Nasal priming with immunobiotic lactobacilli improves the adaptiveimmune response against influenza virus
Fernanda Raya Tonettia,b, Md. Aminul Islamc, Maria Guadalupe Vizoso-Pintoa,b,Hideki Takahashie,f, Haruki Kitazawac,g,⁎, Julio Villenac,d,⁎⁎
a Infection Biology Laboratory, INSIBIO (UNT-CONICET), Tucumán, Argentinab Laboratory of Basic Sciences – Genetics, Faculty of Medicine, National University of Tucuman, Tucumán, Argentinac Food and Feed Immunology Group, Laboratory of Animal Products Chemistry, Graduate School of Agricultural Science, Tohoku University, Sendai, Japand Laboratory of Immunobiotechnology, Reference Centre for Lactobacilli (CERELA-CONICET), Tucumán, Argentinae Laboratory of Plant Pathology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japanf Plant Immunology Unit, International Education and Research Center for Food Agricultural Immunology, Graduate School of Agricultural Science, Tohoku University,Sendai, Japang Livestock Immunology Unit, International Education and Research Center for Food Agricultural Immunology (CFAI), Graduate School of Agricultural Science, TohokuUniversity, Sendai, Japan
The nasal priming with Lactobacillus rhamnosus CRL1505 modulates the respiratory antiviral innate immuneresponse and improves protection against influenza virus (IFV) challenge in mice. However, the potentialbeneficial effect of the CRL1505 strain on the adaptive immune response triggered by IFV infection or vacci-nation was not evaluated before. In this work, we demonstrated that nasally administered L. rhamnosus CRL1505is able to improve both the humoral and cellular adaptive immune responses induced by IFV infection or vac-cination. Higher levels of IFV-specific IgA and IgG as well as IFN-γ were found in the serum and the respiratorytract of CRL1505-treated mice after IFV challenge. Lactobacilli treated mice also showed reduced concentrationsof IL-17 and improved levels of IL-10 during IFV infection. The differential balance of inflammatory and reg-ulatory cytokines induced by L. rhamnosus CRL1505 contributed to the protection against IFV by favoring aneffective effector immune response without inducing inflammatory-mediated lung damage. The optimal im-munomodulatory effect of the CRL1505 strain was achieved with viable bacteria. However, non-viable L.rhamnosus CRL1505 was also efficient in improving the adaptive immune responses generated by IFV challengesand therefore, emerged as an interesting alternative for vaccination of immunocompromised hosts. Similar toother immunomodulatory properties of lactobacilli, it was shown here that the adjuvant effect in the context ofIFV vaccination was a strain dependent ability, since differences were found when L. rhamnosus CRL1505 and theimmunomodulatory strain L. rhamnosus IBL027 were compared. This investigation represents a thorough ex-ploration of the role of immunobiotic lactobacilli in improving humoral and cellular adaptive immune responsesagainst IFV in the context of both infection and vaccination.
1. Introduction
Influenza virus (IFV) is responsible of a highly contagious diseasethat has a substantial impact on global health. This virus is a majorrespiratory pathogen that causes a high degree of morbidity and mor-tality, especially in high-risk populations such as infants, elderly andimmunocompromised hosts. Annual vaccination is the most efficient
and cost-effective strategy to prevent and control influenza epidemics[1]. Most of the currently available influenza vaccines are strong in-ducers of antibody responses against viral surface proteins like he-magglutinin and neuraminidase, but are poor inducers of cell-mediatedimmune responses against conserved internal proteins [2]. In addition,the most widely used IFV vaccine, an inactivated virus vaccine ad-ministered by subcutaneous/intramuscular injection, is limited to the
https://doi.org/10.1016/j.intimp.2019.106115Received 27 October 2019; Received in revised form 3 December 2019; Accepted 4 December 2019
⁎ Corresponding author at: Food and Feed Immunology Group, Laboratory of Animal Products Chemistry, Graduate School of Agricultural Science, TohokuUniversity, Sendai, Japan.
⁎⁎ Corresponding author at: Laboratory of Immunobiotechnology, Reference Centre for Lactobacilli (CERELA-CONICET), Tucumán, Argentina.E-mail addresses: [email protected] (H. Kitazawa), [email protected] (J. Villena).
improvement of systemic immunity, with no effect on the mucosalimmune system. Although the live attenuated IFV vaccine administeredby the nasal route is able to stimulate the production of not only sys-temic IgG antibodies but also local secretory IgA, this vaccine is re-stricted to immunocompetent healthy children and adults and cannotbe applied to high-risk populations [3]. Then, there is still a need todevelop a new generation of IFV vaccines with the ability to inducemucosal and systemic immune responses, stimulate both humoral andcellular adaptive immunity and be safely administered to populationswith poor immune responses [1].
Research have reported that beneficial microbes with the capacityto modulate the mucosal immune system (referred to as immunobiotics)have the ability to improve the outcome of IFV infection (For a reviewsee [4]). Studies have evaluated the effect of immunobiotic strains inthe respiratory and systemic antiviral adaptive immune responsesagainst IFV infection [4]. Well-known immunomodulatory probioticstrains including Bifidobacterium breve YIT4064 [5], L. casei Shirota[6,7] and L. rhamnosus GG [8] have been proved to be able to differ-entially modulate the humoral and cellular adaptive immune responsesagainst IFV and confer protection against this respiratory pathogen.Additionally, a growing number of human trials have examined theeffect of immunobiotics on the incidence and severity of IFV infection.Those clinical studies have evaluated principally the potential adjuvanteffects of immunobiotics on IFV vaccination (Reviewed in [4]) and havedemonstrated that immunomodulatory lactobacilli and bifidobacteriaare able to improve the humoral and cellular immune responses trig-gered by IFV vaccines.
Previously, we reported that the oral [9] or nasal [10] administra-tion of the immunobiotic strain Lactobacillus rhamnosus CRL1505 is ableto modulate the respiratory antiviral innate immune response and im-prove protection against IFV challenge in mice. The mucosal primingwith L. rhamnosus CRL1505 enhanced the respiratory antiviral state byincreasing the production of type I interferons (IFNs) and IFN-γ, whichhelped to significantly reduce the viral replication. In addition, theCRL1505 strain differentially regulated the levels and kinetics of in-flammatory cells and cytokines in mice after IFV challenge. In our ex-perimental model, we observed increased levels of respiratory TNF-α,IL-6, neutrophils, and macrophages in CRL1505-treated mice early afterthe challenge with IFV. Later in the course of the infection, pro-in-flammatory cytokines and infiltrated cells started to decrease in im-munobiotic-treated animals in contrast to control mice, in which thoseparameters continued increasing. The trend toward lower inflammatoryfactors and cells registered later during IFV infection in L. rhamnosusCRL1505-treated mice correlated with a reduced severity of pulmonarydamage when compared to control mice [10,9]. However, the potentialbeneficial effect of L. rhamnosus CRL1505 strain on the adaptive im-mune response triggered by IFV infection or vaccination was notevaluated before.
Research from the last decade demonstrated that the im-munomodulatory effects of immunobiotic bacteria are the consequenceof complex interactions between several bacterial molecules and hostreceptors located in different immune and non-immune cells [11,12]. Ithas also been shown that the immunomodulatory properties of im-munobiotics are dependent on the strains. Therefore, studies carried outwith certain strains cannot be easily extrapolated to other bacteria,even those of the same genus and species [13,14]. Consequently, it isstill necessary to carry out deeper studies to find out whether L.rhamnosus CRL1505 is able to beneficially influence the respiratoryantiviral adaptive immunity in the context of IFV infection. Therefore,in this work we investigated the influence of nasally administered vi-able and non-viable L. rhamnosus CRL1505 on the humoral and cellularadaptive immune responses triggered by IFV infection or IFV vaccina-tion.
2. Materials and methods
2.1. Microorganisms
Lactobacillus rhamnosus CRL1505 (Lr1505) was obtained from theReference Centre for Lactobacilli (CERELA-CONICET) culture collection(San Miguel de Tucumán, Argentina) and Lactobacillus rhamnosusIBL027 (Lr027) was obtained from the Infection Biology Laboratory ofthe Research Institute of Molecular and Cellular Applied Medicine(IMMCA, UNT-CONICET-SIPROSA) culture collection (San Miguel deTucumán, Argentina). Cultures were kept freeze-dried. For experi-ments, cultures were rehydrated using a medium containing 5 g of meatextract, 10 g tryptone, and 15 g of peptone in 1 L of distilled water, pH7. Then, lactobacilli were cultured for 12 h at 37 °C (final log phase) inMan–Rogosa–Sharpe broth (MRS, Oxoid, UK). Bacteria suspensionswere prepared as described before [10,9]. Briefly, lactobacilli wereharvested the by centrifugation at 3000g for 10 min and washed threetimes with sterile 0.01 mol/l phosphate buffer saline (PBS), pH 7.2.
Heat-killed Lr1505 (HK1505) and heat-killed Lr027 (HK027) wereprepared as described previously [15,16]. Briefly, lactobacilli werekilled by tyndallization in a water bath at 80 °C for 30 min and the lackof bacterial growth was confirmed using MRS agar plates.
2.2. Animals and immunization protocols
Six-week-old BALB/c mice were obtained from a closed colony keptat CERELA-CONICET (Tucuman, Argentina). They were housed inplastic cages at room temperature with a 12 h light/dark cycle.Parameters were studied in 5–6 mice per group for every time point. Allgroups were fed a conventional balanced diet ad libitum.
Two sets of experiments with different treatments protocols wereused in this work. In the first set of experiments, Lr1505 or HK1505were administered for two consecutive days at a final dose of 108 cells/mouse/day inoculated via nostrils. One day after the last adminstration,tretated animals and untreated controls were challenged with IFV asdescribed below, and the specific cellular and humoral adaptive im-mune responses were evaluated.
In a second set of experiments mice were nasally vaccinated with20 μl of the commercial IFV vaccine Istivac® (SANOFI PASTEUR,Argentina) alone or with Lr1505, Lr027, HK1505 or HK027 (108 cells/mouse) as adjuvants. Mice were immunized on days 0, 14 and 28. Sevendays after the last immunization, the specific humoral and cellularimmune responses and the resistance to IFV infection were evaluated asdescribed below.
This study was carried out in strict accordance with the re-commendations in the Guide for the Care and Use of LaboratoryAnimals of the Guidelines for Animal Experimentation of CERELA andall efforts were made to minimize suffering. Endpoints were used insurvival experiments to euthanize animals. IFV infection in this animalmodel induces mortality between days 4 and 9 [9,10]. In that period oftime, animals were checked for sings of suffering and euthanized ifconsidered appropriate, before the end of experiments on day 14. Signsof pain, suffering, and especially moribund conditions were used for thedecision to euthanize mice.
2.3. Virus and infection
Infection with IFV was performed as described previously [10,9].Briefly, IFV A/PR/8/34 (H1N1) was propagated in Madin–Darby caninekidney (MDCK) cells, and virus titers in the stock solution were de-termined by a plaque assay. MDCK cells were grown and maintained inEagle’s minimum essential medium supplemented with 2 and 5% heatinactivated fetal bovine serum, respectively. In the first set of experi-ments, infection was performed on day 3, after the 2 days treatmentswith Lr1505 or HK1505. Mice were intranasally infected with 500 PFUof the A/PR/8/34 strain in 25 μl of PBS. In the second set of
F.R. Tonetti, et al. International Immunopharmacology 78 (2020) 106115
2
experiments mice were intranasally challenged with 1000 PFU of IFV7 days after the last immunization.
2.4. Tissue and fluids sampling
Mice were anesthetized and blood samples were obtained throughcardiac puncture. Bronchoalveolar lavage (BAL) samples were obtainedas described previously [15,17,16]. Briefly, the trachea was exposedand intubated with a catheter. Two sequential BAL were performed ineach mouse by injecting 1 ml of sterile PBS; the recovered fluid wascentrifuged for 10 min at 900g; smears were done with the cellularpellet and stained for determining cell counts. The supernatant fluidswere frozen at −70 °C for subsequent cytokines and chemokines ana-lyses.
Immune cells from mediastinal lymph nodes (MLN) and spleen wereisolated by standard methods. Cell suspension were generated bypressing minced MLN or spleen against the bottom of a petri dishcontaining PBS. After elimination of erythrocytes by 10-second in-cubation in distilled water and five washes in cold PBS, the cells wereresuspended in PBS and counted. The viability was determined bytrypan blue exclusion and only cells with viability higher than 95%were used in subsequent experiments.
2.5. Cytokines analysis
TNF-α, IFN-γ, IL-4, IL-10 and IL-17 in serum and BAL samples weredetermined with commercially available enzyme-linked im-munosorbent assay (ELISA) kits following the manufacturer’s instruc-tions (R&D Systems, MN, USA). IFN-γ, IL-4, and IL-17 secretion by invitro IFV vaccine-restimulated splenocytes or MLN immune cells wasmeasured in culture supernatants.
2.6. IFV specific antibodies
The antibody response to the IFV infection was determined usingenzyme-linked immunosorbent assays (ELISA). Plates were coatedovernight at 4 °C with UV-killed IFV A/PR/8/34 (H1N1) virus. Theplates were blocked with albumin. Appropriate dilutions of the samples(serum 1:20; BAL 1:2) were incubated for 1 h at 37 °C. Peroxidaseconjugated anti-mouse IgG, IgG1, IgG2a or IgA antibodies (1:500)(Sigma-Aldrich) were added and incubated for 1 h at 37 °C. The reac-tion was developed with TMB Substrate Reagent (Sigma-Aldrich). Theoptical density (OD) at 450 nm was determined in an ELISA plater-eader. The concentration of antibodies was measured with reference tostandard curves using known amounts of the respective murine Ig(Sigma-Aldrich). Detection of antibodies against IFV vaccine wereevalauted in a similar way. Plates were coated overnight at 4 °C with1 μg/μl of influenza split vaccine Istivac® (SANOFI PASTEUR) and
0 5 10 15 200
100
200
300
400
0 5 10 15 200
200
400
600
0 5 10 15 200
100
200
300
400
0 5 10 15 200
100
200
300
Days post-infection
ng/m
L
BAL IgA
Days post-infection
ng/m
L
BAL IgG
Days post-infection
ng/m
L
BAL IgG1
Days post-infection
ng/m
L
BAL IgG2a
Control Lr1505 HK1505
*
** *
**
*
*
*
**
*
Fig. 1. Effect of lactobacilli on the respiratory humoral immune response induced by influenza virus (IFV) infection. Mice were nasally treated with viable (Lr1505)or heat-killed (HK1505) Lactobacillus rhamnosus CRL1505 for two consecutive days and challenged with IFV on day 3. Untreated mice infected with IFV were used ascontrols. The concentrations of anti-IFV IgA, IgG, IgG1 and IgG2a were determined in broncho-alveolar lavage (BAL) samples at the indicated time points after viralinfection. The results represent data from three independent experiments. Results are expressed as mean ± SD. Asterisks indicate significant differences(*P < 0.05) when compared to control mice.
F.R. Tonetti, et al. International Immunopharmacology 78 (2020) 106115
3
ELISA was performed according the procedure described before.
2.7. Flow cytometry
Cell suspensions obtained from lung [15,16] were incubated withanti-mouse CD32/CD16 monoclonal antibody (Fc block) for 15 min at4 °C. Then, cells were incubated with the respective antibody mixes(anti-mouse CD4-PE and anti-mouse IFN-γ-APC, anti-mouse IL-4-APC oranti-mouse IL-17-APC, BD PharMingen, USA) for further 30 min at 4 °Cand washed with FACS buffer. After staining, cells were acquired on aBD FACSCalibur TM flow cytometer (BD Biosciences) and the data wereanalyzed with FlowJo software (TreeStar). The total number of cells ineach population was determined by multiplying the percentages ofsubsets within a series of marker negative or positive gates by the totalcell number determined for each tissue.
2.8. Statistical analysis
Experiments were done in triplicate and results were expressed asmean ± standard deviation (SD). Data were normally distributed, andtherefore, a 2-way ANOVA was used. Student t-test (for pairwise com-parisons of the means) was used to test for differences among groups.Differences were considered significant at p < 0.05 or p < 0.01.
3. Results
3.1. Viable and heat-killed L. Rhamnosus CRL1505 improve respiratoryadaptive immune response against IFV infection
In order to study whether Lr1505 and HK1505 treatments differ-entially modulated the respiratory anti-IFV adaptive immune response,mice were treated with lactobacilli by the nasal route for 2 days.Treated and untreated control mice were nasally challenged with IFV.Respiratory anti-IFV antibodies were detected from day 5 post-infectionand the levels of BAL IgA, IgG, IgG1, and IgG2a increased until day 15(Fig. 1). Lr1505 and LrHK1505 treated mice showed higher levels ofrespiratory anti-IFV IgA and IgG antibodies when compared to controlmice. In addition, the values of respiratory anti-IFV IgG1 and IgG2awere significantly enhanced in Lr1505 and HK1505 groups whencompared to controls (Fig. 1). Lr1505 and HK1505 were equally ef-fective to improve the levels of all the anti-IFV antibodies studied. Inaddition, the levels of respiratory IFN-γ, IL-4, IL-17 and IL-10 werestudied in order to evaluate the cellular immune response. As shown inFig. 2, the challenge of mice with IFV significantly increased the levelsof the four cytokines in the respiratory tract. Both, Lr1505 and HK1505significantly increased the concentration of IFN-γ, IL-4 and IL-10 whencompared to controls, being Lr1505 more effective than HK1505 toaugment IFN-γ and IL-10 levels (Fig. 2). No differences were observedin the concentrations of IL-17 in mice treated with Lr1505 and HK1505when compared to controls on the first days post-infection. However,the levels of this cytokine were significantly lower on day 5 post-in-fection in the groups of mice that received Lr1505 or HK1505 (Fig. 2).In order to analyze further the cellular response, the numbers of dif-ferent lung CD4+ T cells populations were evaluated after IFV infection(Fig. 3). It was observed that the numbers of lung CD4+IFN-γ+,CD4+IL-4+, and CD4+IL-17+ T cells augmented after the challengewith IFV. The administration of Lr1505 or HK1505 significantly in-creased the numbers of lung CD4+IFN-γ+ T cells, whereas the numbersof CD4+IL-4+ and CD4+IL-17+ T cells in infected mice pre-treatedwith lactobacilli did not suffer modifications when compared to thecontrol group (Fig. 3).
3.2. Viable and heat-killed L. Rhamnosus CRL1505 improve systemicadaptive immune response against IFV infection
The levels of anti-IFV IgA, IgG, IgG1, and IgG2a antibodies were
analyzed in serum to study the systemic adaptive immune response(Fig. 4). As expected, the levels of the four types of antibodies increasedin serum after the challenge with IFV. Lr1505 and HK1505 were able toimprove serum anti-IFV IgA, IgG, IgG1, and IgG2a levels when com-pared with control mice (Fig. 4). The levels of serum IFN-γ, IL-4, IL-17and IL-10 were also studied in order to evaluate the systemic cellularimmune response. The infection with IFV significantly augmented thelevels of the four cytokines in serum as observed in Fig. 5. In addition,both Lr1505 and HK1505 significantly increased the levels of IFN-γ, IL-4 and IL-10 when compared to controls. The treatment with Lr1505 wasmore effective than HK1505 to increase the serum levels of IFN-γ andIL-10 (Fig. 5). The concentration of IL-17 in mice treated with Lr1505and HK1505 was significantly lower than in control mice on days 5 and7 post-infection.
3.3. Viable and heat-killed immunobiotic lactobacilli improve adaptiveimmune response induced by IFV vaccine
Taking into consideration the both Lr1505 and HK1505 improvedthe adaptive immune response against IFV infection, we wonder whe-ther these tretaments were able to enhance the immune responseagainst an IFV vaccine. In addition, we aimed to find out whether theeffect of L. rhamnosus CRL1505 on the adpative immune response isstrain specific or if it is sheared with other immunomodulatory lacto-bacilli of the same species. Then, we performed a second set of ex-periments to evaluate the ability of Lr1505, and HK1505 as well asviable and non-viable immunomodulatory L. rhamnosus IBL027 (Lr027and HK027, respectively) [47] to act as mucosal adjuvants. For thatpurpose, mice were nasally immunized with a commercial IFV vaccinealone or with live or heat-killed lactobacilli as adjuvant on days 0, 14and 28. The levels of systemic and respiratory anti-IFV vaccine anti-bodies were determined seven days after the last immunization. Spe-cific IgG in serum and IgA in BAL were detected in mice vaccinated withthe commercial IFV vaccine indicating that the vaccine induced mu-cosal and systemic humoral immune responses in our animal model(Fig. 6). No significant differences were observed when serum IgGspecific antibodies from control and lactobacilli-treated mice werecompared. Interestingly, the four treatments significantly improved thelevels of BAL IgA specific antibodies, being the HK1505 more efficientthan Lr1505, Lr027 and HK027 treatments to achieve this effect(Fig. 6). For the evaluation of the specific cellular immune response,immune cells from MLN and spleen were isolated, cultured, stimulatedwith IFV vaccine and the concentrations of IFN-γ, IL-17 and IL-4 weredetermined in culture supernatants (Fig. 7). Immune cells isolated fromboth MLNs and spleens of control vaccinate mice were able to producethe three cytokines evaluated. It was also observed that the levels ofIFN-γ and IL-4 produced by immune cells from MLN and spleen oflactobacilli-treated mice were significantly higher than those observedin control groups. The higher levels of IFN-γ production in response tovaccine stimulation were observed in cells isolated from mice treatedwith Lr1505 (Fig. 7). On the other hand, HK1505 and Lr027 treatmentsinduced the highest levels of IL-4. No significant differences were ob-served between control and lactobacilli-treated mice when the levels ofIL-17 produced by MLN or spleen immune cells were compared (Fig. 7).
3.4. Vaccination with viable and heat-killed immunobiotic lactobacilli andIFV vaccine improve resistance against viral infection
Finally, we aimed to evaluate whether the improved adaptive im-mune response induced by the vaccination with IFV vaccine and lac-tobacilli as adjuvants increased the resistance to the viral challenge.Then, vaccinated mice were nasally challenged with IFV seven daysafter the last immunization. As shown in Fig. 8, 50% of the mice in thecontrol group died until day 4 post-infection and only 30% of this groupsurvived up to day 14. The nasal immunization with IFV vaccine andlive and heat-killed lactobacilli significantly improved the survival of
F.R. Tonetti, et al. International Immunopharmacology 78 (2020) 106115
4
infected mice. The most effective treatment to improve survival was theimmunization with IFV vaccine and Lr1505. In this group, 100% ofanimals survived to the IFV infection (Fig. 8). In addition, 90% of micein the HK1505 and Lr027 groups survived the infection while the
HK027 group showed 80% of survival.In order to evaluate whether the increased survival in lactobacilli-
treated mice was related to a better control of virus replication, lungsfrom these mice and controls were recovered to evaluate virus titers
0 2 4 6 80
100
200
300
0 2 4 6 80
100
200
300
0 2 4 6 80
50
100
150
200
0 2 4 6 80
200
400
600
800
Days post-infection
pg/m
L
BAL I -
Days post-infection
pg/m
L
BAL IL-4
Days post-infection
pg/m
L
BAL IL-17
Days post-infection
pg/m
L
BAL IL-10
Control Lr1505 HK1505
*
*** * *
** *
*
* **
*
Fig. 2. Effect of lactobacilli on the respiratory cellular immune response induced by influenza virus (IFV) infection. Mice were nasally treated with viable (Lr1505) orheat-killed (HK1505) Lactobacillus rhamnosus CRL1505 for two consecutive days and challenged with IFV on day 3. Untreated mice infected with IFV were used ascontrols. The concentrations of IFN-γ, IL-4, IL-17 and IL-10 were determined in broncho-alveolar lavage (BAL) samples before IFV challenge (day 0) and at theindicated time points after viral infection. The results represent data from three independent experiments. Results are expressed as mean ± SD. Asterisks indicatesignificant differences (*P < 0.05, **P < 0.01) when compared to control mice.
0 2 4 80
50
100
150
0 2 4 6 80
20
40
60
80
0 2 4 6 80
10
20
30
40
Control Lr1505 HK1505
Days post-infection
Num
ber
of c
ell(
x 10
4 )
Lung CD4+IFN- + cells
Days post-infection
Num
ber
of c
ell(
x 10
4 )
Lung CD4+IL-4+ cells
Days post-infection
Num
ber
of c
ell(
x 10
4 )
Lung CD4+IL-17+ cells
*
*
Fig. 3. Effect of lactobacilli on the respiratory cellular immune response induced by influenza virus (IFV) infection. Mice were nasally treated with viable (Lr1505) orheat-killed (HK1505) Lactobacillus rhamnosus CRL1505 for two consecutive days and challenged with IFV on day 3. Untreated mice infected with IFV were used ascontrols. The numbers of CD4+IFN-γ+, CD4+IL-4+, and CD4+IL-17+ T cells were determined in lung samples before IFV challenge (day 0) and at the indicated timepoints after viral infection. The results represent data from three independent experiments. Results are expressed as mean ± SD. Asterisks indicate significantdifferences (*P < 0.05) when compared to control mice.
F.R. Tonetti, et al. International Immunopharmacology 78 (2020) 106115
5
(Fig. 8). IFV was detected in the lungs of control mice from day 1 to 5post-infection, being the highest titers observed on day 4. The fourlactobacilli treatments significantly reduced IFV titers in lungs on days1, 2 and 3 post-infection, while the virus was not recovered from lungsamples on days 4 and 5 in those experimental groups (Fig. 8). Theconcentrations of BAL IFN-γ, TNF-α and IL-10 were also evaluated afterIFV challenge in vaccinated mice (Fig. 9). In control mice, the levels ofBAL IFN-γ increased until day 5 post-infection and then started to de-crease. Mice vaccinated with IFV vaccine plus Lr027, HK1505 or HK027showed a similar kinetics in IFN-γ production, however, the levels ofthis cytokine in the Lr027 group were significantly higher when com-pared to the other experimental groups. Interestingly, mice vaccinatedwith IFV vaccine plus Lr1505 showed a kinetics in IFN-γ productionthat was different from the other groups. In the Lr1505 mice, IFN-γlevels were significantly higher than the other groups in the first dayspost-infection while this cytokine was significantly reduced by the endof the period studied (Fig. 9). The respiratory levels of TNF-α con-tinuously increased after IFV challenge in control mice. In mice vacci-nated with IFV vaccine plus lactobacilli treatments, the levels of thispro-inflammatory cytokine were significantly higher than the controlsin the first 4 days post-infection while this cytokine was significantlyreduced on day 7 (Fig. 9). In the Lr1505 group, TNF-α was significantlyhigher than the other groups on day 1 post-infection, and the levels ofthis cytokine decreased earlier than the other lactobacilli-treated
groups (Fig. 9). In control mice, the levels of BAL IL-10 were increasedfrom day 5 post-infection in all the groups, however, mice vaccinatedwith IFV vaccine plus Lr1505 showed significantly higher concentra-tions of this cytokine when compared with the other groups (Fig. 9).
4. Discussion
Influenza infection typically initiates at mucosal surfaces. If theantiviral mechanisms mediated by innate immunity are exceeded byIFV, both the humoral and cellular adaptive immune responses aretriggered in the respiratory tract. Upper airway exposure results pri-marily in an IgA response while the contact of IFV with the deep lunginduce an increased production of pathogen-specific IgG [18]. IgAprevents IFV from adhering to the epithelial respiratory surface by in-ducing viral agglutination, and masking adhesion epitopes. Moreover,secretory IgA has a non-inflammatory protective function since theseantibodies can bind to virus without activating complement or stimu-lating the release of inflammatory mediators by innate immune cells[19,18]. In the deep lung, IFV stimulates the differentiation and ex-pansion of antibody-secreting plasma cells that are committed to theproduction of IgG. Induction of neutralizing respiratory and serum IgGantibodies is a key event in the defense against influenza infection sinceIgG prevents systemic spread [20]. On the other hand, influenza in-fection in the lungs also activates the cellular adaptive immune
0 5 10 15 200
20
40
60
80
100
0 5 10 15 200
200
400
600
800
0 5 10 15 200
100
200
300
400
500
0 5 10 15 200
100
200
300
400
500
Days post-infection
ng/m
L
Serum IgA
Days post-infection
ng/m
L
Serum IgG
Days post-infection
ng/m
L
Serum IgG1
Days post-infection
ng/m
L
Serum IgG2a
Control Lr1505 HK1505
*
**
* **
**
*
**
*
Fig. 4. Effect of lactobacilli on the systemic humoral immune response induced by influenza virus (IFV) infection. Mice were nasally treated with viable (Lr1505) orheat-killed (HK1505) Lactobacillus rhamnosus CRL1505 for two consecutive days and challenged with IFV on day 3. Untreated mice infected with IFV were used ascontrols. The concentrations of anti-IFV IgA, IgG, IgG1 and IgG2a were determined in serum samples at the indicated time points after viral infection. The resultsrepresent data from three independent experiments. Results are expressed as mean ± SD. Asterisks indicate significant differences (*P < 0.05) when compared tocontrol mice.
F.R. Tonetti, et al. International Immunopharmacology 78 (2020) 106115
6
response by stimulating the production of IFN-γ by Th1 cells that ef-fectively activate CD8+ T cells and macrophages, which clear virus andinfected cells from the lungs [21]. In this work, we were able to re-capitulate those findings in our animal experimental model. Infection
with IFV stimulated both the humoral and cellular adaptive immuneresponses since increases in respiratory and systemic specific antibodiesas well as lung CD4+IFN-γ+, CD4+IL-4+, and CD4+IL-17+ T cells werefound in mice after the challenge with the respiratory pathogen.
0 2 4 6 80
100
200
300
0 2 4 6 80
100
200
300
0 2 4 6 80
50
100
150
200
0 2 4 6 80
200
400
600
800
Days post-infection
pg/m
L
Serum IFN-
Days post-infection
pg/m
L
Serum IL-4
Days post-infection
pg/m
L
Serum IL-17
Days post-infection
pg/m
L
Serum IL-10
Control Lr1505 HK1505
*
* ***
** *
***
*
Fig. 5. Effect of lactobacilli on the systemic cellular immune response induced by influenza virus (IFV) infection. Mice were nasally treated with viable (Lr1505) orheat-killed (HK1505) Lactobacillus rhamnosus CRL1505 for two consecutive days and challenged with IFV on day 3. Untreated mice infected with IFV were used ascontrols. The concentrations of IFN-γ, IL-4, IL-17 and IL-10 were determined in serum samples before IFV challenge (day 0) and at the indicated time points after viralinfection. The results represent data from three independent experiments. Results are expressed as mean ± SD. Asterisks indicate significant differences(*P < 0.05) when compared to control mice.
0
200
400
600
800Serum IgG
ng/m
l
BAL IgA
ng/m
l
0
200
400
600
800
* * *
**
Control Lr1505 Lr027 HK027HK1505
+ INFLUENZA VACCINE
Control Lr1505 Lr027 HK027HK1505
+ INFLUENZA VACCINE
Fig. 6. Effect of lactobacilli on the humoral immune response induced by the immunization with influenza virus (IFV) vaccine. Mice were nasally immunized with acommercial IFV vaccine alone or with viable or heat-killed Lactobacillus rhamnosus CRL1505 (Lr1505 and HK1505, respectively) or Lactobacillus rhamnosus IBL027(Lr027 and HK027, respectively) as adjuvants, on days 0, 14 and 28. Seven days after the last immunization, the concentrations of anti-IFV vaccine antibodies inbroncho-alveolar lavage (BAL) and serum samples were evaluated. The results represent data from three independent experiments. Results are expressed asmean ± SD. Asterisks indicate significant differences (*P < 0.05, **P < 0.01) when compared to control mice.
F.R. Tonetti, et al. International Immunopharmacology 78 (2020) 106115
7
Interestingly, we demonstrated here for the first time that the nasaladministration of the immunobiotic strain L. rhamnosus CRL1505 sig-nificantly improved the adaptive immune response against IFV infec-tion.
Both, viable and non-viable L. rhamnosus CRL1505 were able toimprove the production of respiratory and systemic specific antibodiesafter IFV challenge. The treatments were equally effective for enhan-cing the humoral adaptive immune response since the levels of anti-IFVBAL IgA and serum IgG were significantly higher than the observed incontrol animals and, with no differences between them. The improved
humoral response induced by the CRL1505 strain could be associated tothe significant reduction of viral titers, body weight loss, and a decreaseof the alterations of physical conditions induced by IFV reported pre-viously [9,10]. Our results are in agreement with early studies of Yasuiet al. [5], which reported that the oral administration of B. breveYIT4064 improved the production of anti-IFV IgG antibodies in serumof IFV-infected mice and reduced viral titers, improved the survivalrate, and decreased the severity of the symptoms associated to the IFVinfection. Similarly, it was shown that orally administered non-viable L.pentosus b240 [22], viable L. brevis KB290 [23] or viable L. plantarum
0
200
400
600
Control Lr1505 Lr027 HK027HK1505
LN IFN-
pg/m
l
0
200
400
600
800
1000
Control Lr1505 Lr027 HK027HK1505
Spleen IFN-
pg/m
l
0
50
100
150
200 MLN IL-17
pg/m
l
0
100
200
300 Spleen IL-17
pg/m
l
0
100
200
300
400MLN IL-4
pg/m
l
0
100
200
300
400
500Spleen IL-4
pg/m
l
+ INFLUENZA VACCINE + INFLUENZA VACCINE
Control Lr1505 Lr027 HK027HK1505 Control Lr1505 Lr027 HK027HK1505
+ INFLUENZA VACCINE + INFLUENZA VACCINE
Control Lr1505 Lr027 HK027HK1505 Control Lr1505 Lr027 HK027HK1505
+ INFLUENZA VACCINE + INFLUENZA VACCINE
*** *
***
* * *
** ** **
** ** *
Fig. 7. Effect of lactobacilli on the cellular immune response induced by the immunization with influenza virus (IFV) vaccine. Mice were nasally immunized with acommercial IFV vaccine alone or with viable or heat-killed Lactobacillus rhamnosus CRL1505 (Lr1505 and HK1505, respectively) or Lactobacillus rhamnosus IBL027(Lr027 and HK027, respectively) as adjuvants, on days 0, 14 and 28. Seven days after the last immunization, mononuclear cells from spleen and mesenteric lymphoidnodes (MLN) were isolated and re-stimulated in vitro with the IFV vaccine. The concentrations of IFN-γ, IL-4, and IL-17 were determined in culture supernatants. Theresults represent data from three independent experiments. Results are expressed as mean ± SD. Asterisks indicate significant differences (*P < 0.05, **P < 0.01)when compared to control mice.
F.R. Tonetti, et al. International Immunopharmacology 78 (2020) 106115
8
AYA [24] were able to improve the levels of respiratory specific IgA andIgG antibodies of mice challenged with IFV and limit viral replication. Itshould be noted that in those studies, the immunobiotic treatmentswere administered by the oral route. Then, to our knowledge, this is thefirst study demonstrating that the nasal administration of lactobacillibeneficially influence the humoral immune response against IFV. Itwould be interesting to find out whether orally administered L. rham-nosus CRL1505 is able to induce this beneficial effect and whether it isas effective as the nasal treatment.
It was reported that immunobiotics are also able to enhance thecellular immune response against IFV. Orally administered L. caseiShirota improved the outcomes of IFV infection of aged [6] and infantmice [7] by increasing systemic and respiratory NK cell activity and theproduction of IFN-γ and TNF-α by respiratory lymphocytes. Severalother studies corroborated those findings by showing similar effects fororally administered lactobacilli. L. gasseri TMC0356, L. rhamnosus GG,or L. plantarum 06CC2 beneficially modulated NK cells activity and Th1response against IFV, diminished virus titers and reduced lung patho-logical changes [25,26]. In addition, it was suggested that the nasaladministration of immunobiotics would be a more efficient alternativeto improve the respiratory cellular immune response against influenza
infection [8,27,28]. Hori et al. [27] observed that BALB/c mice nasallytreated with non-viable L. casei Shirota had increased levels of IL-12,IFN-γ and TNF-α in MLN and lungs. This improved cellular respiratoryimmunity correlated with a beneficial clinical outcome to IFV chal-lenge. Similar observations were performed with nasally administeredL. pentosus S-PT84 [28] and L. rhamnosus GG [8]. In line with thoseprevious findings, we showed that L. rhamnosus CRL1505 was able toimprove the cellular immune response against IFV infection. CRL1505-treated mice had higher levels of IFN-γ in serum and the respiratorytract after IFV challenge.
Interestingly, lactobacilli treated mice showed reduced concentra-tions of IL-17 and improved levels of IL-10 during IFV infection. IL-17 isa potent pro-inflammatory cytokine that plays a crucial role in hostdefenses against diverse pulmonary pathogens. It has been reportedthat IL-17 may act as a ‘double-edged sword' contributing to bothprotection and pulmonary immunopathology during IFV infection.Elevated IL-17 serum levels have been found in patients with severelypandemic H1N1 IFV infections, which may potentiate lung inflamma-tion and its fatal consequence [29]. On the other hand, induction of IL-10 was described following infection with IFV [30]. It has been re-ported that effector CD4+ T cells co-producing IFN-γ in the lung
Survival
Days post-infection
Perc
enta
ge
0 5 10 150
50
100
150
Control Lr1505 Lr027 HkLr1505 HkLr027
0 2 4 60
2
4
6
8
Days post-infection
Virus Titre
PFU
/g
Fig. 8. Effect of lactobacilli on the resistance to influenza virus (IFV) infection induced by the immunization with IFV vaccine. Mice were nasally immunized with acommercial IFV vaccine alone or with viable or heat-killed Lactobacillus rhamnosus CRL1505 (Lr1505 and HK1505, respectively) or Lactobacillus rhamnosus IBL027(Lr027 and HK027, respectively) as adjuvants, on days 0, 14 and 28. Seven days after the last immunization, mice were challenged with IFV. Survival and IFV titers inlungs were determined at the indicated time points. The results represent data from three independent experiments. Results are expressed as mean ± SD. Asterisksindicate significant differences (*P < 0.05) when compared to control mice.
0 2 4 6 80
100
200
300
400
Days post-infection
pg /
mL
BAL IFN-
0 2 4 6 80
50
100
150
200
250
Days post-infection
pg /
mL
BAL TNF-
0 2 4 6 8250
300
350
400
450
500
Days post-infection
pg /
mL
BAL IL-10
Control Lr1505 Lr027 HK027HK1505
**
**
*
*
***
**
** **
**
Fig. 9. Effect of lactobacilli on the respiratory immune response to influenza virus (IFV) infection induced by the immunization with IFV vaccine. Mice were nasallyimmunized with a commercial IFV vaccine alone or with viable or heat-killed Lactobacillus rhamnosus CRL1505 (Lr1505 and HK1505, respectively) or Lactobacillusrhamnosus IBL027 (Lr027 and HK027, respectively) as adjuvants, on days 0, 14 and 28. Seven days after the last immunization, mice were challenged with IFV. Theconcentrations of IFN-γ, TNF-α and IL-10 were determined in broncho-alveolar lavage (BAL) samples at the indicated time points. The results represent data fromthree independent experiments. Results are expressed as mean ± SD. Asterisks indicate significant differences (*P < 0.05, **P < 0.01) when compared to controlmice.
F.R. Tonetti, et al. International Immunopharmacology 78 (2020) 106115
9
represent the most abundant source of IL-10 during primary infection,and that IL-10-producing cells amongst effector T cells serve to dampenproduction of Th17-associated cytokines [30]. Blocking IL-10 signalingcaused increased morbidity in IFV-infected mice, and administration ofIL-10 blocking antibodies correlated with a broad increase in lung in-flammation [31]. Then, the differential balance of inflammatory andregulatory cytokines induced by the CRL1505 strain could contribute tothe protection against IFV by favoring an effective effector immuneresponse without inducing inflammatory-mediated lung damage.
Previously, we reported that nasally administered L. rhamnosusCRL1505 is able to improve the levels of type I IFNs in the respiratorytract and to increase the expression of activation markers in respiratorydendritic cells (DCs) [15,10,9]. Studies have demonstrated that DCsboth produce IFN-α/β and undergo maturation in response to type IIFNs. Moreover, IFN-α/β have been shown to potently enhance immuneresponses in vivo through the stimulation of DCs serving as a signallinking between innate and adaptive immunity [32]. Treatment withtype I IFNs activates in vitro derived DCs, enhancing and modulatingtheir ability to initiate T-cell responses. Type I IFNs-treated DCs haveenhanced expression of MHC-II, CD40 and CD86 molecules, and ahigher ability to stimulate CD4+ and CD8+ T cells that produce IFN-γ[33,34]. In addition, it has been reported that type I IFNs promoteantibody responses in vivo and that stimulation of DCs by type I IFNsplays a role in this adjuvant activity [35]. Type I IFNs were shown toenhance the production of antigen-specific antibodies of all subclassesof IgG and induced IgG2a and IgG3 antibodies far more effectively thanwidely used adjuvants. Type I IFN have been used in combination withIFV vaccines to immunize mice via intranasal administration, and thisvaccination protocols induced an effective humoral response [36].Then, it is tempting to speculate that the ability of L. rhamnosusCRL1505 to stimulate the adaptive immunity would be related to itsability to modulate antigen presentation in respiratory DCs through theimprovement of type I IFNs. Moreover, this hypothesis prompted us tostudy whether nasally administered CRL1505 strain was able to modifythe immune response to an IFV vaccine.
There is an urgent need to develop novel influenza vaccines that caninduce efficient mucosal immunity in the respiratory tract in order toprovide some level of protection against the first replication of the IFVwhen it reaches a new host [2]. Most of the marketed influenza vaccinescan be administered safely via nasal route to the immunocompetentpopulation without risk. Disadvantages of these vaccines are that theyhave shown to be poorly immunogenic when administered via thismucosal route and that they can have detrimental effects in im-munocompromised host [37,38]. Then, identification of novel ad-juvants with the potential to improve mucosal immune responses to IFVvaccines has been one of the major goals of vaccine researchers. Weshowed here that the nasal administration of a commercially availableinactivated IFV vaccine together with viable or non-viable lactobacillisignificantly improved the adaptive immune response and the protec-tion against the respiratory pathogen. Immunization protocols usingIFV vaccine co-administered with viable or non-viable im-munomodulatory lactobacilli enhanced both specific antibodies pro-duction and the cellular-mediated immunity. Of note, viable L. rham-nosus CRL1505 was more efficient than non-viable lactobacilli toimprove IFN-γ production in response to vaccine and IFV challenge.This effect could be related to the higher ability of viable bacteria tointeract and stimulate antigen presenting cells in the respiratory tract,and the subsequent generation of Th1 response.
The levels of anti-IFV antibodies in serum whether elicited bynaturally acquired infection or through vaccination, have long beenconsidered a correlate of protection against clinical influenza. In ad-dition, it has been demonstrated that specific cellular-mediated im-munity plays a significant role in the protection against community-acquired clinical IFV influenza virus infection, especially in youngchildren [39]. Moreover, in studies characterizing the immune responsefollowing intranasal administration of monovalent live attenuated IFV
vaccines, cellular-mediated immunity has been considered to have arole in protection in adults and children that could not be entirely ex-plained by mucosal or serum antibody responses [40]. Then, our resultsindicate that lactobacilli could have a possible application in the de-velopment of new IFV vaccine formulations because of their beneficialeffects of the induction of antibody-mediated and T-cell-mediated im-munity by vaccination and in the amelioration of influenza infectionseverity.
Studies in which the ability of the intramuscular inactivated vaccineand the intranasal live attenuated influenza vaccine were compareddemonstrated that both types of vaccines were safe in im-munocompetent children and able to elicit similar humoral immuneresponses directed against IFV antigens [41–44]. In contrast, only liveattenuated influenza vaccination was shown to induce IFV-specific Tcell responses relevant for cell-mediated immune protection. Studies inelderly individuals also have shown that attenuated IFV vaccine inducesbetter heterosubtypic immunity than the inactivated vaccine, in termsof both humoral and cellular immune responses [45]. However, itshould considered that live attenuated IFV vaccine is not recommendedin elderly, immunosuppressed subjects or children under 2 years of age,as, in early investigations, the administration of this vaccine to this age-group promoted the onset of wheezing (For a review see [46]). Of in-terest, we observed that L. rhamnosus CRL1505 and L. rhamnosusIBL027 improved the generation of cell-mediated immunity induced bymucosal administered inactivated IFV vaccine. Considering that, lac-tobacilli are recognized as safe microorganisms, vaccine formulationsusing them as adjuvants together with inactivated IFV emerge as aninteresting alternative for the protection of immunocompromised hostsagainst influenza. Moreover, although our results showed that viableand non-viable lactobacilli may have different adjuvant abilities,especially when the induction of T cell responses were evaluated, theuse of non-viable lactobacilli represents an alternative that can improvethe response to vaccination and which can also reduce the concernsassociated with the use of live microorganisms in immunocompromisedhosts.
In conclusion, we demonstrated in this work that the immunobioticstrain L. rhamnosus CRL1505 is able to improve both the humoral andcellular adaptive immune responses induced by IFV infection or vac-cination. The optimal immunomodulatory effect of the CRL1505 strainwas achieved with viable bacteria. However, non-viable L. rhamnosusCRL1505 was also efficient in improving the adaptive immune re-sponses generated by IFV challenges and therefore, emerged as an in-teresting alternative for vaccination of immunocompromised hosts.Similar to other immunomodulatory properties of lactobacilli, it wasshown here that the adjuvant effect in the context of IFV vaccinationwas a strain dependent ability, since differences were found when L.rhamnosus CRL1505 and L. rhamnosus IBL027 were compared. Theseresults imply that thorough studies should be carried out to find thelactobacilli strains with the best adjuvant properties. This investigationrepresents a thorough exploration of the role of immunobiotic lacto-bacilli in improving humoral and cellular adaptive immune responsesagainst IFV in the context of both infection and vaccination.
F.R. Tonetti, et al. International Immunopharmacology 78 (2020) 106115
10
Acknowledgements
This study was supported by ANPCyT–FONCyT Grants PICT-2013-3219 and PICT-2016-0410 to Julio Villena. This work was also sup-ported by a Grant-in-Aid for Scientific Research (A) (19H00965) andOpen Partnership Joint Projects of JSPS Bilateral Joint ResearchProjects from the Japan Society for the Promotion of Science (JSPS),and by grants from the project of NARO Bio-oriented TechnologyResearch Advancement Institution (Research Program on developmentof innovation technology, No. 01002A) to Haruki Kitazawa, and bygrants for “Scientific Research on Innovative Areas” from the Ministryof Education, Culture, Science, Sports and Technology (MEXT) of Japan(16H06429, 16K21723 and 16H06435) to Hideki Takahashi. MdAminul Islam was supported by JSPS (Postdoctoral Fellowship forForeign Researchers, Program No. 18F18081). This study was alsosupported by the JSPS Core-to-Core Program Advanced ResearchNetworks “Establishment of international agricultural immunology re-search-core for a quantum improvement in food safety”.
Declaration of Competing Interest
The authors declare that they have no competing interests.
[2] S.V. Vemula, E.E. Sayedahmed, S. Sambhara, S.K. Mittal, Vaccine approachesconferring cross-protection against influenza viruses, Expert. Rev. Vaccines 16 (11)(2017) 1141–1154, https://doi.org/10.1080/14760584.2017.1379396.
[3] A. Ainai, T. Suzuki, S.I. Tamura, H. Hasegawa, Intranasal administration of wholeinactivated influenza virus vaccine as a promising influenza vaccine candidate,Viral Immunol. 30 (6) (2017) 451–462, https://doi.org/10.1089/vim.2017.0022.
[4] H. Zelaya, S. Alvarez, H. Kitazawa, J. Villena, Respiratory antiviral immunity andimmunobiotics: beneficial effects on inflammation-coagulation interaction duringinfluenza virus infection, Front. Immunol. 7 (2016) 633, https://doi.org/10.3389/fimmu.2016.00633.
[5] H. Yasui, J. Kiyoshima, T. Hori, K. Shida, Protection against influenza virus infec-tion of mice fed Bifidobacterium breve YIT4064, Clin. Diagn. Lab. Immunol. 6 (2)(1999) 186–192.
[6] T. Hori, J. Kiyoshima, K. Shida, H. Yasui, Augmentation of cellular immunity andreduction of influenza virus titer in aged mice fed Lactobacillus casei strain Shirota,Clin. Diagn. Lab. Immunol. 9 (1) (2002) 105–108, https://doi.org/10.1128/cdli.9.1.105-108.2002.
[7] H. Yasui, J. Kiyoshima, T. Hori, Reduction of influenza virus titer and protectionagainst influenza virus infection in infant mice fed Lactobacillus casei Shirota, Clin.Diagn. Lab. Immunol. 11 (4) (2004) 675–679, https://doi.org/10.1128/CDLI.11.4.675-679.2004.
[8] G. Harata, F. He, N. Hiruta, M. Kawase, A. Kubota, M. Hiramatsu, H. Yausi,Intranasal administration of Lactobacillus rhamnosus GG protects mice from H1N1influenza virus infection by regulating respiratory immune responses, Lett. Appl.Microbiol. 50 (6) (2010) 597–602, https://doi.org/10.1111/j.1472-765X.2010.02844.x.
[9] H. Zelaya, K. Tsukida, E. Chiba, G. Marranzino, S. Alvarez, H. Kitazawa, G. Aguero,J. Villena, Immunobiotic lactobacilli reduce viral-associated pulmonary damagethrough the modulation of inflammation-coagulation interactions, Int.Immunopharmacol. 19 (1) (2014) 161–173, https://doi.org/10.1016/j.intimp.2013.12.020.
[10] H. Zelaya, A. Tada, M.G. Vizoso-Pinto, S. Salva, P. Kanmani, G. Aguero, S. Alvarez,H. Kitazawa, J. Villena, Nasal priming with immunobiotic Lactobacillus rhamnosusmodulates inflammation-coagulation interactions and reduces influenza virus-as-sociated pulmonary damage, Inflamm. Res. 64 (8) (2015) 589–602, https://doi.org/10.1007/s00011-015-0837-6.
[11] M. Bermudez-Brito, J. Plaza-Diaz, S. Munoz-Quezada, C. Gomez-Llorente, A. Gil,Probiotic mechanisms of action, Ann. Nutr. Metab. 61 (2) (2012) 160–174, https://doi.org/10.1159/000342079.
[12] S. Lebeer, J. Vanderleyden, S.C. De Keersmaecker, Host interactions of probioticbacterial surface molecules: comparison with commensals and pathogens, Nat. Rev.Microbiol. 8 (3) (2010) 171–184, https://doi.org/10.1038/nrmicro2297.
[13] H. Kitazawa, J. Villena, Modulation of respiratory TLR3-anti-viral response byprobiotic microorganisms: lessons learned from Lactobacillus rhamnosus CRL1505,Front. Immunol. 5 (2014) 201, https://doi.org/10.3389/fimmu.2014.00201.
[14] J. Villena, H. Kitazawa, Modulation of intestinal TLR4-inflammatory signalingpathways by probiotic microorganisms: lessons learned from Lactobacillus jenseniiTL2937, Front. Immunol. 4 (2014) 512, https://doi.org/10.3389/fimmu.2013.00512.
[15] P. Clua, P. Kanmani, H. Zelaya, A. Tada, A. Kober, S. Salva, S. Alvarez, H. Kitazawa,
J. Villena, Peptidoglycan from Immunobiotic Lactobacillus rhamnosus ImprovesResistance of Infant Mice to Respiratory Syncytial Viral Infection and SecondaryPneumococcal Pneumonia, Front. Immunol. 8 (2017) 948, https://doi.org/10.3389/fimmu.2017.00948.
[16] Y. Tomosada, E. Chiba, H. Zelaya, T. Takahashi, K. Tsukida, H. Kitazawa,S. Alvarez, J. Villena, Nasally administered Lactobacillus rhamnosus strains dif-ferentially modulate respiratory antiviral immune responses and induce protectionagainst respiratory syncytial virus infection, BMC Immunol 14 (2013) 40, https://doi.org/10.1186/1471-2172-14-40.
[17] P. Kanmani, P. Clua, M.G. Vizoso-Pinto, C. Rodriguez, S. Alvarez, V. Melnikov,H. Takahashi, H. Kitazawa, J. Villena, respiratory commensal bacteria cor-ynebacterium pseudodiphtheriticum improves resistance of infant mice to re-spiratory syncytial virus and Streptococcus pneumoniae Superinfection, Front.Microbiol. 8 (2017) 1613, https://doi.org/10.3389/fmicb.2017.01613.
[18] A. Iwasaki, Exploiting mucosal immunity for antiviral vaccines, Annu. Rev.Immunol. 34 (2016) 575–608, https://doi.org/10.1146/annurev-immunol-032414-112315.
[19] L. Favre, F. Spertini, B. Corthesy, Secretory IgA possesses intrinsic modulatoryproperties stimulating mucosal and systemic immune responses, J. Immunol. 175(5) (2005) 2793–2800, https://doi.org/10.4049/jimmunol.175.5.2793.
[20] C. Chiu, A.H. Ellebedy, J. Wrammert, R. Ahmed, B cell responses to influenza in-fection and vaccination, Curr. Top. Microbiol. Immunol. 386 (2015) 381–398,https://doi.org/10.1007/82_2014_425.
[21] T.J. Braciale, J. Sun, T.S. Kim, Regulating the adaptive immune response to re-spiratory virus infection, Nat. Rev. Immunol. 12 (4) (2012) 295–305, https://doi.org/10.1038/nri3166.
[22] N. Kobayashi, T. Saito, T. Uematsu, K. Kishi, M. Toba, N. Kohda, T. Suzuki, Oraladministration of heat-killed Lactobacillus pentosus strain b240 augments protec-tion against influenza virus infection in mice, Int. Immunopharmacol. 11 (2) (2011)199–203, https://doi.org/10.1016/j.intimp.2010.11.019.
[23] N. Waki, N. Yajima, H. Suganuma, B.M. Buddle, D. Luo, A. Heiser, T. Zheng, Oraladministration of Lactobacillus brevis KB290 to mice alleviates clinical symptomsfollowing influenza virus infection, Lett. Appl. Microbiol. 58 (1) (2014) 87–93,https://doi.org/10.1111/lam.12160.
[24] Y. Kikuchi, A. Kunitoh-Asari, K. Hayakawa, S. Imai, K. Kasuya, K. Abe, Y. Adachi,S. Fukudome, Y. Takahashi, S. Hachimura, Oral administration of Lactobacillusplantarum strain AYA enhances IgA secretion and provides survival protectionagainst influenza virus infection in mice, PLoS One 9 (1) (2014) e86416, , https://doi.org/10.1371/journal.pone.0086416.
[25] M. Kawase, F. He, A. Kubota, G. Harata, M. Hiramatsu, Oral administration oflactobacilli from human intestinal tract protects mice against influenza virus in-fection, Lett. Appl. Microbiol. 51 (1) (2010) 6–10, https://doi.org/10.1111/j.1472-765X.2010.02849.x.
[26] S. Takeda, M. Takeshita, Y. Kikuchi, B. Dashnyam, S. Kawahara, H. Yoshida,W. Watanabe, M. Muguruma, M. Kurokawa, Efficacy of oral administration of heat-killed probiotics from Mongolian dairy products against influenza infection in mice:alleviation of influenza infection by its immunomodulatory activity through in-testinal immunity, Int. Immunopharmacol. 11 (12) (2011) 1976–1983, https://doi.org/10.1016/j.intimp.2011.08.007.
[27] T. Hori, J. Kiyoshima, K. Shida, H. Yasui, Effect of intranasal administration ofLactobacillus casei Shirota on influenza virus infection of upper respiratory tract inmice, Clin. Diagn. Lab. Immunol. 8 (3) (2001) 593–597, https://doi.org/10.1128/CDLI.8.3.593-597.2001.
[28] T. Izumo, T. Maekawa, M. Ida, A. Noguchi, Y. Kitagawa, H. Shibata, H. Yasui,Y. Kiso, Effect of intranasal administration of Lactobacillus pentosus S-PT84 oninfluenza virus infection in mice, Int. Immunopharmacol. 10 (9) (2010) 1101–1106,https://doi.org/10.1016/j.intimp.2010.06.012.
[29] J.F. Bermejo-Martin, R. Ortiz de Lejarazu, T. Pumarola, J. Rello, R. Almansa,P. Ramirez, I. Martin-Loeches, D. Varillas, M.C. Gallegos, C. Seron, D. Micheloud,J.M. Gomez, A. Tenorio-Abreu, M.J. Ramos, M.L. Molina, S. Huidobro, E. Sanchez,M. Gordon, V. Fernandez, A. Del Castillo, M.A. Marcos, B. Villanueva, C.J. Lopez,M. Rodriguez-Dominguez, J.C. Galan, R. Canton, A. Lietor, S. Rojo, J.M. Eiros,C. Hinojosa, I. Gonzalez, N. Torner, D. Banner, A. Leon, P. Cuesta, T. Rowe,D.J. Kelvin, Th1 and Th17 hypercytokinemia as early host response signature insevere pandemic influenza, Crit. Care 13 (6) (2009) R201, https://doi.org/10.1186/cc8208.
[30] K.K. McKinstry, T.M. Strutt, A. Buck, J.D. Curtis, J.P. Dibble, G. Huston, M. Tighe,H. Hamada, S. Sell, R.W. Dutton, S.L. Swain, IL-10 deficiency unleashes an influ-enza-specific Th17 response and enhances survival against high-dose challenge, J.Immunol. 182 (12) (2009) 7353–7363, https://doi.org/10.4049/jimmunol.0900657.
[31] J. Sun, R. Madan, C.L. Karp, T.J. Braciale, Effector T cells control lung inflammationduring acute influenza virus infection by producing IL-10, Nat. Med. 15 (3) (2009)277–284, https://doi.org/10.1038/nm.1929.
[32] A. Le Bon, D.F. Tough, Links between innate and adaptive immunity via type Iinterferon, Curr. Opin. Immunol. 14 (4) (2002) 432–436, https://doi.org/10.1016/s0952-7915(02)00354-0.
[33] M. Montoya, G. Schiavoni, F. Mattei, I. Gresser, F. Belardelli, P. Borrow, D.F. Tough,Type I interferons produced by dendritic cells promote their phenotypic andfunctional activation, Blood 99 (9) (2002) 3263–3271, https://doi.org/10.1182/blood.v99.9.3263.
[35] A. Le Bon, G. Schiavoni, G. D'Agostino, I. Gresser, F. Belardelli, D.F. Tough, Type iinterferons potently enhance humoral immunity and can promote isotype switching
F.R. Tonetti, et al. International Immunopharmacology 78 (2020) 106115
by stimulating dendritic cells in vivo, Immunity 14 (4) (2001) 461–470, https://doi.org/10.1016/s1074-7613(01)00126-1.
[36] L. Bracci, I. Canini, S. Puzelli, P. Sestili, M. Venditti, M. Spada, I. Donatelli,F. Belardelli, E. Proietti, Type I IFN is a powerful mucosal adjuvant for a selectiveintranasal vaccination against influenza virus in mice and affects antigen capture atmucosal level, Vaccine 23 (23) (2005) 2994–3004, https://doi.org/10.1016/j.vaccine.2004.12.006.
[37] R.L. Atmar, W.A. Keitel, T.R. Cate, F.M. Munoz, F. Ruben, R.B. Couch, A dose-response evaluation of inactivated influenza vaccine given intranasally and in-tramuscularly to healthy young adults, Vaccine 25 (29) (2007) 5367–5373, https://doi.org/10.1016/j.vaccine.2007.05.002.
[38] J.E. Eyles, E.D. Williamson, H.O. Alpar, Intranasal administration of influenzavaccines: current status, BioDrugs 13 (1) (2000) 35–59, https://doi.org/10.2165/00063030-200013010-00005.
[39] B.D. Forrest, M.W. Pride, A.J. Dunning, M.R. Capeding, T. Chotpitayasunondh,J.S. Tam, R. Rappaport, J.H. Eldridge, W.C. Gruber, Correlation of cellular immuneresponses with protection against culture-confirmed influenza virus in young chil-dren, Clin. Vaccine Immunol. 15 (7) (2008) 1042–1053, https://doi.org/10.1128/CVI.00397-07.
[40] R.B. Belshe, W.C. Gruber, P.M. Mendelman, H.B. Mehta, K. Mahmood, K. Reisinger,J. Treanor, K. Zangwill, F.G. Hayden, D.I. Bernstein, K. Kotloff, J. King, P.A. Piedra,S.L. Block, L. Yan, M. Wolff, Correlates of immune protection induced by live, at-tenuated, cold-adapted, trivalent, intranasal influenza virus vaccine, J. Infect. Dis.181 (3) (2000) 1133–1137, https://doi.org/10.1086/315323.
[41] X.S. He, T.H. Holmes, C. Zhang, K. Mahmood, G.W. Kemble, D.B. Lewis,C.L. Dekker, H.B. Greenberg, A.M. Arvin, Cellular immune responses in childrenand adults receiving inactivated or live attenuated influenza vaccines, J. Virol. 80
(23) (2006) 11756–11766, https://doi.org/10.1128/JVI.01460-06.[42] D.F. Hoft, E. Babusis, S. Worku, C.T. Spencer, K. Lottenbach, S.M. Truscott,
G. Abate, I.G. Sakala, K.M. Edwards, C.B. Creech, M.A. Gerber, D.I. Bernstein,F. Newman, I. Graham, E.L. Anderson, R.B. Belshe, Live and inactivated influenzavaccines induce similar humoral responses, but only live vaccines induce diverse T-cell responses in young children, J. Infect. Dis. 204 (6) (2011) 845–853, https://doi.org/10.1093/infdis/jir436.
[43] R.A. Subbramanian, S. Basha, M.T. Shata, R.C. Brady, D.I. Bernstein, Pandemic andseasonal H1N1 influenza hemagglutinin-specific T cell responses elicited by sea-sonal influenza vaccination, Vaccine 28 (52) (2010) 8258–8267, https://doi.org/10.1016/j.vaccine.2010.10.077.
[44] A.M. Zeman, T.H. Holmes, S. Stamatis, W. Tu, X.S. He, N. Bouvier, G. Kemble,H.B. Greenberg, D.B. Lewis, A.M. Arvin, C.L. Dekker, Humoral and cellular immuneresponses in children given annual immunization with trivalent inactivated influ-enza vaccine, Pediatr. Infect. Dis. J. 26 (2) (2007) 107–115, https://doi.org/10.1097/01.inf.0000253251.03785.9b.
[45] G.J. Gorse, R.B. Belshe, Enhancement of anti-influenza A virus cytotoxicity fol-lowing influenza A virus vaccination in older, chronically ill adults, J. Clin.Microbiol. 28 (11) (1990) 2539–2550.
[47] L.P. Arce, M.F. Raya Tonetti, M.P. Raimondo, M.F. Müller, S. Salva, S. Alvarez,A. Baiker, J. Villena, M.G. Vizoso Pinto, Oral vaccination with Hepatitis E Viruscapsid protein and immunobiotic bacterium-like particles induce intestinal andsystemic immunity in mice, In press, Probiotics Antimicrob. Proteins (2019),https://doi.org/10.1007/s12602-019-09598-7.
F.R. Tonetti, et al. International Immunopharmacology 78 (2020) 106115
