This work was supported by National Institutes of Health Grants R01 AI138203-01, AI109317-04, AI101973-01, and AI097532-01A1 to M. W. The University of North Dakota Core Facilities were supported by NIH grants (INBRE P20GM103442 and COBRE P20GM113123). This work was also supported by the Key Program of National Nature Science Foundation of China (81530063) to Jianxin Jiang. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Marvin Leier (Center for Rural Health, University of North Dakota) for making the video.

All methods described here were performed in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals and approved by the University of North Dakota Institutional Animal Care and Use Committee (IACUC).
1. Cecal ligation and puncture
NOTE: Female C57BL/6 mice (weight, 18-22 g; age, 6-8 weeks) are randomly divided into six groups: control group (Ctrl), infection group (SA for S. aureus), two sham groups, and two CLP groups. Ctrl animals are left without surgery and secondary infection injuries. SA animals are subjected to S. aureus lung infection without the operation. Sham-operated animals undergo the same laparotomy with an exposition of the cecum (except the cecum is neither ligated nor punctured). Eight mice per group are used for survival analysis, and three to five mice are used for assessment of inflammation at various timepoints.
<ol>
	<li>Prior to surgery, sterilize all surgical instruments and materials by autoclaving for 20 min. Clean and disinfect the operating table with disinfectant wipes. To establish sterile conditions during the surgery, cover the entire operative fields with proper sterile surgical drapes, and wear a hair cover, surgical mask, protective eyewear, surgical gown, and sterile gloves.</li>
	<li>Weigh and anesthetize the mouse using 0.1 mL/mouse of ketamine (80 mg/kg), xylazine (10 mg/kg) mixture intraperitoneally administered. With the thumb and index finger of the left hand forming a U-shaped position, approach the neck of the mouse from behind smoothly and gently, then hold the neck immediately behind the ears so that the mouse head is highly restrained. Use a pinky to hold the mouse&#39;s right back leg and use the middle and ring fingers (all fingers from left hand) to hold the body.</li>
	<li>Check the intensity of anesthesia by toe pinch until no flexion of the extremity.</li>
	<li>Shave the lower quadrants of the abdomen using an electric razor and disinfect the area with iodine.</li>
	<li>Place the mouse onto the sterile surface in a supine position, with the head oriented away from the operator. Drape the mouse with sterile towel with hole over the planned surgical incision to maintain sterile fields.</li>
	<li>Make a longitudinal skin incision (about 0.5 cm long) with a scalpel in the left lower abdomen. Use small scissors to extend the incision (1-2 cm). 
	&#8203;CAUTION: Be careful not to penetrate the peritoneal cavity.</li>
	<li>Make the intramuscular incision to gain access into the peritoneal cavity and allow exposure of the cecum with the adjoining intestine.</li>
	<li>Isolate the cecum on the left side of the abdomen (in most cases, this is done by using blunt anatomical forceps) and remove it on a sterile drape, leaving the small and large intestines in the peritoneal cavity. 
	CAUTION: Avoid damaging the mesenteric blood vessels.</li>
	<li>Ligate the cecum at the distal 25% position to create a prolonged infection with relatively low mortality. 
	CAUTION: Make sure not to ligate the ileocecal valve.</li>
	<li>Perforate the cecum with a 21 G 1 &#189; needle by single through-and-through puncture (two holes) midway between the ligation and tip of the cecum at the least-vascularized area. 
	CAUTION: Make sure not to puncture blood vessels.</li>
	<li>Remove the needle. Gently extrude a small amount of feces from the penetration holes to ensure full thickness perforation. 
	CAUTION: Take care not to obstruct the bowel continuity during the procedure.</li>
	<li>Replace the cecum into the abdominal cavity.</li>
	<li>Close the abdomen in two layers with 4-0 nylon surgical sutures. Close the abdominal musculature by applying simple running sutures and close the skin by applying simple interrupted sutures. Clean the skin with Iodine.</li>
	<li>Inject 1 mL of prewarmed (37 &#176;C) 0.9% sterile normal saline (NS) solution on the back subcutaneously to replace third space loss and place it on a warm pad to recover.</li>
	<li>Return the mice in cage in a temperature-controlled room (22 &#176;C) with 12 h light/dark cycles and free access to food and water. Monitor the mice every 0.5 hours for at least 2 h, then every 6 h for at least 2 d, then every 12 h for 3 days, or euthanize them when moribund for survival analysis. 
	NOTE: Pre- and post-operative analgesia (buprenorphine, 50 &#181;g/kg subcutaneously every 12 h) are recommended for 24 h before surgery until 48 h after surgery9,10.</li>
</ol>
2. Secondary lung infection with S. aureus
NOTE: Except for the Ctrl and SA groups, surviving mice at 3 days post-CLP in the sham and CLP groups should be administered intranasally with 30 &#181;L of a bacterial suspension or NS, respectively. The surviving mice in the SA group should be instilled intranasally with the bacterial suspension.
<ol>
	<li>Anesthetize the mouse by intraperitoneally injecting 100 &#181;L solution of ketamine (45mg/kg) and xylazine (10 mg/kg) and wait until the mouse breaths slowly. The puncturing angle between the needle and abdominal wall is less than 30&#176; for slow injection.</li>
	<li>With the help of a micropipette, slowly and intranasally instill with 30 &#181;L of 1 x 107 colony forming unit (CFU) of S. aureus to be aspirated on inhalation. Holding the mouse by its ears in an upright position, slowly drop 2-3 &#181;L of bacterial suspension into both nostrils each time. Adjust the rate of release to allow the mouse to inhale without forming bubbles and dropping off.</li>
	<li>Raise the mouse up and down, with its head up and tail down, to help the bacteria enter the trachea. Move the mouse up quickly and strongly, while moving it down gently and slowly to increase the gravity. Repeat instillation about 15x until all bacteria are inhaled, which should take 10-15 min for each mouse.</li>
	<li>Lay the mouse on its back and head up on the angled bedding (about 35&#176;) and watch the mouse until its breathing returns to normal and it is completely recovered from the anesthesia and procedures.</li>
	<li>Place the mouse back in the cage in a temperature-controlled room (22 &#176;C) with 12 h light/dark cycles and unlimited access to food and water.</li>
	<li>Check mouse conditions every 2 h for the first day and every 12 h afterwards for 4 days after the surgery. Euthanize them when moribund for survival analysis or 24 h after infection for assessment of inflammation.</li>
</ol>
3. Analyzed parameters
<ol>
	<li>Mice survival
	<ol>
		<li>Monitor the mice for 7 days. Euthanize when moribund and generate survival curves using Kaplan-Meier methods11.</li>
	</ol>
	</li>
	<li>Bacterial counts
	<ol>
		<li>24 h after SA injection, harvest the blood, bronchoalveolar lavage fluid (BALF), and peritoneal lavage fluid (PLF) from the mice. Subsequently, add 100 &#181;L of the diluents to nutrient agar plates and culture them at 37 &#176;C for 24 h to determine the bacterial colony counts11,12,13.</li>
	</ol>
	</li>
	<li>Cytokines 
	&#8203;NOTE: Pro-inflammatory cytokine concentrations of IL-1&#946;, IL-6, and TNF&#945; in serum and BALF from mice should be assessed using ELISA kits according to the manufacturer&#39;s instructions.
	<ol>
		<li>Coat the 96 well ELISA plate with 100 &#181;L/well of different capture antibody working solution to run the standards in duplicate and samples in triplicate. Seal the plate and leave it overnight at 4 &#176;C.</li>
		<li>Wash the plate three times with 255 &#181;L/well wash buffer (1x PBS, 0.05% Tween-20).</li>
		<li>To block nonspecific binding, add 200 &#181;L/well diluent. Seal the plate and incubate at room temperature (RT) for 1 h.</li>
		<li>Prepare standard solution: Eight two-fold serial dilutions from 1000 pg/mL.</li>
		<li>Add 100 &#181;L/well of standards or samples to the appropriate wells and 100 &#181;L/well of diluent to the blank wells. Seal the plate and incubate at RT for 2 h or overnight at 4 &#176;C.</li>
		<li>Wash the plate 5x with 255 &#181;L/well wash buffer.</li>
		<li>Add 100 &#181;L/well of different detection antibody working solution to the appropriate wells. Seal the plate and incubate at RT for 1 h.</li>
		<li>Wash the plate 5x with 255 &#181;L/well wash buffer.</li>
		<li>Add 100 &#181;L/well of diluted HRP solution to all wells. Seal the plate and incubate at RT for 30 min.</li>
		<li>Wash the plate 5x with 255 &#181;L/well wash buffer.</li>
		<li>Add 100 &#181;L/well diluted TMB solution. Incubate the plate away from light at RT for 15 min.</li>
		<li>Add 50 &#181;L/well stop solution (1 M H3PO4). Read absorbance at 450 nm with a plate reader.</li>
	</ol>
	</li>
	<li>Flow cytometry
	<ol>
		<li>Obtain the total number of cells from BALF. Lyse the erythrocytes using ACK lysing buffer and wash 2x using PBS.</li>
		<li>Analyze then the single-cell suspensions by flow cytometry as previously described11. For surface staining, stain neutrophils (CD11b+GR-1+) with APC anti-mouse CD11b and anti-mouse Gr-1 (Ly-6G/Ly-6C) antibodies. Collect a minimum of 3 x 104 live, non-debris cells for analysis.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Singer, 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=The+Third+International+Consensus+Definitions+for+Sepsis+and+Septic+Shock+(Sepsis-3).">The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3).</a> JAMA. 315 (8), 801-810 (2016).</li><li>Delano, M. J., Ward, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+system's+role+in+sepsis+progression,+resolution,+and+long-term+outcome.">The immune system's role in sepsis progression, resolution, and long-term outcome.</a> Immunological Reviews. 274 (1), 330-353 (2016).</li><li>Mayr, F. B., Yende, S., Angus, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+severe+sepsis.">Epidemiology of severe sepsis.</a> Virulence. 5 (1), 4-11 (2014).</li><li>Fleischmann, 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=Assessment+of+Global+Incidence+and+Mortality+of+Hospital-treated+Sepsis.+Current+Estimates+and+Limitations.">Assessment of Global Incidence and Mortality of Hospital-treated Sepsis. Current Estimates and Limitations.</a> American Journal of Respiratory and Critical Care Medicine. 193 (3), 259-272 (2016).</li><li>Reinhart, K., 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=Recognizing+Sepsis+as+a+Global+Health+Priority+-+A+WHO+Resolution.">Recognizing Sepsis as a Global Health Priority - A WHO Resolution.</a> The New England Journal of Medicine. 377 (5), 414-417 (2017).</li><li>Boomer, J. 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=Immunosuppression+in+patients+who+die+of+sepsis+and+multiple+organ+failure.">Immunosuppression in patients who die of sepsis and multiple organ failure.</a> JAMA. 306 (23), 2594-2605 (2011).</li><li>Hotchkiss, R. S., Monneret, G., Payen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sepsis-induced+immunosuppression:+from+cellular+dysfunctions+to+immunotherapy.">Sepsis-induced immunosuppression: from cellular dysfunctions to immunotherapy.</a> Nature Reviews Immunology. 13 (12), 862-874 (2013).</li><li>Dejager, L., Pinheiro, I., Dejonckheere, E., Libert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cecal+ligation+and+puncture:+the+gold+standard+model+for+polymicrobial+sepsis?.">Cecal ligation and puncture: the gold standard model for polymicrobial sepsis?.</a> Trends in Microbiology. 19 (4), 198-208 (2011).</li><li>Rittirsch, D., Huber-Lang, M. S., Flierl, M. A., Ward, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunodesign+of+experimental+sepsis+by+cecal+ligation+and+puncture.">Immunodesign of experimental sepsis by cecal ligation and puncture.</a> Nature Protocols. 4 (1), 31-36 (2009).</li><li>Hugunin, K. M. S., Fry, C., Shuster, K., Nemzek, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+tramadol+and+buprenorphine+on+select+immunologic+factors+in+a+cecal+ligation+and+puncture+model.">Effects of tramadol and buprenorphine on select immunologic factors in a cecal ligation and puncture model.</a> Shock. 34 (3), 250-260 (2010).</li><li>He, 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=Annexin+A2+Modulates+ROS+and+Impacts+Inflammatory+Response+via+IL-17+Signaling+in+Polymicrobial+Sepsis+Mice.">Annexin A2 Modulates ROS and Impacts Inflammatory Response via IL-17 Signaling in Polymicrobial Sepsis Mice.</a> PLoS Pathogens. 12 (7), 23 (2016).</li><li>Pu, Q. Q., 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=Atg7+Deficiency+Intensifies+Inflammasome+Activation+and+Pyroptosis+in+Pseudomonas+Sepsis.">Atg7 Deficiency Intensifies Inflammasome Activation and Pyroptosis in Pseudomonas Sepsis.</a> Journal of Immunology. 198 (8), 3205-3213 (2017).</li><li>Zanotti-Cavazzoni, S. L., 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=Fluid+resuscitation+influences+cardiovascular+performance+and+mortality+in+a+murine+model+of+sepsis.">Fluid resuscitation influences cardiovascular performance and mortality in a murine model of sepsis.</a> Intensive Care Medicine. 35 (4), 748-754 (2009).</li><li>Chin, W., 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=A+macromolecular+approach+to+eradicate+multidrug+resistant+bacterial+infections+while+mitigating+drug+resistance+onset.">A macromolecular approach to eradicate multidrug resistant bacterial infections while mitigating drug resistance onset.</a> Nature Communications. 9 (1), 917 (2018).</li><li>Nascimento, D. 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=IL-33+contributes+to+sepsis-induced+long-term+immunosuppression+by+expanding+the+regulatory+T+cell+population.">IL-33 contributes to sepsis-induced long-term immunosuppression by expanding the regulatory T cell population.</a> Nature Communications. 8, 14919 (2017).</li><li>Deng, 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=Systematic+investigation+on+the+turning+point+of+over-inflammation+to+immunosuppression+in+CLP+mice+model+and+their+characteristics.">Systematic investigation on the turning point of over-inflammation to immunosuppression in CLP mice model and their characteristics.</a> International Immunopharmacology. 42, 49-58 (2017).</li></ol>

The authors have no financial conflicts of interest.

As the gold standard model for sepsis research, CLP has a combination of three insults, including tissue trauma caused by the laparotomy, necrosis due to ligation of the cecum, and infection as a result of microbial leakage that causes peritonitis with translocation of bacteria into blood8. Therefore, CLP mimics the complexity of human sepsis better than many other models. However, a major limitation of current CLP model is the inability to reflect the more prolonged phase of sepsis seen in patien...

Sepsis initiates a complex interplay of host pro-inflammatory and anti-inflammatory processes and is characterized by a hyperinflammatory response and subsequent immune dysfunction1,2. Sepsis represents a global health priority and causes a high number of deaths in intensive care units (ICUs)3. The incidence of sepsis is estimated to exceed 30 million cases worldwide per year, with mortality rates as high as 30% despite advances in ICU management4,5. In 2017, the World Health Organization adopted a resolution to improve the prevention, diagnosis, and management of this deadly disease5. However, recent studies have illustrated that death does not result from primary infection in severe septic patients but rather from secondary nosocomial infection (particularly pneumonia) that caused by immunosuppression6,7. Therefore, understanding the mechanisms of why septic patients develop secondary infection and discovering more effective treatments are urgently required. Herein, a dual model, also known as a double-hit model, to study the immunosuppressive phenomenon occurring in patients with protracted sepsis is described. 
As the gold standard experimental model in research on polymicrobial sepsis, cecal ligation and puncture (CLP) is a surgery characterized by cecum ligation and perforation, which contributes to polymicrobial peritonitis and sepsis8,9. The pathophysiological process and cytokine profiles, along with the kinetics and magnitude, are similar to clinical sepsis. The position of the ligation, needle size used for the puncture, and number of cecal punctures are major factors that impact the mortality following CLP.
The nosocomial pneumonia is the leading cause of mortality among critically ill patients with sepsis. The major type of organisms causing severe sepsis includes Staphylococcus aureus (20.5%), Pseudomonas species (19.9%), Enterobacteriacae (mainly E. coli, 16.0%), and fungi (19%). Meanwhile, recent studies have suggested an increasing incidence of gram-positive organisms, which are now almost as common as gram-negative infections3.
The method described in this protocol involves CLP, performed as the "first hit" to induce sublethal polymicrobial peritonitis, which manifests an immunosuppression condition. The procedure also involves subsequent intranasal instillation of S. aureus as the "second hit" to provide a clinically relevant research platform.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">21 G 1 &#189; Needle</td>
			<td style="width:191px;">BD</td>
			<td style="width:201px;">BD305167</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ACK lysing buffer</td>
			<td>Gibco</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:355px;">Anti-mouse CD11b antibody</td>
			<td style="width:191px;">Biolegend</td>
			<td style="width:201px;">101201</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Anti-mouse Ly-6G/Ly-6C (Gr-1) antibody</td>
			<td style="width:191px;">Biolegend</td>
			<td style="width:201px;">108401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">C57BL/6 mice&#160;</td>
			<td style="width:191px;">Harlan (Indianapolis)</td>
			<td style="width:201px;">C57BL/6NHsd</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Desk light</td>
			<td style="width:191px;">General Supply</td>
			<td style="width:201px;">General Supply</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Disinfecting wipes</td>
			<td style="width:191px;">Clorox</td>
			<td style="width:201px;">B07NV5JMCS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:355px;">Electric razor</td>
			<td style="width:191px;">General Supply</td>
			<td style="width:201px;">General Supply</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:355px;">ELISA kits (mouse IL-1&#946;, IL-6 and TNF&#945;)</td>
			<td>Invitrogen</td>
			<td style="width:201px;">88-7013, 88-7064, and 88-7324</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Iodine</td>
			<td style="width:191px;">Dynarex</td>
			<td style="width:201px;">B003U463PY</td>
			<td>PVP Iodine Wipes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Ketamine</td>
			<td style="width:191px;">FORT DODGE</td>
			<td style="width:201px;">NDC 0856-2013-01</td>
			<td>Amine hydrochloride injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Laboratory scale</td>
			<td style="width:191px;">General Supply</td>
			<td style="width:201px;">General Supply</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">LB Agar, Miller</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:201px;">BP1425-500</td>
			<td>Molecular genetics, powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Micropipette</td>
			<td style="width:191px;">ErgoOne</td>
			<td style="width:201px;">7100-1100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Normal saline</td>
			<td style="width:191px;">General Supply</td>
			<td style="width:201px;">General Supply</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Polylined towel</td>
			<td>CardinalHealth, Convertors</td>
			<td>3520</td>
			<td style="width:263px;">Surgical drape, sterile, for single use only</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:355px;">Silk suture, 4-0</td>
			<td style="width:191px;">DAVIS &#38; GECK</td>
			<td style="width:201px;">1123-31</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Small animal needle holder</td>
			<td style="width:191px;">General Supply</td>
			<td style="width:201px;">General Supply</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Small animal surgery scissors</td>
			<td style="width:191px;">General Supply</td>
			<td style="width:201px;">General Supply</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Small animal surgical forceps</td>
			<td style="width:191px;">General Supply</td>
			<td style="width:201px;">General Supply</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Staphylococcus aureus</td>
			<td>ATCC</td>
			<td style="width:201px;">13301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Warm pad</td>
			<td style="width:191px;">General Supply</td>
			<td style="width:201px;">General Supply</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Xylazine</td>
			<td style="width:191px;">Alfa Aesar</td>
			<td style="width:201px;">7361-61-7</td>
			<td></td>
		</tr>
	</tbody>
</table>

design of cecal ligation and puncture and intranasal infection dual model of sepsis-induced immunosuppression

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

Depending on the experimental design and procedures, C57BL/6 mice were subjected to CLP, and after 3 days, they were administered bacteria intranasally (Figure 1). As shown in Figure 2, the mice began to die at ~12 h after induction of peritonitis. Two mice in the CLP+SA group and three mice in the CLP+NS group died before intranasal S. aureus instillation. No mortality was detected in uninfected non- or sham-operated mi...

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

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

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

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Research

JoVE Journal

Immunology and Infection

9.8K Views.  Sichuan University. This protocol describes techniques to measure infectious outcomes underlying secondary hospital-acquired infections in the immunosuppressive condition, first by establishing cecal ligation/puncture mice then challenging them with intranasal infection to create a clinically relevant model of immunosuppression sepsis. 

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

Particle Agglutination Method for Poliovirus Identification

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute flaccid paralysis (AFP) cases. In the World Health Organization (WHO) Global Polio Laboratory Network, PV isolation and identification are currently being performed by using cell culture system and real-time RT-PCR, respectively. In the post-eradication era of PV, simple and rapid identification procedures would be helpful for rapid confirmation of polio cases at the national laboratories. In the present study, we will show the procedure of novel PA assay developed for PV identification. This PA assay utilizes interaction of PV receptor (PVR) molecule and virion that is specific and uniform affinity to all the serotypes of PV. The procedure is simple (one step procedure in reaction plates) and rapid (results can be obtained within 2 h of reaction), and the result is visually observed (observation of agglutination of gelatin particles).

A recently developed novel particle agglutination (PA) assay utilizing virus receptor molecule allowed a rapid and easy identification of poliovirus (PV). In this article, we will show the procedure for the PA assay for PV identification.

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

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

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

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

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

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

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

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 ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...

Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits (Oryctolagus cuniculus)

Due to similarities in placentation and antibody transfer with humans, rabbits are an excellent model of maternal immunization. Additional advantages of this research model are the ease of breeding and sample collection, relatively short gestation period, and large litter sizes. Commonly assessed routes of immunization include subcutaneous, intramuscular, intranasal, and intradermal. Nonterminal sample collection for the chronological detection of the immunologic responses to these immunizations include the collection of blood, from both dams and kits, and milk from the lactating does. In this article, we will demonstrate techniques our lab has utilized in studies of maternal immunization in New Zealand White rabbits (Oryctolagus cuniculus), including intranasal immunization and milk collection.

Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits Oryctolagus cuniculus

This article describes and demonstrates the administration of intranasal vaccines and the collection of milk from lactating rabbits (Oryctolagus cuniculus) as a means to assess mucosal immunity in a translationally appropriate model of maternal immunization.

Due to similarities in placentation and antibody transfer with humans, rabbits are an excellent model of maternal immunization. Additional advantages of ...

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

Summary

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Chapters in this video

Particle Agglutination Method for Poliovirus Identification

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

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

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits (Oryctolagus cuniculus)