Induction of Nod1 and Nod2 intracellular pattern recognition receptors in murine osteoblasts following bacterial challenge

Abstract

Osteoblasts produce an array of immune molecules following bacterial challenge that could recruit leukocytes to sites of infection and promote inflammation during bone diseases, such as osteomyelitis. Recent studies from our laboratory have shed light on the mechanisms by which this cell type can perceive and respond to bacteria by demonstrating the functional expression of members of the Toll-like family of cell surface pattern recognition receptors by osteoblasts. However, we have shown that bacterial components fail to elicit immune responses comparable with those seen following challenge with the intracellular pathogens salmonellae and Staphylococcus aureus. In the present study, we show that UV-killed bacteria and invasion-defective bacterial strains elicit significantly less inflammatory cytokine production than their viable wild-type counterparts. Importantly, we demonstrate that murine osteoblasts express the novel intracellular pattern recognition receptors Nod1 and Nod2. Levels of mRNA encoding Nod molecules and protein expression are significantly and differentially increased from low basal levels following exposure to these disparate bacterial pathogens. In addition, we have shown that osteoblasts express Rip2 kinase, a critical downstream effector molecule for Nod signaling. Furthermore, to begin to establish the functional nature of Nod expression, we show that a specific ligand for Nod proteins can significantly augment immune molecule production by osteoblasts exposed to either UV-inactivated bacteria or bacterial lipopolysaccharide. As such, the presence of Nod proteins in osteoblasts could represent an important mechanism by which this cell type responds to intracellular bacterial pathogens of bone.

FIG. 1.

UV-inactivated bacteria and invasion-defective bacterial strains are weaker stimuli for immune molecule production by osteoblasts than viable wild-type bacteria. Panel A: osteoblasts (2 × 10⁶ per well) were untreated (0) or exposed to either viable or UV-inactivated S. aureus (multiplicity of infection [MOI] of 25, 75, and 250 bacteria per cell) or salmonellae (MOI of 1, 3, and 10 bacteria per cell). Panel B: osteoblasts (2 × 10⁶ per well) were untreated (0) or exposed to either wild-type or mutant staphylococci (S. aureus and S. carnosus at MOIs of 25 and 75 bacteria per cell) or salmonellae (S. enterica serovar Typhimurium SB300 and S. enterica serovar Typhimurium SB136 at MOIs of 1 and 3 bacteria per cell). At 24 h posttreatment, culture supernatants were taken and assayed for the presence of IL-6 by specific capture ELISA. Data are shown as mean values of triplicate determinations of three separate experiments ± standard error of the mean. Asterisks indicate a significant difference between responses to viable wild-type bacteria and attenuated and inactivated bacteria (P < 0.05).

FIG. 2.

Expression of mRNA encoding Nod1 and Nod2 in murine osteoblasts following bacterial exposure. Osteoblasts (2 × 10⁶ per well) were untreated (0) or exposed to salmonellae (MOIs of 1, 3, and 10 bacteria per cell), S. aureus (MOIs of 25, 75, and 250 bacteria per cell), or LPS (100 ng/ml). RNA was isolated at 4 or 8 h posttreatment, and RT-PCR was performed for the presence of mRNA encoding Nod1 or Nod2. PCR amplification of mRNA encoding G3PDH was performed to ensure that similar amounts of input RNA and similar efficiencies of reverse transcription were being compared. For comparison purposes, RT-PCR was performed on RNA isolated from a similar number of LPS-activated peritoneal macrophages (mφ). Below, densitometric analysis of this representative experiment is shown as arbitrary densitometric units for Nod1 and Nod2 mRNA expression normalized to G3PDH mRNA levels. These studies were performed three times with similar results.

FIG. 3.

Elevated Nod1 and Nod2 protein expression in osteoblasts following exposure to S. aureus and salmonellae. Osteoblasts were either untreated (0) or exposed to S. aureus (MOIs of 25, 75, and 250 bacteria per cell) or salmonellae (MOIs of 1, 3, and 10 bacteria per cell). After 24 h, protein isolates were subjected to Western blot analysis for Nod1 expression (panel A) or after 12 h for Nod2 expression (panel B). Representative immunoblots for each are shown. For comparison purposes, Nod2 protein expression in samples from a similar number of LPS-activated peritoneal macrophages (mφ) was also analyzed. Below, densitometric analyses of Nod1 (n = 4) and Nod2 (n = 3) protein bands are shown as arbitrary densitometric units corrected for background intensity in each lane, ± standard error of the mean. Asterisks indicate a significant difference from unstimulated osteoblasts (P < 0.05).

FIG. 4.

Expression of Rip2 kinase in murine osteoblasts following exposure to bacterial stimuli. Panel A: Osteoblasts (2 × 10⁶ per well) were untreated (0) or exposed to S. aureus (S. aur) (MOIs of 25, 75, and 250 bacteria per cell), salmonellae (Sal) (MOIs of 1, 3, and 10 bacteria per cell), or lipopolysaccharide (LPS) (1, 10, and 100 ng/ml). RNA was isolated at 8 h postinfection and RT-PCR was performed for the presence of mRNA encoding Rip2. PCR amplification of mRNA encoding IL-6 was performed to confirm osteoblast responsiveness to bacterial challenge. PCR amplification of mRNA encoding G3PDH was performed to ensure that similar amounts of input RNA and similar efficiencies of reverse transcription were being compared. These studies were performed three times with similar results. Panel B: Osteoblasts (2 × 10⁶ per well) were untreated (0) or exposed to S. aureus (S. aur) (MOIs of 25, 75, and 250 bacteria per cell) or salmonellae (Sal) (MOIs of 1, 3, and 10 bacteria per cell). After 24 h, protein isolates were subjected to Western blot analysis for Rip2 kinase expression. The migration of protein standards of known size is indicated to the left. A representative immunoblot from three separate experiments is shown.

FIG. 5.

MDP significantly augments LPS or UV killed bacteria-mediated IL-6 production by osteoblasts. Panel A: Cells (10⁷ per well) were untreated (0) or exposed to lipopolysaccharide (LPS) (10 and 100 ng/ml) in the presence or absence of MDP (1 μg/ml). Panel B: Cells (2 × 10⁶ per well) were untreated (0) or exposed to UV-killed salmonellae (MOIs of 1, 3, and 10 bacteria per cell) in the presence or absence of MDP (1 μg/ml). At 24 h posttreatment, culture supernatants were taken and assayed for the presence of IL-6 by specific capture ELISA. For comparison purposes, levels of IL-6 secretion by osteoblasts exposed to UV-killed S. aureus (75:1, bacteria to cells) in the absence or presence of MDP are shown. Data are shown as mean values of triplicate determinations of four separate experiments ± standard error of the mean. Asterisks indicate a significant difference in IL-6 production between activated cells in the absence and the presence of MDP (P < 0.05).