RESUMO
Nearly six million people worldwide have died from the coronavirus disease (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Although COVID-19 vaccines are largely successful in reducing the severity of the disease and deaths, the decline in vaccine-induced immunity over time and the continuing emergence of new viral variants or mutations underscore the need for an alternative strategy for developing broad-spectrum host-mediated therapeutics against SARS-CoV-2. A key feature of severe COVID-19 is dysregulated innate immune signaling, culminating in a high expression of numerous pro-inflammatory cytokines and chemokines and a lack of antiviral interferons (IFNs), particularly type I (alpha and beta) and type III (lambda). As a natural host defense, the myeloid differentiation primary response protein, MyD88, plays pivotal roles in innate and acquired immune responses via the signal transduction pathways of Toll-like receptors (TLRs), a type of pathogen recognition receptors (PRRs). However, recent studies have highlighted that infection with viruses upregulates MyD88 expression and impairs the host antiviral response by negatively regulating type I IFN. Galectin-3 (Gal3), another key player in viral infections, has been shown to modulate the host immune response by regulating viral entry and activating TLRs, the NLRP3 inflammasome, and NF-κB, resulting in the release of pro-inflammatory cytokines and contributing to the overall inflammatory response, the so-called "cytokine storm". These studies suggest that the specific inhibition of MyD88 and Gal3 could be a promising therapy for COVID-19. This review presents future directions for MyD88- and Gal3-targeted antiviral drug discovery, highlighting the potential to restore host immunity in SARS-CoV-2 infections.
Assuntos
Antivirais , COVID-19 , Galectina 3 , Fator 88 de Diferenciação Mieloide , SARS-CoV-2 , Humanos , Fator 88 de Diferenciação Mieloide/metabolismo , Fator 88 de Diferenciação Mieloide/genética , SARS-CoV-2/fisiologia , SARS-CoV-2/imunologia , COVID-19/imunologia , COVID-19/virologia , Antivirais/uso terapêutico , Antivirais/farmacologia , Galectina 3/metabolismo , Tratamento Farmacológico da COVID-19 , Imunidade Inata , Transdução de Sinais , AnimaisRESUMO
PURPOSE OF REVIEW: Burkholderia mallei is a facultative intracellular pathogen that causes the highly contagious and often the fatal disease, glanders. With its high rate of infectivity via aerosol and recalcitrance toward antibiotics, this pathogen is considered a potential biological threat agent. This review focuses on the most recent literature highlighting host innate immune response to B. mallei. RECENT FINDINGS: Recent studies focused on elucidating host innate immune responses to the novel mechanisms and virulence factors employed by B. mallei for survival. Studies suggest that pathogen proteins manipulate various cellular processes, including host ubiquitination pathways, phagosomal escape, and actin-cytoskeleton rearrangement. Immune-signaling molecules such as Toll-like receptors, nucleotode-binding oligomerization domain, myeloid differentiation primary response protein 88, and proinflammatory cytokines such as interferon-gamma and tumor necrosis factor-α, play key roles in the induction of innate immune responses. Modifications in B. mallei lipopolysaccharide, in particular, the lipid A acyl groups, stimulate immune responses via Toll-like receptor4 activation that may contribute to persistent infection. SUMMARY: Mortality is high because of septicemia and immune pathogenesis with B. mallei exposure. An effective innate immune response is critical to controlling the acute phase of the infection. Both vaccination and therapeutic approaches are necessary for complete protection against B. mallei.
Assuntos
Burkholderia mallei/imunologia , Mormo/imunologia , Imunidade Inata , Animais , Burkholderia mallei/patogenicidade , Citocinas/imunologia , Mormo/terapia , Humanos , Lipopolissacarídeos/imunologia , Receptores Toll-Like/imunologia , Fatores de Virulência/imunologiaRESUMO
Burkholderia pseudomallei infection causes melioidosis and is often characterized by severe sepsis. Although rare in humans, Burkholderia mallei has caused infections in laboratory workers, and the early innate cellular response to B. mallei in human and nonhuman primates has not been characterized. In this study, we examined the primary cellular immune response to B. mallei in PBMC cultures of non-human primates (NHPs), Chlorocebus aethiops (African Green Monkeys), Macaca fascicularis (Cynomolgus macaque), and Macaca mulatta (Rhesus macaque) and humans. Our results demonstrated that B. mallei elicited strong primary pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1ß, and IL-6) equivalent to the levels of B. pseudomallei in primary PBMC cultures of NHPs and humans. When we examined IL-1ß and other cytokine responses by comparison to Escherichia coli LPS, African Green Monkeys appears to be most responsive to B. mallei than Cynomolgus or Rhesus. Characterization of the immune signaling mechanism for cellular response was conducted by using a ligand induced cell-based reporter assay, and our results demonstrated that MyD88 mediated signaling contributed to the B. mallei and B. pseudomallei induced pro-inflammatory responses. Notably, the induced reporter activity with B. mallei, B. pseudomallei, or purified LPS from these pathogens was inhibited and cytokine production was attenuated by a MyD88 inhibitor. Together, these results show that in the scenario of severe hyper-inflammatory responses to B. mallei infection, MyD88 targeted therapeutic intervention may be a successful strategy for therapy.
Assuntos
Burkholderia mallei/imunologia , Mormo/imunologia , Imunidade Inata , Leucócitos Mononucleares/imunologia , Animais , Burkholderia mallei/fisiologia , Chlorocebus aethiops , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Mormo/genética , Mormo/microbiologia , Humanos , Imunidade Celular , Leucócitos Mononucleares/microbiologia , Macaca fascicularis , Macaca mulattaRESUMO
Toxic shock syndrome (TSS) is a clinical consequence of the profound amplification of host pro-inflammatory cytokine signaling that results from staphylococcal enterotoxin (SE) exposure. We recently reported that MyD88(-/-) mice were resistant to SEA or SEB toxic shock and displayed reduced levels of pro-inflammatory cytokines in their serum. Here we report that SEB stimulation of total mononuclear cells up-regulated MyD88 in monocytes and T cells. Further, MyD88 gene silencing in primary human cells using siRNA prevented SEB or SEB plus lipopolysaccharide (LPS) induction of interleukin-1ß (IL-1ß) transcriptional activation, suggesting that MyD88-mediated signaling is an essential component of SEB toxicity. We synthesized small molecules that mimic the conserved BB-loop in the Toll/IL-1 receptor (TIR) domain of MyD88. In primary human cells, these mimetics attenuated SEB-induced pro-inflammatory cytokine production. SEB stimulation of primary cells with mimetic affected newly synthesized MyD88 and downstream signaling components. Furthermore, LPS-induced MyD88 signaling was likewise inhibited in a cell-based reporter assay. More importantly, administration of mimetic reduced cytokine responses and increased survivability in a murine SEB challenge model. Collectively, these results suggest that MyD88 BB-loop mimetics interfere with SEB-induced pro-inflammatory signaling and toxicity, thus offering a potential approach in the therapy of toxic shock.
Assuntos
Citocinas/biossíntese , Enterotoxinas/antagonistas & inibidores , Mimetismo Molecular , Fator 88 de Diferenciação Mieloide/química , Peptídeos/farmacologia , Choque Séptico/tratamento farmacológico , Animais , Células Cultivadas , Enterotoxinas/toxicidade , Humanos , Inflamação/prevenção & controle , Camundongos , Camundongos Knockout , Peptídeos/química , Peptídeos/uso terapêutico , Receptores de Interleucina-1/químicaRESUMO
The continuous emergence of infectious pathogens along with antimicrobial resistance creates a need for an alternative approach to treat infectious diseases. Targeting host factor(s) which are critically involved in immune signaling pathways for modulation of host immunity offers to treat a broad range of infectious diseases. Upon pathogen-associated ligands binding to the Toll-like/ IL-1R family, and other cellular receptors, followed by recruitment of intracellular signaling adaptor proteins, primarily MyD88, trigger the innate immune responses. But activation of host innate immunity strongly depends on the correct function of MyD88 which is tightly regulated. Dysregulation of MyD88 may cause an imbalance that culminates to a wide range of inflammation-associated syndromes and diseases. Furthermore, recent reports also describe that MyD88 upregulation with many viral infections is linked to decreased antiviral type I IFN response, and MyD88-deficient mice showed an increase in survivability. These reports suggest that MyD88 is also negatively involved via MyD88-independent pathways of immune signaling for antiviral type I IFN response. Because of its expanding role in controlling host immune signaling pathways, MyD88 has been recognized as a potential drug target in a broader drug discovery paradigm. Targeting BB-loop of MyD88, small molecule inhibitors were designed by structure-based approach which by blocking TIR-TIR domain homo-dimerization have shown promising therapeutic efficacy in attenuating MyD88-mediated inflammatory impact, and increased antiviral type I IFN response in experimental mouse model of diseases. In this review, we highlight the reports on MyD88-linked immune response and MyD88-targeted therapeutic approach with underlying mechanisms for controlling inflammation and antiviral type I IFN response. HIGHLIGHTS: ⢠Host innate immunity is activated upon PAMPs binding to PRRs followed by immune signaling through TIR domain-containing adaptor proteins mainly MyD88. ⢠Structure-based approach led to develop small-molecule inhibitors which block TIR domain homodimerization of MyD88 and showed therapeutic efficacy in limiting severe inflammation-associated impact in mice. ⢠Therapeutic intervention of MyD88 also showed an increase in antiviral effect with strong type I IFN signaling linked to increased phosphorylation of IRFs via MyD88-independent pathway. ⢠MyD88 inhibitors might be potentially useful as a small-molecule therapeutics for modulation of host immunity against inflammatory diseases and antiviral therapy. ⢠However, prior clinical use of more in-depth efforts should be focused for suitability of the approach in deploying to complex diseases including COPD and COVID-19 in limiting inflammation-associated syndrome to infection.
Assuntos
Sistemas de Liberação de Medicamentos , Imunidade Inata/efeitos dos fármacos , Fator 88 de Diferenciação Mieloide , Viroses , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Fator 88 de Diferenciação Mieloide/antagonistas & inibidores , Fator 88 de Diferenciação Mieloide/imunologia , Viroses/tratamento farmacológico , Viroses/imunologiaRESUMO
Host exposure to pathogens engage multiple pathogen recognition receptors (PRRs) including toll-like receptors (TLRs); recruit intracellular signaling adaptor proteins primarily myeloid differentiation primary response protein 88 (MyD88) for activating downstream signaling cascades, which culminate in the production of type I interferons (IFNs), proinflammatory cytokines, and chemokines; and impede pathogen replication and dissemination. However, recent studies highlight that absence of MyD88 increased antiviral type I IFN induction, and MyD88-/- mice showed a higher survival rate compared with the low survival rate of the MyD88+/+ mice, implicating MyD88 limits antiviral type I IFN response. As a single infectious agent may harbor multiple PRR agonists, which trigger different sets of TLR-initiated immune signaling, we examined whether MyD88 inhibition during stimulation of cells with more than one TLR-ligand would augment type I IFN. We stimulated human U87- and TLR3-transfected HEK293-TLR7 cells with TLR-ligands, such as lipopolysaccharides (LPS) (TLR4-ligand) plus poly I:C (TLR3-ligand) or imiquimod (R837, TLR7-ligand) plus poly I:C, in the presence of compound 4210, a previously reported MyD88 inhibitor, and measured IFN-ß response using an enzyme-linked immunosorbent assay. Our results showed that when U87- or TLR3-transfected HEK293-TLR7 cells were stimulated with TLR-ligands, such as poly I:C plus LPS or poly I:C plus R837, IFN-ß production was significantly increased with MyD88 inhibition in a dose-dependent manner. Collectively, these results indicate that during more than one TLR-ligand-induced immune signaling event, impairment of antiviral type I IFN response was restored by inhibition of MyD88 through MyD88-independent pathway of type I IFN signaling, thus, offer a MyD88-targeted approach for type I IFN induction.
Assuntos
Interferon beta , Fator 88 de Diferenciação Mieloide , Animais , Células HEK293 , Humanos , Interferon beta/metabolismo , Ligantes , Lipopolissacarídeos/farmacologia , Camundongos , Transdução de Sinais , Receptores Toll-LikeRESUMO
Staphylococcal enterotoxin (SE) -induced toxic shock is triggered by inflammatory cytokine signal amplification after SE binding to major histocompatibility complex class II molecules on antigen-presenting cells and T-cell receptors. Identifying host cellular elements contributing to this pro-inflammatory signal amplification is critical for developing a strategy for therapeutic intervention. Myeloid differentiation primary-response protein 88 (MyD88) is an intracellular signalling adaptor protein primarily known for mediating pro-inflammatory cytokine responses. We investigated the role of MyD88 in staphylococcal enterotoxin A (SEA) -treated cell cultures and mouse models of toxic shock. Our results demonstrated that elevated levels of tumour necrosis factor-alpha, interferon-gamma, interleukin-1alpha/beta (IL-1alpha/beta), IL-2 and IL-6 production correlated with up-regulation of MyD88 after treatment of spleen cells and mice with SEA alone or in combination with lipopolysaccharide (LPS). The SEA-induced lethality was also observed in (LPS-independent) D-galactosamine-sensitized mice. While LPS potentiated SEA-induced cytokine responses, D-galactosamine treatment had no additive effect. Most importantly, our results demonstrated that MyD88(-/-) mice were resistant to SEA-induced toxic shock and had reduced pro-inflammatory cytokine responses. These results suggest that SEA-induced lethality is primarily dependent on MyD88. Our findings offer an important insight on potential therapeutic treatment of SEA-induced toxic shock targeting MyD88.
Assuntos
Citocinas/imunologia , Enterotoxinas/toxicidade , Fator 88 de Diferenciação Mieloide/imunologia , Staphylococcus/imunologia , Animais , Células Cultivadas , Citocinas/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/deficiência , NF-kappa B/imunologia , NF-kappa B/metabolismo , Baço/imunologia , Staphylococcus/efeitos dos fármacosRESUMO
Introduction. Melioidosis, caused by Burkholderia pseudomallei, in endemic areas, poses a challenge for treating the diseased populations without accurate diagnosis, and the disease-specific biomarkers linked with the infection have yet to be reported. Due to the invasive nature of the causative agent, Burkholderia pseudomallei, host innate effector mechanisms, including autophagy are known to be activated, resulting in differential expression of cellular proteins and immune markers. Identification of a disease-specific biomarker associated with B. pseudomallei infection will be helpful to facilitate rapid confirmation of melioidosis, which would enable early treatment and therapeutic success.Aim. We aimed to assess the levels of a host autophagy component, p62/NBR1, which function as a cargo-receptor in the process of autophagy activation leading to the degradation of ubiquitin-coated intracellular bacteria in which p62/NBR1 itself is degraded in the clearance of the pathogen. We further probed the extent of intracellular p62/NBR1 degradation and assessed its potential as a melioidosis biomarker.Methodology. We analysed peripheral blood mononuclear cell (PBMC) lysates using an ELISA-based assay for detecting cytosolic autophagy-related proteins p62/NBR1. We measured p62/NBR1 levels in diseased (confirmed B. pseudomallei infection) and non -diseased populations and utilized receiver operating characteristic (ROC) curve and max Youden index analysis for evaluating potential disease biomarker characteristics.Results. Our results revealed a three to fivefold increase in p62/NBR1 levels confirmed melioidosis cases compared to uninfected healthy donors. Comparable to p62/NBR1, levels of cytosolic LC3-I levels also increased, whereas the levels of degraded membrane bound form LC3-II was low, suggesting autophagy deficiency. Proinflammatory serum cytokine response, particularly IL-6, was consistently higher alongside B. pseudomallei infection in comparison to healthy controls.Conclusions. ROC curve and max Youden index analysis suggest that increased p62/NBR1 levels in diseased populations display characteristics of a potential disease biomarker in melioidosis and illustrates that an elevated p62/NBR1 level, in conjunction with B. pseudomallei infection associated with autophagy deficiency.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Melioidose/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adulto , Autofagia/fisiologia , Biomarcadores/metabolismo , Burkholderia pseudomallei/metabolismo , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Melioidose/microbiologia , Sri LankaRESUMO
Recent studies highlight that infection with Coxsackievirus B3, Venezuelan equine encephalitis virus (VEEV), Marburg virus, or stimulation using poly I:C (dsRNA), upregulates the signaling adaptor protein MyD88 and impairs the host antiviral type I interferon (IFN) responses. In contrast, MyD88 deficiency (MyD88-/-) increases the type I IFN and survivability of mice implying that MyD88 up regulation limits the type I IFN response. Reasoning that MyD88 inhibition in a virus-like manner may increase type I IFN responses, our studies revealed lipopolysaccharide stimulation of U937 cells or poly I:C stimulation of HEK293-TLR3, THP1 or U87 cells in the presence of a previously reported MyD88 inhibitor (compound 4210) augmented IFN-ß and RANTES production. Consistent with these results, overexpression of MyD88 decreased IFN-ß, whereas MyD88 inhibition rescued IFN-ß production concomitant with increased IRF3 phosphorylation, suggesting IRF-mediated downstream signaling to the IFN-ß response. Further, compound 4210 treatment inhibited MyD88 interaction with IRF3/IRF7 indicating that MyD88 restricts type I IFN signaling through sequestration of IRF3/IRF7. In cell based infection assays, compound 4210 treatment suppressed replication of VEEV, Eastern equine encephalitis virus, Ebola virus (EBOV), Rift Valley Fever virus, Lassa virus, and Dengue virus with IC50 values ranging from 11 to 42 µM. Notably, administration of compound 4210 improved survival, weight change, and clinical disease scores in mice following challenge with VEEV TC-83 and EBOV. Collectively, these results provide evidence that viral infections responsive to MyD88 inhibition lead to activation of IRF3/IRF7 and promoted a type I IFN response, thus, raising the prospect of an approach of host-directed antiviral therapy.
Assuntos
Antivirais/farmacologia , Interferon Tipo I/genética , Fator 88 de Diferenciação Mieloide/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Vírus/efeitos dos fármacos , Animais , Células HEK293 , Humanos , Concentração Inibidora 50 , Fator Regulador 3 de Interferon/metabolismo , Camundongos , Fator 88 de Diferenciação Mieloide/genética , Fosforilação , Poli I-C/farmacologia , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/síntese química , Células THP-1 , Viroses/tratamento farmacológico , Replicação Viral/efeitos dos fármacos , Vírus/classificaçãoRESUMO
INTRODUCTION: Burkholderia mallei (B. mallei) and Burkholderia pseudomallei (B. pseudomallei), causative agents of glanders and melioidosis, respectively, are invasive intracellular pathogens that actively multiply in phagocytic and non-phagocytic cells. Activation of cell-autonomous autophagy mechanism eliminate intracellular pathogens in which p62 a cytosolic cargo protein is selectively degraded, and an accumulation of this marker occurs if autophagy is deficient. Recurrent, relapsed and reinfection of B. pseudomallei in melioidosis patients in endemic area indicative of lack of complete of clearance and persistence of the pathogen. Reasoning that abundance in the levels of p62 may provide an indication of the intracellular infection, we sought to examine whether increase in intracellular p62 and bacterial burden with Burkholderia infection are linked to autophagy deficiency. METHODS: In this study, we investigated cell culture and mouse models of disease to identify an association between autophagy biomarkers (p62/NBR1) accumulation and intracellular persistence of B. mallei and B. pseudomallei. RESULTS: We demonstrate, that elevated levels of intracellular p62/NBR1 correlated with bacterial persistence, while pre-treatment with a pharmacological inducer of autophagy, rapamycin, reduced both intracellular p62, and bacterial survival. Our results showed an elevated p62 levels (2-5 fold) in spleen and liver cells of Burkholderia-infected BALB/c mice, as well as in spleen cells of Burkholderia-infected C57BL/6 mice, suggesting that an increase in p62/NBR1 was due to an autophagy deficiency. Similar to p62, cytosolic LC3-I levels were also elevated, while the characteristic conversion to the autophagosome-associated membrane bound form LC3-II was low in spleens of the infected mice further supporting the conclusion that autophagy was deficient. CONCLUSION: Taken together, our results suggest that an increase in intracellular p62/NBR1 may be a potential host cell biomarker of B. mallei or B. pseudomallei infections, and identifying autophagy manipulation may potentially aid to therapeutic approach for complete clearance of the pathogen.
Assuntos
Autofagia/genética , Burkholderia mallei/fisiologia , Burkholderia pseudomallei/fisiologia , Dessensibilização Imunológica/métodos , Mormo/metabolismo , Melioidose/metabolismo , Animais , Quimases/metabolismo , Modelos Animais de Doenças , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas/genética , Proteínas/metabolismo , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismoRESUMO
A correction has been published and is appended to both the HTML and PDF versions of this paper. The error has not been fixed in the paper.
RESUMO
ES-62 is a protein secreted by the parasitic worm Acanthocheilonema viteae that is anti-inflammatory by virtue of covalently attached phosphorylcholine. Previously we have reported that drug-like Small Molecule Analogues (SMAs) of its phosphorylcholine moiety can mimic ES-62 in protecting against disease development in certain mouse models of autoimmune and allergic conditions, due to them causing partial degradation of the TLR/IL-1R adaptor MyD88. We have now taken a molecular modelling approach to investigating the mechanism underlying this effect and this predicts that the SMAs interact directly with the MyD88 TIR domain. Further support for this is provided by assay of LPS-induced MyD88/NF-κB-driven secreted alkaline phosphatase (SEAP) reporter activity in commercially-available stably transfected (TLR4-MD2-NF-κB-SEAP) HEK293 cells, as SMA12b-mediated inhibition of such SEAP activity is blocked by its pre-incubation with recombinant MyD88-TIR domain. Direct binding of SMA12b to the TIR domain is also shown to inhibit homo-dimerization of the adaptor, an event that can explain the observed degradation of the adaptor and inhibition of subsequent downstream signalling. Thus, these new data identify initial events by which drug-like ES-62 SMAs, which we also demonstrate are able to inhibit cytokine production by human cells, homeostatically maintain "safe" levels of MyD88 signalling.
Assuntos
Acanthocheilonema/metabolismo , Anti-Inflamatórios/farmacologia , Proteínas de Helminto/farmacologia , Inflamação/tratamento farmacológico , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Acanthocheilonema/crescimento & desenvolvimento , Células HEK293 , Humanos , Inflamação/metabolismo , Inflamação/patologia , Fator 88 de Diferenciação Mieloide/genética , NF-kappa B/genética , Domínios Proteicos , Fator de Necrose Tumoral alfaRESUMO
Small molecules were developed to attenuate proinflammatory cytokines resulting from activation of MyD88-mediated toll-like receptor (TLR) signaling by Francisella tularensis. Fifty-three tripeptide derivatives were synthesized to mimic a key BB-loop region involved in toll-like/interleukin-1 receptor recognition (TIR) domain interactions. Compounds were tested for inhibition of TNF-α, IFN-γ, IL-6, and IL-1ß in human peripheral blood mononuclear cells (PBMCs) and primary human bronchial epithelial cells exposed to LPS extracts from F. tularensis. From 53 compounds synthesized and tested, ten compounds were identified as effective inhibitors of F. tularensisLPS-induced cytokines. Compound stability testing in the presence of human liver microsomes and human serum resulted in the identification of tripeptide derivative 7 that was a potent, stable, and drug-like small molecule. Target corroboration using a cell-based reporter assay and competition experiments with MyD88 TIR domain protein supported that the effect of 7 was through MyD88 TIR domain interactions. Compound 7 also attenuated proinflammatory cytokines in human peripheral blood mononuclear cells and bronchial epithelial cells challenged with a live vaccine strain of F. tularensis at a multiplicity of infection of 1:5. Small molecules that target TIR domain interactions in MyD88-dependent TLR signaling represent a promising strategy toward host-directed adjunctive therapeutics for inflammation associated with biothreat agent-induced sepsis.
Assuntos
Desenho de Fármacos , Francisella tularensis/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Peptídeos/química , Receptores Toll-Like/metabolismo , Sequência de Aminoácidos , Células Cultivadas , Citocinas/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Francisella tularensis/fisiologia , Genes Reporter , Células HEK293 , Meia-Vida , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/microbiologia , Lipopolissacarídeos/toxicidade , Microssomos Hepáticos/metabolismo , Fator 88 de Diferenciação Mieloide/química , NF-kappa B/genética , NF-kappa B/metabolismo , Peptídeos/metabolismo , Peptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/antagonistas & inibidores , Ativação Transcricional/efeitos dos fármacosRESUMO
Both Gram-positive and Gram-negative pathogens or pathogen-derived components, such as staphylococcal enterotoxins (SEs) and endotoxin (LPS) exposure, activate MyD88-mediated pro-inflammatory cellular immunity for host defense. However, dysregulated MyD88-mediated signaling triggers exaggerated immune response that often leads to toxic shock and death. Previously, we reported a small molecule compound 1 mimicking BB-loop structure of MyD88 was capable of inhibiting pro-inflammatory response to SEB exposure in mice. In this study, we designed a dimeric structure compound 4210 covalently linked with compound 1 by a non-polar cyclohexane linker which strongly inhibited the production of pro-inflammatory cytokines in human primary cells to SEB (IC50 1-50 µm) or LPS extracted from Francisella tularensis, Escherichia coli, or Burkholderia mallei (IC50 10-200 µm). Consistent with cytokine inhibition, in a ligand-induced cell-based reporter assay, compound 4210 inhibited Burkholderia mallei or LPS-induced MyD88-mediated NF-kB-dependent expression of reporter activity (IC50 10-30 µm). Furthermore, results from a newly expressed MyD88 revealed that 4210 inhibited MyD88 dimer formation which is critical for pro-inflammatory signaling. Importantly, a single administration of compound 4210 in mice showed complete protection from lethal toxin challenge. Collectively, these results demonstrated that compound 4210 inhibits toxin-induced inflated pro-inflammatory immune signaling, thus displays a potential bacterial toxin therapeutic.
Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Inflamação/tratamento farmacológico , Fator 88 de Diferenciação Mieloide/antagonistas & inibidores , Fator 88 de Diferenciação Mieloide/química , Animais , Anti-Inflamatórios/síntese química , Materiais Biomiméticos/síntese química , Citocinas/antagonistas & inibidores , Citocinas/biossíntese , Citocinas/imunologia , Desenho de Fármacos , Enterotoxinas/farmacologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/metabolismo , Choque Séptico/tratamento farmacológico , Relação Estrutura-AtividadeRESUMO
In this study, we used high-throughput computational screening to discover drug-like inhibitors of the host MyD88 protein-protein signaling interaction implicated in the potentially lethal immune response associated with Staphylococcal enterotoxins. We built a protein-protein dimeric docking model of the Toll-interleukin receptor (TIR)-domain of MyD88 and identified a binding site for docking small molecules. Computational screening of 5 million drug-like compounds led to testing of 30 small molecules; one of these molecules inhibits the TIR-TIR domain interaction and attenuates pro-inflammatory cytokine production in human primary cell cultures. Compounds chemically similar to this hit from the PubChem database were observed to be more potent with improved drug-like properties. Most of these 2(nd) generation compounds inhibit Staphylococcal enterotoxin B (SEB)-induced TNF-α, IFN-γ, IL-6, and IL-1ß production at 2-10 µM in human primary cells. Biochemical analysis and a cell-based reporter assay revealed that the most promising compound, T6167923, disrupts MyD88 homodimeric formation, which is critical for its signaling function. Furthermore, we observed that administration of a single dose of T6167923 completely protects mice from lethal SEB-induced toxic shock. In summary, our in silico approach has identified anti-inflammatory inhibitors against in vitro and in vivo toxin exposure with promise to treat other MyD88-related pro-inflammatory diseases.
Assuntos
Biologia Computacional/métodos , Simulação por Computador , Descoberta de Drogas/métodos , Fator 88 de Diferenciação Mieloide/química , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais , Animais , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Enterotoxinas/imunologia , Enterotoxinas/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Concentração Inibidora 50 , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
The filoviruses Ebola virus (EBOV) and Marburg virus (MARV) cause severe hemorrhagic fever in humans for which no vaccines are available. Previously, a priming dose of a DNA vaccine expressing the glycoprotein (GP) gene of MARV followed by boosting with recombinant baculovirus-derived GP protein was found to confer protective immunity to guinea pigs (Hevey et al., 2001. Vaccine 20, 568-593). To determine whether a similar prime-boost vaccine approach would be effective for EBOV, we generated and characterized recombinant baculoviruses expressing full-length EBOV GP (GP(1,2)) or a terminally-deleted GP (GPa-) and examined their immunogenicity in guinea pigs. As expected, cells infected with the GPa- recombinant secreted more GP(1) than those infected with the GP(1,2) recombinant. In lectin binding studies, the insect cell culture-derived GPs were found to differ from mammalian cell derived virion GP, in that they had no complex/hybrid N-linked glycans or glycans containing sialic acid. Despite these differences, the baculovirus-derived GPs were able to bind monoclonal antibodies to five distinct epitopes on EBOV GP, indicating that the antigenic structures of the proteins remain intact. As a measure of the ability of the baculovirus-derived proteins to elicit cell-mediated immune responses, we evaluated the T-cell stimulatory capacity of the GPa- protein in cultured human dendritic cells. Increases in cytotoxicity as compared to controls suggest that the baculovirus proteins have the capacity to evoke cell-mediated immune responses. Guinea pigs vaccinated with the baculovirus-derived GPs alone, or in a DNA prime-baculovirus protein boost regimen developed antibody responses as measured by ELISA and plaque reduction neutralization assays; however, incomplete protection was achieved when the proteins were given alone or in combination with DNA vaccines. These data indicate that a vaccine approach that was effective for MARV is not effective for EBOV in guinea pigs.
Assuntos
Baculoviridae/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Baculoviridae/genética , Células Dendríticas/imunologia , Ebolavirus/genética , Ebolavirus/metabolismo , Cobaias , Humanos , Testes de Neutralização , Vacinação , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologiaRESUMO
Staphylococcal enterotoxin B (SEB) exposure triggers an exaggerated pro-inflammatory cytokine response that often leads to toxic shock syndrome (TSS) associated with organ failure and death. MyD88 mediates pro-inflammatory cytokine signaling induced by SEB exposure and MyD88(-/-) mice are resistant to SEB intoxication, suggesting that MyD88 may be a potential target for therapeutic intervention. We targeted the BB loop region of the Toll/IL-1 receptor (TIR) domain of MyD88 to develop small-molecule therapeutics. Here, we report that a synthetic compound (EM-163), mimic to dimeric form of BB-loop of MyD88 attenuated tumor necrosis factor (TNF)- α, interferon (IFN)-γ, interleukin (IL)-1ß, IL-2 and IL-6 production in human primary cells, whether administered pre- or post-SEB exposure. Results from a direct binding assay, and from MyD88 co-transfection/co-immunoprecipitation experiments, suggest that EM-163 inhibits TIR-TIR domain interaction. Additional results indicate that EM-163 prevents MyD88 from mediating downstream signaling. In an NF-kB-driven reporter assay of lipopolysaccharide-stimulated MyD88 signaling, EM-163 demonstrated a dose-dependent inhibition of reporter activity as well as TNF-α and IL-1ß production. Importantly, administration of EM-163 pre- or post exposure to a lethal dose of SEB abrogated pro-inflammatory cytokine responses and protected mice from toxic shock-induced death. Taken together, our results suggest that EM-163 exhibits a potential for therapeutic use against SEB intoxication.
Assuntos
Materiais Biomiméticos/farmacologia , Enterotoxinas/toxicidade , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Materiais Biomiméticos/química , Citocinas/genética , Citocinas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transdução de Sinais/genéticaRESUMO
Ligands binding to Toll-like receptor (TLR), interleukin 1 receptor (IL-1R), or IFN-γR1 are known to trigger MyD88-mediated signaling, which activates pro-inflammatory cytokine responses. Recently we reported that staphylococcal enterotoxins (SEA or SEB), which bind to MHC class II molecules on APCs and cross link T cell receptors, activate MyD88- mediated pro-inflammatory cytokine responses. We also reported that MyD88(-/-) mice were resistant to SE- induced toxic shock and had reduced levels of serum cytokines. In this study, we investigated whether MHC class II- SE interaction by itself is sufficient to activate MyD88 in MHC class II(+) cells and induce downstream pro-inflammatory signaling and production of cytokines such as TNF-α and IL-1ß. Here we report that human monocytes treated with SEA, SEB, or anti-MHC class II monoclonal antibodies up regulated MyD88 expression, induced activation of NF-kB, and increased expression of IL-1R1 accessory protein, TNF-α and IL-1ß. MyD88 immunoprecipitated from cell extracts after SEB stimulation showed a greater proportion of MyD88 phosphorylation compared to unstimulated cells indicating that MyD88 was a component of intracellular signaling. MyD88 downstream proteins such as IRAK4 and TRAF6 were also up regulated in monocytes after SEB stimulation. In addition to monocytes, primary B cells up regulated MyD88 in response to SEA or SEB stimulation. Importantly, in contrast to primary B cells, MHC class II deficient T2 cells had no change of MyD88 after SEA or SEB stimulation, whereas MHC class II-independent activation of MyD88 was elicited by CpG or LPS. Collectively, these results demonstrate that MHC class II utilizes a MyD88-mediated signaling mechanism when in contact with ligands such as SEs to induce pro-inflammatory cytokines.
Assuntos
Enterotoxinas/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais/imunologia , Animais , Linfócitos B , Citocinas/biossíntese , Enterotoxinas/imunologia , Regulação da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Inflamação , Camundongos , Camundongos Knockout , Monócitos/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Ligação Proteica/imunologiaRESUMO
An elevated pro-inflammatory cytokine response is the primary cause of death by toxic shock after exposure to staphylococcal enterotoxin B (SEB). Identifying an intracellular signal mediator that predominantly controls the pro-inflammatory response is important for developing a therapeutic strategy. We examined the role of the signaling adaptor MyD88 in cell culture and in a mouse model of toxic shock. Our results indicated that elevated tumor necrosis factor-α, interferon-γ, interleukin (IL)-1α/ß and IL-6 production from mouse spleen cells treated with SEB alone or in combination with lipopolysaccharide (LPS) was regulated by MyD88. Elevated levels of MyD88 protein in spleen cells, as well as in CD11c(+) or Mac3(+) cells, and activation of nuclear factor-κB in spleen cells were observed in mice treated with SEB. An SEB-dose dependent lethality was observed in LPS-potentiated and in D-galactosamine-sensitized mice. D-Galactosamine treatment of spleen cells had no effect in cytokine induction but rather increased the sensitivity to toxic shock in mice. Our results demonstrated an impaired pro-inflammatory cytokine production by spleen cells of MyD88(-/-) mice in response to SEB or SEB plus LPS. Most importantly, MyD88(-/-) mice were resistant to SEB-induced death. These results demonstrate that MyD88-dependent pro-inflammatory signaling is responsible for SEB intoxication. In addition, our studies also demonstrated that LPS potentiation, in comparison to D-galactosamine sensitization, contributes to a stronger SEB-induced lethality. This is due to the pro-inflammatory cytokine response elicited by MyD88 after exposure to SEB and LPS. These findings offer an important insight upon SEB intoxication and subsequent therapy targeting MyD88.
Assuntos
Enterotoxinas/administração & dosagem , Fator 88 de Diferenciação Mieloide/metabolismo , Choque Séptico/imunologia , Baço/metabolismo , Infecções Estafilocócicas/imunologia , Animais , Células Cultivadas , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Enterotoxinas/efeitos adversos , Feminino , Galactosamina/administração & dosagem , Regulação da Expressão Gênica , Humanos , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Choque Séptico/induzido quimicamente , Choque Séptico/etiologia , Baço/imunologia , Baço/microbiologia , Baço/patologia , Infecções Estafilocócicas/complicaçõesRESUMO
Interleukin-15 (IL-15) is generally considered to sustain T-cell memory and to be a growth factor for natural killer cells. Previous data from our laboratory demonstrated that IL-15 is also an important factor for developing human dendritic cells. For this study, we investigated the effects of IL-15 on antibody responses in mice to a recombinant staphylococcal enterotoxin B (SEB) vaccine (STEBVax) in a preclinical model of toxic shock syndrome induced by SEB. We observed that mouse spleen cells treated with IL-15 in ex vivo culture gained a dendritic cell-like phenotype. Administration of IL-15 to mice also resulted in an increased number of mature CD11c+ dendritic cells in mouse spleens. A significant, IL-15 dose-dependent increase in antigen-specific antibody was observed after coadministration with the vaccine and an aluminum-based adjuvant (alhydrogel). Furthermore, the coadministration of IL-15 with STEBVax and alhydrogel also protected mice from lethal toxic shock above the levels that obtained without IL-15. Thus, the vaccine response enhanced by IL-15 appears to be mediated by mature dendritic cells and results in prevalent seroconversion to Th2-dependent antibodies. This suggests a potential use of IL-15 as an adjuvant for antibody-dependent responses to vaccines.