Misurare phagosome pH Raziometrico microscopia a fluorescenza

Summary

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

Abstract

Fagocitosi è un processo fondamentale attraverso il quale le cellule immunitarie innate fagocitare batteri, cellule apoptotiche o altre particelle estranee al fine di uccidere o neutralizzare il materiale ingerito, o di presentarlo come antigeni e di avviare le risposte immunitarie adattive. Il pH di fagosomi è un parametro critico regolazione fissione o fusione con endomembrane e l'attivazione degli enzimi proteolitici, eventi che permettono al vacuolo fagocitosi di maturare in un organello degradativa. Inoltre, è richiesto di traslocazione H ⁺ per la produzione di elevati livelli di specie reattive dell'ossigeno (ROS), che sono essenziali per l'uccisione efficiente e segnalazione ad altri tessuti dell'ospite. Molti patogeni intracellulari sovvertono uccisione fagocitico limitando phagosomal acidificazione, sottolineando l'importanza del pH in phagosome biologia. Qui si descrive un metodo per misurare raziometrica phagosomal pH nei neutrofili con fluoresceina isotiocianato (FITC) -labeled zymosan come phagocytic targETS e live-cell imaging. Il saggio si basa sulle proprietà di fluorescenza di FITC, che si spegne da pH acido quando eccitato a 490 nm ma non quando eccitato a 440 nm, permettendo la quantificazione di un rapporto pH-dipendente, piuttosto che di fluorescenza assoluta, di un singolo colorante. Viene inoltre fornito un protocollo dettagliato per l'esecuzione in loco di calibrazione tintura e la conversione del rapporto di valori reali di pH. Single-dye metodi raziometriche sono generalmente considerati superiori a singola lunghezza d'onda o protocolli pseudo-raziometrico dual-dye, in quanto sono meno sensibili alle perturbazioni, come lo sbiancamento, concentrarsi modifiche, variazioni laser, e l'etichettatura irregolare, che distorcono il segnale misurato. Questo metodo può essere facilmente modificato per misurare il pH in altri tipi di cellule fagocitiche e zymosan può essere sostituita da qualsiasi altra particella contenente ammina, da perline inerti ai microrganismi viventi. Infine, questo metodo può essere adattato a fare uso di altre sonde fluorescenti sensibili a diversi intervalli di pH o altri phagosomattività al, rendendolo un protocollo generale per l'imaging funzionale di fagosomi.

Introduction

Phagocytosis, the process through which innate immune cells engulf large particles, evolved from the eating mechanism of single-celled organisms, and involves binding to a target, enveloping it with a membrane and pinching the membrane off to form a vacuole within the cytosol called a phagosome. While the phagosomal membrane is derived from the plasma membrane, active protein and lipid sorting, as well as fusion with endomembranes during phagosome formation, transform the phagosome into a distinct organelle within the cell with degradative properties that allow the killing, neutralization and breakdown of the ingested material^1-3. This process, called phagosomal maturation, relies on the delivery of a host of proteolytic and microbicidal enzymes, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase which transfers electrons into phagosomes producing the strong oxidant O₂^– and its derivative reactive oxygen species (ROS) ^2,4.

The luminal pH of phagosomes has a profound influence on several events required for phagosome function. First, pH influences trafficking of endomembranes in general, as pH-dependent conformational changes of transmembrane trafficking regulators alters the recruitment of trafficking determinants such as Arfs, Rabs and vesicular coat-proteins, which in turn define which vesicles may fuse with phagosomes ^5-8. Second, the ionic composition of the phagosomal lumen is also transformed as phagosomes mature, and some ion transporters, such as the Na⁺/H⁺ exchanger or ClC family Cl^–/H⁺ antiporters, which promote phagocytic function, rely on H⁺ translocation ^9,10. Similarly, ROS production is intimately linked with phagosomal pH. ROS and its toxic oxidant byproducts have long been recognized as crucial for phagosomal killing in neutrophils ^4,11,12, and have been shown to play critical roles in other phagocytes including macrophages, dendritic cells (DCs) and amoeba ^13-16. The NADPH oxidase is an electrogenic enzyme that releases H⁺ in the cytosol as NADPH is consumed, and that requires the simultaneous transfer of H⁺ through companion HVCN1 channels alongside the transported electrons into the phagosomal lumen, in order to alleviate the massive depolarization that would otherwise lead to self-inhibition of the enzyme ^17-21. Finally, several proteolytic enzymes have optimal activity at different pH, so time-dependent phagosomal pH changes can influence which enzymes are active and when. The importance of phagosomal pH is highlighted by organisms such as Mycobacterium tuberculosis, Franciscella tularensis and Salmonella typherium that subvert phagocytic killing at least in part by altering phagosomal pH ^22-24.

In mammals the main phagocytes are neutrophils, macrophages and dendritic cells, and depending on cell type, time-dependent phagosomal pH changes can vary widely, and appear to play different roles. In macrophages a strong and rapid acidification mediated by the ATP-dependent proton pump vacuolar ATPase (V-ATPase) is one of the key factors mediating killing ^25-27, resembling the mechanisms present in amoeba that use phagocytosis as an eating mechanism ²⁸. In these cells activation of acidic proteases is thought to play a key role. In contrast, neutrophil killing relies more on ROS as well as HOCl produced by myeloperoxidase (MPO)¹¹, and the pH remains neutral or alkaline during the first 30 min acidifying only later ^29,30. Neutral pH has been suggested to favor the activity of oxidative proteases such as certain cathepsins. In DCs phagosomal pH is controversial, with some reporting acidification and others neutral or alkaline pH ^31,32, but ROS and pH may profoundly influence the ability of these cells to present antigens to T cells, one of their main functions ³³.

Importantly, hormones, chemokines and cytokines may produce signaling events that induce maturation and changes in phagocyte behavior, and in turn influence phagosomal pH ^34,35. Similarly, drugs, for example the antimalarial chloroquine, which is also considered for anti-cancer therapies ³⁶, may affect phagosomal pH and therefore immune outcomes. Thus, a variety of cell biologists, immunologists, microbiologists and drug developers may be interested in measuring phagosomal pH when seeking to understand the mechanisms underlying the effects of a particular genetic disruption, bioactive compound or microorganism on innate and adaptive immune responses.

Here we describe a method for measuring phagosomal pH in neutrophils that allowed us to show the importance of the HVCN1 channel in regulating neutrophil phagosomal pH ¹⁹. The method is based on the ratiometric property of fluorescein isothiocyanate (FITC) whose fluorescence emission at 535 nm is pH sensitive when excited at 490 nm but not 440 nm ³⁷. When this dye is chemically coupled to a target, in this case zymosan, it can be followed using wide-field fluorescence microscopy, where cells are imaged as they phagocytose, and phagosomal pH changes are measured in real time as the phagosome matures. The actual pH is then gleaned by performing a calibration experiment where cells that have phagocytosed are exposed to solutions of different pH that contain the ionophores nigericin and monensin, that allow the rapid equilibration of the pH within phagosomes with the external solution. Ratio values are then compared to the known pH of solutions, a calibration curve is constructed by nonlinear regression and the resulting equation used to calculate pH from the ratio value.

Protocol

Etica Dichiarazione: Tutte le manipolazioni sugli animali sono state eseguite in stretta conformità con le linee guida del Comitato delle Università di Ginevra Animal Research. 1. Preparazione di obiettivi fagocitiche Aggiungere 20 mg di zimosan essiccata a 10 ml di fosfato sterile salina tamponata (PBS). Vortex e calore in un bagno di acqua bollente per 10 min. Raffreddare e centrifugare a 2000 xg per 5 min. Rimuovere il surnatante, risospendere in 1 ml di P…

Representative Results

Di seguito sono riportati i risultati rappresentativi per un esperimento in cui il pH phagosomal dei neutrofili topo primari isolati dal midollo osseo di wild-type o Hvcn1 – / – sono stati confrontati topi. Per un esperimento riuscito, è importante ottenere abbastanza fagosomi all'interno del campo visivo durante tutta la durata del film accelerato, evitando troppi fagosomi, che sarà poi essere più difficile segmento durante l'analisi delle immagini. La figura 1 mostra</…

Discussion

Sebbene più tempo rispetto metodi alternativi, come la spettroscopia e FACS, che impiegano una simile strategia di utilizzo di un colorante sensibile al pH accoppiato obiettivi ma misurano il pH medio di una popolazione di fagosomi, microscopia offre diversi vantaggi. Prima è che interno ed esterno vincolati, ma non interiorizzato, le particelle possono essere facilmente distinguibili, senza dover aggiungere altre sostanze chimiche, come trypan blu o di anticorpi, per placare o etichettare particelle esterne, rispetti…

Materials

Zymosan A powder	Sigma-Aldrich	Z4250	Various providers exist
Fluorescein isothiocyanate	Sigma-Aldrich	F7250	Various providers exist
Anti-zymosan antibody (Zymosan A Bioparticles opsonizing reagent)	Life Technologies	Z2850	Sigma-Aldrich O6637 is an equivalent product. Alternatively 25% serum can be used as an opsonizing reagent.
4-Aminobenzoic hydrazide (4-ABH)	Santa Cruz	sc-204107	Toxic, use gloves, various providers exist
Diphenyleneiodonium chloride (DPI)	Sigma-Aldrich	D2926	Toxic, use gloves, various providers exist
Concanamycin A (ConcA)	Sigma-Aldrich	27689	Toxic, use gloves, various providers exist
Nigericin	Sigma-Aldrich	N7143	Toxic, use gloves, various providers exist
Monensin	Enzo	ALX-380-026-G001	Toxic, use gloves, various providers exist
Phosphate buffered saline (PBS)	Life Technologies	14200-075	Various providers exist
Hank's balance salt solution	Life Technologies	14025092	Ringer's balanced salt solution or other clear physiological buffers may be substituted.
Sodium carbonate (Na2CO3)	Sigma-Aldrich	S7795	Various providers exist
2-(N-Morpholino)ethanesulfonic acid (MES)	Sigma-Aldrich	M3671	Various providers exist
4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)	Sigma-Aldrich	H3375	Various providers exist
N-Methyl-D-glucamine (NMDG)	Sigma-Aldrich	M2004	Various providers exist
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA)	Sigma-Aldrich	3777	Various providers exist
Tris(hydroxymethyl)aminomethane (Tris)	Sigma-Aldrich	T1503	Various providers exist
Potassium chloride (KCl)	Sigma-Aldrich	P9333	Various providers exist
Sodium chloride (NaCl)	Sigma-Aldrich	S7653	Various providers exist
Magnesium chloride (MgCl2)	Sigma-Aldrich	M8266	Various providers exist
Absolute Ethanol (EtOH)	Sigma-Aldrich	2860	Various providers exist
Glass-bottom 35 mm petri dishes (Fluorodish)	World Precision Instruments	FD35-100	Ibidi µ-clear dishes or coverslips with appropriate imaging chambers may be sustituted
Sonicating water bath	O. Kleiner AG		A sonicator may be used instead, various instrument providers exist
Heamocytometer	Marienfeld GmbH		Various instrument providers exist
Widefield live imaging microscope	Carl Zeiss AG		Various instrument providers exist, but the microscope must be able to image 440/535 and 490/535 excitation/emission respective. Spinning disk confocal set-ups with brightfield capabilities may substituted, but zymosan tend to go out of focus more often.
Peristaltic pump (Dynamax RP-1)	Rainin		Various instrument providers exist
pH meter	Schott Gerate GmbH		Various instrument providers exist
Manual Counter	Milian SA		Various instrument providers exist