This work was supported by "Spanish Ministry of Economy and Competitiveness" [grant number BIO2014-5258P], "European Commission" by the H2020 programs [grant numbers TBVAC2020 643381].

All mice were kept under controlled conditions and observed for any sign of disease. Experimental work was conducted in agreement with European and national directives for protection of experimental animals and with approval from the competent local ethics committees.
1. Preparation of Quantified Glycerol Stocks of BCG Danish and Mycobacterium tuberculosis H37Rv
NOTE: All the protocols described were performed under BSL3 conditions.

<ol>
	<li>Thaw frozen glycerol stocks of BCG Danish or H37Rv strains and inoculate 100 &#956;l in 10 ml of 7H9 medium10 supplemented with Tween-80 0.05% (v/v) and ADC (Albumin, Dextrose, Catalase) 10% (v/v).</li>
	<li>Incubate under static conditions for one week at 37 &#176;C until the culture is in log-phase growth.</li>
	<li>Scale up bacterial culture by transferring the 10-ml culture to 200 ml of fresh medium. Incubate for 20 additional days under static conditions.</li>
	<li>Transfer to 50-ml centrifuge tubes and centrifuge for 15 min at 2500 x g.</li>
	<li>Discard supernatants and resuspend bacterial pellet in the residual volume. Add to each tube sterile 3-mm-diameter glass beads (10 - 15 per tube).</li>
	<li>Vortex vigorously during 1 min in order to dissociate bacterial clumps. Resuspend each pellet in 10 ml of PBS with Tween-80 0.05% (v/v).</li>
	<li>Leave tubes for 10 min allowing the greatest bacterial aggregates and glass beads to settle down.</li>
	<li>Recover 9.5 ml of supernatant from each of the 4 tubes containing small clumps and single bacteria and transfer volumes to a single fresh 50-ml centrifuge tube (final recovered volume 38 ml). Centrifuge for 10 min at 400 x g.</li>
	<li>Recover 35 ml of supernatant (containing mainly single bacteria) in a single fresh 50-ml centrifuge tube and add 15 ml of PBS with glycerol 50% (v/v), obtaining a final glycerol concentration of 15% (v/v).</li>
	<li>Mix gently and distribute in 1-ml aliquots in appropriate tubes for freezing. Store at -80 &#176;C (one lot provides approximately 50 aliquots glycerol stock).</li>
	<li>To quantify glycerol stocks, thaw a glycerol the day after freezing and perform seven ten-fold serial dilutions in separate 1.5-ml tubes containing 900 &#956;l of PBS.</li>
	<li>Plate 100 &#956;l of each dilution in 7H10 solid-agar medium10 supplemented with ADC 10% (v/v) prepared in 55-mm diameter Petri dishes.</li>
	<li>Carefully add 3 mm-diameter glass beads (approx. 10 per plate) and gently shake plate to equally distribute volume over agar plate. Discard beads and seal agar plate with parafilm.</li>
	<li>Incubate for 21 days at 37 &#176;C and determine bacterial concentration by counting colony-forming units (CFU) in the dilutions where single colonies can be accurately distinguished.</li>
</ol>
2. Mouse Vaccination
<ol>
	<li>Thaw a pre-quantified BCG glycerol and dilute it in PBS to prepare two suspensions. Calculate final volume considering the dose per animal is 100 &#956;l for subcutaneous administration (107 CFU per ml for subcutaneous vaccination) and 40 &#956;l for intranasal administration (2.5 x 107 CFU for intranasal vaccination).</li>
	<li>Use a specialized anesthesia workstation for rodents to anesthetize mice with a mixture of isoflurane and oxygen. Use isoflurane 5% to induce anesthesia and 2% to maintain the mice in the anesthesia chamber.&#160;(Other accepted anesthesia method can be used).</li>
	<li>For subcutaneous vaccine administration fill a 1-ml syringe with a 26 G needle with 1 ml of bacterial suspension (107 CFU/ml). Remove air bubbles.</li>
	<li>Place an anesthetized mouse on a flat surface in prone position inside a laminar flow hood and inoculate subcutaneously 100 &#956;l (106 CFU/dose) of vaccine in a flank of the mouse back.&#160;Return the mouse to the cage and monitor it to ensure that it properly recovers from anesthesia.</li>
	<li>Change the syringe needle between each mouse administration. Removed air bubbles as described above.</li>
	<li>For intranasal administration, place an anesthetized mouse in supine position inside the hood.</li>
	<li>With a micropipette take 20 &#956;l of the suspension with 2.5 x 107 CFU/ml. Place the inoculum drop-by-drop between the two nostrils until the whole volume has been deposited, leaving time between drops to allow the mouse breathe the volume in.&#160;Note: mice can suffer from respiratory distress during this procedure so they should be left to assimilate each drop prior to administrate the next one to prevent it as much as possible.</li>
	<li>Re-fill the micropipette with another 20 &#956;l and repeat the operation. If the mouse starts to wake up from the anesthesia after the first inoculation, place it in the anesthesia chamber prior to the second one.&#160;Return the mouse to the cage and ensure that it properly recovers from anesthesia.</li>
</ol>
3. Mouse Intranasal Challenge with H37Rv Strain
<ol>
	<li>Thaw a pre-quantified H37Rv glycerol and dilute it in PBS to prepare a bacterial suspension of 2,500 CFU per ml. Calculate final volume considering the dose per animal is 40 &#956;l.</li>
	<li>H37Rv is inoculated intranasally as described in 2.5 - 2.7. Final challenge dose per mouse is 102 CFU/40 &#181;l.</li>
</ol>
4. Analysis of Induced Immune Response in Lungs
<ol>
	<li>Lung cellular suspension 
	NOTE: Assessment of vaccine-induced adaptive immune response in lungs requires the generation of single cell suspension. For this purpose, we use a tissue dissociator such as GentleMACS .
	<ol>
		<li>Sacrifice the mouse by cervical dislocation (or other accepted humane methodology) and ensure mouse is dead touching the eyeball to confirm absent of blink reflex.</li>
		<li>Inside a laminar flow hood, extract the lungs using sterile scissors and forceps and place them on a clean surface. Clean lungs removing trachea and connective tissue, and transfer the lungs to a 1.5-ml tube with PBS (maintain on ice until processing).</li>
		<li>To generate a cellular suspension, place lungs in a proper tube for organ dissociation (C-dissociator tube) containing 5 ml of 10 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2 and 1.8 mM CaCl2 buffer.</li>
		<li>Add 100 &#956;l of Collagenase D 100 mg/ml (2 mg/ml final concentration), and 40 &#956;l of DNAse I 20,000 IU/ml (160 IU/ml final concentration. 
		NOTE: Stock solutions of Collagenase D or DNaseI are prepared dissolving lyophilized enzymes in PBS or glycerol 50% (v/v), 20 mM Tris-HCl pH 7.5 y MgCl2 1 mM, respectively.</li>
		<li>Place the tube in the tissue dissociator and use the predefined program m_lung_01 for lung fragmentation. Incubate the tube at 37 &#176;C in a water bath for 30 min.</li>
		<li>Place the tube in the dissociator and use the predefined program m_lung_02 for complete lung homogenization into single cellular suspension.</li>
		<li>Pass the cellular suspension through a 70-&#956;m nylon cell strainer fitted to a 50-ml centrifuge tube. Wash the strainer with 10 ml of RPMI 1640 medium.</li>
		<li>Centrifuge cells for 5 minutes at 400 x g and discard supernatant.</li>
		<li>Add 1 ml of erythrocytes lysing buffer, mix by vortexing and incubate for 1 min at room temperature.</li>
		<li>Add 10 ml of RPMI 1640 medium and centrifuge cells for 5 minutes at 400 x g.</li>
		<li>Resuspend cells in 0.5 ml of complete RPMI 1640 medium supplemented with 10% heat-inactivated Foetal Calf Serum (FCS), 2 mM L-Glutamin, 100 &#956;g/ml Streptomycin, 100 IU/ml Penicillin and 50 &#956;M 2-mercaptoethanol. 
		NOTE: FCS is inactivated by incubation at 56&#176;C for 30 minutes.</li>
		<li>Count cells and add complete RPMI 1640 medium to reach final cellular density of 107 cells/ml. 
		NOTE: For cell count, we use the automated cell counter.</li>
		<li>Distribute 100 &#956;l of cellular suspension in U-bottom 96-well sterile plate (106 cells/well).</li>
		<li>Add 50 &#956;l/well of complete RPMI 1640 medium with 15 &#956;g/ml of commercial H37Rv Purified Protein Derivative (PPD) (5 &#956;g/ml final concentration).</li>
	</ol>
	</li>
	<li>Supernatant collection for cytokine determination by ELISA
	<ol>
		<li>Incubate plate from 4.1.14 for 48 hr at 37 &#176;C and 5% CO2.</li>
		<li>Centrifuge plate for 5 min at 400 x g and recover 100 &#956;l of supernatant.</li>
		<li>Store at -80 &#176;C.</li>
		<li>Dilute supernatants 1/10 and 1/2 in assay buffer for IFN&#947; and IL17A determination, respectively. Determine cytokine concentrations with commercial ELISA kits, according to manufacturer instructions (ELISA Development kits).</li>
	</ol>
	</li>
	<li>Intracellular staining for determination of cytokine-producing CD4+ cells by flow cytometry
	<ol>
		<li>Incubate plate from 4.1.14 for 18 hr at 37 &#176;C and 5% CO2.</li>
		<li>Add 1.5 &#956;l of Brefeldin A 1 mg/ml (stock dissolved in DMSO) to each well (10 &#956;g/ml final concentration) and incubate for six additional hours.</li>
		<li>Centrifuge the plate from 4.3.2 for 1 min at 3200 x g and discard supernatant.</li>
		<li>Resuspend cells in 50 &#956;l of anti-mouse CD4-FITC diluted in RPMI 1640 with 10% FCS (1/500) and incubate for 10 min at 4 &#176;C.</li>
		<li>Centrifuge the plate from 4.3.4 for 1 min at 3,200 x g, discard supernatant and add 100 &#956;l/well of fixation solution. Incubate at 4 &#176;C for 20 min.</li>
		<li>Wash cells twice with 150 &#956;l/wash of permeabilization solution. 
		NOTE: 10x concentrated stock is diluted in deionized water to prepare 1x work solution.</li>
		<li>Resuspend cells in 50 &#956;l of anti mouse IFN&#947;-APC and anti-mouse IL17A-APC.Cy7 diluted in permeabilization solution (1/250 both antibodies).</li>
		<li>Incubate for 1 hr at RT.</li>
		<li>Wash cells twice with 150 &#956;l/wash of permeabilization solution.</li>
		<li>Resuspend cells in 200 &#956;l of PBS for flow cytometry acquisition.</li>
	</ol>
	</li>
</ol>
5. Analysis of Immunoglobulins in Bronchoalveolar Lavage (BAL)
<ol>
	<li>Obtainment of BAL samples
	<ol>
		<li>Sacrifice the mouse by cervical dislocation (or other accepted humane methodology) and ensure mouse is dead touching the eyeball to confirm absent of blink reflex.</li>
		<li>Expose the trachea using proper precision sterile forceps and scissors and perform a small cut in the trachea, making sure not to excise it completely. Take care not to cause bleeding that could contaminate BAL sample.</li>
		<li>Introduce into the trachea a cannula connected to a sterile 1-ml syringe with 800 &#956;l of ice-cold PBS.</li>
		<li>Inoculate slowly the PBS into the lungs and then recover BAL volume pulling back the syringe&#39;s piston very slowly in order to avoid lung collapse. 
		NOTE: Recovery of 500 to 600 &#956;l is satisfactory.</li>
		<li>Transfer BAL volume into a 1.5-ml tube and confirm BAL remains uncoloured, indicating that is not contaminated with blood (maintain on ice until processing).</li>
		<li>Centrifuge BAL samples for 5 min at 400 x g, and transfer supernatant to a new tube.</li>
		<li>Store supernatants at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Determination of IgA in BAL supernatants 
	NOTE: All reagents used are at RT.
	<ol>
		<li>Coat high-protein binding polystyrene flat-bottom 96-well plates with 100 &#956;l of PPD diluted in PBS (10 &#956;g/ml) for M. tuberculosis-specific IgA determination, or with 100 &#956;l of BAL supernatant diluted 10 times in PBS for total IgA determination.</li>
		<li>Incubate overnight at 4 &#176;C.</li>
		<li>Discard supernatant and tap the plate on paper towel to dry. Wash with 200 &#956;l/well of PBS.</li>
		<li>Block with 200 &#956;l/well of a bovine serum albumin (BSA) 1% (w/v) in PBS-Tween-80 0.05% (v/v) (Blocking buffer).</li>
		<li>Incubate 1 hr at RT.</li>
		<li>Wash once with 200 &#956;l/well of PBS-Tween-80 0.05% (v/v) (Washing buffer).</li>
		<li>Add 100 &#956;l of undiluted BAL supernatant in the PPD-coated wells. Incubate two hours at room temperature. 
		NOTE: If the total IgA determination is done in parallel with the PPD-specific IgA, leave total IgA wells with 100 &#956;l washing buffer during this incubation.</li>
		<li>Wash three times with 200 &#956;l/well of washing buffer.0170004</li>
		<li>Add 100 &#956;l of horseradish peroxidase (HRP)-conjugated anti-mouse IgA diluted in blocking buffer (1/10,000).</li>
		<li>Incubate 1 hr RT.</li>
		<li>Wash five times with 200 &#956;l/well of washing buffer.</li>
		<li>Add 100 &#956;l/well of 3,3&#8242;,5,5&#8242;-Tetramethylbenzidine (TMB). Incubate 20 min at room temperature in the dark.</li>
		<li>Add 100 &#956;l/well of H2SO4 0.5N. Read the plate in a spectrophotometer at a wavelength of 450 nm.</li>
	</ol>
	</li>
</ol>
6. Bacterial load determination in lungs
<ol>
	<li>Lung homogenization and plating
	<ol>
		<li>Four weeks after challenge, sacrifice the mouse by cervical dislocation (or other accepted humane methodology) and ensure mouse is dead touching the eyeball to confirm absent of blink reflex.</li>
		<li>Place the mouse in supine position fixed on a flat and disinfected surface inside a laminar flow hood.</li>
		<li>Make an incision and remove the skin from chest region to leave visible the thoracic area.</li>
		<li>Cut out the ribs and harvest the lungs and heart using sterile scissors and forceps. Place organs on a clean surface.</li>
		<li>Clean out lungs by removing the heart, trachea and connective tissue and transfer the lungs to a 1.5-ml tube with 1 ml of deionized water (maintain on ice until processing).</li>
		<li>Use sterile forceps to transfer the lungs of each mouse into a proper tube for organ homogenization (M - dissociator tube), containing 1 ml of deionized water.</li>
		<li>Place the tube in the tissue dissociator and use the predefined program RNA_1 to homogenise the lungs.</li>
		<li>Make five ten-fold serial dilutions in 1.5-ml tubes, starting with 100 &#956;l of lung homogenate diluted in 900 &#956;l.</li>
		<li>Plate dilutions as described in 1.11-1.14.</li>
	</ol>
	</li>
	<li>Differentiation between BCG and H37Rv by PCR 
	NOTE: In the plates from 6.1.6 corresponding to the group vaccinated with BCG by the intranasal route, we analyzed a representative number of colonies by a discerning PCR between BCG and H37Rv to ensure that the CFU considered to assess vaccine-conferred protective efficacy corresponded uniquely to H37Rv. This PCR targets amplification of RD9, a genomic region different in BCG and M. tuberculosis, giving a fragment of 2618 bp for H37Rv, and 465 bp for BCG 11.
	<ol>
		<li>Prepare a master mix stock for all samples with volumes per sample as indicated, and then distribute in PCR tubes (9 &#956;l/sample). 
		NOTE: Buffer 5x: 2 &#956;l. Forward primer 25 &#956;M (GTGTAGGTCAGCCCCATCC): 0.32 &#956;l, Reverse primer 25 &#956;M (GCCCAACAGCTCGACATC): 0.32 &#956;l, Taq polymerase 5 units/&#956;l: 0.04 &#956;l, Deionized H2O: 6.32 &#956;l.</li>
		<li>Touch a colony with a sterile toothpick and immerse the toothpick with the sample in a PCR tube containing the master mix.</li>
		<li>Run the PCR with the following program: 10 min 95 &#176;C (this step is to trigger bacterial disruption), 10 min 95 &#176;C (this step is to trigger bacterial disruption), 35 cycles ( 30 seconds at 95 &#176;C, 1 min at 58 &#176;C and 4 min at 72 &#176;C).</li>
		<li>Load the PCR samples in a 1% agarose gel with ethidium bromide and run samples to visualize the fragments.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Although current vaccine against tuberculosis, BCG, is the most widely administered vaccine in history, tuberculosis remains one of the leading causes of death and morbidity from infectious diseases worldwide. This paradox is explained by the lack of protection of this vaccine against pulmonary tuberculosis, the responsible form of transmission. New vaccination approaches effective against pulmonary forms of the disease are urgently needed, as they would have the greatest impact on disease transmission globally.
Our data clearly show that a change in the route of administration of BCG to mimic the natural route of infection could be a successful strategy to prevent pulmonary tuberculosis. Our results are in accordance with other authors showing equivalent data in different animal models, including guinea pigs and non-human primates (NHP) 6,9,12.
Remarkably, our unpublished data indicated that the volume of administration by the intranasal route is a critical step of the protocol. These results revealed that following intranasal delivery of 100 H37Rv bacteria resuspended in 10 &#956;l (instead of 40 &#956;l), we only recovered lung CFUs from around 20% of the animals (data not shown. 
Importantly, one of the possible limitations of intranasal administration as a vaccine delivery route in clinic could be its proximity with the central nervous system13,14. In this regard, aerosol immunization might be safer as delivery route for pulmonary tuberculosis vaccines in humans.
This protocol describes a standardized methodology that can be adapted to other studies, as comparison of pulmonary vaccines, or different routes of pulmonary immunization, including aerosol.
Our data suggest that the analysis in parallel of protective efficacy and immune response, using lung samples from the same animal, could be a useful tool to identify biomarkers of protection. In this regard, our results reveal that lung IL17, but not IFN&#947;, seems to correlate with a better vaccine protective efficacy. These data highlight a possible role of IL17 in the protective response induced by intranasal BCG, which is in accordance with data reported by other authors using mucosal subunit TB vaccines 7,15. Our data also indicate that presence of IgA (both total and MTB-specific) in BAL samples correlates with protection conferred by intranasal BCG. Importantly, we described previously, in agreement with other works, that IL17 contributes to traslocation of IgA to respiratory airways and gut lumen.9,16,17

Tuberculosis (TB) is one of the leading infectious diseases causing more associated deaths than HIV in the world and combined with rising increase of multidrug resistant strains makes TB an alarming global health problem1. New diagnostic tools, more effective and less toxic drugs, and new safe and effective TB vaccines are an urgent need, especially in the developing world.
Live attenuated Bacille Calmette-Guerin (BCG) is currently the only licenced vaccine against TB, which has been administered intradermally at birth since the 1970s worldwide. BCG is considered effective in preventing severe forms of the disease (meningitis and miliary TB) in children, but has shown inconsistent efficacy against pulmonary TB responsible of disease transmission 2.
Pulmonary vaccination, which mimics natural route of TB infection, represents an attractive approach for priming local host immune responses. In this regard, various preclinical works in different relevant TB animal models have demonstrated greater vaccine efficacy following pulmonary immunization as compared to the subcutaneous or intradermal route 3-6. Nevertheless, the protective mechanisms triggered by pulmonary vaccination are not well understood. In the last years, several works have pointed towards IL17-mediated response as an important factor of TB-specific mucosal immune response, as in mouse models deficient for IL17 mucosal vaccine-induced protective efficacy is impaired 7,8.
Recently we demonstrated for the first time that intranasal BCG administration protected DBA/2 mice, a mouse strain characterized by the lack of protection after subcutaneous BCG immunization 9. These results suggested that respiratory TB vaccination could be more effective in reducing rate of TB in endemic countries, where intradermal BCG is considered ineffective against pulmonary TB.

<table border="0" cellpadding="0" cellspacing="0" width="895">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Middlebrook 7H9 broth</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">271310</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Middlebrook ADC Enrichment</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">211887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Tween 80</td>
			<td style="width:176px;">Scharlau</td>
			<td style="width:119px;">TW00800250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">3-mm diameter Glass Beads</td>
			<td style="width:176px;">Scharlau</td>
			<td style="width:119px;">038-138003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Middlebrook 7H10 Agar</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">262710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">1-ml syringe 26GA 0.45x10 mm</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">301358</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">GentleMACS dissociator</td>
			<td style="width:176px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">C tubes</td>
			<td style="width:176px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">M tubes</td>
			<td style="width:176px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Collagenase D</td>
			<td style="width:176px;">Roche</td>
			<td style="width:119px;">11088882001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">DNaseI</td>
			<td style="width:176px;">Applichem</td>
			<td style="width:119px;">A3778,0100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Falcon 70&#181;m Cell Strainer</td>
			<td style="width:176px;">Corning</td>
			<td style="width:119px;">352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">RPMI 1640</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">R0883</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Red Blood Cell Lysing Buffer</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">R7757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">GlutaMAX Supplement</td>
			<td style="width:176px;">Gibco</td>
			<td style="width:119px;">35050-061</td>
			<td>100X concentrated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Penicillin-Streptomycin Solution</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">P4333</td>
			<td>100X concentrated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Fetal Calf Serum</td>
			<td style="width:176px;">Biological Industries</td>
			<td style="width:119px;">04-001-1A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">2-Mercaptoethanol</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">M3148-25ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Scepter 2.0 Handheld Automated Cell Counter</td>
			<td style="width:176px;">Millipore</td>
			<td style="width:119px;">PHCC20040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Scepter Cell Counter Sensors, 40 &#181;m</td>
			<td style="width:176px;">Millipore</td>
			<td style="width:119px;">PHCC40050</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:409px;">Mycobacterium Tuberculosis - Tuberculin PPD</td>
			<td style="width:176px;">Statens Serum Institut (SSI)</td>
			<td style="width:119px;">2390</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Mouse IFN-&#947; ELISA development kit&#160;</td>
			<td style="width:176px;">Mabtech</td>
			<td>3321-1H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Mouse IL17A ELISA development kit&#160;</td>
			<td style="width:176px;">Mabtech</td>
			<td>3521-1H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Brefeldin A</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">B7651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">FITC Rat Anti-Mouse CD4</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">553047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">BD Cytofix/Cytoperm Kit</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">555028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">APC-Cy7 Rat Anti-mouse IL-17A</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">560821</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">APC Mouse Anti-mouse IFNg</td>
			<td style="width:176px;">BD</td>
			<td style="width:119px;">554413</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">LACHRYMAL OLIVE LUER LOCK 0.60 x 30 mm. 23G x 1 1/4&#8221;</td>
			<td style="width:176px;">UNIMED</td>
			<td style="width:119px;">27.134</td>
			<td>Used as trachea cannula for BAL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">high-protein binding polystyrene flat-bottom 96-well plates MAXISORP</td>
			<td style="width:176px;">NUNC</td>
			<td style="width:119px;">430341</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Albumin, from bovine serum</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">A4503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Goat Anti-Mouse IgA (&#945;-chain specific)&#8722;Peroxidase antibody</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">A4789</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">3,3&#8242;,5,5&#8242;-Tetramethylbenzidine (TMB)&#160;</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:119px;">T0440</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">MyTaq DNA Polymerase</td>
			<td style="width:176px;">Bioline</td>
			<td style="width:119px;">BIO-21107</td>
			<td>The kit Includes Buffer 5x</td>
		</tr>
	</tbody>
</table>

protective efficacy and pulmonary immune response following subcutaneous and intranasal bcg administration in mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data have encouraged pulmonary tuberculosis vaccines as a promising strategy to prevent pulmonary disease, which is responsible for transmission. In this work, we describe the methodology used to demonstrate in the mouse model the benefits of intranasal BCG vaccination when compared to subcutaneous. Our data revealed greater protective efficacy following intranasal BCG administration. In addition, our results indicate that pulmonary vaccination triggers a higher immune response in lungs, including Th1 and Th17 responses, as well as an increase of immunoglobulin A (IgA) concentration in respiratory airways. Our data show correlation between protective efficacy and the presence of IL17-producing cells in lungs post-Mycobacterium tuberculosis challenge, suggesting a role for this cytokine in the protective response conferred by pulmonary vaccination. Finally, we detail the global workflow we have developed to study respiratory vaccination in the mouse model, which could be extrapolated to other tuberculosis vaccines, apart from BCG, targeting the mucosal response or other pulmonary routes of administration such as the intratracheal or aerosol.

This work describes the comparison of two routes of administration of BCG: subcutaneous and intranasal. Subcutaneous route is comparable to the intradermal, which is the current clinical route for BCG worldwide. Intranasal route of vaccination aims to mimic the natural route of infection of M. tuberculosis, with the objective to induce immune response directly in the lungs, the primary target organ of this pathogen.
Figure 1 describes the workflow followed. Eight to ten week-old female DBA/2 mice are vaccinated with 106 CFU of BCG Danish by the subcutaneous or intranasal route of administration. Eight weeks later, a group of mice is sacrificed to analyze lung immune response induced by vaccination. BAL samples are first obtained and then we harvest lungs. In order to study vaccine-conferred protective efficacy, we inoculate a different group of mice with a low-dose intranasal challenge of M. tuberculosis H37Rv strain. One month later, we sacrifice animals and harvest lungs. In this case, for each animal we use the left lung to determine bacterial load and the right lung to assess vaccine-induced immune response post-challenge. The objective is to generate bacterial load and immune response data in lungs for each animal in order to study potential correlates of protection.
As shown in Figure 2, lungs were dissociated to obtain either an organ homogenate or a cellular suspension. Homogenized lungs were plated on solid agar medium to determine bacterial load four weeks post-challenge. Cellular suspension to study vaccine-induced immune response was obtained following lung enzymatic digestion with collagenase D and DNaseI.
Our results clearly indicate that, when compared to the subcutaneous route, the intranasal route of vaccination confers a much greater protective efficacy in lungs four weeks after challenge&#160;&#160;(Figure 3A). In addition, we confirmed that bacterial load in lungs from the intranasal BCG-vaccine group correspond to H37Rv and not to BCG vaccine. To this end, we analyzed a representative number of colonies from this group by specific PCR for the RD9 genome region, which amplifies different length fragments in BCG and M. tuberculosis (Figure 3B). Figure clearly showed that all the colonies analyzed provided a fragment of 0.4 kbp, which corresponded to H37Rv.
Our data revealed correlation between protective efficacy conferred by intranasal BCG vaccination and vaccine-induced immune response in lungs prior to challenge. Intranasal BCG clearly triggered higher IL17 and IFN&#947; production in lungs, measured by ELISA (Figure 4A). These data were confirmed by intracellular staining (ICS) and flow cytometry (data not shown). In addition, we also found a higher concentration of both total and PPD-specific IgA concentration in BAL samples (Figure 4B), indicating that pulmonary BCG vaccination induces production of IgA and translocation to respiratory airways.
Finally, we studied BCG-induced immune response in lungs after challenge (Figure 5). Our data revealed differences between IL17 and IFN&#947;; IL17A-producing CD4+ cells were only detected in the intranasal BCG group, whereas IFN&#947;-producing cells were found in all groups infected with H37Rv regardless of vaccination. Representation of data of each animal corresponding to IL17-producing cells and bacterial load showed a significant correlation between presence of IL17 and lung bacterial load reduction, which was not observed in the case of IFN&#947;.
<img alt="Figure 1" src="/files/ftp_upload/54440/54440fig1.jpg" /> 
Figure 1. Workflow to Compare Intranasal and Subcutaneous Route of BCG Vaccination. Eight weeks post BCG immunization, a set of mice (6 per experimental group) is used to harvest lungs and perform a BAL, and analyze pulmonary immune response induced by vaccination. Another set of mice (6/group), is inoculated with a low-dose intranasal challenge of M. tuberculosis H37Rv strain (100 CFU). One month later, animals are sacrificed and bacterial load is analyzed in the left lung and PPD-specific immune response in the right lung. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54440/54440fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54440/54440fig2.jpg" /> 
Figure 2. Processing of Lung Samples. A tissue dissociator was used to process lungs. In the experiments to determine bacterial load, lungs were homogenised prior to plate them in solid agar medium. In experiments that require a lung cellular suspension, this was generated following enzymatic digestion with collagenase D and DNaseI. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54440/54440fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54440/54440fig3.jpg" /> 
Figure 3. Protective Efficacy Conferred by BCG Immunization. Groups of 6 DBA/2 mice were vaccinated by the subcutaneous (BCG sc), intranasal (BCG in) route, or non-vaccinated (Unvacc) with BCG Danish vaccine 106 CFU. At two months post-vaccination, mice were inoculated intranasally with a low dose (100 CFU) H37Rv challenge, and one-month later bacterial burden in lungs was determined. A representative experiment of two independent is shown. (A) Data in the graphs are represented as mean+SD. One-way ANOVA test with Bonferroni post analysis was performed to calculate statistical significance. (B) A representative number of single colonies from the BCG intranasal group were analyzed by PCR specific for RD9 region (different in BCG and H37Rv genomes) to discern BCG and H37Rv colonies. (Previously published 9). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54440/54440fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54440/54440fig4.jpg" /> 
Figure 4. Vaccine-specific Pulmonary Immune Response Analyzed Prior to Challenge with H37Rv. Groups of 6 DBA/2 mice were vaccinated by the subcutaneous (BCG sc) or intranasal (BCG in) route, or non-vaccinated (Unvacc) with BCG Danish vaccine 106 CFU. (A) At two months post-vaccination, a cellular suspension from harvested lungs was obtained. Cells were stimulated with PPD as described in methods section and IL17A (left panel) and IFN&#947; (right panel) production were analyzed by ELISA. (B) Total IgA, and M. tuberculosis (MTB)-specific IgA were analyzed from BAL samples by ELISA. Pooled data from two independent experiments are shown. Data in the graphs are represented as mean + SD. (Previously published 9). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54440/54440fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54440/54440fig5.jpg" /> 
Figure 5. Vaccine-specific Pulmonary Immune Response Analyzed prior to Challenge with H37Rv. Groups of 6 DBA/2 mice were vaccinated by the subcutaneous (BCG sc) or intranasal (BCG in) route, or non-vaccinated (NV) with BCG Danish vaccine 106 CFU. A control group of non-vaccinated, non-infected mice was also included (NV/NI). At two months post-vaccination, mice were challenged intranasally with a low H37Rv dose (100 CFU), and one-month later animals were euthanized. Left and right lungs from the same animal were used to determine bacterial load and IL17A- (A) or IFN&#947;- (B) producing CD4+ cells, respectively. Data in left panels correspond to percentage of cytokine-producing cells measured by flow cytometry, and are represented as mean + SD. Right panels represent data from bacterial load and cytokine- producing CD4+ cells obtained for each mouse. Linear regression was calculated and the p-value obtained in each case is shown in the case of IL17A. Pooled data from two independent experiments are shown in the figure. (Previously published 9). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54440/54440fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice at JoVE.com

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

protective-efficacy-pulmonary-immune-response-following-subcutaneous

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.5K Views.  University of Zaragoza. We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

Video: Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

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 ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

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 ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Summary

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Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

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

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

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

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice