https://doi.org/10.1007/s12602-021-09822-3

Human Systemic Immune Response to Ingestion of the Oral Probiotic

Streptococcus salivarius BLIS K12

Gemma L. Laws1 · John D. F. Hale2 · Roslyn A. Kemp1

Accepted: 8 July 2021

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021

Abstract

Streptococcus salivarius K12 is an oral probiotic known to contribute to protection against oral pathogenic bacteria in humans. Studies of immune responses to S. salivarius K12 have focused on the oral cavity, and systemic immune responses have not yet been reported. The aim of this study was to identify acute systemic immune responses to the commercial product,

1. salivarius BLIS K12, in a double-blinded, placebo-controlled human clinical trial. It was hypothesised that consumption of S. salivarius BLIS K12 would induce an anti-inflammatory response and a decrease in pro-inflammatory cytokines. Blood samples were obtained from participants prior to a single dose of S. salivarius BLIS K12 or a placebo and then second- ary blood samples were obtained 24 h and 7 days post-consumption. Samples were analysed using multi-parametric flow cytometry, to quantify immune cell frequency changes, and by a LEGENDplex assay of human inflammatory cytokines. Consumption of S. salivarius BLIS K12 was associated with increased levels of IL-8 at 24 h. The frequency of Tregs increased in samples taken 7 days after probiotic consumption, and IL-10 concentrations were higher at 7 days than 24 h after consumption. There was no difference in the frequency and/or activation of CD4 T cells, CD8 T cells, B cells and NK cells. Interestingly, there was an increase in IL-12, 7 days after the consumption of S. salivarius BLIS K12. Collectively, this research demonstrates that ingestion of the probiotic S. salivarius K12 can induce changes in the systemic immune response. The implications of the generation and type of immune response warrant further study to determine potential health benefits.

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Keywords Probiotic · Immune · Oral tolerance · Cytokines · S. salivarius · Regulatory T cells

Introduction

Probiotics have been consumed for thousands of years in fermented foods such as cheeses, yoghurts, wine and bread [1]. Consumption of probiotics can lead to health benefits and modulate intestinal health through one or more modes of action: colonisation of target site result- ing in physical elimination or exclusion of pathogens

[2]; anti-competitor behaviour through the production of inhibitory molecules, such as bacteriocins [3]; enhance- ment of epithelial cell function[4]; and modulation of the microbiota and of the immune system [5, 6]. More difficult to define is the effect of probiotics on the host immune

response. Evidence using cell lines suggests that commen- sal bacteria can influence immune responses and benefit the host by suppressing inflammation, for example, IL-8 secretion by a human bronchial cell line decreased when co-cultured with Streptococcus salivarius K12 [7]. Cer- tain Bifidobacterium strains have been shown to stimulate the inflammatory cytokines IL-12 and TNF in a murine macrophage-like cell line [8, 9]. Probiotic consumption has been implicated in increased host protection from viral infection, with studies showing a reduced occurrence of cold and influenza-like symptoms in children [10], and a significantly shortened duration and degree of severity of viral respiratory tract infections in adults [11, 12]. Stud- ies investigating peripheral immune cells give an indi-

      cation of the systemic immune responses to probiotics;

• Roslyn A. Kemp roslyn.kemp@otago.ac.nz

1      Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand

2      Blis Technologies, Dunedin, New Zealand

for example, human peripheral blood mononuclear cells (PBMCs) cultured with heat-killed Lactobacillus casei Shirota had enhanced natural killer cell (NK) activity and increased IL-12 production compared to PBMCs without stimulation [13]. However, an analysis of the effect of

probiotic administration that incorporates the complexity of the peripheral human immune response has yet to be undertaken.

Streptococcus salivarius is a human commensal bacte- rium that is an early coloniser of epithelial surfaces in the human mouth and nasopharynx [7]. While predominantly an oral bacterium, S. salivarius also colonises the jejunum and stomach [14, 15] and is found in the gut of newborn infants [16]. Streptococcus salivarius BLIS K12 is a com- mercially available probiotic that specifically colonises the oral cavity. Bacteriocins produced by S. salivarius K12 include the lantibiotics, salivaricin A2 (Sal A2) and sali- varicin B (Sal B) [17, 18]. These bacteriocins are effective at interfering with the growth of nasopharygeal and oral cavity bacterial pathogens, especially Streptococcus pyo- genes [19]. S. salivarius BLIS K12 can colonise the oral microbiota of human subjects following oral cavity administration [20–22]. Culture of S. salivarius K12 with a human bronchial epithelial cell line led to down- regulated genes of the NF-κB pathway [7] and decreased secretion of IL-8. Other strains of S. salivarius have been shown to have anti-inflammatory properties when cultured in vitro with human intestinal epithelial cell lines, specifi- cally interfering with TNF-induced NF-κB activation [23]. In contrast, when S. salivarius was cultured with human PBMCs; there was an increase in IL-10, IL-12, IFN-γ and TNF cytokine production measured by enzyme-linked immunosorbent assay (ELISA) [23]. These studies have provided evidence of the effects of S salivarius on individ- ual components or molecules of the immune system. How- ever, when studied in isolation, it is difficult to determine an overall immune effect, for example, a decrease in IL-8 production indicates a reduction in inflammatory capac- ity of cells, beneficial if the goal is to inhibit inflamma- tion. In contrast, increased production of TNF and IFN-γ indicates a pro-inflammatory response. The concomitant increase in anti-inflammatory IL-10 concentrations with that of pro-inflammatory IL-12 further highlights the diffi- culty in identifying an overall immune effect. The immune system is large and complex, and it is difficult to extrapo- late results from studies in cell lines, or measurements of immune parameters in isolation, to determine a size or type of immune response in people.

The aim of this study was to determine the acute sys-

temic immune response to S. salivarius K12 in a double- blinded, placebo-controlled human clinical trial. We focused on analysis of immune cell frequencies, including activated and regulatory cells, as well as detailed cytokine production. It was hypothesised that consumption of S. sal- ivarius K12 would induce an anti-inflammatory response, and there would be either no change or a decrease in pro- inflammatory cytokines, due to the induction of an oral tolerance response.

Methods

Participants

This study was approved by the Human Ethics Committee of the University of Otago (Ref#H18/054). Healthy indi- viduals were invited to participate in the study. All subjects involved received an information sheet and gave written consent. Exclusion criteria included individuals with flu- like symptoms, dietary intolerances, immune disorders or adverse reactions to dairy products. Pregnant women and individuals who had either consumed probiotic products in the two previous weeks or who were currently taking antibiotics were also excluded. Participants and research- ers were blinded to the intervention. Of the 60 initially enrolled participants, 53 completed the trial (Fig. 1). Study size was determined based on data from an identical but smaller trial.

Probiotic Intervention

A 20-mL blood sample was collected from participants immediately prior to the intervention. We have used the following nomenclature throughout the manuscript: Strep- tococcus salivarius K12 when referring to the bacteria spe- cies, and Streptococcus salivarius BLIS K12 when refer- ring to the specific probiotic product. The subjects received either a single 1 g dose of non-irradiated, freeze-dried S. salivarius BLIS K12 (1 × 1010 cfu/g) or gamma-irradiated placebo maltodextrin powder. Both powders were identical in colour, mixed with 30 mL water and swallowed directly. The S. salivarius BLIS K12 and placebo powders were sourced from Blis Technologies Ltd (Dunedin, New Zea- land). Participants returned and provided further blood sam- ples 24 h and 7 days following the intervention. All blood samples were collected at the same time of day to account for natural circadian fluctuations in PBMC cell frequencies [24, 25]. No adverse events were observed or reported by any subjects.

Isolation of PBMCs

PBMCs were isolated from whole blood samples via a Ficoll-Paque density gradient in a SepMate tube (STEM- CELL Technologies, Canada) and then frozen at − 80 °C in 10% dimethyl sulfoxide (DMSO, Sigma-Aldrich, St Louis, MO, USA) and 90% foetal bovine serum (FBS, New Zea- land origin, Gibco, Grand Island, NY, USA). The follow- ing day, the PBMCs were transferred to liquid nitrogen storage. All samples were gathered and frozen in this man- ner to allow simultaneous analysis of the samples gathered on different days.

Cell Culture Stimulation for Flow Cytometry

PBMCs were suspended in RPMI (Gibco) supplemented with 0.1% 2 mercaptoethanol (Life Technologies, Carlsbad, CA, USA), 1% penicillin streptomycin (Life Technologies) and 5% FBS at 1 × 106 cells/mL in 1 mL in a 24-well plate. Cells were stimulated with phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) and 500 ng/mL iono- mycin (Sigma-Aldrich) for 2 h; then, 1 μL/mL of brefeldin A (Sigma-Aldrich) was added and the cells were incubated for a further 2 h. PBMCs were then incubated with antibodies for the following proteins: CD3, CD4, CD8, CD19, CD56, CD25, CD127, CD69, IL-2, IL-17A, IFN-γ and live/dead zombie NIR (viability dye). All flow cytometry data was acquired on an LSRFortessa (Becton Dickinson, Franklin Lakes, NJ, USA) using FACSDiva software (version 8.0, Becton Dickinson). Raw data was exported as flow cytometry standard 3.1 files and analysed using FlowJo software (version 10.0.7, Tree Star, Ashland, OR, USA). The gating strategy used for cell analy- sis is shown in Supplementary Fig. 1; fluorescence minus one controls were used to set gates.

Cell Culture Stimulation for LEGENDplex Assay

Activation of PBMC samples was performed using Dyna- beads Human T-Activator CD3/CD28 (Thermo Fisher Sci- entific, Waltham, MA, USA). PBMCs were suspended at a concentration of 8 × 104 cells in 100 µL RPMI in a 96-well tissue culture plate. Washed Dynabeads were added to each sample at a bead-to-cell ratio of 1:1. Samples were

incubated at 37 °C in 5% CO2 for 24 h. Dynabeads were removed from the solution, and the supernatant was col- lected for analysis with LEGENDplex Human Inflamma- tion Panel, 13-plex (BioLegend, San Diego, CA, USA; IL-1β, IFN-α2, IFN-γ, TNF, MCP-1, IL-6, IL-8, IL-10, IL12p70, IL-17A, IL-18, IL-23, IL-33). Assay of culture

supernatant was performed as per LEGENDplex proto- col instructions. PBMC culture supernatants were either incubated undiluted or diluted 1:10. All samples were ana- lysed using an LSRFortessa. Cytokine data points that fell below the minimum detectable limit are plotted at this limit (indicated by the dotted line) only if that individual had detectable levels in that parameter at any other time point to enable calculation of differences between time points.

Statistical Analysis

A D’Agostino-Pearson omnibus normality test was per- formed to test if values came from a Gaussian distribution. However, the values were not normally distributed and therefore were transformed Log10 prior to analysis. Within each of the S. salivarius BLIS K12 and placebo groups, a one-way ANOVA was performed to assess differences between individuals from their baseline immune responses, to 24 h, and 7 days post-consumption. Significant differences are indicated with #p < 0.05. However, to indicate if changes observed were due to S. salivarius BLIS K12, a two-way ANOVA was performed, comparing S. salivarius BLIS K12 to the placebo group. Significant differences are indicated with *p < 0.05.

Fig. 1 Cohrt flow diagram indicating the flow of partici- pants through each stage of the randomised trial. S. salivarius BLIS K12 versus placebo

Table 1 Participant characteristics

Participant characteristics          Intervention groups (n = 53)

1. salivarius BLIS K12 Placebo

Gender

Male                                             14                                                17

Female                                         13                                                10

Age range

18–30 years                                 22                                                20

31–50 years                                 5                                                  4

50–60 years                                 0                                                  0

60 + years                                     1                                                  1

Results

Clinical Information

Of the 60 potential participants assessed for eligibility, 53 completed the trial; 28 in the S. salivarius BLIS K12 group; and 25 in the placebo group (Fig. 1). Both the participants and researchers were blinded to intervention allocation. Table 1 indicates the participant characteristics.

Changes in Cytokine Concentrations Were Observed in PBMC Samples 24 h post‑Consumption of S. salivarius BLIS K12

Blood samples were obtained 24 h after consumption of either the probiotic or the placebo (Fig. 2). We measured

a                      IL-6                          b

IL-8

ns                                                           ns

#

105

104

103

102

101

100

p=0.0558

105

104

103

102

101

100

c

102

101

100

10-1

IL-18

ns

p=0.0558

Placebo Streptococcus salivarius BLIS K12

Fig. 2 Consumption of S. salivarius BLIS K12 changes cytokine concentrations 24 h after consumption. PBMCs was collected from healthy participants pre-, 24 h post- and 7 days post-consumption of placebo (white) or S. salivarius BLIS K12 at 1 × 1010 cfu/serve (grey). PBMCs were stimulated for 24 h with anti-CD3/CD28 Dyna- beads. The culture supernatant was collected, and concentration of cytokines was measured by LEGENDplex assay. Concentrations in pg/mL of a IL-6, b IL-8 and c IL-18. The middle line represents the

median. Whiskers extend to the min and max points. Some cytokine concentrations were below the detectable level, indicated by the dot- ted line. Each dot represents one participant. Placebo group (n = 25);

1. salivarius BLIS K12 group (n = 28). Statistical analysis to deter- mine changes within each group was completed using a one-way ANOVA #p < 0.05. Overall statistical analysis to compare S. salivar- ius BLIS K12 to placebo was completed using two-way ANOVA

a

104

103

102

101

100

10-1

IL-10

ns

b

102

101

100

10-1

IL-12

ns

Placebo Streptococcus salivarius BLIS K12

Fig. 3 Consumption of S. salivarius BLIS K12 increased cytokines 7 days post-consumption. PBMCs was collected from healthy par- ticipants pre-, 24 h post- and 7 days post-consumption of either pla- cebo (white) or S. salivarius BLIS K12 at 1 × 1010 cfu/ serve (grey). PBMCs were stimulated for 24 h with anti-CD3/CD28 Dynabeads. The culture supernatant was collected, and concentration of cytokines was measured by LEGENDplex assay. Concentrations in pg/mL of a IL-10 and b IL-12. The middle line represents the median. Whisk-

ers extend to the min and max points. Some cytokine concentrations were below the detectable level, indicated by the dotted line. Each dot represents one participant. Placebo group (n = 25); S. salivarius BLIS K12 group (n = 28). Statistical analysis to determine changes within each group was completed using a one-way ANOVA #p < 0.05. Over- all statistical analysis to compare S. salivarius BLIS K12 to placebo was completed using two-way ANOVA

a panel of cytokines known to be involved in inflamma- tion (LEGENDplex Human Inflammation Panel) and pre- sent the data for those cytokines that showed differences in the placebo versus probiotic groups at the described time points (data for all other cytokines at all three time points are shown in Supplementary Fig. 2). There was a median decrease of 245.41 pg/mL in IL-6 concentration

time points. Surprisingly, there was also an increase in the inflammatory cytokine IL-12, by an average concentration of

Tregs

ns

24 h after the consumption of S. salivarius BLIS K12;            15

however, this was not statistically significant (p = 0.0558;

Fig. 2A). The group that consumed S. salivarius BLIS            10

K12 had a statistically significant increase in IL-8 pro- duction after 24 h (p = 0.0195; Fig. 2B), when com-

pared to the placebo group. Similarly, there was a non-             5

significant decrease in the pro-inflammatory cytokine IL-18 24 h after consumption of S. salivarius BLIS K12

(p = 0.0558; Fig. 2C).                                                             0

Placebo Streptococcus salivarius BLIS K12

Changes in Cytokine Concentrations Were Observed Within PBMC Samples 7 Days Post‑Consumption

of S. salivarius BLIS K12

Blood samples were obtained again at 7 days after the initial consumption of the intervention. The concentration of the anti-inflammatory cytokine IL-10 increased by an average of

186.84 pg/mL between 24 h and 7 days after the consumption of S. salivarius BLIS K12 (p = 0.0432) (Fig. 3A). Analysis within the placebo showed no significant differences over the

Fig. 4 Consumption of S. salivarius BLIS K12 may increase the frequency of Treg cells. PBMCs was collected from healthy partici- pants, pre-, 24 h post- and 7 days post-consumption of placebo (grey) or S. salivarius BLIS K12 at 1 × 1010 cfu/ serve (black). PBMCs were stimulated for 4 h with PMA and ionomycin. They were then ana- lysed by flow cytometry. The frequency of Tregs was determined by the gating strategy shown in Supplementary Fig. 1. Each dot repre- sents one participant. The middle line represents the median. Placebo group (n = 25); S. salivarius K12 group (n = 28). Statistical analysis to determine changes within each group was completed using a one-way ANOVA #p < 0.05. Overall statistical analysis to compare S. salivar- ius K12 to placebo was completed using two-way ANOVA. *p < 0.05

2.83 pg/mL from baseline to 7 days post-consumption of S. salivarius K12 (p = 0.0152; Fig. 3B), and no difference in the group who received the placebo. There were no significant differences in the probiotic versus placebo groups for any other cytokines tested (Supplementary Fig. 2).

Consumption of S. salivarius BLIS K12 Increased the Frequency of Tregs But No Other Immune Cells

PBMCs collected at each time point were incubated with a flow cytometry antibody panel, specifically designed to detect a wide range of immune cells, including CD8+ T cells, CD4+ T cells, NK cells, B cells and T regulatory cells (Tregs) (Supplementary Fig. 1). The immune cell frequen- cies were compared at the three time points. There was an increase in the frequency of CD25hi CD127low CD4+ T cells (Tregs) between baseline and 7 days (but not after 24 h) post-consumption of S. salivarius BLIS K12 (p = 0.0497; Fig. 4), which was not observed in the placebo group. There were no significant differences between the probiotic and placebo groups in the frequency of any other immune cells (Supplementary Figs. 3–5).

Discussion

The aim of this study was to identify any acute systemic immune response to the ingestion of a single dose of

1. salivarius BLIS K12 in a double-blinded, placebo- controlled human clinical trial. It was hypothesised that consumption of S. salivarius BLIS K12 would induce an anti-inflammatory response and that there would be a decrease in pro-inflammatory cytokines. Investigating the acute peripheral immune response at both 24 h and 7 days indicated the immediate effect of S. salivarius K12 on the systemic immune response. While the majority of immune cells and cytokines were unaffected by consumption of the probiotic, we observed an increase in Treg frequency and increased levels of IL-10 production, indicating that
2. salivarius BLIS K12 may stimulate a regulatory, anti- inflammatory peripheral immune response. Interestingly, we observed a decrease in IL-6 and an increase in IL-8 24 h after consumption, compared to baseline. IL-12 was increased after 7 days—this finding may suggest an ongo- ing activated immune response. Taken together, our data indicate that S. salivarius BLIS K12 can induce peripheral immune responses, and the balance and implications of these immune responses are worthy of further study.

Probiotics have been reported to play a role in promoting oral tolerance, a phenomenon that allows orally delivered food antigens and commensal bacteria to be ingested with- out evoking mucosal and systemic antigen-specific immune responses [26]. Immune regulation can be influenced by

probiotic consumption when measured by changes in the frequency of immune cells, such as Tregs [27, 28]. Our data showed an increase in frequency of Tregs and concentrations of IL-10 7 days following consumption of the probiotic. Each of these immune factors has been associated with a reduction in inflammation in mouse models [29]. These find- ings warrant further investigation into mechanistic effects of

1. salivarius BLIS K12 on the immune response.

After the consumption of S. salivarius BLIS K12, there was a lower concentration of IL-6 detected. IL-6 can act as both a pro- and an anti-inflammatory cytokine; it is primarily produced by epithelial cells and macrophages in response to bacterial pathogen associated molecular patterns [30]. IL-6 is also antagonistic to Tregs; it has been shown to inhibit the proliferation of Tregs when cultured with cytokine-induced killer cells generated from PBMCs [31]. The decrease in IL-6 here may be important to allow the activation and pro- liferation of Tregs that was observed after the consumption of S. salivarius BLIS K12.

There were no changes in the frequency of CD8+ T cells, total CD4+ T cells, B cells, or NK cells. There was also no increase in inflammatory cytokine secretion detected after S. salivarius BLIS K12 consumption, and this inabil- ity of effector peripheral T cells to produce inflammatory cytokines may be due to gut-derived Tregs functioning sys- temically. These observations support the hypothesis that the consumption of S. salivarius BLIS K12 may result in an anti-inflammatory systemic immune response, although no statistically significant differences were seen when compar- ing the placebo to the probiotic groups.

However, it is worth noting that there was a statistically significant increase in IL-12, 7 days post-consumption of S. salivarius BLIS K12. IL-12 is secreted by antigen presenting cells and activates T cells and NK cells and stimulates the production of IFN-γ [32]. Unfortunately, it cannot be deter- mined which cells produced the cytokines in the LEGEND- plex assay. The PBMCs were stimulated with anti-CD3/28 Dynabeads, which selectively stimulate T cells, so it is pos- sible that the higher level of IL-12 was from APCs in the PBMC culture that was in a more activated state at a baseline level; however, this requires further study. Some studies of probiotics have observed an increase in NK cell frequency and activity after oral administration [33]. For example, con- tinued ingestion of L. casei Shirota over 3 weeks increased NK cell frequency and enhanced NK cell activity. This response was mediated by IL-12 [13, 34]. It would there- fore be interesting to investigate if the observed increase in IL-12 mediates the enhancement of cytotoxic activity (NK cells and CD8+ T cells) at a time point beyond the 7 day sample. The balance of IL-10 /IL-12 secretion by dendritic cells stimulated with probiotic strains in vitro appeared to be strain-specific and dose-dependent [35]. The inflamma- tory effect of IL-12 from APCs may also be counteracted by

a concurrent increase in IL-10, a potent anti-inflammatory cytokine. It is difficult to interpret the increases in both IL-12 and IL-10 observed in this study without more direct, mechanistic, experiments.

The current study provides a comprehensive investigation into a wide range of immune parameters. It has demonstrated that immune modulation by S. salivarius K12 affects mul- tiple components of the immune response. The relevance of using clinical trials to study the effects of probiotics is highlighted by the difference in production of IL-8 - in vitro studies using cell lines showed an increase in the produc- tion of IL-8 in response to S. salivarius K12 [7], but no differences were seen in the participants in this trial. A vari- ety of methods were used to minimise bias in this trial. For example, the participants were randomly allocated to either the probiotic and placebo interventions. Participants and researchers were blinded to which intervention was admin- istered (S. salivarius BLIS K12, or placebo), and all data analyses were completed prior to the unblinding process. In order to increase the relevance of the findings of the current study to a broad age spectrum of potential consumers of S. salivarius BLIS K12, the participants were of a wide age range (18–75 years).

In this study, systemic immune responses were measured

after a single dose of probiotic rather than as a continuous supplement, which many traditional probiotic trials have modelled. Since participant compliance typically decreases over longer time periods, we chose to examine the immune responses following a single dose of probiotic. This strategy may more accurately reflect real-world use of probiotics.

1. salivarius BLIS K12 is typically sold in a chewable tablet (suckable lozenge) format at a concentration of 1 × 10 cfu/lozenge. A dose of 1 × 10 cfu S. salivarius BLIS K12 was chosen for use in this study as this was con- sidered to be a relatively high, but safe, dose to administer [36].

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The majority of experimental research into the health benefits of probiotics has been conducted on groups of diseased subjects, measuring the effect of the probiotic on disease-related symptoms. These studies are often designed purely for methodological reasons, as significant positive results are easy to identify. Studies of the benefits of probi- otics for the healthy population are more complicated, as it is difficult to detect significant physiological benefits when functional abnormalities are not present [37]. However, pro- biotics are principally marketed for use by healthy consum- ers. The results from clinical trials on diseased populations are not directly generalizable to the healthy population [37]. We attempted to address the research gap of immune effects of probiotics on a healthy population with our study design. However, a limitation of the current trial was the difficulty in measuring significant differences in a population of healthy individuals where there is wide natural variation in immune

parameters. This trial also did not aim to assess any clinical responses to the consumption of probiotics or include mul- tiple doses of probiotics.

It is important to consider how the use of the probiotic S. salivarius K12 stimulates the systemic immune response and the possible mechanism of any such activity. The mechanism of action by which S. salivarius K12 stimulates the immune response is unknown. S. salivarius K12 produces several bacteriocin molecules, and these are known to be effec- tive anti-competitor molecules. Kaci et al. indicated that the anti-inflammatory effect of the S. salivarius strain JIM8772 observed in both in vitro and in vivo experiments occurred only with the use of viable, metabolically active S. salivarius, and not heat-killed bacteria [23]. This suggests that it is a product from the metabolic activity of the bacteria that are responsible for the observed effects. To fully under- stand how S. salivarius K12 stimulates the immune system, future experiments could focus on the mechanism of action of immune stimulation by testing immune responses to heat- killed bacteria, or even to purified bacteriocin in human par- ticipants. S. salivarius K12 supernatant could also be tested in in vitro cultures with epithelial cells [38], or in an organoid model mimicking the gut microenvironment [39]. Increased understanding of probiotic interactions with the host’s immune system is imperative if probiotics are to be prescribed in a clinical setting.

This study aimed to investigate the acute systemic

immune response to the oral probiotic S. salivarius BLIS K12. The results support the hypothesis that the consump- tion of S. salivarius K12 may result in an anti-inflammatory systemic immune response, although follow-up studies using a larger cohort are now indicated. Studies into the mecha- nistic role of probiotics will become increasingly important to increase our understanding of their contributions to the health of the consumer.

Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1007/s12602-021-09822-3.

Acknowledgements The authors thank Janet Rhodes and Jackson Treece for phlebotomy services and Nic West for advice on data analysis.

Author Contributions JH conceived the project, provided probiotics and contributed to writing. GL performed all experiments and analysis and wrote the manuscript. RAK conceived the project, had oversight of the project, contributed to data analysis and wrote the manuscript.

Funding This work was made possible by a Research and Development Student grant from Callaghan Innovation, New Zealand.

Data Availability Statement The datasets generated during and/or analysed during the current study are not publicly available due to limitations in the ethical consent process but are available from the corresponding author on reasonable request.

Declarations

Ethics Approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Human Ethics Committee of the University of Otago (Ref#H18/054).

Consent to Participate Informed consent was obtained from all indi- vidual participants included in the study.

Consent for Publication Informed consent to publish was obtained from all individual participants in the study.

Conflict of Interest JDFH is an employee of Blis Technologies, the manufacturer of Streptococcus salivarius BLIS K12 probiotics.

GLL was an employee of Blis Technologies prior to commencing this work. Blis Technologies supported student fees for GL.

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