Lactobacilli and Streptococci Activate NF-κB and STAT Signaling Pathways in Human Macrophages

Minja Miettinen, Anne Lehtonen, Ilkka Julkunen and Sampsa Matikainen
J Immunol April 1, 2000, 164 (7) 3733-3740; DOI: https://doi.org/10.4049/jimmunol.164.7.3733

Abstract

Gram-positive bacteria induce the production of several cytokines in human leukocytes. The molecular mechanisms involved in Gram-positive bacteria-induced cytokine production have been poorly characterized. In this work we demonstrate that both nonpathogenic Lactobacillus rhamnosus GG and pathogenic Streptococcus pyogenes (group A streptococci) induce NF-κB and STAT DNA-binding activity in human primary macrophages as analyzed by EMSA. NF-κB activation was rapid and was not inhibited by a protein synthesis inhibitor cycloheximide, suggesting that these bacteria could directly activate NF-κB. STAT1, STAT3, and IFN regulatory factor-1 DNA binding was induced by both bacteria with delayed kinetics compared with NF-κB. In addition, streptococci induced the formation of IFN-α-specific transcription factor complex and IFN-stimulated gene factor-3 (ISGF3). STAT1 and STAT3 activation and ISGF3 complex formation were inhibited by cycloheximide or by neutralization with IFN-α/β-specific Abs. Streptococci were more potent than lactobacilli in inducing STAT1, ISGF3, and IFN regulatory factor-1 DNA binding. Accordingly, only streptococci induced IFN-α production. The activation of the IFN-α signaling pathway by streptococci could play a role in the pathogenesis of these bacteria. These results indicate that extracellular Gram-positive bacteria activate transcription factors involved in cytokine signaling by two mechanisms: directly, leading to NF-κB activation, and indirectly via cytokines, leading to STAT activation.

Macrophages have a central role in initiating the innate immune response, which leads to activation of the adaptive response. Macrophages phagocytose infected cells, present Ags to T and B cells, and produce cytokines and chemokines that modulate immune responses (1). These cytokines include TNF-α and IL-6, which are among the first produced during the innate immune response toward bacteria. TNF-α and IL-6 have pleiotropic effects such as induction of the acute phase response and activation of macrophages (2, 3). An innate immune response to viruses is characterized by rapid production of IFN-α in macrophages. In addition to its direct antiviral functions, IFN-α has a range of immunoregulatory functions, including NK cell activation and enhancement of Th1-type immunity (4, 5, 6, 7). Although the role of IFN-α in viral and intracellular bacterial infections is well established, in extracellular bacterial infections it remains poorly characterized.

Cytokines initiate signaling cascades through their receptors, leading to activation of transcription factors and target gene expression. NF-κB and STATs are both latent cytoplasmic transcription factors activated by Ag or cytokine stimulation. They regulate transcription of genes encoding proteins involved in immune, acute phase, and inflammatory responses. In humans, activated NF-κB dimers consist mainly of the Rel family proteins p50 and p65 subunits. NF-κB binds to responsive κB sites in the promoters and enhancers of target genes, including TNF-α and IL-6. TNF-α is known to initiate an autoregulatory feedback loop, where the activation of NF-κB is followed by the production of TNF-α and further activation of NF-κB (8, 9). STAT activation occurs via tyrosine phosphorylation; binding of cytokines to their receptors results in autophosphorylation of receptor-associated JAK kinases that phosphorylate and activate STATs. Activated STATs form homo- or heterodimers that translocate into the nucleus and bind specific target elements in the promoters of cytokine-inducible genes (10, 11, 12). STAT1 and STAT3 can be activated by several cytokines, including IFN-α and IL-6, and bind to the IFN-γ activation site (GAS)3 element. IFN-α is the only known activator of STAT2 that, together with STAT1 and p48, form the IFN-stimulated gene factor-3 (ISGF3) (3) complex that binds to the IFN response element (ISRE) (13, 14).

Lactobacilli are nonpathogenic Gram-positive inhabitants of human normal microflora (15); some of them have been postulated to have health beneficial effects, such as stimulation of the immune system (16). Strain Lactobacillus rhamnosus GG has been extensively studied regarding safety and clinical effects (17). Streptococcus pyogenes (group A streptococci) is a major Gram-positive human pathogen causing a wide range of infections. Since the 1980s, highly invasive strains associated with shock and organ failure have emerged (18). To analyze the mechanisms by which Gram-positive bacteria induce immune responses we have compared nonpathogenic and pathogenic strains. We have previously shown that L. rhamnosus GG and S. pyogenes induce the production of several cytokines in human PBMC (19). In this work we have compared and analyzed the role of live L. rhamnosus GG and S. pyogenes in activation of NF-κB and STATs in human primary macrophages.

Materials and Methods

Bacterial strains

Lactobacillus rhamnosus GG (American Type Culture Collection 53103) was obtained from Valio R&D (Helsinki, Finland), and Streptococcus pyogenes serotype T1 (IH32030), isolated from a child with bacteremia, was obtained from the collection of the National Public Health Institute (Helsinki, Finland). Bacteria were stored in skimmed milk at −70°C and passaged three times as previously described (20) before their use in stimulation experiments. Lactobacilli were grown in MRS medium (Difco, Detroit, MI) and streptococci in TY medium supplemented with 0.2% glucose (21). For stimulation experiments bacteria were grown to logarithmic growth phase, and the number of bacterial cells was determined by counting in a Petroff Hausser counting chamber.

Cell culture

Freshly collected leukocyte-rich buffy coats from healthy blood donors were supplied by the Finnish Red Cross Blood Transfusion Service (Helsinki, Finland). PBMC were isolated by a density gradient centrifugation over Ficoll-Paque gradient (Pharmacia, Uppsala, Sweden). After washing, the cells were resuspended in RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 0.6 μg/ml penicillin, 60 μg/ml streptomycin, 2 mM l-glutamine, and 20 mM HEPES. For monocyte differentiation, PBMC were allowed to adhere to plastic six-well plates (Falcon, Becton Dickinson, Franklin Lakes, NJ) for 1 h at 37°C in RPMI 1640 medium supplemented with antibiotics, glutamine, and HEPES without FCS (10 × 106 cells/well). After incubation nonadherent cells were removed, and the wells were washed twice with PBS (pH 7.4). Adherent cells were then grown for 7 days in macrophage/serum-free medium (Life Technologies, Grand Island, NY) supplemented with antibiotics and recombinant GM-CSF at 10 ng/ml (Leucomax, Schering-Plough, Innishannon, Ireland). More than 90% of the cultured cells were macrophages as determined by their morphology and CD14 expression (data not shown).

Stimulation experiments

To minimize interindividual variation all experiments were performed with cells obtained from four to six blood donors. Stimulation experiments were conducted in RPMI 1640 medium either with or without 10% heat-inactivated FCS (Integro, Zaandam, The Netherlands). Macrophages were stimulated with live bacteria at a 1:1 ratio. Cycloheximide (CHX; Sigma) was used to inhibit protein synthesis at concentration of 10 μg/ml. It was added to cell culture medium 0.5 h after the beginning of stimulations. Neutralizing sheep anti-IFN-α/β (22) and goat anti-IL-6 (R & D Systems, Abingdon, U.K.) Abs were used at concentrations of 2400/165 neutralizing IU/ml and 1 μg/ml, respectively. Cell culture supernatants and cells were collected at different times after stimulation and pooled. Cells were used for preparing nuclear extracts, isolation of total cellular RNA, or SDS-PAGE sample preparation. Supernatants stored at −20°C were used for cytokine determinations.

EMSA

Nuclear extracts were prepared as previously described (23). Nuclear protein/DNA-binding reactions were performed in a volume of 20 μl containing 5 μg of nuclear extract protein, 10 mM HEPES-KOH (pH 7.9), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, and 2 μg poly(dI-dC) as a nonspecific competitor. NF-κB (5′-AGTTGAGGGGACTTTCCCAGG-3′), IRF1-GAS (5′-AGCTTCAGCCTGATTTCCCCGAAATGACGGCA-3′), SIE (5′-GATCTAGGGATTTCCGGGAAATGAAGCT-3′), ISRE15 (5′-AGCTTGATCGGGAAAGGGAAACCGAAACTGAAGCCA-3′), and PRDI (5′-GATCAAGTGAAAGTGAAAGTGA-3′) oligonucleotides were synthesized with an IBI oligonucleotide synthesizer (Foster City, CA) and purified on PAGE in the presence of 8 mol/l urea. The probes for NF-κB, IRF1-GAS, SIE, and ISRE15 were end labeled with [γ-32P]dATP (3000 Ci/mol; Amersham, Aylesbury, U.K.) by T4 polynucleotide kinase and the probe for PRDI with the Klenow fill-in method. The binding reaction was performed at room temperature for 30 min. For supershift assays, nuclear protein extracts were incubated with Abs against NF-κB p50 (sc-1190 x), NF-κB p65 (sc-372 x), STAT1α p91 (sc-345 x), STAT5 recognizing both STAT5A and STAT5B (sc-835 x; all from Santa Cruz Biotechnology, Santa Cruz, CA), and STAT3 (71-0900, Zymed, San Francisco, CA) for 1 h on ice before addition of the radiolabeled probe. Nondenaturing low ionic strength PAGE gels (6%) in 0.25 × Tris-borate-EDTA buffer were used. Gels were dried, and bands were visualized by autoradiography.

Western blotting

SDS-PAGE was conducted by using the Laemmli buffer system (24) on 10% polyacrylamide gels. Proteins separated on gels were transferred to Immobilon-P (polyvinylidene difluoride) membranes (Millipore, Bedford, MA) with an Isophor electrotransfer device (Hoefer Scientific Instruments, San Francisco, CA) at 200 mA for 2 h. Binding of the primary and secondary Abs was performed in PBS (pH 7.4) containing 5% nonfat milk for 1 h at room temperature. Primary Abs used in immunoblotting were guinea pig anti-human IRF-1 (1/2000 dilution) (25) and guinea pig anti-human MxA (1/2000 dilution) (26). Peroxidase-conjugated rabbit anti-guinea pig Igs (Dako, Copenhagen, Denmark) were used as secondary Abs (1/2000 dilution). The bands were visualized on Amersham HyperMax film using the ECL chemiluminescence system according to the manufacturer’s (Amersham) instructions. Protein concentrations in the samples were determined with the Bio-Rad protein assay kit (Hercules, CA).

RNA isolation and analysis

For isolation of total cellular RNA, stimulated cells were collected, washed once with cold PBS (pH 7.4), and lysed in guanidium isothiocyanate (stored at −70°C) (27) followed by centrifugation through a CsCl cushion (28). RNA was quantitated photometrically, and samples containing equal amounts (20 μg) of total cellular RNA were size-fractionated on 1% formaldehyde-agarose gels, transferred to Hybond-N nylon membranes (Amersham), and hybridized with human IRF-1 (29) and MxA (30) cDNA probes. To control for equal RNA loading, ethidium bromide staining or hybridization with β-actin cDNA probe was used. The probes were labeled with [α-32P]dCTP (3000 Ci/mmol; Amersham) using a random primed DNA labeling kit (Roche, Indianapolis, IN). Hybridizations were performed in a solution containing 50% formamide, 5× Denhardt’s solution, 5× SSPE, and 0.5% SDS at 42°C. Membranes were washed twice with 1× SSC/0.1% SDS at 42°C for 30 min and once at 65°C for 30 min. The membranes were exposed to Kodak X-OMAT AR films (Eastman Kodak, Rochester, NY) at −70°C with intensifying screens.

Cytokine-specific ELISAs and biological assay for IFN-α/β

TNF-α and IL-6 levels in cell culture supernatants were determined by ELISA methods as described previously (19) with sensitivities of 20 pg/ml. The IFN-α/β assay was performed as previously described (31) with a detection limit of 3 IU/ml. Briefly, cell culture supernatants were harvested and dialyzed against acidic glycine buffer (pH 2) followed by two dialyses in PBS. IFN-α titers in samples were assayed by VSV plaque reduction in Hep2 cells.

Results

Activation of NF-κB DNA binding in human macrophages by Gram-positive bacteria

To study lactobacilli- and streptococci-induced activation of transcription factors, differentiated macrophages were stimulated with live bacteria. Nuclear extracts from stimulated macrophages were prepared and analyzed by EMSA using NF-κB oligonucleotide. Both bacteria activated NF-κB DNA binding rapidly at 1 h after stimulation, and this activation increased with time for up to 24 h (Fig. 1⇓A). As shown by the supershift experiment, the NF-κB DNA binding complex containing p50 and p65 was activated at 1 h after bacterial stimulation and was most prominent at the 24 h point (Fig. 1⇓B).

FIGURE 1.
FIGURE 1.

Activation of NF-κB DNA binding by lactobacilli and streptococci. Macrophages were stimulated with live lactobacilli (LAB) or streptococci (GAS) for 1, 3, 6, 9, or 24 h, and nuclear extracts were prepared and analyzed by EMSA using the NF-κB oligonucleotide. A, Kinetics of NF-κB activation by lactobacilli and streptococci. B, Supershift experiment with anti-p50 and anti-p65 Abs. Results are representative of three separate experiments.

Activation of STAT1, STAT3, and ISGF3 complex formation in human macrophages by Gram-positive bacteria

Both NF-κB and STATs are involved in cytokine signaling during immune responses. We therefore studied the possible activation of STATs induced by Gram-positive bacteria. STAT activation was analyzed using nuclear extracts prepared from live bacteria-stimulated macrophages by EMSA with IRF1-GAS, SIE, and ISRE15 oligonucleotides. STAT binding to IRF1-GAS in response to streptococci was detected at 3 h after stimulation, and the intensity of this complex increased up to 24 h. Lactobacilli-induced STAT binding to IRF1-GAS was weakly detectable at 9 and 24 h after stimulation (Fig. 2⇓A). Supershift experiments with anti-STAT1, anti-STAT3 and anti-STAT5 Abs showed that both bacteria activated STAT1 binding (Fig. 2⇓B). As analyzed using the SIE element, streptococci apparently activated the lowest of the three STAT DNA binding complexes at 3 h after stimulation. Both lactobacilli and streptococci activated the STAT DNA binding complex at 6 h after stimulation, with the intensity of the complexes increasing at least up to 24 h (Fig. 3⇓A). The SIE binding complexes activated by bacteria containing STAT1 and STAT3 homodimers (the lowest and the uppermost of the three bands, respectively) as well as STAT1/3 heterodimers (the middle band). Also, STAT5 binding to the SIE element induced by lactobacilli was seen at 24 h after stimulation (Fig. 3⇓B). ISGF3 complex binding to the ISRE15 element was detectable at 6 and 9 h after stimulation with streptococci, and the complex started to disappear thereafter (Fig. 4⇓).

FIGURE 2.
FIGURE 2.

Activation of STAT DNA binding to IRF1-GAS element by lactobacilli and streptococci. Macrophages were stimulated with live lactobacilli (LAB) and streptococci (GAS) for 1, 3, 6, 9, or 24 h, and nuclear extracts were prepared and analyzed by EMSA using IRF1-GAS oligonucleotide. A, Kinetics of STAT activation by lactobacilli and streptococci. B, Supershift experiment with anti-STAT1, anti-STAT3, and anti-STAT5 Abs. Results are representative of two separate experiments.

FIGURE 3.
FIGURE 3.

Activation of STAT DNA binding to the SIE element by lactobacilli and streptococci. Macrophages were stimulated with live lactobacilli (LAB) or streptococci (GAS) for 1, 3, 6, 9, or 24 h, and nuclear extracts were prepared and analyzed by EMSA using SIE oligonucleotide. A, Kinetics of STAT activation by lactobacilli and streptococci. B, Supershift experiment with anti-STAT1, anti-STAT3, and anti-STAT5. Results are representative of two separate experiments.

FIGURE 4.
FIGURE 4.

Activation of ISGF3 DNA binding by lactobacilli and streptococci. Macrophages were stimulated with live lactobacilli (LAB) or streptococci (GAS), and the kinetics of ISGF3 complex formation and binding to ISRE15 element at 1, 3, 6, 9 or 24 h after stimulation were analyzed by EMSA. Results are representative of two separate experiments.

Effect of protein synthesis inhibition on the activation of NF-κB and STATs by Gram-positive bacteria

To analyze the requirement of ongoing protein synthesis for NF-κB and STAT activation, macrophages were stimulated with live bacteria for 3 and 6 h in the presence or the absence of CHX. NF-κB activation by lactobacilli or streptococci was not inhibited by CHX at either time point (Fig. 5⇓A). Streptococci-induced STAT1 DNA binding to IRF1-GAS or SIE elements was inhibited by CHX (Fig. 5⇓, B and C). Lactobacilli activated weak STAT1 and STAT3 binding that was also inhibited by CHX (Fig. 5⇓C). Streptococci-induced ISGF3 complex formation was similarly blocked by CHX (Fig. 5⇓D).

FIGURE 5.
FIGURE 5.

Effect of CHX treatment on NF-κB and STAT DNA binding activated by lactobacilli and streptococci. Macrophages were stimulated with live lactobacilli (LAB) or streptococci (GAS), and CHX (10 μg/ml) was added at 0.5 h after the beginning of bacterial stimulation. Nuclear extracts were prepared and analyzed by EMSA using NF-κB (A), IRF1-GAS (B), SIE (C), and ISRE15 (D) oligonucleotides. Results are representative of four separate experiments.

Production of proinflammatory cytokines and IFN-α in human macrophages stimulated with Gram-positive bacteria

As CHX experiments showed, ongoing protein synthesis was required for bacteria-induced STAT activation. This suggested that activation of STATs is likely to be cytokine mediated. The cytokines involved in STAT1 and STAT3 activation include macrophage-derived IFN-α and IL-6, while TNF-α is the most prominent activator of NF-κB. To quantitate TNF-α and IL-6 production we used ELISA, whereas IFN-αβ levels were measured by a biological assay. Both lactobacilli and streptococci induced the production of TNF-α at 3 h and IL-6 at 6 h after stimulation. Bacteria-induced TNF-α and IL-6 production increased up to 24 h after stimulation. Detectable amounts of IFN-α were found only after 24 h of stimulation with streptococci (Fig. 6⇓).

FIGURE 6.
FIGURE 6.

Kinetics of cytokine production in macrophages stimulated with lactobacilli and streptococci. Macrophages were stimulated with live lactobacilli (LAB) and streptococci (GAS) for 1, 3, 6, 9, or 24 h; cell culture supernatants were collected; and the amounts of secreted TNF-α, IL-6, and IFN-α were measured. Results are representative of several separate experiments.

Anti-IFN-αβ Abs inhibit Gram-positive bacteria-induced STAT activation

To study the role of IFN-α and IL-6 in bacteria-induced STAT activation, we used neutralizing Abs. Macrophages were stimulated with live bacteria for 8 h in the presence or the absence of neutralizing Abs to IFN-αβ or IL-6. STAT1 binding to IRF1-GAS by both bacteria was reduced by anti-IFN-αβ Abs, while anti-IL-6 Abs had no detectable effect on STAT1 or STAT3 DNA binding (Fig. 7⇓A). Similarly, bacteria-induced SIE DNA binding was reduced, while ISGF3 DNA binding activity was completely blocked by anti-IFN-αβ Abs (Fig. 7⇓, B and C).

FIGURE 7.
FIGURE 7.

Effect of anti-IFN-α and anti-IL-6 Abs on activation of STAT1, STAT3, and ISGF3 DNA binding by lactobacilli and streptococci. Macrophages were stimulated with live lactobacilli (LAB) or streptococci (GAS) or with IFN-α (20 IU/ml) and/or IL-6 (5 ng/ml). Abs to IFN-α (2400 IU/ml) and/or IL-6 (1 μg/ml) were added at 1 and 4 h after the beginning of bacterial stimulation. Nuclear extracts were prepared and analyzed by EMSA following binding to IRF1-GAS (A), SIE (B), and ISRE15 (C)oligonucleotides. Results are representative of three separate experiments.

Induction of IRF-1 and MxA mRNA and protein expression, and activation of IRF-1 DNA binding by Gram-positive bacteria

To characterize in more detail bacteria-induced IFN-α production and IFN-α-mediated target gene activation, we studied bacteria-induced IRF-1 and MxA mRNA and protein expression as well as activation of IRF-1 DNA binding. Both lactobacilli and streptococci induced IRF-1 mRNA at 2 h and MxA mRNA at 6 h after stimulation (Fig. 8⇓A). Streptococci induced IRF-1 and MxA mRNAs more strongly than lactobacilli. Expression of IRF-1 and MxA proteins by streptococci was first detectable at 6 h after stimulation, continuing up to 24 h (Fig. 8⇓B). Increasing the streptococcal dose to 10-fold slightly potentiated the expression of IRF-1, but decreased that of MxA. Both lactobacilli and streptococci induced IRF-1 DNA binding to PRDI oligonucleotide at 3 h after stimulation. However, streptococci induced IRF-1 DNA binding more efficiently than lactobacilli at all time points studied (Fig. 9⇓).

FIGURE 8.
FIGURE 8.

Expression of IRF-1 and MxA in lactobacilli- and streptococci-stimulated macrophages. Macrophages were stimulated with live lactobacilli (LAB) and streptococci (GAS) for 2, 6, 12, or 24 h; total cellular RNA was collected; and expression of IRF-1 and MxA mRNAs was analyzed by Northern blotting (A). Macrophages were stimulated in a 1:1 or 1:10 ratio with live streptococci (GAS) for 6, 12, or 24 h, and the expression of IRF-1 and MxA proteins was analyzed by Western blotting (B).

FIGURE 9.
FIGURE 9.

Activation of IRF-1 DNA binding by lactobacilli and streptococci. Macrophages were stimulated with live lactobacilli (LAB) or streptococci (GAS), and the kinetics of IFR-1 binding to PRDI oligonucleotide at 1, 3, 6, 9, or 24 h after stimulation were analyzed by EMSA. Results are representative of three separate experiments.

Discussion

Transcriptional regulation of the genes involved in inflammatory responses such as TNF-α and IL-6 is accomplished by NF-κB. We have analyzed and compared the ability of live nonpathogenic Lactobacillus rhamnosus GG and pathogenic Streptococcus pyogenes to induce NF-κB activation in human primary macrophages. Lactobacilli and streptococci similarly induced NF-κB DNA binding with fast kinetics, and the intensity of the DNA binding complex increased with time. As shown by the supershift experiment, bacteria-induced NF-κB consisted of at least of p50 and p65 proteins, which form the most common active dimer in human cells (8). Our data are in line with previous studies where Streptococcus pneumoniae and its cell wall (32), Listeria monocytogenes and cell wall lipoteichoic acid (33), and Lactobacillus crispatus (34) were shown to activate NF-κB in several cell lines. Lactobacilli and streptococci induced the production of TNF-α and IL-6 in macrophages with similar kinetics. These cytokines were produced only after the initial activation of NF-κB DNA binding, and their production increased with time. The increase in the intensity of NF-κB DNA binding signal as a function of time could thus result from the additive activating effect of TNF-α produced by macrophages stimulated with bacteria.

The rapid activation of NF-κB DNA binding without measurable cytokine production suggested that lactobacilli and streptococci were able to directly activate NF-κB in macrophages. NF-κB DNA binding was activated by both bacteria in the presence of protein synthesis inhibitor, CHX, supporting this observation. Likewise, rapid NF-κB activation after binding of pathogenic bacteria to their target cells has been observed with listeria, mycobacteria, Escherichia coli, and salmonella (33, 35, 36). The possible direct activation of NF-κB as a result of binding of lactobacilli and streptococci to macrophages raises the question of the nature of the receptor binding these bacteria and mediating the signal. CD14 receptor has been implicated in NF-κB activation by Gram-positive bacteria and their cell wall components (37). However, other receptors have been suggested to mediate binding and signaling (38, 39, 40). Human Toll-like receptor 2 (TLR2) and TLR4 were first demonstrated to mediate signaling by LPS leading to NF-κB activation, and this response was shown to be enhanced by CD14 (41, 42, 43). TLR2 and TLR4 were then shown to have a role in Gram-positive bacteria-mediated signaling, leading to NF-κB activation (44, 45, 46). Most recent data indicate that TLR2 is mainly involved in responses to Gram-positive bacteria, while TLR4 has a role in recognition and signaling in response to Gram-negative bacteria (47, 48). It is thus likely that in our experimental system with human primary macrophages TLRs in addition to CD14 may play a role in mediating lactobacilli- and streptococci-induced NF-κB activation. TLR2 and TLR4 mRNAs are expressed in human primary macrophages (data not shown), making our hypothesis plausible. The marked similarity in the ability of lactobacilli and streptococci regardless of their pathogenicity to induce NF-κB activation could be explained by the similarity in the components and structure of the cell wall. Pepetidoglycan that is present in abundance in the cell walls of all Gram-positive bacteria and has been shown to induce NF-κB activation through CD14 (37) and TLR2 (44, 45, 48) receptors is a potential component in the cell walls of lactobacilli and streptococci mediating NF-κB activation.

STATs are activated as a result of cytokine binding to their receptors and, like NF-κB, are involved in activating immune responses. Streptococci-induced STAT1 DNA binding was more pronounced compared with that induced by lactobacilli. In contrast, STAT3 was more strongly activated by lactobacilli than by streptococci. Bacteria-induced activation of STAT1 and STAT3 appeared to be dependent on protein synthesis. This suggested that STAT activation by Gram-positive bacteria was cytokine mediated. The kinetics of STAT activation and IL-6 production induced by bacteria coincided, suggesting that IL-6 has a role in bacteria-induced STAT activation. Interestingly, neutralization of IL-6 did not have any effect on bacteria-induced activation of these STATs. Although the role of STAT1 in intracellular bacterial infection (listeria) in mice is well established (49), to our knowledge the only reported work on bacteria-induced STAT activation in human cells concerns Listeria monocytogenes-induced IL-6-dependent activation of STAT3 in hepatocytes (50). It could be that neutralization of IL-6 in our experimental setting was incomplete, and a small amount of IL-6 was sufficient to exert an activating effect. Neutralization of IFN-α clearly reduced STAT activation. IFN-α thus appears to have a significant role in both STAT1 and STAT3 activation, even though we were unable to detect IFN-α production in 8 h supernatants (data not shown). However, we were able to measure IFN-α in 24 h supernatants of streptococci-stimulated macrophages.

IRF-1 is a transcriptional regulator participating in the up-regulation of IFN-α- and IFN-inducible genes by binding to their ISRE elements. The expression of IRF-1 is induced, e.g., by viruses, IFNs, TNF-α, and IL-6 (51). While acting as a positive regulator of innate immune responses, IRF-1 also mediates Th1-type immune responses, thus being of importance in both viral and bacterial infections (52, 53). Lactobacilli and streptococci induced rapid expression of IRF-1 mRNA that coincided with TNF-α and IL-6 mRNA expression (data not shown). Based on our data it remains open whether bacteria-induced TNF-α and IL-6 have a role in IRF-1 induction. Likewise, IFN-α production at early time points after bacterial stimulation could not be detected. It is thus possible that these bacteria directly induce the expression of IRF-1, as has been shown to be the case with LPS (54). Rapid induction of IRF-1 production could also suggest the involvement of NF-κB in bacteria-induced IRF-1 activation. Since it is known that IRF-1 gene expression is regulated by both STAT1 and NF-κB (55, 56), it is likely that IFN-α, TNF-α, and IL-6 produced at later time points after bacterial stimulation may then contribute to activation of IRF-1 gene expression. Both bacteria induced IRF-1 DNA binding. However, streptococci activated IRF-1 DNA binding more efficiently than lactobacilli, which correlated to IFN-α production. Bacteria-induced IRF-1 activation was inhibited by CHX (data not shown), suggesting that the activation of IRF-1 DNA binding is not induced directly by these bacteria but, instead, could involve bacteria-induced cytokines, IFN-α, TNF-α, or IL-6.

Formation of ISGF3 complex is specifically induced by IFN-α (57). Streptococci induced the formation of ISGF3 complex, which was inhibited by CHX and abolished with anti-IFN-α Ab, while lactobacilli-induced ISGF3 complex formation was variably detectable. These results together with those regarding bacteria-induced STAT1 and STAT3 activation, strongly suggest that despite the lack of measurable IFN-α production, bacteria-induced IFN-α is involved in STAT activation. In some experiments we have been able to measure the production of 3–10 IU/ml IFN-α induced by lactobacilli; however, streptococci was always a better inducer. In previous studies in mice, it has been shown that nonpathogenic lactobacilli are able to induce the production of IFN-α (58, 59). It is known that as little as 0.3 IU/ml of IFN-α has biological effects in human PBMC (60). Thus, it is likely that a small amount of IFN-α is produced and used up by the macrophages at early time points after stimulation with both bacteria. The observation that both bacteria induced the expression of MxA, that is strictly under regulation of IFN-α (61, 62), further supports the idea that, in addition to streptococci, lactobacilli induce the production of small, yet biologically significant, amounts of IFN-α.

Analysis of the activation of transcription factors involved in immune responses sheds light on the molecular mechanisms of interaction between bacteria and immune cells, especially those between Gram-positive bacteria and human leukocytes, which have been poorly characterized. This work elucidates factors involved in cytokine signaling and regulation of immune responses by nonpathogenic and pathogenic Gram-positive bacteria. We show that lactobacilli and streptococci similarly induce the activation of transcription factor NF-κB in primary macrophages. We are also the first to report that both nonpathogenic and pathogenic extracellular bacteria can induce the activation of STATs. Our results suggest that NF-κB activation is direct, while STAT activation is mediated mainly by IFN-α. Nonpathogenic and pathogenic bacteria, or at least the studied strains, differ with regard to STAT activation. Pathogenic streptococci potently induce IFN-α production, which is likely to contribute to the activation of STATs and IRF-1. It can be speculated that the efficient induction of IFN-α production and transcription factor activation by streptococci would lead to fast and effective immune responses and could play a role in the pathogenesis. The factors in lactobacilli and streptococci mediating different interactions with macrophages remain uncharacterized; however, it could be that streptococci have surface components, such as lipoteichoic acids or proteins, that more avidly interact with macrophages. We have previously shown that S. pyogenes is a better inducer of Th1 type cytokines IL-12 and IFN-γ than L. rhamnosus GG (19). The ability of streptococci to induce IFN-α production better than lactobacilli could further contribute to its ability to direct immune responses toward the Th1 type.

Acknowledgments

We thank Dr. Tapani Hovi for critically reading the manuscript. We also thank Katja Moilanen, Valma Mäkinen, Teija Westerlund, and Marika Yliselä for their excellent technical assistance.

Footnotes

  • ↵1 This work was supported by the University of Helsinki 350th Anniversary Fund, the Medical Research Council of the Academy Finland, the Sigrid Juselius Foundation, and the Jenny and Antti Wihuri Foundation.

  • ↵2 Address correspondence and reprint requests to Dr. Minja Miettinen, Department of Virology, National Public Health Institute, Mannerheimintie 166, 00300 Helsinki, Finland. E-mail address: minja.miettinen{at}ktl.fi

  • ↵3 Abbreviations used in this paper: GAS, IFN-γ activation site; ISGF3, IFN-stimulated gene factor-3; ISRE, IFN response element; CHX, cycloheximide; IRF, IFN regulatory factor; TLR, Toll-like receptor; GAS, group A streptococci; SIE, c-sis-inducible element.

  • Received July 19, 1999.
  • Accepted January 21, 2000.

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The Journal of Immunology: 164 (7)
The Journal of Immunology
Vol. 164, Issue 7
1 Apr 2000
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