Cytokine and chemokine production by PBMCs of NTMPD patients and healthy household contacts in response to MAC antigens. We cultured PBMCs from 15 NTMPD patients and 15 healthy household contacts with heat-killed MAC antigens. After 72 h, culture supernatants were collected, and 34 cytokine and chemokine levels were measured by multiplex immunoassay (Supplementary Fig. 1). PBMCs from NTMPD patients produced significantly less IL-1β, IL-18, IL-1α and IL-10 than PBMCs from their healthy household contacts in response to heat-killed MAC antigens (Fig. 1). In response to heat-killed M. intracellulare antigen, PBMCs of NTMPD patients produced 4187.0 ± 681.1 pg/ml IL-1β, 46.9 ± 4.3 pg/ml IL-18, 52.4 ± 7.0 pg/ml IL-1α, and 1178.0 ± 167.9 pg/ml IL-10, whereas PBMCs of their healthy household contacts produced 6819.0 ± 895.7 pg/ml IL-1β (P < 0.01; Fig. 1A), 76.0 ± 11.5 pg/ml IL-18 (P < 0.05; Fig. 1B), 67.2 ± 8.9 pg/ml IL-1α (P < 0.01; Fig. 1C), and 1715.0 ± 242.0 pg/ml IL-10 (P < 0.01; Fig. 1D). Similarly, in response to heat-killed M. avium antigen, PBMCs of NTMPD patients produced 4073.0 ± 698.6 pg/ml IL-1β, 45.4 ± 4.6 pg/ml IL-18, 55.2 ± 7.6 pg/ml IL-1α, and 789.0 ± 126.2 pg/ml IL-10, whereas PBMCs of their healthy household contacts produced 6105.0 ± 883.9 pg/ml IL-1β (P < 0.01; Fig. 1A), 69.7 ± 10.7 pg/ml IL-18 (P < 0.05; Fig. 1B), 65.0 ± 9.1 pg/ml IL-1α (P = 0.052; Fig. 1C), and 1082.0 ± 174.4 pg/ml IL-10 (P < 0.01; Fig. 1D). Our findings demonstrate that PBMCs of NTMPD patients produce less IL-1β, IL-18, IL-1α and IL-10 in response to MAC antigens than PBMCs of their healthy household contacts.
Cytokine and chemokine levels in the plasma of patients and healthy household contacts. We also measured cytokine and chemokine levels by multiplex immunoassay in the plasma of patients and healthy household contacts (Supplementary Fig. 2). Among 34 cytokines and chemokines, RANTES levels were significantly higher in NTMPD patient plasma (22.6 ± 1.2 pg/ml) than in healthy household contact plasma (15.5 ± 1.2 pg/ml) (P < 0.001; Fig. 2A).
Effect of RANTES on IL-1β production by macrophages. RANTES is required for IFN-γ production by T cells 16 and promotes nitric oxide production by macrophages 17. The essential role of IL-1β in protection against mycobacterial diseases has been well demonstrated 18–20. Since RANTES was the only chemokine significantly elevated in the plasma of patients, we determined whether RANTES can affect IL-1β production by macrophages. Monocyte-derived macrophages (MDM) were infected with M. intracellulare and incubated in the presence of various concentrations of recombinant RANTES or plasma from NTMPD patients (in some cases, plasma was pretreated with an anti-RANTES antibody or isotype antibody) at 37°C in 5% CO2. Cell culture supernatants were collected after 24 h of incubation, and IL-1β concentrations were measured by ELISA. M. intracellulare-induced IL-1β (397.1 ± 130.8 pg/ml) was not significantly changed by recombinant RANTES (443.0 ± 155.8 pg/ml at 100 ng/ml RANTES, 440.5 ± 138.4 pg/ml at 300 ng/ml RANTES) or plasma from NTMPD patients (559.0 ± 82.5 pg/ml after addition of plasma, 559.0 ± 82.5 pg/ml after addition of plasma pretreated with 0.5 ng/ml anti-RANTES antibody, 492.3 ± 94.0 pg/ml after addition of plasma pretreated with 1 ng/ml anti-RANTES antibody, 524.3 ± 112.9 pg/ml after addition of plasma pretreated with 1 ng/ml anti-RANTES antibody). These data suggest that RANTES has no effect on IL-1β production by macrophages infected with M. intracellulare.
NTMPD patients have a defective expression of TLR2 and TWIK2. TLR2, P2X7R and TWIK2 are involved in mycobacteria-induced proinflammatory signaling, especially IL-1β production 21–23. To determine whether any of these molecules are involved in reduced IL-1β, IL-18, IL-1α and IL-10 production, we cultured PBMCs of NTMPD patients and their healthy household contacts with or without heat-killed M. intracellulare. We determined the relative mRNA expression levels of TLR2, P2X7R and two-pore domain potassium (K+) efflux channels, including TWIK2, THIK2 and TREK1 (Fig. 3). Upon stimulation with heat-killed M. intracellulare antigen, the relative mRNA expression of TLR2 and TWIK2 in PBMCs was significantly reduced in NTMPD patients compared to their healthy household contacts (Fig. 3A,C). In contrast, there was no difference in the expression of P2X7R and THIK2 in these PBMCs. (Fig. 3B,D). Of note, the relative mRNA expression level of TWIK2 in PBMCs was significantly reduced in NTMPD patients compared to their healthy household contacts even without stimulation (Fig. 3C). TREK1 expression was not detected in any samples until 40 cycles of real-time PCR amplification (data not shown). These data suggest that the expression of TLR2 and TWIK2 is impaired in NTMPD patients.
TLR2 inhibition reduces IL-1β, IL-18 IL-1α and IL-10 production by PBMCs and monocytes in response to MAC. We next determined whether reduced expression of TLR2 is related to reduced production of IL-1β, IL-18, IL-1α and IL-10 by PBMCs stimulated with heat-killed MAC antigens. PBMCs from 4 healthy donors were cultured with or without heat-killed MAC antigens in the presence or absence of a TLR2-selective inhibitor (C29). The TLR2-selective inhibitor significantly reduced IL-1β, IL-18, IL-1α and IL-10 production by heat-killed MAC antigen-stimulated PBMCs in a dose-dependent manner (Fig. 4A-D). Monocytes and macrophages are known as the major sources of IL-1β, IL-18 and IL-1α. We next determined whether TLR2 is involved in the production of IL-1β, IL-18, IL-1α and IL-10 by monocytes infected with live MAC. Monocytes from 6 healthy donors were infected with or without MAC in the presence or absence of a TLR2-selective inhibitor (C29). The TLR2-selective inhibitor significantly reduced the production of IL-1β, IL-18, IL-1α and IL-10 by MAC-infected CD14 + monocytes in a dose-dependent manner (Fig. 4E-H). The TLR2-selective inhibitor (C29) had no effect on cell viability (Supplementary Fig. 3A,B). These data suggest that TLR2 is involved in IL-1β, IL-18, IL-1α and IL-10 production by PBMCs and monocytes in response to MAC.
TWIK2 inhibition reduces IL-1β, IL-18 and IL-1α but not IL-10 production by PBMCs and monocytes in response to MAC. We next determined whether the reduced expression of TWIK2 is related to the reduced production of IL-1β, IL-18, IL-1α and IL-10 by PBMCs stimulated with heat-killed MAC antigens. PBMCs from 4 healthy donors were cultured with or without heat-killed MAC antigens in the presence or absence of a TWIK2-selective inhibitor (quinine). The TWIK2-selective inhibitor significantly reduced IL-1β, IL-18 and IL-1α, but not IL-10, production by heat-killed MAC antigen-stimulated PBMCs in a dose-dependent manner (Fig. 5A-D). We next determined whether TWIK2 is involved in the production of IL-1β, IL-18, IL-1α and IL-10 by monocytes infected with live MAC. Monocytes from 7 healthy donors were infected with or without MAC in the presence or absence of a TWIK2-selective inhibitor (quinine). The TWIK2-selective inhibitor significantly reduced IL-1β, IL-18 and IL-1α, but not IL-10, production by MAC-infected CD14 + monocytes in a dose-dependent manner (Fig. 5E-H). The TWIK2-selective inhibitor (quinine) had no effect on cell viability (Supplementary Fig. 3C,D). These data suggest that TWIK2 is involved in IL-1β, IL-18 and IL-1α, but not IL-10, production by PBMCs and monocytes in response to MAC.