Modulation of human respiratory immunity by immunobiotics

A targeted combination of the beneficial bacterium Lacticaseibacillus rhamnosus CRL1505, beta-glucans and vitamin D

The combination of the lactic acid bacterium Lacticaseibacillus rhamnosus CRL1505 (Lr1505) and beta-glucans was selected on the basis of their scientifically and clinically demonstrated ability to regulate the immune response outside the gastrointestinal tract, including the respiratory mucosa.

In vivo studies

Several current studies have demonstrated the considerable influence of the gut microbiota on the innate antiviral mechanisms of respiratory immunity through its effect on a) respiratory epithelial cells, b) respiratory dendritic cells (DC) and c) lung macrophages. The influence of the gut microbiota on innate immunity also modifies humoral adaptive immune responses (Fig. 1) (1).

In order to study the mechanisms involved in strengthening Lr1505-mediated respiratory antiviral immunity, studies were carried out in animal models using two respiratory viruses: respiratory syncytial virus (RSV) and influenza virus (IFV) (2,3).

In order to study the mechanisms involved in strengthening Lr1505-mediated intestinal as well as respiratory antibacterial immunity, studies were carried out in animal models using the bacteria Salmonella enterica serovar typhimurium and Streptococcus pneumoniae (4).

1) Respiratory innate antiviral immune response and L. rhamnosus CRL1505

The results of the study showed that the orally administered strain Lr1505 was able to significantly reduce viral titres in the lungs, reduce lung damage and increase the survival of mice in response to RSV (2) or IFV (3) infections. The protective effect induced by Lr1505 was associated with its ability to increase type I IFN levels in the respiratory tract (1,2,3).

The probiotic strain Lr1505 induced the mobilisation of CD4(+)IFN-γ(+) T cells from the gut to the respiratory tract, which increased local production of IFN-γ, a key activator of the innate and adaptive immune system, and stimulated antigen-presenting cells (2, 5) (Fig. 1).

2) Respiratory adaptive antiviral immune response and L. rhamnosus CRL1505

Evaluation of the population of lung dendritic cells (DC) in mice after oral administration of Lr1505 pointed to increased levels of CD11c(+)CD103(+) and CD11c(+)CD11b(high) DC.

Improved antigen presentation mediated by Lr1505 was also demonstrated, which could have a beneficial effect on the respiratory humoral immune response (1).

3) Intestinal antiviral immune response and L. rhamnosus CRL1505

Further studies carried out on intestinal epithelial cells showed that Lr1505 is able to increase the production of type 1 IFN (6), improve the expression of IFN-α and IFN-β as well as various antiviral factors (Nplr3, Oas1, etc.) needed for an effective defence against viruses (7).

4) Antibacterial immune response and L. rhamnosus CRL1505

Oral supplementation with Lr1505 has also proved effective against enteropathogenic bacteria, and stimulation of the immune system of the respiratory mucosa has also been demonstrated. By increasing IFN-γ levels in the serum and intestinal fluid as a result of taking Lr1505, it was possible to activate peritoneal macrophages, which led to increased elimination of the enteropathogens S. typhimurium (Salmonella enterica serovar Typhimurium) (8).

Administration of Lr1505 also significantly increased the levels of anti-pneumococcal respiratory and serum IgA and IgG antibodies, which points to a beneficial effect on the antibody response in the case of infection with Streptococcus pneumoniae (4).

Clinical studies

• 6-month (July–December), in a cohort of 298 children with Lr1505
• the mode of immunomodulatory action also demonstrated in an in vivo and clinical 8-week study, in a cohort of 60 individuals with heat-killed Lr1505 (HkLr1505)
• randomised
• double-blind
• placebo-controlled

The results of the first study, carried out in a group of children aged 2–5 years, suggest a marked easing of severity and a reduction in the incidence of gastrointestinal and respiratory infections, particularly in young children, through a direct antimicrobial effect and a strengthening of mucosal barrier function resulting from the effects of the bacterium Lr1505 on both innate and adaptive immunity.

The results of the mechanism-of-action study, carried out in an adult population of 60 healthy individuals, also pointed to the ability of the heat-killed bacterium Lr1505 to maintain an optimal state of the immune system as well as physical condition by acting on and activating pDC cells, which act on the whole immune function, which confirmed the immunomodulatory potential of the live strain Lr1505.

Introduction

Infectious diseases such as acute otitis media, pharyngitis and tonsillitis, upper respiratory tract infections, pneumonia, bronchitis and diarrhoea remain one of the main causes of death in pre-school children (9-11).

The rise in antibiotic resistance and the need for new and improved strategies for tackling infectious diseases have led to an examination of the therapeutic potential of induced modulation of the mucosal immune response with probiotics.

As a result, certain probiotic lactic acid bacteria (LAB) have been found to have protective effects against bacterial and viral infections in the gastrointestinal tract (12).

Considerable attention has focused on the role of probiotics in protecting the gut from pathogens. However, there is growing evidence that orally administered probiotics are able to regulate immune responses outside the gastrointestinal tract, including the respiratory mucosa (13).

The first clinical study revealed that the probiotic Lr1505 added to yoghurt strengthened and improved mucosal immunity and reduced the incidence and severity of intestinal and respiratory infections in children (14).

In 2023 clinical studies based on a food supplement were completed in child and adult populations, and the results correspond to the conclusions of the probiotic yoghurt study. This means that the carrier for the bacteria is not key and that their effect is transferable. The studies will be published during 2024.

Methods

Healthy children attending day-care centres five days a week were included in the study. The probiotic strain Lr1505 was added to sweetened yoghurt containing at least 1x10^8 colony-forming units (CFU)/100 g (14).

The placebo group was given the same yoghurt without Lr1505.

Both groups were monitored by nutrition specialists, paediatricians and immunologists throughout the study (6 months).

Stool samples were collected at the beginning and at the end of the study to determine the number of lactic acid bacteria. In addition, salivary IgA levels were determined (14).

Results

1) Incidence of infections

Of the total of 298 children who took part in the study, 132 (45%) had an infectious disease during the study. The most common infectious diseases were upper respiratory tract infections, followed by pharyngitis and tonsillitis, and diarrhoea. In the group of children who received yoghurt with the probiotic strain L. rhamnosus CRL1505, a significant decrease in infectious diseases was observed compared with the placebo group (34% vs. 66%). The analysis subsequently revealed significant differences between the placebo group and the Lr1505 group in the incidence of infectious diseases, namely: upper respiratory tract infections (31% vs. 69%)(Fig. 2A), pharyngitis and tonsillitis (28% vs. 72%)(Fig. 2B) and acute diarrhoea (26% vs. 74%)(Fig. 2C) (14).

The clinical effects of probiotic supplementation on the incidence of fever and the need for antibiotic treatment were also evaluated. Subjects in the group receiving the probiotic Lr1505 in yoghurt were found to have a markedly lower incidence of fever (Fig. 3A). In addition, the need for antibiotic treatment in children consuming the probiotics was markedly lower than in the placebo group (Fig. 3B) (14).

2) Salivary immunoglobulin A (IgA) levels

Salivary IgA was also determined in order to establish the level of mucosal immunity. Samples were taken the day before the start of the intervention (baseline levels) and at the end of administration of the probiotics or placebo (post-supplementation levels). When the probiotic yoghurt was compared with placebo, no significant differences in baseline IgA levels were observed.

By contrast, a significant increase in IgA levels was observed in the children who received the probiotic yoghurt, whereas post-supplementation IgA levels in the placebo group were similar to those measured at the start of the study. Consumption of probiotics containing the strain Lr1505 is therefore able to improve mucosal immunity, as is also evidenced by the salivary IgA level (Fig. 4) (14).

The results of the study suggest that the probiotic Lr1505 helps to reduce the incidence of upper respiratory tract and gastrointestinal tract infections, supports the immune system and reduces the need for antibiotics.

Mechanism of action

The human immune system is an effective defence mechanism against the ubiquitous bacteria and viruses to which we are exposed every day, and its vitality needs to be maintained and supported.

Plasmacytoid dendritic cells (pDC) are thought to be important in the activation and regulation of immune functions. Activation of pDCs activates various immune cells responsible for innate and acquired immunity by increasing IFN-α, HLA-DR and other important immune molecules, which leads to the normal maintenance of overall immune function (16).

This clinical study also revealed the potential of the heat-killed bacterium Lr1505 (HkLr1505) to support the maintenance of normal immune function by activating pDC cells as well as to reduce disease symptoms at the time of infection (15), which reinforces the findings of the previous clinical study (14).

Methods

The clinical study was carried out over 8 weeks in healthy individuals of both sexes aged 20–64 years who were not being treated for, and did not have, a serious or chronic illness, had never had intestinal diseases affecting digestion or absorption, and had had no infectious respiratory disease in the previous 2 years. Participants in the study were given a tablet a) containing HkLr1505 or b) without HkLr1505 (placebo group) (15).

pDC activity was evaluated by measuring the expression levels of HLA-DR and CD86. Overall health status was assessed by a series of health questionnaires and monitored by a doctor (15).

Results

1) HLA-DR levels

When immune function was assessed, HLA-DR fell in both groups after 8 weeks of consumption, but remained significantly higher in the HkLr1505 group than in the placebo group (15).

2) Overall physical condition

When physical condition was assessed, the cumulative numbers of days with partial symptoms (such as a blocked nose, discomfort in the throat, chest heaviness, hoarseness, headaches, nausea, joint and muscle pain, stomach pain during the intake period) were markedly reduced in the HkLr1505 group compared with the placebo group (15).

3) PG05 – a key aspect of the immunomodulatory activity of the bacterium Lr1505

One of the key elements identified in the interaction of Lr1505 and HkLr1505 with the host is its strain-specific peptidoglycan (PG05; a component of the cell wall of both the live and the heat-killed bacterium), which has been shown to have immunomodulatory activity. PG05 significantly increases the production of specific antibodies and the number of alveolar macrophages (AM) producing IFN-β. These cells play a key role in the beneficial modulation of the innate immune response during viral and bacterial infections (17).

Conclusion

The conclusions of the study on the mechanism of action of the immunomodulatory activity of heat-killed Lr1505 confirm and reinforce the results of the previous clinical study carried out in the child population as well as the results of the clinical study carried out in the adult population, which will be published during 2024.

The immunomodulatory effect of beta-glucans

At present, most prebiotics on the market are derived from indigestible oligosaccharides, but the latest research is also focusing on indigestible long-chain complex carbohydrates such as beta-glucans, obtained from various food sources, which have shown not only health-supporting effects but also the potential of a new source of prebiotics. Most recently, the effect of consuming long-chain (1,3)-(1,6)-ß-D-glucan isolated from the yeast Saccharomyces cerevisiae on the number of episodes of the common cold in healthy individuals was investigated in a placebo-controlled, double-blind, randomised, multicentre clinical study, which demonstrated an increase in the potential of the human immune system to defend itself against invading pathogens (18).

The beta-glucan (1,3)-(1,6)-ß-D-glucan with a long chain isolated from the yeast Saccharomyces cerevisiae stimulates immune cells in the gut to trigger a non-specific or specific immune response that activates the so-called “scavenger cells” (macrophages) in Peyer’s patches. These “scavenger” cells begin by destroying pathogens and activate other defence cells such as B and T cells or dendritic cells. By supporting white blood cells, this beta-glucan contributes to a great extent to the fight against bacteria, viruses, fungi and parasites that cause disease (19).

Vitamin D

Vitamin D-metabolising enzymes and vitamin D receptors are also present in immune cells (antigen-presenting cells, T cells, B cells, monocytes). In vitro data show that, in addition to modulating innate immune cells, vitamin D also promotes a more tolerogenic immunological state. In vivo data from animals and from vitamin D supplementation studies in humans have demonstrated beneficial effects of vitamin D on immune function, particularly in the context of autoimmunity (20).

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