Lactobacillus acidophilus LA-5 Ameliorates Inflammation and Alveolar Bone Loss Promoted by A. actinomycetemcomitans and S. gordonii in Mice and Impacts Oral and Gut Microbiomes.

The benefits of probiotics on dysbiotic microbiomes and inflammation are dependent on the tested strain, host factors, and the resident microbiome. There is limited knowledge on the effects of probiotics in A. actinomycetemcomitans-associated periodontitis. Thus, Lactobacillus acidophilus LA5 (LA5) was orally inoculated for 30 days in C57Bl/6 mice infected with A. actinomycetemcomitans JP2 (Aa) and S. gordonii (Sg). Alveolar bone loss, gingival gene expression, and oral and gut microbiomes were determined. LA5 controlled bone loss in Aa+Sg-infected mice, downregulated the expression of Il-1ß and upregulated Il-10 in gingival tissues, and altered the oral and gut microbiomes. LA5 increased the diversity of the oral microbiome of Aa+Sg infected mice, and Aa+Sg and Aa+Sg+LA5 oral or gut microbiomes clustered apart. LA5 induced shifts in Aa+Sg infected mice by increasing the abundance of Muribaculaceae and decreasing Bifidobacteriaceae in the oral cavity and increasing the abundance of Verrucomicrobiae and Eggerthellales in the gut. In conclusion, LA5 oral administration controls experimental Aa-associated periodontitis by altering inflammatory gene expression and the oral and gut microbiomes.

Lactobacilli Attenuate the Effect of Aggregatibacter actinomycetemcomitans Infection in Gingival Epithelial Cells.

Probiotics may be considered as an additional strategy to achieve a balanced microbiome in periodontitis. However, the mechanisms underlying the use of probiotics in the prevention or control of periodontitis are still not fully elucidated. This in vitro study aimed to evaluate the effect of two commercially available strains of lactobacilli on gingival epithelial cells (GECs) challenged by Aggregatibacter actinomycetemcomitans. OBA-9 GECs were infected with A. actinomycetemcomitans strain JP2 at an MOI of 1:100 and/or co-infected with Lactobacillus acidophilus La5 (La5) or Lacticaseibacillus rhamnosus Lr32 (Lr32) at an MOI of 1:10 for 2 and 24 h. The number of adherent/internalized bacteria to GECs was determined by qPCR. Production of inflammatory mediators (CXCL-8, IL-1ß, GM-CSF, and IL-10) by GECs was determined by ELISA, and the expression of genes encoding cell receptors and involved in apoptosis was determined by RT-qPCR. Apoptosis was also analyzed by Annexin V staining. There was a slight loss in OBA-9 cell viability after infection with A. actinomycetemcomitans or the tested probiotics after 2 h, which was magnified after 24-h co-infection. Adherence of A. actinomycetemcomitans to GECs was 1.8 × 107 (± 1.2 × 106) cells/well in the mono-infection but reduced to 1.2 × 107 (± 1.5 × 106) in the co-infection with Lr32 and to 6 × 106 (± 1 × 106) in the co-infection with La5 (p < 0.05). GECs mono-infected with A. actinomycetemcomitans produced CXCL-8, GM-CSF, and IL-1ß, and the co-infection with both probiotic strains altered this profile. While the co-infection of A. actinomycetemcomitans with La5 resulted in reduced levels of all mediators, the co-infection with Lr32 promoted reduced levels of CXCL-8 and GM-CSF but increased the production of IL-1ß. The probiotics upregulated the expression of TLR2 and downregulated TLR4 in cells co-infected with A. actinomycetemcomitans. A. actinomycetemcomitans-induced the upregulation of NRLP3 was attenuated by La5 but increased by Lr32. Furthermore, the transcription of the anti-apoptotic gene BCL-2 was upregulated, whereas the pro-apoptotic BAX was downregulated in cells co-infected with A. actinomycetemcomitans and the probiotics. Infection with A. actinomycetemcomitans induced apoptosis in GECs, whereas the co-infection with lactobacilli attenuated the apoptotic phenotype. Both tested lactobacilli may interfere in A. actinomycetemcomitans colonization of the oral cavity by reducing its ability to interact with gingival epithelial cells and modulating cells response. However, L. acidophilus La5 properties suggest that this strain has a higher potential to control A. actinomycetemcomitans-associated periodontitis than L. rhamnosus Lr32.

Bifidobacterium Strains Present Distinct Effects on the Control of Alveolar Bone Loss in a Periodontitis Experimental Model.

Periodontitis is an inflammatory disease induced by a dysbiotic oral microbiome. Probiotics of the genus Bifidobacterium may restore the symbiotic microbiome and modulate the immune response, leading to periodontitis control. We evaluated the effect of two strains of Bifidobacterium able to inhibit Porphyromonas gingivalis interaction with host cells and biofilm formation, but with distinct immunomodulatory properties, in a mice periodontitis model. Experimental periodontitis (P+) was induced in C57Bl/6 mice by a microbial consortium of human oral organisms. B. bifidum 1622A [B+ (1622)] and B. breve 1101A [B+ (1101)] were orally inoculated for 45 days. Alveolar bone loss and inflammatory response in gingival tissues were determined. The microbial consortium induced alveolar bone loss in positive control (P + B-), as demonstrated by microtomography analysis, although P. gingivalis was undetected in oral biofilms at the end of the experimental period. TNF-α and IL-10 serum levels, and Treg and Th17 populations in gingiva of SHAM and P + B- groups did not differ. B. bifidum 1622A, but not B. breve 1101A, controlled bone destruction in P+ mice. B. breve 1101A upregulated transcription of Il-1ß, Tnf-α, Tlr2, Tlr4, and Nlrp3 in P-B+(1101), which was attenuated by the microbial consortium [P + B+(1101)]. All treatments downregulated transcription of Il-17, although treatment with B. breve 1101A did not yield such low levels of transcripts as seen for the other groups. B. breve 1101A increased Th17 population in gingival tissues [P-B+ (1101) and P + B+ (1101)] compared to SHAM and P + B-. Administration of both bifidobacteria resulted in serum IL-10 decreased levels. Our data indicated that the beneficial effect of Bifidobacterium is not a common trait of this genus, since B. breve 1101A induced an inflammatory profile in gingival tissues and did not prevent alveolar bone loss. However, the properties of B. bifidum 1622A suggest its potential to control periodontitis.

Distinct Signaling Pathways Between Human Macrophages and Primary Gingival Epithelial Cells by Aggregatibacter actinomycetemcomitans.

In aggressive periodontitis, the dysbiotic microbial community in the subgingival crevice, which is abundant in Aggregatibacter actinomycetemcomitans, interacts with extra- and intracellular receptors of host cells, leading to exacerbated inflammation and subsequent tissue destruction. Our goal was to understand the innate immune interactions of A. actinomycetemcomitans with macrophages and human gingival epithelial cells (HGECs) on the signaling cascade involved in inflammasome and inflammatory responses. U937 macrophages and HGECs were co-cultured with A. actinomycetemcomitans strain Y4 and key signaling pathways were analyzed using real-time PCR, Western blotting and cytokine production by ELISA. A. actinomycetemcomitans infection upregulated the transcription of TLR2, TLR4, NOD2 and NLRP3 in U937 macrophages, but not in HGECs. Transcription of IL-1ß and IL-18 was upregulated in macrophages and HGECs after 1 h interaction with A. actinomycetemcomitans, but positive regulation persisted only in macrophages, resulting in the presence of IL-1ß in macrophage supernatant. Immunoblot data revealed that A. actinomycetemcomitans induced the phosphorylation of AKT and ERK1/2, possibly leading to activation of the NF-κB pathway in macrophages. On the other hand, HGEC signaling induced by A. actinomycetemcomitans was distinct, since AKT and 4EBP1 were phosphorylated after stimulation with A. actinomycetemcomitans, whereas ERK1/2 was not. Furthermore, A. actinomycetemcomitans was able to induce the cleavage of caspase-1 in U937 macrophages in an NRLP3-dependent pathway. Differences in host cell responses, such as those seen between HGECs and macrophages, suggested that survival of A. actinomycetemcomitans in periodontal tissues may be favored by its ability to differentially activate host cells.

Probiotics alter the immune response of gingival epithelial cells challenged by Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Although previous studies revealed the potential use of probiotics in the control of periodontitis, little is known about their interactions with gingival epithelial cells (GECs). Since GECs comprise the first defense in the subgingival microenvironment, the aim of this study was to evaluate the effect of probiotic lactobacilli and bifidobacteria strains on OBA-9 cells challenged with Porphyromonas gingivalis. METHODS: Immortalized human GECs (OBA-9) were challenged with live P. gingivalis (strains W83 and ATCC33277) and co-infected with one of 12 tested probiotic strains at a multiplicity of infection (MOI) of 1:1000 for 2 hours. Bacterial adhesion and invasion were determined by antibiotic exclusion analysis and CFU counting. OBA-9 viability was assessed by MTT assay, and levels of inflammatory mediators (TNF-α, IL-1ß, and CXCL8) in the supernatants were determined by ELISA. The expression of genes encoding Toll-like receptors (TLR2, TLR4) was evaluated by RT-qPCR. RESULTS: Both strains of P. gingivalis were able to adhere and invade OBA-9 cells, with significant loss in cell viability, increase in the levels of TNF-α and IL-1ß, and upregulation of TLR4. However, co-infection with probiotics attenuated these effects in P. gingivalis challenged GECs. Most probiotics maintained OBA-9 viability and reduced pathogens adhesion and invasion. Furthermore, probiotics were able to adhere to GECs, which was enhanced for most strains in the presence of P. gingivalis. The synthesis of IL-1ß and TNF-α by P. gingivalis in challenged GECs was reduced in co-culture with most of the tested probiotics, whereas the secretion of CXCL8 increased, and TLR4 was downregulated. CONCLUSION: Probiotics can alter the interaction of GECs with P. gingivalis by modulating the pathogen's ability to adhere and invade these cells, as well as by regulating the innate immune response. Such properties are strain-specific and may indicate the most efficient probiotics to control periodontitis.

Probiotic Bacteria Alter Pattern-Recognition Receptor Expression and Cytokine Profile in a Human Macrophage Model Challenged with Candida albicans and Lipopolysaccharide.

Probiotics are live microorganisms that confer benefits to the host health. The infection rate of potentially pathogenic organisms such as Candida albicans, the most common agent associated with mucosal candidiasis, can be reduced by probiotics. However, the mechanisms by which the probiotics interfere with the immune system are largely unknown. We evaluated the effect of probiotic bacteria on C. albicans challenged human macrophages. Macrophages were pretreated with lactobacilli alone (Lactobacillus rhamnosus LR32, Lactobacillus casei L324m, or Lactobacillus acidophilus NCFM) or associated with Escherichia coli lipopolysaccharide (LPS), followed by the challenge with C. albicans or LPS in a co-culture assay. The expression of pattern-recognition receptors genes (CLE7A, TLR2, and TLR4) was determined by RT-qPCR, and dectin-1 reduced levels were confirmed by flow cytometry. The cytokine profile was determined by ELISA using the macrophage cell supernatant. Overall probiotic lactobacilli down-regulated the transcription of CLEC7A (p < 0.05), resulting in the decreased expression of dectin-1 on probiotic pretreated macrophages. The tested Lactobacillus species down-regulated TLR4, and increased TLR2 mRNA levels in macrophages challenged with C. albicans. The cytokines profile of macrophages challenged with C. albicans or LPS were altered by the probiotics, which generally led to increased levels of IL-10 and IL-1ß, and reduction of IL-12 production by macrophages (p < 0.05). Our data suggest that probiotic lactobacilli impair the recognition of PAMPs by macrophages, and alter the production of pro/anti-inflammatory cytokines, thus modulating inflammation.

Brazilian red propolis effects on peritoneal macrophage activity: Nitric oxide, cell viability, pro-inflammatory cytokines and gene expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Propolis has been used in folk medicine since ancient times and it presented inhibitory effect on neutrophil recruitment previously. However, its effect on macrophage obtained from mice remains unclear. AIM OF THE STUDY: To demonstrate BRP effects on LPS activated peritoneal macrophage. MATERIALS AND METHODS: Peritoneal macrophages, obtained from C57BL6 mice and activated with LPS, were treated with 50-80µg/mL of crude extract of Brazilian red propolis (BRP) during 48h. Cell viability, levels of NO, 20 cytokines and expression of 360 genes were evaluated. RESULTS: BRP 60µg/mL reduced NO production by 65% without affecting the cell viability and decreased production IL1α, IL1ß, IL4, IL6, IL12p40, Il12p70, IL13, MCP1 and GM-CSF. Molecular mechanism beyond the anti-inflammatory activity may be due to BRP-effects on decreasing expression of Mmp7, Egfr, Adm, Gata3, Wnt2b, Txn1, Herpud1, Axin2, Car9, Id1, Vegfa, Hes1, Hes5, Icam1, Wnt3a, Pcna, Wnt5a, Tnfsf10, Ccl5, Il1b, Akt1, Mapk1, Noxa1 and Cdkn1b and increasing expression of Cav1, Wnt6, Calm1, Tnf, Rb1, Socs3 and Dab2. CONCLUSIONS: Therefore, BRP has anti-inflammatory effects on macrophage activity by reducing NO levels and diminished release and expression of pro-inflammatory cytokine and genes, respectively.

Alteration of Homeostasis in Pre-osteoclasts Induced by Aggregatibacter actinomycetemcomitans CDT.

The dysbiotic microbiota associated with aggressive periodontitis includes Aggregatibacter actinomycetemcomitans, the only oral species known to produce a cytolethal distending toxin (AaCDT). Give that CDT alters the cytokine profile in monocytic cells, we aimed to test the hypothesis that CDT plays a role in bone homeostasis by affecting the differentiation of precursor cells into osteoclasts. Recombinant AaCDT was added to murine bone marrow monocytes (BMMC) in the presence or absence of RANKL and the cell viability and cytokine profile of osteoclast precursor cells were determined. Multinucleated TRAP(+) cell numbers, and relative transcription of genes related to osteoclastogenesis were also evaluated. The addition of AaCDT did not lead to loss in cell viability but promoted an increase in the average number of TRAP(+) cells with 1-2 nuclei in the absence or presence of RANKL (Tukey, p < 0.05). This increase was also observed for TRAP(+) cells with ≥3nuclei, although this difference was not significant. Levels of TGF-ß, TNF-α, and IL-6, in the supernatant fraction of cells, were higher when in AaCDT exposed cells, whereas levels of IL-1ß and IL-10 were lower than controls under the same conditions. After interaction with AaCDT, transcription of the rank (encoding the receptor RANK), nfatc1 (transcription factor), and ctpK (encoding cathepsin K) genes was downregulated in pre-osteoclastic cells. The data indicated that despite the presence of RANKL and M-CSF, AaCDT may inhibit osteoclast differentiation by altering cytokine profiles and repressing transcription of genes involved in osteoclastogenesis. Therefore, the CDT may impair host defense mechanisms in periodontitis.

Brazilian Red Propolis Attenuates Inflammatory Signaling Cascade in LPS-Activated Macrophages.

Although previous studies suggested an anti-inflammatory property of Brazilian red propolis (BRP), the mechanisms involved in the anti-inflammatory effects of BRP and its activity on macrophages were still not elucidated. This study aimed to evaluate whether BRP attenuates the inflammatory effect of LPS on macrophages and to investigate its underlying mechanisms. BRP was added to RAW 264.7 murine macrophages after activation with LPS. NO production, cell viability, cytokines profile were evaluated. Activation of inflammatory signaling pathways and macrophage polarization were determined by RT-qPCR and Western blot. BRP at 50 µg/ml inhibited NO production by 78% without affecting cell viability. Cd80 and Cd86 were upregulated whereas mrc1 was down regulated by BRP indicating macrophage polarization at M1. BRP attenuated the production of pro-inflammatory mediators IL-12, GM-CSF, IFN-Ɣ, IL-1ß in cell supernatants although levels of TNF- α and IL-6 were slightly increased after BRP treatment. Levels of IL-4, IL-10 and TGF-ß were also reduced by BRP. BRP significantly reduced the up-regulation promoted by LPS of transcription of genes in inflammatory signaling (Pdk1, Pak1, Nfkb1, Mtcp1, Gsk3b, Fos and Elk1) and of Il1ß and Il1f9 (fold-change rate > 5), which were further confirmed by the inhibition of NF-κB and MAPK signaling pathways. Furthermore, the upstream adaptor MyD88 adaptor-like (Mal), also known as TIRAP, involved in TLR2 and TLR4 signaling, was down- regulated in BRP treated LPS-activated macrophages. Given that BRP inhibited multiple signaling pathways in macrophages involved in the inflammatory process activated by LPS, our data indicated that BRP is a noteworthy food-source for the discovery of new bioactive compounds and a potential candidate to attenuate exhacerbated inflammatory diseases.

IgG sera levels against a subset of periodontopathogens and severity of disease in aggressive periodontitis patients: a cross-sectional study of selected pocket sites.

AIMS: To evaluate the association among serum immunoglobulin G (IgG) responses to Aggregatibacter actinomycetemcomitans (Aa) serotypes a, b and c, Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf) and clinical parameters in Aggressive Periodontitis (AP) subjects. Associations between periodontal pathogens and clinical and immunological parameters were also evaluated. METHODS: Thirty-eight subjects diagnosed with generalized AP (GAP) and localized AP (LAP) were included. Ten healthy controls were also evaluated. Clinical parameters were assessed and percentages of subgingival levels of Aa, Pg and Tf (beyond bacterial load), were determined by quantitative real-time polymerase chain reaction. Serum IgG antibody levels against Aa, Pg and Tf were evaluated by enzyme-linked immunosorbent assay. RESULTS: Percentages of Aa, Pg and Tf were significantly higher in AP than in controls. The response to Aa serotype c was higher in LAP subjects than in controls. There were no differences in microbial composition or antibodies responses between GAP and LAP, except for IgG response to Tf. Pg levels were correlated with probing depth (PD), BoP and CAL in GAP but not in LAP subjects. Tf levels correlated with PD and CAL in GAP subjects. In GAP, the infection levels of Aa and Pg correlated with the corresponding IgG levels to Aa serotype c and Pg. CONCLUSION: Given the evidences that IgG response in AP patients correlated with bacterial infection level in GAP, but not in LAP, and that LAP patients lack a response to Tf, despite harbouring this species, our data suggest a difference in host immune defence between these two forms of aggressive periodontitis.