Nonpathogenic Lactobacillus rhamnosus activates the inflammasome ...

Research paper

Gut Microbes 3:6, 510-522; November/December 2012; © 2012 Landes Bioscience

Nonpathogenic Lactobacillus rhamnosus activates the inflammasome and antiviral responses in human macrophages 4

1 Valio R&D; Helsinki, Finland; 2University of Helsinki; Department of Veterinary Sciences; Helsinki, Finland; 3University of Helsinki; Institute of Biomedicine; Helsinki, Finland; National Institute for Health and Welfare (THL); Department of Infectious Disease Surveillance and Control; Helsinki, Finland; 5University of Helsinki; Institute of Biomedicine; Pharmacology, Medical Nutrition Physiology; Helsinki, Finland

Keywords: inflammasome, influenza A virus, innate immunity, interferon, lactobacilli, macrophages, microarray, probiotics Abbreviations: CT, comparative threshold; GO, gene ontology; GG, L. rhamnosus strain GG; MDP, muramyl dipeptide; MRS, de Man, Sharpe and Rogosa; Mx, myxovirus resistance protein; M1, matrix protein 1 ; NALP3, nucleotide-binding oligomerization domain-like protein 3; NF-κB, nuclear factor kappa B; NP, nucleoprotein ; NS1, non-structural protein 1; RIG-I, retinoic acidinducible gene 1; STAT, signal transducer and activator of transcription; TLR, Toll-like receptor

In this study, we have utilized global gene expression profiling to compare the responses of human primary macrophages to two closely related, well-characterized Lactobacillus rhamnosus strains GG and LC705, since our understanding of the responses elicited by nonpathogenic bacteria in human innate immune system is limited. Macrophages are phagocytic cells of the innate immune system that perform sentinel functions to initiate appropriate responses to surrounding stimuli. Macrophages that reside on gut mucosa encounter ingested and intestinal bacteria. Bacteria of Lactobacillus genus are nonpathogenic and used in food and as supplements with health-promoting probiotic potential. Our results demonstrate that live GG and LC705 induced quantitatively different gene expression profiles in macrophages. A gene ontology analysis revealed functional similarities and differences in responses to GG and LC705 that were reflected in host defense responses. Both GG and LC705 induced interleukin-1β production in macrophages that required caspase-1 activity. LC705, but not GG, induced type I interferon -dependent gene activation that correlated with its ability to prevent influenza A virus replication and production of viral proteins in macrophages. Our results indicate that nonpathogenic bacteria are able to activate the inflammasome. In addition, our results suggest that L. rhamnosus may prime the antiviral potential of human macrophages.

Introduction Macrophages are key operators of the innate immune system due to their sentinel functions.1 Macrophages are the most abundant population of phagocytic cells in the intestine2 where the contact between ingested bacteria including Lactobacillus and the human immune system takes place. Macrophages are equipped with pattern-recognition receptors (PRR) for efficient detection of microbes. Macrophages are able to sense threat levels posed by the encountered microbes by combining signals from several membrane-bound and cytoplasmic PRRs, and respond accordingly.3 Activation of the NALP3 inflammasome is an example of controlled activation of the inflammatory response where both transcriptional and translational regulation are required. For NALP3 inflammasome activation, the priming signal for interleukin (IL)-1β induction is generated via PRR, and the second danger signal leads to caspase-1

activation followed by the production of biologically active IL-1β and IL-18.4,5 Bacteria of Gram-positive Lactobacillus genus are among the first to colonize the newborn human intestine. Lactobacillus species are nonpathogenic endogenous bacteria that benefit the human host. Various Lactobacillus species are commonly used in health-promoting, probiotic products and generally regarded as safe.6 The requirement for a probiotic bacterium is to remain viable in the host. Lactobacillus rhamnosus GG is of human origin, and is able to survive passage through the gut and transiently colonize human gastrointestinal tract.7,8 GG is a thoroughly studied probiotic bacterium currently available in foods and supplements in the US, Europe, and Asia.9,10 Lactobacillus has been occasionally associated with bacteremia in hospitalized patients with severe underlying diseases.11 However, the introduction of GG into dairy products in Finland followed by increase in GG consumption has not increased the number of bacteremia cases

*Correspondence to: Minja Miettinen; Email: [email protected] Submitted: 01/18/12; Revised: 06/28/12; Accepted: 08/05/12 http://dx.doi.org/10.4161/gmic.21736 510

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©2012 Landes Bioscience. Do not distribute

Minja Miettinen,1,* Taija E. Pietilä,2 Riina A. Kekkonen,1 Matti Kankainen,3 Sinikka Latvala, 4 Jaana Pirhonen,4 Pamela Österlund,4 Riitta Korpela5 and Ilkka Julkunen4

due to Lactobacillus.12 Since the effects on the host immune system are suggested to be unique for each Lactobacillus strain, it is of great importance to compare different, well-characterized bacteria in the same experimental setting. L. rhamnosus LC705 originates from a dairy source and contributes to the commercial food industry (e.g., cheese production).13 Recently, the complete genomes of GG and LC705 were sequenced and annotated7 making GG and LC705 attractive candidates for analyzing the details of interactions between nonpathogenic bacteria and human immune system. Lactobacillus species have shown promise in ameliorating the symptoms and duration of infections in humans.14 GG has been demonstrated to moderately reduce the duration and severity of respiratory tract infections.15 Understanding the mechanisms by which GG affects the human innate immune response is fundamental for gaining proof-of-concept for any observed in vivo effect. Knowledge of the molecular interactions between GG and human innate immune cells including macrophages is currently limited. We have previously shown in human primary macrophages that GG induces weaker production of inflammatory cytokines and chemokines and less signal transducer and activator of transcription (STAT) activation than the extracellular Gram-positive pathogen Streptococcus pyogenes,16,17 but activates nuclear factor kappa B (NF-κB) as efficiently as S. pyogenes via Toll-like receptor (TLR) 2.18 In the present study, we have utilized global gene expression profiling to gain novel information on the effects of live GG on human primary macrophages by comparing macrophage responses between two closely related strains GG and LC705. We show that nonpathogenic GG and LC705 similarly activate the inflammasome leading to caspase-1 activation and IL-1β production. However, GG and LC705 differently induce type I interferon (IFN) response which correlates with a reduction in influenza A virus replication and viral protein production in macrophages. Results L. rhamnosus GG and LC705 induce quantitatively different but functionally similar transcription profiles in human primary macrophages. The global gene expression of human leukocytes after stimulation with nonpathogenic Lactobacillus has not been reported to date. Probiotic bacteria need to be viable when consumed. Therefore, we used live GG or LC705 to stimulate human primary macrophages for 6 h and 24 h. Changes in gene expression profiles were measured by using the Affymetrix U132 Plus 2.0 Array. Quantitative analysis showed that GG and LC705 differed in their ability to alter macrophage gene expression after 6 h and 24 h stimulation. In total, GG altered the expression of 1,375 genes and LC705 that of 2016 genes. At both time points, the expression of 401 genes was affected by GG and LC705. However, there were genes whose expression was affected exclusively by the other bacterium only at either time point. Among those genes, there were 161 and 124 specific to LGG, at 6 h and 24 h stimulation, respectively, whereas LC705 changed the expression of 20 and 902 genes at these time points.

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The functional categorization of significantly differently expressed genes into GO-classes revealed similarities and differences between GG and LC705-induced biological processes (Fig. 1). The complete list of GO-classes affected is available in the Supplemental Material. Both GG and LC705 significantly affected several macrophage responses relating to their immunological functions. After 6 h stimulation, genes related to response to stress, cytokine activity and chemotaxis were induced strongly by both GG and LC705. After 24 h stimulation, genes most significantly induced by both GG and LC705 were associated with host defense and inflammatory responses. Interestingly, LC705induced genes associated with response to virus more significantly than GG-induced genes. In addition, GG but not LC705, was able to significantly affect genes associated with negative regulation of apoptosis. GG and LC705 induce transcription of inflammatory and innate immune response genes, while only LC705 upregulates type I IFN-inducible genes. The array data was manually investigated to obtain a more detailed view of the ability of GG and LC705 to alter primary macrophage responses. The complete microarray data are available at GEO (GSE20940). Hierarchical clustering of selected individual genes with altered expression after GG or LC705 stimulation is shown in Figure 2. The expression of inflammatory cytokine and chemokine genes was strongly inducible by both GG and LC705 after 6 h and 24 h stimulation. Among the upregulated cytokine genes were IL1B, IL18, IL15, IL12B, IL23A, EBI3 and IL27. The genes encoding subunits of NF-κB and NF-κB-associated signaling, MAPK genes, and signaling components downstream of TLRs were also similarly induced by GG and LC705, albeit at a lower level than the genes for inflammatory cytokines. The expression of genes for various macrophage receptors was also affected by GG and LC705. The receptor genes that were upregulated at either or both time points included those for CARD15, CD14, CD48, ITGB3, SIGLEC9, TLR2 and TLR7 genes. Only LC705 enhanced TLR3 gene expression. Gene expression of CD36 and TLR5 was downregulated by GG and LC705. GG and LC705 similarly affected the gene expression of components of the NALP3/NLRP3 inflammasome. NALP3 was moderately upregulated by GG and LC705, while PYCARD/ASC was downregulated. CASP1 gene was induced only by LC705 after 24 h stimulation. Most strikingly, LC705 but not GG was able to strongly upregulate the expression of type I IFN-inducible genes CXCL10, DDX58, IFI27, IRF7, ISG15, MX1, MX2, OAS1, OAS2, SIGLEC1, STAT2 and TRIM25 after 24 h stimulation. L. rhamnosus GG and LC705 activate the inflammasome and IL-1β and IL-18 processing in human macrophages. Since the array data suggested that genes associated with NALP3 inflammasome were regulated by both GG and LC705, we wanted to analyze the possible inflammasome activation by these two nonpathogenic bacteria in more detail. We performed qRT-PCR for caspase-1, IL-1β and NALP3 mRNA detection from the samples used for the microarray analysis. Results with qRT-PCR regarding GG and LC705-induced

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research paper

Research paper

IL-1β and NALP3 expression were similar with the array results (Fig. 3A). Both GG and LC705 similarly increased the expression of NALP3 after 24 h. The mRNA expression of IL-1β was upregulated after 6 h and 24 h stimulation by both GG and LC705. In addition, LC705-induced IL-1β mRNA expression was higher after 24 h stimulation. The array data suggested that LC705 was more potent than GG in upregulating caspase-1 mRNA. As shown by the qRT-PCR analysis, GG and

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LC705-induced caspase-1 mRNA expression was low with no significant difference. The activation of NALP3 inflammasome leads to self-cleavage and activation of procaspase-1 into its proteolytically active form, caspase-1. The production of mature IL-1β and IL-18 is indicative of caspase-1 activity.19 We used specific antibodies in western blotting to analyze whether GG and LC705 stimulation induced IL-1β, IL-18 and caspase-1 protein expression and

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Figure 1. Functional annotation of the biological processes affected in macrophages stimulated by live GG or LC705. Depicted are immunologically relevant GO-classes that are significantly affected by either GG or LC705. P-values below or equal to 0.01 were considered as significant. Dotted lines indicate p value of 0.01. David software was used in the assessment of categories as described in Materials and Methods. The complete list of GOclasses affected is available in the Supplemental Materials.

Figure 2. Expression profiles of selected genes significantly modulated by GG or LC705 stimulation in macrophages. Heat map shows genes that were significantly differentially expressed (p ≤ 0.01), and were also present in functional GO-classes activated by GG or LC705. Clustering and visualization were obtained with TMEV-tool.


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processing in macrophages (Fig. 3B). GG and LC705 similarly induced the production of pro-IL-1β and mature IL-1β. ProIL18 was basally expressed in macrophages, and was not induced by GG or LC705. Both GG and LC705 slightly increased the levels of mature IL-18 protein. Both GG and LC705 increased the production of procaspase-1 and 20 kDa form of caspase-1 (Fig. 3B).20 Both live and dead L. rhamnosus GG and LC705 induce the production of IL-1β in human macrophages. To rule out that the NALP3-dependent induction of IL-1β and IL-18 was due to the presence of endogenous ligands or lactic acid produced by metabolically active lactobacilli, we stimulated human macrophages with different doses of live and heat-killed bacteria. The production of pro-IL-1β and mature IL-1β occurred similarly in macrophages stimulated with either live or heat-killed L. rhamnosus GG and LC705 (Fig. 4A). Moreover, secretion of IL-1β occurred in a similar fashion in response to stimulation of cells with live or dead bacteria (Fig. 4B). In addition, both processing and secretion of IL-1β were bacterial dose-dependent events (Fig. 4.). Caspase-1 activation by GG and LC705 is required for IL-1β production in macrophages. Next we investigated the role of caspase-1 in GG and LC705-induced secretion of IL-1β in human primary macrophages. We used a caspase-1-specific inhibitory peptide YVAD-CHO.21 The blockage of IL-1β secretion by YVAD-CHO correlates with inactivation of caspase-1 enzymatic activity in human monocytes.22 Inhibition of caspase-1 activity reduced IL-1β production after 6 h and 24 h GG and LC705 stimulation, but the reduction in IL-1β secretion was significant only after 24 h. Bacteria-induced TNF-α production remained unaffected at both time points (Fig. 5A). To further analyze the mechanisms involved in GG and LC705-induced caspase-1 activation, we used dynasore, a cell-permeable dynamin inhibitor that blocks endocytosis.23 Dynasore has also been suggested to inhibit bacterial MDPprimed caspase-1 activation in mouse macrophages and IL-1β production in human monocytes.24 As prolonged dynasore exposure affects primary cell viability,24 we analyzed caspase-1 and IL-1β expression after 6 h GG or LC705 stimulation. Dynasore treatment diminished both the expression of pro-IL-1β and the formation of mature IL-1β protein after GG and LC705 stimulation as analyzed by western blotting (Fig. 5B). Dynasore also diminished GG and LC705-induced procaspase-1 and caspase-1 p20 production. IL-1β secretion from macrophages stimulated with GG or LC705 was also inhibited by dynasore as measured by ELISA (Fig. 5C), but the reduction was not statistically significant (p = 0.35). L. rhamnosus LC705 elicits more pronounced type I IFNdependent antiviral response than GG. According to our microarray data LC705 was a more potent inducer of type I IFNdependent gene expression than GG. ELISA analysis showed that LC705, but not GG, was able to induce IFN-α production after 24 h stimulation (Fig. 6A). We compared the ability of LC705 and GG to induce CXCL10 production whose expression is at least partially type I IFN-dependent.17,25 LC705 induced CXCL10 production better than GG (Fig. 6A).

Functional categorization of the genes induced in macrophages by LC705 and GG suggested that LC705 was more potent than GG in activating macrophage antiviral responses (Fig. 1). This striking difference in the upregulation of type I IFN-dependent antiviral genes is also depicted in Figure 2. To analyze whether LC705 was better in activating type I IFN-dependent antiviral protein production, we focused on RIG-I (DDX58), MxA (MX1) and MxB (MX2) that participate in the recognition of viruses and inhibiting virus replication, respectively.26,27 LC705 induced RIG-I, MxA and MxB protein expression after 24 h stimulation (Fig. 6B). GG was able to induce only weak MxA production after 24 h stimulation. No RIG-I or MxB production was detectable after GG stimulation. To assess the biological relevance of L. rhamnosus-induced RIG-I and/or Mx protein production, we tested the ability of GG and LC705 pretreatment to inhibit influenza A virus replication in primary human macrophages. Stimulation of macrophages for 24 h with LC705 prior to influenza A infection resulted in reduction of viral replication as measured by NP, M1 and NS1 mRNA levels after 6 h infection (Fig. 6C). Priming macrophages with LC705 prior to influenza A infection also diminished viral NP, M1 and NS1 protein production (Fig. 6D and E). GG priming had no effect on influenza A mRNA production (Fig. 6C) even though some reduction in NP, M1, and NS1 protein production was observed at low MOI of influenza A (Fig. 6D and E). No effect on viral protein

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production was observed after 24 h virus infection (data not shown). Discussion Various Lactobacillus species are used in food fermentations and as supplements in probiotic products. GG has been shown to induce favorable effects on the human immune system as documented in several clinical trials.14 Yet, the details of interactions between any particular Lactobacillus including GG and the innate immune system remain elusive. In this study, we show that two closely related L. rhamnosus strains, GG and LC705 induced similar but not identical global gene expression in human primary macrophages. Our results demonstrate that both live nonpathogenic L. rhamnosus strains activated the inflammasome as measured by caspase-1-mediated IL-1β production. Interestingly, LC705 was more efficient than GG in inducing type I IFNmediated responses, and in limiting influenza A virus replication. Global gene expression profiling of purified populations of human primary leukocytes following live Lactobacillus stimulation has not been reported before. We wanted to obtain a broad view of human macrophage responses to GG and LC705 that have been recently sequenced and annotated7 making these L. rhamnosus strains attractive candidates for studying the interactions between nonpathogenic bacteria and human immune system. While GG and LC705 elicited quantitatively

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Figure 3. Expression of NALP3 inflammasome components in macrophages is activated by both GG and LC705. (A) Human primary macrophages were stimulated at a 10:1 (bacteria:macrophage) ratio with GG or LC705, and total RNA was isolated for qRT-PCR analysis. Results were normalized to β-actin levels, and relative fold induction was determined by using the 2-ΔΔCT formula. Results are presented as means ± SD from three independent experiments, each performed with cells from six donors (B) Human macrophages were treated as described above, and cell lysates were prepared. Proteins were separated on 15% SDS-PAGE, blotted, and visualized with indicated antibodies. Results are representative of four independent experiments.

different responses, the nature of macrophage response was similar with strongest induction in innate immune responserelated genes. The results are in line with our previous observations in macrophages on the ability of GG to moderately activate the production of inflammatory cytokines and the transcription factors involved.16-18 Reflecting previous findings, LC705 and GG upregulated genes for inflammatory cytokines IL-6, IL-8, IL-12 and TNF-α, anti-inflammatory IL-10, genes for transcription factors NF-κB, Stat1, Stat3, ISGF3 and IRF1 and the gene for GG receptor TLR2.18 Upregulation of inflammatory genes including those encoding proteins required for prostaglandin synthesis, MyD88, MAPKs and IL-23, genes for Gram-positive bacterial receptors including CD48, ITGβ3 as well as downregulation of genes for bacterial receptors CD36 and TLR5 after GG and LC705 stimulation, indicate that GG


and LC705 are able to activate macrophage innate immune responses broadly. In vivo, GG and LC705 encounter human macrophages in the intestine. The studies performed with human intestinal macrophages originating from subepithelial lamina propria are few, and limited to observations made in vitro.28 Human intestinal macrophages have been demonstrated to display inability to mount an inflammatory response while retaining the ability to phagocytose microbes.29 However, CD14 + lamina propria macrophages have been shown to induce pro-inflammatory cytokines including IL-1β and IL-630,31 indicating that intestinal macrophage population is not exclusively anti-inflammatory. This newly isolated intestinal macrophage type is hypothesized to represent monocyte-derived macrophages that have exited circulation at sites of infection.32 Global gene expression profiling

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Figure 4. Processing and secretion of IL-1β occurs upon stimulation of macrophages by live and dead lactobacilli. (A) Human primary macrophages were stimulated at 2:1, 10:1 and 25:1 (bacteria:macrophages) ratios with GG or LC705. Proteins were separated with 15% SDS-PAGE, blotted, and visualized with indicated antibodies. (B) Production of IL1β was measured from cell culture supernatants by ELISA at 6 and 24 h after stimulation. The results are the means ± SD from a representative experiment performed with cells from six donors.

of intestinal biopsies of humans has shown that GG consumption induces genes involved in immune response and inflammation.33 Taken together, the data in the present study and those obtained with intestinal biopsies33 suggest that GG has the ability to induce a moderate inflammatory response. However, experiments using human intestinal macrophages are needed to validate these findings. Comparison of gene expression profiles and functions of inflammatory CD14 + and anti-inflammatory CD14macrophages from human intestinal mucosa during stimulation

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with probiotic bacteria would thus promote our understanding of probiotic actions in vivo environment. Inflammasome activation is characteristic for an inflammatory response, and NALP3 is one of the inflammasome components that links microbial and endogenous danger signals to caspase-1 activation.4,5 Interestingly, both nonpathogenic GG and LC705 induced the expression of IL-1β, IL-18 and NALP3 mRNAs in macrophages. Gene array data suggested that LC705 was more potent than GG in inducing caspase-1 mRNA, even

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Figure 5. Caspase-1 activity is required for GG and LC705 induced IL-1β production in macrophages. (A) Human primary macrophages were treated for 30 min with 50 μM Ac-YVAD-CHO caspase-1 inhibitor peptide before stimulation with live GG or LC705 for 6 or 24 h. Supernatants were collected, and the amounts of mature IL1β and TNF-α were determined by ELISA. The results at 6 h are the means ± SD of four donors and the results at 24 h are representative of four independent experiments each performed with cells from four donors. (B) Dynasore was used at 80 μM to pretreat macrophages 30 min prior to 6 h GG or LC705 stimulation. The presence of pro-IL-1β, mature IL-1β, pro-caspase-1 and mature caspase-1 was analyzed by western blotting. (C) Secreted IL-1β was measured from 6 h cell culture supernatants by ELISA. Results are the means ± SD of two independent experiments, each performed with cells from four donors. Calculations for p values were performed using Anova, and group-wise differences were analyzed by paired t-test followed by FDR-test (*p ≤ 0.1). Only statistically significant differences between groups are marked in the figure.

though the difference was not significant. Caspase-1 mRNA is type I IFN-inducible in macrophages.34 Hence, LC705-induced type I IFNs are likely contributing to the more efficient caspase-1 mRNA induction as compared with GG stimulated cells. GG


and LC705 also increased the production of procaspase-1 and active caspase-1 p20 equally well. Somewhat higher caspase-1 mRNA levels after 24 h LC705 stimulation were not reflected in procaspase-1 protein expression. This could be explained by the

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Figure 6. Antiviral responses are activated by live GG and LC705. (A) Macrophage supernatants were collected at 6 and 24 h after stimulation with GG or LC705, and the amounts of IFN-α and CXCL10 were measured by ELISA. (B) Macrophages were stimulated with live GG and LC705, after which the expression of RIG-I, MxA and MxB was analyzed by western blotting with specific antibodies. The results are representative of four independent experiments, each performed with cells from four blood donors. (C) Macrophages were stimulated with live GG or LC705 for 24 h, followed by influenza A infection for 6 h. Viral replication was measured by using qRT-PCR specific for influenza NP, M1 or NS1 mRNA. The experiment was repeated three times with similar results. (D) Macrophages primed with GG or LC705 for 24 h were infected with indicated doses of influenza A, and cell lysates were collected at 6 h. Influenza A NP, M1 and NS1 proteins were detected by western blotting by using specific antibodies. (E) Western blot band intensities from panel D were scanned separately after which NP, M1 and NS1 signal intensities were normalized to the levels of β-actin.

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effects were demonstrated to be mediated by two GG proteins p40 and p75.48 These proteins may be responsible and explain the ability of GG to induce anti-apoptotic gene expression in human macrophages. Genes for p40 and p75 are also present in the LC705 genome.7 Our results suggest that LC705 p40 and p75 are not secreted or do not have a strong anti-apoptotic effect (our unpublished data). It was surprising that LC705 was more potent than GG in upregulating the expression of several type I IFN-regulated genes in macrophages. Most of these type I IFN-inducible genes including OAS gene variants and signaling molecules TLR3, TLR7, IRF7, STAT1, STAT2 and TRIM25 are crucial for defense against virus infection.49 RIG-I, MxA and MxB are also type I IFN-inducible genes50,51 and at least RIG-I and MxA are required for host defense against influenza A virus.26,52 LC705 activated the production of IFN-α, RIG-I, MxA and MxB proteins in macrophages. GG induced only little MxA production, providing indirect evidence of type I IFN production. The difference in the ability of LC705 and GG to induce IFN-α production most likely contributes to the observed difference between these bacteria to activate type I IFN-inducible gene expression and protein production. LC705 was able to partially inhibit influenza A virus replication in macrophages as evidenced by reduced viral mRNA and protein expression. LC705 prestimulation restricted influenza A virus replication better than GG, which correlated with the ability of LC705 to induce type I IFN response better than GG. In a recent mouse study, GG feeding alleviated symptoms following influenza A infection as well as reduced virus titers in the lungs.53 In addition to reducing viral titers in the lungs, L. plantarum administration before influenza A infection was shown to concomitantly increase type I IFN levels in serum at early stages of infection.54 RIG-I participates in inflammasome activation during viral infection.55 This could provide LC705-stimulated RIGI-expressing macrophages an advantage over GG-stimulated macrophages in limiting influenza A and possibly other virus replication. Since IFNs have a primary protective role during early steps of influenza A infection,49 type I IFN-dependent mechanisms are likely to be responsible for the ability of L. rhamnosus to improve early macrophage defenses against influenza A virus. The comparative genome annotation of GG and LC705 revealed differences between GG and LC705 that could account for the more robust macrophage activation by LC705. LC705 encodes for a long adhesion protein missing in GG.7 In addition, LC705 genome encodes for lipoproteins and hydrolases that are not present in GG. Whether any of these LC705 components are involved in inducing macrophage type I IFN responses remains to be investigated. GG has been shown to colonize the human gastrointestinal tract and to persist there at higher levels and longer than LC705. This intestinal binding may be mediated by the GG-specific mucus-binding SpaC pilin,7 possibly facilitated by other binding factors such as mucus binding factor (MBF)56 and modulator of adhesion and biofilm (MabA).57 It seems logical that GG, which is of human origin, provokes a quantitatively and qualitatively more limited immune response in human macrophages than the dairy product-originating LC705.

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lag period in protein synthesis, or by the insignificant difference in mRNA levels. Furthermore, GG and LC705-stimulated macrophages expressed IL-1β and IL-18 proteins and secreted IL-1β similarly, suggesting that caspase-1 was equally well activated by both bacteria. In the presence of caspase-1-specific inhibitor peptide, GG and LC705-induced IL-1β production was significantly reduced. Our results thus demonstrate that nonpathogenic GG and LC705 activate caspase-1 which leads to IL-1β production. To our knowledge, no reports linking nonpathogenic Lactobacillus to inflammasome activation in human cells have been published. Recently, Lactobacillus delbrueckii subsp bulgaricus and Lactobacillus gasseri were shown to induce NALP3 expression in porcine gut-associated lymphoid tissues, but this study was not extended to inflammasome-dependent caspase-1, IL-1β and IL-18 analyses.35 Extracellular Gram-positive pathogens including S. pyogenes, S. pneumoniae, Staphylococcus aureus and their toxins have been demonstrated to activate the NALP3 inflammasome leading to IL-1β production.5,36-38 Inflammasome activation and IL-1β production have been linked to phagocytosis.39,40 MDP is a degradation product of phagocytosed bacteria and provides a priming signal for inflammasome activation.41,42 As macrophages are professional phagocytes, it is likely that degradation products of GG and LC705 including MDP are present in the cytosol, and provide a signal for inflammasome activation. Dynamin-2 is required for phagosome maturation43 and its inhibition by dynasore impairs MDP-primed caspase-1 activation and IL-1β secretion.24 By using dynasore we were able to see inhibition of caspase-1 p20 and IL-1β production in GG and LC705-stimulated macrophages. Even though inhibition of IL-1β secretion by dynasore did not reach high statistical significance, our results suggest that the intrinsic ability of macrophages to phagocytose GG and LC705 is involved in inflammasome activation in these cells. The ability of GG and LC705 to similarly and moderately activate the inflammasome and the expression of several cytokine and signaling genes involved in macrophage defense responses could be attributed to their high genomic identity and synteny. Both GG and LC705 were described to contain pilus gene clusters for spaFED 7 that could account for their immunostimulatory effects. The Lactobacillus components involved in the activation of human immune responses have remained largely uncharacterized. Only lipoteichoic acid (LTA) of L. plantarum has been shown to mediate inflammatory cytokine production in human peripheral blood mononuclear cells.44 It is likely that the yet to be described components of Lactobacillus important for macrophage interactions are partly overlapping with those described for Gram-positive pathogens.45 It is, however, interesting that live and heat-killed GG and LC705 induced macrophage inflammasome responses equally well suggesting that preferentially the ingested whole bacteria, whether they are live or dead, are able to trigger signals leading to efficient activation of host inflammatory responses. GG had a significant effect on the expression of anti-apoptotic genes in human macrophages (Figs. 1 and 2). A similar effect was reported in mouse intestinal tissue after GG administration.46 GG has been shown to prevent cytokine-induced47 and chemical-induced46 apoptosis in mouse intestinal cells. Anti-apoptotic

Materials and Methods Bacteria and viruses. L. rhamnosus GG (GG; ATCC 53103) and L. rhamnosus LC705 (DSM 7061) were obtained from Valio Ltd. R&D. Bacteria were grown on MRS medium (Lab M Ltd.) to late logarithmic growth phase as previously described.58,59 Heatkilled bacteria were obtained by treating late logarithmic phase GG and LC705 at +120°C, 5 min. Killing was verified by MRS plate counting method. Human pathogenic influenza A virus (strain A/Beijing/353/89 H3N2) was grown in embryonated chicken eggs.60 The infectivity of the virus stock in human macrophages has been determined previously.61 Human primary macrophages. Peripheral blood mononuclear cells from healthy blood donors were isolated by Ficoll Paque (GE Healthcare) density gradient centrifugation, and monocytes were purified by adherence on six-well cell culture plates (Falcon Becton Dickinson) as previously described.16 To obtain macrophages, monocytes were cultured for 7 d in serumfree medium (Gibco Invitrogen) supplemented with 10 ng/ml recombinant human GM-CSF (R&D Systems). Macrophage stimulation experiments. Macrophages of four to six blood donors in each experiment were stimulated with live GG or LC705 at 10:1 bacteria:macrophage ratio in RPMI-1640 medium (Sigma-Aldrich) without serum.18 Preceding influenza A virus infection macrophages were mock treated or stimulated with live GG or LC705 for 24 h. After stimulation, macrophages were infected with indicated doses of influenza A virus. The virus was allowed to adsorb for 1 h, followed by washing with PBS, and addition of fresh RPMI-1640 medium containing 10% FCS (Integro BV).62 Macrophage stimulations were performed for times indicated in the figure legends. Each experiment was performed at least three times unless specified separately. Microarray study design, methodology and data analysis. A total of 18 Affymetrix Human Genome U132 Plus 2.0 Array GeneChips (Affymetrix) were used to analyze gene expression profiles of mock, GG or LC705 stimulated human macrophages. Three independent macrophage stimulation experiments were performed, each with cells from six different donors at 6 h and 24 h time points. Total RNA preparation and microarray gene expression measurements were performed at Biomedicum Genomics using standard protocols recommended by the manufacturer (Affymetrix). Microarray data are available at GEO (GSE20940). The robust multiarray averaging algorithm [26] in the Bioconductor simpleaffy package63,64 was used to calculate expression estimates from GeneChip signal intensity data. To provide better precision and accuracy and to overcome interpretation


problems related to conflicting id gene references,63 we used updated probe set definitions65 based on Ensemble gene information. In contrast to the default Affymetrix chip description file with 54,675 probe sets, the used custom chip description file (version 11.0.1) contained 17,491 unique Ensemble gene probe sets. The significance of differential expression between biological replicate sets was assessed using the empirical Bayes moderated paired t statistics (eBayes-function) in the limma package, followed by Intensity-based hierarchical Bayes analysis.66,67 In the analysis, a moderated paired t-test was computed by constructing cell line effects in the linear model. All p values were adjusted for multiple hypotheses testing using Benjamini and Hochberg’s FDR-test in the stats package.68 Genes with p values less or equal to 0.01 were identified as significantly differentially expressed. David software69 was used to assess biological processes enriched within significantly differentially expressed gene sets. Functional categories found among the differentially expressed genes were compared against the frequency of functional categories of all Ensemble genes. GO-classes with a p value equal to or below 0.01 after applying Benjamini and Hochberg’s FDR-test were considered as significant.68 The gene set enrichment analyses of genes ranked based on their moderated t values were performed using Gorilla software70 with the default human gene set as a background. Clustering and visualization of the gene expression differences were done using TMEV-tool and hierarchical clustering.71,72 Inhibitor treatments. Caspase-1 inhibitor peptide Ac-YVADCHO (Bachem, Bubendorf, Switzerland) was used at a 50 μM concentration to pretreat macrophages for 30 min before GG or LC705 stimulation.60 Likewise, macrophages were treated for 30 min with 80 μM dynamin inhibitor dynasore (D7693, Sigma-Aldrich) prior to GG or LC705 stimulation.73 The optimal concentration of dynasore for macrophage stimulations has been determined previously.74 Macrophage inhibitor experiments were performed in RPMI-1640 media without serum. ELISA and western blot analysis. Cytokine levels were measured by ELISA method58 with antibodies and standards for IL-1β (R&D Systems), CXCL10 and tumor necrosis factor (TNF-α) (BD PharMingen). Determination of IFN-α was performed by using Verikine™ Human IFN-α Multi Subtype kit (PBL Biomedical Laboratories). Cellular and viral proteins were detected by western blotting.51 Membranes were stained with antibodies against caspase-1 (C4851; Sigma-Aldrich), IL-1β,34 IL-18,60 MxA,51 MxB,75 RIG-I,50 influenza A NP76 and NS1.77 Polyclonal antibody against purified baculovirus-expressed influenza A M1 was a kind gift from Dr K. Melén (National Institute for Health and Welfare, Helsinki, Finland) and produced according to previously described protocols.75 Scanning of band intensities was performed with AIDA software. Quantitative real-time PCR. Total RNA extraction, cDNA synthesis and qRT-PCR were performed as described.18 The primers used were: caspase-1 (Hs00354832_m1), IL-1β (Hs00174097_m1) and NALP3 (Hs00918092_m1), all from Applied Biosystems. Influenza-specific primers were: M1,78 NS1 forward primer 5'-tga aag cga att tca gtg tga t, NS1 reverse primer 5'-ctg gaa aag aag gca atg gt, NS1 probe 5'-CY5-cta agg gct ttc acc gaa gag gg, and NP forward primer 5'-cca taa gga cca gga gtg ga,

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Our results demonstrate that nonpathogenic L. rhamnosus GG and LC705 are able to activate the inflammasome and IFN response in human macrophages, even though LC705 was clearly better than GG to induce IFN and IFN induced gene expression. It remains to be seen whether this is a feature shared by other nonpathogenic bacteria. The results of our study also provide one mechanistic explanation for the ability of Lactobacillus GG to reduce the duration and severity of viral infections in humans.

Disclosure of Potential Conflicts of Interest

Biosciences and Environment Grants 119065 and 129954 (to M.M.), the Academy of Finland Research Council for Health and the Sigrid Juselius Foundation (to I.J.), and the Finnish Funding Agency for Technology and Innovation (Tekes) Grant 2243/31/05 (to R.A.K.). Authorship: M.M. designed the study, performed laboratory experiments, analyzed the results, and wrote the paper, T.E.P. designed the study, analyzed the results, and wrote the paper, R.A.K. contributed to the study’s conception, and performed laboratory experiments, M.K. analyzed the microarray data, performed statistical analysis, and wrote the paper, S.L. performed laboratory experiments and analyzed the results, J.P. designed the study and wrote the paper, P.Ö. designed the study and analyzed the results, R.K. contributed to the study’s conception, and I.J. designed the study and wrote the paper.

No potential conflicts of interest were disclosed.

Supplemental Materials

Acknowledgments

The authors thank Dr Krister Melén for providing anti-influenza virus M1 antibodies. Mari Aaltonen and Johanna Lahtinen are thanked for their skillful technical assistance. This work was supported by the Academy of Finland Research Council for References 1. Martinez FO, Helming L, Gordon S. Alternative activation of macrophages: an immunologic functional perspective. Annu Rev Immunol 2009; 27:451-83; PMID:19105661; http://dx.doi.org/10.1146/annurev. immunol.021908.132532. 2. Lee SH, Starkey PM, Gordon S. Quantitative analysis of total macrophage content in adult mouse tissues. Immunochemical studies with monoclonal antibody F4/80. J Exp Med 1985; 161:475-89; PMID:3973536; http://dx.doi.org/10.1084/jem.161.3.475. 3. Zak DE, Aderem A. Systems biology of innate immunity. Immunol Rev 2009; 227:264-82; PMID:19120490; http://dx.doi.org/10.1111/j.1600-065X.2008.00721.x. 4. Franchi L, Eigenbrod T, Muñoz-Planillo R, Nuñez G. The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis. Nat Immunol 2009; 10:241-7; PMID:19221555; http://dx.doi.org/10.1038/ni.1703. 5. Martinon F, Mayor A, Tschopp J. The inflammasomes: guardians of the body. Annu Rev Immunol 2009; 27:229-65; PMID:19302040; http://dx.doi. org/10.1146/annurev.immunol.021908.132715. 6. Kleerebezem M, Vaughan EE. Probiotic and gut lactobacilli and bifidobacteria: molecular approaches to study diversity and activity. Annu Rev Microbiol 2009; 63:269-90; PMID:19575569; http://dx.doi. org/10.1146/annurev.micro.091208.073341. 7. Kankainen M, Paulin L, Tynkkynen S, von Ossowski I, Reunanen J, Partanen P, et al. Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a human- mucus binding protein. Proc Natl Acad Sci U S A 2009; 106:17193-8; PMID:19805152; http://dx.doi.org/10.1073/pnas.0908876106. 8. Goldin BR, Gorbach SL, Saxelin M, Barakat S, Gualtieri L, Salminen S. Survival of Lactobacillus species (strain GG) in human gastrointestinal tract. Dig Dis Sci 1992; 37:121-8; PMID:1728516; http:// dx.doi.org/10.1007/BF01308354. 9. Saxelin M, Tynkkynen S, Mattila-Sandholm T, de Vos WM. Probiotic and other functional microbes: from markets to mechanisms. Curr Opin Biotechnol 2005; 16:204-11; PMID:15831388; http://dx.doi. org/10.1016/j.copbio.2005.02.003.

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The complete list of GO-classes affected by GG and LC705 stimulation in macrophages is available as part of supplemental materials. Supplemental material may be downloaded here: http://w w w.landesbioscience.com/journals /gutmicrobes / article/21736/

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NP reverse primer 5'-ccc tcc gta ttt cca gtg aa, NP probe 5'-CY5cag gcc aaa tca gtg tgc aac cta c. Normalization between samples was performed by PCR amplification of β-actin (Hs99999903_ m1; Applied Biosystems). Relative quantification was determined by standard 2-ΔΔCT calculations. Samples from experiments containing virus were normalized by PCR amplification of 18S rRNA using ribosomal control RNA reagents (4308329; Applied Biosystems), and relative quantification was done by using the standard curve method as instructed by the manufacturer. Statistical analyses. The results from qRT-PCR and ELISA were analyzed by two-way Anova for repeated measurements using AOV-function in R. Group-wise differences were analyzed by paired t-test followed by the FDR-test.


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