Probiotics in livestock feed supplements are considered a replacement for antibiotics that enhance gastrointestinal immunity. Although bacterial cell wall components have been proposed to be ...associated with probiotic function, little evidence demonstrates that they are responsible for probiotic functions in livestock. The present study demonstrated that lipoteichoic acid (LTA) of
(Lp.LTA) confers anti-inflammatory responses in porcine intestinal epithelial cell line, IPEC-J2. A synthetic analog of viral double-stranded RNA, poly I:C, dose-dependently induced IL-8 production at the mRNA and protein levels in IPEC-J2 cells. Lp.LTA, but not lipoprotein or peptidoglycan from
, exclusively suppressed poly I:C-induced IL-8 production. Compared with LTAs from other probiotic
strains including
,
, and
GG, Lp.LTA had higher potential to suppress poly I:C-induced IL-8 production. Dealanylated or deacylated Lp.LTA did not suppress poly I:C-induced IL-8 production, suggesting that D-alanine and lipid moieties in the Lp.LTA structure were responsible for the inhibition. Furthermore, Lp.LTA attenuated the phosphorylation of ERK and p38 kinase as well as the activation of NF-κB, resulting in decreased IL-8 production. Taken together, these results suggest that Lp.LTA acts as an effector molecule to inhibit viral pathogen-induced inflammatory responses in porcine intestinal epithelial cells.
is an extremely resistant bacterium against extracellular stress owing to on its unique physiological functions and the structure of its cellular constituents. Interestingly, it has been reported ...that the pattern of alteration in
proportion on the skin is negatively correlated with skin inflammatory diseases, whereas the proportion of
was increased in patients with chronic skin inflammatory diseases. However, the biological mechanisms of deinococcal interactions with other skin commensal bacteria have not been studied. In this study, we hypothesized that deinococcal cellular constituents play a pivotal role in preventing
colonization by inhibiting biofilm formation. To prove this, we first isolated cellular constituents, such as exopolysaccharide (DeinoPol), cell wall (DeinoWall), and cell membrane (DeinoMem), from
and investigated their inhibitory effects on
colonization and biofilm formation
and
. Among them, only DeinoPol exhibited an anti-biofilm effect without affecting bacterial growth and inhibiting staphylococcal colonization and inflammation in a mouse skin infection model. Moreover, the inhibitory effect was impaired in the Δ
strain, a mutant that cannot produce DeinoPol. Remarkably, DeinoPol not only interfered with
biofilm formation at early and late stages but also disrupted a preexisting biofilm by inhibiting the production of poly-
-acetylglucosamine (PNAG), a key molecule required for
biofilm formation. Taken together, the present study suggests that DeinoPol is a key molecule in the negative regulation of
biofilm formation by
. Therefore, DeinoPol could be applied to prevent and/or treat infections or inflammatory diseases associated with
biofilms.
Periodontitis is caused by multi-bacterial infection and Aggregatibacter actinomycetemcomitans and Enterococcus faecalis are closely associated with inflammatory periodontal diseases. Although ...lipopolysaccharide (LPS) of A. actinomycetemcomitans (Aa.LPS) and lipoteichoic acid of E. faecalis (Ef.LTA) are considered to be major virulence factors evoking inflammatory responses, their combinatorial effect on the induction of chemokines has not been investigated. In this study, we investigated the interaction between Aa.LPS and Ef.LTA on IL-8 expression in human periodontal ligament (PDL) cells. Aa.LPS, but not Ef.LTA, substantially induced IL-8 expression at the protein and mRNA levels. Interestingly, Ef.LTA suppressed Aa.LPS-induced IL-8 expression without affecting the binding of Aa.LPS to Toll-like receptor (TLR) 4. Ef.LTA reduced Aa.LPS-induced phosphorylation of mitogen-activated protein kinases, including ERK, JNK and p38 kinase. Furthermore, Ef.LTA inhibited the Aa.LPS-induced transcriptional activities of the activating protein 1, CCAAT/enhancer-binding protein and nuclear factor-kappa B transcription factors, all of which are known to regulate IL-8 gene expression. Ef.LTA augmented the expression of IL-1 receptor-associated kinase-M (IRAK-M), a negative regulator of TLR intracellular signaling pathways, in the presence of Aa.LPS at both the mRNA and protein levels. Small interfering RNA silencing IRAK-M reversed the attenuation of Aa.LPS-induced IL-8 expression by Ef.LTA. Collectively, these results suggest that Ef.LTA down-regulates Aa.LPS-induced IL-8 expression in human PDL cells through up-regulation of the negative regulator IRAK-M.
Abstract Although mastitis caused by Staphylococcus aureus is a problematic inflammatory disease in lactating cows, the innate immunity to S. aureus in the mammary gland is poorly understood. In the ...present study, we observed that heat-killed S. aureus (HKS) induced IL-1β expression at both the mRNA and protein levels in the mammary gland epithelial cell-line, MAC-T. IL-1β production was suppressed by inhibitors of lipid rafts, ERK, JNK, and p38 kinases. Furthermore, HKS augmented the activities of the AP-1, CRE, and NF-κB transcription factors that regulate IL-1β gene expression. Among staphylococcal cell-wall components with inflammatory potential, Pam2CSK4 (a representative model for diacylated lipoproteins) enhanced IL-1β mRNA expression, while lipoteichoic acid and peptidoglycan did not. Collectively, we suggest that S. aureus -induced IL-1β production requires lipid raft formation, activation of MAP kinases, and activation of transcription factors AP-1, CRE, and NF-κB. Lipoprotein seems to be a major cell-wall component for the S. aureus -induced IL-1β production in bovine mammary gland epithelial cells.
Abstract Although intestinal epithelial cells (IECs) are continuously exposed to high densities of enteric bacteria, they are not highly responsive to microbe-associated molecular patterns (MAMPs). ...However, inflammatory cytokines such as interferon-γ (IFN-γ) are potentially capable of priming IECs to enhance responsiveness to MAMPs. In this study, we observed that heat-killed Vibrio cholerae (HKVC) and its lipopolysaccharide (LPS) poorly induced IL-8 production in a human IEC line, HT-29. However, both HKVC and the LPS showed a substantial induction of IL-8 production in IFN-γ-primed HT-29 cells. LPS-induced IL-8 production was proportional to the IFN-γ-priming period and LPS could not induce IL-8 production in the presence of polymyxin B. Moreover, LPS-induced IL-8 production in the IFN-γ-primed HT-29 cells was mediated through signaling pathways requiring p38 kinase and ERK, but not the JNK/SAPK pathway. Since deleted in malignant brain tumor 1 (DMBT1) is known to interact with and antagonize the action of LPS, we hypothesized that IFN-γ enhanced the responsiveness to LPS in HT-29 through down-regulation of DMBT1. We found that IFN-γ indeed attenuated DMBT1 expression at both the mRNA and protein levels in HT-29 cells. Conversely, when the cells were transfected with small interfering RNA to specifically silence DMBT1, IL-8 expression was augmented even in the absence of IFN-γ and the augmentation was further enhanced by treatment with V. cholerae LPS. Since IFN-γ is known to increase IFN-β expression in the IECs, we examined if IFN-β functioned similar to IFN-γ. Although IFN-β alone was able to induce IL-8 expression, it failed to render HT-29 cells responsive to V. cholerae LPS. In conclusion, our study suggests that IFN-γ primes IECs to become responsive to V. cholerae and its LPS by suppressing the expression of DMBT1.
A Gram-positive bacterium, Staphylococcus aureus is known to be one of the major pathogenic bacteria responsible for causing bovine mastitis. Among the various cell wall components of S. aureus, ...lipoteichoic acid (LTA) and peptidoglycan (PGN) are closely associated with inflammatory responses. However, the role of LTA and PGN derived from S. aureus in bovine mastitis has not been clearly elucidated. In this study, we characterized the gene expression profile of a bovine mammary gland epithelial cell line, MAC-T cells, in the presence of LTA and PGN from S. aureus. LTA plus PGN, but not LTA or PGN alone, activated MAC-T cells. The analysis of transcriptional profiles using an Affymetrix genechip microarray showed that stimulation with LTA plus PGN produced a total of 2019 (fold change >1.2) differentially expressed genes (DEGs), with 801 up-regulated genes and 1218 down-regulated genes. Of the up-regulated genes, 14 inflammatory mediator-related DEGs, 22 intra-cellular signaling molecule-related DEGs, and 15 transcription factor-related DEGs were observed, whereas among the down-regulated DEGs 17 inflammation-related DEGs were found. The microarray results were confirmed using real-time RT-PCR of 18 genes with substantial changes in expression (9 each from the up-regulated and down-regulated DEGs). These results provide a comprehensive analysis of gene-expression profiles elicited by S. aureus LTA and PGN in MAC-T cells, contributing to an understanding of the pathogenesis for S. aureus-induced bovine mastitis.
•The bovine mammary gland epithelial cell, MAC-T expresses TLR2 and NOD2.•S. aureus LTA plus PGN, but not LTA or PGN alone, activates NF-kB in MAC-T cells.•S. aureus LTA plus PGN up-regulates inflammation-related genes.•S. aureus LTA plus PGN also down-regulates immuno-regulating genes.•The gene expression profiles provide insight into the pathogenesis for S. aureus-induced bovine mastitis.
TLR2 is essential for immune responses to cholera vaccination, where OmpU is the major immunostimulatory component of cholera vaccines.
Vibrio cholerae can cause severe diarrhea and dehydration ...leading to high mortality and morbidity. Current cholera vaccines are formulated with KVC. Although the innate immune responses following vaccination deeply influence the induction of adaptive immunity, the initial recognition of cholera vaccines by the host innate immune system is not well characterized. In this study, the ability of KVC to induce innate immune responses was investigated. Unlike typical Gram‐negative bacteria stimulating TLR2 and TLR4, KVC activated TLR2 but hardly TLR4. However, purified V. cholerae LPS preferentially stimulated TLR4, although not as potently as LPS of other Gram‐negative bacteria, implying that LPS is not a major immunostimulatory component of KVC. Instead, MPFs were similar to KVC in the capacity to activate TLR2, transcription factors, and cytokine expression. Furthermore, OmpU is an abundant membrane protein of V. cholerae and could interact with TLR2 for inducing cytokine expression. Notably, cholera vaccine‐induced immune responses are impaired in TLR2−/− mice. Conclusively, TLR2 is essential for the immune responses to cholera vaccination, and OmpU is the major immunostimulatory component of cholera vaccines.
The vibriocidal assay using guinea pig complement is widely used for the evaluation of immune responses to cholera vaccines in human clinical trials. However, it is unclear why guinea pig complement ...has been used over human complement in the measurement of vibriocidal activity of human sera and there have not been comparison studies for the use of guinea pig complement over those from other species. Therefore, we comparatively investigated the effects of complements derived from human, guinea pig, rabbit, and sheep on vibriocidal activity. Complements from guinea pig, rabbit, and human showed concentration-dependent vibriocidal activity in the presence of quality control serum antibodies. Of these complements, guinea pig complement was the most sensitive and effective over a wide concentration range. When the vibriocidal activity of complements was measured in the absence of serum antibodies, human, sheep, and guinea pig complements showed vibriocidal activity up to 40-fold, 20-fold, and 1-fold dilution, respectively. For human pre- and post-vaccination sera, the most potent vibriocidal activity was observed when guinea pig complement was used. In addition, the highest fold-increases between pre- and post- vaccinated sera were obtained with guinea pig complement. Furthermore, human complement contained a higher amount of
V. cholerae
- and its lipopolysaccharide-specific antibodies than guinea pig complement. Collectively, these results suggest that guinea pig complements are suitable for vibriocidal assays due to their high sensitivity and effectiveness to human sera.
Abstract
Serum vibriocidal antibody assay has been used to evaluate the immunogenicity of cholera vaccines formulated with killed whole-cell of Vibrio cholerae. However, serum antibodies representing ...the vibriocidal activity are not fully characterized. In this study, we examined which isotype is associated with the vibriocidal activity using human convalescent sera with high virbiocidal activity and 20 clinical serum samples obtained from human subjects who had been vaccinated with a killed, whole-cell cholera vaccine. Total IgG, IgA or IgM antibody levels were not correlated with the vibriocidal activity in the 20 human clinical sera. When IgG, IgA and IgM antibodies specific to each of the heat-killed V. cholerae, lipopolysaccharide (LPS), or outer membrane protein U were measured in the 20 clinical serum samples, anti-LPS IgM antibody showed the highest correlation with the vibriocidal activity (Pearson correlation coefficient r = 0.818). Depletion of IgM antibodies in the convalescent sera produced a significant (P<0.05) decrease in the vibriocidal activity (16-fold decrease), whereas little change was observed with depletion of IgG and/or IgA. Furthermore, size-exclusion column chromatography followed by vibriocidal assay and analysis of antibody isotypes demonstrated the co-elution of vibriocidal activity and IgM antibody. Collectively, these results suggest that LPS-specific IgM could be a potential serologic marker for the vibriocidal antibody assay.
Although intestinal epithelial cells (IECs) are continuously exposed to high densities of enteric bacteria, they are not highly responsive to microbe-associated molecular patterns (MAMPs). However, ...inflammatory cytokines such as interferon-I3 (IFN-I3) are potentially capable of priming IECs to enhance responsiveness to MAMPs. In this study, we observed that heat-killed Vibrio cholerae (HKVC) and its lipopolysaccharide (LPS) poorly induced IL-8 production in a human IEC line, HT-29. However, both HKVC and the LPS showed a substantial induction of IL-8 production in IFN-I3-primed HT-29 cells. LPS-induced IL-8 production was proportional to the IFN-I3-priming period and LPS could not induce IL-8 production in the presence of polymyxin B. Moreover, LPS-induced IL-8 production in the IFN-I3-primed HT-29 cells was mediated through signaling pathways requiring p38 kinase and ERK, but not the JNK/SAPK pathway. Since deleted in malignant brain tumor 1 (DMBT1) is known to interact with and antagonize the action of LPS, we hypothesized that IFN-I3 enhanced the responsiveness to LPS in HT-29 through down-regulation of DMBT1. We found that IFN-I3 indeed attenuated DMBT1 expression at both the mRNA and protein levels in HT-29 cells. Conversely, when the cells were transfected with small interfering RNA to specifically silence DMBT1, IL-8 expression was augmented even in the absence of IFN-I3 and the augmentation was further enhanced by treatment with V. cholerae LPS. Since IFN-I3 is known to increase IFN-I2 expression in the IECs, we examined if IFN-I2 functioned similar to IFN-I3. Although IFN-I2 alone was able to induce IL-8 expression, it failed to render HT-29 cells responsive to V. cholerae LPS. In conclusion, our study suggests that IFN-I3 primes IECs to become responsive to V. cholerae and its LPS by suppressing the expression of DMBT1.
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