Assaying Legionella pneumophila Replication in Macrophages Pretreated with Outer Membrane Vesicles

Samenvatting

Source: Jung, A. L. et. al., Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages. J. Vis. Exp. (2017)

This video demonstrates the pre-treatment of macrophages with Legionella pneumophila outer membrane vesicles (OMVs) to examine their effect on bacterial replication. By altering host immune signaling, OMVs make macrophages more permissive to infection. Higher colony counts in treated cells, compared to untreated ones, indicate that OMVs promote L. pneumophila replication inside macrophages.

Protocol

1. Pre-treat Macrophages

1. Prepare THP-1 (Tohoku Hospital Pediatrics-1 cells, a human monocytic leukemia cell line) cells.
   NOTE: THP-1 is a monocytic cell line derived from a leukemia patient.
   1. Add 2x10⁵ THP-1 cells per 24 wells and differentiate them by adding 20 nM phorbol 12-myristate 13-acetate (PMA) into macrophage-like cells. Incubate for 24 h at 37 °C.
   2. Replace the medium with 500 µL of fresh medium and incubate for another 24 h; the optimal medium for THP-1 cells comprises Roswell Park Memorial Institute Medium (RPMI) 1640 high glucose supplemented with 10% fetal calf serum.
2. Isolate the murine bone marrow-derived macrophages (mBMDM).
3. Treat THP-1-derived macrophages or mBMDM with outer membrane vesicles (OMVs).
   1. Thaw the OMVs and add them to the human or murine macrophages according to their protein amount (0.1, 1, and 10 µg/mL). Incubate the macrophages with OMVs at 37 °C for at least 20 h.

2. Infect the Macrophages and Assess Bacterial Replication with a Colony-Forming Unit (CFU) Assay

1. Use Legionella pneumophila, pre-treated THP-1 cells or mBMDM from step 4.3, and not pre-treated macrophages as controls (2x10⁵/24 wells). Do not exchange the medium.
2. Infect THP-1 cells with L. pneumophila Corby WT and mBMDM with a flagellin-lacking mutant of L. pneumophila Corby (both with a multiplicity of infection (MOI) of 0.5; 1x10⁵ L. pneumophila/24 wells) and incubate for 24 and 48 h, respectively.
3. Lyse the cells in their medium by the addition of saponin (final concentration: 0.1%) and incubate at 37 °C for 5 min.
4. Resuspend the bacteria by pipetting and transfer the suspension to a reaction vessel. Prepare serial dilutions of the L. pneumophila-containing media in sterile phosphate-buffered saline (PBS).
5. Streak 50 µL of the required dilutions on Buffered charcoal yeast extract (BCYE) agar plates and incubate for 3 days at 37 °C.
6. Visually count the formed colonies. Calculate the CFU

Normalize the CFU count result to not pre-treated but infected macrophages, which are set to 100%.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
10 cm petridish	Sarstedt AG & Co KG (Nuembrecht, Germany)	82.1473
Activated charcoal	Carl Roth GmbH & Co KG (Karlsruhe, Germany)	X865.2
Agar-agar, Kobe I	Carl Roth GmbH & Co KG (Karlsruhe, Germany)	5210.2
Columbia agar with 5 % sheep blood	Becton Dickinson GmbH (Heidelberg, Germany)	254005
Ferric nitrate nonahydrate	Carl Roth GmbH & Co KG (Karlsruhe, Germany)	5632.1
Fetal calf serum (FCS)	Life Technologies GmbH (Darmstadt, Germany)	10270-106
Heracell 240i Carbon dioxide incubator	Thermo Fisher Scientific Germany BV & Co KG (Braunschweig, Germany)	40830469
Heraeus Multifuge X3R	Thermo Fisher Scientific Germany BV & Co KG (Braunschweig, Germany)	75004515
Inoculation loop	Sarstedt AG & Co KG (Nuembrecht, Germany)	86.1567.010
L. pneumophila Corby	---	---	Kindly provided by Prof Dr Antje Flieger (RKI, Berlin, Germany)
L. pneumophila Corby ΔflaA	---	---	Kindly provided by Prof Dr Klaus Heuner (RKI, Berlin, Germany)
mBMDM	---	---	Kindly provided by Prof Dr Markus Schnare (Philipps Univeristy Marburg, Marburg, Germany) and Prof Dr Carsten Kirschning (University Duisburg Essen, Essen, Germany)
PBS	Biochrom GmbH (Berlin, Germany)	L 1825
Phorbol 12-myristate 13-acetate	Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany)	P8139-1MG
Rotating shaker (MaxQ 6000)	Thermo Fisher Scientific Germany BV & Co KG (Braunschweig, Germany)	SHKE6000
RPMI 1640 high glucose	Life Technologies GmbH (Darmstadt, Germany)	11875-093
Saponin	Carl Roth GmbH & Co KG (Karlsruhe, Germany)	9622.1
Ultrospec 10	Biochrom Ltd (Cambridge, England)	80-2116-30
THP-1	Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany)	88081201-1VL
Sorvall Discovery 100 SE	Thermo Fisher Scientific Germany BV & Co KG (Braunschweig, Germany)
Yeast extract	Carl Roth GmbH & Co KG (Karlsruhe, Germany)	2363.2