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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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><tbody><tr><td>1-200 &amp;micro;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&amp;nbsp;Snap-Cap Microcentrifuge Biopur Safe-Lock Tubes</td><td>Fisherscientific</td><td>05-402-24C</td><td>Shape:&amp;nbsp;Round, Length (Metric):&amp;nbsp;38mm, Diameter (Metric) Outer:&amp;nbsp;10mm, Capacity (Metric):&amp;nbsp;2mL</td></tr><tr><td>70 &amp;micro;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>&amp;nbsp;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&amp;nbsp;</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, &amp;kappa;</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, &amp;kappa;;</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&amp;nbsp;</td><td>Bio-rad</td><td>1450017</td><td>For TC20 Cell Counter</td></tr><tr><td>Cell strainer 70 &amp;micro;L Nylon</td><td>Falcon</td><td>198718</td><td>REF 352350</td></tr><tr><td>Collagenase Type1&amp;nbsp;</td><td>Worthington Biochemical Corporation</td><td>LS004197</td><td></td></tr><tr><td>Culti-Loop Streptococcus pneumoniae&amp;nbsp;</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 &amp;micro;L</td></tr><tr><td>DMEM (Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;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&amp;nbsp;</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&quot;</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&amp;nbsp;</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&amp;nbsp;</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&quot;(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&amp;nbsp;</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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/files/ftp_upload/63925/63925fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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 ...

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1.1K Views.  Johns Hopkins University School of Medicine. 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.

Video: Experimental Model to Evaluate Resolution of Pneumonia

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. ...

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. ...

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 ...

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 Precise Pathogen Delivery and Recovery System for Murine Models of Secondary Bacterial Pneumonia

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, murine models of co-infection have been the primary tool developed to explore the pathologies of both the primary and secondary infections. Despite the prevalence of this model, considerable discrepancies regarding instillation procedures, dose volumes, and efficacies are prevalent among studies. Furthermore, these efforts have been largely incomplete in addressing how the pathogen may be directly influencing disease progression post-infection. Herein we provide a precise method of pathogen delivery, recovery, and analysis to be used in murine models of secondary bacterial pneumonia. We demonstrate that intratracheal instillation enables an efficient and accurate delivery of controlled volumes directly and evenly into the lower respiratory tract. Lungs can be excised to recover and quantify the pathogen burden. Following excision of the infected lungs, we describe a method to extract high quality pathogen RNA for subsequent transcriptional analysis. This procedure benefits from being a non-surgical method of delivery without the use of specialized laboratory equipment and provides a reproducible strategy to investigate pathogen contributions to secondary bacterial pneumonia.

Here, we present methods to improve secondary bacterial pneumonia studies by providing a non-invasive route of instillation into the lower respiratory tract followed by pathogen recovery and transcript analysis. These procedures are reproducible and can be performed without specialized equipment such as cannulas, guide wires, or fiber optic cables.

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, ...

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 ...

Murine Oropharyngeal Aspiration Model of Ventilator-associated and Hospital-acquired Bacterial Pneumonia

Murine infection models are critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics designed to combat causative pathogens. Infectious pneumonia is among the most common infections presented by patients in the clinic and thus warrants an appropriate in vivo model. Typical pneumonia models use intranasal inoculation, which deposits excessive organisms outside the lung, causing off-target complications and symptoms, such as sinusitis, gastritis, enteritis, physical trauma, or microparticle misting to mimic aerosol spread more typical of viral, tuberculous, or fungal pneumonia. These models do not accurately reflect the pathogenesis of typical community- or healthcare-acquired bacterial pneumonia. In contrast, this murine model of oropharyngeal aspiration pneumonia mimics the droplet route in healthcare-acquired pneumonia. Inoculating 50 &#181;L of the bacteria suspension into the oropharynx of anesthetized mice causes reflexive aspiration, which results in pneumonia. With this model, one can examine the pathogenesis of pneumonia-causing pathogens and new treatments to combat these diseases.

Infectious pneumonia is among the most common infections in human. An appropriate in vivo model is critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics. With this murine oropharyngeal aspiration pneumonia model, one can examine the pathogenesis and new treatments against these deadly infections.

Murine infection models are critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics designed to combat causative ...

Medicine

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic pathogen upon influenza A virus (IAV) infection. Advanced age enhances host susceptibility to secondary pneumococcal pneumonia and is associated with worsened disease outcomes. The host factors driving those processes are not well defined, in part due to a lack of animal models that reproduce the transition from asymptomatic colonization to severe clinical disease. 
This paper describes a novel mouse model that recreates the transition of pneumococci from asymptomatic carriage to disease upon viral infection. In this model, mice are first intranasally inoculated with biofilm-grown pneumococci to establish asymptomatic carriage, followed by IAV infection of both the nasopharynx and lungs. This results in bacterial dissemination to the lungs, pulmonary inflammation, and obvious signs of illness that can progress to lethality. The degree of disease is dependent on the bacterial strain and host factors. 
Importantly, this model reproduces the susceptibility of aging, because compared to young mice, old mice display more severe clinical illness and succumb to disease more frequently. By separating carriage and disease into distinct steps and providing the opportunity to analyze the genetic variants of both the pathogen and the host, this S. pneumoniae/IAV co-infection model permits the detailed examination of the interactions of an important pathobiont with the host at different phases of disease progression. This model can also serve as an important tool for identifying potential therapeutic targets against secondary pneumococcal pneumonia in susceptible hosts.

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

This paper describes a novel mouse model for the transition of pneumococcus from an asymptomatic colonizer to a disease-causing pathogen during viral infection. This model can be readily adapted to study polymicrobial and host-pathogen interactions during the different phases of disease progression and across various hosts.

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic ...

Establishing a Lung-on-Chip Model for Respiratory Infection Studies

Immunocompetent Alveolus-on-Chip Model for Studying Alveolar Mucosal Immune Responses

We introduce an advanced immunocompetent lung-on-chip model designed to replicate the human alveolar structure and function. This innovative model employs a microfluidic-perfused biochip that supports an air-liquid interface mimicking the environment in the human alveoli. Tissue engineering is used to integrate key cellular components, including endothelial cells, macrophages, and epithelial cells, to create a representative tissue model of the alveolus. The model facilitates in-depth examinations of the mucosal immune responses to various pathogens, including viruses, bacteria, and fungi, thereby advancing our understanding of lung immunity. The primary goal of this protocol is to provide details for establishing this alveolus-on-chip model as a robust in vitro platform for infection studies, enabling researchers to closely observe and analyze the complex interactions between pathogens and the host's immune system within the pulmonary environment. This is achieved through the application of microfluidic-based techniques to simulate key physiological conditions of the human alveoli, including blood flow and biomechanical stimulation of endothelial cells, alongside maintaining an air-liquid interface crucial for the realistic exposure of epithelial cells to air. The model system is compatible with a range of standardized assays, such as immunofluorescence staining, cytokine profiling, and colony-forming unit (CFU)/plaque analysis, allowing for comprehensive insights into immune dynamics during infection. The Alveolus-on-chip is composed of essential cell types, including human distal lung epithelial cells (H441) and human umbilical vein endothelial cells (HUVECs) separated by porous polyethylene terephthalate (PET) membranes, with primary monocyte-derived macrophages strategically positioned between the epithelial and endothelial layers. The tissue model enhances the ability to dissect and analyze the nuanced factors involved in pulmonary immune responses in vitro. As a valuable tool, it should contribute to the advancement of lung research, providing a more accurate and dynamic in vitro model for studying the pathogenesis of respiratory infections and testing potential therapeutic interventions.

Author Spotlight: Developing a Microfluidic Lung-on-Chip Model for In-Depth Study of Human Immune Response and Infection Mechanisms

Lung-on-chip models surpass traditional 2D cultures by mimicking the air-liquid interface and endothelial cell perfusion, simulating blood flow and nutrient exchange crucial for lung physiology studies. This enhances lung research relevance, offering a dynamic, physiologically accurate environment to advance the understanding and treatment of respiratory infections.

We introduce an advanced immunocompetent lung-on-chip model designed to replicate the human alveolar structure and function. This innovative model employs ...

Ex Vivo Porcine Experimental Model for Studying and Teaching Lung Mechanics

Mechanical ventilation is widely used and requires specific knowledge for understanding and management. Health professionals in this field may feel insecure and lack knowledge because of inadequate training and teaching methods. Therefore, the objective of this article is to outline the steps involved in generating an ex vivo porcine lung model to be used in the future, to study and teach lung mechanics. To generate the model, five porcine lungs were carefully removed from the thorax following the guidelines of the Animal Research Ethics Committee with adequate care and were connected to the mechanical ventilator through a tracheal cannula. These lungs were then subjected to the alveolar recruitment maneuver. Respiratory mechanics parameters were recorded, and video cameras were used to obtain videos of the lungs during this process. This process was repeated for five consecutive days. When not used, the lungs were kept refrigerated. The model showed different lung mechanics after the alveolar recruitment maneuver every day; not being influenced by the days, only by the maneuver. Therefore, we conclude that the ex vivo lung model can provide a better understanding of lung mechanics and its effects, and even of the alveolar recruitment maneuver through visual feedback during all stages of the process.

Ex Vivo Porcine Experimental Model for Studying and Teaching Lung Mechanics

We present an ex vivo pig lung model for the demonstration of pulmonary mechanics and alveolar recruitment maneuvers for teaching purposes. The lungs can be used for more than one day (up to five days) with minimal changes in pulmonary mechanics variables.

Mechanical ventilation is widely used and requires specific knowledge for understanding and management. Health professionals in this field may feel ...

Experimental Model to Evaluate Resolution of Pneumonia

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A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

Immunocompetent Alveolus-on-Chip Model for Studying Alveolar Mucosal Immune Responses

Ex Vivo Porcine Experimental Model for Studying and Teaching Lung Mechanics