Osteoblasts participate in the innate immunity of the bone by producing human beta defensin-3

Abstract

Gram-positive bacterial bone infections are an important cause of morbidity particularly in immunocompromised patients. Antimicrobial peptides (AP) are effectors of the innate immune system and directly kill microorganisms in the first hours after microbial infection. The aim of the present investigation was to study the expression and regulation of gram-positive specialized human beta-defensin-3 (HBD-3) in bone. Samples of healthy and osteomyelitic human bone were assessed for the expression of HBD-3. Using primary and immortalized osteoblasts (SAOS-2 cells), release and regulation of HBD-3 was evaluated after exposure to Staphylococcus aureus supernatant and/or corticosteroids using PCR, immunohistochemistry, Western blot and ELISA. To determine the role of toll-like-receptors-2 and -4 (TLR-2/-4), shRNA was used to downregulate TLRs. An osteomyelitis mouse model was created performed to investigate the release of murine beta-defensins using immunohistochemistry and RT-PCR. Cultured osteoblasts and human bone produce HBD-3 under standard conditions. The release increases within hours of bacterial supernatant exposure in cultured osteoblasts. This observation was not made in chronically infected bone samples. The shRNA-technology revealed the necessity of TLR-2 and -4 in HBD-3 induction in osteoblasts. Blocking protein synthesis with cycloheximide showed that the rapid release of HBD-3 is not dependent on a translational de novo synthesis and is not affected by glucocorticoids. The murine osteomyelitis model confirmed the in vivo release uptake of mouse beta-defensins-4 (MBD-4) in bone. This report shows the bacterial induction of HBD-3 via TLR-2 and -4 in osteoblasts and suggests a central role of antimicrobial peptides in the prevention of bacterial bone infection. The rapid and effective induction of HBD-3 in osteoblasts incubated with conditioned media from bacteria is more likely a result of a rapid secretion of preformed HBD-3 by osteoblasts rather than a result of enhanced biosynthesis. The increased incidence of gram-positive bacterial bone infection in patients with regular intake of glucocorticoids does not seem to be caused by a deranged HBD-3 release in osteoblasts.

Staphylococcus aureus regulates secretion of interleukin-6 and monocyte chemoattractant protein-1 through activation of nuclear factor kappaB signaling pathway in human osteoblasts

“…28,31,32 The different TLRs recognize different ligands in a pathogen-associated molecular pattern. It has been reported that a number of these TLR homologues are present on osteoblasts, such as TLR2, 33,34 TLR4, 34,35 TLR5, 36 and TLR9. 37 We are now in the process of exploring if the pathogen associated molecular pattern between TLRs of S. aureus-infected human osteoblasts and S. aureus cell components is a predominant transcriptional factor that regulates the activation of NF-κB.…”

Defensins defend against bone loss

“…In an earlier study, samples of healthy and osteomyelitic human bone were assessed for the expression of HBD3. In that study, bacteria rapidly and effectively induced osteoblastic HBD3 production [6]. However, much remains to be learned about the mechanisms involved and whether or not HBD3 requires the presence of a bacterial presence for its effects.…”

Human Defensins: Potential Tools for Clinical Applications

“…Human defensins respond to various inflammatory stimuli, like tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), lipopolysaccharide (LPS) as well as a number of bacterial surface antigens and toxins [12,37,[45][46][47][48][49][50][51][52][53]. Besides their direct antimicrobial activity, defensins prevent the activation of toll-like receptors (TLRs) by blocking binding of LPS to LPS binding protein [54].…”