This work is supported by funding from the National Institutes of Health, Bethesda, Maryland, United States (KP) K08EY0123998, (KP) R01EY030431, (KP) R21 EY02939, UW vision research core grant (NEI P30EY01730), gifts from the Mark Daily, MD Research Fund and the Christopher and Alida Latham Research fund, an unrestricted departmental grant from Research to Prevent Blindness, and career development award from Research to Prevent Blindness (KP). The work conducted in Bristol was supported by additional funding from Sight Research UK and The Underwood Trust.

All procedures performed were approved locally by the Animal Care and Use Committee at the University of Washington (animal study protocol # 4481-02) or in concordance with the United Kingdom Home Office license (PPL 30/3281) and the University of Bristol Ethical Review Group. Experiments conducted at both institutions were concordant with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Visual Research. PMU was generated in 6-10-week-old C57BL/6J mice; all mice weighed at least 18 g at the time of uveitis induction and were confirmed negative for&#160;the rd8 mutation of the Crb1 gene29. Mice were maintained with standard chow and medicated water (acetaminophen 200-300 mg/kg/day) ad libitum under specific pathogen-free conditions. Animal euthanasia was performed using a standard carbon dioxide inhalation method30.
1. Antigen preparation for subcutaneous injection
<ol>
	<li>Perform all procedures in this section inside a chemical fume hood to prevent inhalation or skin contact with the Mtb H37Ra powder. Handle according to your institutional policies for Complete Freund&#39;s Adjuvant (typically BSL-1). This includes using chemical-resistant gloves, safety glasses, and protective work clothing (lab coat).</li>
	<li>Use a good sterile technique to prevent contamination of reagents that will be introduced into experimental animals.</li>
	<li>Make the Mtb suspension in PBS by mixing 5 mg of lyophilized, heat-killed M. tuberculosis H37Ra powder with 2.5 mL of cold PBS in a 5 mL microcentrifuge tube. Vortex once for 30 s and then place on ice.</li>
	<li>To generate a fine suspension of the H37Ra in PBS, sonicate the suspension on ice for 5 min.
	<ol>
		<li>Unclamp the body of the converter unit and clean the probe with a 70% (v/v) alcohol swab.</li>
		<li>Switch on the sonicator, adjust the power setting to 4 by turning the power control knob and immerse the probe&#39;s tip into the PBS-containing mycobacterial powder. Ensure that the probe tip is immersed to at least half the depth of the sample and that the probe tip is not touching the wall of the microcentrifuge tube.</li>
	</ol>
	</li>
	<li>Sonicate the mixture on ice for 30 s, pause 30 s and repeat for a total of 5 min to fully disperse the powder into an even suspension without heating the liquid.</li>
	<li>Add 2.5 mL of Freund&#39;s Incomplete Adjuvant to the mixture and repeat the sonication process on ice until the emulsion forms a toothpaste-like consistency.</li>
	<li>Set the power to 0 using the control knob and turn off the unit to end the sonication. Remove the tip from the suspension and wipe the probe with an alcohol swab.</li>
	<li>Store the antigen emulsion at 4 &#176;C. Making batches of the emulsion will help ensure consistency across experiments. The emulsion can be stored at 4 &#176;C for up to 3 months.</li>
</ol>
2. Subcutaneous injection
<ol>
	<li>Perform subcutaneous injection a week prior to the intravitreal injection (designated as day -7).</li>
	<li>Load a 1 mL syringe (no needle attached) with the mycobacterial emulsion. Due to the viscosity and opacity of the emulsion, difficult-to-see air bubbles can fill the syringe.
	<ol>
		<li>To prevent airbubbles in the syringe, after loading 0.2-0.3 mL of emulsion, invert the syringe (tip facing up) and gently tap the syringe repeatedly on the edge of a counter to bring the bubbles to the surface.</li>
	</ol>
	</li>
	<li>Expel the air from the syringe and continue filling the syringe. Invert and intermittently tap until filled.</li>
	<li>Place a 25 G needle on the syringe and advance the emulsion to fill the needle. Store the syringe on ice until used.</li>
	<li>To perform the subcutaneous injection safely, either anesthetize the mouse or utilize humane restraint methods that allow easy access to the animal hindquarters31.</li>
	<li>To anesthetize for subcutaneous injection, place the animal in an isoflurane induction chamber (3%-4% for induction and 1%-3% for maintenance). Once anesthetized, ensure that the mouse has a slow respiratory rate and exhibits no signs of respiratory distress.</li>
	<li>Place the subcutaneous injections on either the dorsal surface of the hips, or on the ventral surface of the legs proximal to the region of the inguinal lymph nodes.
	<ol>
		<li>Carefully insert the needle to prevent injecting into the muscle. Inject 0.05 mL of the Mtb emulsion into the subcutaneous space. Do not remove the needle immediately in order to allow the thick emulsion to be fully injected.</li>
		<li>Repeat the injection on both left and right sides for a total of 0.1 mL per animal.</li>
	</ol>
	</li>
	<li>If anesthetized, place the mouse on a warm heating pad until complete recovery. Do not leave the mouse unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
	<li>Return the mouse to its cage upon complete recovery and label the cage card with the date of subcutaneous injection.</li>
	<li>Provide analgesia with oral acetaminophen (200 mg/kg/day), but not NSAIDs as anti-inflammatory agents can impact the induction of uveitis.</li>
</ol>
3. Antigen stock preparation for intravitreal injection
<ol>
	<li>Perform all procedures in this section under appropriate sterile conditions to prevent contamination of the intravitreal Mtb suspension.</li>
	<li>Make the intravitreal suspensions.
	<ol>
		<li>For induction of mild to moderate panuveitis, make the intravitreal suspension at a 5 mg/mL concentration by adding 5 mg of the mycobacteria extract to 1 mL of 1x PBS.</li>
		<li>For induction of moderate to severe panuveitis, make the intravitreal suspension at a 10 mg/mL concentration by adding 10 mg of the mycobacteria extract to 1 mL of 1x PBS.</li>
	</ol>
	</li>
	<li>Vortex once for 30 s and then place on ice.</li>
	<li>To generate a fine suspension of the H37Ra in PBS, sonicate the suspension on ice for 10 min as described in step 1.4. Aliquot this stock solution in 100 &#181;L volumes and store at -20 &#176;C.</li>
	<li>Prior to use, thaw at room temperature and vortex on high for 1 min. Keep the aliquots on ice while transport to the animal facility.</li>
</ol>
4. Intravitreal injection procedure on day 0
<ol>
	<li>Animal preparation
	<ol>
		<li>Wear fitted examination gloves, place the mouse on a weighing balance to obtain its weight in grams.</li>
		<li>Give an intraperitoneal injection of 0.02 mL/g bodyweight of a solution containing 100 mg/mL Ketamine and 20 mg/mL Xylazine mixed with sterile water to anesthetize the animal. An alternative approach includes induction using ~1.5% isoflurane (inhaled).</li>
		<li>Wait approximately 2 min for the mouse to fall asleep, and then place the mouse in a warming box and cover the lid. Perform pain reflex tests like ear, toe, and tail pinch to assess the depth of anesthesia for the procedure32.</li>
		<li>Once asleep, anesthetize the cornea with 1 drop of 0.5% (v/v) tetracaine. Avoid getting tetracaine near the nose or mouth of the mouse. After 10 s, dab off the excess liquid. 
		NOTE: It has been observed that iris dilation and anterior chamber (AC) visualization are improved when topical anesthesia is administered, possibly due to improved corneal reflex suppression with the combined systemic and topical anesthesia. However, this step could be omitted if desired.</li>
		<li>Dilate the pupil with 1 drop of 2.5% (v/v) phenylephrine. Use caution to avoid any excess droplets that might enter the nose or mouth. After 2-3 min, dab off the excess liquid.</li>
		<li>To decrease the risk of endophthalmitis, add 1 drop of 5% betadine to the eye surface and surrounding hair. Leave on the eye for 2-3 min. 
		NOTE: Perform all procedures in this section under appropriate sterile conditions to prevent endophthalmitis.</li>
		<li>Remove betadine and cover the eye with hypromellose (0.3%) or carbomer eye gel 0.2% w/w) to prevent dryness under anesthesia. This will also help prevent cataract formation.</li>
	</ol>
	</li>
	<li>Setting up the microinjection system
	<ol>
		<li>Perform the intravitreal injection using a micropump connected to a microsyringe pump controller and an injection syringe (Figure 1A-C). Alternatively, inject with a 33 G needle attached to a Hamilton syringe as described in subsection 4.4.</li>
		<li>Connect a 34 G needle to the injection holder to assemble the injector. Loosen the silver screw cap at the front end of the injection holder and slide the needle into the body of the holder about halfway. Tighten the silver screw cap finger tight.</li>
		<li>Connect the tubing to the injection holder as mentioned in steps 4.2.4-4.2.5.</li>
		<li>To insert the tubing on the injection holder, loosen the plastic screw on the back end of the holder, slide the tubing through the gasket inside and tighten the screw.</li>
		<li>Maintain a slight gap with the end of the tubing to prevent tubing damage during the injection. Refer to Figure 1C.</li>
		<li>Thaw a 100 &#181;L aliquot of the mycobacterium stock suspension.</li>
		<li>Add 3 &#181;L of a 1% fluorescein sodium (AK-Fluor) solution and vortex well.</li>
		<li>Load a 10 &#181;L syringe with the antigen and fluorescein mix without including any air bubbles.</li>
		<li>Remove the loading needle from the syringe, and slide the tubing through the silver screw cap gasket until the tip reaches the zero mark in the syringe body.</li>
		<li>Once the tip of the tubing is correctly aligned to the desired position, tighten the screw cap finger tight.</li>
		<li>Flush the solution in the syringe through the injection tubing to fully load the system. Then repeat steps 4.2.8-4.2.11 to reload the syringe for injection.</li>
		<li>To install the loaded syringe on the micropump, press the clamp release button at the end of the micro-pump to open the syringe clamps.</li>
		<li>Position the cap of the plunger into the plunger cap holder at the rear end of the micropump.</li>
		<li>Then slide the syringe collar onto the collar stop and the syringe body into the syringe clamp.</li>
		<li>Release the clamp button and tighten the plunger retaining screw. Refer to Figure 1D.</li>
		<li>Slide the injection holder and the needle through the o-clamp on the stereotactic injection apparatus. This is a custom platform; alternatively, the syringe can be held and positioned manually.</li>
		<li>Set the infusion volume and the rate of infusion volume on the microsyringe pump controller to inject 500 nL per cycle at a rate of 40 nL/s, respectively. 
		NOTE: Faster injection rates can be used, however, more reflux may be experienced before needle repositioning can be achieved.</li>
		<li>Test the system to ensure correct functioning prior to performing an intravitreal injection. 
		NOTE: When the injection system is functioning correctly, activation of an injection cycle using the foot pedal or the control pad will produce visible movement of the plunger cap holder and a small droplet of greenish liquid will be seen at the tip of the needle. In the case that no liquid is produced, activate additional cycles or flush and reload the syringe.</li>
		<li>Prior to injecting the eye, gently wipe the needle with a 95% ethanol pad.</li>
	</ol>
	</li>
	<li>Intravitreal injection procedure
	<ol>
		<li>The mouse is placed on a stereotaxic apparatus to perform the injection procedure.</li>
		<li>Keep the stage/platform on which the mouse rests warm by attaching 2-3 paper towels on its surface.</li>
		<li>Place the mouse in a prone position on the platform. Use the right and left ear bars to gently fix the animal&#39;s head. Refer to Figure 1E.</li>
		<li>Position the mouse and orient under the scope so that the superior nasal aspect of the right eye is visible.</li>
		<li>Use a 30 G needle to displace the eyelashes and expose the sclera. Visualize the limbus and the radial blood vessel.</li>
		<li>Use a sterile 30 G needle to make a guide hole in the sclera 1-2 mm posterior to the limbus.</li>
		<li>Insert the 34 G needle attached to the injection holder into the eye through the guide hole at an angle that will avoid the lens, but place the needle tip into the vitreous cavity.</li>
		<li>Using the microsyringe pump controller, carefully inject 1 &#181;L of the Mtb extract into the vitreous cavity. In case of consistent reflux, increase the injection volume to 1.5 &#181;L to ensure adequate dose delivery. 
		NOTE: For sham controls, inject 1 &#181;L of PBS into the eye of the animal.</li>
		<li>Verify intravitreal placement by visualization of a greenish reflex in the eye. Refer to Figure 1F.</li>
		<li>After 10 s, withdraw the needle from the eye. Note any reflux.</li>
		<li>Remove the mouse from the platform, place 0.3% hypromellose or 0.2% w/w carbomer eye ointment on both eyes for corneal protection, and move to the recovery warming box.</li>
		<li>Do not leave the mouse unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return to the company of other animals until fully recovered.</li>
		<li>When the mouse is fully awake, return to the cage and add acetaminophen (200-300 mg/kg/day) medicated water bottle. Label the cage card with the date of IVT injection.</li>
		<li>Intravitreal injections are generally well tolerated. Clinical signs that may indicate pain and the need for removal from the study include periocular alopecia (indicative of self-trauma), corneal ulceration, weight loss, and hunched posture.</li>
	</ol>
	</li>
	<li>Alternative method for intravitreal injection 
	&#8203;NOTE: This procedure is performed using an operating microscope and a 33 G needle on a microsyringe.
	<ol>
		<li>Proptose the eye and hold it in position with a pair of forceps.</li>
		<li>Then apply carbomer eye gel 0.2 % w/w or 0.3% hypromellose eye gel and place a circular coverslip (7 mm diameter) over the eye.</li>
		<li>Mount a 33 G hypodermic needle on a 5 &#181;L Hamilton syringe and insert it approximately 2 mm circumferential to the corneal limbus with a ~45&#176; injection angle.</li>
		<li>Guide the needle bevel into the vitreous, stopping between the lens and the optic disc (from the relative viewpoint of the surgeon, this is above/covering the optic disc - approximately 1.5 mm from the site of insertion), and inject 2 &#181;L of Mtb (at 2.5 ug/&#181;L in PBS) slowly.</li>
		<li>Hold the needle in place briefly (to reduce the amount of reflux of injectate) and then remove it.</li>
		<li>Post-injection, reposit the globe by releasing the forceps.&#160;A drop of 1% w/w chloramphenicol ointment can be administered to the eye at this time to provide additional protection from post-injection endophthalmitis.&#160;</li>
		<li>Post-injection, move to the recovery warming box as mentioned in steps 4.3.11-4.3.13.</li>
	</ol>
	</li>
</ol>
5. OCT imaging to detect and quantify uveitis
<ol>
	<li>Animal preparation
	<ol>
		<li>Anesthetize the mouse as described in steps 4.1.2-4.1.4</li>
		<li>Dilate the pupil with 1 drop of 2.5% phenylephrine. Use caution to avoid any excess droplets that might enter the nose or mouth. After 2-3 mins, dab off the excess liquid.</li>
		<li>Place 0.3% hypromellose or 0.2% w/w carbomer gel on the eye to prevent dryness while under anesthesia. This will also help to prevent cataract formation.</li>
		<li>Wrap the mouse in a layer of surgical gauze to maintain body warmth and place it on the animal cassette. Position the head with the bite bar.</li>
	</ol>
	</li>
	<li>Acquire the OCT images of the anterior and posterior chambers. 
	NOTE: If obtaining anterior and posterior chamber images, obtain the posterior chamber (PC) images first to prevent image degradation following cataract formation. Cataract formation can be prevented with frequent lubrication and applying 0.3% hypromellose or 0.2% w/w carbomer eye gel. For extended imaging (&#62;10 min), keeping the mouse warm (through the use of a heat pad) also helps.
	<ol>
		<li>After turning on the OCT imaging system, secure the correct imaging lens and adjust the reference arm position as needed.</li>
		<li>Open the imaging software, create the unique mouse ID and begin imaging per OCT manufacturer&#39;s protocol.</li>
		<li>Using the Free Run option with the fast scan protocol, position the eye with the optic nerve centered on posterior chamber images or the apex of the cornea on anterior chamber images. 
		NOTE: Table 1 contains imaging protocol parameters for two commercially available small animal imaging systems. Refer to the&#160;Table of Materials for product specifications.</li>
		<li>For posterior chamber imaging, bring the OCT close to the surface of the eye. Use caution to avoid bringing the surface of the lens in contact with the eye.</li>
		<li>Once the eye is correctly positioned, stop the fast scan and select the volume scan protocol, and activate the scan with the Aim option.</li>
		<li>For posterior segment images, adjust untilthe optic nerve is centered in the Horizontal B-Scan Alignment image and that the retina is aligned with the Vertical Alignment axis</li>
		<li>For anterior segment images, adjust the position to center the apex of the cornea in both the Horizontal B-Scan Alignment image and Vertical Alignment B-Scan Alignment image. The presence of a reflection artifact in both images will confirm proper alignment. Then shift the horizontal image enough to remove the reflection artifact. Refer to Figure 2.</li>
		<li>Click on Snapshot to capture the volume scan image and then click on Save.</li>
		<li>Next, obtain the averaged central line scan. Open the scan protocol and click on Aim followed by Snapshot. Right-click on the same panel and then click on Average.</li>
		<li>Repeat steps 5.2.1-5.2.9 for each eye with both lenses.</li>
		<li>After all images are collected, remove the mouse from the cassette and provide corneal protection during recovery as listed in steps 4.3.11-4.3.13.</li>
	</ol>
	</li>
</ol>
6. Scoring inflammation by OCT
<ol>
	<li>Score the OCT images with the help of graders who are masked to the treatment condition. 
	NOTE: For PMU in the mouse model, the scoring system provided in Table 2 is recommended.</li>
	<li>If both anterior chamber (AC) and posterior chamber (PC) images were obtained, combine these scores to obtain the final score for each eye. 
	&#8203;NOTE: Anterior chamber inflammation resolves before posterior chamber inflammation.</li>
</ol>
7. Scoring inflammation by post-mortem histology
<ol>
	<li>At the end of the experiment, collect individual eyes by enucleation, fix in 4% formaldehyde overnight, and proceed for paraffin embedding, sectioning, and H&#38;E staining33. 
	NOTE: Multiple 4-8 &#181;m sections along the pupillary-optic nerve axis are recommended. 
	NOTE: Three sections per eye are scored by a masked grader using the scoring system provided in Table 3, and the average score of the three sections is reported as the final histology inflammation score.</li>
</ol>

The authors have no financial conflicts to disclose.

Animal models of uveitis have been instrumental in understanding the mechanisms of ocular inflammation and homeostasis as well as enabling preclinical evaluation of medical and surgical therapies for patients with uveitis37. Both rabbit and rat variants of the PMU model have demonstrated their value in preclinical therapy via proof of concept studies38,39,40. Due to the availability of a diverse range of transgenic strains in mice, establishing the mouse PMU model system now permits more detailed mechanistic studies to identify specific cell types, pathways, and genes that contribute to the pathology of this disease.
Animal models of uveitis can demonstrate animal to animal variability in the incidence and intensity of inflammation41. In the C57BL/6 mouse strain, PMU is reliably generated using the protocol outlined here. Strain-specific variations in uveitis course and intensity have been reported for both EAU and EIU42,43. While strain-specific impacts on severity and course of PMU have not been measured experimentally, this model has been used in wild-type C57BL/6J as well as in albino mice (B6(Cg)-Tyrc-2J/J) and produced similar inflammatory responses. In generating the PMU model, controlling the considerations listed below can help new researchers limit variability and produce the most consistent and reproducible uveitis.
Ensure consistency in the subcutaneous injections: 
To provide a consistent subcutaneous injection, ensure that all air bubbles are removed from the emulsion. Considerations include a short centrifuge (30 s at 400 x g) of the premade emulsion prior to loading the syringe. This will remove air trapped in the emulsion. Also, when loading the syringe, periodically invert (tip-up) and tap the syringe to remove any air bubbles. While injecting, do not place the syringe too deep in order to avoid intramuscular injection. Conversely, a shallow (intradermal) injection can result in erosion of the emulsion through the skin. Remember to pause briefly before removing the syringe from the injection site to ensure complete injection of the thick viscous emulsion and to prevent reflux from the skin.
Seven days after placing the subcutaneous injection, confirm the presence of palpable nodules on either side of the hind legs. If no nodules can be identified, it is possible that air was injected rather than emulsion. In this case, acute inflammation may not be as robust, and chronic inflammation may not develop.
Prevent the development of infectious endophthalmitis: 
Bacterial or fungal endophthalmitis will generate a confounding variable if not prevented44. In order to prevent bacterial endophthalmitis, always practice good aseptic technique when making the intravitreal suspension, handling, and cleaning all reusable tools that will come in contact with the eye. Using sterile single-use items, autoclaving, or cleaning with 95% alcohol washes or wipes is important. Appropriate use of betadine applied to the ocular surface, lids, and periocular fur will also help prevent endophthalmitis45. It is straightforward to recognize an eye with infection as the ocular structures will be obliterated by extreme inflammation during the post-injection course. This is not typical for PMU. The presence of intraocular bleeding can also suggest endophthalmitis or trauma from the injection. In such cases, exclude these animals from the study.
Ensure consistency in the intravitreal injection: 
The intravitreal injection is a critical step in the induction of reliable and reproducible inflammation in PMU. Providing a consistent amount of Mtb suspension with each injection, avoiding trauma, and preventing reflux of the suspension are all factors that should be considered when performing the injections. To ensure a consistent suspension, vortex the stock suspension thoroughly upon thawing and before loading it in the syringe. Since this Mtb extract used does not form a solution, the suspension can undergo sedimentation over time. To ensure uniform concentration of the Mtb extract in each injection, use or expel and reload the syringe within 15 minutes of loading. Phenylephrine is used for dilation to provide a larger field of view to the posterior eye and reduce the risk of trauma to the eye during the injection. This drop generates natural lid retraction and slight proptosis of the globe, allowing good visualization of area 1-2 mm posterior to the limbus without the need to grasp the eye with forceps. Using forceps to restrain the eye could cause potential trauma and transiently increase intraocular pressure and the risk of reflux of the Mtb suspension. Trauma can also be caused by attempting to inject too much volume into the eye. The injection volume is limited to 2 &#181;L to prevent significant and prolonged elevation of intraocular pressure and trauma to the eye. Additionally, younger animals will have eyes that are smaller than adult mice. Typically 6-8 week mice (20-25 g) provide a uniform eye size and ensure greater consistency in the inflammation following injection of Mtb. A higher frequency of post-injection reflux of the mycobacterial suspension was observed in smaller mice. This, in turn, leads to a less-than-expected acute inflammation. A dilute fluorescein solution is used to provide the novice injector visual feedback on the success of their injection technique. Dilation at the time of injection will allow for direct visualization of the injected material in the vitreous cavity and the opportunity to note any evidence of lens trauma.&#160;In the case of lens trauma, it can cause a change in lens clarity that will cause a cataract which can be visualized on OCT. In the case of ocular trauma, the eyes need to be excluded from the study due to the possibility of lens-induced uveitis46. We recommend pausing for 10 s before removing the syringe from the eye to allow dispersion of the Mtb suspension within the eye and decrease reflux.
The PMU model can be modified to change the intensity of acute inflammation by varying the concentration of the Mtb in the intravitreal injection. Different dosages ranging from 2.5 &#181;g/&#181;L to 15 &#181;g/&#181;L have been tested previously in our lab. However, doses higher than 10 &#181;g/&#181;L were found to cause severe eye damage, including spontaneous lens rupture, severe corneal edema and scarring, and hyphema. This degree of severity is not typical in human patients with post-infectious uveitis, and therefore, these concentrations are not recommended. A 5 &#181;g/&#181;L dose was found to reliably produce mild to moderate acute inflammation and mild chronic uveitis; the 10 &#181;g/&#181;L dose produces a reliably moderate to severe acute disease and more notable chronic disease. Thus, varying the intravitreal concentration can provide alternative disease severities for use as needed based on the experimental question. Controls should be selected to ensure results are due to the response to mTB and not trauma associated with the subcutaneous or intravitreal injections. In the sham injection controls, PBS can be used in place of the mTB extract. For comparisons to unexposed animals, true naive samples should be considered as fellow eyes are not always equivalent.
Due to the small size of the mouse eye, OCT can be a more sensitive assay to detect inflammation in the anterior chamber than direct visualization or microscopic bright field photography. Prior work with PMU in rats25 determined that more cells can be detected by histology than by OCT but that there is a good correlation between the two modalities. OCT has the added advantage that it can be used to monitor the inflammation longitudinally in the same animal. Other major mouse models of uveitis, such as EAU and EIU, have also employed OCT for quantitative analysis12,47,48. In the PMU model of mice, anterior chamber cells are only visible on OCT and cannot be seen on clinical exams unless a large hypopyon is present. Vitreous inflammation (vitritis) can be observed with color fundus imaging, but detecting quantitative change is possible only with OCT imaging. Other aspects of the model, such as retinal vascular inflammation and retinal damage, can be easily identified with either OCT and microscopic brightfield fundus photography.
When using OCT, it is important to consider how localized imaging can be impacted by regional differences in the degree of inflammation. Prior reports have identified an uneven distribution of cells in the anterior chamber of humans, with more cells located inferiorly49. In mice, a similar predisposition is common. Thus, vertical or radial scans through the AC will help ensure images that capture the range of inflammation. Additionally, performing imaging in the same place will also provide consistency to images collected in the same eye longitudinally. To obtain images in the same part of the eye, use stable landmarks and a systematic approach. For anterior chamber images, the image is centered immediately adjacent to the apex of the cornea and oriented vertically so that the presence of a hypopyon can be detected in the inferior angle. For posterior segment images, the image is centered on the optic nerve. It is recommended to consider using at least 3 line scans for scoring to ensure regional variability is captured. In cases where inflammation is restricted to peripheral locations, acquiring volume scans can be helpful. The collection of volume scans can also help capture regional variations but will increase data storage requirements.
Other in vivo assays that can be used to characterize inflammation in the PMU mouse model include bioluminescence imaging13,35. Post-mortem assays like multi-parameter flow cytometric analysis can be performed to identify and quantify infiltrating immune cell type populations in the aqueous and posterior chamber of the eye12,26. In the PMU model, acute inflammation is characterized by an innate response with a predominant neutrophil infiltrate, followed by a chronic and persistent adaptive T cell dominant response that persists for over a month35. Other assays of immune function that can be performed on post-mortem tissues include ocular fluid cytokine analysis. Additionally, other downstream assays like mRNA sequencing and immunofluorescence imaging can be used to assess gene and protein expression patterns of retinal immune cell populations in uveitis50,51.
The PMU model can be replicated in other rodent systems using adaptations appropriate for the different species. PMU model has been previously used in rats and rabbits38,39,40. In rats, acute panuveitis develops following intravitreal injection that resolves spontaneously over 14 days without developing signs of chronic inflammation by histology24. In rabbits, induction of uveitis utilizes two rounds of subcutaneous injection prior to intravitreal injection but also generates a robust panuveitis. One of the advantages of using the mouse model is the ready availability of numerous transgenic and knockout strains that can help understand the basic mechanism of uveitis52. All rodent models can be used for preclinical therapy testing if the agent is administered systemically or as a topical drop. However, due to their larger size, rat and rabbit eyes are better models for use in preclinical studies of implantable or local injection treatment options for uveitis.
In summary, this protocol provides researchers interested in studying the mechanisms of chronic ocular inflammation with a new tool that is not dependent on prior immunization with ocular antigens.

The term &#39;uveitis&#39; describes a heterogeneous set of conditions that all feature intraocular inflammation1. Animal models of uveitis are important for understanding disease mechanisms and for preclinical testing of new therapies. A number of animal models of uveitis have been established2. The two that have been studied most extensively are experimental autoimmune uveitis (or uveoretinitis; EAU) and endotoxin-induced uveitis (EIU). EAU is typically generated by immunization with ocular antigens or can occur spontaneously when central tolerance is disrupted in the absence of the AIRE gene3,4. Other variants of the model have since been developed5,6,7 to include different uveitogenic peptides; these have been reviewed extensively8,9,10. EAU is the primary model for forms of T cell-dependent autoimmune uveitis such as Vogt-Koyanagi-Harada disease and birdshot chorioretinitis in humans. EIU is generated by systemic or local injection of bacterial lipopolysaccharide (LPS)10,11. EIU has been used as a model of acute uveitis generated by activation of innate immune signaling pathways12. Both models have been instrumental to the current understanding of ocular immunology, but neither are effective models for post-infectious chronic uveitis. Recently established in mice, the Primed Mycobacterial Uveitis (PMU) model now provides an approach to interrogate and evaluate clinical and cellular aspects of this form of uveitis13.
There is a high prevalence of mycobacterial infection worldwide, with over 10 million new cases and more than 1.4 million deaths reported by the World Health Organization in 201914. Extrapulmonary manifestation of active tuberculosis (TB) infection includes uveitis and is a well-recognized cause of infectious uveitis15,16. The manifestations of TB-associated uveitis are protean, which likely reflects multiple distinct mechanisms of disease to include direct ocular infection as well as less well-understood immune-mediated inflammation17,18,19. The proposed mechanisms for these post-infectious sequelae include a chronic inflammatory response stimulated by the persistence of a pauci-bacillary infection in the retinal pigment epithelium (RPE), a chronic inflammatory response stimulated by the presence of residual pathogen-associated molecular patterns (PAMPs) from a successfully cleared ocular infection, and inappropriate activation of the adaptive immune response against ocular antigens through a process of molecular mimicry or antigen spread caused by systemic TB infection20,21,22,23.
In order to gain a better mechanistic understanding of chronic post-infectious uveitis and study the role of mycobacterial antigens in the initiation of disease, the PMU model was developed for use in mice13,24. Accordingly, to elicit inflammation, the mouse first receives a subcutaneous injection of antigen from the heat-killed Mycobacterium tuberculosis H37Ra strain to mimic systemic infection, followed seven days later by intravitreal injection of the same antigen administered to the left or right eye to mimic local ocular infection. The intensity and duration of the ensuing uveitis are monitored by longitudinal in vivo Optical Coherence Tomography (OCT) and fundal imaging of the eye25. PMU is characterized by an acute, myeloid-dominant panuveitis that develops into a chronic T cell-dominant posterior uveitis with vitritis, perivascular retinal inflammation, and focal areas of outer retinal damage26. The presence of granulomatous inflammation in the posterior segment of the eye suggests that the PMU model can be used to study some forms of anterior (granulomatous and non-granulomatous) and intermediate uveitis, seen in patients with immunological evidence of past Mtb infection27. Additionally, the components of heat-killed Mtb used in the PMU model have been suggested to trigger immune responses underlying the aspects of recurrent uveitis in patients with ocular tuberculosis who respond to anti-tubercular therapy (ATT)28. Due to the differences in disease initiation and inflammatory course when compared to EAU and EIU, PMU represents a new animal model of uveitis that is not dependent on immunization with ocular antigens and may help elucidate mechanisms of disease in patients with chronic uveitis. This protocol outlines the methods for generating PMU, monitoring the clinical course of inflammation, and collecting ocular samples for post-mortem analysis with flow cytometry.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AK-FLUOR</td>
			<td>Akorn Pharmaceuticals, IL, USA</td>
			<td></td>
			<td>10% Fluorescein sodium 100 mg/mL in 5 mL vial</td>
		</tr>
		<tr>
			<td>AnaSed</td>
			<td>Akorn Animal Health, IL, USA</td>
			<td>NDC 59399-110-20</td>
			<td>Xylazine 20 mg/mL</td>
		</tr>
		<tr>
			<td>Betadine 5% Sterile Ophthalmic Prep Solution</td>
			<td>Alcon, TX, USA</td>
			<td>8007-1</td>
			<td></td>
		</tr>
		<tr>
			<td>B-D Precision Glide Needles -25 G</td>
			<td>Becton, Dickinson and Company, NJ, USA</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>B-D Precision Glide needle -30-G</td>
			<td>Becton, Dickinson and Company, NJ, USA</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>Bond MAX, Bond Rx</td>
			<td>Leica Biosystems, IL,USA</td>
			<td></td>
			<td>Automated IHC staining system</td>
		</tr>
		<tr>
			<td>Chloramphenicol ointment</td>
			<td>Martindale Pharma, Romford, UK</td>
			<td></td>
			<td>1% w/w Chloramphenicol</td>
		</tr>
		<tr>
			<td>EG1150H</td>
			<td>Leica Biosystems, IL,USA</td>
			<td></td>
			<td>Tissue Embedding</td>
		</tr>
		<tr>
			<td>Envisu R2300</td>
			<td>Bioptigen/Leica</td>
			<td></td>
			<td>OCT Machine</td>
		</tr>
		<tr>
			<td>Freund&#39;s Incomplete Adjuvant</td>
			<td>BD Difco, NJ, USA</td>
			<td>263910</td>
			<td></td>
		</tr>
		<tr>
			<td>GenTeal lubricant eye ointment</td>
			<td>Alcon, TX, USA</td>
			<td>---</td>
			<td></td>
		</tr>
		<tr>
			<td>GenTeal lubricant eye gel</td>
			<td>Alcon, TX, USA</td>
			<td>---</td>
			<td></td>
		</tr>
		<tr>
			<td>H37Ra lyophilized Mycobacteria extract</td>
			<td>BD Difco, NJ, USA</td>
			<td>231141</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton RN Needle (33/12/2)S</td>
			<td>Hamilton, Reno, NV</td>
			<td>7803-05(33/12/2)</td>
			<td>33 G</td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton, Reno, NV</td>
			<td>CAL7633-01</td>
			<td>5 &#181;L</td>
		</tr>
		<tr>
			<td>Insulin needle</td>
			<td>Exel International, USA</td>
			<td>26029</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketaset</td>
			<td>Zoetis, USA</td>
			<td>377341</td>
			<td>Ketamine HCL 100 mg/mL</td>
		</tr>
		<tr>
			<td>Microinjection Syringe Pump and Micro4Controller</td>
			<td>World Precision Instruments, FL, USA</td>
			<td>UMP3</td>
			<td></td>
		</tr>
		<tr>
			<td>Micron IV</td>
			<td>Phoenix Research Laboratories, Pleasanton, CA</td>
			<td></td>
			<td>Alternative Imaging/OCT Machine</td>
		</tr>
		<tr>
			<td>Nanofil 10 &#181;L syringe</td>
			<td>World Precision Instruments, FL, USA</td>
			<td>NANOFIL</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanofil Intraocular Injection Kit</td>
			<td>World Precision Instruments, FL, USA</td>
			<td>IO-KIT</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus SZX10</td>
			<td>Olympus</td>
			<td></td>
			<td>Dissection scope</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylephrine Hydrochloride Ophthalmic Solution USP 2.5% Sterile 15 mL</td>
			<td>Akorn Pharmaceuticals, IL, USA</td>
			<td>17478020115</td>
			<td></td>
		</tr>
		<tr>
			<td>RM2255</td>
			<td>Leica Biosystems, IL,USA</td>
			<td></td>
			<td>Tissue Sectioning</td>
		</tr>
		<tr>
			<td>TB Syringe</td>
			<td>Becton, Dickinson and Company, NJ, USA</td>
			<td>309602</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Tetracaine 0.5%</td>
			<td>Alcon, TX, USA</td>
			<td>1041544</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Tek VIP series</td>
			<td>Sakura Finetek USA, Inc.,CA.</td>
			<td></td>
			<td>Histology Tissue Processing</td>
		</tr>
		<tr>
			<td>Tropicamide 1%</td>
			<td>Chauvin Pharmaceuticals, Romford, UK</td>
			<td></td>
			<td>Minims</td>
		</tr>
		<tr>
			<td>Tylenol</td>
			<td>Johnson &#38; Johnson Consumer Inc, PA, USA</td>
			<td>NDC 50580-614-01</td>
			<td>Acetaminophen</td>
		</tr>
		<tr>
			<td>Viscotears</td>
			<td>Novartis Pharmaceuticals, Camberley, UK</td>
			<td></td>
			<td>Carbomer eye gel 0.2% w/w</td>
		</tr>
	</tbody>
</table>

primed mycobacterial uveitis (pmu) as a model for post-infectious uveitis

The term 'uveitis' describes a heterogeneous set of conditions that all feature intraocular inflammation. Broadly, uveitis is defined by etiology: infection or autoimmunity. Infectious uveitis requires treatment with the appropriate antimicrobial agents, while autoimmune uveitis requires treatment with corticosteroids or other immunosuppressive agents. Post-infectious uveitis is a form of chronic uveitis that requires corticosteroids to control immune sequela following the initial infection. Uveitis associated with Mycobacterium tuberculosis (Mtb) infection is a well-recognized form of post-infectious uveitis, but the mechanisms of disease are not fully understood. To understand the role mycobacterial antigens and innate ligands play in stimulating chronic ocular inflammation following mTB infection, the model Primed Mycobacterial Uveitis (PMU) was developed for use in mice. This manuscript outlines the methods for generating PMU and monitoring the clinical course of inflammation using color fundus and optical coherence tomography (OCT) imaging. PMU is induced by immunization with heat-killed mycobacterial extract followed by intravitreal injection of the same extract into one eye seven days later. Ocular inflammation is monitored longitudinally using in vivo imaging and followed by sample collection for a wide range of assays, including histology, flow cytometry, cytokine analysis, qPCR, or mRNA sequencing. The mouse model of PMU is a useful new tool for studying the ocular responses to mTB, the mechanism of chronic uveitis, and for preclinical effectiveness tests of new anti-inflammatory therapies.

Watch this Scientific Journal Video about Primed Mycobacterial Uveitis PMU as a Model for Post-Infectious Uveitis at JoVE.com

Primed Mycobacterial Uveitis PMU as a Model for Post-Infectious Uveitis

This protocol outlines the steps for inducing&#160;Primed Mycobacterial Uveitis (PMU) in mice. This method outlines the steps to help produce reliable and robust ocular inflammation in the mouse model system. Using this protocol, we generated uveitic eyes and uninflamed fellow eyes from single animals for further evaluation with immunologic, transcriptomic, and proteomic assays.

Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis

The term 'uveitis' describes a heterogeneous set of conditions that all feature intraocular inflammation. Broadly, uveitis is defined by etiology:  ...

primed-mycobacterial-uveitis-pmu-as-model-for-post-infectious

Research

JoVE Journal

Immunology and Infection

2.5K Views.  University of Washington. This protocol outlines the steps for inducing Primed Mycobacterial Uveitis (PMU) in mice. This method outlines the steps to help produce reliable and robust ocular inflammation in the mouse model system. Using this protocol, we generated uveitic eyes and uninflamed fellow eyes from single animals for further evaluation with immunologic, transcriptomic, and proteomic assays.

Video: Primed Mycobacterial Uveitis PMU as a Model for Post-Infectious Uveitis

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.
G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 &deg;C to 30 &deg;C) and infection studies can be conducted between 15 &deg;C to above 37 &deg;C12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014, measurement of bacterial survival15,16 and examination of the infection process17. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19.

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, gains a pathogenic advantage over in vitro cultured bacteria when first harvested from protozoan cells prior to infection of mammalian macrophages. This suggests that important virulence factors may not be properly expressed in vitro. We have developed a tractable system for priming L. pneumophila through its natural protozoan host Acanthamoeba castellanii prior to mammalian cell infection. The contribution of any virulence factor can be examined by comparing intracellular growth of a mutant strain to wild-type bacteria after protozoan priming. GFP-expressing wild-type and mutant L. pneumophila strains are used to infect protozoan monolayers in a priming step and allowed to reach late stages of intracellular growth. Fluorescent bacteria are then harvested from these infected cells and normalized by spectrophotometry to generate comparable numbers of bacteria for a subsequent infection into mammalian macrophages. For quantification, live bacteria are monitored after infection using fluorescence microscopy, flow cytometry, and by colony plating. This technique highlights and relies on the contribution of host cell-dependent gene expression by mimicking the environment that would be encountered in a natural acquisition route. This approach can be modified to accommodate any bacterium that uses an intermediary host as a means for gaining a pathogenic advantage.

This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

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 ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

Experimental Infection with &lt;em&gt;Listeria monocytogenes&lt;/em&gt; as a Model for Studying Host Interferon-&amp;#947; Responses

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...