RESUMO
Lymphatics proliferate, become enlarged, or regress in multiple inflammatory lung diseases in humans. Lymphatic growth and remodeling is known to occur in the mouse trachea in sustained inflammation, but whether intrapulmonary lymphatics exhibit similar plasticity is unknown. We examined the time course, distribution, and dependence on vascular endothelial growth factor receptor (VEGFR)-2/VEGFR-3 signaling of lung lymphatics in sustained inflammation. Lymphatics in mouse lungs were examined under baseline conditions and 3 to 28 days after Mycoplasma pulmonis infection, using prospero heomeobox 1-enhanced green fluorescence protein and VEGFR-3 as markers. Sprouting lymphangiogenesis was evident at 7 days. Lymphatic growth was restricted to regions of bronchus-associated lymphoid tissue (BALT), where VEGF-C-producing cells were scattered in T-cell zones. Expansion of lung lymphatics after infection was reduced 68% by blocking VEGFR-2, 83% by blocking VEGFR-3, and 99% by blocking both receptors. Inhibition of VEGFR-2/VEGFR-3 did not prevent the formation of BALT. Treatment of established infection with oxytetracycline caused BALT, but not the lymphatics, to regress. We conclude that robust lymphangiogenesis occurs in mouse lungs after M. pulmonis infection through a mechanism involving signaling of both VEGFR-2 and VEGFR-3. Expansion of the lymphatic network is restricted to regions of BALT, but lymphatics do not regress when BALT regresses after antibiotic treatment. The lung lymphatic network can thus expand in sustained inflammation, but the expansion is not as reversible as the accompanying inflammation.
Assuntos
Brônquios/patologia , Linfangiogênese , Vasos Linfáticos/patologia , Tecido Linfoide/patologia , Pneumonia/patologia , Animais , Anticorpos Bloqueadores/farmacologia , Brônquios/efeitos dos fármacos , Brônquios/microbiologia , Humanos , Linfangiogênese/efeitos dos fármacos , Vasos Linfáticos/efeitos dos fármacos , Vasos Linfáticos/microbiologia , Tecido Linfoide/efeitos dos fármacos , Tecido Linfoide/microbiologia , Camundongos Endogâmicos C57BL , Infecções por Mycoplasma/complicações , Infecções por Mycoplasma/microbiologia , Infecções por Mycoplasma/patologia , Mycoplasma pulmonis/efeitos dos fármacos , Mycoplasma pulmonis/fisiologia , Pneumonia/complicações , Pneumonia/microbiologia , Transdução de Sinais/efeitos dos fármacos , Organismos Livres de Patógenos Específicos , Fatores de Tempo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Blood vessel leakiness is an early, transient event in acute inflammation but can also persist as vessels undergo remodeling in sustained inflammation. Angiopoietin/Tie2 signaling can reduce the leakiness through changes in endothelial cells. The role of pericytes in this action has been unknown. We used the selective PDGF-B-blocking oligonucleotide aptamer AX102 to determine whether disruption of pericyte-endothelial crosstalk alters vascular leakiness or remodeling in the airways of mice under four different conditions: i) baseline, ii) acute inflammation induced by bradykinin, iii) sustained inflammation after 7-day infection by the respiratory pathogen Mycoplasma pulmonis, or iv) leakage after bradykinin challenge in the presence of vascular stabilization by the angiopoietin-1 (Ang1) mimic COMP-Ang1 for 7 days. AX102 reduced pericyte coverage but did not alter the leakage of microspheres from tracheal blood vessels at baseline or after bradykinin; however, AX102 exaggerated leakage at 7 days after M. pulmonis infection and increased vascular remodeling and disease severity at 14 days. AX102 also abolished the antileakage effect of COMP-Ang1 at 7 days. Together, these findings show that pericyte contributions to endothelial stability have greater dependence on PDGF-B during the development of sustained inflammation, when pericyte dynamics accompany vascular remodeling, than under baseline conditions or in acute inflammation. The findings also show that the antileakage action of Ang1 requires PDGF-dependent actions of pericytes in maintaining endothelial stability.
Assuntos
Angiopoietina-1/metabolismo , Inflamação/patologia , Pericitos/patologia , Traqueia/irrigação sanguínea , Traqueia/patologia , Actinas/metabolismo , Animais , Aptâmeros de Nucleotídeos/farmacologia , Bradicinina/farmacologia , Contagem de Células , Forma Celular/efeitos dos fármacos , Desmina/metabolismo , Inflamação/complicações , Camundongos , Camundongos Endogâmicos C57BL , Microesferas , Infecções por Mycoplasma/complicações , Infecções por Mycoplasma/patologia , Mycoplasma pulmonis/efeitos dos fármacos , Mycoplasma pulmonis/fisiologia , Pericitos/efeitos dos fármacos , Pericitos/microbiologia , Proteínas Proto-Oncogênicas c-sis/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-sis/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Traqueia/microbiologiaRESUMO
We investigated the mode of action underlying the anti-mycoplasma activity of cationic antimicrobial peptides (AMPs) using four known AMPs and Mycoplasma pulmonis as a model mycoplasma. Scanning electron microscopy revealed that the integrity of the M. pulmonis membrane was significantly damaged within 30 min of AMPs exposure, which was confirmed by measuring the uptake of propidium iodine into the mycoplasma cells. The anti-mycoplasma activity of AMPs was found to depend on the binding affinity for phosphatidylcholine, which was incorporated into the mycoplasma membrane from the growth medium and preferentially distributed in the outer leaflet of the lipid bilayer.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Mycoplasma pulmonis/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Cromatografia em Camada Fina , Canamicina/farmacologia , Microscopia Eletrônica de Varredura , Mycoplasma pulmonis/metabolismo , Mycoplasma pulmonis/ultraestrutura , Fosfolipídeos/químicaRESUMO
We showed in a previous study that associations of antimicrobial peptides (AMPs), which are key components of the innate immune systems of all living species, with the fluoroquinolone enrofloxacin can successfully cure HeLa cell cultures of Mycoplasma fermentans and M. hyorhinis contamination. In the present work, the in vitro susceptibility of M. pulmonis, a murine pathogen, to enrofloxacin and four AMPs (alamethicin, globomycin, gramicidin S, and surfactin) was investigated, with special reference to synergistic associations and the effect of the mycoplasma cell concentration. Enrofloxacin and globomycin displayed the lowest MICs (0.4 microM), followed by gramicidin S (3.12 microM), alamethicin (6.25 microM), and surfactin (25 microM). When the mycoplasma cell concentration was varied from 10(4) to 10(8) CFU/ml, the MICs of enrofloxacin and globomycin increased while those of the three other molecules remained essentially constant. The minimal bactericidal concentration of enrofloxacin (0.8 microM) was also lower than those of the peptides (6.25 to 100 microM), but the latter killed the mycoplasma cells much faster than enrofloxacin (2 h versus 1 day). The use of the AMPs in association with enrofloxacin revealed synergistic effects with alamethicin and surfactin. Interestingly, the mycoplasma-killing activities of the two combinations enrofloxacin (MIC/2) plus alamethicin (MIC/4) and enrofloxacin (MIC/2) plus surfactin (MIC/16) were about 2 orders of magnitude higher than those of the three molecules used separately. These results support the interest devoted to AMPs as a novel class of antimicrobial agents and pinpoint their ability to potentiate the activities of conventional antibiotics, such as fluoroquinolones.
Assuntos
Anti-Infecciosos/farmacologia , Fluoroquinolonas/farmacologia , Mycoplasma pulmonis/efeitos dos fármacos , Alameticina/farmacologia , Animais , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Enrofloxacina , Células HeLa , Humanos , Camundongos , Infecções por Mycoplasma/tratamento farmacológico , Infecções por Mycoplasma/microbiologia , Mycoplasma pulmonis/citologia , Peptídeos Cíclicos/farmacologia , Fatores de TempoRESUMO
The length of the tandem repeat region of the Vsa protein of Mycoplasma pulmonis has previously been shown to modulate the susceptibility of mycoplasmas to killing by complement: cells that produce a short form of the Vsa protein are highly sensitive, and cells producing the long Vsa protein are resistant. In contrast to their differing susceptibilities to complement, the mycoplasmas were highly sensitive to gramicidin irrespective of the length of the Vsa protein produced. We show here that when encased within a biofilm, cells of M. pulmonis producing a short form of the Vsa protein were more resistant to complement and gramicidin than mycoplasmas that were dispersed. The resistance appeared to be localized to those mycoplasmas within tower structures of the biofilms. Biofilm formation may be a mechanism that protects mycoplasmas from host immunity.
Assuntos
Antibacterianos/farmacologia , Biofilmes , Proteínas do Sistema Complemento/imunologia , Gramicidina/farmacologia , Viabilidade Microbiana , Mycoplasma pulmonis/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Biofilmes/efeitos dos fármacos , Contagem de Colônia Microbiana , Farmacorresistência Bacteriana , Microscopia de Fluorescência , Mycoplasma pulmonis/efeitos dos fármacos , Mycoplasma pulmonis/imunologiaRESUMO
Antimicrobial peptides are widely distributed in nature, and in vertebrates, they play a key function in the innate immune defense system. It is generally agreed that these molecules may provide new antibiotics with therapeutic value. However, there are still many unsolved questions regarding the mechanisms underlying their antimicrobial activity as well as the mechanisms of resistance evolved by microorganisms against these molecules. The second point was addressed in this study. After determining the activity of 10 antimicrobial peptides against Mycoplasma pulmonis, a murine respiratory pathogen, the development of resistance was investigated. Following in vitro selection using subinhibitory concentrations of peptides, clones of this bacterium showing increased resistance to melittin or gramicidin D were obtained. For some of the clones, a cross-resistance was observed between these two peptides, in spite of their deep structural differences, and also with tetracycline. A proteomic analysis suggested that the stress response in these clones was constitutively activated, and this was confirmed by finding mutations in the hrcA gene; in mycoplasmas, bacteria which lack alternative sigma factors, the HrcA protein is supposed to play a key role as a negative regulator of heat shock proteins. By complementation of the hrcA mutants with the wild-type gene, the initial MICs of melittin and gramicidin D decreased to values close to the initial ones. This indicates that the resistance of M. pulmonis to these two antimicrobial peptides could result from a stress response involving HrcA-regulated genes.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Farmacorresistência Bacteriana/genética , Proteínas de Choque Térmico/metabolismo , Mycoplasma pulmonis/efeitos dos fármacos , Células Clonais , Análise Mutacional de DNA , Resistência a Múltiplos Medicamentos/genética , Genes Bacterianos , Teste de Complementação Genética , Gramicidina/farmacologia , Proteínas de Choque Térmico/genética , Meliteno/farmacologia , Testes de Sensibilidade Microbiana , Mutação , Mycoplasma pulmonis/genética , Proteoma/análise , Proteômica , Seleção GenéticaRESUMO
The Vsa proteins are associated with the virulence of the murine respiratory pathogen Mycoplasma pulmonis. The antigens consist of a conserved N-terminal region that is combined with one of several different variable C-terminal regions comprised of tandem repeats. M. pulmonis strains that produce VsaA with about 40 tandem repeats do not adhere to polystyrene or erythrocytes and are highly resistant to complement killing. Strains that produce VsaA with three tandem repeats adhere strongly to polystyrene and erythrocytes and are highly susceptible to complement killing. We report here that the resistance to complement lysis was not due to a lack of activation of the complement cascade. Isolation and analysis of M. pulmonis strains that produced Vsa proteins other than VsaA (VsaG and VsaI) with either long or short repeat regions indicated that adherence to polystyrene and resistance to complement were dependent on the length of the repeat region but not on the Vsa type. Furthermore, M. pulmonis Vsa variants were susceptible to the polypeptide pore-forming molecule gramicidin D, independent of the Vsa type and length. Collectively, the data indicate the Vsa proteins nonspecifically mediate M. pulmonis surface interactions and function to sterically hinder access of complement to the mycoplasma cell membrane while permitting access of smaller molecules.