A Mycobacterium avium subsp. paratuberculosis relA deletion mutant and a 35 kDa major membrane protein elicit development of cytotoxic T lymphocytes with ability to kill intracellular bacteria.

Efforts to develop live attenuated vaccines against Mycobacterium avium subspecies paratuberculosis (Map), using indirect methods to screen Map deletion mutants for potential efficacy, have not been successful. A reduction in the capacity to survive in macrophages has not predicted the ability of mutants to survive in vivo. Previous studies for screening of three deletion mutants in cattle and goats revealed one mutant, with a deletion in relA (ΔMap/relA), could not establish a persistent infection. Further studies, using antigen presenting cells (APC), blood dendritic cells and monocyte derived DC, pulsed with ΔMap/relA or a 35 kDa Map membrane protein (MMP) revealed a component of the response to ΔMap/relA was directed towards MMP. As reported herein, we developed a bacterium viability assay and cell culture assays for analysis and evaluation of cytotoxic T cells generated against ΔMap/relA or MMP. Analysis of the effector activity of responding cells revealed the reason ΔMap/relA could not establish a persistent infection was that vaccination elicited development of cytotoxic CD8 T cells (CTL) with the capacity to kill intracellular bacteria. We demonstrated the same CTL response could be elicited with two rounds of antigenic stimulation of APC pulsed with ΔMap/relA or MMP ex vivo. Cytotoxicity was mediated through the perforin granzyme B pathway. Finally, cognate recognition of peptides presented in context of MHC I and II molecules to CD4 and CD8 T cells is required for development of CTL.

Bovine monocyte TLR2 receptors differentially regulate the intracellular fate of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium.

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.

A peptide-based vaccine for Mycobacterium avium subspecies paratuberculosis.

Recent efforts to develop a live attenuated vaccine against Mycobacterium avium subsp. paratuberculosis (Map), the causative agent of Johne's disease (JD), revealed relA is important in Map virulence. Deletion of the relA gene impairs the ability of Map to establish a persistent infection. Analysis of the basis for this observation revealed infection with a relA deletion mutant (ΔrelA) elicits development of cytotoxic CD8 T cells (CTL) with the ability to kill intracellular bacteria. Further analysis of the recall response elicited by ΔrelA vaccination showed a 35 kDa membrane peptide (MMP) is one of the targets of the immune response, suggesting it might be possible to develop a peptide-based vaccine based on MMP. To explore this possibility, ex vivo vaccination studies were conducted with MMP alone and incorporated into a nanoparticle (NP) vector comprised of poly (D, L-lactide-co-glycolide) and monophosphoryl lipid A (PLGA/MPLA). As reported, ex vivo vaccination studies showed CD8 CTL were elicited with classic and monocyte derived dendritic cells (cDC and MoDC) pulsed with MMP alone and incorporated into a PGLA/MPLA vector. Incorporation of MMP into a NP vector enhanced the ability of CD8 CTL to kill intracellular bacteria. The findings indicate incorporation of MMP into a PGLA/MPLA nanoparticle vector is one of the possible ways to develop a MMP based vaccine for Johne's disease.

Effects of nuclear factor-kappaB on regulation of cytokine expression and apoptosis in bovine monocytes exposed to Mycobacterium avium subsp paratuberculosis.

OBJECTIVE: To evaluate the role of the nuclear factor-kappaB (NF-kappaB) in the response of bovine monocytes to exposure to Mycobacterium avium subsp paratuberculosis (MAP). SAMPLE POPULATION: Monocytes from healthy adult Holstein cows that were known to be negative for MAP infection. PROCEDURES: Monocytes were incubated with MAP organisms with or without a specific inhibitor of the NF-kappaB pathway (pyrrolidine dithiocarbamate), and activation of the NF-kappaB pathway was detected by use of an electrophorectic mobility shift assay. The capacities of monocytes to express tumor necrosis factor (TNF)-alpha, interleukin (IL)-10, and IL-12; to acidify phagosomes; to phagocytize and kill MAP organisms; and to undergo apoptosis were evaluated. RESULTS: Addition of MAP organisms to monocytes activated the NF-kappaB pathway as indicated by increased NF-kappaB-DNA binding. Addition of pyrrolidine dithiocarbamate prevented nuclear translocation of NF-kappaB, decreased expression of TNF-alpha and IL-10, and increased IL-12 expression. Treatment of MAP-exposed monocytes with pyrrolidine dithiocarbamate increased the rate of apoptosis but failed to alter phagosome acidification, organism uptake, or organism killing by those cells. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that NF-kappaB rapidly translocated to the nucleus after exposure of bovine monocytes to MAP organisms. These data suggest that NF-kappaB is involved in initiation of inflammatory cytokine transcription and inhibition of apoptosis but that it is not directly involved in phagosome acidification or organism killing.

Role of the mitogen-activated protein kinase pathway in the differential response of bovine monocytes to Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium.

We compared the kinetics of activation and antimicrobial activities of MAPK-p38 and MAPK-ERK in bovine monocytes infected with Mycobacterium avium subsp. paratuberculosis (MAP) and Mycobacterium avium subsp. avium (Maa). Monocytes were incubated with MAP or Maa organisms with or without a specific inhibitor of the MAPK-p38 pathway (SB203580), and MAPK phosphorylation and antimicrobial functions of monocytes were evaluated. At early time points MAPK-p38 phosphorylation was greater in MAP-infected bovine monocytes than in Maa-infected monocytes. At later time points MAPK-p38 phosphorylation by both organisms was similar. MAPKp38 phosphorylation in MAP-infected monocytes was similar to negative control cells, whereas in Maa-infected this activation remained greater than negative control cells. Increase phosphorylation MAPK-ERK was similar at all time points for both organisms. Bovine monocytes had minimal capacity to kill MAP organisms, to acidify MAP-containing phagosomes, or to form phagolysosome. Alternatively, bovine monocytes were able to kill Maa organisms. Addition of SB203580 to monocyte cultures increased phagosome acidification, phagolysosome formation, and killing of MAP and Maa organisms. Taken together these data indicate that early transient activation of MAPK-p38 in bovine mononuclear phagocytes by MAP organisms may be a key mechanism involved in the capacity of MAP to survive in bovine monocytes.

Role of the MAPK(ERK) pathway in regulation of cytokine expression by Mycobacterium avium subsp paratuberculosis-exposed bovine monocytes.

OBJECTIVE: To evaluate the role of the mitogen-activated protein kinase extracellular signal-regulated kinase (MAPK(ERK)) pathway in the interaction between Mycobacterium avium subsp paratuberculosis (MAP) organisms and bovine monocytes. SAMPLE POPULATION: Monocytes obtained from healthy adult Holstein dairy cows that were not infected with MAP organisms. PROCEDURES: Monocytes and MAP organisms were incubated together with or without a specific inhibitor of the MAPK(ERK) pathway (PD98059), and the capacity of monocytes to express tumor necrosis factor alpha (TNF)-alpha and interleukin (IL)-10 and -12, produce nitric oxide, acidify phagosomes, kill MAP organisms, and undergo apoptosis was evaluated. RESULTS: The MAPK(ERK) pathway was activated within 10 minutes after addition of MAP organisms to monocytes. Addition of PD98059 to monocyte-MAP mixtures decreased monocyte TNF-alpha and IL-12 mRNA expression but had no effect on IL-10 mRNA expression. Treatment with PD98059 failed to induce significant alterations in phagosome acidification, organism killing, nitric oxide production, or apoptosis of MAP-exposed monocytes. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that the MAPK(ERK) pathway was activated during the interaction of MAP organisms with monocytes, which initiated TNF-alpha and IL-12 mRNA expression but failed to initiate antimicrobial activity. The MAPK(ERK) pathway may be involved in initiating proinflammatory and proimmune responses in MAP infection in cattle.

Cell membrane receptors on bovine mononuclear phagocytes involved in phagocytosis of Mycobacterium avium subsp paratuberculosis.

OBJECTIVE: To determine cell membrane receptors involved in phagocytosis of Mycobacterium avium subsp paratuberculosis (MAP) organisms. SAMPLE POPULATION: Monocytes were obtained from healthy adult Holstein dairy cows that were test negative for MAP infection on the basis of bacteriologic culture of feces and serologic test results. PROCEDURES: Monocytes or bovine macrophage cell line (BoMac) cells were incubated with MAP organisms for 30, 60, or 120 minutes with or without inhibitors of integrins, CD14, or mannose receptors. Phagocytosis was evaluated by light microscopy or by flow cytometry. CD11a/CD18, CD11b, and CD14 expression on monocytes and BoMac cells was evaluated by use of flow cytometry. RESULTS: Monocytes and BoMac cells rapidly phagocytized MAP organisms. However, compared with BoMac cells, monocytes had a greater total capacity to phagocytize MAP organisms. Addition of neutralizing anti-integrin antibodies (anti-CD11a/CD18 and anti-CD11b) substantially inhibited phagocytosis by monocytes during the first 60 minutes of incubation with MAP organisms, but were less effective at 120 minutes of incubation. Anti-CD11a/CD18 and anti-CD11b antibodies were less effective in inhibiting phagocytosis by BoMac cells. Addition of inhibitors of CD14 or mannose receptors also inhibited phagocytosis of MAP by monocytes. Addition of a combination of integrin and mannose inhibitors had an additive effect in reducing phagocytosis, but addition of integrin and CD14 inhibitors did not have an additive effect. CONCLUSIONS AND CLINICAL RELEVANCE: Multiple receptors are involved in phagocytosis of MAP organisms. Although CD11/CD18 receptors appear to be the major receptors used by MAP at early time points, mannose receptors and CD14 also contribute substantially to phagocytosis.

Regulation by Jun N-terminal kinase/stress activated protein kinase of cytokine expression in Mycobacterium avium subsp paratuberculosis-infected bovine monocytes.

OBJECTIVE: To evaluate activation of Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway in bovine monocytes after incubation with Mycobacterium avium subsp paratuberculosis (Mptb) organisms. SAMPLE POPULATION: Bovine monocytes obtained from 4 healthy adult Holstein dairy cows. PROCEDURES: Bovine monocytes were incubated with Mptb organisms with or without a specific inhibitor of the JNK/SAPK pathway (SP600125) for 2, 6, 24, or 72 hours. Expression of interleukin (IL)-1beta, IL-10, IL-12, IL-18; transforming growth factor-beta (TGF-beta); and tumor necrosis factor-alpha (TNF-alpha) and the capacity of Mptb-infected monocytes to acidify phagosomes and kill Mptb organisms were evaluated. Phosphorylation status of JNK/SAPK was evaluated at 10, 30, and 60 minutes after Mptb incubation. RESULTS: Compared with uninfected control monocytes, Mptb-infected monocytes had increased expression of IL-10 at 2 and 6 hours after incubation and had increased expression of TNF-alpha, IL-1beta, IL-18, and TGF-beta at 2, 4, and 6 hours. Additionally, Mptb-infected monocytes had increased expression of IL-12 at 6 and 24 hours. Addition of SP600125 (specific chemical inhibitor of JNK/SAPK) resulted in a decrease in TNF-alpha expression at 2, 6, and 24 hours, compared with untreated Mptb-infected cells. Addition of SP600125 resulted in a decrease in TGF-beta expression at 24 hours and an increase in IL-18 expression at 6 hours. Addition of SP600125 failed to alter phagosome acidification but did enhance the capacity of monocytes to kill Mptb organisms. CONCLUSIONS AND CLINICAL RELEVANCE: Activation of JNK/SAPK may be an important mechanism used by Mptb to regulate cytokine expression in bovine monocytes for survival and to alter inflammatory and immune responses.

In Vitro Evaluation of the Biological Responses of Canine Macrophages Challenged with PLGA Nanoparticles Containing Monophosphoryl Lipid A.

Poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) have been considerably studied as a promising biodegradable delivery system to induce effective immune responses and to improve stability, safety, and cost effectiveness of vaccines. The study aimed at evaluating early inflammatory effects and cellular safety of PLGA NPs, co-encapsulating ovalbumin (PLGA/OVA NPs), as a model antigen and the adjuvant monophosphoryl lipid A (PLGA/MPLA NPs) as an adjuvant, on primary canine macrophages. The PLGA NPs constructs were prepared following the emulsion-solvent evaporation technique and further physic-chemically characterized. Peripheral blood mononuclear cells were isolated from canine whole blood by magnetic sorting and further cultured to generate macrophages. The uptake of PLGA NP constructs by macrophages was demonstrated by flow cytometry, transmission electron microscopy and confocal microscopy. Macrophage viability and morphology were evaluated by trypan blue exclusion and light microscopy. Macrophages were immunophenotyped for the expression of MHC-I and MHC-II and gene expression of Interleukin-10 (IL-10), Interleukin-12 (IL-12p40), and tumor necrosis factor alpha (TNF-α) were measured. The results showed that incubation of PLGA NP constructs with macrophages revealed effective early uptake of the PLGA NPs without altering the viability of macrophages. PLGA/OVA/MPLA NPs strongly induced TNF-α and IL-12p40 expression by macrophages as well as increase relative expression of MHC-I but not MHC-II molecules. Taken together, these results indicated that PLGA NPs with addition of MPLA represent a good model, when used as antigen carrier, for further, in vivo, work aiming to evaluate their potential to induce strong, specific, immune responses in dogs.

Expression of interleukin-10 and suppressor of cytokine signaling-3 associated with susceptibility of cattle to infection with Mycobacterium avium subsp paratuberculosis.

OBJECTIVE: To determine functional characteristics of monocytes obtained from cows with subclinical infection with Mycobacterium avium subsp paratuberculosis (MAP) that may have predisposed those cows to becoming infected with MAP SAMPLE POPULATION: Monocytes obtained from 5 uninfected cows and 5 cows subclinically infected with MAP in a herd with a high prevalence of paratuberculosis (ie, Johne's disease). PROCEDURES: Monocytes from uninfected and subclinically infected cows were incubated with MAP for 2, 6, 24, 72, or 96 hours. Variables measured included expression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-10, IL-12, transforming growth factor-beta, and suppressor of cytokine signaling-3 (SOCS-3); apoptosis of monocytes; acidification of phagosomes; and killing of MAP. RESULTS: Monocytes from infected cows had greater expression of IL-10 and SOCS-3 at 2 hours of coincubation with MAP and lower expression of TNF-alpha and IL-12 when results for all incubation times were combined. Monocytes from infected cows had a greater capacity to acidify phagosomes. No differences were observed in the rate of apoptosis or capacity of monocytes to kill MAP organisms. CONCLUSIONS AND CLINICAL RELEVANCE: Monocytes obtained from cows with subclinical infection with MAP had upregulated expression of IL-10 and SOCS-3 within the first 2 hours after exposure to MAP organisms. Although this did not inhibit acidification of phagosomes, apoptosis of monocytes, or attenuation of the capacity to kill MAP organisms, it may have attenuated the capacity of mononuclear phagocytes to initiate inflammatory and adaptive immune responses.

Blocking the mitogen activated protein kinase-p38 pathway is associated with increase expression of nitric oxide synthase and higher production of nitric oxide by bovine macrophages infected with Mycobacterium avium subsp paratuberculosis.

This study evaluated the role of the mitogen-activated protein kinase (MAPK)-p38 pathway in the nitric oxide synthase (iNOS) expression and nitric oxide (NO) production by bovine monocyte-derived macrophages ingesting Mycobacterium avium subsp. paratuberculosis (MAP) organisms in vitro. Bovine monocyte-derived macrophages were incubated with MAP organisms with or without a specific inhibitor of the MAPKp38 pathway and activation of the MAPKp38, interleukin - (IL) IL-10, IL-12, iNOS mRNA expression and NO production were evaluated. Incubation of macrophages with MAP organisms activates the MAPKp38 pathway at early time points post infection. Chemically inhibition of MAPKp38 before incubation of bovine macrophages with MAP resulted in increased expression of IL-12 mRNA at 2, 6 and 24h, decreased expression of IL-10 mRNA at 2, 6 and 24h and increased expression of iNOS mRNA at 2 and 6h. Nitric oxide was evaluated to indirectly determine the effects of MAPKp38 pathway on the anti-microbial activity of bovine macrophages. Incubation of bovine macrophages with MAP resulted in modest increased production of NO at 4 and 6h post infection. Pretreatment of bovine macrophages with the MAPKp38 inhibitor SB203580 before addition of MAP organisms resulted in increased production of NO at 2, 4, 6 and 24h post infection. This study expanded our knowledge of the importance of the MAPKp38 pathway in limiting an appropriate macrophage response to MAP and suggested how activation of MAPKp38 pathway may be a target of this organism to disrupt earlier antimicrobial mechanisms of macrophages. These findings raises the interesting possibility that the cellular manipulation of MAPKp38 may be useful in designing novel vaccines against MAP.

Mycobacterium avium Subspecies paratuberculosis Recombinant Proteins Modulate Antimycobacterial Functions of Bovine Macrophages.

It has been shown that Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) activates the Mitogen Activated Protein Kinase (MAPK) p38 pathway, yet it is unclear which components of M. paratuberculosis are involved in the process. Therefore, a set of 42 M. paratuberculosis recombinant proteins expressed from coding sequences annotated as lipoproteins were screened for their ability to induce IL-10 expression, an indicator of MAPKp38 activation, in bovine monocyte-derived macrophages. A recombinant lipoprotein, designated as MAP3837c, was among a group of 6 proteins that strongly induced IL-10 gene transcription in bovine macrophages, averaging a 3.1-fold increase compared to non-stimulated macrophages. However, a parallel increase in expression of IL-12 and TNF-α was only observed in macrophages exposed to a subset of these 6 proteins. Selected recombinant proteins were further analyzed for their ability to enhance survival of M. avium within bovine macrophages as measured by recovered viable bacteria and nitrite production. All 6 IL-10 inducing MAP recombinant proteins along with M. paratuberculosis cells significantly enhanced phosphorylation of MAPK-p38 in bovine macrophages. Although these proteins are likely not post translationally lipidated in E. coli and thus is a limitation in this study, these results form the foundation of how the protein component of the lipoprotein interacts with the immune system. Collectively, these data reveal M. paratuberculosis proteins that might play a role in MAPK-p38 pathway activation and hence in survival of this organism within bovine macrophages.

Role of cell membrane receptors in the suppression of monocyte anti-microbial activity against Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP), the agent of paratuberculosis, is a slow growing mycobacteria that survives within ruminant mononuclear phagocytes by preventing cell activation and phagosome maturation. We investigated interactions between MAP and monocyte membrane receptors that result in activation of the mitogen-activated protein kinase (MAPK) p38 pathway and suppression of monocyte antimicrobial activity. Bovine monocytes were treated with blocking antibodies or specific chemical inhibitors of toll-like receptor 2 (TLR2), CD14 and CR3 receptor before infection with MAP organisms. MAPKp38 pathway activation, IL-10 expression and production, phagosome acidification, and MAP survival were determined. Our results indicated that MAP organisms-induced MAPKp38 activation occurs through partial interaction with TLR2. Blocking TLR2 receptors decreased IL-10 mRNA expression but not IL-10 protein production, increased phagosome acidification, and increased the capacity of monocytes to kill MAP organisms. Furthermore, blocking CR3 receptors increased phagosome acidification but did not alter MAP killing. These finding suggest that phagosome acidification is dependent on a complex interaction between MAP and the phagosome wall that may involve multiple receptors but that organism killing is dependent on specific signaling involving TLR2 receptors.

Mitogen activated protein kinase p38 pathway is an important component of the anti-inflammatory response in Mycobacterium avium subsp. paratuberculosis-infected bovine monocytes.

We investigated the role of cell signaling through the mitogen-activated protein kinase-p38 (MAPK p38) pathway on the antimicrobial functions and cytokine expression by bovine monocytes after ingestion of Mycobacterium avium subsp. paratuberculosis. We evaluated the dynamic secretion of interleukin (IL)-10, IL-12 and tumor necrosis factor-alpha (TNF-alpha) as well as phagosome acidification and organism killing at several time points after in vitro infection of bovine monocytes with M. avium subsp. paratuberculosis. Monocytes treated with M. avium subsp. paratuberculosis had a significant increase in IL-10 expression at 2, 4, and 6h post-infection and an increase expression of TNF-alpha at 2, 4, 6, and 24h post-infection. In contrast, IL-12 expression did not increase at any time point post-infection. Moreover, MAPK p38 was rapidly phosphorylated at 10 and 60 min after M. avium subsp. paratuberculosis ingestion. Chemical inhibition of the MAPK p38 signaling pathway (SB203580) resulted in decreased expression of IL-10 and increased expression of IL-12 at 6h post-infection. Chemically blocking the MAPK p38 pathway also increased acidification of phagosomes as well as increasing the capacity of macrophages to kill organisms. Taken together, these results indicated that selective activation of MAPK p38 may be a major mechanism exploited by M. avium subsp. paratuberculosis to circumvent the antimycobacterial effects of mononuclear phagocytes.