A probiotic provides protection against acute salmonellosis in mice: Possible role of innate lymphid NKP46+ cells

Journal of Functional Foods 23 (2016) 329–338

Available online at www.sciencedirect.com

ScienceDirect j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j ff

A probiotic provides protection against acute salmonellosis in mice: Possible role of innate lymphid NKP46+ cells Firoz Mian a,b, Nalaayini Kandiah a, Marianne Chew b, Ali Ashkar b, John Bienenstock a,b, Paul Forsythe a,c, Khalil Karimi a,b,c,* a

The McMaster Brain – Body Institute, McMaster University, Hamilton, Ontario L8N 4A6, Canada Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario L8N 4A6, Canada c Department of Medicine, McMaster University, Hamilton, Ontario L8N 4A6, Canada b

A R T I C L E

I N F O

A B S T R A C T

Article history:

A subset of innate lymphoid cells (ILCs), which produce IL-22 and function in protection

Received 14 October 2015

against microbial infection, is generated via a local process in gut that is conditioned by

Received in revised form 4 February

commensal bacteria. We investigated the possibility that modulation of ILCs may contrib-

2016

ute to the protective effect of certain bacteria in relation to pathogen infection. We fed BALB/c

Accepted 8 February 2016

mice with L. rhamnosus JB-1 and characterized Peyer’s patches NKp46+ cells. Additionally,

Available online

the effect of feeding L. rhamnosus JB-1 on S. enterica serovar Typhimurium infection was de-

Keywords:

in the Peyer’s patches, mesenteric lymph nodes and colon; and was associated with high

Functional foods

levels of IL-22, RORγt, and CD127 expression by NKp46+ cells indicating an increase in ILC3s.

Innate lymphoid cells

Our findings indicate that Lactobacillus feeding regulates the innate lymphoid cell popula-

Lactobacilli

tion that may contribute to host defensive function during Salmonella infection.

termined. L. rhamnosus JB-1-feeding resulted in lower bacterial loads of Salmonella Typhimurium

NKp46+ cells

© 2016 Elsevier Ltd. All rights reserved.

Probiotics Salmonella

1.

Introduction

Probiotics are defined as live organisms which, when administered in adequate amounts, confer a health benefit on the host (De Santis, Cavalcanti, Mastronardi, Jirillo, & Chieppa, 2015) and have been considered as an important category of food supplement. The safety and regulation of probiotics are

important for both consumers and nutrition professionals (Lin, 2003). Most probiotic products contain lactic-acid-producing bacteria, which mainly belong to the genera Lactobacillus and Bifidobacterium. Lactobacilli possess the potential to institute a homeostatic balance in the GI tract in part through regulation of cytokine production (Reid, Jass, Sebulsky, & McCormick, 2003) in various immune cells (Bron, van Baarlen, & Kleerebezem, 2012). Indeed, the intestinal mucosa consists of

* Corresponding author. The McMaster Brain-Body Institute, St. Joseph’s Healthcare, 50 Charlton Avenue East, T3304, Hamilton, Ontario L8N 4A6, Canada. Tel.: +905 522 1155 ex 33820; fax: +905 540 6593. E-mail address: [email protected] (K. Karimi). Abbreviations: ILC3s, group 3 innate lymphoid cells; GI, gastrointestinal; NK, natural killer cells; NCR, natural cytotoxicity triggering receptor; L. rhamnosus JB-1, Lactobacillus rhamnosus JB-1; LB, Luria–Bertani; MRS, Man–Rogosa–Sharpe; CFU, colony forming units; PMA, phorbol-12-myristate-13-acetate; FMO, fluorescence-minus-one http://dx.doi.org/10.1016/j.jff.2016.02.020 1756-4646/© 2016 Elsevier Ltd. All rights reserved.

330

Journal of Functional Foods 23 (2016) 329–338

a vast number of immune cells and has developed dynamic mechanisms to work against microbial pathogens found in the intestines (Sanos & Diefenbach, 2010). Such immune cells, including natural killer (NK) cells, T regulatory cells, CD4+ T cells, macrophages and dendritic cells, are found in all gut tissues, particularly in various organized structures in the gut, such as cryptopatches, Peyer’s patches, isolated lymph follicles and mesenteric lymph nodes (Sanos & Diefenbach, 2010). Much attention has been paid to the various component cells of the mucosal immune system in providing protection against luminal pathogens. However, relatively incomplete investigation has occurred in relation to innate lymphoid cells (ILCs) populations. ILCs are members of the lymphoid lineage that have emerging roles in mediating immune responses and in regulating tissue homeostasis and inflammation (Artis & Spits, 2015). ILCs can be grouped (Leavy, 2013) based on the resemblance of their cytokine-expression profile to that of T helper 1 (TH1) cells (group 1 ILCs; includes NK cells), TH2 cells (group 2 ILCs) and TH17 and TH22 cells (group 3 ILCs). One of the IL-22-producing ILC3 populations is defined by the expression of natural cytotoxicity triggering receptor (NCR) NKp46 and is termed NCR+ILC3s (Spits et al., 2013). As mentioned above, NCR+ILC3s express NKp46 (Sanos & Diefenbach, 2010), which is a common phenotypic definition of NK cells in all mammalian species (Walzer, Jaeger, Chaix, & Vivier, 2007). However, they are distinguished from classical NK cells by their limited IFN-γ production (Satoh-Takayama et al., 2008). This subset of ILCs are generated via a local process, conditioned by commensal bacteria, and requires the transcription factor RORγt for their development (Buela, Omenetti, & Pizarro, 2015; Spits & Di Santo, 2011). We therefore, sought to study the modulation and function of these cells in mice upon feeding with a strain of commensal bacteria, L. rhamnosus JB-1, which we demonstrated previously to possess the ability to induce immunomodulatory effects (Karimi, Inman, Bienenstock, & Forsythe, 2009). We further examined L. rhamnosus JB-1 induced enhancement of immune protection against Salmonella.

850 xg for 15 minutes at 20 °C and washed twice with sterile phosphate-buffered saline (PBS) to a concentration of 6 × 108 bacteria/ml as determined by a Vitek colorimeter (bioMérieux, Hazelwood, MO). Bacterial suspensions were centrifuged in 15ml tubes at 850 xg for 15 minutes at 20 °C, supernatants discarded, and bacteria re-suspended in MRS broth to give a concentration of 5 × 109/ml.

2.2.

Adult male BALB/c mice (20–25g) were obtained from Harlan (Indianapolis, IN, USA), and housed either in the St. Joseph’s Hospital or McMaster University animal facilities for 1 week before experimentation. 6–8 week old mice were used in all experiments, which were performed in accordance with the guidelines of the Canadian Council for Animal Care.

2.3.

Materials and methods

2.1.

Bacteria

Lactobacillus rhamnosus JB-1 and Salmonella enterica serovar Typhimurium strain SL1344 were purchased from the American Type Culture Collection (ATCC, USA). The strains were prepared from frozen glycerol stocks (−80 °C). Salmonella enterica serovar Typhimurium strain SL1344 were routinely cultured in Luria broth (LB) supplemented with streptomycin at 50 µg/ml. Prior to infection, SL1344 was cultured overnight in LB with shaking at 225 rpm. Overnight cultures were washed in 100 mM HEPES (pH 8.0)–0.9% NaCl and then re-suspended in the same buffered solution. From frozen stocks (−80 °C), Lactobacillus rhamnosus JB-1 were suspended in Man–Rogosa–Sharpe liquid medium (MRS broth; Difco Laboratories, Detroit, MI) plated in MRS agar, cultured anaerobically at 37 °C for 24 hours, then inoculated in fresh MRS broth and grown at 37 °C under anaerobic conditions for 48 hours in 50-ml tubes. After 2 days, tubes were centrifuged at

Bacterial treatment

Naïve mice received 1 × 109 L. rhamnosus JB-1 suspended in Man– Rogosa–Sharpe (MRS) broth (Difco Laboratories, Detroit, MI) via oral gavaging daily for 5 days. Control animals were fed daily with a similar volume of broth alone. Where indicated, mice were fed on day 6 with 1 × 107 colony forming units (CFU) of S. enterica serovar Typhimurium (re-suspended in 0.9% NaCL in 100 mM HEPES, pH 8.0) and administered by oral gavage. In each experiment, 5 of 10 mice were sacrificed after 48 h of infection to determine Salmonella bacterial colony forming units (CFU) and the other five were monitored for body weight and general health conditions. Spleen, Peyer’s patches, mesenteric lymph nodes and colon were obtained from mice after 48 h of infection and pooled in 1 ml sterile PBS. The tissues were weighed, homogenized in a mixer mill machine and 10-fold serial dilutions in PBS were plated onto Luria–Bertani (LB) agar plates containing 50 µg/ml of streptomycin. Plates were then incubated overnight at 37°C to obtain counts of bacterial CFU per gram of tissue.

2.4.

2.

Animals

Preparation of single cell suspension

Peyer’s patches were carefully excised from the wall of the small intestine, pooled, and dissociated into single-cell suspensions by enzymatic digestion with collagenase type IV (250 U/ ml; Sigma-Aldrich, Oakville, Ontario, Canada) (Gilbert, Kobayashi, Sekine, & Fujihashi, 2011). Following incubation at 37 °C for 20 min, the collected patches were mechanically dissociated using 1 ml syringes. This cell suspension was washed by centrifugation (100 g × 10 min), and the pellet was re-suspended in RPMI 1640 containing 10% foetal bovine serum (FBS, Gibco, USA). Mesenteric lymph nodes were aseptically removed and pooled and single-cell suspensions were prepared by gentle passage of the tissue through sterile cell strainer (BD Falcon™, Canada).

2.5.

FACS analysis

Single cell suspensions from Peyer’s patches and mesenteric lymph nodes were re-suspended in FACS buffer (PBS-1% foetal calf serum) and stained for surface markers CD3 (Cluster of Differentiation 3)-APC-Cy7 (BD Pharmingen, San Diego, CA)

Journal of Functional Foods 23 (2016) 329–338

NKp46 (Natural Cytotoxicity Receptors)-Alexa Fluor 647, and CD127 (Interleukin-7 Receptor)-FITC (eBiosciences, San Diego, CA). Thereafter, the cells were fixed and permeabilized with Cytofix/Cytoperm according to the manufacturer’s directions (BD Bioscience, Mississauga, Canada) and stained for RORγt (RAR-related orphan receptor gamma)-PE (eBiosciences, San Diego, CA). Data were acquired with flow cytometry (Becton Dickinson, Oakville, Canada) and analysed with FlowJo program (TreeStar, Ashland, OR) employing the gating strategies based on unstained controls and/or fluorescence-minus-one (FMO) controls (Herzenberg, Tung, Moore, & Parks, 2006; Tung et al., 2007).

2.6.

Cell stimulation and degranulation assay

Single cell suspensions from Peyer’s patches or mesenteric lymph nodes were re-suspended at a concentration of 106 cells/ ml in RPMI 1640 containing 10% FBS (Gibco, USA), 2 mM L-glutamine, 50 IU/ml penicillin. Cells were stimulated with phorbol-12-myristate-13-acetate (PMA) (250 ng/ml) and ionomycin (10 ng/ml). CD107α (Cluster of Differentiation 107α) antibody was added (1:50 dilutions) at the time of stimulation. Cells were incubated for 5 h at 37 °C in 5% CO2 after which the protein transport inhibitor Golgi Plug (BD Bioscience) was added at a final concentration of 10 µg/mL together with 6 µl of Golgi-Stop (BD Bioscience) at a final concentration of 6 µg/ mL and incubated for an additional 4 h at 37 °C in 5% CO2 (Alter, Malenfant, & Altfeld, 2004). For IL-22 and IFN-γ expressions, cells initially stimulated with PMA and ionomycin were incubated for 6 h in the presence of GolgiStop™ (BD Biosciences). Thereafter, the cells were stained for surface antigens prior to fixation and permeabilization (Cytofix/Cytoperm, BD Bioscience, Mississauga, Canada). The cytokine antibodies IL-22 (eBiosciences, San Diego, CA) and IFN-γ (BD Bioscience) were used to detect intracellular cytokine expression.

2.7.

IL-22 ELISA

Single cell suspension from Peyer’s patches were seeded at 106 cells/ml in RPMI 1640 containing 10% FBS (Gibco, Grand Island, NY, USA), 2 mM L-glutamine, 50 IU/ml penicillin. Cells were stimulated as before either with PMA and ionomycin or in a plate-bound NKp46 antibody assay, as previously described (Sun, Beilke, Bezman, & Lanier, 2011). Briefly, flatbottom 96-well tissue culture plates were coated with antibody against NKp46 (10 µg/ml in PBS (Orr, Beilke, Proekt, & Lanier, 2010), clone 29A1.4 eBiociences) and kept overnight at 4 °C. Then 2 × 105 cells from Peyer’s patches were incubated on these antibody-coated plates for 20 h at 37 °C. Cell free supernatants were then assayed for IL-22 production by DuoSet ELISA (R & D systems).

2.8. Co-cultivation of S. Typhimurium and L. rhamnosus JB-1 1 × 107 CFU of S. enterica serovar Typhimurium and 1 × 107 CFU L. rhamnosus JB-1 were co-cultured in 2 ml of LB broth (antibiotic free), at 37°C with shaking (225 rpm) for 0, 2, 4, 6 and 24 hours. S. Typhimurium alone (in LB broth, antibiotic free) or

331

L. rhamnosus JB-1 alone (in MRS broth) were cultured separately as controls for the same duration. The bacterial cultures were then plated at each time point onto LB agar plus streptomycin (50 µg/ml) plates overnight, and S. Typhimurium CFU were enumerated. S. Typhimurium from co-cultures was compared with S. Typhimurium grown alone in LB broth at each time point to evaluate if L. rhamnosus JB-1 has any influence on S. Typhimurium growth.

2.9.

Statistics

Results were expressed as means ± the standard errors of the means. Means were compared using one-way ANOVA, employing Tukey’s post-hoc tests. Survival plots were analysed using the log-rank (Mantel–Cox) test. All data were analysed using GraphPad PRISM version 5.0. P-values of less than 0.05 were considered statistically significant.

3.

Results

3.1. Feeding mice with L. rhamnosus JB-1 increases Peyer’s patches RORγt+ NKp46+ cells The effects of feeding L. rhamnosus JB-1 on NCR+ILC3s in the Peyer’s patches and the mesenteric lymph nodes were assessed by determining the percentage of NKp46+ cells that lack the CD3 surface marker but express RORγt in Peyer’s patches and mesenteric lymph nodes. After 5-day treatment with L. rhamnosus JB-1, the number of RORγt expressing CD3- NKp46+ cells dramatically increased in the Peyer’s patches when compared with the vehicle-fed mice (Fig. 1A–C). This increase in RORγt expression was not observed in the mesenteric lymph nodes (Fig. 1B,D, and E). A previous study demonstrated that RORγt+ NKp46+ cells conditioned by commensal bacteria express CD127, the α-chain of the IL-7 receptor (Spits & Di Santo, 2011). We further determined that feeding with L. rhamnosus JB-1 led to changes in the phenotype of RORγt+ NKp46+ cells in the Peyer’s patches. After daily feeding of L. rhamnosus JB-1 for 5 consecutive days, the percentage of CD127 expressing CD3-NKp46+ RORγt+ cells in the Peyer’s patches was increased in the L. rhamnosus JB-1 fed mice compared with the vehicle fed mice (Fig. 1F).

3.2. RORγt+NKp46+ cell degranulation and IFN-γ expression are low and remain unchanged in Peyer’s patches of mice fed with L. rhamnosus JB-1 Lysosomal-associated membrane protein-1, LAMP-1 or CD107α marker has previously been shown by FACS to be expressed at the cell surface of CD8+ T cells and NK cells as a consequence of degranulation of lytic granules (Uhrberg, 2005). Therefore, measuring CD107α surface expression can assess the levels of degranulation and cytotoxicity. We examined degranulation and IFN-γ levels in RORγt+ cells in the Peyer’s patches to evaluate the cytotoxic capacity of the NKp46+RORγt+ cell population. After 5 days of treatment with L. rhamnosus JB-1, the Peyer’s patches were harvested and the cells were stimulated with PMA and ionomycin for 5 h. The percentage of

332

Journal of Functional Foods 23 (2016) 329–338

Fig. 1 – Increase in RORγt+ NKp46+ cells that express CD127 in Peyer’s patches following L. rhamnosus JB-1 administration. L. rhamnosus JB-1 or vehicle (MRS media) was orally administered to 6–8 week old BALB/c mice for 5 consecutive days. Single cell suspensions of Peyer’s patches or mesenteric lymph nodes were stained for CD3, NKp46, RORγt, and CD127 and analysed by flow cytometry. Representative dot plots (n = 5) (A) and mean ± SEM values (n = 5; *p < 0.05) (B and C) of RORγt+ cells on day 5 among the Peyer’s patches CD3-NKp46+ in lactobacillus and vehicle-fed mice are depicted. Representative dot plot of NKp46+ cells (n = 5) (D) and NKp46+RORγt+ cells (n = 5) (E) in mesenteric lymph nodes and Peyer’s patches in L. Rhamnosus JB-1-fed animals are shown. Representative dot plots (n = 5) (F) of CD127 expression on day 5 among the Peyer’s patches CD3-NKp46+RORγt+ cells in L. Rhamnosus JB-1 and vehicle-fed mice are depicted. CD107α+ cells or IFN-γ+ cells among the CD3-NKp46+RORγt+ populations suggested that a very low numbers of CD107α+ and IFNγ+ cells are found in the RORγt+ population of NKp46+ cells (Fig. 2A) as reported previously (Cooper, Colonna, & Yokoyama, 2009). There are no significant changes in degranulation and IFN-γ expression in mice treated with L. rhamnosus JB-1 compared with the vehicle-treated mice (Fig. 2B).

3.3. Association of elevated IL-22 expression in RORγt+ + NKp46 cells in Peyer’s patches with L. rhamnosus JB-1 feeding To further evaluate the function of the RORγt+ NKp46+ cell population, we assessed the IL-22 production in Peyer’s patches of L. rhamnosus JB-1 fed mice. Following stimulation with PMA and

Journal of Functional Foods 23 (2016) 329–338

%CD107 + in CD3-NKp46+

30 20

*

10 0

ROR t-

ROR t+

%IFN + in CD3-NKp46+

7.5

5.0

2.5

* 0.0

ROR t-

ROR t+

%CD107 + in CD3-NKp46+ROR t+

B 40

20 15 10 5 0

Vehicle-Fed JB-1-Fed

%IFN + in CD3-NKp46+ROR t+

A

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

Vehicle-Fed JB-1-Fed

Fig. 2 – RORγt+NKp46+ cell degranulation and IFNγ expression are low and remain unchanged in Peyer’s patches of mice fed with L. rhamnosus JB-1. L. rhamnosus JB-1 was orally administered to 6–8 week old BALB/c mice for 5 consecutive days. Peyer’s patches were harvested after oral administration and single cell suspensions were stimulated with PMA/Ionomycin for 6 hr. Anti-CD107α antibody was added at the time of stimulation. The cells were stained for CD3, NKp46, RORγt, and IFNγ. IFNγ and CD107α expression were analysed by flow cytometry. (A) Percentage of CD3-NKp46+CD107α+ cells or CD3-NKp46+IFNγ+ cells in RORγ+/- populations, in Peyer’s patches in JB-1-fed mice are depicted. (B) Percentage of CD107α+and IFNγ+ cells in RORγt+NKp46+ cells in L. rhamnosus JB-1-fed mice compared to vehicle fed mice are shown. Data are presented as mean ± SEM (n = 5).

ionomycin, there was a significant increase in the percentage (Fig. 3A and B) and total number (Fig. 3C) of IL-22 expressing CD3-NKP46+ cells in Peyer’s patches following L. rhamnosus JB-1 feeding. The higher IL-22 expression was found in the RORγt+ cell population rather than in the RORγt- population (Fig. 3D). The L. rhamnosus JB-1 induced regulation of IL-22 r was further confirmed by the observation that PMA and ionomycin stimulation of Peyer’s patch cells from L. rhamnosus JB-1 fed mice (Fig. 3E) resulted in 2 fold greater release of IL-22 (466.8 ± 22.5 pg/ ml (n = 5)) than from vehicle fed mice (260 ± 20 pg/ml (n = 5)).

(Satoh-Takayama et al., 2008). Moreover, both T-cell- and innate immune-dependent induction of IL-22 have been demonstrated during immunity to Salmonella infection (Godinez et al., 2008, 2009; Siegemund et al., 2009). Based on this previous literature and our observation of L. rhamnosus JB-1 induced upregulation of IL-22, we tested the effect of L. rhamnosus JB-1 pretreatment on S. Typhimurium infection. Mice were treated for 5 days with L. rhamnosus JB-1 prior to oral administration of S. Typhimurium, and, 48 h post-infection, expression of IL22 in Peyer’s patches and bacterial load in several tissues including colon were evaluated. As with uninfected animals, mice fed with L. rhamnosus JB-1 had higher IL-22 expression by RORγt+ NKp46+ cells in the Peyer’s patches compared with the controls (Fig. 4A). Furthermore, no IFN-γ expression was detected in the RORγt+ cells (Fig. 4B). We then sought to examine whether feeding L. rhamnosus JB-1 was associated with protection of mice against Salmonella infection. To achieve this, we first monitored the animals for decline in the body temperature and weight loss. 48 h after the challenge, the body temperature increased in both groups due to an immune response. L. rhamnosus JB-1 fed mice showed a mild increase in the body temperature that dropped to the normal level within next two days. In contrast, the vehicle fed mice demonstrated to have a sharp increase in the body temperature that abruptly declined and was accompanied by a huge weight loss in the animals (Figs 5A and B). Feeding of L. rhamnosus JB-1 to mice markedly prolonged the survival time compared to the vehicle-fed animals following challenge with S. Typhimurium (Fig. 5C). The increased survival of infected mice that were fed with L. rhamnosus JB-1suggests a protective effect mediated by improving the innate control of early pathogen colonization. In agreement with other studies, we also previously showed that in the absence of antibiotic pretreatment, this serovar of S. Typhimurium reaches a low but significant bacterial density in the murine cecum and colon (Ashkar, Reid, Verdu, Zhang, & Coombes, 2009). Therefore, we examined the bacterial density in the mice pretreated with L. rhamnosus JB-1. These mice had a significant decrease in bacterial load compared to control vehicle-fed mice (Fig. 5D). To exclude the possibility of microbe–microbe interactions and the presence of antimicrobial activity of L. rhamnosus JB-1 against Salmonella, we co-cultivated both bacteria at a range of ratios and determined the Salmonella growth during pure and mixed strain batch cultures at different time points. The growth of S. Typhimurium in the mixed cultures were stable and yielded a high cell count of ~109 CFU ml−1 after a very short incubation time of 2 h and up to ~5.6 × 109 CFU ml−1 after 6 h (data not shown). No inhibition of bacterial growth was detected in the co-cultivation system.

4. 3.4. High IL-22 expression in Peyer’s patches and low bacterial load in colon of mice fed with L. rhamnosus JB-1 prior to infection with S. Typhimurium IL-22 increases innate immunity and gut microbes have been demonstrated to drive IL-22 production in the intestine

333

Discussion

It is suggested that the immunomodulatory effects of probiotics are associated with induction of certain types of regulatory cells (Karimi et al., 2009; Karimi, Kandiah, Chau, Bienenstock, & Forsythe, 2012). We demonstrated that feeding of L. rhamnosus JB-1 to mice led to an increase in RORγt-expressing NKp46+ cells

334

Journal of Functional Foods 23 (2016) 329–338

Fig. 3 – Association of elevated IL-22 production in NKp46+ cells in Peyer’s patches with L. rhamnosus JB-1 feeding. L. rhamnosus JB-1 was orally administered to 6–8 week old BALB/c mice for 5 consecutive days. Peyer’s patches were harvested after oral administration, and single cell suspensions were stained for CD3, NKp46, RORγt, and IL-22. IL-22 expression was analysed by flow cytometry. (A) Percentage (n = 5); (B) representative dot plots (n = 5); and (C) total cell number (n = 5) of IL22+NKp46+ cells in Peyer’s patches in JB-1-fed mice compared to vehicle fed mice; and (D) percentage and representative dot plots (n = 5) of CD3-NKp46+IL22+ in RORγ+/- cell population in Peyer’s patches in L. rhamnosus JB-1-fed mice are shown. Data are presented as mean ± SEM. (E) Single cell suspension from Peyer’s patches were stimulated (see Methods section), and IL-22 release was measured using ELISA. Data are presented as mean ± SEM (n = 5).

with a high capacity of IL-22 production in the Peyer’s patches. Moreover, the oral administration of L. rhamnosus JB-1 to mice contributed to innate immune protection against Salmonella infection, illustrating the impact of the commensal microbiota on host immune defence and immune homeostasis. It has been shown that RORγt (Sanos et al., 2009) and the commensal microbiota (Satoh-Takayama et al., 2008) drive IL22 producing NKp46+ cells in mucosa. However, which particular microbial species regulates the homeostasis and function of these cells is not yet known. Our study clearly demonstrates that the ingestion of a single strain of bacteria, L. rhamnosus JB-1, resulted in profound elevation of RORγt-expressing NKp46+ cells. Interestingly, feeding of L. rhamnosus JB-1 was not accompanied by the production of inflammatory cytokine IFN-γ in NKp46+ cells, but by increases in IL-22 production, a cytokine known to limit intestinal inflammation. Our observation, in accord with existing studies suggests that these cells are distinct from conventional NK cells, as they are non-cytotoxic, produce little, if any, IFN-γ, and express CD127α-chain of the

IL-7 receptor (Spits & Di Santo, 2011). Indeed, feeding L. rhamnosus JB-1 to mice appears to be driving a RORγt expressing NKp46+ IL-22high/ IFN-γnegative/low cell population in the gut, which are classified as NCR+ILC3s (Spits et al., 2013). NKp46+ cells have been shown to release IL-22 upon stimulation with cytokines, such as combination of IL-12 and IL-18, but not in response to stimulation by cross-linking of cell surface receptor NKp46 (Satoh-Takayama et al., 2009). We also demonstrated the generation of high levels of IL-22 by these cells following activation by PMA and ionomycin but not by stimulating the cells in a plate-bound NKp46 antibody. RORγt+ cells, including Th17 cells and IL-22 producing NKp46+ cells have been suggested to limit intestinal inflammatory disease by strengthening antibacterial immunity (Guo et al., 2014; Lochner et al., 2011; Satoh-Takayama et al., 2008) such as the production of antibacterial peptides by epithelial cells (Liang et al., 2006). Moreover, the essential role of innate IL22 during early host defence against bacterial infection has been demonstrated (Zheng et al., 2008). IL-22 receptors are broadly

Journal of Functional Foods 23 (2016) 329–338

335

Fig. 4 – NKp46+RORγt+ cells produce high amount of IL-22 but no IFN-γ in Peyer’s patches of mice fed with L. rhamnosus JB-1 prior to infection with Salmonella. L. rhamnosus JB-1 was orally administered to 6–8 week old BALB/c mice for 5 consecutive days. On day 6, a group of mice received Salmonella or vehicle. A group of naïve mice also received Salmonella. Peyer’s patches were harvested 48 hr after oral administration of Salmonella and single cell suspensions were stained for CD3, NKp46, RORγt, IFN-γ, and IL-22. IL-22 and IFN-γ expression was analysed by flow cytometry. Representative mean ± SEM values (A) and dot plots (B) of IL-22 + cells on day 5 among the Peyer’s patches CD3-NKp46+RORγt+ cells in lactobacillus and control mice are depicted (n = 5; *p < 0.05.).

expressed in many types of epithelial cells (Gurney, 2004), and are important to protect mucosal surfaces from bacterial infection. Increased IL-22 production following L. rhamnosus JB-1 may be related to the role that NCR+ILC3s may play in innate defence against bacterial infection. Our demonstration that the number of IL-22 producing NKp46+ cells increased in Peyer’s patches could account for the participation of these cells in innate immune defence against Salmonella infection and is strongly supported by the striking results of our survival studies. However, our study does not show whether the increase in the number of cells in the RORγt+ population in the Peyer’s patches after Lactobacillus treatment is an induction of RORγt in the cells or an increased recruitment of the NCR+ILC3s RORγt-expressing cell subset. Further studies will examine this issue in more detail. The current study lacks direct evidence that NKp46+ cellderived IL-22 is involved in the survival of mice infected with Salmonella. However, in Salmonella infection, IL-22 is an important part of the immune response (Denning & Parkos, 2013; Godinez et al., 2008, 2009; Mayuzumi, Inagaki-Ohara, Uyttenhove, Okamoto, & Matsuzaki, 2010; Siegemund et al., 2009), and is critical for clearing Salmonella in the absence of IL-12 (Schulz et al., 2008). Therefore, we could not test effects of IL-22 neutralization, as this would be expected to aggravate the pathogenicity of the Salmonella even in vehicle treated mice. Our study adds IL-22 to the cytokines identified as contributing to (Coburn, Grassl, & Finlay, 2007; de LeBlanc Ade, Castillo, & Perdigon, 2010) protection against salmonella infection, and the data complement the conclusions of previous publications showing beneficial effects of two lactobacilli in a C. rodentium model of colitis (Gareau, Wine, Reardon, & Sherman, 2010; Johnson-Henry et al., 2005; Rodrigues, Sousa, Johnson-Henry, Sherman, & Gareau, 2012). It has been shown that L. salivarius, a probiotic strain of human origin, produces a bacteriocin in vivo that can significantly protect mice against infection with the invasive food borne pathogen, Listeria monocytogenes (Colonna, 2009; Corr et al.,

2007). Our in vitro co-culture studies of S. Typhimurium and L. rhamnosus JB-1 clearly show that L. rhamnosus JB-1 had no direct inhibitory effects on growth or direct killing ability against S. Typhimurium.

5.

Conclusions

For the first time, it was demonstrated that induction of IL22 production by the host likely contributes to the ability of certain probiotic organisms to protect against Salmonella infection. Given the now well described, strain specific properties and immunomodulatory activities of commensal organisms, it is crucial to select a particular species or strain of a probiotic for a particular health-maintenance. Our study of a lethal Salmonella infection in mice supports the selection of IL-22 inducing Lactobacillus strains, or indeed prebiotic supplements that encourage the growth of such organisms, for clinical evaluation as possible adjunct therapy or protective treatments against pathogenic gut infections.

Conflict of interest The authors have no financial or personal conflicts of interest.

Acknowledgements The research was supported by unrestricted grants from Concetta and Giovanni Giulietti Family, St Joseph’s Hospital Foundation and the McMaster Brain Body Institute at St Joseph’s Healthcare Hamilton.

336

Journal of Functional Foods 23 (2016) 329–338

vehicle-fed JB-1-fed

100 80 60 40 20 0

8 10 12 14 16 18 Day

Days

D 25

0 3 6 9 12 15 18 21

Vehicle fed

1250

JB-1 fed

1000

20 15

colon

750 500 250

10

*

1250

CFU/organ (x103)

6

B

0

PP

1000 750 500 250

*

0 Vehicle-fed JB-1-fed

Vehicle-fed JB-1-fed

5 1250

2 4 6 8 10 12 14 16 Days

CFU/organ (x103)

0

MLNs

spleen

1000

1250

750 500 250

*

0

CFU/organ (x103)

Body weight loss (%)

C

Vehicle fed JB-1 fed

Percent survival

39.5 39.0 38.5 38.0 37.5 37.0 36.5 36.0 35.5 35.0 34.5

CFU/organ (x103)

Body temperature

A

1000 750 500 250 0

Vehicle-fed JB-1-fed

Vehicle-fed JB-1-fed

Fig. 5 – Significant survival prolongation and low bacterial load in the gut of mice fed with L. rhamnosus JB-1 prior to infection with Salmonella. L. rhamnosus JB-1 was orally administered to 6–8 week old BALB/c mice for 5 consecutive days. On day 6, a group of mice received Salmonella or vehicle. (A) Body temperature; (B) body weight loss; and (C) survival plot of L. rhamnosus JB-1-fed and vehicle-fed mice. Data represent two independent experiments (n = 5 per treatment group). (D). Tissues were harvested from mice after 48 hours of infection and bacterial CFU per gram of tissues were obtained. Data are presented as mean ± SEM (n = 5 per treatment group).

REFERENCES

Alter, G., Malenfant, J. M., & Altfeld, M. (2004). CD107a as a functional marker for the identification of natural killer cell activity. Journal of Immunological Methods, 294(1–2), 15–22. doi:10.1016/j.jim.2004.08.008; S0022-1759(04)00292-3 [pii]. Artis, D., & Spits, H. (2015). The biology of innate lymphoid cells. Nature, 517(7534), 293–301. doi:10.1038/nature14189. Ashkar, A. A., Reid, S., Verdu, E. F., Zhang, K., & Coombes, B. K. (2009). Interleukin-15 and NK1.1+ cells provide innate protection against acute Salmonella enterica serovar Typhimurium infection in the gut and in systemic tissues. Infection and Immunity, 77(1), 214–222. doi:10.1128/IAI.01066-08; IAI.01066-08 [pii]. Bron, P. A., van Baarlen, P., & Kleerebezem, M. (2012). Emerging molecular insights into the interaction between probiotics and the host intestinal mucosa. Nature Reviews. Microbiology, 10(1), 66–78. doi:10.1038/nrmicro2690.

Buela, K. A., Omenetti, S., & Pizarro, T. T. (2015). Cross-talk between type 3 innate lymphoid cells and the gut microbiota in inflammatory bowel disease. Current Opinion in Gastroenterology, 31(6), 449–455. doi:10.1097/ MOG.0000000000000217. Coburn, B., Grassl, G. A., & Finlay, B. B. (2007). Salmonella, the host and disease: A brief review. Immunology and Cell Biology, 85(2), 112–118. doi:10.1038/sj.icb.7100007. Colonna, M. (2009). Interleukin-22-producing natural killer cells and lymphoid tissue inducer-like cells in mucosal immunity. Immunity, 31(1), 15–23. doi:10.1016/j.immuni.2009.06.008; S1074-7613(09)00280-5 [pii]. Cooper, M. A., Colonna, M., & Yokoyama, W. M. (2009). Hidden talents of natural killers: NK cells in innate and adaptive immunity. EMBO Reports, 10(10), 1103–1110. doi:10.1038/ embor.2009.203; embor2009203 [pii]. Corr, S. C., Li, Y., Riedel, C. U., O’Toole, P. W., Hill, C., & Gahan, C. G. (2007). Bacteriocin production as a mechanism for the antiinfective activity of Lactobacillus salivarius UCC118.

Journal of Functional Foods 23 (2016) 329–338

Proceedings of the National Academy of Sciences of the United States of America, 104(18), 7617–7621. doi:10.1073/ pnas.0700440104; 0700440104 [pii]. de LeBlanc Ade, M., Castillo, N. A., & Perdigon, G. (2010). Antiinfective mechanisms induced by a probiotic Lactobacillus strain against Salmonella enterica serovar Typhimurium infection. International Journal of Food Microbiology, 138(3), 223– 231. doi:10.1016/j.ijfoodmicro.2010.01.020. De Santis, S., Cavalcanti, E., Mastronardi, M., Jirillo, E., & Chieppa, M. (2015). Nutritional keys for intestinal barrier modulation. Front in Immunology, 6, 612. doi:10.3389/fimmu.2015.00612. Denning, T. L., & Parkos, C. A. (2013). Neutrophils enlist IL-22 to restore order in the gut. Proceedings of the National Academy of Sciences of the United States of America, 110(31), 12509–12510. doi:10.1073/pnas.1310907110. Gareau, M. L. G., Wine, E., Reardon, C., & Sherman, P. M. (2010). Probiotics prevent death caused by citrobacter rodentium infection in neonatal mice. Journal of Infectious Diseases, 201(1), 81–91. doi:10.1086/648614. Gilbert, R. S., Kobayashi, R., Sekine, S., & Fujihashi, K. (2011). Functional transforming growth factor-beta receptor type II expression by CD4+ T cells in Peyer’s patches is essential for oral tolerance induction. PLoS ONE, 6(11), e27501. doi:10.1371/ journal.pone.0027501. Godinez, I., Haneda, T., Raffatellu, M., George, M. D., Paixao, T. A., Rolan, H. G., Santos, R. L., Dandekar, S., Tsolis, R.M., & Baumler, A. J. (2008). T cells help to amplify inflammatory responses induced by Salmonella enterica serotype Typhimurium in the intestinal mucosa. Infection and Immunity, 76(5), 2008–2017. doi:10.1128/IAI.01691-07; IAI.01691-07 [pii]. Godinez, I., Raffatellu, M., Chu, H., Paixao, T. A., Haneda, T., Santos, R. L., Bevins, C.L., Tsolis, R. M., & Baumler, A. J. (2009). Interleukin-23 orchestrates mucosal responses to Salmonella enterica serotype Typhimurium in the intestine. Infection and Immunity, 77(1), 387–398. doi:10.1128/IAI.00933-08; IAI.00933-08 [pii]. Guo, X., Qiu, J., Tu, T., Yang, X., Deng, L., Anders, R. A., Zhou, L., & Fu, Y. X. (2014). Induction of innate lymphoid cell-derived interleukin-22 by the transcription factor STAT3 mediates protection against intestinal infection. Immunity, 40(1), 25–39. doi:10.1016/j.immuni.2013.10.021. Gurney, A. L. (2004). IL-22, a Th1 cytokine that targets the pancreas and select other peripheral tissues. International Immunopharmacology, 4(5), 669–677. doi:10.1016/ j.intimp.2004.01.016; S1567-5769(04)00012-8 [pii]. Herzenberg, L. A., Tung, J., Moore, W. A., & Parks, D. R. (2006). Interpreting flow cytometry data: A guide for the perplexed. Nature Immunology, 7(7), 681–685. doi:10.1038/ni0706-681. Johnson-Henry, K. C., Nadjafi, M., Avitzur, Y., Mitchell, D. J., Ngan, B.-Y., Galindo-Mata, E., Jones, N. L., & Sherman, P. M. (2005). Amelioration of the effects of citrobacter rodentium infection in mice by pretreatment with probiotics. Journal of Infectious Diseases, 191(12), 2106–2117. doi:10.1086/430318. Karimi, K., Inman, M. D., Bienenstock, J., & Forsythe, P. (2009). Lactobacillus reuteri-induced regulatory T cells protect against an allergic airway response in mice. American Journal of Respiratory and Critical Care Medicine, 179(3), 186–193. doi:10.1164/rccm.200806-951OC; 200806-951OC [pii]. Karimi, K., Kandiah, N., Chau, J., Bienenstock, J., & Forsythe, P. (2012). A lactobacillus rhamnosus strain induces a heme oxygenase dependent increase in foxp3+ Regulatory T Cells. PLoS ONE, 7(10), e47556. doi:10.1371/journal.pone.0047556. Leavy, O. (2013). Innate-like lymphocytes: Will the real ILC1 please stand up? Nature Reviews. Immunology, 13(2), 67. doi:10.1038/nri3397. Liang, S. C., Tan, X. Y., Luxenberg, D. P., Karim, R., DunussiJoannopoulos, K., Collins, M., & Fouser, L. A. (2006). Interleukin

337

(IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides. The Journal of Experimental Medicine, 203(10), 2271–2279. doi:10.1084/jem.20061308; jem.20061308 [pii]. Lin, D. C. (2003). Probiotics as functional foods. Nutrition in Clinical Practice, 18(6), 497–506. Lochner, M., Ohnmacht, C., Presley, L., Bruhns, P., Si-Tahar, M., Sawa, S., & Eberl, G. (2011). Microbiota-induced tertiary lymphoid tissues aggravate inflammatory disease in the absence of ROR{gamma}t and LTi cells. The Journal of Experimental Medicine, 208(1), 125–134. doi:10.1084/ jem.20100052; jem.20100052 [pii]. Mayuzumi, H., Inagaki-Ohara, K., Uyttenhove, C., Okamoto, Y., & Matsuzaki, G. (2010). Interleukin-17A is required to suppress invasion of Salmonella enterica serovar Typhimurium to enteric mucosa. [Research Support, Non-U.S. Gov’t]. Immunology, 131(3), 377–385. doi:10.1111/j.13652567.2010.03310.x. Orr, M. T., Beilke, J. N., Proekt, I., & Lanier, L. L. (2010). Natural killer cells in NOD.NK1.1 mice acquire cytolytic function during viral infection and provide protection against cytomegalovirus. Proceedings of the National Academy of Sciences of the United States of America, 107(36), 15844–15849. doi:10.1073/pnas.1010685107. Reid, G., Jass, J., Sebulsky, M. T., & McCormick, J. K. (2003). Potential uses of probiotics in clinical practice. Clinical Microbiology Reviews, 16(4), 658–672. Rodrigues, D. M., Sousa, A. J., Johnson-Henry, K. C., Sherman, P. M., & Gareau, M. L. G. (2012). Probiotics are effective for the prevention and treatment of citrobacter rodentium, äìinduced colitis in mice. Journal of Infectious Diseases, 206(1), 99–109. doi:10.1093/infdis/jis177. Sanos, S. L., Bui, V. L., Mortha, A., Oberle, K., Heners, C., Johner, C., & Diefenbach, A. (2009). RORgammat and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp46+ cells. Nature Immunology, 10(1), 83–91. doi:10.1038/ni.1684; ni.1684 [pii]. Sanos, S. L., & Diefenbach, A. (2010). Isolation of NK cells and NKlike cells from the intestinal lamina propria. Methods in Molecular Biology, 612, 505–517. doi:10.1007/978-1-60761-3626_32. Satoh-Takayama, N., Dumoutier, L., Lesjean-Pottier, S., Ribeiro, V. S., Mandelboim, O., Renauld, J. C., Vosshenrich, C. A., & Di Santo, J. P. (2009). The natural cytotoxicity receptor NKp46 is dispensable for IL-22-mediated innate intestinal immune defense against Citrobacter rodentium. Journal of Immunology (Baltimore, Md. : 1950), 183(10), 6579–6587. doi:10.4049/ jimmunol.0901935. Satoh-Takayama, N., Vosshenrich, C., Lesjean-Pottier, S., Sawa, S., Lochner, M., Rattis, F., Mention, J. J., Thiam, K., CerfBensussan, N., Mandelboim, O., Eberl, G., & Di Santo, J. (2008). Microbial flora drives interleukin 22 production in intestinal NKp46+ cells that provide innate mucosal immune defense. Immunity, 29(6), 958–970. doi:10.1016/j.immuni.2008.11.001. Schulz, S. M., Kohler, G., Schutze, N., Knauer, J., Straubinger, R. K., Chackerian, A. A., Witte, E., Wolk, K., Sabat, R., Iwakura, Y., Holscher, C., Müller, U., Kastelein, R. A., & Alber, G. (2008). Protective immunity to systemic infection with attenuated Salmonella enterica serovar enteritidis in the absence of IL-12 is associated with IL-23-dependent IL-22, but not IL-17. [Research Support, Non-U.S. Gov’t]. Journal of Immunology, 181(11), 7891–7901. Siegemund, S., Schutze, N., Schulz, S., Wolk, K., Nasilowska, K., Straubinger, R. K., Sabat, R., & Alber, G. (2009). Differential IL23 requirement for IL-22 and IL-17A production during innate immunity against Salmonella enterica serovar Enteritidis. International Immunology, 21(5), 555–565. doi:10.1093/intimm/ dxp025; dxp025 [pii].

338

Journal of Functional Foods 23 (2016) 329–338

Spits, H., Artis, D., Colonna, M., Diefenbach, A., Di Santo, J. P., Eberl, G., Koyasu, S., Locksley, R. M., McKenzie, A. N., Mebius, R. E., Powrie, F., & Vivier, E. (2013). Innate lymphoid cells – a proposal for uniform nomenclature. Nature Reviews. Immunology, 13(2), 145–149. doi:10.1038/nri3365. Spits, H., & Di Santo, J. P. (2011). The expanding family of innate lymphoid cells: Regulators and effectors of immunity and tissue remodeling. Nature Immunology, 12(1), 21–27. doi:10.1038/ni.1962; ni.1962 [pii]. Sun, J. C., Beilke, J. N., Bezman, N. A., & Lanier, L. L. (2011). Homeostatic proliferation generates long-lived natural killer cells that respond against viral infection. The Journal of Experimental Medicine, 208(2), 357–368. doi:10.1084/ jem.20100479; jem.20100479 [pii]. Tung, J. W., Heydari, K., Tirouvanziam, R., Sahaf, B., Parks, D. R., & Herzenberg, L. A. (2007). Modern flow cytometry: A practical

approach. Clinics in Laboratory Medicine, 27(3), 453–468, v. doi:10.1016/j.cll.2007.05.001. Uhrberg, M. (2005). The CD107 mobilization assay: Viable isolation and immunotherapeutic potential of tumor-cytolytic NK cells. Leukemia, 19(5), 707–709. doi:10.1038/sj.leu.2403705; 2403705 [pii]. Walzer, T., Jaeger, S., Chaix, J., & Vivier, E. (2007). Natural killer cells: From CD3(-)NKp46(+) to post-genomics meta-analyses. Current Opinion in Immunology, 19(3), 365–372. doi:10.1016/ j.coi.2007.04.004; S0952-7915(07)00056-8 [pii]. Zheng, Y., Valdez, P. A., Danilenko, D. M., Hu, Y., Sa, S. M., Gong, Q., Abbas, A. R., Modrusan, Z., Ghilardi, N., de Sauvage, F.J., & Ouyang, W. (2008). Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens. Nature Medicine, 14(3), 282–289. doi:10.1038/nm1720; nm1720 [pii].