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Badger macrophages fail to produce nitric oxide, a key anti-mycobacterial effector molecule

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ABSTRACT

The European badger is recognised as a wildlife reservoir for bovine tuberculosis (bTB); the control of which is complex, costly and controversial. Despite the importance of badgers in bTB and the well-documented role for macrophages as anti-mycobacterial effector cells, badger macrophage (bdMφ) responses remain uncharacterised. Here, we demonstrate that bdMφ fail to produce nitric oxide (NO) or upregulate inducible nitric oxide synthase (iNOS) mRNA following Toll-like receptor (TLR) agonist treatment. BdMφ also failed to make NO after stimulation with recombinant badger interferon gamma (bdIFNγ) or a combination of bdIFNγ and lipopolysaccharide. Exposure of bdMφ to TLR agonists and/or bdIFNγ resulted in upregulated cytokine (IL1β, IL6, IL12 and TNFα) mRNA levels indicating that these critical pathways were otherwise intact. Although stimulation with most TLR agonists resulted in strong cytokine mRNA responses, weaker responses were evident after exposure to TLR9 agonists, potentially due to very low expression of TLR9 in bdMφ. Both NO and TLR9 are important elements of innate immunity to mycobacteria, and these features of bdMφ biology would impair their capacity to resist bTB infection. These findings have significant implications for the development of bTB management strategies, and support the use of vaccination to reduce bTB infection in badgers.

No MeSH data available.


Badger macrophages do not produce NO.(a) Badger and ferret peripheral blood monocyte-derived macrophages, and mouse and chicken macrophage cell lines, were treated with LPS and supernatants assayed for NO after 48 hours (MC: media control). Error bars indicate standard error of the mean. (b) QRT-PCR was used to measure iNOS RNA in bdMφ following TLR agonist treatment. GAPDH was detected at a mean of 1.1 × 106 copies/well. (c) Badger macrophages were treated with recombinant badger IFNγ and induction of TNFα and iNOS measured by QRT-PCR. QRT-PCR results are given as copy number/well. GAPDH = 1.5 × 105 copies/well. Difference from media control (MC): *p < 0.05, **p < 0.01.
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f1: Badger macrophages do not produce NO.(a) Badger and ferret peripheral blood monocyte-derived macrophages, and mouse and chicken macrophage cell lines, were treated with LPS and supernatants assayed for NO after 48 hours (MC: media control). Error bars indicate standard error of the mean. (b) QRT-PCR was used to measure iNOS RNA in bdMφ following TLR agonist treatment. GAPDH was detected at a mean of 1.1 × 106 copies/well. (c) Badger macrophages were treated with recombinant badger IFNγ and induction of TNFα and iNOS measured by QRT-PCR. QRT-PCR results are given as copy number/well. GAPDH = 1.5 × 105 copies/well. Difference from media control (MC): *p < 0.05, **p < 0.01.

Mentions: Forty-eight hour cultured badger blood monocyte-derived adherent cells exhibited classical macrophage morphology and were phagocytic (Supplementary Fig. 1). Badger macrophages did not produce NO detectable by Griess assay following 48 hours exposure to lipopolysaccharide (LPS, Fig. 1a). In contrast, similar treatment of murine and chicken macrophages resulted in substantial NO production (Fig. 1a). Despite extensive analyses with macrophages isolated from wild badgers (n = 33) NO was never detected in response to a wide range of TLR agonists (Pam3CSK4, PAM; lipoarabinomannan, LAM; poly (I:C), PIC; flagellin, FGN; R848; ODN M362) or heat killed TB, BCG or Listeria monocytogenes (LM). To test whether this lack of NO phenotype was badger-specific or found in another Mustelid, we examined macrophages derived from ferret peripheral blood monocytes (Fig. 1a), or spleen and neither produced detectable NO after exposure to LPS, or other TLR agonists.


Badger macrophages fail to produce nitric oxide, a key anti-mycobacterial effector molecule
Badger macrophages do not produce NO.(a) Badger and ferret peripheral blood monocyte-derived macrophages, and mouse and chicken macrophage cell lines, were treated with LPS and supernatants assayed for NO after 48 hours (MC: media control). Error bars indicate standard error of the mean. (b) QRT-PCR was used to measure iNOS RNA in bdMφ following TLR agonist treatment. GAPDH was detected at a mean of 1.1 × 106 copies/well. (c) Badger macrophages were treated with recombinant badger IFNγ and induction of TNFα and iNOS measured by QRT-PCR. QRT-PCR results are given as copy number/well. GAPDH = 1.5 × 105 copies/well. Difference from media control (MC): *p < 0.05, **p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5382539&req=5

f1: Badger macrophages do not produce NO.(a) Badger and ferret peripheral blood monocyte-derived macrophages, and mouse and chicken macrophage cell lines, were treated with LPS and supernatants assayed for NO after 48 hours (MC: media control). Error bars indicate standard error of the mean. (b) QRT-PCR was used to measure iNOS RNA in bdMφ following TLR agonist treatment. GAPDH was detected at a mean of 1.1 × 106 copies/well. (c) Badger macrophages were treated with recombinant badger IFNγ and induction of TNFα and iNOS measured by QRT-PCR. QRT-PCR results are given as copy number/well. GAPDH = 1.5 × 105 copies/well. Difference from media control (MC): *p < 0.05, **p < 0.01.
Mentions: Forty-eight hour cultured badger blood monocyte-derived adherent cells exhibited classical macrophage morphology and were phagocytic (Supplementary Fig. 1). Badger macrophages did not produce NO detectable by Griess assay following 48 hours exposure to lipopolysaccharide (LPS, Fig. 1a). In contrast, similar treatment of murine and chicken macrophages resulted in substantial NO production (Fig. 1a). Despite extensive analyses with macrophages isolated from wild badgers (n = 33) NO was never detected in response to a wide range of TLR agonists (Pam3CSK4, PAM; lipoarabinomannan, LAM; poly (I:C), PIC; flagellin, FGN; R848; ODN M362) or heat killed TB, BCG or Listeria monocytogenes (LM). To test whether this lack of NO phenotype was badger-specific or found in another Mustelid, we examined macrophages derived from ferret peripheral blood monocytes (Fig. 1a), or spleen and neither produced detectable NO after exposure to LPS, or other TLR agonists.

View Article: PubMed Central - PubMed

ABSTRACT

The European badger is recognised as a wildlife reservoir for bovine tuberculosis (bTB); the control of which is complex, costly and controversial. Despite the importance of badgers in bTB and the well-documented role for macrophages as anti-mycobacterial effector cells, badger macrophage (bdM&phi;) responses remain uncharacterised. Here, we demonstrate that bdM&phi; fail to produce nitric oxide (NO) or upregulate inducible nitric oxide synthase (iNOS) mRNA following Toll-like receptor (TLR) agonist treatment. BdM&phi; also failed to make NO after stimulation with recombinant badger interferon gamma (bdIFN&gamma;) or a combination of bdIFN&gamma; and lipopolysaccharide. Exposure of bdM&phi; to TLR agonists and/or bdIFN&gamma; resulted in upregulated cytokine (IL1&beta;, IL6, IL12 and TNF&alpha;) mRNA levels indicating that these critical pathways were otherwise intact. Although stimulation with most TLR agonists resulted in strong cytokine mRNA responses, weaker responses were evident after exposure to TLR9 agonists, potentially due to very low expression of TLR9 in bdM&phi;. Both NO and TLR9 are important elements of innate immunity to mycobacteria, and these features of bdM&phi; biology would impair their capacity to resist bTB infection. These findings have significant implications for the development of bTB management strategies, and support the use of vaccination to reduce bTB infection in badgers.

No MeSH data available.