Bone loss and aggravated autoimmune arthritis in HLA-DRβ1-bearing humanized mice following oral challenge with Porphyromonas gingivalis

BACKGROUND: The linkage between periodontal disease and rheumatoid arthritis is well established. Commonalities among the two are that both are chronic inflammatory diseases characterized by bone loss, an association with the shared epitope susceptibility allele, and anti-citrullinated protein antibodies. METHODS: To explore immune mechanisms that may connect the two seemingly disparate disorders, we measured host immune responses including T-cell phenotype and anti-citrullinated protein antibody production in human leukocyte antigen (HLA)-DR1 humanized C57BL/6 mice following exposure to the Gram-negative anaerobic periodontal disease pathogen Porphyromonas gingivalis. We measured autoimmune arthritis disease expression in mice exposed to P. gingivalis, and also in arthritis-resistant mice by flow cytometry and multiplex cytokine-linked and enzyme-linked immunosorbent assays. We also measured femoral bone density by microcomputed tomography and systemic cytokine production. RESULTS: Exposure of the gingiva of DR1 mice to P. gingivalis results in a transient increase in the percentage of Th17 cells, both in peripheral blood and cervical lymph nodes, a burst of systemic cytokine activity, a loss in femoral bone density, and the generation of anti-citrullinated protein antibodies. Importantly, these antibodies are not produced in response to P. gingivalis treatment of wild-type C57BL/6 mice, and P. gingivalis exposure triggered expression of arthritis in arthritis-resistant mice. CONCLUSIONS: Exposure of gingival tissues to P. gingivalis has systemic effects that can result in disease pathology in tissues that are spatially removed from the initial site of infection, providing evidence for systemic effects of this periodontal pathogen. The elicitation of anti-citrullinated protein antibodies in an HLA-DR1-restricted fashion by mice exposed to P. gingivalis provides support for the role of the shared epitope in both periodontal disease and rheumatoid arthritis. The ability of P. gingivalis to induce disease expression in arthritis-resistant mice provides support for the idea that periodontal infection may be able to trigger autoimmunity if other disease-eliciting factors are already present

Positive feedback between Cdc42 activity and H + efflux by the Na-H exchanger NHE1 for polarity of migrating cells

A fundamental feature of cell polarity in response to spatial cues is asymmetric amplification of molecules generated by positive feedback signaling. We report a positive feedback loop between the guanosine triphosphatase Cdc42, a central determinant in eukaryotic cell polarity, and H+ efflux by Na-H+ exchanger 1 (NHE1), which is necessary at the front of migrating cells for polarity and directional motility. In response to migratory cues, Cdc42 is not activated in fibroblasts expressing a mutant NHE1 that lacks H+ efflux, and wild-type NHE1 is not activated in fibroblasts expressing mutationally inactive Cdc42-N17. H+ efflux by NHE1 is not necessary for release of Cdc42–guanosine diphosphate (GDP) from Rho GDP dissociation inhibitor or for the membrane recruitment of Cdc42 but is required for GTP binding by Cdc42 catalyzed by a guanine nucleotide exchange factor (GEF). Data indicate that GEF binding to phosphotidylinositol 4,5–bisphosphate is pH dependent, suggesting a mechanism for how H+ efflux by NHE1 promotes Cdc42 activity to generate a positive feedback signal necessary for polarity in migrating cells

Misclocalized scaffolding by the Na-H exchanger NHE1 inhibits fibronectin production and assembly

Secretion and assembly of the extracellular matrix protein fibrobnectin (FN) regulates a plethora of normal cell and tissue functions, including development, cell growth, differentiation and cell migration, and is dysregulated in diseases such as fibrosis, diabetes, and cancer. My thesis work revealed that mislocalized scaffolding by the plasma membrane Na-H exchanger NHE1 dominantly suppresses FN expression, secretion, and assembly, and inhibits cleavage of latent to active TGF-beta;. By catalyzing an electroneutral exchange of extracellular Na+ and intracellular H+, NHE1 has recognized functions in intracellular pH and osmotic homeostasis. Recent evidence indicates NHE1 also functions as a plasma membrane anchor for the actin cytoskeleton by binding directly to ERM (ezrin, moesin, radixin) proteins and a PI(4,5)P2-binding scaffold, which are independent of ion translocation by NHE1. Endogenous NHE1 localizes to the distal margin of membrane protrusions in lamellipodia, however a mutantNHE1-KRA2, lacking binding sites for PI(4,5)P2 and the ERM proteins ezrin, radixin and moesin, is mislocalized and distributed uniformly along the plasma membrane. Fibroblasts expressing NHE1-KRA2 but not wild-type NHE1 have impaired FN expression, secretion, and assembly, and reduced active but not latent TGF-beta;. We found that FN production is not regulated by changes in intracellular pH, nor is it attenuated in NHE1-deficient cells, indicating FN expression and TGF-beta; activation are independent of NHE1 activity. However, treating NHE1-KRA2 fibroblasts with recombinant TGF-beta; restores FN secretion and assembly. These data suggest that scaffolding by NHE1-KRA2 sequesters and mislocalizes signals necessary for FN synthesis and TGF-beta; activation. Although the precise signals sequestered by NHE1-KRA2 remain unknown, these findings suggest that NHE1-KRA2 could be a valuable tool for obtaining a mechanistic understanding of how FN production and TGF-beta; activation are regulated, and more speculatively for therapeutic control of increased FN production in pathological conditions such as fibrosis and inflammation

Positive feedback between Cdc42 activity and H+ efflux by the Na-H exchanger NHE1 for polarity of migrating cells.

A fundamental feature of cell polarity in response to spatial cues is asymmetric amplification of molecules generated by positive feedback signaling. We report a positive feedback loop between the guanosine triphosphatase Cdc42, a central determinant in eukaryotic cell polarity, and H(+) efflux by Na-H(+) exchanger 1 (NHE1), which is necessary at the front of migrating cells for polarity and directional motility. In response to migratory cues, Cdc42 is not activated in fibroblasts expressing a mutant NHE1 that lacks H(+) efflux, and wild-type NHE1 is not activated in fibroblasts expressing mutationally inactive Cdc42-N17. H(+) efflux by NHE1 is not necessary for release of Cdc42-guanosine diphosphate (GDP) from Rho GDP dissociation inhibitor or for the membrane recruitment of Cdc42 but is required for GTP binding by Cdc42 catalyzed by a guanine nucleotide exchange factor (GEF). Data indicate that GEF binding to phosphotidylinositol 4,5-bisphosphate is pH dependent, suggesting a mechanism for how H(+) efflux by NHE1 promotes Cdc42 activity to generate a positive feedback signal necessary for polarity in migrating cells

A comparison of two types of electrospun chitosan membranes and a collagen membrane in vivo

Background: Electrospun chitosan membranes subjected to post-spinning processes using either triethylamine/tert-butyloxycarbonyl (TEA/tBOC) or butyryl-anhydride (BA) modifications to maintain nanofiber structure have exhibited potential for use in guided bone regeneration applications. The aim of this study was to evaluate ability of the modified membranes to support healing of bone-grafted defects as compared to a commercial collagen membrane. Method: TEA/tBOC-treated and BA-treated chitosan membranes were characterized for fiber morphology by electron microscopy, residual trifluoroacetic acid by19F NMR and endotoxin level using an endotoxin quantitation kit (ThermoScientific, US). Chitosan membranes were cut into 12 mm diameter disks. An 8 mm calvarial defect was created in each of 48 male rats and then filled with Bio-Oss (Geistlich, US) bone graft. The grafted defects were covered with either (1) TEA/tBOC-treated chitosan membrane (2) BA-treated chitosan membrane or (3) the control BioMend Extend (Zimmer Biomet, US) collagen membrane. After 3 and 8 weeks, the rats were euthanized and calvaria was retrieved for microCT and histological analyses (n = 8/group/time points). Results: Both TEA/tBOC-treated and BA-treated membranes were composed of nanofibers in the ∼231 to ∼284 nm range respectively, exhibited no TFA salt residue and low endotoxin levels (≤0.1 ± 0.01 EU/membrane). All membranes supported increased bone growth from 3 weeks to 8 weeks though there was no significant difference among the membrane types. However, TEA/tBOC treated and BA treated chitosan membranes both showed significantly greater bone density (∼6% greater at 3 weeks and ∼8% greater at 8 weeks) as compared to BioMend Extend collagen membrane at both time points (p = 0.0002). Conclusions: Chitosan membranes supported better bone healing based on bone density than the collagen membrane

Influence of the shared epitope on the elicitation of experimental autoimmune arthritis biomarkers.

Our previous studies have shown that inoculation of the oral cavity of "humanized" B6.DR1/4 mice with the periodontal pathogen Porphyromonas gingivalis results in an increase in the percentage of circulating Th17 cells, loss of bone and an exacerbation of experimental autoimmune arthritis. The aim of this study was to assess the role played by the human HLA-DRβ molecule containing the shared epitope supplied as a transgene to I-A˚ (murine class II null) C57BL/6 (B6) mice in driving these findings. We compared various immune response parameters as well as alveolar and peri-articular bone loss between humanized B6.DR1 (or B6.DR4) mice and their WT (B6) counterparts. We found that the presence of the shared epitope in the context of inoculation with P. gingivalis enhanced the percentage of Th17 cells generated, dramatically enhanced bone loss and importantly allowed for the generation of CCP2⁺ ACPAs that are not found in C57BL/6 or DBA/1 arthritic mouse serum. Due to the exceedingly complex nature of environmental factors impacting on genetic elements, it has been difficult to unravel mechanisms that drive autoimmune arthritis in susceptible individuals. The findings in this study may provide one small piece of this puzzle that can help us to better understand part of this complexity

Mechanically stable surface-hydrophobilized chitosan nanofibrous barrier membranes for guided bone regeneration

The use of chitosan based nanofiber membranes in guided bone regeneration (GBR) is limited by its uncontrolled swelling and mechanical instability in aqueous environments. This paper describes the significantly improved stability and properties of surface butyrylated chitosan nanofiber (BCSNF) membranes that greatly enhance their potential in GBR. The BCSNF membranes exhibited an overall degree of substitution of 1.61, an average diameter of 99.3 ±33.7 nm, and a 75% decrease in swelling with an approximate doubling in suture pull out strengths as compared to unmodified fibers in aqueous environment. In a five week phosphate-buffered saline-lysozyme degradation study, it was found that the remaining mass fraction of BCSNF membranes was 11.5% more than that of unmodified fibers. In vitro, the BCSNF membranes were found to support the adhesion and proliferation of fibroblasts and were cell occulusive. In vivo, the BCSNF membranes were found to significantly improve the regeneration of a rat calvarial critical size defect in a 12 week healing period and showed better barrier function than commercially available collagen membranes with little soft tissue penetration through the membranes. Taken together, these data provide strong scientific evidence for use of BCSNF membranes in GBR applications