Caterina Romano Carratelli

Immunomodulatory effects of Lactobacillus plantarum on human colon cancer cells

Probiotics, defined as live microbial food supplements which improve the health of the host, have obtained increasing medical importance. In the intestine they may prevent the overgrowth of pathogenic bacteria, increase the resistance of the gut to invasion by pathogens and ameliorate disease processes by inducting the secretion of soluble factors such as cytokines and antimicrobial beta-peptides. One important class of human antimicrobial peptides is the family of defensins. Human beta-defensin 2 (HBD-2) is a major inducible peptide which plays an important role in host defense and represents a link between innate and adaptive immune responses. This linkage is in part mediated through the recognition of conserved bacterial products or bacteria by Toll-like receptors (TLRs). The aim of this study was to investigate the effects of Lactobacillus plantarum on intestinal epithelial cells. We found that Caco-2 cells exposed to L. plantarum bacteria significantly induced HBD-2 mRNA expression and HBD-2 secretion in a dose- (16+/-1.4 pg/ml and 31.5+/-2.3 pg/ml at MOI 10 and 50, respectively) and time-dependent manner, but not HBD-3, compared to controls; in addition, when LPS was added to cells for 48 h, the interleukin (IL)-23 secretion (850+/-5.4 pg/ml) and IL-23 mRNA expression increased; while it was reduced when LPS was cocultured with L. plantarum (330+/-4.2 pg/ml). The L. plantarum-induced increase in HBD-2 expression is inhibited by anti-TLR-2 neutralizing antibodies, in the same way the pre-treatment with the anti-TLR-2 antibody inhibited the production of IL-23 induced by LPS in Caco-2 cells. The results of our study help to achieve a better understanding of how the intestinal epithelium participates in the innate immune response to commensal bacteria and pathogens in the gut.

Lactobacillus crispatus modulates epithelial cell defense against Candida albicans through Toll-like receptors 2 and 4, interleukin 8 and human β-defensins 2 and 3.

Lactobacilli are members of the normal mucosal microflora of most animals. Probiotic bacteria, such as Lactobacilli, play a major role in the maintenance of a healthy urogenital tract by preventing the colonization of pathogenic bacteria. The potentially probiotic strain Lactobacillus crispatus (ATCC 33820) was investigated for its capacity to influence the innate immune response of HeLa epithelial cells to Candida albicans. In addition, its capacity to modulate the toll-like receptor (TLR) expression of HeLa cells was investigated by Western blot. When HeLa cells were pre-treated with the L. crispatus and infected with C. albicans, the interleukin-8 levels were significantly lower than without pre-treatment. Also, the effect of L. crispatus on innate immunity was enhanced by its capacity to increase the effect of human β-defensin 3 against C. albicans growth. Pre-treating HeLa cells with L. crispatus attenuated the yeasts virulence, as demonstrated by its reduced adhesion and growth on human epithelial cells. Our findings indicated, also, that after contact with C. albicans, epithelial cells expressed more TLR2/4 than non-infected cells, whereas pre-treatment with L. crispatus downregulated the TLR2/4 expression by epithelial cells stimulated with C. albicans. In conclusion, our results show that L. crispatus promotes epithelial cell defense against C. albicans infection through the involvement of TLR2/4, IL-8 and human β-defensin 2 and 3, thus suggesting a probiotic potential of this Lactobacillus as an anti-infective agent against C. albicans.

Toll‐like receptor‐4 (TLR4) mediates human β‐defensin‐2 (HBD‐2) induction in response to Chlamydia pneumoniae in mononuclear cells

Monocytes are pivotal effector cells of the innate immune system that are vital for recognizing and eliminating invasive microbial pathogens. When microbial products bind to pathogen-recognition receptors, monocytes are activated and release a broad array of cytokines and defensins that orchestrate the host innate and adaptive immune responses. The aim of the present study is to investigate whether Toll-like receptor-4 (TLR4) mediates human beta-defensin-2 (HBD-2) induction in response to Chlamydia pneumoniae in mononuclear cells. We showed that TLR4 is expressed in U937 cells and monocytes infected with viable microorganisms in a time-dependent fashion, while heat-inactivated microorganisms induced a lesser expression, albeit still significant, of TLR4 compared with viable organisms; flow cytometric analysis, in particular, revealed a higher level of TLR4 expression at 48 and 72 h postinfection. In addition, U937 cells and monocytes responded to C. pneumoniae in a TLR4-dependent manner with induction of mRNA and protein of the antimicrobial peptide HBD-2. The treatment of cells with TLR4-neutralizing antibody resulted in a decrease in C. pneumoniae-induced HBD-2 production. This study reveals that TLRs not only recognize ligands but also the types of effector molecules induced, namely, antimicrobial peptides. An understanding of the importance of the TLR-mediated antimicrobial mechanisms may provide new avenues for the development of therapeutic regimens aimed at activating the bodys own defenses by stimulating TLR-dependent pathways.

Effect of resveratrol and modulation of cytokine production on human periodontal ligament cells.

Periodontitis is a multifactorial polymicrobial infection characterized by a destructive inflammatory process. Porphyromonas gingivalis, a Gram-negative anaerobic black-pigmented rod, which produces several virulence factors that stimulate human periodontal ligament cells (HPLCs) to produce various inflammatory mediators, has been implicated as a crucial etiologic agent in the initiation and progression of periodontitis. Since natural polyphenols such as resveratrol have growth-inhibitory effects on some bacterial pathogens and have shown chemo-preventive, anti-inflammatory and antioxidant activity, in the present study we used an HPLC model stimulated with lipopolysaccharide (LPS) of P. gingivalis to simulate the in vivo conditions such as those found in diseased periodontal sites. To determine whether resveratrol interferes with P. gingivalis LPS-activity and reduces the production of pro-inflammatory molecules, we investigated its effect on the cytokines IL-1β, IL-6, IL-8, IL-12 and TNF-α and NO production of HPLCs. The results showed that resveratrol treatment decreased in a dose- and time-dependent manner the NO expression induced by P. gingivalis LPS, correlated to an increased viability of infected HPLCs, and decreased the production of pro-inflammatory cytokines in HPLCs stimulated by P. gingivalis LPS. These results suggest that the ability of resveratrol to determine immunomodulatory effects could provide possible therapeutic applications for the treatment of periodontitis.

Effect of resveratrol and quercetin in experimental infection by Salmonella enterica serovar Typhimurium

Flavonoids are phenolic compounds widely distributed in almost every plant and act as pharmacologically active constituents in many herbal medicines. They have multiple biological, pharmacological, and medicinal properties including anti-inflammatory and cytoprotective effects. In the present study, the experiments were performed to evaluate the effect of resveratrol and quercetin on proliferation, viability, nitric oxide (NO) production, and apoptosis in Salmonella enterica serovar Typhimurium-infected U937 cells and monocytes (MN). The results showed in a time- and dose-dependent manner that both resveratrol and quercetin reduced S. enterica serovar Typhimurium-induced NO production. In addition, the vegetable extracts resveratrol and quercetin inhibited cell viability and proliferation in S. enterica serovar Typhimurium-infected cells. S. enterica serovar Typhimurium-induced apoptosis was also blocked by resveratrol and quercetin. The results obtained indicate that flavonoids modulate the host response during salmonellosis by protecting the host cells from the toxic effects of bacterial infection and also by decreasing programmed cell death. Hence, these polyphenols can be considered potential candidates against S. enterica serovar Typhimurium-related gastric pathogenic processes, and further attention should be given to their application as a treatment for infectious diseases.

Modulation of cytokine and β-defensin 2 expressions in human gingival fibroblasts infected with Chlamydia pneumoniae

Human beta-defensin 2 is an antimicrobial peptide that is produced by several epithelial cells after stimulation with micro-organisms and inflammatory mediators. Gram-negative bacteria, which are typically detected in periodontal pockets in periodontitis, elicit a stronger antibacterial peptide response of human beta-defensin 2 by epithelial cells. In this study, we investigated whether Chlamydia pneumoniae is able both to enter and grow in human gingival fibroblasts (HGF), to modify the production of cytokines, and is involved in regulation of beta-defensin 2 expression. Gingival fibroblasts discarded from periodontal procedures on healthy young individuals were infected with viable C. pneumoniae or with heat- or ultraviolet-inactivated organisms at a multiplicity of infection of 4 inclusion-forming units per cell. Our results demonstrate that after 48 h of incubation with viable C. pneumoniae, gingival fibroblasts showed a proliferative response as seen by both colorimetric assay and direct cell count (40% and 45%, respectively). Moreover, cells incubated with viable or ultraviolet light-inactivated C. pneumoniae organisms showed an increase in the levels of interleukin-6, interleukin-10 and human beta-defensin 2 in a time-dependent fashion, while the cells infected with heat-killed bacteria did not show a significant production either of the cytokines or beta-defensin 2 at any time. In conclusion, we demonstrate the correlation between multiplication of C. pneumoniae in human gingival fibroblasts and release of interleukin-6, interleukin-10 and up-regulation of beta-defensin 2, suggesting that gingival fibroblasts may be a periodontium niche for obligate intracellular C. pneumoniae and may play a role in innate gingival immune system and inflammatory response mechanisms of periodontitis.

Lactobacillus plantarum reduces Streptococcus pyogenes virulence by modulating the IL-17, IL-23 and Toll-like receptor 2/4 expressions in human epithelial cells

Streptococcus pyogenes is a common colonizer of the mucosal layers in the mouth, nose, and pharynx but it is also a major Gram-positive human pathogen that causes infections ranging from pharyngitis to severe systemic diseases. The lactobacilli colonize the oral tracts and are known to protect against colonization by many pathogens. Epithelial cells participate in the innate host defense by expressing a variety of proinflammatory cytokines and TLRs in the interaction with microorganisms. The potentially probiotic strain Lactobacillus plantarum was investigated for its capacity to influence the innate immune response of HEp-2 and A549 epithelial cells to S. pyogenes infection. In both epithelial cell types, pre-treatment with L. plantarum showed inhibition of S. pyogenes growth and a greater decrease in IL-17 and IL-23 levels compared to the control. Pre-treatment with the anti-TLR2/4 antibody abolished the inhibitory effects of L. plantarum on IL-17 and IL-23 production following S. pyogenes infection, indicating that L. plantarum downregulates TLR2/4-dependent IL-17 and IL-23 production. Overall, our findings suggest that in epithelial cell cultures with S. pyogenes, cytokine responses are modulated by the presence of L. plantarum through the induction of TLR2/TLR4.

Induction of proinflammatory cytokines in human osteoblastic cells by Chlamydia pneumoniae

Chlamydia pneumoniae is an obligate intracellular Gram-negative bacterium that causes recurrent pharyngitis, pneumonia and chronic inflammation induced by cycles of persistence and productive infection that might also explain the association with chronic diseases. The aim of this study was to determine whether C. pneumoniae can invade and survive within human osteoblasts and whether this infection elicits the secretion of proinflammatory cytokines. Our results demonstrated that C. pneumoniae was able to infect the SaOS-2 osteoblastic cell line and to replicate in the osteoblasts in a time-dependent manner and was associated to an increase in the cell number and cell viability. In addition, infection of the SaOS-2 cell line with C. pneumoniae at MOI of 4 is correlated to a proinflammatory response. Infected osteoblasts produced increased levels of cytokines IL-6, IL-8, IL-17, and IL-23. The production of cytokines increased with subsequent interaction between osteoblasts and monocytes and the maximum levels of cytokines released were detected 72 h after infection with C. pneumoniae. Thus, controlling the release of chemokines, e.g., IL-23, may be a therapeutic strategy for preventing inflammatory bone disease and counteract inflammation and bone destruction.

Modulation of apoptosis in mice treated with Echinacea and St. John's wort.

Apoptosis, or programmed cell death (PCD), is a physiological active cellular suicide process that occurs in non-contiguous cells, and is usually not associated with inflammation. The apoptotic process can be modulated by various stimuli, including hormones, cytokines, growth factors, and some chemotherapeutic agents. To determine whether Echinacea purpurea and Hypericum perforatum are able to regulate the process of apoptosis in vivo and to define the role of the Fas-Ag and Bcl-2 signal transduction cascade, we have orally treated groups of mice with these vegetable drugs for 14 days. The splenic lymphocytes from mice treated with E. purpurea and H. perforatum at the two dose levels used (30 and 100 mg kg(-1) per day) were shown to be significantly more resistant to apoptosis than those from mice treated only with the vehicle. In addition, mice treated with the natural substances showed a decrease in Fas-Ag expression and an increase in Bcl-2 expression. In conclusion, our results shown that in vivo the studied drugs modulate apoptosis in mice splenic lymphocytes and that this action could be mediated in part by a decrease in Fas-Ag expression and in part by an increase in Bcl-2 expression.

Cardiac and skeletal muscle expression of mutant β-myosin heavy chains, degree of functional impairment and phenotypic heterogeneity in hypertrophic cardiomyopathy.

Several mutations in distinct genes, all coding for sarcomeric proteins, have been reported in unrelated kindreds with familial hypertrophic cardiomyopathy (FHC). We have identified nine individuals from three families harboring two distinct mutations in one copy of the β‐myosin heavy chain (β‐MHC) gene. In this study, the expression of the mutant β‐myosin protein isoform, isolated from slow‐twitch fibers of skeletal muscle, was demonstrated by Northern and Western blot analysis; this myosin showed a decreased in vitro motility activity and produced a lower actin‐activated ATPase activity. Isometric tension, measured in single slow‐twitch fibers isolated from the affected individuals, also showed a significant decrease. The degree of impairment of β‐myosin function, as well as the loss in isometric tension development, were strictly dependent on the amount of the isoform transcribed from the mutated allele. Interestingly, a strong correlation was also demonstrated between mutant β‐myosin content and clinical features of FHC. On the other hand, we were unable to detect any correlation between mutant β‐myosin expression and degree of cardiac hypertrophy, thereby strengthening the hypothesis that hypertrophy, one of the hallmarks of FHC, might not necessarily be related to the clinical evolution of this disease. These findings lend support to the notion that additional factors rather than the mutated gene may play a pathogenetic role in cardiac wall thickening, whereas the prognosis appears to be strongly related to the amount of mutant protein. J. Cell. Physiol. 227: 3471–3476, 2012.