We thank Prof. Dr. Markus Schnare for providing us with TLR2-/- and TLR2/4-/- mice and Prof. Dr. Carsten Kirschning for TRIF/MyD88-/- mice. Parts of this work was funded by Bundesministerium f&#252;r Bildung und Forschung (e:bio miRSys - FKZ 0316175B, e:Med CAPSYS - FKZ 01X1304E; http://www.bmbf.de/), Deutsche Forschungsgemeinschaft (SFB/TR-84; http://www.sfb-tr84.de/), and Hessisches Ministerium f&#252;r Wissenschaft und Kunst (LOEWE Medical RNomics - FKZ 519/03/00.001-(0003); http://www.proloewe.de/medicalrnomics), all to BS.

1. Prepare Medium and Agar Plates
<ol>
	<li>Prepare 1 L of broth medium (YEB). Dissolve 10 g of ACES and 10 g of yeast extract in 900 mL of distilled water. Adjust the pH to 6.9 with KOH (5 N). Add 10 mL of L-cysteine (0.4 g in 10 mL of distilled water) and 10 mL of Fe(NO3)3x9H2O (0.25 g in 10 mL of distilled water). Fill up to 1 L with distilled water and filter sterilize the solution (pore size: 0.22 &#181;m). Store at 4 &#176;C.</li>
	<li>Prepare buffered charcoal yeast extract (BCYE) agar plates. Dissolve 10 g of ACES and 10 g of yeast extract in 900 mL of distilled water. Adjust the pH to 6.9 with KOH (5 N). Add 15 g of agar and 2.5 g of activated charcoal. Fill up to 1 L with distilled water and autoclave.
	<ol>
		<li>Add 10 mL of L-cysteine (0.4 g in 10 mL of distilled water) and 10 mL of Fe(NO3)3x9H2O (0.25 g in 10 mL of distilled water, both sterilized by filtration through 0.22-&#181;m pores) to cooled BCYE (approximately 50 &#176;C). Pour plates and store at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Cultivate L. pneumophila
<ol>
	<li>Spread L. pneumophila strain Corby (wild type, WT) on BCYE agar plates and incubate them at 37 &#176;C for 3 days. Inoculate 10 mL of YEB at an OD600 of 0.3 with L. pneumophila from the preculture plate; incubate the bacteria at 37 &#176;C on a rotating shaker (150 rpm) for 6 h.</li>
	<li>Verify the purity of the liquid culture by spreading 100 &#181;L of the suspension on a blood agar plate. Incubate overnight at 37 &#176;C.</li>
	<li>Add the remaining liquid culture to 90 mL of fresh YEB medium and incubate on a rotating shaker (37 &#176;C and 150 rpm) to reach an OD600 of 3.0-3.5, which takes approximately 16-20 h.</li>
</ol>
3. Prepare and Quantify L. pneumophila OMVs
NOTE: Carry out all of the following centrifugation steps under sterile conditions and at 4 &#176;C.
<ol>
	<li>Centrifuge the liquid culture at 4,000 x g for 20 min to pellet the bacteria. Transfer the supernatant to fresh centrifuge tubes, discard the bacterial pellet, and repeat the centrifugation (4,000 x g for 20 min). Repeat this step once.</li>
	<li>Sterile-filter the remaining supernatant twice (pore size: 0.22 &#181;m). Transfer the bacteria-free supernatant to ultracentrifuge tubes and ultracentrifuge at 100,000 x g for 3 h.</li>
	<li>Decant the supernatant and discard it. Resuspend the OMV pellet in sterile phosphate-buffered saline (PBS) and ultracentrifuge (100,000 x g for 3 h) to remove contaminating proteins and LPS.</li>
	<li>Discard the supernatant and resuspend the OMV pellet in 500 &#181;L of sterile PBS. Streak 20 &#181;L on a blood agar plate and on a BCYE agar plate to exclude bacterial contamination of the prepared vesicles. Incubate the blood agar plate overnight and the BCYE agar plate for 3 days (both at 37 &#176;C).</li>
	<li>Quantify the protein amount obtained from the OMV preparation using a bicinchoninic acid assay according to the manufacturer&#39;s instructions. 
	NOTE: The concentration of 100 mL of L. pneumophila culture is usually 1 &#181;g/&#181;L. Store the prepared and quantified OMVs at -20 &#176;C.</li>
</ol>
4. Pre-treat Macrophages
<ol>
	<li>Prepare THP-1 cells. 
	NOTE: THP-1 is a monocytic cell line derived from a leukemia patient.
	<ol>
		<li>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 &#176;C.</li>
		<li>Replace the medium with 500 &#181;L of fresh medium and incubate for another 24 h; the optimal medium for THP-1 cells is composed of RPMI 1640 high glucose supplemented with 10% fetal calf serum.</li>
	</ol>
	</li>
	<li>Isolate the murine bone marrow-derived macrophages (mBMDM), as described in Reference 20.</li>
	<li>Treat THP-1-derived macrophages or mBMDM with OMVs.
	<ol>
		<li>Thaw the OMVs prepared in step 3 and add them according to their protein amount (0.1, 1, and 10 &#181;g/mL) to the human or murine macrophages. Incubate the macrophages with OMVs at 37 &#176;C for at least 20 h. Use the supernatant for ELISA or move on with step 5.</li>
	</ol>
	</li>
</ol>
5. Infect the Macrophages and Assess Bacterial Replication with a Colony-Forming Unit (CFU) Assay
<ol>
	<li>Use L. pneumophila from step 2.1, 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.</li>
	<li>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. Prepare both L. pneumophila Corby (WT or flagellin-lacking mutant) as described in step 2.1.</li>
	<li>Lyse the cells in their medium by the addition of saponin (final concentration: 0.1%) and incubate at 37 &#176;C for 5 min.</li>
	<li>Resuspend the bacteria by pipetting and transfer the suspension to a reaction vessel. Prepare serial dilutions of the L. pneumophila-containing media in sterile PBS.</li>
	<li>Streak 50 &#181;L of the required dilutions on BCYE agar plates and incubate for 3 days at 37 &#176;C.</li>
	<li>Visually count the formed colonies. Calculate the CFU <img alt="Equation 1" src="/files/ftp_upload/55146/55146eq1.jpg" />&#160; Normalize the CFU count result to not pre-treated but infected macrophages, which are set to 100%.</li>
</ol>

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Novartis.com Available from: <a target="_blank" href="https://www.novartis.com/news/media-releases/novartis-bexsero%C2%AE-vaccine-approved-fda-prevention-meningitis-b-leading-cause">https://www.novartis.com/news/media-releases/novartis-bexsero%C2%AE-vaccine-approved-fda-prevention-meningitis-b-leading-cause</a> (2016)</li></ol>

The authors have nothing to disclose.

The OMVs of bacterial pathogens and the impact of these membrane vesicles on their target cells are currently being intensively studied. For example, Clostridium perfringens-derived OMVs induce cytokine secretion in macrophages, B lymphocytes can be activated by OMVs from Borrelia burgdorferi, and Helicobacter pylori-released membrane vesicles can act on gastric epithelial cells21,22,23. L. pneumophila, an intracellular pathogen that can induce a severe form of atypical pneumonia, also releases OMVs that are able to activate lung epithelial cells and macrophages16,19. Here, we present a detailed protocol for the small-scale isolation of L. pneumophila OMVs from liquid culture to study the potential role of OMVs in pneumonia. It is critical to work under sterile conditions and to rule out contamination from other bacteria in order to obtain a pure L. pneumophila-derived OMV preparation. The isolation of OMVs includes a filtration step through 0.22-&#181;m pores in order to prevent the contamination of the obtained OMV pellet with L. pneumophila, even though this reduces the OMV yield, since the largest OMVs are lost by this filtration step.
Furthermore, we tested the response of human and murine macrophages to those isolated vesicles and infected cells with L. pneumophila to more closely approximate the situation in Legionella pneumonia, where OMVs are released inside the LCV by extracellular bacteria15. The employed OMV doses have been estimated according to the free OMV amount in an in vitro infection of human macrophages after 24 h of incubation (described in Reference 20). For the stimulation of other recipient cells or in vivo experiments, other OMV doses might be necessary and must be established. The analysis of the effect of L. pneumophila OMVs represents an advancement to the protocol described by Jager and Steinert24.
Here, PMA-differentiated THP-1 cells serve as a model for alveolar macrophages due to the limited availability of primary human material. The addition of PMA differentiates the monocytic THP-1 cells into macrophage-like cells25. Furthermore, they are a well-known model cell line for L. pneumophila studies26. Besides this human monocytic cell line, mBMDM cells are used. mBMDM are widely accepted for the study of the effects of L. pneumophila27,28,29. The possibility of using genetic knockouts for different TLRs or other proteins make them a valuable tool for studying OMV effects. In order to lower the amount of mice per experiment, mBMDM are used instead of alveolar macrophages due to the limitations of the macrophages. Key experiments might require alveolar macrophages for validation.
Besides the herein-described protocol of ultracentrifugation to purify OMVs, it is possible to perform a density gradient centrifugation, which is included in the protocol by Chutkan et al.30. This could improve the purity of the obtained OMV preparation and reduce the amount of co-purified protein aggregates, flagellin, and LPS. The purity of the obtained OMV preparation can be analyzed by transmission electron microscopy or by nanoparticle tracking analysis as a supplementary step in quality control. This can provide an additional means of quantification, beyond the protein measurement procedure presented here. Optionally, the LPS concentration can be analyzed by a limulus amebocyte lysate test. If the OMV yield is low, an additional concentration step via centrifugal filters could be performed, which was not done here. If the yield was lower than expected, the OMVs were discarded.
As part of the ongoing effort to elucidate the biological mechanisms and functions behind OMVs, the influence of different stress conditions on OMV production could be tested. Nutrient deprivation, changes in incubation temperature, or exposure to harmful agents might have an impact on OMV secretion31. Possible stress conditions are discussed in the protocol by Klimentova and Stulik32. Moreover, hyper- or hypovesiculating L. pneumophila mutants could be generated. The different OMV preparations could then be analyzed in infection experiments with macrophages, human lung tissue explants (described in Reference 17), or even in in vivo models. Besides the role of OMVs in innate immune signaling, their influence in bacterial communication can be addressed experimentally. Furthermore, the impact of various innate immune signaling cascades might be analyzed by the use of murine knockout cells or the generation of CRISPR/Cas9 knockouts in human cell lines. This basic research in OMVs will assist in the development of new vaccine strategies, which already exist for meningitis B transmitted by Neisseria meningitides33.
Starting from the protocol on OMV isolation and characterization, one can apply this to other Gram-negative bacteria and to other host cells; it only needs to be adjusted to the growth of the bacteria in liquid culture. The protocol published by Chutkan et al. provides detailed information on the generation of OMVs from Escherichia coli and Pseudomonas aeruginosa30. The culture should not reach the late stationary phase in order to avoid increases in lysed bacteria and contaminating proteins and membranes. Additionally, the OMV dose used for stimulation of the host cells needs to be determined according to the amount of OMVs present during in vivo infections, while still ensuring a low rate of cytotoxicity. In this way, the pathological role of OMVs, their impact on inter-species communication, and host-pathogen interactions could be examined.
To further study the role of L. pneumophila OMVs in pneumonia, standardized OMV preparations with sufficient yields and comparable infection experiments are needed. This protocol will help to standardize isolation procedures and to extend OMV studies to other Gram-negative bacteria and to other host cells. Furthermore, research will benefit from the detailed in vitro knowledge, which can be used to extend experiments to in vivo settings. In the future, this protocol could be extended to the isolation of OMVs from primary biological material, such as serum or bronchoalveolar lavage fluid, to gain insight into the composition of OMVs released under physiological conditions. This will help to determine key parameters of OMV composition and to understand the properties of in vitro-generated OMVs.

Bacteria can secrete virulence factors via different mechanisms1. Besides the well-known secretory systems, Gram-negative bacteria can exchange information and deliver virulence factors via outer membrane vesicles (OMVs), which are small, spheroid vesicles 10-300 nm in diameter and with a bilayered membrane structure. They are secreted in a variety of growth environments (liquid culture, solid culture, and biofilms) and in all growth phases2,3. OMVs are an important means of transportation (e.g., for proteins, adhesins, toxins, and enzymes, as well as for LPS, which is found on the OMV surface)4. The intraluminal cargo is protected from proteolytic degradation, so it is able to act over long distances, and the vesicles can be found in body fluids and distant organs5,6,7,8. They can not only be recognized and taken up by eukaryotic cells9,10, but furthermore, they are able to facilitate the binding of bacteria and their invasion into host cells4. Legionella pneumophila (L. pneumophila) is a Gram-negative bacterium that can release OMVs. In the human lung, it primarily infects alveolar macrophages, even though its natural host are freshwater amoebae11. An L. pneumophila infection can cause Legionnaires&#39; disease, a severe form of pneumonia12. It blocks phagosome-lysosome fusion in the host cell. It also recruits host organelles, whereby a replication niche, the Legionella-containing vacuole (LCV), is formed13,14. Lysosomal degradation is inhibited not only by effector protein translocation via the type IV secretion system, but also by the release of OMVs15.
The purification of OMVs from bacterial cultures is required to analyze their effect on recipient cells. Earlier studies focused on the protein content of L. pneumophila OMVs and on the influence of the vesicles on alveolar epithelial cells16, but later studies with human lung tissue transplants demonstrated that L. pneumophila OMVs are taken up by alveolar macrophages17.
As OMVs present pathogen-associated molecular patterns (PAMPs) and other bacterial antigens, they might have an impact on the infection of eukaryotic cells and modulate the host immune response18. L. pneumophila OMVs rapidly fuse with host cell membranes and, moreover, they activate the membranous TLR219. As it is known that L. pneumophila OMVs stimulate macrophages and epithelial cells in a pro-inflammatory manner16,17, we analyzed the impact of OMVs on the infection process in human and murine macrophages.
Here, we describe a protocol for the cultivation of L. pneumophila in liquid culture to isolate the secreted OMVs by differential ultracentrifugation and to assess the impact of the vesicles on eukaryotic host cells, either directly or following an infection.

<table><tbody><tr><td>10 cm Petri dish</td><td>Sarstedt AG &amp;amp; Co KG (Nuembrecht, Germany)</td><td>82.1473</td><td></td></tr><tr><td>70 Ti rotor</td><td>Beckman Coulter Incorporation (California, USA)</td><td>337922</td><td></td></tr><tr><td>ACES</td><td>Carl Roth GmbH &amp;amp; Co KG (Karlsruhe, Germany)</td><td>9138.2</td><td></td></tr><tr><td>activated charcoal</td><td>Carl Roth GmbH &amp;amp; Co KG (Karlsruhe, Germany)</td><td>X865.2</td><td></td></tr><tr><td>agar-agar, Kobe I</td><td>Carl Roth GmbH &amp;amp; Co KG (Karlsruhe, Germany)</td><td>5210.2</td><td></td></tr><tr><td>Columbia agar with 5% sheep blood</td><td>Becton Dickinson GmbH (Heidelberg, Germany)</td><td>254005</td><td></td></tr><tr><td>cuvettes</td><td>Sarstedt AG &amp;amp; Co KG (Nuembrecht, Germany)</td><td>67.742</td><td></td></tr><tr><td>ELISA (human)</td><td>BD OptEIA&amp;trade;; Becton Dickinson GmbH (Heidelberg, Germany)</td><td>IL-8: 555244 IL-6: 550799</td><td></td></tr><tr><td>ELISA (murine)</td><td>DuoSet, R&amp;amp;D (Minneapolis, USA)</td><td>CXCL1: DY453-05</td><td></td></tr><tr><td>Fe(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;3&lt;/sub&gt;x9H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth GmbH &amp;amp; Co KG (Karlsruhe, Germany)</td><td>5632.1</td><td></td></tr><tr><td>Fetal calf serum (FCS)</td><td>Life Technologies GmbH (Darmstadt, Germany)</td><td>10270-106</td><td></td></tr><tr><td>Heracell 240i CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo Fisher Scientific Germany BV &amp;amp; Co KG (Braunschweig, Germany)</td><td>40830469</td><td></td></tr><tr><td>Heraeus Multifuge X3R</td><td>Thermo Fisher Scientific Germany BV &amp;amp; Co KG (Braunschweig, Germany)</td><td>75004515</td><td></td></tr><tr><td>Inoculation loop&amp;nbsp;</td><td>Sarstedt AG &amp;amp; Co KG (Nuembrecht, Germany)</td><td>86.1567.010</td><td></td></tr><tr><td>KOH</td><td>Carl Roth GmbH &amp;amp; Co KG (Karlsruhe, Germany)</td><td>6751.1</td><td></td></tr><tr><td>&lt;em&gt;L. pneumophila&lt;/em&gt; Corby</td><td>---</td><td>---</td><td>kindly provided by Prof Dr Antje Flieger (RKI, Berlin, Germany)</td></tr><tr><td>&lt;em&gt;L. pneumophila&lt;/em&gt; Corby &amp;Delta;flaA</td><td>---</td><td>---</td><td>kindly provided by Prof Dr Klaus Heuner (RKI, Berlin, Germany)</td></tr><tr><td>L-cystein</td><td>Carl Roth GmbH &amp;amp; Co KG (Karlsruhe, Germany)</td><td></td><td></td></tr><tr><td>mBMDM</td><td>---</td><td>---</td><td>kindly provided by Prof Dr Markus Schnare (Philipps Univeristy Marburg, Marburg, Germany) and Prof Dr Carsten Kirschning (University Duisburg Essen, Essen, Germany)</td></tr><tr><td>PBS</td><td>Biochrom GmbH (Berlin, Germany)</td><td>L 1825</td><td></td></tr><tr><td>phorbol 12-myristate 13-acetate</td><td>Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany)</td><td>P8139-1MG</td><td></td></tr><tr><td>rotating shaker (MaxQ 6000)</td><td>Thermo Fisher Scientific Germany BV &amp;amp; Co KG (Braunschweig, Germany)</td><td>SHKE6000</td><td></td></tr><tr><td>RPMI 1640 high glucose</td><td>Life Technologies GmbH (Darmstadt, Germany)</td><td>11875-093</td><td></td></tr><tr><td>saponin</td><td>Carl Roth GmbH &amp;amp; Co KG (Karlsruhe, Germany)</td><td>9622.1</td><td></td></tr><tr><td>Ultrospec 10</td><td>Biochrom Ltd (Cambridge, England)</td><td>80-2116-30</td><td></td></tr><tr><td>sterile filter (pore size: 0.22 &amp;micro;m)</td><td>Corning Incorporated (new York, USA)</td><td>431096</td><td></td></tr><tr><td>THP-1</td><td>Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany)</td><td>88081201-1VL</td><td></td></tr><tr><td>Sorvall Discovery 100 SE</td><td>Thermo Fisher Scientific Germany BV &amp;amp; Co KG (Braunschweig, Germany)</td><td></td><td></td></tr><tr><td>yeast extract</td><td>Carl Roth GmbH &amp;amp; Co KG (Karlsruhe, Germany)</td><td>2363.2</td><td></td></tr><tr><td>Pierce BCA protein assay kit</td><td>Thermo Fisher Scientific Germany BV &amp;amp; Co KG (Braunschweig, Germany)</td><td>23225</td><td></td></tr></tbody></table>

legionella pneumophila outer membrane vesicles: isolation and analysis of their pro-inflammatory potential on macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

The experimental setup to prepare L. pneumophila OMVs and to analyze their influence on the pro-inflammatory response of macrophages following infection is depicted in Figure 1. The pro-inflammatory potential of the prepared OMVs can be analyzed on PMA-differentiated THP-1 cells, which is shown in Figure 2. THP-1 cells respond with a time- and dose-dependent increase of IL-8 and IL-6 secretion. Additionally, the influence of different TLRs on L. pneumophila OMV recognition can be analyzed by using mBMDM from different genetic backgrounds, as presented by the CXCL1 ELISA in Figure 3. mBMDM from WT mice secreted CXCL1 after OMV stimulation, while mBMDM TLR2/4-/- secreted significantly less. To study the impact of L. pneumophila OMVs on bacterial replication in THP-1 macrophages, cells were pre-incubated with OMVs and then additionally infected with L. pneumophila (Figure 4 A). The pre-stimulation of THP-1-derived macrophages first reduces the bacterial replication after 24 h of infection, but it leads to a doubling in CFU count at the later time point (48 h p.i.). The impact of Toll-like receptor (TLR) signaling on OMV recognition following the infection of the macrophages can be assessed by mBMDM, as presented in Figure 4 B. Bacterial replication increases by tenfold in mBMDM from WT animals after OMV pre-incubation, while TLR2-/- and TRIF/MyD88-/-cells do not show this increase in L. pneumophila replication.
<img alt="Figure 1" src="/files/ftp_upload/55146/55146fig1.jpg" /> 
Figure 1: Experimental procedure. (A) L. pneumophila Corby WT from 10-cm BCYE agar plates are used to inoculate a small liquid culture (10 mL), which is transferred into 90 mL of fresh YEB medium after 6 h. A small volume is also plated on a blood agar plate to check for purity. Bacteria are incubated at 37 &#176;C until the early stationary phase (OD600 = 3.0-3.5). (B) The liquid culture is centrifuged and sterile-filtered to remove the bacteria. The Legionella-free supernatant is then ultracentrifuged to obtain an OMV pellet, which is resuspended in PBS and ultracentrifuged again. The isolated vesicles are resuspended, checked for purity, and quantified for the protein amount. The scale bar represents 2.5 cm. (C) Human or murine macrophages are stimulated with the quantified OMVs. The cell culture supernatant can be used for ELISA, or macrophages can be infected with L. pneumophila to determine bacterial replication by CFU assay on 10-cm BCYE agar plates. <a href="http://cloudflare.jove.com/files/ftp_upload/55146/55146fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55146/55146fig2.jpg" /> 
Figure 2: Pro-inflammatory activation of THP-1 cells by L. pneumophila OMVs. (A) Here, the monocytic THP-1 cell line is used as a model for alveolar macrophages. PMA-differentiated THP-1 cells were treated with increasing doses of L. pneumophila OMVs (0.01-25 &#181;g/mL) for 24 and 48 h, respectively. The cell-free supernatant was used for IL-8 ELISA. The mean values of three independent experiments &#177; SEM are shown. THP-1 cells responded to as little as 0.01 &#181;g/mL L. pneumophila OMVs with significant IL-8 secretion, which was time- and dose-dependent. (B) L. pneumophila OMVs (0.1-10 &#181;g/mL) were used to stimulate PMA-differentiated THP-1 cells. The supernatant was collected after 24 and 48 h of incubation, and the released IL-6 was measured in the supernatant via ELISA. The mean values of three independent experiments &#177; SEM are shown. THP-1 cells secreted significant amounts of IL-6, even with the lowest dose of OMVs (0.1 &#181;g/mL). The secretion of IL-6 increased with increasing OMV doses and with prolonged incubation times. Statistics: Mann-Whitney test; *p &#60; 0.05 and **p &#60; 0.01 compared to the corresponding 0 &#181;g/mL OMV. Reprinted with permission from Reference 20. <a href="http://cloudflare.jove.com/files/ftp_upload/55146/55146fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55146/55146fig3.jpg" /> 
Figure 3: The pro-inflammatory activation of macrophages depends on TLR2/4. mBMDM from WT and TLR2/4-/- mice were incubated with L. pneumophila OMVs (0.1 or 1 &#181;g/mL). CXCL1 secretion was analyzed by ELISA after 24 and 48 h, respectively. The mean values &#177; SEM of three independent experiments are shown. mBMDM from WT mice responded with a dose-dependent CXCL1 secretion after L. pneumophila OMV incubation. TLR2/4-/- mBMDM secreted significantly less CXCL1 compared to WT mBMDM, and this secretion did not increase dose-dependently. Statistics: Mann-Whitney test; *p &#60; 0.05 compared to the corresponding 0 &#181;g/mL OMV; #p &#60; 0.05 compared to an equally treated WT sample. Reprinted with permission from Reference 20. <a href="http://cloudflare.jove.com/files/ftp_upload/55146/55146fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55146/55146fig4.jpg" /> 
Figure 4: L. pneumophila OMV pre-incubation increases bacterial replication in macrophages. (A) Differentiated THP-1 cells were pre-incubated with OMVs (0.1, 1, or 10 &#181;g/mL) or LPS/IFN-&#947; (200 ng/mL each) or were left untreated for control. After pre-incubation (20 h), THP-1 cells were infected with L. pneumophila Corby WT (MOI 0.5) for 2, 24, and 48 h, respectively. THP-1 cells were lysed by the addition of saponin, and the bacteria were plated on BCYE agar plates. CFUs were calculated relative to 0 &#181;g/mL OMV after every time point. The bars represent the mean values &#177; SEM of three independent experiments, each performed in technical duplicates. There were no differences in bacterial uptake (2 h post-infection (p.i.)) in comparison to not pre-treated cells. Alterations in bacterial replication were determined after 24 and 48 h, respectively. LPS/IFN-&#947; pre-treated THP-1 cells showed a reduction in bacterial load 24 h p.i. This was also observed dose-dependently for L. pneumophila OMV pre-treated cells. At the later time point (48 h p.i.), OMV pre-treated THP-1 cells showed a doubling in L. pneumophila replication, whereas LPS/IFN-&#947; pre-treated macrophages showed a further reduction of bacterial load. Statistics: Mann-Whitney test; *p &#60; 0.05 and **p &#60; 0.01 compared to the corresponding 0 &#181;g/mL OMV. (B) mBMDM from mice with different genetic backgrounds (WT, TLR2-/-, and TRIF/MyD88-/-) were pre-incubated with 0.1 &#181;g/mL L. pneumophila OMVs for 20 h and were then infected with a flagellin-deficient mutant of L. pneumophila Corby (MOI 0.5) for 48 h. mBMDM were lysed by the addition of saponin, and the Legionella were plated on BCYE agar plates. The CFU were calculated relative to 0 &#181;g/mL OMV, indicated by the solid line. The bars represent the mean values &#177;&#177; SEM of three independent experiments, each performed in duplicates. mBMDM from WT mice showed an increase in L. pneumophila replication after OMV pre-treatment. TLR2-/- macrophages showed significantly reduced Legionella replication, which was comparable to TRIF/MyD88-/- mBMDM. Statistics: Mann-Whitney test; p &#60; 0.05 compared to the WT sample. Reprinted with permission from Reference 20. <a href="http://cloudflare.jove.com/files/ftp_upload/55146/55146fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages at JoVE.com

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

legionella-pneumophila-outer-membrane-vesicles-isolation-analysis

Research

JoVE Journal

Immunology and Infection

11.1K Views.  Philipps-University Marburg.  We beschrijven de zuivering van Legionella pneumophila (L. pneumophila) buitenmembraan vesicles (OMV) van vloeibare kweken. Deze gezuiverde blaasjes worden vervolgens gebruikt voor de behandeling van macrofagen aan de pro-inflammatoire potentieel analyseren. 

Video: Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

Cancer Research

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

Biochemistry

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, gains a pathogenic advantage over in vitro cultured bacteria when first harvested from protozoan cells prior to infection of mammalian macrophages. This suggests that important virulence factors may not be properly expressed in vitro. We have developed a tractable system for priming L. pneumophila through its natural protozoan host Acanthamoeba castellanii prior to mammalian cell infection. The contribution of any virulence factor can be examined by comparing intracellular growth of a mutant strain to wild-type bacteria after protozoan priming. GFP-expressing wild-type and mutant L. pneumophila strains are used to infect protozoan monolayers in a priming step and allowed to reach late stages of intracellular growth. Fluorescent bacteria are then harvested from these infected cells and normalized by spectrophotometry to generate comparable numbers of bacteria for a subsequent infection into mammalian macrophages. For quantification, live bacteria are monitored after infection using fluorescence microscopy, flow cytometry, and by colony plating. This technique highlights and relies on the contribution of host cell-dependent gene expression by mimicking the environment that would be encountered in a natural acquisition route. This approach can be modified to accommodate any bacterium that uses an intermediary host as a means for gaining a pathogenic advantage.

This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

Flow Cytometry Analysis of Intracellular and Emergent Extracellular Listeria monocytogenes In Vitro

Source: Gayle, P. et al., <a href="https://app.jove.com/v/58775">Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses.</a> J. Vis. Exp. (2019)
In this video, we quantify intracellular and extracellular Listeria monocytogenes, an intracellular pathogenic bacteria, following their infection of cultured macrophages in vitro using flow cytometry.

Source: Gayle, P. et al., Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses. J. Vis. Exp. (2019)
In this video, we ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

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