Nicotine Modulates MyD88-Dependent Signaling Pathway in Macrophages during Mycobacterial Infection

Abstract

Recently, we reported that cigarette smoking, and especially nicotine, increases susceptibility to mycobacterial infection and exacerbates inflammation in patients with Crohn's disease (CD). The macrophagic response to Mycobacterium avium subspecies paratuberculosis (MAP) in CD and Mycobacteria tuberculosis (MTB) continues to be under investigation. The role of toll-like-receptors (TLRs) and cytoplasmic adaptor protein (MyD88) in proinflammatory response during Mycobacterial infection has been suggested. However, the mechanism of how nicotine modulates macrophage response during infection in CD and exacerbates inflammatory response remain unclear. In this study, we elucidated the mechanistic role of nicotine in modulating MyD88-dependent/TLR pathway signaling in a macrophage system during mycobacterial infection. The data demonstrated that MAP infection in THP-1 derived macrophages was mediated through TLR2 and MyD88 leading to increase in IL-8 in expression and production. On the other hand, LPS-representing, Gram-negative bacteria mediated macrophage response through TLR4. Blocking TLR2 and TLR4 with antagonists voided the effect of MAP, and LPS, respectively in macrophages and reversed response with decrease in expression of iNOS, TNF-α and IL-8. Interestingly, nicotine in infected macrophages significantly (1) downregulated TLR2 and TLR4 expression, (2) activated MyD88, (3) increased M1/M2 ratio, and (4) increased expression and secretion of proinflammatory cytokines especially IL-8, as seen in CD smokers. We also discovered that blocking macrophages during MAP infection with MyD88 antagonist significantly decreased response which illustrates the key role for MyD88 during infection. Surprisingly, dual treatment of MAP-infected macrophages with MyD88 antagonist and nicotine absolutely impaired immune response and decreased MAP viability, which clearly validate the inflammatory role of nicotine in macrophages through TLR2/MyD88 pathway during infection. This is the first report to describe the mechanism by which nicotine modulates TLR2/MyDD88 and exacerbates inflammation in CD smokers associated with infection.

Keywords: Crohn’s disease; MAP; Macrophages; MyD88; Nicotine; TLR2.

Figure 1

Effect of nicotine on the TLR2/TLR4 expression on infected macrophages. THP-1 PMA differentiated macrophages were infected with MAP and MTB, or treated with LPS for 24 h, then treated with nicotine (4 µg/mL). Expression of TLR2 (A,B) and TLR4 (C,D)were measured by RT-PCR and ELISA, respectively. All experiments were performed in duplicates. MAP: Mycobacterium avium paratuberculosis. MTB: Mycobacteria tubercalusis. LPS: lipopolysaccharide derived from Escherichia coli ATCC 8739. * p < 0.05, ** p < 0.001.

Figure 2

Effect of nicotine on MyD88 signaling and subsequent IL-8 expression in macrophages during infection. THP-1-PMA-differentiated macrophages were infected with MAP and MTB, or treated with LPS for 24 h, then treated with nicotine (4 µg/mL). Expression of MyD88 (A) and IL-8 (B) were measured by RT-PCR. All experiments were performed in duplicates. MAP: Mycobacterium avium paratuberculosis. MTB: Mycobacteria tubercalusis. LPS: lipopolysaccharide derived from Escherichia coli ATCC 8739. * p < 0.05.

Figure 3

TLR4 antagonist abrogates the macrophage response against LPS but not MAP: M1, M2, TNF-α, and IL-8 studies. THP-1 PMA differentiated macrophages were infected with MAP or treated with LPS for 24 h after pretreatment with 0, 2.5, 5 and 10 µg/mL of TLR4-IN-C34 (Anti-TLR4). Expression of iNOS (A), MMR (B), TNF-α (C) and IL-8 (D) were measured by RT-PCR. All experiments were performed in duplicate. * p < 0.05.

Figure 4

TLR2 antagonist decreases the macrophage response against MAP infection: M1, M2, TNF-α, and IL-8 studies. THP-1-PMA-differentiated macrophages were infected with MAP for 24 h after pretreatment with 0, 2.5, 5 and 10 µg/mL of MMG11 (Anti-TLR2). Expression of iNOS (A), MMR (B), TNF-α (C) and IL-8 (D) were measured by RT-PCR. All experiments were performed in duplicates. * p < 0.05.

Figure 5

MyD88 antagonist cancels the macrophage response against MAP infection: M1, M2, TNF-α, and IL-8 studies. THP-1 PMA differentiated macrophages were infected with MAP for 24 h after pretreatment with 0, 2.5, 5 and 10 µg/mL of T6167923 (Anti-MyD88). Expression of iNOS (A), MMR (B), TNF-α (C) and IL-8 (D) were measured by RT-PCR. All experiments were performed in duplicate. * p < 0.05.

Figure 6

Effect of nicotine/MAP infection on inflammatory response following TLR2 and MyD88 inhibition in vitro. MAP-infected macrophages were preincubated with 10 µg/mL of T6167923 or 5 µg/mL of MMG11, following by nicotine treatment for 24 h. Expression of iNOS (A), IL-8 (B), MMR (C) and IL-10 (D) were measured by RT-PCR. All experiments were performed in duplicates. (+): Presence, (-): Absence. * p < 0.05.

Figure 7

Effect of nicotine on MAP survival in macrophages blocked with TLR2/MyD88 antagonists. MAP viability was measured in infected macrophages preincubated with 10 µg/mL of T6167923 or 5 µg/mL of MMG11 after 48 hours without nicotine (A) and with nicotine treatment (B). All experiments were performed in duplicates. (+): Presence, (-): Absence.