Name: th17 inflammation model of oropharyngeal candidiasis in immunodeficient mice
Uploaded: 2015-02-18T15:00:00.000+00:00
Duration: 8 min 2 s
Description: Watch this Scientific Journal Video about Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice at JoVE.com

We thank Dr. Helene Bernstein for providing us with access to her flow cytometer. We also thank CFAR flow cytometry facility for the flow cytometry services. This project was in part supported by CTSC core utilization funding and STERIS corporation/University Hospitals-Division of Infectious diseases grant to PP.

NOTE: The experiments using mice were performed in accordance to the institutional animal welfare committee (IACUC) guidelines.
1. Reconstituting the Rag-1-/- Mice with In Vitro Cultured Th17 cells (Three days prior to Infection)
<ol>
	<li>For establishing Th17 cells, culture CD4+&#160;CD44low&#160;CD62Lhigh&#160;CD25-&#160;na&#239;ve T cells (3 x 104) in U-bottom 96 well plates alone, or co-culture them along with 3 x 104&#160;CD4+CD25+Foxp3+Tregs&#160;in the presence of soluble&#160;&#160;&#945;-CD3 (1 &#181;g/ml),&#160;&#945;-CD28 (2 &#181;g/ml) antibodies and antigen presenting cells, under Th17 conditions (polarized using IL-6 (25 ng/ml), TGF- &#946;&#160;(2 ng/ml),&#160;&#160;&#945;-IFN-&#947;&#160;(2&#160;&#181;g/ml) and&#160;&#945;&#160;-IL-4 (2&#160;&#181;g/ml)) for three to four days.&#160; 
	NOTE: Na&#239;ve cells and Tregs were sorted using fluorescence activated cell sorting (FACS) and magnetic cell isolation procedures and cultured as described previously19-22.</li>
	<li>On day 4, after resuspending the Th17 cells using the pipette, collect them from cultures, and centrifuge them under sterile conditions.
	<ol>
		<li>Take a small aliquot of these cells to examine their viability and for phenotyping. Resuspend them in 500 &#181;l of RPMI medium, add propidium iodide (200 ng/ml) and assess their viability immediately by flow cytometry. Perform phenotyping by flow cytometry assessment of IL-17A production after restimulation (see section 6.3).</li>
	</ol>
	</li>
	<li>Centrifuge and wash the main bulk of the cells in sterile PBS at 480 x g for 6 min at 4&#176; C in all the steps unless otherwise specified.</li>
	<li>Use these cells for adoptive transfer into 6-8 week old CD45.2 Rag1-/- immunodeficient mice.
	<ol>
		<li>Resuspend the cells at 1 x 107 cells/ml cell density using cold sterile PBS.</li>
		<li>Inject 100 &#956;l of the cells by intraperitoneal injection, using 25 G needles on 1 ml tuberculin syringes, such that one mouse receives 1 x 106 cells. Some mice will receive PBS or Th17 cells only, and other mice will receive Th17 cells that were co-cultured with Treg cells. 
		NOTE: Perform all the steps aseptically.</li>
	</ol>
	</li>
</ol>
2. Growing C. albicans for infection (one day prior to Infection)
<ol>
	<li>Before inoculation, disperse five colonies of the CAF2-1 C. albicans laboratory strain in 100 &#181;l of sterile PBS suspension, and add the suspension to the sterile broth.</li>
	<li>Inoculate 5 colonies of the C. albicans in 50 ml of the Yeast nitrogen base (YNB without amino acids)/Peptone/Dextrose broth medium and incubate in a shaker incubator at 30 &#176;C for 15&#8211;18 hr at 130 rpm.</li>
	<li>Monitor the broth for cloudiness, as that is an indication for the growth of the fungus.</li>
</ol>
3. Candida Harvesting and Counting Procedure (On the Day of Infection)
<ol>
	<li>Collect 10 ml of Candida broth in 15 ml tubes. For larger volumes, collect Candida in 50 ml tubes.</li>
	<li>Centrifuge the blastospores at 1900 x g for 5 min at RT&#160;in all the steps unless otherwise specified. Pellet the blastospores by centrifugation, followed by the removal of the supernatant.</li>
	<li>If there is more than one tube, pool the Candida blastospores from multiple tubes, adding sterile PBS to the pellets and resuspend them in 10 ml of PBS for counting.</li>
	<li>Take 20 &#956;l of blastospores in 1.5 ml microcentrifuge tubes, add 20 &#956;l of the 2X paraformaldehyde and incubate at RT for 15-20 min.&#160;Count the fixed blastospores using the hemocytometer under the microscope. 
	NOTE: Paraformaldehyde is carcinogenic. Open the undiluted solution only in the fume hood.</li>
	<li>In the meantime, repeat washing the blastospores at least twice, by centrifuging them in PBS, and pelleting the blastospores . Keep the pellets in 15 ml tubes at RT, in the ABSL1 hood, ready for infection.</li>
	<li>Leave some sterile PBS at RT for resuspending the blastospores for infection.</li>
	<li>After counting, add sterile PBS to the blastospore pellet to adjust the yeast blastospore cell numbers to 2 x108 of yeast cells/ml. This is the blastospore suspension that will be used to infect the mice. 
	NOTE: Perform all the steps aseptically.</li>
</ol>
4. Mice Infection (3-5 Days after Cell Transfer)
NOTE: The basic infection procedure is performed as described previously19,23-25.
<ol>
	<li>Weigh the Rag-1-/- mice that received PBS or the cells three days earlier.</li>
	<li>Calculate the dose of anesthetic agents using their body weight. Anesthetize the mice by administering ketamine/xylazine mixture (16.1 mg/ml and 1.6 mg/ml), using 25 G needles on 1 ml tuberculin syringes, by intraperitoneal injection.
	<ol>
		<li>Administer 50 &#181;l per 10 grams of body weight. (i.e., 0.8 mg of ketamine and 0.08 mg of xylazine/10 g of body weight or 80 mg of ketamine and 8 mg of xylazine per kg of body weight respectively). Observe for toe pinch response every 15 min after anesthesia. 
		NOTE: This dosage will induce 60-90 min of anesthesia, which is enough for the infection procedure.</li>
		<li>Apply the ophthalmic lubricant ointment in the eyes of the mice to prevent the corneas from drying out. As the eye lubricant may dry out, repeat ophthalmic lubrication every 45 min.</li>
	</ol>
	</li>
	<li>Inject 1 ml of saline (0.9% NaCl) subcutaneously on the back adjacent to the forelimb, to help rehydration of the mice during anesthesia.</li>
	<li>Obtain a new, clean cage. Follow the infection procedure one mouse at a time, placing each mouse into the new cage once infected. Perform the PBS/sham infection first,and then proceed to the Candida infection groups.</li>
	<li>Pick up the anesthetized mouse and open the mouth wide to reveal the base of the tongue.
	<ol>
		<li>Place a 3 mm diameter cotton ball saturated with 50 &#181;l of PBS or blastospore suspension, sublingually in the oral cavity for 90 min. Flip the mice every 15 min front and back to prevent lung congestion, ensuring that the cotton balls don&#39;t move.</li>
	</ol>
	</li>
	<li>Set up a timer for every mouse to ensure 90 min of Sham or Candida inoculation in the oral cavity.</li>
	<li>Keep them in a cage under the heat lamp (4 feet away), and each mouse on heat gel pads.</li>
	<li>Make sure that the tongue is withdrawn inside and away from the teeth, to avoid teeth lacerating the tongue.</li>
	<li>At the end of 90 min, remove the cotton ball from the mouth of the mouse. Watch for any mouse that may recover from anesthesia sooner than 75 minutes. In such a case, anesthetize them again with ketamine (40 mg/kg) only.</li>
</ol>
5. Post Procedure Monitoring (during the 90 min Anesthesia)
<ol>
	<li>Use heat gel pads during the 90 min anesthesia.
	<ol>
		<li>To maintain the body temperature and to prevent suffocation, do not allow mice to recover on the regular corn-cob mouse bedding.</li>
	</ol>
	</li>
	<li>After the recovery from anesthesia, administer an additional 1 ml of sterile 0.9% NaCl subcutaneously in two locations on the back.</li>
	<li>Keep up to five PBS (sham) inoculated mice per cage. House only 1-2 infected mice per cage.</li>
	<li>Fill out the procedure card with initials every day after the procedure to note down the changes in the body weight, grooming habits and overall health.</li>
</ol>
6. Assessing the Inflammation
6.1) Weight loss
<ol>
	<li>Weigh the mice every day during the infection, starting on the day prior to infection procedure. 
	NOTE: Typically, the immunodeficient mice injected with Th17 cells and with no Tregs succumb to 20-30% weight loss due to increased fungal burden19 and exacerbated inflammation.</li>
</ol>
6.2) Histology scoring of the tongue
<ol>
	<li>Sacrifice the mice by CO2 asphyxiation followed by cervical dislocation.</li>
	<li>Open the jaws and dissect out the tongue with scissors and forceps. Use blunt forceps to hold the tongue, and using the scissors reach out to the back of the mouth to incise the back end of the tongue.</li>
	<li>For immunocytochemical hematoxylin and eosin (H&#38;E) staining of the tongue tissues, rinse the tongue tissues with ice cold PBS. Add 5 ml of 10% formalin for 2-3 tongues and fix them O/N in 15 ml tubes.
	<ol>
		<li>The next day, remove the formalin and immerse the tissues in 5 ml of 70% ethanol to prevent hyper-fixation. Send them out to commercial service to continue with paraffin embedding, sectioning and staining of paraffin sections. 
		NOTE: Commercial histology service is used to perform these steps.</li>
	</ol>
	</li>
	<li>Once the slides are back from the commercial histology service, grade the inflammation by observing the tissue sections under a light microscope.
	<ol>
		<li>Score from 0 to 5, with 0 being no inflammation, and 5 being the most severe inflammation. Score the inflammation using Table 1.</li>
	</ol>
	</li>
</ol>
6.3) Cytokine production by Th17 cells
NOTE: Excessive TNF-&#945; production is one of the readouts for excessive immunopathology. When mice are adoptively transferred with Th17 cells only, it causes immunopathology that is associated with excessive TNF-&#945; production in Th17 cells.
<ol>
	<li>Collect a single cell suspension from spleen, cervical lymph nodes, axillary lymphnodes, and tongue, using previously described methods19,26.</li>
	<li>Restimulate the cells with phorbol myristate acetate (PMA) (50 ng/ml) and Ionomycin (500 ng/ml) for 4 hr with Brefeldin A (10 &#181;g/ml) added in the last 2 hr. Wash the cells with PBS and fix them using a commercial Fixation/permeabilization kit according to manufacturer&#8217;s instructions.</li>
	<li>Perform intra cellular cytokine staining using the fluorochrome conjugated anti-TNF-&#945;, anti-IL-17A and ROR-&#947;t antibodies as described previously19.</li>
	<li>Briefly, resuspend the cells in the 1X permeabilization buffer with the cocktail of anti-TNF-&#945;, anti-IL-17A and ROR-&#947;t antibodies, each antibody at 2 - 3 &#181;g/ml final concentration.</li>
	<li>Incubate the cells for 1 hr at RT.</li>
	<li>After the incubation, wash the cells with 1X permeabilization buffer.</li>
	<li>Resuspend the cell pellet in 500 &#181;l of PBS with 0.5% bovine serum albumin for flow cytometry analysis.</li>
</ol>

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The authors declare that they have no competing financial interests.

This model is based on inducing oral C. albicans infection dependent Th17 inflammation. Because of the absence of Tregs, the Th17 cell induced inflammation is unrestrained and leads to poorly resolved immunopathology. In vitro derived na&#239;ve CD4 cells polarized as Th17 cells were used for the adoptive transfer. 40 - 50% of the cultured CD4+ cells show detectable IL-17A expression around day 3 (Th17 cells), and therefore were used for the injection in mice. The major advantage of the model...

Oral infections and inflammation have been related to cancer and cardiovascular diseases, and have dramatic impact on overall human health2,3. Opportunistic infections and inflammation caused by C. albicans are associated with primary immunodeficiencies (PID)4,5, inflammatory disorders such as periodontitis 6,7, Sjogren&#8217;s syndrome, and salivary gland disease8,9, as well as oral squamous cell carcinoma 10-12. C. albicans is a dimorphic commensal fungus that colonizes the mouths of 60% of healthy humans asymptomatically, yet it is the most common fungal pathogen causing infections when the host defense is weakened13-15. It causes recurring and chronic infections and inflammation in patients with AIDS and PID, and also in other immunocompromised individuals. As a commensal, its colonization load is associated with the change in the diversity of the overall oral microbiome16. As a pathogen it causes several forms of oropharyngeal candidiasis such as acute pseudomembranous, acute atrophic, chronic atrophic, chronic hypertrophic/hyperplastic, and angular cheilitis. 
Protection against C. albicans is determined not only by host immune resistance, but also by the ability to appropriately control Candida-induced immunopathology. Although commensals such as C. albicans contribute to modulation and exacerbation of other oral inflammatory conditions, the mechanisms by which dysbiosis occur during opportunistic infections are unclear. Besides the known role of adaptive Th17 cells in memory response to C. albicans17, their role in initiation and perpetuation of inflammation pathology during chronic infections remain unclear. Furthermore, oral inflammatory diseases such as Sjogren&#8217;s syndrome and periodontitis are associated with Th17 mediated pathology. Interestingly, these diseases are also strongly associated with frequent OPC. However, the interactions among Th17 cells, oral immunopathology of OPC and other oral inflammatory diseases are unstudied. 
Although mouse models of primary and secondary infection of oral candidiasis are available, a mouse model to study Candida infection associated Th17 inflammation, especially in the context of immunodeficiency is unavailable. This study presents a method for establishing oral Th17 inflammation associated with oral Candida infection in mice. Candida infection in mice is characterized by fungal lesions, inflammation in the tongue, decreased food intake, weight loss and eventually a moribund state. Oral pathology resembles chronic candidal infection lesions, as well as epithelial dysplasia in mouse oral cancer models12,18.

<table><tbody><tr><td>CAF-2</td><td>University of Pittsburgh (Sarah Gaffen)</td><td>-</td><td>Candida culture</td></tr><tr><td>U-bottom 96 well plates&amp;nbsp;</td><td>Fisher</td><td>055588</td><td>Used for cell culture</td></tr><tr><td>a-CD3&amp;nbsp;</td><td>eBiosciences</td><td>16-0031-85</td><td>Polarization of cells to Th17 conditions</td></tr><tr><td>a-CD28&amp;nbsp;</td><td>eBiosciences</td><td>16-0281-85</td><td>Polarization of cells to Th17 conditions</td></tr><tr><td>m-IL-6</td><td>Bio Basic</td><td>RC232</td><td>Polarization of cells to Th17 conditions</td></tr><tr><td>h-TGF-b&amp;nbsp;</td><td>R&amp;amp;D</td><td>240-B</td><td>Polarization of cells to Th17 conditions</td></tr><tr><td>a-IFN-g&amp;nbsp;</td><td>eBiosciences</td><td>16-7311-85</td><td>Polarization of cells to Th17 conditions</td></tr><tr><td>a-IL-4&amp;nbsp;</td><td>eBiosciences</td><td>16-7041-85</td><td>Polarization of cells to Th17 conditions</td></tr><tr><td>&amp;alpha;-IL17A ef660</td><td>eBiosciences</td><td>50-7177-82</td><td>Used for cell culture - 1:50</td></tr><tr><td>&amp;alpha;-TNFa-PE-Cy7</td><td>eBiosciences</td><td>25-7423-41</td><td>Used for cell culture - 1:100</td></tr><tr><td>&amp;alpha;-RORgt PE&amp;nbsp;</td><td>eBiosciences</td><td>12-6981-82</td><td>Used for cell culture - 1:50</td></tr><tr><td>YNB w/o amino acids)/Peptone/Dextrose broth medium&amp;nbsp;</td><td>Bio Basic</td><td>S507.SIZE</td><td>Mediium for candida growth</td></tr><tr><td>Shaker incubator&amp;nbsp;</td><td>New Brunswick Scientific</td><td>Innova 4300</td><td>Incubation growth for candida</td></tr><tr><td>15 ml tubes</td><td>Bio Basic</td><td>BT888-SY</td><td>Used for cell culture and candida growth</td></tr><tr><td>50 ml tubes</td><td>Bio Basic</td><td>CT 788-YS</td><td>Used for cell culture and candida growth</td></tr><tr><td>Table top Centrifuge</td><td>VWR International LLC</td><td>82017-654</td><td>For pelleting candida -900 g</td></tr><tr><td>Allegra Centrifuge</td><td>Beckman Coulter</td><td>392302</td><td>Cell culture - 480 g</td></tr><tr><td>1.5 ml eppendorf tubes</td><td>Bio Basic</td><td>BT620-NS</td><td>Preparing final concentration of candida</td></tr><tr><td>2x paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Fixing candida for count</td></tr><tr><td>Hemocytometer</td><td>VWR International LLC</td><td>15170-172</td><td>Counting cells</td></tr><tr><td>PBS - Phosphate Buffered Saline</td><td>Bio Basic</td><td>PD8117</td><td>Preparation of buffers</td></tr><tr><td>Ketamine/xylazine&amp;nbsp;</td><td>Case Western Reserve University - Animal Resource Center</td><td>-</td><td>Obtained from ARC approved protocol for anesthesia</td></tr><tr><td>Ophthalmic lubricant ointment&amp;nbsp;</td><td>Allergan</td><td>-</td><td>Eye ointment for animals to prevent dryness</td></tr><tr><td>Saline (0.9% NaCl)</td><td>G Biosciences</td><td>786-561</td><td>Administerd to animals to prevent dehydration</td></tr><tr><td>3-mm-diameter cotton wool ball&amp;nbsp;</td><td>VWR International LLC</td><td>BP7603</td><td>3 mm balls for candida infection</td></tr><tr><td>Heat gel pads</td><td>Case Western Reserve University - Animal Resource Center</td><td>-</td><td>for maintaining the body temperaturre and fast recovery</td></tr><tr><td>Hematoxylin and Eosin staining</td><td>Histoserv - MD</td><td>-</td><td>Tissue histology</td></tr><tr><td>10% formalin&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>JC1111/MC</td><td>Tissue histology</td></tr><tr><td>70% ethanol&amp;nbsp;</td><td>VWR International LLC</td><td>97064-490</td><td>Sterilization purpose</td></tr><tr><td>PMA - Phorbol 12-myristate 13-acetate</td><td>Sigma-Aldrich</td><td>P1585-1MG</td><td>Restimulation of cells</td></tr><tr><td>Ionomycin</td><td>Life Technologies</td><td>124222 1mg</td><td>Restimulation of cells</td></tr><tr><td>Fixation permeabilization kit</td><td>eBiosciences</td><td>E16913-106</td><td>Fixing cells for Flow cytometry</td></tr><tr><td>Tuberculin syringes</td><td>BD Biosciences</td><td>309659</td><td>Mice injection</td></tr><tr><td>25 G x 3/8 needles</td><td>BD Biosciences</td><td>309626</td><td>Mice injection</td></tr><tr><td>Rag1 -/- mice</td><td>Jackson laboratories</td><td>Stock no: 002216</td><td>Recipient mice</td></tr><tr><td>CD45.1 congenic mice</td><td>Jackson laboratories</td><td>Stock no:002014&amp;nbsp;</td><td>Donor Th17 cells</td></tr><tr><td>CB17-SCID mice</td><td>Jackson laboratories</td><td>Stock no: 001303</td><td>Recipient mice</td></tr><tr><td>Balb/c mice</td><td>Jackson laboratories</td><td>Stock no: 000651</td><td>Donor Th17 cells</td></tr></tbody></table>

th17 inflammation model of oropharyngeal candidiasis in immunodeficient mice

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of infection-induced immunopathology. Although Th17 cells are implicated in antifungal defense, their role in immunopathology is unclear. This study presents a method for establishing oral Th17 immunopathology associated with oral candidal infection in immunodeficient mice. The method is based on reconstituting lymphopenic mice with in vitro cultured Th17 cells, followed by oral infection with Candida albicans (C. albicans). Results show that unrestrained Th17 cells result in inflammation and pathology, and is associated with several measurable read-outs including weight loss, pro-inflammatory cytokine production, tongue histopathology and mortality, showing that this model may be valuable in studying OPC immunopathology. Adoptive transfer of regulatory cells (Tregs) controls and reduces the inflammatory response, showing that this model can be used to test new strategies to counteract oral inflammation. This model may also be applicable in studying oral Th17 immunopathology in general in the context of other oral diseases.

In this model, both the Candida-infected mice and uninfected mice in Rag-1-/- immunodeficient background were adoptively transferred with Th17 cells 3-5 days prior to the infection. A total of 10-12 mice were used for the experiments, with 2 mice in each Sham infected groups, and 4-5 in each of the Candida infected groups. Naive cells were derived from congenic Thy1.1 or CD45.1 mice, so that injected Th17 cells were tracked in vivo using Thy1.1 or CD45.1 staining respectively (<stron...

Watch this Scientific Journal Video about Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice at JoVE.com

Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice

Although Candida infection models are available to study host immune resistance, a model to study T cell mediated immunopathology in the context of Candida infection is absent. Here we describe a method to establish Th17 immunopathology associated with oral Candida infection in immunodeficient mice.

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of ...

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9.9K Views.  Case Western Reserve University. Although Candida infection models are available to study host immune resistance, a model to study T cell mediated immunopathology in the context of Candida infection is absent. Here we describe a method to establish Th17 immunopathology associated with oral Candida infection in immunodeficient mice. 

Video: Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of otherwise healthy women during their reproductive years18,32-34. Predisposing factors include antibiotic usage, uncontrolled diabetes and disturbance in reproductive hormone levels due to pregnancy, oral contraceptives or hormone replacement therapies33,34. Recurrent VVC (RVVC), defined as three or more episodes per year, affects a separate 5 to 8% of women with no predisposing factors33.
An experimental mouse model of VVC has been established and used to study the pathogenesis and mucosal host response to Candida3,4,11,16,17,19,21,25,37. This model has also been employed to test potential antifungal therapies in vivo13,24. The model requires that the animals be maintained in a state of pseudoestrus for optimal Candida colonization/infection6,14,23. Under such conditions, inoculated animals will have detectable vaginal fungal burden for weeks to months. Past studies show an extremely high parallel between the animal model and human infection relative to immunological and physiological properties3,16,21. Differences, however, include a lack of Candida as normal vaginal flora and a neutral vaginal pH in the mice.
Here, we demonstrate a series of key methods in the mouse vaginitis model that include vaginal inoculation, rapid collection of vaginal specimens, assessment of vaginal fungal burden, and tissue preparations for cellular extraction/isolation. This is followed by representative results for constituents of vaginal lavage fluid, fungal burden, and draining lymph node leukocyte yields. With the use of anesthetics, lavage samples can be collected at multiple time points on the same mice for longitudinal evaluation of infection/colonization. Furthermore, this model requires no immunosuppressive agents to initiate infection, allowing immunological studies under defined host conditions. Finally, the model and each technique introduced here could potentially give rise to use of the methodologies to examine other infectious diseases of the lower female genital tract (bacterial, parasitic, viral) and respective local or systemic host defenses.

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Key techniques to be used in the evaluation of Candida vaginitis in an experimental animal model are described. The methods will allow rapid collection of vaginal specimens and lymphocytes from draining lumbar lymph nodes. These techniques could give rise to mouse models of other diseases in the female lower genital tract.

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of ...

Immunology and Infection

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

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 ...

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...

In vivo Function of Differential Subsets of Cutaneous Dendritic Cells to Induce Th17 Immunity in Intradermal Candida albicans Infection

The skin is the outermost barrier organ in the body, which contains several types of dendritic cells (DCs), a group of professional antigen-presenting cells. When the skin encounters invading pathogens, different cutaneous DCs initiate a distinct T cell immune response to protect the body. Among the invading pathogens, fungal infection specifically drives a protective interleukin-17-producing Th17 immune response. A protocol was developed to efficiently differentiate Th17 cells by intradermal Candida albicans infection to investigate a subset of cutaneous DCs responsible for inducing Th17 immunity. Flow cytometry and gene expression analyses revealed a prominent induction of Th17 immune response in skin-draining lymph nodes and infected skin. Using diphtheria toxin-induced DC subset-depleting mouse strains, CD301b+ dermal DCs were found to be responsible for mounting optimal Th17 differentiation in this model. Thus, this protocol provides a valuable method to study in vivo function of differential subsets of cutaneous DCs to determine Th17 immunity against deep skin fungal infection.

In vivo Function of Differential Subsets of Cutaneous Dendritic Cells to Induce Th17 Immunity in Intradermal Candida albicans Infection

Here, we demonstrate the in vivo function of cutaneous dendritic cell subsets in Th17 immunity of deep dermal Candida albicans infection.

The skin is the outermost barrier organ in the body, which contains several types of dendritic cells (DCs), a group of professional antigen-presenting ...

Effective aPDT Using Curcuminoids for Oral Candidiasis in Mice

Curcuminoid-Mediated Antimicrobial Photodynamic Therapy on a Murine Model of Oral Candidiasis

Antimicrobial Photodynamic Therapy (aPDT) has been extensively investigated in vitro, and preclinical animal models of infections are suitable for evaluating alternative treatments prior to clinical trials. This study describes the efficacy of aPDT in a murine model of oral candidiasis. Forty mice were immunosuppressed with subcutaneous injections of prednisolone, and their tongues were inoculated using an oral swab previously soaked in a C. albicans cell suspension. Tetracycline was administered via drinking water during the course of the experiment. Five days after fungal inoculation, mice were randomly distributed into eight groups; a ninth group of untreated uninfected mice was included as a negative control (n = 5). Three concentrations (20 &#181;M, 40 &#181;M, and 80 &#181;M) of a mixture of curcuminoids were tested with a blue LED light (89.2 mW/cm2; ~455 nm) and without light (C+L+ and C+L- groups, respectively). Light alone (C-L+), no treatment (C-L-), and animals without infection were evaluated as controls. Data were analyzed using Welch&#39;s ANOVA and Games-Howell tests (&#945; = 0.05). Oral candidiasis was established in all infected animals and visualized macroscopically through the presence of characteristic white patches or pseudomembranes on the dorsum of the tongues. Histopathological sections confirmed a large presence of yeast and filaments limited to the keratinized layer of the epithelium in the C-L- group, and the presence of fungal cells was visually decreased in the images obtained from mice subjected to aPDT with either 40 &#181;M or 80 &#181;M curcuminoids. aPDT mediated by 80 &#181;M curcuminoids promoted a 2.47 log10 reduction in colony counts in comparison to those in the C-L- group (p = 0.008). All other groups showed no statistically significant reduction in the number of colonies, including photosensitizer (C+L-) or light alone (C-L+) groups. Curcuminoid-mediated aPDT reduced the fungal load from the tongues of mice.

Author Spotlight: Exploring Photodynamic Therapy with Curcumin in a Murine Model for Oral Candidiasis

This protocol describes the application of antimicrobial Photodynamic Therapy (aPDT) in a murine model of oral candidiasis. aPDT was performed using a water-soluble mixture of curcuminoids and blue LED light.

Antimicrobial Photodynamic Therapy (aPDT) has been extensively investigated in vitro, and preclinical animal models of infections are suitable for ...

Establishing a Mouse Model for Candida albicans Biofilm-Related PJI

A Periprosthetic Joint Candida albicans Infection Model in Mouse

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and scientists. Generally, biofilms that can shield C. albicans from antibiotics and immune clearance are formed at the infection site. Surgery involving the removal of the infected implant, debridement, antimicrobial treatment, and reimplantation is the gold standard for the treatment of PJI. Thus, establishing animal PJI models is of great significance for the research and development of new drugs or therapeutics for PJI. In this study, a smooth nickel-titanium alloy wire, a widely used implant in orthopedic clinics, was inserted into the femoral joint of a C57BL/6 mouse before the C. albicans were inoculated into the articular cavity along the wire. After 14 days, mature and thick biofilms were observed on the surface of implants under a scanning electronic microscope (SEM). A significantly reduced bone trabecula was found in the H&amp;E staining of the infected joint specimens. To sum up, a mouse PJI model with the advantages of easy operation, high successful rate, high repeatability, and high clinical correlation was established. This is expected to be an important model for clinical studies of C. albicans biofilm-related PJI prevention.

Author Spotlight: Advancing Research on Candida albicans Biofilm-Associated Prosthetic Joint Infections 

Periprosthetic joint infection (PJI) caused by dangerous pathogens is common in clinical orthopedics. Existing animal models cannot accurately simulate the actual situation of PJI. Here, we established a Candida albicans biofilm-associated PJI mouse model to research and develop new therapeutics for PJI.

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and ...

Establishing a Mouse Model for Candida albicans-Associated Catheter-Related Infection

A Catheter-Related Candida albicans Infection Model in Mouse

Catheter-related infection (CRI) is a common nosocomial infection caused by candida albicans during catheter implantation. Typically, biofilms are formed on the outer surface of the catheter and lead to disseminated infections, which are fatal to patients. There are no effective prevention and treatment management in clinics. Therefore, it is urgent to establish an animal model of CRI for the preclinical screening of new strategies for its prevention and treatment. In this study, a polyethylene catheter, a widely used medical catheter, was inserted into the back of the BALB/c mice after hair removal. Candida albicans ATCC MYA-2876 (SC5314) expressing enhanced green fluorescent protein was subsequently inoculated on the skin's surface along the catheter. Intense fluorescence was observed on the surface of the catheter under a fluorescent microscope 3 days later. Mature and thick biofilms were found on the surface of the catheter via scanning electron microscopy. These results indicated the adhesion, colonization, and biofilm formation of candida albicans on the surface of the catheter. The hyperplasia of the epidermis and the infiltration of inflammatory cells in the skin specimens indicated the histopathological changes of the CRI-associated skin. To sum up, a mouse CRI model was successfully established. This model is expected to be helpful in the research and development of therapeutic management for candida albicans associated CRI.

Author Spotlight: Enhancing Candida albicans Detection in Catheter Infections Using Fluorescent Protein Tagging 

We establish a mouse model of C.albicans-associated catheter-related infection (CRI), in which biofilm forms on the catheter, and the interaction between C.albicans and host correlates well with the clinical CRI. This model helps screen therapies for C.albicans biofilm-associated CRI, laying a foundation for clinical transformation.

Catheter-related infection (CRI) is a common nosocomial infection caused by candida albicans during catheter implantation. Typically, biofilms are formed ...

Th17 Cell Differentiation from Naive CD4+ T-Cells Using Flow Cytometry

In Vitro Differentiation of Naive CD4+ T Cells into Pathogenic Th17 Cells in Mouse

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have been linked to a wide range of diseases in recent times, including multiple sclerosis (MS), rheumatoid arthritis, acute respiratory distress syndrome (ARDS), sepsis, and other autoimmune disorders. However, the currently known in vitro differentiation protocols have difficulty achieving high purity of pathogenic Th17 cells, with the induction efficiency often below 50%, which is a key challenge in in vitro experiments. In this protocol, we propose an enhanced in vitro culture and differentiation protocol for pathogenic Th17 cells, which is used to directly differentiate naive CD4+ T cells isolated from mouse spleens into pathogenic Th17 cells. This protocol provides detailed instructions on splenocyte isolation, purification of naive CD4+ T cells, and differentiation of pathogenic Th17 cells. Through this protocol, we can achieve a differentiation purity of approximately 90% for pathogenic Th17 cells, which meets the basic needs of many cellular experiments.

Author Spotlight: Achieving High-Purity In Vitro Differentiation of Th17 Cells Using Cytokine Concentration Modulation

This protocol describes the differentiation of na&#239;ve CD4+ T cells into pathogenic Th17 cells in vitro. Specifically, when combined with a multi-parameter flow cytometry-based approach, 90% purity of pathogenic Th17 cells can be obtained from na&#239;ve CD4+ T cells using this differentiation method.

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have ...

Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice

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Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Isolation and Th17 Differentiation of Naïve CD4 T Lymphocytes

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

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

In vivo Function of Differential Subsets of Cutaneous Dendritic Cells to Induce Th17 Immunity in Intradermal Candida albicans Infection

Curcuminoid-Mediated Antimicrobial Photodynamic Therapy on a Murine Model of Oral Candidiasis

A Periprosthetic Joint Candida albicans Infection Model in Mouse

A Catheter-Related Candida albicans Infection Model in Mouse

In Vitro Differentiation of Naive CD4⁺ T Cells into Pathogenic Th17 Cells in Mouse