RESUMO
IFN-γ, a proinflammatory cytokine produced primarily by T cells and NK cells, activates macrophages and engages mechanisms to control pathogens. Although there is evidence of IFN-γ production by murine macrophages, IFN-γ production by normal human macrophages and their subsets remains unknown. Herein, we show that human M1 macrophages generated by IFN-γ and IL-12- and IL-18-stimulated monocyte-derived macrophages (M0) produce significant levels of IFN-γ. Further stimulation of IL-12/IL-18-primed macrophages or M1 macrophages with agonists for TLR-2, TLR-3, or TLR-4 significantly enhanced IFN-γ production in contrast to the similarly stimulated M0, M2a, M2b, and M2c macrophages. Similarly, M1 macrophages generated from COVID-19-infected patients' macrophages produced IFN-γ that was enhanced following LPS stimulation. The inhibition of M1 differentiation by Jak inhibitors reversed LPS-induced IFN-γ production, suggesting that differentiation with IFN-γ plays a key role in IFN-γ induction. We subsequently investigated the signaling pathway(s) responsible for TLR-4-induced IFN-γ production in M1 macrophages. Our results show that TLR-4-induced IFN-γ production is regulated by the ribosomal protein S6 kinase (p70S6K) through the activation of PI3K, the mammalian target of rapamycin complex 1/2 (mTORC1/2), and the JNK MAPK pathways. These results suggest that M1-derived IFN-γ may play a key role in inflammation that may be augmented following bacterial/viral infections. Moreover, blocking the mTORC1/2, PI3K, and JNK MAPKs in macrophages may be of potential translational significance in preventing macrophage-mediated inflammatory diseases.
Assuntos
Interferon gama/biossíntese , Macrófagos/efeitos dos fármacos , Poli I-C/farmacologia , COVID-19/imunologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/imunologia , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/imunologia , Macrófagos/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 70-kDa/imunologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/imunologia , Receptor 4 Toll-Like/agonistasRESUMO
The inflammatory and anti-inflammatory MÏs have been implicated in many diseases including rheumatoid arthritis, multiple sclerosis, and leprosy. Recent studies suggest targeting MÏ function and activation may represent a potential target to treat these diseases. Herein, we investigated the effect of second mitochondria-derived activator of caspases (SMAC) mimetics (SMs), the inhibitors of apoptosis (IAPs) proteins, on the killing of human pro- and anti-inflammatory MÏ subsets. We have shown previously that human monocytes are highly susceptible whereas differentiated MÏs (M0) are highly resistant to the cytocidal abilities of SMs. To determine whether human MÏ subsets are resistant to the cytotoxic effects of SMs, we show that M1 MÏs are highly susceptible to SM-induced cell death whereas M2a, M2b, and M2c differentiated subsets are resistant, with M2c being the most resistant. SM-induced cell death in M1 MÏs was mediated by apoptosis as well as necroptosis, activated both extrinsic and intrinsic pathways of apoptosis, and was attributed to the IFN-γ-mediated differentiation. In contrast, M2c and M0 MÏs experienced cell death through necroptosis following simultaneous blockage of the IAPs and the caspase pathways. Overall, the results suggest that survival of human MÏs is critically linked to the activation of the IAPs pathways. Moreover, agents blocking the cellular IAP1/2 and/or caspases can be exploited therapeutically to address inflammation-related diseases.
Assuntos
Apoptose , Inibidores de Caspase/farmacologia , Polaridade Celular , Macrófagos/citologia , Oligopeptídeos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Polaridade Celular/efeitos dos fármacos , Citocinas/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Janus Quinases/metabolismo , Cinética , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , Necroptose/efeitos dos fármacos , Fenótipo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Resistance to apoptosis is an important characteristic that human macrophages acquire during differentiation from monocytes. However, the intracellular mechanisms that mediate the development of resistance are not well understood. We have used M-CSF-stimulated primary human monocytes and PMA-treated THP1 cells to study apoptosis resistance during differentiation of human macrophages. Our results indicate that PI3K/Akt distinctively regulates survival of macrophages during and after differentiation. More specifically, a signaling pathway consisting of PI3K/Akt-NF-κB-Bcl-xL regulates cell survival during the differentiation process. PI3K/Akt-mediated activation of NF-κB plays a key role in survival of differentiating macrophages by specifically sustaining antiapoptotic Bcl-xL expression. With the use of pharmacological inhibitors and siRNA for Akt and Bcl-xL, we show that in the absence of Akt-dependent Bcl-xL expression during differentiation, cells undergo caspase-mediated apoptosis. In contrast, in differentiated macrophages, Bcl-xL expression is independent of PI3K/Akt activation. Taken together, these results suggest that survival of macrophages is distinctly regulated during and after differentiation. Our results also suggest new, potential therapeutic targets to modulate differentiation and survival of this cell type.