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ATPase cation transporting 13A2 (ATP13A2) is an endolysosomal P-type ATPase known to be a polyamine transporter, explored mostly in neurons. As endolysosomal functions are also crucial in innate immune cells, we aimed to explore the potential role of ATP13A2 in the human immunocellular compartment. We found that human plasmacytoid dendritic cells (pDCs), the professional type I IFN-producing immune cells, especially have a prominent enrichment of ATP13A2 expression in endolysosomal compartments. ATP13A2 knockdown in human pDCs interferes with cytokine induction in response to TLR9/7 activation in response to bona fide ligands. ATP13A2 plays this crucial role in TLR9/7 activation in human pDCs by regulating endolysosomal pH and mitochondrial reactive oxygen generation. This (to our knowledge) hitherto unknown regulatory mechanism in pDCs involving ATP13A2 opens up a new avenue of research, given the crucial role of pDC-derived type I IFNs in protective immunity against infections as well as in the immunopathogenesis of myriad contexts of autoreactive inflammation.
Assuntos
Células Dendríticas , Endossomos , Lisossomos , Receptor Toll-Like 9 , Humanos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Lisossomos/metabolismo , Lisossomos/imunologia , Receptor Toll-Like 9/metabolismo , Receptor Toll-Like 9/imunologia , Endossomos/metabolismo , Endossomos/imunologia , ATPases Translocadoras de Prótons/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/imunologia , Células Cultivadas , Interferon Tipo I/metabolismo , Interferon Tipo I/imunologia , Receptor 7 Toll-LikeRESUMO
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing coronavirus disease 2019 (COVID-19), has led to significant morbidity and mortality. While most suffer from mild symptoms, some patients progress to severe disease with acute respiratory distress syndrome (ARDS) and associated systemic hyperinflammation. METHODS: First, to characterize key cytokines and their dynamics in this hyperinflammatory condition, we assessed abundance and correlative expression of a panel of 48 cytokines in patients progressing to ARDS as compared to patients with mild disease. Then, in an ongoing randomized controlled trial of convalescent plasma therapy (CPT), we analyzed rapid effects of CPT on the systemic cytokine dynamics as a correlate for the level of hypoxia experienced by the patients. RESULTS: We identified an anti-inflammatory role of CPT independent of its neutralizing antibody content. CONCLUSIONS: Neutralizing antibodies, as well as reductions in circulating interleukin-6 and interferon-γ-inducible protein 10, contributed to marked rapid reductions in hypoxia in response to CPT. CLINICAL TRIAL REGISTRY OF INDIA: CTRI/2020/05/025209. http://www.ctri.nic.in/.
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COVID-19/imunologia , COVID-19/terapia , SARS-CoV-2/imunologia , Adulto , Anti-Inflamatórios/uso terapêutico , Anticorpos Neutralizantes/imunologia , COVID-19/epidemiologia , COVID-19/virologia , Citocinas/sangue , Citocinas/imunologia , Feminino , Humanos , Imunização Passiva/métodos , Índia/epidemiologia , Masculino , Pessoa de Meia-Idade , Plasma , RNA Viral/isolamento & purificação , Síndrome do Desconforto Respiratório/tratamento farmacológico , Síndrome do Desconforto Respiratório/imunologia , SARS-CoV-2/isolamento & purificação , Carga Viral , Tratamento Farmacológico da COVID-19 , Soroterapia para COVID-19RESUMO
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease, characterized by loss of tolerance toward self nuclear Ags. Systemic induction of type I IFNs plays a pivotal role in SLE, a major source of type I IFNs being the plasmacytoid dendritic cells (pDCs). Several genes have been linked with susceptibility to SLE in genome-wide association studies. We aimed at exploring the role of one such gene, α/ß-hydrolase domain-containing 6 (ABHD6), in regulation of IFN-α induction in SLE patients. We discovered a regulatory role of ABHD6 in human pDCs through modulating the local abundance of its substrate, the endocannabinoid 2-arachidonyl glycerol (2-AG), and elucidated a hitherto unknown cannabinoid receptor 2 (CB2)-mediated regulatory role of 2-AG on IFN-α induction by pDCs. We also identified an ABHD6High SLE endophenotype wherein reduced local abundance of 2-AG relieves the CB2-mediated steady-state resistive tuning on IFN-α induction by pDCs, thereby contributing to SLE pathogenesis.
Assuntos
Células Dendríticas/imunologia , Endocanabinoides/metabolismo , Interferon gama/biossíntese , Lúpus Eritematoso Sistêmico/imunologia , Monoacilglicerol Lipases/imunologia , Adulto , Ácidos Araquidônicos/imunologia , Ácidos Araquidônicos/metabolismo , Células Dendríticas/metabolismo , Endocanabinoides/imunologia , Endofenótipos , Feminino , Regulação da Expressão Gênica/imunologia , Glicerídeos/imunologia , Glicerídeos/metabolismo , Humanos , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Masculino , Pessoa de Meia-Idade , Monoacilglicerol Lipases/genética , Receptor CB2 de Canabinoide/imunologia , Receptor CB2 de Canabinoide/metabolismoRESUMO
Understanding the dynamic changes in gene expression during Acute Respiratory Distress Syndrome (ARDS) progression in post-acute infection patients is crucial for unraveling the underlying mechanisms. Study investigates the longitudinal changes in gene/transcript expression patterns in hospital-admitted severe COVID-19 patients with ARDS post-acute SARS-CoV-2 infection. Blood samples were collected at three time points and patients were stratified into severe and mild ARDS, based on their oxygenation saturation (SpO2/FiO2) kinetics over 7 d. Decline in transcript diversity was observed over time, particularly in patients with higher severity, indicating dysregulated transcriptional landscape. Comparing gene/transcript-level analyses highlighted a rather limited overlap. With disease progression, a transition towards an inflammatory state was evident. Strong association was found between antibody response and disease severity, characterized by decreased antibody response and activated B cell population in severe cases. Bayesian network analysis identified various factors associated with disease progression and severity, viz. humoral response, TLR signaling, inflammatory response, interferon response, and effector T cell abundance. The findings highlight dynamic gene/transcript expression changes during ARDS progression, impact on tissue oxygenation and elucidate disease pathogenesis.
Assuntos
COVID-19 , Síndrome do Desconforto Respiratório , Humanos , COVID-19/genética , Estudos Longitudinais , Teorema de Bayes , SARS-CoV-2 , Síndrome do Desconforto Respiratório/genética , Imunidade , Unidades de Terapia Intensiva , Progressão da DoençaRESUMO
T cells are crucial for efficient antigen-specific immune responses and thus their migration within the body, to inflamed tissues from circulating blood or to secondary lymphoid organs, plays a very critical role. T cell extravasation in inflamed tissues depends on chemotactic cues and interaction between endothelial adhesion molecules and cellular integrins. A migrating T cell is expected to sense diverse external and membrane-intrinsic mechano-physical cues, but molecular mechanisms of such mechanosensing in cell migration are not established. We explored if the professional mechanosensor Piezo1 plays any role during integrin-dependent chemotaxis of human T cells. We found that deficiency of Piezo1 in human T cells interfered with integrin-dependent cellular motility on ICAM-1-coated surface. Piezo1 recruitment at the leading edge of moving T cells is dependent on and follows focal adhesion formation at the leading edge and local increase in membrane tension upon chemokine receptor activation. Piezo1 recruitment and activation, followed by calcium influx and calpain activation, in turn, are crucial for the integrin LFA1 (CD11a/CD18) recruitment at the leading edge of the chemotactic human T cells. Thus, we find that Piezo1 activation in response to local mechanical cues constitutes a membrane-intrinsic component of the 'outside-in' signaling in human T cells, migrating in response to chemokines, that mediates integrin recruitment to the leading edge.
Assuntos
Quimiocinas , Canais Iônicos , Linfócitos T , Humanos , Adesão Celular , Movimento Celular , Quimiotaxia , Antígeno-1 Associado à Função Linfocitária , Canais Iônicos/metabolismoRESUMO
Visceral leishmaniasis is a potentially fatal disease caused by infection by the intracellular protist pathogens Leishmania donovani or Leishmania infantum. Present therapies are ineffective because of high costs, variable efficacy against different species, the requirement for hospitalization, toxicity and drug resistance. Detailed analysis of previously published hit molecules suggested a crucial role of 'guanidine' linkage for their efficacy against L. donovani. Here we report the design of 2-aminoquinazoline heterocycle as a basic pharmacophore-bearing guanidine linkage. The introduction of various groups and functionality at different positions of the quinazoline scaffold results in enhanced antiparasitic potency with modest host cell cytotoxicity using a physiologically relevant THP-1 transformed macrophage infection model. In terms of the ADME profile, the C7 position of quinazoline was identified as a guiding tool for designing better molecules. The good ADME profile of the compounds suggests that they merit further consideration as lead compounds for treating visceral leishmaniasis.
Assuntos
Leishmania donovani , Leishmania infantum , Leishmaniose Visceral , Humanos , Leishmaniose Visceral/tratamento farmacológico , Antiparasitários/farmacologia , Quinazolinas/farmacologia , Quinazolinas/uso terapêuticoRESUMO
OBJECTIVE: Visceral adipose tissue (VAT) inflammation contributes to metabolic dysregulation in obesity. VAT recruitment and activation of plasmacytoid dendritic cells (pDCs) through toll-like receptor 9 (TLR9) recognition of self-DNA, leading to induction of type I interferons, are crucial innate triggers for this VAT inflammation. It was hypothesized that mitochondrial DNA (mtDNA) can contribute to TLR9 activation in VAT-recruited pDCs in obesity, and this study aimed to identify the carrier protein for ligand access to TLR9 and to explore whether this also provides for a source of autoantigens in this context. METHODS: VAT samples, used for gene expression studies as well as adipose explant cultures, were collected from patients with obesity (n = 54) and lean patients (n = 10). Supernatants from human pDC cultures, treated with adipose explant culture supernatants, were used for interferon α ELISA. Venous plasma, from patients with (n = 114) and without (n = 45) obesity, was used for an ELISA for autoantibodies. RESULTS: MtDNA from VAT in obesity, in complex with mitochondrial transcription factor A protein (TFAM), acts as interferogenic ligands for pDCs. Humoral autoreactivity against TFAM is also induced in obesity. CONCLUSIONS: Interferogenic ligands and an autoantigen can be sourced from dysfunctional mitochondria in VAT of humans with obesity. Further therapeutic and prognostic potential for this immune mechanism in obesity warrants exploration.
Assuntos
Autoantígenos , Receptor Toll-Like 9 , Humanos , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo , Ligantes , Autoantígenos/metabolismo , Obesidade/metabolismo , Inflamação/metabolismo , Mitocôndrias/metabolismo , DNA Mitocondrial/metabolismo , Células Dendríticas/metabolismoRESUMO
hERG is considered to be a primary anti-target in the drug development process, as the K+ channel encoded by hERG plays an important role in cardiac re-polarization. It is desirable to address the hERG safety liability during early-stage development to avoid the expenses of validating leads that will eventually fail at a later stage. We have previously reported the development of highly potent quinazoline-based TLR7 and TLR9 antagonists for possible application against autoimmune disease. Initial experimental hERG assessment showed that most of the lead TLR7 and TLR9 antagonists suffer from hERG liability rendering them ineffective for further development. The present study herein describes a coordinated strategy to integrate the understanding from structure-based protein-ligand interaction to develop non- hERG binders with IC50 >30â µM with retention of TLR7/9 antagonism through a single point change in the scaffold. This structure-guided strategy can serve as a prototype for abolishing hERG liability during lead optimization.
Assuntos
Receptor 7 Toll-Like , Receptor Toll-Like 9 , Receptor Toll-Like 9/metabolismo , Canais de Potássio Éter-A-Go-GoRESUMO
Cases of rhino-orbital mucormycosis in patients suffering from severe coronavirus disease 2019 (COVID-19) were reported in different parts of the world, especially in India. However, specific immune mechanisms that are linked to susceptibility to COVID-19-associated mucormycosis (CAM) remain largely unexplored. We aimed to explore whether the differential regulation of circulating cytokines in CAM patients had any potential pathogenic links with myeloid phagocyte function and susceptibility to mucormycosis. A small cohort of Indian patients suffering from CAM (N = 9) as well as COVID-19 patients with no evidence of mucormycosis (N = 5) were recruited in the study. Venous blood was collected from the patients as well as from healthy volunteers (N = 8). Peripheral blood mononuclear cells and plasma were isolated. Plasma samples were used to measure a panel of 48 cytokines. CD14+ monocytes were isolated and used for a flow cytometric phagocytosis assay as well as a global transcriptome analysis via RNA-sequencing. A multiplex cytokine analysis of the plasma samples revealed reduction in a subset of cytokines in CAM patients, which is known to potentiate the activation, migration, or phagocytic activity of myeloid cells, compared to the COVID-19 patients who did not contract mucormycosis. Compared to monocytes from healthy individuals, peripheral blood CD14+ monocytes from CAM patients were significantly deficient in phagocytic function. The monocyte transcriptome also revealed that pathways related to endocytic pathways, phagosome maturation, and the cytoskeletal regulation of phagocytosis were significantly downregulated in CAM patients. Thus, the study reports a significant deficiency in the phagocytic activity of monocytes, which is a critical effector mechanism for the antifungal host defense, in patients with CAM. This result is in concordance with results regarding the specific cytokine signature and monocyte transcriptome. IMPORTANCE A number of cases of mucormycosis, often fatal, were reported among severe COVID-19 patients from India as well as from some other parts of the world. However, specific immunocellular mechanisms that underlie susceptibility to this fungal infection in COVID-19 remain largely unexplored. Our study reports a deficiency in phagocytosis by monocytes in COVID-19 patients who are concomitantly afflicted with mucormycosis, with this deficiency being linked to a characteristic monocyte transcriptome as well as a circulating cytokine signature. The functional phenotype and cytokine signature of the monocytes may provide useful biomarkers for detecting potential susceptibility to mucormycosis in COVID-19 as well as in other viral infections.
Assuntos
COVID-19 , Mucormicose , Humanos , Monócitos , Leucócitos Mononucleares , Fagocitose , CitocinasRESUMO
Severe COVID-19 frequently features a systemic deluge of cytokines. Circulating cytokines that can stratify risks are useful for more effective triage and management. Here, we ran a machine-learning algorithm on a dataset of 36 plasma cytokines in a cohort of severe COVID-19 to identify cytokine/s useful for describing the dynamic clinical state in multiple regression analysis. We performed RNA-sequencing of circulating blood cells collected at different time-points. From a Bayesian Information Criterion analysis, a combination of interleukin-8 (IL-8), Eotaxin, and Interferon-γ (IFNγ) was found to be significantly linked to blood oxygenation over seven days. Individually testing the cytokines in receiver operator characteristics analyses identified IL-8 as a strong stratifier for clinical outcomes. Circulating IL-8 dynamics paralleled disease course. We also revealed key transitions in immune transcriptome in patients stratified for circulating IL-8 at three time-points. The study identifies plasma IL-8 as a key pathogenic cytokine linking systemic hyper-inflammation to the clinical outcomes in COVID-19.
Assuntos
COVID-19 , Interleucina-8 , Humanos , Teorema de Bayes , Citocinas , Progressão da DoençaRESUMO
BACKGROUND: Severe coronavirus disease 2019 (COVID-19) is associated with systemic hyper-inflammation. An adaptive interaction between gut microbiota and host immune systems is important for intestinal homeostasis and systemic immune regulation. The association of gut microbial composition and functions with COVID-19 disease severity is sparse, especially in India. We analysed faecal microbial diversity and abundances in a cohort of Indian COVID-19 patients to identify key signatures in the gut microbial ecology in patients with severe COVID-19 disease as well as in response to different therapies. The composition of the gut microbiome was characterized using 16Sr RNA gene sequences of genomic DNA extracted from faecal samples of 52 COVID-19 patients. Metabolic pathways across the groups were predicted using PICRUSt2. All statistical analyses were done using Vegan in the R environment. Plasma cytokine abundance at recruitment was measured in a multiplex assay. RESULTS: The gut microbiome composition of mild and severe patients was found to be significantly different. Immunomodulatory commensals, viz. Lachnospiraceae family members and Bifidobacteria producing butyrate and short-chain fatty acids (SCFAs), were under represented in patients with severe COVID-19, with an increased abundance of opportunistic pathogens like Eggerthella. The higher abundance of Lachnoclostridium in severe disease was reduced in response to convalescent plasma therapy. Specific microbial genera showed distinctive trends in enriched metabolic pathways, strong correlations with blood plasma cytokine levels, and associative link to disease outcomes. CONCLUSION: Our study indicates that, along with SARS-CoV-2, a dysbiotic gut microbial community may also play an important role in COVID-19 severity through modulation of host immune responses.
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Disruption in the composition of the gut microbial flora, the so-called gut dysbiosis, has been associated with and pathogenically implicated in a number of metabolic disorders such as type 2 diabetes mellitus, non-alcoholic fatty liver disease as well as atherosclerosis. We describe the key finding in this domain in terms of the gut dysbiotic effects of obesogenic diets linked to metabolic dysregulations, the cross-talk between host genetics and gut ecology in preclinical models of metabolic syndrome as well as in humans, effect of circadian rhythm on gut microbiome, the regulation of systemic immunocellular response, participating in the metabolic disorder-associated systemic inflammation, by gut microbiome and metabolites derived from them. Finally, we collate the evidence gathered till date on specific gut dysbiotic features documented in different components of metabolic syndrome in humans. Understanding gut dysbiosis in metabolic syndrome offers new insights into the pathogenesis of the specific clinical contexts as well as provide potential new therapeutic targets that warrant further exploration.
Assuntos
Diabetes Mellitus Tipo 2 , Microbioma Gastrointestinal , Doenças Metabólicas , Síndrome Metabólica , Humanos , Disbiose , Síndrome Metabólica/complicações , Diabetes Mellitus Tipo 2/complicaçõesRESUMO
Undesirable activation of endosomal toll-like receptors TLR7 and TLR9 present in specific immune cells in response to host-derived ligands is implicated in several autoimmune diseases and other contexts of autoreactive inflammation, making them important therapeutic targets. We report a drug development strategy identifying a new chemotype for incorporating relevant structural subunits into the basic imidazopyridine core deemed necessary for potent TLR7 and TLR9 dual antagonism. We established minimal pharmacophoric features in the core followed by hit-to-lead optimization, guided by in vitro and in vivo biological assays and ADME. A ligand-receptor binding hypothesis was proposed, and selectivity studies against TLR8 were performed. Oral absorption and efficacy of lead candidate 42 were established through favorable in vitro pharmacokinetics and in vivo pharmacodynamic studies, with IC50 values of 0.04 and 0.47 µM against TLR9 and TLR7, respectively. The study establishes imidazopyridine as a viable chemotype to therapeutically target TLR9 and TLR7 in relevant clinical contexts.
Assuntos
Receptor 7 Toll-Like , Receptor Toll-Like 9 , Imidazóis/farmacologia , Ligantes , Piridinas/farmacologia , Receptor 7 Toll-Like/metabolismoRESUMO
Objective: To assess the clinical and immunological benefits of passive immunization using convalescent plasma therapy (CPT). Materials and Methods: A series of subclass analyses were performed on the previously published outcome data and accompanying clinical metadata from a completed randomized controlled trial (RCT) (Clinical Trial Registry of India, number CTRI/2020/05/025209). The subclass analyses were performed on the outcome data and accompanying clinical metadata from a completed RCT (patient recruitment between May 15, 2020 and October 31, 2020). Data on the plasma abundance of a large panel of cytokines from the same cohort of patients were also used to characterize the heterogeneity of the putative anti-inflammatory function of convalescent plasma (CP) in addition to passively providing neutralizing antibodies. Results: Although the primary clinical outcomes were not significantly different in the RCT across all age groups, significant immediate mitigation of hypoxia, reduction in hospital stay, and significant survival benefit were registered in younger (<67 years in our cohort) patients with severe coronavirus disease 2019 and acute respiratory distress syndrome on receiving CPT. In addition to neutralizing the antibody content of CP, its anti-inflammatory proteome, by attenuation of the systemic cytokine deluge, significantly contributed to the clinical benefits of CPT. Conclusion: Subgroup analyses revealed that clinical benefits of CPT in severe coronavirus disease 2019 are linked to the anti-inflammatory protein content of CP apart from the anti-severe acute respiratory syndrome coronavirus 2 neutralizing antibody content.
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A single center open label phase 2 randomised control trial (Clinical Trial Registry of India No. CTRI/2020/05/025209) was done to assess clinical and immunological benefits of passive immunization using convalescent plasma therapy. At the Infectious Diseases and Beleghata General Hospital in Kolkata, India, 80 patients hospitalized with severe COVID-19 disease and fulfilling the inclusion criteria (aged more than 18 years, with either mild ARDS having PaO2/FiO2 200-300 or moderate ARDS having PaO2/FiO2 100-200, not on mechanical ventilation) were recruited and randomized into either standard of care (SOC) arm (N = 40) or the convalescent plasma therapy (CPT) arm (N = 40). Primary outcomes were all-cause mortality by day 30 of enrolment and immunological correlates of response to therapy if any, for which plasma abundance of a large panel of cytokines was quantitated before and after intervention to assess the effect of CPT on the systemic hyper-inflammation encountered in these patients. The secondary outcomes were recovery from ARDS and time taken to negative viral RNA PCR as well as to report any adverse reaction to plasma therapy. Transfused convalescent plasma was characterized in terms of its neutralizing antibody content as well as proteome. The trial was completed and it was found that primary outcome of all-cause mortality was not significantly different among severe COVID-19 patients with ARDS randomized to two treatment arms (Mantel-Haenszel Hazard Ratio 0.6731, 95% confidence interval 0.3010-1.505, with a P value of 0.3424 on Mantel-Cox Log-rank test). No adverse effect was reported with CPT. In severe COVID-19 patients with mild or moderate ARDS no significant clinical benefit was registered in this clinical trial with convalescent plasma therapy in terms of prespecified outcomes.
Assuntos
COVID-19/terapia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/uso terapêutico , Doadores de Sangue , COVID-19/imunologia , COVID-19/virologia , Citocinas/sangue , Feminino , Hospitais Gerais , Humanos , Imunidade Humoral , Imunização Passiva , Índia , Inflamação , Masculino , Filogenia , Síndrome do Desconforto Respiratório/imunologia , Síndrome do Desconforto Respiratório/terapia , Síndrome do Desconforto Respiratório/virologia , SARS-CoV-2/classificação , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Análise de Sobrevida , Resultado do Tratamento , Carga Viral , Soroterapia para COVID-19RESUMO
Aberrant activation of the endosomal Toll-like receptor 7 (TLR7) has been implicated in myriad autoimmune diseases and is an established therapeutic target in such conditions. Development of diverse TLR7 antagonists is mainly accomplished through random screening. To correlate human TLR7 (hTLR7) antagonistic activity with the structural features in different chemotypes, we derived a hypothetical binding model based on molecular docking analysis along with molecular dynamics (MD) simulations study. The binding hypothesis revealed different pockets, grooves and a central cavity where ligand-receptor interaction with specific residues through hydrophobic and hydrogen bond interactions take place, which correlate with TLR7 antagonistic activity thus paving the way for rational design using varied chemotypes. Based on the structural insight thus gained, TLR7 antagonists with quinazoline were designed to understand the effect of engagement of these pockets as well as boundaries of the chemical space associated with them. The newly synthesized most potent hTLR7 antagonist, i.e. compound 63, showed IC50 value of 1.03 ± 0.05 µM and was validated by performing primary assay in human plasmacytoid dendritic cells (pDC) (IC50pDC: 1.42 µM). The biological validation of the synthesized molecules was performed in TLR7-reporter HEK293 cells as well as in human plasmacytoid dendritic cells (pDCs). Our study provides a rational design approach thus facilitating further development of novel small molecule hTLR7 antagonists based on different chemical scaffolds.
Assuntos
Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Quinazolinas/farmacologia , Receptor 7 Toll-Like/antagonistas & inibidores , Sítios de Ligação/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Estrutura Molecular , Quinazolinas/síntese química , Quinazolinas/química , Relação Estrutura-Atividade , Receptor 7 Toll-Like/metabolismoRESUMO
Several toll-like receptors (TLRs) reside inside endosomes of specific immune cells-among them, aberrant activation of TLR7 and TLR9 is implicated in myriad contexts of autoimmune diseases, making them promising therapeutic targets. However, small-molecule TLR7 and TLR9 antagonists are not yet available for clinical use. We illustrate here the importance of C2, C6, and N9 substitutions in the purine scaffold for antagonism to TLR7 and TLR9 through structure-activity relationship studies using cellular reporter assays and functional studies on primary human immune cells. Further in vitro and in vivo pharmacokinetic studies identified an orally bioavailable lead compound 29, with IC50 values of 0.08 and 2.66 µM against TLR9 and TLR7, respectively. Isothermal titration calorimetry excluded direct TLR ligand-antagonist interactions. In vivo antagonism efficacy against mouse TLR9 and therapeutic efficacy in a preclinical murine model of psoriasis highlighted the potential of compound 29 as a therapeutic candidate in relevant autoimmune contexts.
Assuntos
Purinas/farmacologia , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor Toll-Like 9/antagonistas & inibidores , Administração Oral , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Purinas/administração & dosagem , Purinas/química , Ratos , Relação Estrutura-AtividadeRESUMO
Disease caused by SARS-CoV-2 coronavirus (COVID-19) led to significant morbidity and mortality worldwide. A systemic hyper-inflammation characterizes severe COVID-19 disease, often associated with acute respiratory distress syndrome (ARDS). Blood biomarkers capable of risk stratification are of great importance in effective triage and critical care of severe COVID-19 patients. Flow cytometry and next-generation sequencing were done on peripheral blood cells and urokinase-type plasminogen activator receptor (suPAR), and cytokines were measured from and mass spectrometry-based proteomics was done on plasma samples from an Indian cohort of COVID-19 patients. Publicly available single-cell RNA sequencing data were analyzed for validation of primary data. Statistical analyses were performed to validate risk stratification. We report here higher plasma abundance of suPAR, expressed by an abnormally expanded myeloid cell population, in severe COVID-19 patients with ARDS. The plasma suPAR level was found to be linked to a characteristic plasma proteome, associated with coagulation disorders and complement activation. Receiver operator characteristic curve analysis to predict mortality identified a cutoff value of suPAR at 1,996.809 pg/ml (odds ratio: 2.9286, 95% confidence interval 1.0427-8.2257). Lower-than-cutoff suPAR levels were associated with a differential expression of the immune transcriptome as well as favorable clinical outcomes, in terms of both survival benefit (hazard ratio: 0.3615, 95% confidence interval 0.1433-0.912) and faster disease remission in our patient cohort. Thus, we identified suPAR as a key pathogenic circulating molecule linking systemic hyperinflammation to the hypercoagulable state and stratifying clinical outcomes in severe COVID-19 patients with ARDS.
Assuntos
COVID-19/sangue , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , SARS-CoV-2 , Adulto , Idoso , Transtornos da Coagulação Sanguínea/sangue , Transtornos da Coagulação Sanguínea/imunologia , Proteínas Sanguíneas/análise , COVID-19/imunologia , Citocinas/sangue , Humanos , Inflamação/sangue , Inflamação/imunologia , Pessoa de Meia-Idade , Células Mieloides/imunologia , Proteoma/análise , Ensaios Clínicos Controlados Aleatórios como Assunto , Síndrome do Desconforto Respiratório/sangue , Síndrome do Desconforto Respiratório/imunologia , Índice de Gravidade de Doença , Adulto JovemRESUMO
Obesity and overweight are a global health problem affecting almost one third of the world population. There are multiple complications associated with obesity including metabolic syndrome that commonly lead to development of type II diabetes and non-alcoholic fatty liver disease. The development of metabolic syndrome and severe complications associated with obesity is attributed to the chronic low-grade inflammation that occurs in metabolic tissues such as the liver and the white adipose tissue. In recent years, nucleic acids (mostly DNA), which accumulate systemically in obese individuals, were shown to aberrantly activate innate immune responses and thus to contribute to metabolic tissue inflammation. This minireview will focus on (i) the main sources and forms of nucleic acids that accumulate during obesity, (ii) the sensing pathways required for their detection, and (iii) the key cellular players involved in this process. Fully elucidating the role of nucleic acids in the induction of inflammation induced by obesity would promote the identification of new and long-awaited therapeutic approaches to limit obesity-mediated complications.
Assuntos
Tecido Adiposo/imunologia , DNA/imunologia , Síndrome Metabólica/imunologia , Obesidade/imunologia , Transdução de Sinais/imunologia , Tecido Adiposo/patologia , Diabetes Mellitus Tipo 2/imunologia , Diabetes Mellitus Tipo 2/patologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Síndrome Metabólica/patologia , Hepatopatia Gordurosa não Alcoólica/imunologia , Hepatopatia Gordurosa não Alcoólica/patologia , Obesidade/patologiaRESUMO
Toll-like receptor 7 (TLR7) is an established therapeutic target in myriad autoimmune disorders, but no TLR7 antagonist is available for clinical use to date. Herein, we report a purine scaffold TLR7 antagonist, first-of-its-kind to our knowledge, which was developed by rationally dissecting the structural requirements for TLR7-targeted activity for a purine scaffold. Specifically, we identified a singular chemical switch at C-2 that could make a potent purine scaffold TLR7 agonist to lose agonism and acquire antagonist activity, which could further be potentiated by the introduction of an additional basic center at C-6. We ended up developing a clinically relevant TLR7 antagonist with favorable pharmacokinetics and 70.8% oral bioavailability in mice. Moreover, the TLR7 antagonists depicted excellent selectivity against TLR8. To further validate the in vivo applicability of this novel TLR7 antagonist, we demonstrated its excellent efficacy in preventing TLR7-induced pathology in a preclinical murine model of psoriasis.