe (free probe). In some experiments, an excess of unlabeled DNA probe was added (free probe [competitor]; negative control treatment). – = Not present. + = Present.

Citation: American Journal of Veterinary Research 69, 6; 10.2460/ajvr.69.6.804

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Nuclear factor-κβ–DNA binding in bovine monocytes alone and in bovine monocytes incubated with MAP organisms for 60 minutes with or without pyrrolidine dithiocarbamate (inhibitor of NF-κβ activation; 25μM) as detected by analysis of nuclear extracts via an electrophoretic mobility shift assay. Similar data were obtained in 3 independent experiments. Arrows indicate NF-κβ–bound biotin-labeled DNA probe (NF-κβ) and excess unlabeled biotin-labeled DNA probe (free probe). In some experiments, an excess of unlabeled DNA probe was added (free probe [competitor]; negative control treatment). – = Not present. + = Present.

Citation: American Journal of Veterinary Research 69, 6; 10.2460/ajvr.69.6.804

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Effect of the NF-κβ on expression of IL-10, TNF-α, and IL-12 mRNAs—Bovine monocytes were or were not treated with pyrrolidine dithiocarbamate for 1 hour at 37°C before exposure to MAP. Treatment with pyrrolidine dithiocarbamate alone had no effect on cytokine expression (data not shown). Subsequently, monocytes were exposed to MAP for 2 or 6 hours, and expression of TNF-α, IL-10, and IL-12 mRNAs were analyzed via real-time PCR assays. Monocytes exposed to MAP had greater expression of TNF-α mRNA at 2 and 6 hours (Figure 2). Mycobacterium avium subsp paratuberculosis–exposed monocytes that were treated with pyrrolidine dithiocarbamate had lower expression of TNF-α mRNA at 2 and 6 hours, compared with findings in MAP-exposed monocytes that were not treated with pyrrolidine dithiocarbamate. Mycobacterium avium subsp paratuberculosis–exposed monocytes had greater expression of IL-10 mRNA at 2 and 6 hours, compared with findings in unexposed cells. Mycobacterium avium subsp paratuberculosis–exposed monocytes that were treated with pyrrolidine dithiocarbamate had lower IL-10 mRNA expression at 2 and 6 hours, compared with MAP-exposed monocytes that were not treated with pyrrolidine dithiocarbamate. Interleukin-12 expression at 2 and 6 hours in MAP-exposed monocytes was greater than findings in unexposed monocytes. Compared with findings in MAP-exposed monocytes that were not treated with pyrrolidine dithiocarbamate, MAP-exposed monocytes that were treated with pyrrolidine dithiocarbamate had greater IL-12 mRNA expression at 2 and 6 hours.

Effect of inhibition of the NF-κβ pathway on expression of TNF-α (A), IL-10 (B), and IL-12 (C) mRNAs by bovine monocytes 2 (black bars) or 6 (gray bars) hours after exposure to MAP. Monocytes were or were not treated with pyrrolidine dithiocarbamate (PDTC; 25μM) for 1 hour before addition of MAP organisms; control samples were composed of monocytes incubated with vehicle alone and monocytes incubated with organisms alone. Results are expressed as mean ± SD relative fold expression by use of the Delta cycle threshold method, and glyceraldehyde-3-phosphate dehydrogenase expression was used to normalize the results. *For this group at this time point, value was significantly (P < 0.05) different from the corresponding value for the MAP-exposed monocytes that were not treated with PDTC.

Citation: American Journal of Veterinary Research 69, 6; 10.2460/ajvr.69.6.804

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Effect of inhibition of the NF-κβ pathway on expression of TNF-α (A), IL-10 (B), and IL-12 (C) mRNAs by bovine monocytes 2 (black bars) or 6 (gray bars) hours after exposure to MAP. Monocytes were or were not treated with pyrrolidine dithiocarbamate (PDTC; 25μM) for 1 hour before addition of MAP organisms; control samples were composed of monocytes incubated with vehicle alone and monocytes incubated with organisms alone. Results are expressed as mean ± SD relative fold expression by use of the Delta cycle threshold method, and glyceraldehyde-3-phosphate dehydrogenase expression was used to normalize the results. *For this group at this time point, value was significantly (P < 0.05) different from the corresponding value for the MAP-exposed monocytes that were not treated with PDTC.

Citation: American Journal of Veterinary Research 69, 6; 10.2460/ajvr.69.6.804

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Effect of inhibition of the NF-κβ pathway on expression of TNF-α (A), IL-10 (B), and IL-12 (C) mRNAs by bovine monocytes 2 (black bars) or 6 (gray bars) hours after exposure to MAP. Monocytes were or were not treated with pyrrolidine dithiocarbamate (PDTC; 25μM) for 1 hour before addition of MAP organisms; control samples were composed of monocytes incubated with vehicle alone and monocytes incubated with organisms alone. Results are expressed as mean ± SD relative fold expression by use of the Delta cycle threshold method, and glyceraldehyde-3-phosphate dehydrogenase expression was used to normalize the results. *For this group at this time point, value was significantly (P < 0.05) different from the corresponding value for the MAP-exposed monocytes that were not treated with PDTC.

Citation: American Journal of Veterinary Research 69, 6; 10.2460/ajvr.69.6.804

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Effect of NF-κβ on phagosome acidification—Bovine monocytes had minimal capacity to acidify MAP-containing phagosomes, as indicated by low colocalization coefficients (Table 1). Pretreatment with pyrrolidine dithiocarbamate had no effect on acidification of MAP-containing phagosomes.

Effect of NF-κβ on organism ingestion and killing—Via light microscopy, the percentage of cells that had ingested MAP organisms was determined after staining of samples with Ziehl-Neelsen carbolfuchsin stain; after 72 hours of incubation of organisms with monocytes, the percentage of dead organisms was determined by use of a live-dead stain. Bovine monocytes rapidly phagocytosed MAP organisms (Table 1). Addition of pyrrolidine dithiocarbamate had no effect on the percentage of monocytes that had ingested organisms after 1.5 hours. Bovine monocytes had negligible capacity to kill MAP organisms after 72 hours of incubation. Addition of pyrrolidine dithiocarbamate had no effect on the capacity of monocytes to kill MAP organisms.

Effect of NF-κβ on apoptosis—Incubation of monocytes with MAP organisms for 24 hours resulted in a modest increase in the percentage of apoptotic monocytes (24 ± 4%), compared with negative control cultures (16 ± 3%). Addition of pyrrolidine dithiocarbamate to MAP-exposed monocytes resulted in an increase in the percentage of apoptotic monocytes (Table 1).

Discussion

Pathogenic mycobacteria have developed specialized mechanisms for survival within mononuclear phagocytes. In our previous studies,^5,6,7 we determined that bovine monocytes incubated with MAP rapidly phosphorylated MAPK-p38, expressed large amounts of IL-10, and failed to acidify phagosomes or kill the mycobacterial organisms. Addition of a neutralizing anti–IL-10 antibody to bovine monocytes before addition of MAP organisms increased phagosome acidification and enabled monocytes to kill MAP organisms.⁶ When MAPK-p38 was inhibited, monocytes produced less IL-10 and phagosome acidification and organism killing was enhanced.⁵ Results of other studies^23,24 indicate that inhibition of other major MAPK pathways, including MAPK^ERK and MAPK-c-jun N-terminal kinase, has no effect on IL-10 expression in bovine monocytes. These data suggest that IL-10 is an important mediator of MAP survival within bovine mononuclear phagocytes and that MAPK-p38 is a major pathway that induces IL-10 transcription.

The NF-κβ pathway is another important pathway in the response of mononuclear phagocytes to mycobacterial infection.^12,13 Both the NF-κβ and MAPK pathways are activated through cell membrane toll-like receptors.^14,25 Several recent studies^12,13,26 have revealed that blocking signaling through the MAPK-p38 pathway decreases activation of NF-κβ. These data indicate that NF-κβ may be a major factor in mediation of transcriptional events induced by the MAPK-p38 pathway.^12,14 In the present study, blockade of NF-κβ activation by use of a specific chemical inhibitor resulted in some but not all of the changes associated with blockade of MAPKp38 activation. Blockade of the activation of the NF-κβ pathway resulted in decreased IL-10 mRNA expression and increased IL-12 mRNA expression. Both of these changes were evident when MAPK-p38 was blocked.⁵ These data indicate that NF-κβ is one of the transcription factors involved in initiating IL-10 transcription. Early production of IL-10 could suppress transcription of IL-12^5,27; therefore, the effect on IL-12 mRNA expression could be indirect. Blockade of NF-κβ also resulted in a decrease in TNF-α mRNA expression in the present study. A similar effect was observed when the MAPK^ERK or MAPK-c-jun N-terminal kinase pathway was blocked in MAP-exposed bovine monocytes but was not evident when the MAPK-p38 pathway was blocked in other studies.^5,23,24 Nuclear factor-κβ has been identified previously as a potential initiator of TNF-α mRNA transcription.¹⁰

As in previous investigations,^5,6,7 bovine monocytes rapidly phagocytosed MAP organisms but failed to acidify phagosomes or kill the organisms in the study of this report. Blockade of NF-κβ–DNA binding failed to alter organism phagocytosis, phagosome acidification, or organism killing by the monocytes. Results of other studies^5,6 had indicated that addition of a neutralizing anti– IL-10 antibody or blockade of the MAPK-p38 pathway during exposure of monocytes to MAP increased the cells' phagosome acidification and MAP killing. These data indicate that NF-κβ may not be the transcription factor involved in mediating these antimicrobial events. This is consistent with evidence that MAPK-p38 is able to activate several transcription factors in addition to NF-κβ, including cAMP response element and activating transcription factors 1 and 2.²⁵

A well-known function of NF-κβ is to block apoptosis in a variety of cells.¹⁴ Nuclear factor-κβ upregulates expression of the caspase 8 inhibitor Fas-associated protein with death domain-like IL-1 B-converting enzyme inhibitory protein, resulting in increased resistance to Fas ligand– or TNF-mediated apoptosis in mouse macrophages.²⁸ In the present study, blockade of the NF-κβ activation pathway resulted in accelerated apoptosis in MAP-exposed bovine monocytes. Delayed apoptosis may provide a survival advantage for mycobacterial organisms because organisms within apoptotic cells are readily phagocytized and organisms are killed by other cells.²⁹

The results of the present study indicated that the NF-κβ signaling pathway is activated in MAP-exposed bovine monocytes, and findings of blocking experiments indicated that NF-κβ may be involved in initiation of the transcription of cytokines, including TNF-α and IL-10. Results of the blocking experiments further indicated that NF-κβ is involved in prevention of apoptosis but does not appear to play an important role in phagosome acidification or organism killing. Results of other recent studies^30,31,32 in bovine monocytes indicate that MAP organisms initiate MAPK-p38 pathway activation through interaction with toll-like receptor-2. Because toll-like receptor-2 is known to activate the NF-κβ pathway, it is a potential mediator of MAP-induced NF-κβ pathway activation.^14,15 The findings of our study taken together with results of other studies suggest that NF-κβ and MAPK-p38 appear to be major pathways involved in initiating early transcription of IL-10 by MAP-exposed bovine monocytes.