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1.
PLoS One ; 17(4): e0267093, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35443015

RESUMO

Short chain fatty acids (SCFAs; including acetate, propionate, and butyrate) are an important class of biological molecules that play a major role in modulating host-microbiome interactions. Despite significant research into SCFA-mediated biological mechanisms, absolute quantification of these molecules in their native form by liquid chromatography mass spectrometry is challenging due to their relatively poor chromatographic properties. Herein, we introduce SQUAD, an isotope-based strategy for absolute quantification of SCFAs in complex biological samples. SQUAD uses aniline derivatization in conjunction with isotope dilution and analysis by reverse-phase liquid chromatography mass spectrometry. We show that SQUAD enables absolute quantification of biologically relevant SCFAs in complex biological samples with a lower limit of detection of 40 nM and a lower limit of quantification ranging from 160 nM to 310 nM. We observed an intra- and inter-day precision under 3% (relative standard deviation) and errors in intra- and inter-day accuracy under 10%. To demonstrate this quantification strategy, we analyzed SCFAs in the caecal contents of germ free versus conventionally raised specific pathogen free (SPF) mice. We showed that acetate was the most abundant SCFA in both types of mice and was present at 200-fold higher concentration in the SPF mice. We also illustrated the use of our quantification strategy in in vitro microbial cultures from five different species of bacteria grown in Mueller Hinton media. This study illustrates the diverse SCFA production rates across microbial taxa with acetate production serving as one of the key differentiating factors across the species. In summary, we introduce an isotope dilution strategy for absolute quantification of aniline-dativized SCFAs and illustrate the utility of this approach for microbiome research.


Assuntos
Cromatografia de Fase Reversa , Ácidos Graxos Voláteis , Acetatos , Cromatografia Líquida/métodos , Ácidos Graxos Voláteis/análise , Espectrometria de Massas em Tandem/métodos
2.
Science ; 346(6212): 1000-3, 2014 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-25414314

RESUMO

Nucleoside reverse transcriptase inhibitors (NRTIs) are mainstay therapeutics for HIV that block retrovirus replication. Alu (an endogenous retroelement that also requires reverse transcriptase for its life cycle)-derived RNAs activate P2X7 and the NLRP3 inflammasome to cause cell death of the retinal pigment epithelium in geographic atrophy, a type of age-related macular degeneration. We found that NRTIs inhibit P2X7-mediated NLRP3 inflammasome activation independent of reverse transcriptase inhibition. Multiple approved and clinically relevant NRTIs prevented caspase-1 activation, the effector of the NLRP3 inflammasome, induced by Alu RNA. NRTIs were efficacious in mouse models of geographic atrophy, choroidal neovascularization, graft-versus-host disease, and sterile liver inflammation. Our findings suggest that NRTIs are ripe for drug repurposing in P2X7-driven diseases.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Inflamassomos/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Elementos Alu , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Apoptose/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Caspase 1/metabolismo , Neovascularização de Coroide/tratamento farmacológico , Modelos Animais de Doenças , Atrofia Geográfica/tratamento farmacológico , Doença Enxerto-Hospedeiro/tratamento farmacológico , Hepatite/tratamento farmacológico , Fígado/efeitos dos fármacos , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Receptores Purinérgicos P2X7/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/fisiologia , Inibidores da Transcriptase Reversa/uso terapêutico
3.
J Innate Immun ; 5(4): 315-23, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23486162

RESUMO

Neutrophils are recruited to a site of infection or injury where they help initiate the acute inflammatory response. In instances of sterile inflammation, where no microbial threats are present, this neutrophil recruitment is mediated by the release of danger signals or damage-associated molecular patterns (DAMPs) from disrupted cells and tissues. At basal state, many of these substances are sequestered and remain hidden within the cell, but are released following the rupture of the plasma membrane. In other instances, these DAMPs are undetected by the innate immune system unless chemically or proteolytically modified by tissue damage. DAMPs may be directly detected by neutrophils themselves and modulate their recruitment to sites of damage or, alternatively, they can act on other cell types which in turn facilitate the arrival of neutrophils to a site of injury. In this review, we outline the direct and indirect effects of a number of DAMPs, notably extracellular ATP, mitochondrial formylated peptides and mitochondrial DNA, all of which are released by necrotic cells. We examine the effect of these substances on the recruitment and behaviour of neutrophils to sites of sterile injury. We also highlight research which suggests that neutrophils are actively involved in triggering the resolution phase of an inflammatory response. This review brings to light a growing body of work that demonstrates that the release of DAMPs and the ensuing influx of neutrophils plays an important functional role in the inflammatory response, even when no pathogens are present.


Assuntos
Citosol/metabolismo , Mitocôndrias/metabolismo , Neutrófilos/imunologia , Trifosfato de Adenosina/imunologia , Motivos de Aminoácidos/imunologia , Animais , Movimento Celular/imunologia , DNA Mitocondrial/imunologia , Humanos , Imunidade Inata , Inflamação/imunologia , Mitocôndrias/imunologia , Receptores de Reconhecimento de Padrão/imunologia
4.
Nat Med ; 18(9): 1386-93, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22922410

RESUMO

Neutrophil extracellular traps (NETs) are released as neutrophils die in vitro in a process requiring hours, leaving a temporal gap that invasive microbes may exploit. Neutrophils capable of migration and phagocytosis while undergoing NETosis have not been documented. During Gram-positive skin infections, we directly visualized live polymorphonuclear cells (PMNs) in vivo rapidly releasing NETs, which prevented systemic bacterial dissemination. NETosis occurred during crawling, thereby casting large areas of NETs. NET-releasing PMNs developed diffuse decondensed nuclei, ultimately becoming devoid of DNA. Cells with abnormal nuclei showed unusual crawling behavior highlighted by erratic pseudopods and hyperpolarization consistent with the nucleus being a fulcrum for crawling. A requirement for both Toll-like receptor 2 and complement-mediated opsonization tightly regulated NET release. Additionally, live human PMNs injected into mouse skin developed decondensed nuclei and formed NETS in vivo, and intact anuclear neutrophils were abundant in Gram-positive human abscesses. Therefore early in infection NETosis involves neutrophils that do not undergo lysis and retain the ability to multitask.


Assuntos
Espaço Extracelular/metabolismo , Movimento/fisiologia , Neutrófilos/imunologia , Dermatopatias Bacterianas/imunologia , Análise de Variância , Animais , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Neutrófilos/metabolismo , Neutrófilos/fisiologia , Proteínas Opsonizantes/metabolismo , Dermatopatias Bacterianas/metabolismo , Receptor 2 Toll-Like/metabolismo
5.
Science ; 330(6002): 362-6, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-20947763

RESUMO

Neutrophils are recruited from the blood to sites of sterile inflammation, where they contribute to wound healing but may also cause tissue damage. By using spinning disk confocal intravital microscopy, we examined the kinetics and molecular mechanisms of neutrophil recruitment to sites of focal hepatic necrosis in vivo. Adenosine triphosphate released from necrotic cells activated the Nlrp3 inflammasome to generate an inflammatory microenvironment that alerted circulating neutrophils to adhere within liver sinusoids. Subsequently, generation of an intravascular chemokine gradient directed neutrophil migration through healthy tissue toward foci of damage. Lastly, formyl-peptide signals released from necrotic cells guided neutrophils through nonperfused sinusoids into the injury. Thus, dynamic in vivo imaging revealed a multistep hierarchy of directional cues that guide neutrophil localization to sites of sterile inflammation.


Assuntos
Inflamação/imunologia , Inflamação/patologia , Hepatopatias/imunologia , Hepatopatias/patologia , Fígado/imunologia , Fígado/patologia , Infiltração de Neutrófilos , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Transporte/metabolismo , Adesão Celular , Quimiocina CXCL2/metabolismo , Quimiocinas/metabolismo , Quimiotaxia de Leucócito , Sinais (Psicologia) , Endotélio Vascular/fisiologia , Inflamação/metabolismo , Cinética , Fígado/irrigação sanguínea , Fígado/metabolismo , Hepatopatias/metabolismo , Antígeno de Macrófago 1/fisiologia , Camundongos , Microscopia/métodos , Microscopia Confocal , Microvasos/fisiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Necrose , Neutrófilos/fisiologia , Peptídeos/metabolismo , Receptores de Formil Peptídeo/metabolismo , Receptores de Interleucina-8B/metabolismo , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X7 , Transdução de Sinais
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