The research conducted is funded by President&#39;s NSERC grant as well as start-up funds from the Sylvia Fedoruk Canadian Center for Nuclear Innovation. The Sylvia Fedoruk Canadian Center for Nuclear Innovation is funded by Innovation Saskatchewan. Fluorescence imaging was performed at the WCVM Imaging Centre, which is funded by NSERC. Jessica Brocos (MSc Student) and Manpreet Kaur (MSc Student) were funded by the start-up funds from the Sylvia Fedoruk Canadian Center for Nuclear Innovation.

<ol>
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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Lung+is+a+Host+Defense+Niche+for+Immediate+Neutrophil-Mediated+Vascular+Protection.">The Lung is a Host Defense Niche for Immediate Neutrophil-Mediated Vascular Protection.</a> Science Immunology. 2 (10), (2017).</li><li>Lee, T. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiostatin+regulates+the+expression+of+antiangiogenic+and+proapoptotic+pathways+via+targeted+inhibition+of+mitochondrial+proteins.">Angiostatin regulates the expression of antiangiogenic and proapoptotic pathways via targeted inhibition of mitochondrial proteins.</a> Blood. 114 (9), 1987-1998 (2009).</li><li>Hawkins, C. L., Davies, M. 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The authors have no conflicts of interest or disclosures to make.

The methods presented in the current study highlight the usefulness of multiple compartment analysis to study multiple cellular events during lung inflammation. We have summarized the findings in Table 2. We and many labs have extensively studied the murine response to intranasal LPS instillation, which is marked by rapid recruitment of lung neutrophils, which peaks between 6-24 h following which, resolution kicks in. And recently, we have shown that sub-clinical O3 (at 0.05 ppm for 2 h) alone...

Acute lung injury (ALI) is a crucial pathologic response of lungs to infectious or other harmful stimuli which is marked by simultaneous activation of coagulative, fibrinolytic and innate immune systems1. Neutrophils promptly sense microbial as well as intracellular damage patterns through the Toll-like receptor (TLR) family2,3,4. Neutrophils release preformed cytokines and cytotoxic granule contents, which can then cause collateral tissue damage. The ensuing alveolar damage is marred with secondary cell death leading to release of molecules such as adenosine triphosphate (ATP)5, thus setting in a vicious cycle of immune-dysregulation.
An unsolved problem in the understanding of ALI relates to the question of how the injury is initiated within the alveolar membrane. The electron transport complex V, F1F0 ATP synthase, is a mitochondrial protein known to be expressed ubiquitously, on cell (including endothelial, leukocyte, epithelial) plasma membrane during inflammation. The cell cytoskeleton which is comprised of actin and tubulin, harbors many cell shape and function modulating as well as mitochondrial proteins, respectively. We have recently shown that blockade of the ATP synthase by an endogenous molecule, angiostatin, silences neutrophil recruitment, activation and lipopolysaccharide (LPS) induced lung inflammation6. Thus, both biochemical (ATP synthase) and immune (TLR4) mechanisms might regulate the alveolar barrier during lung inflammation.
Exposure to ozone (O3), an environmental pollutant, impairs lung function, increases susceptibility to pulmonary infections, and short low-levels of O3 exposures increase the risk of mortality in those with underlying cardiorespiratory conditions7,8,9,10,11,12,13,14. Thus, exposure to physiologically relevant concentrations of O3 provides a meaningful model of ALI to study fundamental mechanisms of inflammation7,8. Our lab has recently established a murine model of low-dose O3 induced ALI15. After performing a dose and time-response to low O3 concentrations, we observed that exposure to 0.05 ppm O3 for 2 h, induces acute lung injury that is marked by lung ATP synthase complex V subunit &#946; (ATP&#946;) and angiostatin expression, similar to the LPS model. Intravital lung imaging revealed disorganization of alveolar actin microfilaments indicating lung damage, and ablation of alveolar septal reactive oxygen species (ROS) levels (indicating abrogation of baseline cell signaling) and mitochondrial membrane potential (indicating acute cell death) after 2 h exposure to 0.05 ppm O315 which correlated with a heterogeneous lung 18FDG retention16, neutrophil recruitment and cytokine release, most notably IL-16 and SDF-1&#945;. The take-home message from our recent studies is that O3 produces exponentially high toxicity when exposed at concentrations below the allowed limits of 0.063 ppm over 8 h (per day) for human exposure. Importantly, no clear understanding exists on whether these sub-clinical O3 exposures can modulate TLR4-mediated mechanisms such as by bacterial endotoxin17. Thus, we studied a dual-hit O3 and LPS exposure model and observed the immune and non-immune cellular adaptations.
We describe a comprehensive fluorescent microscopic analysis of various lung and systemic body compartments, namely the broncho-alveolar lavage fluid (i.e., BAL) which samples the alveolar spaces, the lung vascular perfusate (i.e., LVP) that samples the pulmonary vasculature and the alveolar septal interstitium in the event of a compromised endothelial barrier, left lung cryosections, to look into resident parenchymal and adherent leukocytes left in the lavaged lung tissue, peripheral blood which represents the circulating leukocytes and the sternal and femur bone marrow perfusates that sample the proximal and distal sites of hematopoietic cell mobilization during inflammation, respectively.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>33-plex Bioplex chemokine panel</td>
			<td>Biorad</td>
			<td>12002231</td>
			<td></td>
		</tr>
		<tr>
			<td>63X oil (NA 1.4-0.6) Microscope objectives</td>
			<td>Leica</td>
			<td>HCX PL APO CS (11506188)</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 350 conjugated goat anti-mouse IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A11045</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 488 conjugated goat anti-mouse IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A11002</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 488 conjugated phalloidin</td>
			<td>Invitrogen</td>
			<td>A12370</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 555 conjugated mouse anti-&#945; tubulin clone DM1A</td>
			<td>Millipore</td>
			<td>05-829X-555</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 568 conjugated goat anti-hamster IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A21112</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 568 conjugated goat anti-rat IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A11077</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 633 conjugated goat anti-rabbit IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A21070</td>
			<td></td>
		</tr>
		<tr>
			<td>Armenian hamster anti-CD61 (clone 2C9.G2) IgG1 kappa</td>
			<td>BD Pharmingen</td>
			<td>553343</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 J Mice</td>
			<td>Jackson Laboratories</td>
			<td>64</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Leica</td>
			<td>Leica TCS SP5</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#8242;,6-diamidino-2-phenylindole)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>aliquot in 2 &#181;l stocks and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Inverted fluorescent wide field microscope</td>
			<td>Olympus</td>
			<td>Olympus IX83</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine (Narketan)</td>
			<td>Vetoquinol</td>
			<td>100 mg/ml</td>
			<td>Dilute 10 times to make a 10 mg/ml stock</td>
		</tr>
		<tr>
			<td>Live (calcein)/Dead (Ethidium homodimer-1) cytotoxicity kit</td>
			<td>Invitrogen</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-ATP5A1 IgG2b (clone 7H10BD4F9)</td>
			<td>Invitrogen</td>
			<td>459240</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-ATP5&#946; IgG2b (clone 3D5AB1)</td>
			<td>Invitrogen</td>
			<td>A-21351</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-NK1.1 IgG2a kappa (clone PK136)</td>
			<td>Invitrogen</td>
			<td>16-5941-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce 660 nm protein assay</td>
			<td>Thermoscientific</td>
			<td>22660</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-angiostatin (mouse aa 98-116) IgG</td>
			<td>Abcam</td>
			<td>ab2904</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-CX3CR1 IgG (RRID 467880)</td>
			<td>Invitrogen</td>
			<td>14-6093-81</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-Ki-67 (clone SolA15) IgG2a kappa</td>
			<td>Invitrogen</td>
			<td>14-5698-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-Ly6G IgG2a kappa (clone 1A8)</td>
			<td>Invitrogen</td>
			<td>16-9668-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-Ly6G/Ly6C (Gr1) IgG2b kappa (clone RB6-8C5)</td>
			<td>Invitrogen</td>
			<td>53-5931-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD16/CD32 Fc block (clone 2.4G2)</td>
			<td>BD Pharmingen</td>
			<td>553142</td>
			<td></td>
		</tr>
		<tr>
			<td>Reduced mitotracker orange</td>
			<td>Invitrogen</td>
			<td>M7511</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine (Rompun)</td>
			<td>Bayer</td>
			<td>20 mg/ml</td>
			<td>Dilute 2 times to make a 10 mg/ml stock</td>
		</tr>
	</tbody>
</table>

visualizing lung cellular adaptations during combined ozone and lps induced murine acute lung injury

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or fungal) inflammatory conditions. An overwhelming host response may result in compromised respiration and acute lung injury, which is characterized by lung neutrophil recruitment as a result of the patho-logical host immune, coagulative and tissue remodeling response. Sensitive microscopic methods to visualize and quantify murine lung cellular adaptations, in response to low-dose (0.05 ppm) ozone, a potent environmental pollutant in combination with bacterial lipopolysaccharide, a TLR4 agonist, are crucial in order to understand the host inflammatory and repair mechanisms. We describe a comprehensive fluorescent microscopic analysis of various lung and systemic body compartments, namely the broncho-alveolar lavage fluid, lung vascular perfusate, left lung cryosections, and sternal bone marrow perfusate. We show damage of alveolar macrophages, neutrophils, lung parenchymal tissue, as well as bone marrow cells in correlation with a delayed (up to 36-72 h) immune response that is marked by discrete chemokine gradients in the analyzed compartments. In addition, we present lung extracellular matrix and cellular cytoskeletal interactions (actin, tubulin), mitochondrial and reactive oxygen species, anti-coagulative plasminogen, its anti-angiogenic peptide fragment angiostatin, the mitochondrial ATP synthase complex V subunits, &#945; and &#946;. These surrogate markers, when supplemented with adequate in vitro cell-based assays and in vivo animal imaging techniques such as intravital microscopy, can provide vital information towards understanding the lung response to novel immunomodulatory agents.

Combined O3 and LPS exposure leads to systemic inflammation and bone marrow mobilization at 72 h: Cell counts in different compartments revealed significant changes in peripheral blood and the femur bone marrow total cell counts upon combined O3 and LPS exposures. Although combined O3 and LPS exposures did not induce any changes in the total BAL (Figure 1A) or LVP (Figure 1B</str...

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Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury

Combined ozone and bacterial endotoxin exposed mice show wide-spread cell death, including that of neutrophils. We observed cellular adaptations such as disruption of cytoskeletal lamellipodia, increased cellular expression of complex V ATP synthase subunit &#946; and angiostatin in broncho-alveolar lavage, suppression of the lung immune response and delayed neutrophil recruitment.

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or ...

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Research

JoVE Journal

Immunology and Infection

4.9K Views.  University of Saskatchewan. Combined ozone and bacterial endotoxin exposed mice show wide-spread cell death, including that of neutrophils. We observed cellular adaptations such as disruption of cytoskeletal lamellipodia, increased cellular expression of complex V ATP synthase subunit β and angiostatin in broncho-alveolar lavage, suppression of the lung immune response and delayed neutrophil recruitment.

Video: Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

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.

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.

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

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

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 ...

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...

Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple organs. With the development of therapies, most patients survive the hyperinflammatory phase but progress to an immunosuppressive phase, which increases the emergence of secondary infections. Therefore, improved understanding of the pathogenesis underlying secondary hospital-acquired infections in the immunosuppressive phase during sepsis is of tremendous importance. Reported here is a model to test infectious outcomes by creating double-hit infections in mice. A standard surgical procedure is used to induce polymicrobial peritonitis by cecal ligation and puncture (CLP) and followed by intranasal infection of Staphylococcus aureus to simulate pneumonia occurring in immune suppression that is frequently seen in septic patients. This dual model can reflect the immunosuppressive state occurring in patients with protracted sepsis and susceptibility to secondary infection from nosocomial pneumonia. Hence, this model provides a simple experimental approach to investigate the pathophysiology of sepsis-induced secondary bacterial pneumonia, which may be used for discovering novel treatments for sepsis and its complications.

This protocol describes techniques to measure infectious outcomes underlying secondary hospital-acquired infections in the immunosuppressive condition, first by establishing cecal ligation/puncture mice then challenging them with intranasal infection to create a clinically relevant model of immunosuppression sepsis.

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple ...

A Model of Self-limited Acute Lung Injury by Unilateral Intra-bronchial Acid Instillation

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic findings similar to human acute respiratory distress syndrome (ARDS). The resulting alveolar edema, alveolar-capillary barrier damage, and leukocyte infiltration predominantly affect the left lung, preserving the right lung as an uninjured control and allowing animals to survive. This model of self-limited acute lung injury enables investigation of tissue resolution mechanisms, such as macrophage efferocytosis of apoptotic neutrophils and restitution of alveolar-capillary barrier integrity. This model has helped identify important roles for resolution agonists, including specialized pro-resolving mediators (SPMs), providing a foundation for the development of new therapeutic approaches for patients with ARDS.

Selective intra-bronchial acid instillation to the left lung in mice results in unilateral and self-limited acute lung injury that models human acute respiratory distress syndrome (ARDS) induced by gastric acid aspiration.

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic ...