The present work illustrates the convenience of using sublingual immunotherapy to boost the innate immune response in the lungs and confer protection against acute pneumococcal pneumonia in mouse.
Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung’s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages.
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs’ immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.
The overall goal of this work is to illustrate the benefits of sublingual immunotherapy for the treatment of acute respiratory infections (ARI) and present the advantages of this delivery route compared to other routes of administration, namely intranasal.
ARI cause millions of deaths every year especially in children under five. Streptococcus pneumoniae remains as one of the major etiological agents of bacterial pneumonia in infants and the elderly1,2. To present, the main available treatment relies on the use of antibiotics but resistant strains are continuously arising3,4.
SLIT induces broad responses at systemic and also mucosal level, particularly at the respiratory tract5. It has proven effectiveness against influenza infection, promoting long term protection with production of humoral and cellular responses6,7. Besides, it has been shown that prophylactic treatment with bacterial lysates delivered by sublingual route reduced exacerbations of chronic obstructive bronchitis in the elderly8 and prevented recurrent respiratory infections in children9. SLIT has been widely used for the treatment of allergies and asthma. Clinical studies had not only demonstrated its efficacy to modulate the immune response in the respiratory tract but also its safety10. Despite the growing interest of pharmaceutical companies and researchers in SLIT, the mechanisms involved in the induction of mucosal immune responses after sublingual delivery of compounds remain obscure. Recently, attention has been focused on the mechanisms promoting tolerance associated with allergen desensitization. It has been proposed that resident and recruited cells at the sublingual mucosa, like dendritic cells and macrophages, can promote tolerance after SLIT11-13. Dendritic cells of the oral mucosa can promote IFN-gamma and IL-10 producing T helper cells11 as well as recirculate to the distal genital mucosa and promote CD8+ T cells14. However, little is known about the impact of SLIT on innate cells or its capacity to improve pathogen clearance during acute respiratory infections.
The natural control of pneumococcal infection in the lungs greatly depends on the efficient and swift activation of local innate defences. We previously showed that enhancement of lungs’ innate immunity by a single intranasal dose of flagellin (FliC), a TLR5 and NLRC4 agonist, protects 75-100% of mice challenged with a lethal dose of a clinical isolate of Streptococcus pneumoniae serotype 1. This protection was shown to be dependent on local recruitment of GR1+ cells (likely polymorphonuclear neutrophils, PMNs) and not dependent on antibodies, B or T cells15.
Flagellin is the structural component of the bacterial flagellum. In its monomeric form it is recognized by two Pathogen Recognition Receptors (PRRs), TLR5 that senses extracellular FliC16 and NLRC4/NAIP5 inflammasome that detects intracellular flagellin17,18. When FliC is sensed by the PRRs an important inflammatory response is triggered. We and others have demonstrated that instillation of purified FliC from Salmonella enterica serovar Typhimurium into the lungs drives swift production of chemokines and cytokines specially when recognized by the lungs’ epithelium that in turn orchestrate the recruitment of immune cells into the airways, mainly PMNs15,19-21. Although transient, the substantial neutrophil infiltration that takes place into the airways after nasal delivery of FliC could be a concern if moving towards clinical therapies for human use. Excessive inflammation could be detrimental for the lungs’ function. Moreover, it has been shown that intranasal delivery of immunostimulatory molecules may cause facial paralysis and/or brain toxicity22-24.
Sublingual immunotherapy offers a safer alternative to modulate the immune response in the respiratory tract compared to the intranasal route. It is non-invasive, painless, simple and has good patient compliance25. Furthermore, as mentioned before, it can induce protective responses in the respiratory mucosa without the risks associated to direct intranasal or intrapulmonary delivery of formulations. Sublingual route could be alternatively used to deliver molecules that have great effects onto the lung’s immune system but that have been proven to be toxic or to elicit great inflammation when administered intranasally. Besides these advantages, formulations for sublingual immunotherapy have lower cost of manufacture since non-sterile products can be delivered by this route and endotoxic shock is not a concern for SLIT. On the other hand, it is worth noticing that higher doses of the immunostimulatory compounds compared to those used by intranasal or parenteral routes are necessary to induce an immune response in the lungs; also highly concentrated solutions are needed when using the mouse model of SLIT since the anatomical site where the formulations are deposited is small.
Based on our previous published data, we developed a model of protection using sublingual immunotherapy with flagellin as model immunostimulant. We demonstrated that a single dose of flagellin induced 60% survival against invasive pneumococcal pneumonia caused by the serotype 1 strain while all mice in the control group died of infection within 5 days. Flow cytometry analysis showed that higher numbers of PMN are recruited into the airways of protected animals after sublingual treatment with flagellin suggesting that these cells might be involved in the mechanism of protection induced by sublingual immunotherapy.
This video shows in detail how to perform sublingual immunotherapy and also how to recover relevant tissue from the sublingual mucosa, draining lymph nodes as well as lungs and airways to perform further analysis. Additionally, it illustrates the general technique of cell preparation for FACS analysis and briefly shows how to prepare Streptococcus pneumoniae suspensions and how to perform intranasal infections in mouse to set up the acute infection model.
La administración sublingual de agentes terapéuticos ha demostrado como un medio útil para modular la respuesta inmune en el tracto respiratorio. La principal ventaja de SLIT para el tratamiento de afecciones respiratorias es que no implica la entrega directa de los compuestos en los pulmones o la nariz, siendo más seguro que los tratamientos basados en la administración intranasal 31.
La inmunoterapia sublingual puede utilizarse para modular la respuesta inmune de diferentes maneras, ya sea para la inducción de respuestas reguladoras que pueden mejorar los síntomas de la inflamación alérgica y el asma o 32 para inducir la activación transitoria de mecanismos inmunes innatas para tratar infecciones pulmonares agudas como se muestra aquí.
El modelo de ratón se presenta en este vídeo es un método conveniente para la detección de diferentes compuestos como agentes terapéuticos para SLIT.
Este modelo animal ofrece un medio útil para determinar el impactode SLIT en la respuesta inmune de los pulmones, así como en otros órganos (por ejemplo., ganglios linfáticos de drenaje o de los sitios de la mucosa distales) que no puede ser imitado por el uso de modelos in vitro. Aunque hay varios artículos que describen los resultados obtenidos con la inmunoterapia sublingual, métodos de desarrollo para los procedimientos de administración sublingual no se han facilitado todavía. Además, el modelo se puede utilizar para la evaluación de las vacunas sublinguales con el objetivo de conferir protección sistémica así como local en el tracto respiratorio.
Como se muestra en el vídeo adjunto, la administración sublingual de los compuestos es un procedimiento simple que puede ser realizado fácilmente sin la necesidad de una amplia formación. Por lo general, una persona competente en el manejo de animales requerirá 1 hora para realizar la ITSL en un grupo de 10 ratones utilizando anestésicos inyectables como se describe en este protocolo. Si reto neumocócica se realiza, así, se requerirá 90 min adicional para prepararla suspensión bacteriana y realizar desafío intranasal de los animales.
Los protocolos de FACS presentados aquí permiten la caracterización adecuada de impacto de la ITSL en el sitio local de la administración, ganglios linfáticos de drenaje, así como sus efectos sobre la dinámica celular de los pulmones.
El análisis separado de la broncoalveolar parénquima pulmonar contenido y es importante para discriminar residente inmune de las vías respiratorias y de la infiltración de los tipos de células de las que quedan dentro del tejido. El análisis del contenido BAL permite el estudio de la facturación de los macrófagos alveolares, así como la dinámica de reclutamiento células en los espacios alveolares inducidas por diferentes tratamientos, por ejemplo., PMN, eosinófilos, monocitos. BAL también se puede utilizar para evaluar la presencia de citocinas y quimiocinas secretadas por el ensayo de inmunoabsorción ligado a enzimas (ELISA) o la detección de anticuerpos IgA secretados inducidos después de la vacunación sublingual. Estudio de los tejidos de los pulmonespermitirá caracterización de otros tipos de células, células dendríticas Clásicamente, las células T y células B.
Preparación de muestras de BAL y los ganglios linfáticos para el análisis FACS es simple. Después de la recogida de la muestra, se requieren normalmente de 60 minutos para completar el protocolo de tinción durante 10-20 muestras. En contraste, el aislamiento de las células de los pulmones o el tejido sublingual requerirá más tiempo ya que se requiere la digestión de la matriz extracelular. La absorción del agente terapéutico administrado por vía sublingual puede abordarse mediante el seguimiento de las moléculas con fluorescencia o marcadas radiactivamente utilizando en los sistemas de formación de imágenes in vivo.
La inmunoterapia sublingual es un método atractivo para inducir eficazmente las respuestas inmunes en el tracto respiratorio, así como sistémicamente que puede ser utilizado para tratar o prevenir enfermedades respiratorias. La elucidación de los mecanismos que determinan la activación vs tolerancia de la respuesta inmune en el tracto respiratorio después de SLIT ies crucial para permitir el diseño racional de nuevas estrategias terapéuticas que podrían ser utilizados solos o en combinación con tratamientos disponibles contra diferentes condiciones respiratorias.
The authors have nothing to disclose.
We acknowledge Dr. Jean-Claude Sirard from the Center for Infection and Immunity of Lille, Institute Pasteur de Lille-France, for kindly providing the purified flagellin and Dr. Teresa Camou, Director of the National Reference Laboratory, Ministry of Health of Uruguay for kindly providing the pneumococcal strain.
The authors would like to express their acknowledgement to Mr. Diego Acosta and Mr. Ignacio Turel form BichoFeo Producciones-Uruguay for their commitment and hard work during the entire video production and edition.
This work was supported by the grants PR_FCE_2009_1_2783 and BE_POS_2010_1_2544 from the National Agency of Research and Innovation, ANII from Uruguay, the Program for Development of Basic Sciences, PEDECIBA of Uruguay and Sectoral Commission of Scientific research, CSIC-Universidad de la República, Uruguay.
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
Ketamine solution (50 mg/ml) | Pharma Service, Uruguay | N/A | |
Xilacine solution (2 %) | Portinco S.A., Uruguay | N/A | |
Sterile 1ml syringe | Modern, Uruguay | N/A | |
Sterile 27G needle | Modern, Uruguay | N/A | |
RPMI 1640 | General Electric Health Care | E15885 | |
Fetal Bovine Serum | ATCC | 302020 | |
Penicillin/Streptimycin Solution | SIGMA | P4333 | |
Sterile PBS without Ca2+/Mg2+ | PAA | H21002 | |
Type-I Collagenase | Life Technologies/Gibco | 17100017 | |
Deoxyribonuclease I (DNAse-I) | SIGMA | D4513 | |
Dispase | Life Technologies/Gibco | 17105041 | |
PerCP-Cy5.5 conjugated rat anti mouse IgG2b anti CD11b | BD | 550993 | Clone M1/70 |
APC conjugated hamster anti mouse IgG1 anti CD11c | BD | 550261 | Clone HL3 |
APC-Cy7 conjugated rat anti mouse IgG2a anti Ly6G | BD | 560600 | Clone 1A8 |
Sterile Saline Solution | Laboratorio Farmaco Uruguayo, Uruguay | N/A | |
Tryptic Soy Agar | BD Difco, France | 236950 | |
Defibrinated Sheep Blood | Biokey, Uruguay | N/A | |
Sterile Petri Dishes | Greiner | 633180 | |
p10 Pipette | Gilson | F144802 | |
P20 Pipette | Eppendorf | 3120000097 | |
p200 Pipette | Gilson | F123601 | |
p200 Pipette | Capp | C200 | |
p200 Pipette | Eppendorf | 3120000054 | |
p1000 Pipette | Eppendorf | 3120000062 | |
Sterile Filter Tips P10 | Greiner | 771288 | |
Sterile Filter Tips P200 | Greiner | 739288 | |
Sterile Filter Tips P1000 | Greiner | 750288 | |
Vortex | BIOSAN | V1-plus | |
Stainless steel fine tip forceps | SIGMA | Z168785/Z168777 | curved and straight |
Dressing tissue forceps | SIGMA | F4392 | length 8 inches |
Micro-dissecting forceps | SIGMA | F4017 | straight |
Micro-dissecting forceps | SIGMA | F4142 | Curved |
Mayo Scissors | SIGMA | Z265993 | |
Scalpel | SAKIRA MEDICAL | N/A | |
Sterile Biopsy Punch Ø 3mm | Stiefel Laboratories Ltd. | 2079D | 5mm diameter can also be used |
Sterile 1.5ml Tubes | Deltalab | 200400P | |
Sterile 15ml Tubes | Greiner | 188271 | |
Sterile 50ml Tubes | Greiner | 227261 | |
Sterile serological pipettes 5 ml | Greiner | 606160 | |
Sterile serological pipettes 10 ml | Greiner | 607160 | |
Sterile serological pipettes 25 ml | Greiner | 760180 | |
Biological safety cabinet, class II | Thermo Scientific | 1300 series, type A2 | |
Micro-Isolator Rack | RAIR IsoSystem | 76144W | Super Mouse 1800 AllerZone |
Refrigerated Microcentfifuge | Eppendorf | Legend Micro 21R | |
Microcentfifuge | Heraeus | Biofuge-pico | |
Centrifuge | Thermo Scientific | Sorval ST40R | |
CO2 Incubator | Thermo Scientific | Model 3111 | |
Sterile Thin-tip pasteur pipettes | Deltalab | D210022 | |
Sterile pasteur pipettes | Deltalab | 200007 | |
Sterile 24-well plate | Greiner | 662160 | |
Trypan Blue Solution | Life Technologies | T10282 | |
Automatic Cell Counter – Cuntess | Life Technologies | C10227 | |
Countess Cell Counting Chamber Slides | Life Technologies | C10312 | |
Flow Cytometry Tubes | BD | 343675 | |
Flow Cytometer – FACS Canto-II | BD | N/A | |
Real Time PCR Instrument – Rotor Gene Q or ABI 7900 | Qiagen / Applied Biosystems | N/A | |
Trizol Reagent | Life Technologies | 15596-026 | Molecular Biology Grade |
DNAse-I | Life Technologies | 18068-015 | Molecular Biology Grade |
DNAse-I Buffer 10X | Life Technologies | 18068015 | Molecular Biology Grade |
EDTA 25 mM | Life Technologies | 18068015 | Molecular Biology Grade |
Ultra-Pure Water | Life Technologies | 10977 | Molecular Biology Grade |
RNAse Out | Life Technologies | 100000840 | Molecular Biology Grade |
Rndom Hexamer Primers | Life Technologies | N8080127 | Molecular Biology Grade |
M-MLV-RT buffer | Life Technologies | 18057-018 | Molecular Biology Grade |
M-MLV-RT enzime | Life Technologies | 28025-021 | Molecular Biology Grade |
QuantiTect Syber Green PCR Kit | Qiagen | 204143 | Molecular Biology Grade |
Specific primers | Life Technologies | N/A | Molecular Biology Grade |