Despite advances in medicine, RVI (such as the common cold or flu) continue to cause a considerable economic burden; fortunately, some probiotic strains have been studied for their positive effects on certain infectious diseases, and in the last 5 years, their use to prevent and treat RVI has significantly increased (11
). There is thus a clear interest in the identification and characterization of new candidate strains with well-demonstrated probiotic properties against RVI.
In this study, we determined the immunomodulatory properties of 158 strains of LAB in order to identify the most interesting candidate probiotic strain able to alleviate RVI symptoms. For this, we first performed a large-scale screening of the 158 strains for their resistance to bile salts. Indeed, bile salts may constitute a deleterious factor preventing a given strain from exerting its beneficial properties in vivo. A high-throughput method was used to rapidly characterize strain resistance. Among the tested strains, L. plantarum CNRZ1997 had a delay of 2.9 h. Eighty strains (62.5% of the collection), including VEL12208 and VEL12195, showed moderate resistance, with a growth delay of between 5 and 13 h. Eight strains were not resistant to bile salts. This simple yet robust protocol could also be used to test other properties, such as other stresses or the ability to grow under specific conditions.
The immunomodulatory effects of the bacterial collection were then assessed by using two cellular models: TNF-α-activated HT-29 cells and peripheral blood mononuclear cells (PBMCs). These in vitro
cellular models are commonly used to validate the immunomodulatory properties (anti- or proinflammatory cytokine profile) of a given candidate bacterium before validation in animal models. Indeed, these tests have a predictive value and allow a reduction of the number of bacterial strains to be tested in animal models, which are expensive and time-consuming. One of these predictive in vitro
models is PBMCs, in which the immunomodulation potential of probiotic strains is usually assessed by measuring IL-10 and IL-12 cytokine levels after stimulation with live bacterial strains. In this manner, Foligne et al. (9
) successfully established a correlation between this PBMC model, the in vivo
immunomodulation potential of probiotic strains, and the ability to prevent experimental colitis in mice. Bacteria inducing higher levels of the anti-inflammatory cytokine IL-10 and lower levels of the proinflammatory cytokine IL-12 in vitro
displayed the best protection in the murine colitis model (15
). The human intestinal epithelial cell line HT-29 has been also used successfully to evaluate the immunomodulatory properties of bacteria (25
). Once coincubation with bacterial strains was achieved, IL-8 quantification led to the selection of anti-inflammatory bacteria (reducing IL-8 production) and proinflammatory bacteria (enhancing IL-8 production) (25
). After we tested our bacterial collection with these two cellular models, we selected 3 different responsive strains according to their immunomodulatory profile, one with a highly proinflammatory profile (L. plantarum
CNRZ1997), one with a weakly proinflammatory profile (L. brevis
VEL12208), and one with a markedly anti-inflammatory profile (L. paracasei
VEL12195), for in vivo
experiments using a model of A/PR8/34 strain infection in mice. Based on the preliminary results observed with a high dose of IAV (2,000 PFU), we found that L. plantarum
strain CNRZ1997 (highly proinflammatory profile) was the most interesting candidate among the three candidate strains tested, with beneficial effects against IAV. The results obtained with a less challenging model (lower dose of IAV of 100 PFU) as well as with the five-point scale of visual scores confirmed these observations. Indeed, this strain was effective in preventing body weight loss () and clinical condition alterations by alleviating significantly the symptoms of the infected mice (P
< 0.05) (). Kawase et al. (28
) previously found similar results (i.e., improved clinical symptoms) when studying the oral administration of two LAB strains, L. rhamnosus
GG and L. gasseri
TMC0356, in a murine model of IAV infection.
In addition, when we analyzed virus titration in lungs of infected mice at 10 and 14 days postinfection, we found that strain CNRZ1997 was able to decrease virus titers in these infected mice. This result indicates a positive effect of strain CNRZ1997 in mice by perhaps accelerating IAV elimination from the lungs. These results are similar to those obtained previously by Kawashima et al. (29
), where L. plantarum
strain LpYU (with a proinflammatory profile observed in vitro
) was tested in a model of virus infection with the H1N1 virus (A/NWS/33).
In this work, we decided to use strains GG and DN114-001 as positive controls in our model of IAV, since they have already been successfully studied for their anti-IAV properties in preclinical trials (23
) and in a human clinical trial against RVI (13
). Interestingly, the AUWC results observed for mice treated with either GG or DN114-001 showed that there are no protective effects of these two well-known probiotic strains in our IAV model. These unexpected results can be explained by the fact that strain GG was reported previously to be protective against IAV in mice after “intranasal” administration, in contrast with our study, where we used an “intragastric” route. In order to confirm this, we performed experiments with L. rhamnosus
GG by the intranasal route in our IAV model, and we confirmed a protective effect; however, we cannot favor this route, since the aim of this study was to develop a probiotic oral dietary supplement. Second, most trials using strain DN114-001 against IAV have been done with the fermented final product (which means that it contained L. casei
strain DN114-001 combined with two cultures commonly used in yogurt, Streptococcus thermophilus
and Lactobacillus bulgaricus
), while in our study, we used DN114-001 as a single isolated strain, so we cannot discard a synergistic effect of strain DN114-001 and the fermented final product.
The effects of probiotic bacteria against infectious diseases, such as IAV, have been demonstrated, but the mechanisms of action are not yet fully understood; however, some hypotheses have been advanced. For example, oral administration of acidic exopolysaccharide extracts of L. delbrueckii
OLL1073R-1 to mice infected with IAV provided protection (30
). In another study, the protective properties of L. plantarum
strain LpYU against IAV were dependent on Toll-like receptor 2 (TLR2), which recognizes the peptidoglycan (PG) and the lipoteichoic acids (LTA) of Gram-positive bacteria (29
). These two results seem interesting to explore in order to identify the mechanisms of action of our proinflammatory strain CNRZ1997.
Most of the studies using LAB to prevent and treat IAV infection have been performed with intranasal administration of either live or heat-killed bacteria (15
). In this study, we chose to test live bacteria administered orally, in order to develop a potential probiotic supplement for use in humans. Of note, parallel research demonstrated that the proinflammatory L. plantarum
strain CNRZ1997 is compatible with all manufacturing and formulation technological processes (data not shown), and currently, a double-blind, randomized, controlled trial is in progress in order to investigate whether the consumption of this strain influences the severity of symptoms and the incidence and duration of common cold infections. In conclusion, our results suggest the feasibility of a large in vitro
screening of bacterial strains using two cellular models (TNF-α-activated HT-29 cells and PBMCs), in order to determine their immunomodulatory properties based on a cytokine profile. Oral administration of the most proinflammatory strain (L. plantarum
CNRZ1997) conferred protection against IAV infection. Further research is being conducted in our laboratory to identify the mechanism of action of this new candidate probiotic bacterium.