RESUMO
Bactericidal permeability-increasing protein (BPI) is a multifunctional cationic protein produced by neutrophils, eosinophils, fibroblasts, and macrophages with antibacterial anti-inflammatory properties. In the context of Gram-negative infection, BPI kills bacteria, neutralizes the endotoxic activity of lipopolysaccharides (LPSs), and, thus, avoids immune hyperactivation. Interestingly, BPI increases in patients with Gram-positive meningitis, interacts with lipopeptides and lipoteichoic acids of Gram-positive bacteria, and significantly enhances the immune response in peripheral blood mononuclear cells. We evaluated the antimycobacterial and immunoregulatory properties of BPI in human macrophages infected with Mycobacterium tuberculosis. Our results showed that recombinant BPI entered macrophages, significantly reduced the intracellular growth of M. tuberculosis, and inhibited the production of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-α). Furthermore, BPI decreased bacterial growth directly in vitro. These data suggest that BPI has direct and indirect bactericidal effects inhibiting bacterial growth and potentiating the immune response in human macrophages and support that this new protein's broad-spectrum antibacterial activity has the potential for fighting tuberculosis.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Proteínas Sanguíneas , Macrófagos , Mycobacterium tuberculosis , Fator de Necrose Tumoral alfa , Humanos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/efeitos dos fármacos , Proteínas Sanguíneas/metabolismo , Proteínas Sanguíneas/farmacologia , Macrófagos/metabolismo , Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Tuberculose/microbiologia , Tuberculose/imunologia , Tuberculose/tratamento farmacológicoRESUMO
Severe inflammatory responses are associated with the misbalance of innate and adaptive immunity. TLRs, NLRs, and cytokine receptors play an important role in pathogen sensing and intracellular control, which remains unclear in COVID-19. This study aimed to evaluate IL-8 production in blood cells from COVID-19 patients in a two-week follow-up evaluation. Blood samples were taken at admission (t1) and after 14 days of hospitalization (t2). The functionality of TLR2, TLR4, TLR7/8, TLR9, NOD1, and NOD2 innate receptors and IL-12 and IFN-γ cytokine receptors was evaluated by whole blood stimulation with specific synthetic receptor agonists through the quantification of IL-8, TNF-α, or IFN-γ. At admission, ligand-dependent IL-8 secretion was 6.4, 13, and 2.5 times lower for TLR2, TLR4, and endosomal TLR7/8 receptors, respectively, in patients than in healthy controls. Additionally, IL-12 receptor-induced IFN-γ secretion was lower in COVID-19 patients than in healthy subjects. We evaluated the same parameters after 14 days and observed significantly higher responses for TLR2, TLR4, TLR7/8, TLR9, and NOD1, NOD2, and IFN-γ receptors. In conclusion, the low secretion of IL-8 through stimulation with agonists of TLR2, TLR4, TLR7/8, TLR9, and NOD2 at t1 suggests their possible contribution to immunosuppression following hyperinflammation in COVID-19 disease.
RESUMO
Resolvins and protectins counter inflammation, enhance phagocytosis, induce bactericidal/permeability-increasing protein (BPI) expression, and restore inflamed tissue to homeostasis. Because modulating the inflammation/antiinflammation balance is important in Mycobacterium tuberculosis infection, we evaluated the effects of resolvins and protectins on human macrophages infected in vitro. Monocyte-derived macrophages were infected with M. tuberculosis H37Rv at a multiplicity of infection (MOI) of 5 and treated 1â¯h post-infection in vitro with 100â¯nM LXA4, RvD1, RvD2, PD1 or 150â¯nM Mar1. After 24â¯h, cytokine production was measured by Luminex, and BPI and cathelicidin LL37 expression was determined by real-time PCR. Macrophage bactericidal activity was assessed by colony-forming units (CFUs) 3â¯days posttreatment. Nuclear translocation of Nrf2 was assessed by ELISA, NFκB translocation was determined by imaging cytometry, and BPI production was determined by fluorescence microscopy. We found that all lipids reduced LPS-dependent and M. tuberculosis-induced TNF-α production. RvD1 and Mar1 also induced a significant reduction in M. tuberculosis intracellular growth. RvD1 and Mar1 elicited distinct immunomodulatory patterns. RvD1 induced upregulation of both antimicrobial effector genes (BPI and LL37) and cytokines (GM-CSF and IL-6). Mar1 induced only BPI overexpression. RvD1 and Mar1 induced NFκB nuclear translocation, but only Mar1 induced Nrf2 translocation. Inhibition of G protein-coupled receptor signaling in infected macrophages abrogated the regulatory effects of RvD1. In conclusion, RvD1 and Mar1 modulate the anti-inflammatory and antimicrobial properties of M. tuberculosis-infected human macrophages. Since both proresolving lipids are inducible and synthesized from dietary components, they have immunotherapeutic potential against tuberculosis when inflammation is uncontrolled.
Assuntos
Anti-Inflamatórios/farmacologia , Ácidos Docosa-Hexaenoicos/farmacologia , Inflamação/terapia , Macrófagos/imunologia , Mycobacterium tuberculosis/fisiologia , Tuberculose/terapia , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Processos de Crescimento Celular , Células Cultivadas , Humanos , Imunomodulação , Inflamação/imunologia , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Fagocitose , Tuberculose/imunologia , Fator de Necrose Tumoral alfa/metabolismo , CatelicidinasRESUMO
To determine the role of human beta-defensin 2 (HBD-2) in human tuberculosis, we studied the in vitro induction of HBD-2 gene expression by Mycobacterium tuberculosis H37Rv infection in the human lung epithelial cell line A549, in alveolar macrophages (AM), and in blood monocytes (MN) by reverse transcription-PCR. We also studied the induction of HBD-2 gene expression by mannose lipoarabinomannan (manLAM) from M. tuberculosis. Intracellular production of HBD-2 peptide was detected by immunocytochemistry and electron microscopy. Our results demonstrated that there was induction of HBD-2 mRNA in A549 cells after infection with M. tuberculosis at various multiplicities of infection (MOI) and that there was stimulation with manLAM. AM expressed the HBD-2 gene only at a high MOI with M. tuberculosis. MN did not express HBD-2 at any of the experimental M. tuberculosis MOI. Immunostaining revealed the presence of intracellular HBD-2 peptide in A549 cells following infection with M. tuberculosis, and the staining was more intense in areas where there were M. tuberculosis clusters. By using electron microscopy we also demonstrated production of HBD-2 after M. tuberculosis infection and adherence of HBD-2 to the membranes of M. tuberculosis. Alveolar epithelial cells are among the first cells to encounter M. tuberculosis following aerogenic infection. As HBD-2 has been shown to control growth of M. tuberculosis and has chemotactic activity, our results suggest that HBD-2 induction by M. tuberculosis may have a role in the pathogenesis of human tuberculosis.