Bovine lactoferrin increases the poly(I:C)-induced antiviral response in vitro.

Bovine lactoferrin (bLF) is a naturally occurring glycoprotein with antibacterial and antiviral activities. We evaluated whether bLF can prevent viral infections in the human intestinal epithelial cell line Caco-2. To assess antiviral responses, we measured the levels of interferon (IFN) expression, IFN-stimulated gene expression, and infection with a pseudotyped virus bearing either severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein or vesicular stomatitis virus (VSV)-G protein after treatment of cells with both bLF and polyinosinic-polycytidylic acid, an analog of double-stranded RNA that mimics viral infection. Combination treatment of cells with both bLF and polyinosinic-polycytidylic acid increased mRNA and protein expression of several IFN genes (IFNB, IFNL1, and IFNL2) and IFN-stimulated genes (ISG15, MX1, IFITM1, and IFITM3) in Caco-2 cells. However, treatment with bLF alone did not induce an antiviral response. Furthermore, combination treatment suppressed infection of the SARS-CoV-2 pseudotyped virus more efficiently than did bLF treatment alone, even though combination treatment increased the expression of mRNA encoding ACE2. These results indicate that bLF increases the antiviral response associated with the double-stranded RNA-stimulated signaling pathway. Our results also suggest that bLF and double-stranded RNA analogs can be used to treat viral infections, including those caused by SARS-CoV-2.

RANK-RANKL signaling upregulates Il-10 mRNA expression in mucosal Candida infection in vivo.

Osteoprotegerin (OPG) prevents binding of receptor activator of nuclear factor-kappa B ligand (RANKL) to RANK. Recent studies have reported that immune cell RANK-RANKL interactions are critical to the infection process. Candida albicans is an opportunistic pathogenic fungus and a common cause of candidiasis. This study utilized an orally inoculated mouse model of C. albicans infection to determine whether superficial or systemic candidiasis was associated with alterations in RANK/RANKL/OPG expression. Invasive systemic C. albicans infection increased serum OPG levels in mice. In addition, tongue Opg, Rankl, and Rank mRNA expression were upregulated in mice with superficial oral cavity C. albicans infection. Moreover, administration of exogenous soluble RANKL upregulated Rank and interleukin-10 (Il-10) mRNA in superficially infected tissue, suggesting suppression of localized inflammation. Taken together, these findings suggested that RANK/RANKL/OPG signaling contributes to the pathogenesis of candidiasis. This is the first in vivo study to identify a relationship between this opportunistic infection and the RANK/RANKL/OPG axis.

Gastrointestinal colonisation and systemic spread of Candida albicans in mice treated with antibiotics and prednisolone.

Normally, Candida albicans is a commensal microbe that resides in the human oral cavity, gut and vagina. However, the fungus can cause mucosal and systemic infections in immunocompromised individuals. The mechanism by which local mucosal infections progress to systemic candidiasis is poorly understood. Here, a murine model of gastrointestinal (GI) candidiasis was developed by inoculation of the oral cavity, followed by treatment with tetracycline (TC) and prednisolone (PSL). Temporal progression from a local infection of the oral cavity to a systemic infection was then monitored. Histological analysis of tissues from mice treated with both TC and PSL revealed massive infiltration of the tongue and stomach by hyphae. PSL increased the fungal burden in the tongue, stomach and small intestine, and facilitated dissemination to the spleen, kidney and liver within 3 days post-infection. Treatment with both TC and PSL supressed interferon (IFN)-γ and interleukin (IL)-17 (cytokines that play key roles in host defence against fungal infection) levels in the tongue, which were induced by C. albicans infection. In addition, the mucosal layer of the small intestine of mice treated with both TC and PSL was almost destroyed by the fungal infection; this may be a critical event that allows passage of the fungus across the mucosa and into the systemic circulation. Thus, this mouse model is useful for studying mechanisms underlying progression of C. albicans from a local infection of the oral cavity to a systemic infection in immunocompromised individuals.

Extracellular galectin-1 enhances adhesion to and invasion of oral epithelial cells by Porphyromonas gingivalis.

Galectin-1 and galectin-3 are C-type lectin receptors that bind to lipopolysaccharide in the cell wall of gram-negative bacteria. In this study, we investigated the effects of galectin-1 and galectin-3 on adhesion to and invasion of the human gingival epithelial cell line Ca9-22 by Porphyromonas gingivalis, a periodontal pathogenic gram-negative bacterium. Recombinant galectin-1, but not galectin-3, enhanced P. gingivalis adhesion and invasion, although both galectins bound similarly to P. gingivalis. Flow cytometry also revealed that Ca9-22 cells express low levels of galectin-1 and moderate levels of galectin-3. Ca9-22 cells in which galectin-3 was knocked-down did not exhibit enhanced P. gingivalis adhesion and invasion. Similarly, specific antibodies to galectin-1 and galectin-3 did not inhibit P. gingivalis adhesion and invasion. These results suggest that soluble galectin-1, but not galectin-3, may exacerbate periodontal disease by enhancing the adhesion to and invasion of host cells by periodontal pathogenic bacteria.

Osteoprotegerin induces cytoskeletal reorganization and activates FAK, Src, and ERK signaling in endothelial cells.

Osteoprotegerin (OPG) is a soluble tumor necrosis factor receptor family member that is expressed by a range of cell types in bone as well as in the vasculature. However, the specific role of OPG in the vascular system is unclear. We recently reported that OPG treatment protects endothelial cells from detachment and apoptotic cell death induced by cysteine proteases of Porphyromonas gingivalis, an important pathogen of adult periodontitis. We also found that OPG activates the extracellular signal-regulated kinase (ERK) 1/2, which has been linked to cell survival and angiogenesis. In this study, we demonstrate that exposure to OPG induces a substantial morphological change in human dermal microvascular endothelial cells. Our results show that OPG induced a dose-dependent increase in the length of microtubules, which coincided with the transition of the cells from a polygonal to an elongated shape. Furthermore, we demonstrated that OPG activates signaling pathways that lead to the activation of Src, focal adhesion kinase, and ERK1/2. These findings suggest that OPG regulates at least two distinct pathways: one that induces cell proliferation via ERK signaling and another that induces angiogenesis via Src signaling. The findings of this study suggest that OPG may function as a regulator of angiogenesis.

Role of alphav integrin in osteoprotegerin-induced endothelial cell migration and proliferation.

Osteoprotegerin (OPG) is a decoy receptor for the receptor activator of nuclear factor kappaB ligand (RANKL). However, the role of OPG in the endothelium remains unknown. In this study, we demonstrate that OPG stimulates the proliferation and migration of human microvascular endothelial cells (HMVECs). In addition, we show that treatment with integrin alpha(v)beta(3) or integrin alpha(v)beta(5) blocking antibody inhibits endothelial cell migration. In contrast, treatment with anti-alpha(v)beta(3) antibody or anti-alpha(v)beta(5) antibody alone did not inhibit OPG-induced proliferation. However, OPG-induced proliferation was inhibited when these antibodies were applied simultaneously. Furthermore, OPG evoked activation of extracellular signal-regulated kinase (ERK) 1/2, which has been linked to integrin alpha(v) activity. Taken together, these results suggest that integrins alpha(v)beta(3) and/or alpha(v)beta(5) contribute to endothelial cell proliferation and migration induced by OPG.

Etidronate down-regulates Toll-like receptor (TLR) 2 ligand-induced proinflammatory cytokine production by inhibiting NF-κB activation.

BACKGROUND: Etidronate is a non-nitrogen-containing bisphosphonate (non-NBP) used for anti-bone resorptive therapy as well as having inhibitory effects on atherosclerotic plaques. The present study examined the effects of etidronate on the production of proinflammatory cytokines and chemokines by the macrophage-like cell line, J774.1, incubated with Pam3Cys-Ser-(Lys)4 (Pam3CSK4, a Toll-like receptor (TLR) 2 agonist) and lipid A (a TLR4 agonist). METHODS: J774.1 cells and human monocytic THP-1 cells were pretreated with or without etidronate for 5min, and then incubated with or without Pam3CSK4 or lipid A for 24h. Levels of secreted interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1α (MIP-1α) in culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Cytotoxicity was determined by LDH activity in the supernatants. We also examined the effects of etidronate on the activation of nuclear factor-κB (NF-κB) and p38 mitogen-activated protein kinase (MAPK) in J774.1 cells by ELISA and Western blotting. RESULTS: Treatment of J774.1 cells with etidronate down-regulated TLR2 ligand-induced production of IL-6, TNF-α, MCP-1, and MIP-1α. Etidronate also inhibited Pam3CSK4-induced MCP-1 and TNF-α production by THP-1 cells. However, etidronate did not induce cytotoxicity and reduced lipid A-induced cytotoxicity in J774.1 cells. In addition, this agent did not down-regulate TLR4 ligand-induced proinflammatory cytokine production. Furthermore, etidronate inhibited the translocation of NF-κB but not p38 MAPK in J774.1 cells stimulated with Pam3CSK4 or lipid A. CONCLUSION: Etidronate likely inhibits proinflammatory cytokine production in J774.1 cells by suppressing NF-κB activation in the TLR2 and not the TLR4 pathway.

Osteoprotegerin protects endothelial cells against apoptotic cell death induced by Porphyromonas gingivalis cysteine proteinases.

Osteoprotegerin (OPG) is a key regulator of osteoclastogenesis during the progression of periodontitis. Recent reports suggest that osteoprotegerin may also prevent arterial calcification and contribute to endothelial cell survival. To determine whether the vascular functions of osteoprotegerin are involved in periodontitis, we examined whether osteoprotegerin contributed to the survival of endothelial cells damaged by Porphyromonas gingivalis cysteine proteinases (gingipains). Gingipain proteinases cleave a broad range of host proteins, and are important virulence factors of P. gingivalis, a major causative bacterium of adult periodontitis. Human microvascular endothelial cells (HMVEC) were exposed to activated gingipain extracts from P. gingivalis 381, with and without pretreatment with osteoprotegerin. Cell viability was quantified by the tetrazolium (WST-8) reduction assay, and apoptosis was examined using Hoechst 33342 nuclear staining. After 16 h of treatment with activated gingipain extracts, HMVEC showed near-complete detachment from the tissue culture dish, and apoptosis was evident by 24 h. Pretreatment of HMVEC with osteoprotegerin reduced the extent of both cellular detachment and apoptotic cell death. Our results indicated that osteoprotegerin pretreatment protected HMVEC against detachment and apoptotic cell death induced by gingipain-active bacterial cell extracts. These results also suggest that osteoprotegerin may function as a survival factor for endothelial cells during periodontitis.

Anti-cell-associated glucosyltransferase immunoglobulin Y suppression of salivary mutans streptococci in healthy young adults.

BACKGROUND: The authors evaluated the suppressive effects of lozenges containing egg yolk antibodies (that is, immunoglobulin Y [IgY]) against Streptococcus mutans cell-associated glucosyltransferase (CA-gtf) on oral colonization by mutans streptococci (MS) in healthy young adults. METHODS: In a five-day double-masked placebo-controlled trial, young adult participants self-administered lozenges containing anti-CA-gtf IgY (Ovalgen DC, GHEN, Gifu-City, Japan) or a placebo at prescribed times each day. On the basis of bacterial colony counts of saliva cultures, the authors analyzed the pretrial and posttrial differences in levels of MS and total anaerobic bacteria among participants in the treatment (anti-CA-gtf IgY) and placebo groups and a control group. RESULTS: Salivary MS scores in participants in the treatment group decreased significantly (P < .001), and the mean anaerobic bacterial count in the treatment group was not statistically different before and after the trial. In the placebo and control groups, posttrial changes in median MS scores and total salivary anaerobic bacterial counts were not statistically significant. CONCLUSIONS: The results of the study show that lozenges containing anti-CA-gtf IgY can suppress oral colonization by MS in healthy young adults. CLINICAL IMPLICATIONS: Lozenges containing anti-CA-gtf IgY may help reduce dental caries risk in humans.

Porphyromonas gingivalis modulates the production of interleukin 8 and monocyte chemotactic protein 1 in human vascular endothelial cells.

Porphyromonas gingivalis seems to perturb the cytokine network to maintain periodontal disease. Although endothelial cells are a leading source of interleukin 8 (IL-8) and monocyte chemotactic protein 1 (MCP-1), the effect of P. gingivalis on the cells remains unclear. We used reverse transcription-PCR (RT-PCR) to assess levels of IL-8 and MCP-1 mRNA and enzyme-linked immunosorbent assays (ELISA) to evaluate their protein production by human umbilical vein endothelial cells (HUVEC) challenged with P. gingivalis. IL-8 and MCP-1 protein levels decreased in response to challenge with P. gingivalis, and N-a- p-tosyl- L-lysine chloromethylketone (TLCK) prevented the degradation of these chemokines. Furthermore, the bacteria upregulated expression of the mRNAs of these chemokines. Our results indicate that P. gingivalis proteases degraded IL-8 and MCP-1. Degradation of chemokines secreted from endothelial cells may decrease the recruitment of leukocytes and their migration through the endothelium, thus contributing to a delay in the host immune defense mechanism.

NF-kappaB-dependent induction of osteoprotegerin by Porphyromonas gingivalis in endothelial cells.

Porphyromonas gingivalis is a major etiological pathogen of adult periodontitis characterized by alveolar bone resorption. Vascular endothelial cells supply many inflammatory cytokines into periodontal tissue. However, whether the cells contribute to bone metabolism in periodontitis remains unknown. In this study, we investigated the effect of P. gingivalis on osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) production, both of which are key regulators of bone metabolism, in human microvascular endothelial cells (HMVECs). We showed that P. gingivalis upregulated expression of OPG but not RANKL mRNA in HMVEC. P. gingivalis induced NF-kappaB activation, and the induction of OPG in HMVEC by the pathogen was blocked by the inhibitors of NF-kappaB. In addition, incubation of OPG with P. gingivalis supernatant resulted in loss of the protein. These results indicate that P. gingivalis-stimulated HMVEC secrete OPG via a NF-kappaB-dependent pathway, while the OPG is partly degraded by the bacteria. Thus, microvascular endothelial cells can act as a source of OPG and thereby may play an important role in regulating bone metabolism in periodontitis.