This study was supported by NIH grant R01 HL131812 and R01HL163881.

All the animal protocols described in this study were approved by the Institutional Animal Care and Use Committee (ACUC) at the Johns Hopkins University, School of Medicine for the animal protocol MO21M160. In addition, the experiments were performed following the institutional, state, and federal regulations for animal research.
CAUTION: The replication of all the protocols described below requires to be performed in a Biosafety level 2 (BSL-2) cabinet, following all of the institutional BSL-2 guidelines under the biosafety cabinet.
1. Plating bacterial stocks from commercial loops
NOTE: This protocol can be used for growing bacterial stocks for Spn (ATCC 49619) and Kpn (ATCC 43816), starting with the culti-loops obtained from the provider (see Table of Materials for details).
<ol>
	<li>Warm a 5% sheep blood agar plate for 15 min at 37 &#176;C in an incubator. A cell incubator can be used for this purpose.</li>
	<li>Under the hood, remove the sheath from the bacterial inoculating loop and spread it carefully, streaking into the agar plate, following a zig-zag pattern. Repeat this step using a separate plate as a duplicate. Up to five plates can be streaked with the same loop.</li>
	<li>Incubate the plates overnight at 37 &#176;C for optimal bacteria growth. Passage the bacteria daily for 3 days. Warm the blood agar plates for 15 min at 37 &#176;C, take between 15 to 20 colonies from the first agar plate using a disposable inoculating loop, and spread them into a new, prewarmed agar plate. Label the plate appropriately.</li>
</ol>
2. Bacteria growth and storage for future use
<ol>
	<li>After 3 days, pick up to 30 colonies from the blood agar plate with a streak loop, and place it directly into a 250 mL flask containing Todd Hewitt broth (TH broth). Incubate at 37 &#176;C, shaking at 250 rpm, with 5% CO2 for approximately 4 h.</li>
	<li>Take aliquots every 15 min to measure the OD620nm until it reaches 0.3, which is equivalent to approximately 3 x 108 colony forming units (CFU) per mL.</li>
	<li>Aliquot out 1 mL of the new bacteria stock immediately into 2 mL cryogenic vials. Flash freeze the freshly aliquoted vials in liquid nitrogen for 5 min. Store aliquots of bacteria in a -80 &#176;C freezer (vials can be used for up to 6-8 months before they lose potency).</li>
	<li>After 7 days of freezing, the aliquots can be used for animal studies. Therefore, determine the new concentration of the bacteria.</li>
</ol>
3. Bacteria thawing for intratracheal instillation
<ol>
	<li>Warm a blood agar plate for 10 min at 37 &#176;C in a shaker. Take one of the new stock vials from the freezer and thaw it down by gentle agitation in a 37 &#176;C water bath for approximately 2 min. Avoid touching the O-ring or cap with the warm water.</li>
	<li>Plate on a blood agar plate to manually count the bacterial colonies. Perform a 1 x 10-6 dilution, and plate 200 &#181;L from the last dilution in a prewarmed blood agar plate. Do this in duplicates.</li>
	<li>Incubate the plates overnight at 37 &#176;C for optimal growth of the bacteria. The next day, apply the following formula to determine the new bacterial concentration: CFU/mL = (number of colonies x dilution factor) / volume of stock plated</li>
</ol>
4. Intratracheal instillation of live bacteria
NOTE: This protocol has been optimized to instill a volume of 50 &#181;L intratracheally. Bacterial stocks can be stored for up to 6-9 months. To ensure the bacterial CFU in each vial, make sure to plate it before each experiment, calculate the bacterial stock CFU as described above, and use TH broth for subsequent dilutions.
CAUTION: Under the biosafety cabinet, perform a rodent survival procedure using sterile surgical instruments.
<ol>
	<li>Anesthetize the mouse by injecting intraperitoneally 100 mg/kg of ketamine and 2.5 mg/kg of acetylpromazine. Repeat for the number of mice being injected at a time. 
	NOTE: Mice can be exposed to isoflurane to facilitate animal handling. However, an experienced handler who is capable of injecting intraperitoneal anesthetics without causing significant animal distress can avoid isoflurane exposure. The number of mice to inject depends on the expertise of the surgeon.</li>
	<li>After all the mice are under appropriate anesthesia, confirm anesthesia by tail pinch. Place the anesthetized mouse on a clean and sterilized surface, hang the animal by the incisors, and gently tape the forelimbs (Figure 1A). 
	NOTE: To ensure animal wellness, provide corneal lubrication in all the anesthetized animals. Carboxymethylcellulose eye drops can be used, applying one drop per eye.</li>
	<li>Shave the neck region and disinfect the area&#160;with chlorhexidine and 70% alcohol. Using surgical scissors, make a 1 cm superficial midline neck incision to visualize the trachea (Figure 1B). One small incision should be enough, but if an abundance of fat tissue is seen, carefully dissect the adipose tissue vertically to visualize the trachea.&#160; 
	NOTE: Use sterile gloves and sterile instruments via the tips-only technique.&#160;The risk of infection is minimal if the procedure is performed under sterile conditions.</li>
	<li>Using forceps, intubate each mouse. Pull the tongue outward gently and introduce a 20 G angio-catheter through the mouth, advancing the catheter into the trachea. Apply gentle pressure over the trachea to facilitate intubation. 
	NOTE: Because the trachea is anterior to the mouse body, slightly bend the tip of the catheter, as this will help get the angle to successfully intubate the mouse. The neck incision is performed mainly for visualization purposes; alternatively the operator can intubate the trachea blindly.</li>
	<li>After intubation, connect the mouse to a respirator to confirm intubation. The regular ventilator parameters used in this procedure are 200 &#181;L of stroke volume and 200 strokes per minute. Turn the ventilator rate up and/or down briefly for quick and easy confirmation.</li>
	<li>After confirming intubation, disconnect the mouse from the respirator and carefully instill 50 &#181;L of the bacterial agent through the angio-catheter using a 200 &#181;L pipet gel loading tip. For control mice, inject 50 &#181;L of sterile TH broth.</li>
	<li>After instilling the bacterial agent, connect the mouse to the respirator again to help restart breathing. Leave the mouse on the respirator for 30-60 s. After injecting all the mice, monitor their breathing. If there&#39;s a slow breathing pattern, connect the mice to respirator again.</li>
	<li>Close the incision by adding one small drop of glue on the skin. Bring together the skin folds and apply gentle pressure until the glue dries.</li>
	<li>Place the mice on a heating pad for recovery and closely monitor them until they regain sufficient consciousness. Transfer the mice back in the cage once fully recovered. For post-operative pain/distress management, the mice can be treated with buprenorphine, with a dose of 0.05-0.1mg/kg subcutaneously (SC). 
	&#8203;NOTE: Animals with weight loss greater than 20% or experiencing significant distress post-procedure such as lethargy, an inability to reach water or food, labored breathing, impaired response to external stimuli, or decreased mental alertness should be euthanized.</li>
</ol>
5. Bronchoalveolar lavage and lung harvest
<ol>
	<li>Euthanize the mouse by placing it in a closed container containing 5% isoflurane. Continue isoflurane exposure for 1 min after the mouse stops breathing. Perform thoracotomy to confirm euthanasia. 
	NOTE: Confirm euthanasia by visual and physical examination. The heart must have stopped beating and the mice not breathing. Mucous membranes should be white or pale.</li>
	<li>Lay the mouse in a supine position on a clean surgical board and hang it by the incisors. Spray the mouse skin with 70% ethanol. Using surgical scissors, make a small superficial midline neck incision to visualize the trachea of the mouse.</li>
	<li>Cannulate the trachea with a 20 G catheter. Carefully, add 1 mL of calcium-free phosphate-buffered saline (PBS) intratracheally, using a 1 mL syringe. Allow full lung expansion, and then aspirate the fluid using the same syringe. Repeat this step for a total of 2 mL.</li>
	<li>Transfer the BAL into a 2 mL aliquot. Perform this step twice for a final volume of 2 mL. Do not lavage the lungs with more than 1 mL at a time, due to the high risk of alveolar space disruption.</li>
	<li>Open the thoracic cavity and expose the lung, heart, and trachea using scissors and forceps. Carefully dissect the diaphragm and remove the rib cage. Make sure not to pinch the lung tissue.</li>
	<li>Transect the abdominal aorta to allow exsanguination. Perfuse the lung tissue by making a small incision of approximately 2 mm in the right ventricle using scissors and inject 5 mL of cold PBS using a 20 G catheter. The PBS should perfuse the lung. If an adequate perfusion is performed, the lung tissue will turn white pale and the PBS will leave the intravascular compartment through the abdominal aorta.</li>
	<li>Carefully, extract the lungs and dissect them from the trachea to perform either histology or further processing to single cell suspension.</li>
	<li>If processing for histology, carefully insert a 20 G catheter to inflate the lungs up to 25 cm H2O with formalin solution (neutral buffered 10%). Once the lungs are insufflated, pass a 3.0 suture string about 5 cm long underneath the trachea, and tie it firmly twice to ensure the formalin stays in the lung tissue. Gently, dissect out the lung from the rest of the tissues and place it in a 15 mL conical tube containing 10 mL of formalin solution.</li>
</ol>
6. Bronchoalveolar lavage processing
<ol>
	<li>Centrifuge the BAL at 500 x g at 4 &#176;C for 5 min. Remove the cell-free supernatant in a separate tube and store at -80 &#176;C. 
	NOTE: Further analysis can be performed in the BAL supernatant, including protein quantification (e.g., BCA assay13) or measurement of specific biomarkers or cytokines (ELISA assays and multiple assays with platforms such as MSD and Luminex).</li>
	<li>Lyse the red blood cells by adding 100 &#181;L of lysing buffer for 1 min. Neutralize the lysing reaction by adding 1 mL of PBS. Centrifuge the BAL at 500 x g at 4 &#176;C for 5 min and remove the supernatant.</li>
	<li>Resuspend the cells in PBS (100-300 &#181;L; based on cell pellet size). Perform a cell count with 0.4% trypan blue stain by manual or automated cell counting. Use the remaining pellet for flow cytometry staining and/or cryopreserve (by resuspending in cryopreserving solution) in liquid nitrogen for further testing.</li>
</ol>
7. Lung processing for single cell suspension
<ol>
	<li>Gently dissect the lung from the rest of the tissues and place it in a 15 mL conical tube containing 5 mL of cold PBS. Remove the lung from PBS and dry it using a paper towel.</li>
	<li>Prepare a digestion cocktail by adding 1 mg of DNase and 5 mg of collagenase into 1 mL of low glucose Dulbecco&#39;s modified Eagle&#39;s medium (DMEM). Transfer the lung to a C-tube containing 1 mL of digestion cocktail.</li>
	<li>Transfer the C-tube to the tissue dissociator and follow the standardized protocol for processing lung tissue14.</li>
	<li>Add 10 mL of cold PBS into the C-tube and mix properly. Filter the single cell suspension using a 70 &#181;m cell strainer on top of a 50 mL conical tube. Perform the filtration twice.</li>
	<li>Centrifuge the suspension at 500 x g at 4 &#176;C for 5 min. Carefully decant the supernatant and add 1 mL of lysing buffer for 1 min at room temperature. Add 10 mL of cold PBS to stop the lysing reaction and remove supernatant.</li>
	<li>Centrifuge the suspension at 500 x g at 4 &#176;C for 5 min. Carefully decant the supernatant and add 10 mL of cold PBS.</li>
	<li>Perform a cell count with trypan blue stain by manual or automated cell counting. Use a cell pellet for flow cytometry staining and/or cryopreserve in liquid nitrogen for further testing.</li>
</ol>

<ol>
	<li>Bellani, 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=Epidemiology,+patterns+of+care,+and+mortality+for+patients+with+acute+respiratory+distress+syndrome+in+intensive+care+units+in+50+countries.">Epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in 50 countries.</a> JAMA. 315 (8), 788-800 (2016).</li><li>Hariri, L., Hardin, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covid-19,+angiogenesis,+and+ARDS+endotypes.">Covid-19, angiogenesis, and ARDS endotypes.</a> The New England Journal of Medicine. 383 (2), 182-183 (2020).</li><li>Ferguson, N. D., 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+Berlin+definition+of+ARDS:+an+expanded+rationale,+justification,+and+supplementary+material.">The Berlin definition of ARDS: an expanded rationale, justification, and supplementary material.</a> Intensive Care Medicine. 38 (10), 1573-1582 (2012).</li><li>Tomashefski Jr, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+pathology+of+the+adult+respiratory+distress+syndrome.">Pulmonary pathology of the adult respiratory distress syndrome.</a> Clinics in Chest Medicine. 11 (4), 593-619 (1990).</li><li>Martin, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+cytokines+and+ARDS:+Roger+S.+Mitchell+lecture.">Lung cytokines and ARDS: Roger S. Mitchell lecture.</a> Chest. 116 (1 Suppl), 2S-8S (1999).</li><li>Colletti, L. M., 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=Role+of+tumor+necrosis+factor-alpha+in+the+pathophysiologic+alterations+after+hepatic+ischemia/reperfusion+injury+in+the+rat.">Role of tumor necrosis factor-alpha in the pathophysiologic alterations after hepatic ischemia/reperfusion injury in the rat.</a> The Journal of Clinical Investigation. 85 (6), 1936-1943 (1990).</li><li>Donnelly, S. C., 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+association+between+mortality+rates+and+decreased+concentrations+of+interleukin-10+and+interleukin-1+receptor+antagonist+in+the+lung+fluids+of+patients+with+the+adult+respiratory+distress+syndrome.">The association between mortality rates and decreased concentrations of interleukin-10 and interleukin-1 receptor antagonist in the lung fluids of patients with the adult respiratory distress syndrome.</a> Annals of Internal Medicine. 125 (3), 191-196 (1996).</li><li>Miller, E. J., Cohen, A. B., Matthay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+interleukin-8+concentrations+in+the+pulmonary+edema+fluid+of+patients+with+acute+respiratory+distress+syndrome+from+sepsis.">Increased interleukin-8 concentrations in the pulmonary edema fluid of patients with acute respiratory distress syndrome from sepsis.</a> Critical Care Medicine. 24 (9), 1448-1454 (1996).</li><li>Matute-Bello, 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=An+official+American+Thoracic+Society+workshop+report:+features+and+measurements+of+experimental+acute+lung+injury+in+animals.">An official American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals.</a> American Journal of Respiratory Cell and Molecular Biology. 44 (5), 725-738 (2011).</li><li>Kulkarni, H. S., 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=Update+on+the+features+and+measurements+of+experimental+acute+lung+injury+in+animals:+an+official+American+Thoracic+Society+Workshop+report.">Update on the features and measurements of experimental acute lung injury in animals: an official American Thoracic Society Workshop report.</a> American Journal of Respiratory Cell and Molecular Biology. 66 (2), e1-e14 (2022).</li><li>Borsa, N., Pasquale, M. D., Restrepo, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+pneumococcal+pneumonia.">Animal models of pneumococcal pneumonia.</a> International Journal of Molecular Sciences. 20 (17), 4220 (2019).</li><li>Rubins, J. B., 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=Dual+function+of+pneumolysin+in+the+early+pathogenesis+of+murine+pneumococcal+pneumonia.">Dual function of pneumolysin in the early pathogenesis of murine pneumococcal pneumonia.</a> The Journal of Clinical Investigation. 95 (1), 142-150 (1995).</li><li>Thermo Scientific, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pierce+BCA+Protein+Assay+Kit.">Pierce BCA Protein Assay Kit.</a> , .</li><li>Jungblut, M., Oeltze, K., Zehnter, I., Hasselmann, D., Bosio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+preparation+of+single-cell+suspensions+from+mouse+lung+tissue+using+the+gentleMACS+Dissociator.">Standardized preparation of single-cell suspensions from mouse lung tissue using the gentleMACS Dissociator.</a> Journal of Visualized Experiments. (29), e1266 (2009).</li><li>Tighe, R. M., 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=Improving+the+quality+and+reproducibility+of+flow+cytometry+in+the+lung.+an+official+American+Thoracic+Society+Workshop+report.">Improving the quality and reproducibility of flow cytometry in the lung. an official American Thoracic Society Workshop report.</a> American Journal of Respiratory Cell and Molecular Biology. 61 (2), 150-161 (2019).</li><li>Aeffner, F., Bolon, B., Davis, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+acute+respiratory+distress+syndrome:+a+review+of+analytical+approaches,+pathologic+features,+and+common+measurements.">Mouse models of acute respiratory distress syndrome: a review of analytical approaches, pathologic features, and common measurements.</a> Toxicologic Pathology. 43 (8), 1074-1092 (2015).</li><li>Torres, A., 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=Pneumonia.">Pneumonia.</a> Nature Reviews Disease Primers. 7 (1), 25 (2021).</li><li>Xiong, 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=Estradiol+resolves+pneumonia+via+ER&#946;+in+regulatory+T+cells.">Estradiol resolves pneumonia via ER&#946; in regulatory T cells.</a> JCI Insight. 6 (3), e133251 (2021).</li><li>D'Alessio, F. R., 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=Enhanced+resolution+of+experimental+ARDS+through+IL-4-mediated+lung+macrophage+reprogramming.">Enhanced resolution of experimental ARDS through IL-4-mediated lung macrophage reprogramming.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 310 (8), L733-L746 (2016).</li><li>D'Alessio, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+acute+lung+injury+and+ARDS.">Mouse models of acute lung injury and ARDS.</a> Methods in Molecular Biology. 1809, 341-350 (2018).</li><li>Gotts, J. E., 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=Clinically+relevant+model+of+pneumococcal+pneumonia,+ARDS,+and+nonpulmonary+organ+dysfunction+in+mice.">Clinically relevant model of pneumococcal pneumonia, ARDS, and nonpulmonary organ dysfunction in mice.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 317 (5), L717-L736 (2019).</li></ol>

The authors have nothing to disclose.

Experimental murine models of PNA offer a platform to evaluate the cellular and molecular mechanisms underlying injury and resolution of ARDS. The pathophysiological components that can be evaluated include early inflammatory pathways, bacterial clearance, dynamic immune landscape changes, resolution of inflammation, fibroproliferation, and epithelial and vascular repair16. However, several aspects must be considered when planning to replicate this model of pneumonia-induced lung injury, including age, sex, mouse strain, intrinsic host factors (e.g., immunocompromised state), the specific pathogen and bacterial load used, and the experience of the staff performing the procedure.
PNA is among the leading causes of ARDS. We chose to use live Spn and Kpn,&#160;which are prevalent causes of community and hospital acquired PNA in humans, respectively17. We suggest optimizing bacterial models of PNA by titrating doses of live bacteria to achieve the desired mortality and injury resolution profile that best fits the investigator&#39;s hypothesis. We have optimized an intratracheal bacteria inoculation of 3 x 106 CFU for Spn and 200 CFU for Kpn in mice, which caused alveolar inflammation, disruption of alveolocapillary barrier, and organ dysfunction (Figure 2). However, bacterial batches from different sources or even within duplicate loops can display different degrees of inflammation and injury, even when using the same strain and CFU.
Therefore, to replicate the results presented in this manuscript, researchers should start with the bacteria concentration described here; however, they may need to increase or decrease the dose to obtain a similar model profile. Hence, every new batch of bacteria utilized needs to be optimized for its potential injury resolution effect. We present a robust model of PNA with two different resolution outcomes, one self-resolving (Spn) and another a slowly/non-resolving (Kpn) which can serve as platforms for investigators to evaluate immunological mechanisms and test therapeutic interventions, particularly at or after peak infection (e.g., 2 days after infection).
Age, sex, strain, and genetic factors impact the kinetics of the injury resolution patterns16. For instance, sex renders accelerated resolution in females compared to males18; therefore, an increase in bacterial load leads to increased mortality and delayed resolution in males compared to females. Aging is another factor to consider when titrating the CFU of bacteria utilized. Aging mice displayed 100% mortality when we used the specified Spn dose (not shown here). Young mice are most frequently used in the pneumococcal PNA models (ranging from 6 to 14 weeks), while aged mice (19-26 months old) display an altered immune response and are used to investigate the role of aging in PNA11 . We had to decrease the CFU down to 300% to achieve survival in aging animals (not shown here). Male C57BL/6 mice (8-12 weeks old) were used in this study and were followed during 6 to 10 days post-infection. Significant differences in susceptibility can also be found between strains; inbred strains such BALB/C and C57BL6/J have different responses to infection11,19.
Direct inoculation of bacteria intratracheally allows a more precise delivery of the inoculum (up to 99%) into the lungs12, representing an alternative for less virulent serotypes and decreasing the aerosolization of bacteria11. However, it can be argued that it is an invasive procedure. Intubation can be challenging, requires systemic anesthesia, and can lead to tracheal trauma with subsequent airway edema and stridor. Mice can develop a vasovagal reflex that leads to apnea, for which it is required to have a small mouse ventilator to provide additional ventilator support when needed. The expertise of the surgeon performing the procedure is a critical component to guarantee successful intubation11. In our study, no mice needed to be euthanized due to the appropriate pain management and no greater than 20% loss of body weight. No signs of pain and distress such as lethargy, an inability to reach water or food, labored breathing, or decreased mental alertness were seen. Alternative methods of direct bacterial delivery to the lungs is oropharyngeal aspiration, although the resolution of lung injury appears to occur faster, and some bacteria can end in the stomach and gastrointestinal tract20.
Preclinical models of PNA enable investigators to evaluate the immune landscape. Bronchoalveolar and interstitial compartments can be assessed for dynamic changes in immune cells16. Moreover, cells can be cultured and stimulated ex vivo to determine their specific production of cytokines and chemokines. Here, we focus on exploring the immune cell landscape in the lung and BAL by using multicolor flow cytometry. Single cell RNA sequencing can also be performed to understand the cell specific transcriptomic signatures at different stages of injury resolution.
PNA-ARDS models generate systemic effects that can be detected early by measuring the body weight during the course of the disease10. Although we do not directly measure systemic effects of ARDS, organ dysfunction can also be assessed by blood measurement of chemistry profiles, and by harvesting different tissues such as spleen, kidney, and liver for histology. Systemic effects of pneumococcal PNA-ARDS have been previously described by other groups using the same bacteria strain21.
Here, a model of experimental PNA that resembles some of the key pathophysiological findings underlying human ARDS is described. Although there are no ideal models that completely recapitulate the complexity and heterogeneity of human ARDS9, these models are relevant and reproducible for studying the mechanisms of lung injury and repair, also serving as a platform for the identification of new potential pharmacological targets that focus on accelerating resolution of lung inflammation and promoting lung repair.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/63925">Experimental Model to Evaluate Resolution of Pneumonia</a>. A figure was updated.
Figure 2 was updated from:
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63925/63925fig02.jpg" />
to:
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63925/63925fig02v3.jpg" />

An erratum was issued for:&#160;<a href="https://www.jove.com/t/63925">Experimental Model to Evaluate Resolution of Pneumonia</a>. A figure was updated.

Erratum: Experimental Model to Evaluate Resolution of Pneumonia

Acute respiratory distress syndrome (ARDS) remains a common lethal and disabling syndrome that affects approximately 10% of intensive care unit (ICU) patients and up to 23% of individuals under mechanical ventilation, leading to a hospital mortality rate of 35%-46%1. Moreover, the recent COVID-19 pandemic has re-emphasized the importance of studying ARDS. COVID-19 positive cases have accounted for an increase in ARDS mortality, highlighting the limitation of pharmacological therapies2.
In humans, ARDS is characterized by the rapid onset of hypoxemia (PaO2/FiO2 &#60; 300) and evidence of non-hydrostatic bilateral pulmonary edema due to excessive alveolar-capillary permeability and alveolitis3. Although ARDS has been traditionally described as a pattern of acute lung injury (ALI) secondary to a variety of insults, bacterial and viral pneumonia (PNA) remains among the most common causes. Three main pathophysiological stages, namely the exudative, proliferative, and fibrotic stages, have been described, while the two main cardinal features of ARDS are dysregulated inflammation and alveolocapillary disruption4. During these processes, alveolar injury is produced by the release of inflammatory cytokines (e.g., tumor necrosis factor [TNF-&#945;], interleukin [IL-1&#946;, IL-6, IL-8, etc.]), an influx of neutrophils and inflammatory macrophages, and the flooding of protein-rich fluid. Ultimately, these events lead to patchy and bilateral alveolar damage5,6,7,8.
Although significant progress in understanding early lung injury and inflammation has been made, the mechanisms underlying resolution of PNA are less known and should be the focus of future mechanistic investigations. The main objective of this method paper is to provide investigators with an injury-resolution model of infectious pneumonia that can replicate the main pathophysiological features of ARDS. We propose that this model will help to better understand the biological mechanisms underlying resolution of lung inflammation and repair, thus serving as a platform for rescue therapeutics.
The main physiopathology stages occurring during ARDS can be replicated in preclinical animal models of ALI, which should include a histological evidence of inflammatory response, tissue injury, physiological dysfunction, alveolitis, and alteration of the alveolar-capillary barrier9. A mouse model that induces PNA and ALI is advantageous due to its high reproducibility, fast breeding, and the availability of multiple tools to perform mechanistic and molecular studies. There is no single model that fully recapitulates all the features of human ARDS9.
Models of PNA in mice allow replication of the key pathophysiological mechanisms produced by infectious-ARDS in humans, such as the rapid onset, evidence of tissue injury in histology, alveolo-capillary barrier impairment, evidence of inflammatory response, and physiological dysfunction while producing modest mortality10 . Infectious models can be induced by local or systemic delivery of the pathogen, with intranasal, intratracheal, and intravenous administration being the most frequent routes of administration. The intratracheal route allows direct inoculation of the infectious agent into the lungs, decreasing aerosolization and optimizing the delivery11,12.
The methodology for a preclinical murine model of PNA by intratracheal instillation of either live Streptococcus pneumoniae (Spn) or Klebsiella pneumoniae (Kp) is described here. These models represent a good surrogate of ARDS produced by bacterial PNA, possessing several advantages, including: prevalent causes of human PNA-ARDS (community and hospital-acquired); high reproducibility; mortality and injury can be easily titrated (modeling different degrees of pulmonary inflammation) to exhibit a robust inflammatory response, leading to alveolitis and alveolocapillary dysfunction; the evaluation of early and late phases of lung injury and resolution; and the evaluation of therapeutic strategies at different stages of PNA-ARDS.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-200 &#181;L Round 0.5 mm Thick Gel-Loading Pipet Tips</td>
			<td>Corning</td>
			<td>4853</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Cryogenic vials</td>
			<td>Corning Incorporated</td>
			<td>431420</td>
			<td>2 mL self standing, round bottom, red cap, polypropylene</td>
		</tr>
		<tr>
			<td>2 mL Eppendorf&#160;Snap-Cap Microcentrifuge Biopur Safe-Lock Tubes</td>
			<td>Fisherscientific</td>
			<td>05-402-24C</td>
			<td>Shape:&#160;Round, Length (Metric):&#160;38mm, Diameter (Metric) Outer:&#160;10mm, Capacity (Metric):&#160;2mL</td>
		</tr>
		<tr>
			<td>70 &#181;m Cell Strainer</td>
			<td>Falcon</td>
			<td>352350</td>
			<td>White, Sterile, Individually Packaged</td>
		</tr>
		<tr>
			<td>96-well Clear Round Bottom</td>
			<td>Falcon</td>
			<td>353077</td>
			<td>&#160;TC-treated Cell Culture Microplate, with Lid, Individually Wrapped, Sterile</td>
		</tr>
		<tr>
			<td>Acepromizine Maleate Injection, USP 500 mg/50 mL (10mg/mL)</td>
			<td>Phoenix</td>
			<td>NDC 57319-604-04</td>
			<td>EACH mL CONTAINS: acepromazine maleate 10 mg, sodium citrate 0.36%, Citric acid 0.075%, benzyl alcohol 1% and water for injection.</td>
		</tr>
		<tr>
			<td>Ammonium-Chloride-Potassium (ACK) Lysing Buffer</td>
			<td>Quality Biological</td>
			<td>118-156-721</td>
			<td>4 x 100mL</td>
		</tr>
		<tr>
			<td>Anti-mouse I-A/I-E</td>
			<td>Biolegend</td>
			<td>107628</td>
			<td>APC/Cyanine7 anti-mouse I-A/I-E [M5/114.15.2]; Isotype: Rat IgG2b</td>
		</tr>
		<tr>
			<td>BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Trypticase Soy Agar&#160;</td>
			<td>BD-Biosciences</td>
			<td>90001-276</td>
			<td>5% Sheep Blood Prepared Media Stacker Plates, BD Diagnostics</td>
		</tr>
		<tr>
			<td>Biotix Disposable Reagent Reservoirs</td>
			<td>Biotix</td>
			<td>89511-194</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrih</td>
			<td>A4503</td>
			<td></td>
		</tr>
		<tr>
			<td>CD103</td>
			<td>Invitrogen</td>
			<td>509723</td>
			<td>Integrin alpha E) Armenian Hamster anti-Mouse, FITC, Clone: 2E7</td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>Invitrogen</td>
			<td>RM2817</td>
			<td>PE-Texas Red, Clone: M1/70.15, Invitrogen</td>
		</tr>
		<tr>
			<td>CD11c</td>
			<td>BD Biosciences</td>
			<td>565872</td>
			<td>Hamster anti-Mouse, APC-R700, Clone: N418, BD Horizon</td>
		</tr>
		<tr>
			<td>CD19</td>
			<td>Biolegend</td>
			<td>152410</td>
			<td>APC anti-mouse CD19 [1D3/CD19]; Isotype: Rat IgG2a, &#954;</td>
		</tr>
		<tr>
			<td>CD24</td>
			<td>BD Biosciences</td>
			<td>563450</td>
			<td>Rat anti-Mouse, Brilliant Violet 711, Clone: M1/69</td>
		</tr>
		<tr>
			<td>CD4</td>
			<td>BD Biosciences</td>
			<td>563790</td>
			<td>BUV395; Clone: GK1.5</td>
		</tr>
		<tr>
			<td>CD45</td>
			<td>Biolegend</td>
			<td>103157</td>
			<td>Brilliant Violet 750 anti-mouse CD45 [30-F11]; Isotype: Rat IgG2b, &#954;;</td>
		</tr>
		<tr>
			<td>CD8a</td>
			<td>BD Biosciences</td>
			<td>612759</td>
			<td>Rat anti-Murine, Brilliant Ultraviolet 737, Clone: 53-6.7</td>
		</tr>
		<tr>
			<td>Cell Counting Slides&#160;</td>
			<td>Bio-rad</td>
			<td>1450017</td>
			<td>For TC20 Cell Counter</td>
		</tr>
		<tr>
			<td>Cell strainer 70 &#181;L Nylon</td>
			<td>Falcon</td>
			<td>198718</td>
			<td>REF 352350</td>
		</tr>
		<tr>
			<td>Collagenase Type1&#160;</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LS004197</td>
			<td></td>
		</tr>
		<tr>
			<td>Culti-Loop Streptococcus pneumoniae&#160;</td>
			<td>Thermo Scientific</td>
			<td>R4609015</td>
			<td>ATCC 4961</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas</td>
			<td>Sigma-Aldrih</td>
			<td>DN25</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable inoculation loops/needles</td>
			<td>Fisherbrand</td>
			<td>22-363-603</td>
			<td>Color blue; Volume 1 &#181;L</td>
		</tr>
		<tr>
			<td>DMEM (Dulbecco&#8217;s Modified Eagle&#8217;s Medium)</td>
			<td>Corning</td>
			<td>10-014-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc Block</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td>CD16/CD32 Rat anti-Mouse, Unlabeled, Clone: 2.4G2</td>
		</tr>
		<tr>
			<td>Formalin solution</td>
			<td>Sigma-Aldrih</td>
			<td>HT501640</td>
			<td>Formalin solution, neutral buffered, 10%</td>
		</tr>
		<tr>
			<td>Gauze Sponges, Covidien</td>
			<td>Curity</td>
			<td>2146-</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS C Tubes</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS Dissociator</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td>SN: 4715</td>
		</tr>
		<tr>
			<td>Hema 3 Manual Staining System and Stat Pack</td>
			<td>Thermo Scientific&#160;</td>
			<td>23123869</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane Liquid Inhalation</td>
			<td>Henry Schein</td>
			<td>1182097</td>
			<td></td>
		</tr>
		<tr>
			<td>IV CATHETER JELCO 20GX1.25&#34;</td>
			<td>Hanna Pharmaceutical Supply Co., Inc</td>
			<td>405611</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine HCl Injection</td>
			<td>Henry Schein</td>
			<td>1049007</td>
			<td>Ketamine HCl Injection MDV 100mg/mL 10mL 10/Box</td>
		</tr>
		<tr>
			<td>Klebsiella pneumoniae&#160;</td>
			<td>ATCC</td>
			<td>43816</td>
			<td>subsp. pneumoniae (Schroeter) Trevisan</td>
		</tr>
		<tr>
			<td>Loctite 409</td>
			<td>Electron Microscopy Sciences</td>
			<td>7257009</td>
			<td></td>
		</tr>
		<tr>
			<td>Ly-6C</td>
			<td>Biolegend</td>
			<td>128036</td>
			<td>Brilliant Violet 605 anti-mouse Ly-6C [HK1.4]; Isotype: Rat IgG2c</td>
		</tr>
		<tr>
			<td>Ly-6G</td>
			<td>BD Biosciences</td>
			<td>740157</td>
			<td>Rat anti-Mouse, Brilliant Violet 510, Clone: 1A8, BD Optibuild</td>
		</tr>
		<tr>
			<td>MiniVent Type 845</td>
			<td>Hugo Sachs Elektronik- Harvard Apparatus</td>
			<td>4694</td>
			<td>D-79232 March (Germany)</td>
		</tr>
		<tr>
			<td>NK-1.1</td>
			<td>BD Biosciences</td>
			<td>553165</td>
			<td>Mouse anti-Mouse, PE, Clone: PK136, BD</td>
		</tr>
		<tr>
			<td>Phase Hemacytometer</td>
			<td>Hausser Scientific</td>
			<td>1475</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td>1X without calcium and magnesium,</td>
		</tr>
		<tr>
			<td>Round Bottom</td>
			<td>Sarstedt</td>
			<td>55.476.305</td>
			<td></td>
		</tr>
		<tr>
			<td>Round-Bottom Polystyrene Test Tubes&#160;</td>
			<td>Falcon</td>
			<td>352235</td>
			<td>With Cell Strainer Snap Cap, 5mL</td>
		</tr>
		<tr>
			<td>SealRite 1.5 mL Natural Microcentrifuge Tube</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td>Free of detectable Rnase, DNase, DNA and pyrogens.</td>
		</tr>
		<tr>
			<td>Shandon EZ Single Cytofunnel</td>
			<td>Epredia</td>
			<td>A78710003</td>
			<td></td>
		</tr>
		<tr>
			<td>Siglec-F</td>
			<td>BD Biosciences</td>
			<td>562681</td>
			<td>Anti-Mouse, Brilliant Violet 421, Clone: E50-2440</td>
		</tr>
		<tr>
			<td>Silk Black Braided 30&#34;(75 cm) Sterile, nonabsorbable surgical suture U.S.P.</td>
			<td>Ethicon</td>
			<td>K-834</td>
			<td>0 (3.5 metric)</td>
		</tr>
		<tr>
			<td>Stainless-Steel Slide Clip</td>
			<td>Epredia</td>
			<td>59910052</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Single Use Vacuum Filter Units</td>
			<td>Thermo Scientific</td>
			<td>1660045</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe sterile, single use, 1 mL</td>
			<td>BD-Biosciences</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>TC20 Automatic Cell Counter</td>
			<td>Bio-Rad</td>
			<td>508BR05740</td>
			<td></td>
		</tr>
		<tr>
			<td>TipOne 200 ul yellow pipet tip refill</td>
			<td>USA Scientific</td>
			<td>1111-0706</td>
			<td></td>
		</tr>
		<tr>
			<td>TODD HEWITT BROTH</td>
			<td>RPI</td>
			<td>T47500&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>TPX Sample Chamber</td>
			<td>Epredia</td>
			<td>A78710018</td>
			<td></td>
		</tr>
		<tr>
			<td>TPX Single Sample Chamber, Caps and Filter Cards</td>
			<td>Epredia</td>
			<td>5991022</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Bio-rad</td>
			<td>1450022</td>
			<td></td>
		</tr>
		<tr>
			<td>U-100 Insulin Syringes</td>
			<td>BD-Biosciences</td>
			<td>329461</td>
			<td></td>
		</tr>
		<tr>
			<td>Wet-Proof Multi-Heat Electric Heat Pad</td>
			<td>Cullus</td>
			<td>Model PR7791AB</td>
			<td>120 volst AC; 45 watts; Listed 562B/E26869</td>
		</tr>
	</tbody>
</table>

experimental model to evaluate resolution of pneumonia

Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to high morbidity and mortality. Currently, there are no pre-clinical models that recapitulate the complexity of human ARDS. However, infectious models of pneumonia (PNA) can replicate the main pathophysiological features of ARDS. Here, we describe a model of PNA induced by the intratracheal instillation of live Streptococcus pneumoniae and Klebsiella pneumoniae in C57BL6 mice. In order to evaluate and characterize the model, after inducing injury, we carried out serial measurements of body weight and bronchoalveolar lavage (BAL) for measuring markers of lung injury. Additionally, we harvested lungs for cell count and differentials, BAL protein quantification, cytospin, bacterial colony-forming unit counts, and histology. Lastly, high dimensional flow cytometry was performed. We propose this model as a tool to understand the immune landscape during the early and late resolution phases of lung injury.

The procedures described above allowed modeling the pathophysiological mechanisms underlying bacterial pneumonia-induced lung injury in mice. To begin to model, C57BL/6 wild-type (WT) mice were obtained from the Jackson Laboratory and bred at the institute&#39;s animal facility. Male WT C57BL/6 mice, 8 weeks old, underwent intratracheal inoculation of either TH broth (control), 3 x 106 CFU&#160;of live Spn, or 200 CFU of live Kpn. After the infection, the mice were monitored for 6 and 10 days for Spn and Kpn, respectively. Although the infected groups displayed lower body weight compared to the uninfected control, the Spn group recovered their body weight toward baseline, while Kpn-infected mice displayed slow recovery after 6 days of infection (Figure 2A). During the duration of the study, no mice required undergoing euthanasia due to body weight over 20%, and there was no evidence of pain and distress.
We measured lung injury over different intervals. BAL protein concentration and the total cell count for both the BAL and lungs were remarkedly higher in the infected groups (Figure 2). Representative histological sections displaying the inflammatory process in both models were obtained at day 2, 4, and 6 post-inoculation (Figure 3), showing evidence of persistent alveolar inflammation in the Kpn-infected mice (Figure 4), even at day 10. Kpn-infected mice continued the injury by day 10 (Figure 2 and Figure 4), while Spn-infected mice resolved the lung inflammation by day 6 (Figure 2 and Figure 3).
Lung single cell suspensions were used to discriminate the immune landscape by high-dimensional flow cytometry at day 6 post-infection in the Spn model, using an 18-color panel in fluorescence activated cell sorting (FACS). Using t-distributed stochastic neighbor embedding (t-SNE), overall differences in the immune cell composition can be visualized, being remarkable for an increased number of granulocytes (CD45+, CD11b+, CD24+, MHC-II-), interstitial macrophages (CD45+, CD11b+, MHC-II-, CD24-), monocytes (CD45+, CD11b+, MHC-II-, CD24-, CD64-), B cells (CD45+, CD19+), and T cells (CD45+, CD3+), including natural killer cells (CD45+, CD3+, NK1.1+), as displayed in Figure 5. Gating strategies are shown in Figure 6.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63925/63925fig01.jpg" /> 
Figure 1: Surgical procedure for intratracheal instillation of live bacteria. (A)&#160;Position of the mouse in the sterile surgical area, hanging by the incisors. (B) Incision area and trachea exposure. (C) Intubation process inserting the 20 G catheter. Figure created with Biorender.com. <a href="https://www.jove.com/files/ftp_upload/63925/63925fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63925/63925fig02v3.jpg" /> 
Figure 2: Acute lung injury (ALI) profiles after pneumonia models. (A) Body weight over time relative to baseline, control versus Kpn and Spn (n = 4, per group). (B) BAL total protein quantification by BCA assay (n = 4, per group). (C)&#160;BAL total cell counts in control (n = 3), Kpn (n = 4), and Spn (n = 3). (D) Lung total cell counts in control (n = 3), Kpn (n = 4), and Spn (n = 3). (E-G) BAL cell differential by manual histology count of the BAL cytospin in control (n = 3), Kpn (n = 4), and Spn (n = 3). Statistical tests were done by individual t-test, comparing the uninfected control versus the infected groups. *p &#60; 0.05. Data is displayed using standard error (SE) for each plot. The y axis is days post-infection for all panels. Abbreviations: BAL = bronchoalveolar lavage; Spn = Streptococcus pneumoniae; Kpn = Klebsiella pneumoniae. <a href="https://www.jove.com/files/ftp_upload/63925/63925fig02v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63925/63925fig03.jpg" /> 
Figure 3:&#160;Lung&#160;histopathologic findings during Spn infection. Hematoxylin and eosin (H&#38;E) staining of histological sections of a representative BAL and lung section after the intratracheal infection of Spn and on days 2, 4, and 6. BAL magnification = 100x; lung magnification = 10x. Abbreviations: BAL = bronchoalveolar lavage; Spn = Streptococcus pneumoniae. <a href="https://www.jove.com/files/ftp_upload/63925/63925fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63925/63925fig04.jpg" /> 
Figure 4: Lung histopathologic findings during Kpn infection. Hematoxylin and eosin (H&#38;E) staining of histological sections of a representative BAL and lung section after the intratracheal infection of Kpn on days 2, 4, 6, and 10. Images show a high power magnification (scale bar = 50 &#181;m). Abbreviations: BAL = bronchoalveolar lavage; Kpn = Klebsiella pneumoniae. <a href="https://www.jove.com/files/ftp_upload/63925/63925fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63925/63925fig05.jpg" /> 
Figure 5:&#160;Immune cell landscape by multicolor flow cytometry after 6 days of Spn infection. (A) T-distributed stochastic neighbor embedding (t-SNE) was used to visualize the immune cell populations (CD45+) of the lung comparing the uninfected control versus infected group. (B) Summary of the immune cell frequencies out of the total CD45+ cells in the lung. (C) Total cell count of each individual population. Statistical comparisons were done by t-tests between the control and infected. *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0,001. Data is displayed using standard error (SE) for each plot. Abbreviations: BAL = bronchoalveolar lavage; Spn = Streptococcus pneumoniae. <a href="https://www.jove.com/files/ftp_upload/63925/63925fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63925/63925fig06.jpg" /> 
Figure 6:&#160;Gating strategies for identifying the immune cell subpopulation in the lungs at baseline and after pneumonia. BAL and Lung cell suspension underwent staining for multicolor flow cytometry. Debris was gated out first using SSC-A and FSC-A, and the single cells were gated by two strategies (SSC-W vs. SSC-H and FSC-W vs. FSC-H). Live cells were identified by using a live/dead discriminator versus SSC-A. Subsequent cell populations were identified by previously identified markers 15. Abbreviation: BAL = bronchoalveolar lavage. <a href="https://www.jove.com/files/ftp_upload/63925/63925fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Experimental Model to Evaluate Resolution of Pneumonia at JoVE.com

Experimental Model to Evaluate Resolution of Pneumonia

This manuscript describes the establishment of an infectious model of pneumonia in mice and the respective characterization of injury resolution along with methods for growing bacteria and intratracheal instillation. A novel approach using high-dimensional flow cytometry to evaluate the immune landscape is also described.

Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to ...

experimental-model-to-evaluate-resolution-of-pneumonia

Research

Education

JoVE Core

JoVE Journal

Medical-Surgical Nursing

Immunology and Infection

Unit 1

Lower Respiratory Tract Infections

SCIENTISTS IN ACTION

1.1K Görüntüleme.  Johns Hopkins University School of Medicine. Bu protokol, sağlam, akut akciğer hasarı oluşturan tekrarlanabilir bir pnömoni modeli sağlar. Bu modelin diğer avantajları, farelerde enfeksiyöz ARDS veya akut solunum sıkıntısı sendromunu çoğaltmak için kullanılabilir. Daha önce de belirttiğim gibi, bu model, akut akciğer hasarının erken ve geç aşamalarını ve çözünürlüğünü değerlendirmek için mortalite ve yaralanma seviyesinin kolayca titre edilebildiği ve terapötik stratejileri değerlendirmek için bir...

Video: Experimental Model to Evaluate Resolution of Pneumonia

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments4-6. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs7, 8. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Experimental rat endocarditis model due to methicillin-resistant S. aureus.

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause ...

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to increased susceptibility of smokers to microbial infections and tumor incidence. Ex vivo suppression of receptor-mediated immune responses in human peripheral blood mononuclear cells (PBMCs) treated with smoke constituents is an attractive approach to study mechanisms and evaluate the likely long-term effects of exposure to tobacco products. Here, we optimized methods to perform ex vivo assays using PBMCs stimulated by bacterial lipopolysaccharide, a Toll-like receptor-4 ligand. The effects of whole smoke-conditioned medium (WS-CM), a combustible tobacco product preparation (TPP), and nicotine were investigated on cytokine secretion and target cell killing by PBMCs in the ex vivo assays. We show that secreted cytokines IFN-&#947;, TNF, IL-10, IL-6, and IL-8 and intracellular cytokines IFN-&#947;, TNF-&#945;, and MIP-1&#945; were suppressed in WS-CM-exposed PBMCs. The cytolytic function of effector PBMCs, as determined by a K562 target cell killing assay was also reduced by exposure to WS-CM; nicotine was minimally effective in these assays. In summary, we present a set of improved assays to evaluate the effects of TPPs in ex vivo assays, and these methods could be readily adapted for testing other products of interest.

Using optimized human peripheral blood mononuclear cell (PBMC) ex vivo assays, we showed that a combustible tobacco product preparation markedly suppresses receptor-mediated intracellularly secreted cytokines and cytolytic ability of effector PBMCs. These rapid assays may be useful in product evaluation and understanding the potential long-term effects of tobacco exposure.

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...