U.S. Army Medical Research Institute of Infectious Diseases
Cisney, E. D., Fernandez, S., Hall, S. I., Krietz, G. A., Ulrich, R. G. Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines. J. Vis. Exp. (66), e3960, doi:10.3791/3960 (2012).
The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11.
Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-α knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.
1. NALT Collection and Culturing
2. Surgical Ablation of NALT
3. Preparing NALT for Histology and Assessing the Success of NALT Surgery
4. Collection of Biological Samples from Mice
4.3 Nasal secretions
4.4 Vaginal secretions
5. Antigen-specific antibodies or cytokines in the collected samples can be measured by ELISA or another quantitative method.
6. Representative Results
Figure 1 provides a general schematic of steps involved with processing the NALT-containing tissue for ex vivo analysis. In Figure 2 (A, B), the size of the palate is shown, as well as the location of the incision during ablation surgery (A), as indicated by the dotted line. The location of NALT are indicated by arrows in the premolar area on an excised hematoxylin-stained palate in Figure 2 (C), showing the parallel tissues.
Figure 3 presents steps of the NALT disruption surgery, showing exposure of the upper palate for access to NALT (A, B), ablation (C), and final cauterization of the incision (D). A typical H & E cross-section of the nasal sinus area surrounding the NALT before surgery is shown in Figure 3E, while an image of NALT disruption by the microcurette directly after surgery appears in Figure 3F. Allowing sufficient time for recuperation from surgery, the incisions should be closed and the nasal cavity devoid of NALT (Figure 3G).
Typical experimental results obtained by using these techniques are shown in Figure 4, comparing tissue culture supernatants and biological samples from a study of a staphylococcal subunit vaccine (STEBVax). Mice were administered STEBVax by intranasal (IN) or intraperitoneal (IP) routes. The vaccine was formulated with an adjuvant that activates the Toll-like receptor 4 pathway3,14, and controls were given only saline or vaccine without adjuvant. Cultured NALT obtained from experimental groups secreted antigen-specific immunoglobulins into medium that was measurable by ELISA. In this example (Figure 4A), the results indicate that the greatest amounts of IgA were released by NALT obtained from mice vaccinated IN with a subunit vaccine combined with adjuvant.
Biological samples (such as serum, saliva, nasal secretions, vaginal secretions) acquired from control or NALT-free mice can be used to profile the in vivo immune response to nasal antigens for comparison with the tissue culture results. In Figure 4B, IgA and IgG responses to IN vaccination were significantly decreased without functional NALT. Levels of antigen-specific IgA were generally greater than IgG in mucosal secretions (saliva, nasal washes) of vaccinated mice.
Figure 1. Schematic of NALT collection and ex vivo culturing.
Figure 2. Visualization of the mouse palate indicating the position of NALT and surgery incision. Size and location of upper palate with surgery incision denoted by dotted line (A); upper palate excised (B) or stained in hematoxylin (C) to view the parallel NALT in the premolar area of palate (stained dark purple on anterior side of palate). NALT indicated by arrows.
Figure 3. Surgical NALT disruption. Key stages of NALT disruption surgery: supine view of mouse upper palate before surgery (A); midline incision made on upper palate to access the NALT (B); microcurette probe inserted through midline incision to disrupt NALT structure integrity (C); cauterization of incision at conclusion of surgery (D). Microscopic images of nasal cavities, H&E stained, before surgery (E); immediately after surgery (F); and a successfully healed, NALT-free mouse (G).
Figure 4. Vaccine-specific antibodies from cultured NALT, saliva, and nasal secretions collected from vaccinated mice. NALT-free or normal control mice were vaccinated IN or IP with STEBVax, and biological samples were collected. Antibody levels of triplicate samples were measured using ELISA. (A) NALT were removed from control mice (not surgically manipulated) and cultured to examine antibody responses. The vaccine-specific IgA response in culture for IN vaccinated mice was statistically different from controls (Student's t-test, p ≤ 0.01, compared to no adjuvant or no vaccine). (B) NALT disruption substantially reduced specific antibody responses to IN vaccination. There were significant differences between specific antibody levels of NALT- and NALT+ groups (no surgery or control surgery) for all comparisons except saliva IgG results (Student's t-test, p≤ 0.05).
We have presented collective methods for developing an animal model, obtaining biological samples, and assays for examining NALT-associated immune responses1-4. There are additional factors to consider during the performance of these methods. Standard sterile techniques for surgery and tissue culture should be followed. A combination of antibacterial and antifungal agents used during isolation and culture, as well as maintaining sterilized instruments, work area, and disinfected palates will reduce the risk of contamination. Saliva, nasal washes and similar mucosal secretion samples should be inspected for potential contamination with blood, as serum antibodies are generally found in higher concentrations. Further, mucosal secretions should only be slightly diluted for analysis because lower concentrations of antibodies are present in these samples compared to serum.
Mice must be kept warm directly after surgery to prevent potential anesthesia-induced hypothermia. Alternate resting mice on their sides during post-surgery recuperation to minimize irregular respiration. The surgical procedure is more efficient with three individuals working together to complete these tasks: one performing the surgery, one to assist in holding the mouth open, and one to provide post-surgical care as the mice recover from anesthesia.
It is necessary to use H&E staining of cranial cross-sections at the end of all experimental procedures or studies to verify the success of the surgery for each mouse. Possible surgery outcomes are: complete and bilateral NALT ablation, incomplete ablation, or intact NALT. Because not all surgeries will result in complete loss of the NALT, animals with residual or intact NALT can be used as internal controls. Another potential outcome is that the palate fails to completely heal, leaving an opening connecting the nasal and the oral cavities. Incomplete healing of the palate will result in low weight and failure to thrive, and these individuals should be removed from studies.
Examining vaccine responses first with the mouse model will serve to establish a role for NALT in the intended study outcome (antibody response, survival, etc.). Surgically removing the NALT facilitates the determination of nasal contributions to local and systemic immunity. The surgical approach described here is the most direct method for obtaining a mouse model devoid of NALT. Select knock-out mouse models have been reported to lack NALT, but these animals also are deficient in cytokines or chemokines essential to the development of other secondary lymphoid tissues, and may harbor additional defects12,13. Further, the methods described here were developed for examining several aspects of immune responses originating within the nasal passages. Our experimental results are based on studies using the entire upper palate from the mouse for tissue culture, although it is possible that sections may be used. Finally, the cultured NALT model is useful for performing experiments completely in tissue culture.
No conflicts of interest declared.
Support was provided by Becton Dickinson Technologies. Views expressed in this submission are those of the authors and do not purport to reflect official policy of the U.S. Government. Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996. The facility where this research was conducted is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.
|Stainless Steel Sterile Surgical Blades, No. 11||Miltex||4-311|
|Knife Handle, No. 3||Miltex||4-7|
|48-well Cell Culture Plates||Costar||3548|
|Fetal Bovine Serum, Heat Inactivated||Gibco/Invitrogen||16000-044||Final Conc: 10% by volume in culture media|
|Streptomycin Sulfate||Sigma||S9137||Final Conc: 100 μg/mL|
|Penicillin||Sigma||P7794||Final Conc: 100 UI/mL|
|Gentamicin||Sigma-Aldrich||G1397-10ML||Final Conc: 50 μg/mL|
|Fungizone||Gibco/Invitrogen||15290-018||Final Conc: 1 μg/mL|
|HEPES||Sigma-Aldrich||H0887||Final Conc: 10 mM|
|Eppendorf Microcentrifuge Tubes||Eppendorf||022364111|
|Nutra-gel Cherry Flavored Mouse Wet Food||Bio-Serve||S4798-TRAY|
|Metacam||Boehringer Ingelheim||601531000||One drop of 1.5 mg/mL Oral Suspension|
|Ketamine||Pfizer||00856440301||Final Conc: 6.06 mg/mL|
|Acepromazine||Vedco||VEDC207||Final Conc: 0.061 mg/mL|
|Xylazine||Lloyd||4811||Final Conc: 0.667 mg/mL|
|Puralube Vet Ointment||Pharmaderm Animal Health||1621|
|0.9% Sodium Chloride Injection USP||Baxter||2B1302|
|0.5 mm Microcurette||Roboz||RS-6350|
|Thermal Cautery Unit||Geiger||150-S/150A-S|
|TCU Replacement Tip, Straight Fine Loop||Geiger||214|
|10% Neutral Buffered Formalin||Sigma||HT501128|
|Formic Acid||Fisher||A119P-4||Mix 426 mL formic acid into 1047 mL tap water, then add 45 mL Rexyn 101 (H)|
|Rexyn 101 (H)||Fisher||R231-500|
|Tissue-Tek VIP E150/E300||Sakura|
|Gill No.1 Hematoxylin||Sigma||GHS116|
|Microtainer Serum Separator||BD Medical||365956|
|Phosphate Buffered Saline||100 mM NaH2PO4, 140 mM NaCl, pH 7.4|
|Protease Inhibitor Cocktail, EDTA-free||Thermo Scientific||78415|