This research was funded, in whole or in part, by the Wellcome Trust Investigator Award [107964/Z/15/Z]. P.R.T is also supported by the UK Dementia Research Institute. M.A.C is supported by the Biotechnology and Biological Sciences Research Council Discovery Fellowship (BB/T009543/1). For the purpose of Open Access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. L.C.D is a lecturer at Swansea University and an honorary research fellow at Cardiff University. This work is supported by work carried out by Ipseiz et al. 202018.

Lentiviral Vector Preparation for In Vivo Gene Modulation 

<ol>
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The authors have nothing to disclose.

Tissue-resident macrophages perform a range of homeostatic and inflammatory tissue-specific functions1,2 dictated by their physiological environment6,7,8,9. In this protocol, an effective method18 for manipulation of peritoneal resident macrophages in vivo using lentivirus particles was introduced to inv...

Tissue-resident macrophages (M&#966;) are a heterogeneous population of phagocytic immune cells that sense and respond to invading pathogens1,2. In addition, they play an essential role in tissue development, remodeling, and maintaining homeostasis1,3. Many tissue M&#966; derive from yolk sac progenitors during embryogenesis and persist in the tissue throughout the life4,5. The phenotype and functions of these cells are dictated by collaborative and hierarchical interactions of specific transcription factors and the local microenvironment6,7,8,9. A growing understanding of this dependency increases the need for effective in vivo methods for gene manipulation of M&#966; within their physiologically relevant niche.
Lentiviral vectors are a frequently employed tool for the manipulation of nucleic acids in specific cell populations in vivo10,11,12, particularly due to their ability to infect both dividing and non-dividing cells and to stably integrate into host genome13,14. Over the last two decades, lentivirus delivery technology has been optimized, and alternative envelopes and synthetic promoters have been investigated to increase lineage-specific targeting8,15. Owing to its broad cell tropism, vesicular stomatitis virus envelop glycoprotein (VSV-G)16,17 has become the &#34;gold-standard&#34; envelope used in lentivirus technology.
In this protocol18, VSV-G pseudotyped lentiviral particles are employed to demonstrate targeted and effective delivery of short hairpin RNA (shRNA) and microRNA (miR) to mouse peritoneal M&#966; (pM&#966;) in vivo, at steady state19. Transgene expression was driven by the spleen focus forming virus (SFFV) promoter. Productive infection of cells was defined by expression of lentivirus-derived enhanced green fluorescent protein (GFP). Utilization of this approach allowed easy readout for in vivo lentivirus experiments to define the optimal dose and the experimental timeframe. Finally, in vivo lentiviral challenge of mice during thioglycolate-induced inflammation revealed the natural propensity for selective pM&#966; infection.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA (1x) (Trypsin 500 mg/L or 0.02 mM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m sterile millex GP filter</td>
			<td>Merck</td>
			<td>SLGS033SS</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 &#956;m sterile millex GP filter</td>
			<td>Merck</td>
			<td>SLHP033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL U-100 insulin syringe with needle, 0.33 mm x 12.7 mm (29 G)</td>
			<td>BD</td>
			<td>324892</td>
			<td></td>
		</tr>
		<tr>
			<td>1 Litre Sharps Container</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>2.4G2 antibody (TruStain FcX anti-mouse CD16/32)</td>
			<td>Biolegend</td>
			<td>101320</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#956;m strainer</td>
			<td>Thermo Fisher Scientific</td>
			<td>22363547</td>
			<td></td>
		</tr>
		<tr>
			<td>AimV medium (research grade), AlbuMax Supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>31035025</td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking buffer</td>
			<td>prepared in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brewer thioglycolate medium</td>
			<td>Sigma-Aldrich</td>
			<td>B2551</td>
			<td>4% stock solution prepared in water, autoclaved and kept frozen.</td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>Biolegend</td>
			<td>101222</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>BD</td>
			<td>550993</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>CD11c</td>
			<td>Biolegend</td>
			<td>117317</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>CD11c</td>
			<td>Biolegend</td>
			<td>117333</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>CD19</td>
			<td>BD</td>
			<td>560375</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>CD19</td>
			<td>Biolegend</td>
			<td>152410</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>CD226</td>
			<td>Biolegend</td>
			<td>128808</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>CD3e</td>
			<td>BD</td>
			<td>560527</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>CD3e</td>
			<td>Biolegend</td>
			<td>152313</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>CD4</td>
			<td>Biolegend</td>
			<td>100412</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>CD73</td>
			<td>eBioscience</td>
			<td>16-0731-82</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>CD8a</td>
			<td>eBioscience</td>
			<td>48-0081-82</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>Cell culture flask (T175 fask, 175 cm2, 550 mL)</td>
			<td>Greiner Bio One</td>
			<td>658175</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes, conical bottom tubes 25 mm x 89 mm</td>
			<td>Beckman Coulter</td>
			<td>358126</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuges</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Ultracentrifuge and TC centrifuge</td>
		</tr>
		<tr>
			<td>Collagenase type IV</td>
			<td>Sigma-Aldrich</td>
			<td>C5138</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical centrifuge tubes (15 mL and 50 mL)</td>
			<td>Greiner Bio One</td>
			<td>11512303 &#38; 11849650</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryotubes</td>
			<td>Greiner Bio One</td>
			<td>123277</td>
			<td>or cryotubes</td>
		</tr>
		<tr>
			<td>DMEM medium (1x) + 4.5g/L D-glucose, 400 &#181;M L-glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>41965-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Sigma-Aldrich</td>
			<td>11284932001</td>
			<td></td>
		</tr>
		<tr>
			<td>Effectene transfection reagent</td>
			<td>Qiagen</td>
			<td>301425</td>
			<td></td>
		</tr>
		<tr>
			<td>F4/80</td>
			<td>Biolegend</td>
			<td>123123</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>F4/80</td>
			<td>Biolegend</td>
			<td>123133</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>F4/80</td>
			<td>Biolegend</td>
			<td>123147</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>FceR1</td>
			<td>eBioscience</td>
			<td>48-5898-80</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>10270-106</td>
			<td>heat inactivated for 30 min at 56 &#176;C and sterile filtered through 0.22 &#956;m filter</td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>&#160;Attune NxT</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometry (FACS) buffer</td>
			<td>prepared in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent tissue culture microscope</td>
			<td>Thermo Fisher Scientific</td>
			<td>EVOS FL</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>User preference</td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution (HBSS)</td>
			<td>Gibco, Life Technologies</td>
			<td>14175-053</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cell line</td>
			<td></td>
			<td></td>
			<td>grown for at least a week prior transfection. Mycoplasma free</td>
		</tr>
		<tr>
			<td>HIV-1 Core antigen</td>
			<td>Beckman Coulter</td>
			<td>6604667</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma-Aldrich</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrex surgical scrub, chlorhexiding gluconate 4% w/v skin cleanser</td>
			<td>Ecolab</td>
			<td>3037170</td>
			<td></td>
		</tr>
		<tr>
			<td>I-A/I-E</td>
			<td>Biolegend</td>
			<td>107625</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>ICAM1</td>
			<td>Becton Dickinson</td>
			<td>554970</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>Jurkat T cell line</td>
			<td></td>
			<td></td>
			<td>grown for at least a week prior use. Mycoplasma free</td>
		</tr>
		<tr>
			<td>LIVE/DEAD fixable near-IR dead cell stain kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>L34975</td>
			<td></td>
		</tr>
		<tr>
			<td>Ly6G</td>
			<td>Biolegend</td>
			<td>127615</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td></td>
			<td></td>
			<td>here used C57BL/6 females, aged 8-12 weeks (Charles Rivers), unless specified differently</td>
		</tr>
		<tr>
			<td>Microcapillary pipettes (volume range 0.5-1,000 &#956;L)</td>
			<td>Fisher Scientific &#38; Starlab</td>
			<td>11963466 &#38; 11943466 &#38; 11973466 &#38; S1111-3700</td>
			<td></td>
		</tr>
		<tr>
			<td>NK1.1</td>
			<td>Biolegend</td>
			<td>108724</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148-500G</td>
			<td>prepared to 2% w/v in PBS</td>
		</tr>
		<tr>
			<td>pCMV-&#916;R8.91 packaging plasmid</td>
			<td></td>
			<td>Zuffrey, R., et al. 1997</td>
			<td>encodes Gag-Pol HIV protein driven by cytomegalovirus promoter. Ampicilin resistance.</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (100x, 10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Greiner Bio One</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>pHR&#39;SIN-cPPT-SEW plasmid</td>
			<td></td>
			<td>Rosas, M. et al. 2014</td>
			<td>modified for shRNA and miR expression studies. Encodes EGFP marker downstream SFFV promoter and upstream of the Woodchuck hepatitiv virus enhancer. Ampicilin resistance.</td>
		</tr>
		<tr>
			<td>pMD2.G plasmid</td>
			<td></td>
			<td>Naldini, L. et al. 1996</td>
			<td>encodes vesicular stomatis virus g-glycoprotein (VSV-G) envelope. Ampicilin resistance.</td>
		</tr>
		<tr>
			<td>Rat IgG1, &#954; isotype control</td>
			<td>Becton Dickinson</td>
			<td>550617</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>Rat serum</td>
			<td>Sigma-Aldrich</td>
			<td>R9759-10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood ACK lysis buffer</td>
			<td>prepared in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium (1x) + 400 uM L-glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>21875-091</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma-Aldrich</td>
			<td>S4521</td>
			<td></td>
		</tr>
		<tr>
			<td>SiglecF</td>
			<td>BD</td>
			<td>562681</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>Sodium hypochlorite Tablets (bleach, 2,000 ppm)</td>
			<td>Guest Medical</td>
			<td>H8818</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 24-well cell culture plate</td>
			<td>Greiner Bio One</td>
			<td>662160</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Dulbecco&#39;s PBS (DPBS) (1x) Mg++ and Ca2+ - free</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile VWR disposable transfer pipets (23.0 mL, 30 cm)</td>
			<td>VWR</td>
			<td>612-4515</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Thermo Fisher Scientific</td>
			<td>15503022</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical scissors</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>User preference</td>
		</tr>
		<tr>
			<td>Syringes (50 mL and 1 0mL)</td>
			<td>Fisher Scientific</td>
			<td>10084450 &#38; 768160</td>
			<td></td>
		</tr>
		<tr>
			<td>Tim4</td>
			<td>Biolegend</td>
			<td>130007</td>
			<td>Refer to Table 1 for the dilution and concentration</td>
		</tr>
		<tr>
			<td>U-bottom 96-well cell culture plate</td>
			<td>Greiner Bio One</td>
			<td>650180</td>
			<td></td>
		</tr>
	</tbody>
</table>

lentiviral vector preparation for efficient gene and microrna modulation of peritoneal cavity tissue&#45;resident macrophages in vivo in mice

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.

When followed fully and correctly, this protocol yields a total of 1.5 mL of high-quality lentivirus stock per single preparation, sufficient for twelve in vivo injections at the optimal volume determined in this study18. The success of the transfection can be evaluated early in the protocol. Healthy and confluent HEK293T cells should display, if present in the plasmids, an easily detectable marker signal (e.g., GFP used in this study) after 48 h post plasmid transfection (<strong class="...

Read this Scientific Article Protocol about Author Spotlight: Exploring Macrophage Immunometabolism Through Lentiviral Vector-Mediated Gene Manipulation at JoVE.com

Author Spotlight: Exploring Macrophage Immunometabolism Through Lentiviral Vector&#45;Mediated Gene Manipulation

We demonstrate a step-by-step protocol for the investigation of gene function in peritoneal tissue-resident macrophages in vivo, using lentiviral vectors.

Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue&#45;Resident Macrophages In Vivo in Mice

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring ...

Compared to other viral approaches for in vivo gene manipulation in macrophages, the use of lentiviral vectors offers a stable integration of the transgenic tissue macrophages, efficient transgene expression, and the largest vector size limit. When employing in vivo lentiviral manipulation of metabolic enzymes, we discovered notable functional changes in tissue resident macrophages. Therefore, we are currently investigating immuno metabolism and its role in preserving tissue specific functions, including the epigenetics and immune function.In the future, we would like to focus on defining how tissue macrophages affect other local cell populations during homeostasis and in disease models, and how these interactions can be employed for disease prevention and resolution.

lentiviral-vector-preparation-for-efficient-gene-microrna-modulation

Research

JoVE Journal

Immunology and Infection

581 Views.  Cardiff University. Compared to other viral approaches for in vivo gene manipulation in macrophages, the use of lentiviral vectors offers a stable integration of the transgenic tissue macrophages, efficient transgene expression, and the largest vector size limit. When employing in vivo lentiviral manipulation of metabolic enzymes, we discovered notable functional changes in tissue resident macrophages. Therefore, we are currently investigating immuno metabolism and its role in preserving tissue specific function...

Video: Author Spotlight: Exploring Macrophage Immunometabolism Through Lentiviral Vector-Mediated Gene Manipulation

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells.

We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, ...

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

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. ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a promising future of using miRNAs in the diagnosis and care of lung diseases. Here, we define a protocol for miRNA profiling to measure the relative abundance of a group of miRNAs predicted to regulate inflammatory genes in the lung tissue from of an ozone-induced airway inflammation mouse model. Because it has been shown that circulating sex hormone levels can affect the regulation of lung innate immunity in females, the purpose of this method is to describe an inflammatory miRNA profiling protocol in female mice, taking into consideration the estrous cycle stage of each animal at the time of ozone exposure. We also address applicable bioinformatics approaches to miRNA discovery and target identification methods using limma, an R/Bioconductor software, and functional analysis software to understand the biological context and pathways associated with differential miRNA expression.

Here we describe a method to assess lung expression of miRNAs that are predicted to regulate inflammatory genes using mice exposed to ozone or filtered air at different stages of the estrous cycle.

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...

Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal failure. In recent years, the involvement of microRNAs (miRNAs) has been reported in the pathogenesis of IgA nephropathy. However, there is no established method for profiling miRNAs in IgA nephropathy using small animal models. Therefore, we developed a reliable method for analyzing miRNA in the kidney of an IgA mouse model (HIGA mouse). The goal of this protocol is to detect the altered expression levels of miRNAs in the kidneys of HIGA mice when compared with the levels in kidneys of control mice. In brief, this method consists of four steps: 1) obtaining kidney samples from HIGA mice; 2) purifying total RNA from kidney samples; 3) synthesizing complementary DNA from total RNA; and 4) quantitative reverse transcription polymerase chain reaction (qRT-PCR) of miRNAs. Using this method, we successfully detected the expression levels of several miRNAs (miR-155-5p, miR-146a-5p, and miR-21-5p) in the kidneys of HIGA mice. This new method can be applied to other studies profiling miRNAs in IgA nephropathy.

microRNAs are involved in the pathogenesis of IgA nephropathy. We have developed a reliable method for detecting microRNA expression levels in the kidneys of an IgA nephropathy mouse model (HIGA mice). This new method will facilitate to check for miRNAs involvement in IgA nephropathy.

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal ...