Dental pulp inflammatory/immune response to a chitosan-enriched fibrin hydrogel in the pulpotomised rat incisor.

Current pulpotomy is limited in its ability to induce regeneration of the dental-pulp (DP) complex. Hydrogels are reported to be well-suited for tissue engineering and are unlikely to induce an inflammatory response that might damage the remaining tissue. The present study investigated the molecular and cellular actors in the early inflammatory/immune response and deciphered M1/M2 macrophage polarisation to a chitosan-enriched fibrin hydrogel in pulpotomised rat incisors. Both fibrin and fibrin-chitosan hydrogels induced a strong increase in interleukin-6 (IL-6) transcript in the DP when compared to the DP of untreated teeth. Gene expression of other inflammatory mediators was not significantly modified after 3 h. In the viable DP cell population, the percentage of leukocytes assessed by flow cytometry was similar to fibrin and fibrin-chitosan hydrogels after 1 d. In this leukocyte population, the proportion of granulocytes increased beneath both hydrogels whereas the antigen-presenting cell, myeloid dendritic cells, T cells and B cells decreased. The natural killer (NK) cell population was significantly decreased only in DPs from teeth treated with fibrin-chitosan hydrogel. Immunolabeling analysis of the DP/hydrogel interface showed accumulation of neutrophil granulocytes in contact with both hydrogels 1 d after treatment. The DP close to this granulocyte area contained M2 but no M1 macrophages. These data collectively demonstrated that fibrin-chitosan hydrogels induced an inflammatory/immune response similar to that of the fibrin hydrogel. The results confirmed the potential clinical use of fibrin-chitosan hydrogel as a new scaffold for vital-pulp therapies.

Immune Cells and Molecular Networks in Experimentally Induced Pulpitis.

Dental pulp is a dynamic tissue able to resist external irritation during tooth decay by using immunocompetent cells involved in innate and adaptive responses. To better understand the immune response of pulp toward gram-negative bacteria, we analyzed biological mediators and immunocompetent cells in rat incisor pulp experimentally inflamed by either lipopolysaccharide (LPS) or saline solution (phosphate-buffered saline [PBS]). Untreated teeth were used as control. Expression of pro- and anti-inflammatory cytokines, chemokine ligands, growth factors, and enzymes were evaluated at the transcript level, and the recruitment of the different leukocytes in pulp was measured by fluorescence-activated cell-sorting analysis after 3 h, 9 h, and 3 d post-PBS or post-LPS treatment. After 3 d, injured rat incisors showed pulp wound healing and production of reparative dentin in both LPS and PBS conditions, testifying to the reversible pulpitis status of this model. IL6, IL1-ß, TNF-α, CCL2, CXCL1, CXCL2, MMP9, and iNOS gene expression were significantly upregulated after 3 h of LPS stimulation as compared with PBS. The immunoregulatory cytokine IL10 was also upregulated after 3 h, suggesting that LPS stimulates not only inflammation but also immunoregulation. Fluorescence-activated cell-sorting analysis revealed a significant, rapid, and transient increase in leukocyte levels 9 h after PBS and LPS stimulation. The quantity of dendritic cells was significantly upregulated with LPS versus PBS. Interestingly, we identified a myeloid-derived suppressor cell-enriched cell population in noninjured rodent incisor dental pulp. The percentage of this population, known to regulate immune response, was higher 9 h after inflammation triggered with PBS and LPS as compared with the control. Taken together, these data offer a better understanding of the mechanisms involved in the regulation of dental pulp immunity that may be elicited by gram-negative bacteria.

Distinct expression of interleukin (IL)-36α, ß and γ, their antagonist IL-36Ra and IL-38 in psoriasis, rheumatoid arthritis and Crohn's disease.

Interleukin (IL)-36α, IL-36ß and IL-36γ are expressed highly in skin and are involved in the pathogenesis of psoriasis, while the antagonists IL-36Ra or IL-38, another potential IL-36 inhibitor, limit uncontrolled inflammation. The expression and role of IL-36 cytokines in rheumatoid arthritis (RA) and Crohn's disease (CD) is currently debated. Here, we observed that during imiquimod-induced mouse skin inflammation and in human psoriasis, expression of IL-36α, γ and IL-36Ra, but not IL-36ß and IL-38 mRNA, was induced and correlated with IL-1ß and T helper type 17 (Th17) cytokines (IL-17A, IL-22, IL-23, CCL20). In mice with collagen-induced arthritis and in the synovium of patients with RA, IL-36α, ß, γ, IL-36Ra and IL-38 were all elevated and correlated with IL-1ß, CCL3, CCL4 and macrophage colony-stimulating factor (M-CSF), but not with Th17 cytokines. In the colon of mice with dextran sulphate sodium-induced colitis and in patients with CD, only IL-36α, γ and IL-38 were induced at relatively low levels and correlated with IL-1ß and IL-17A. We suggest that only a minor subgroup of patients with RA (17-29%) or CD (25%) had an elevated IL-36 agonists/antagonists ratio, versus 93% of patients with psoriasis. By immunohistochemistry, IL-36 cytokines were produced by various cell types in skin, synovium and colonic mucosa such as keratinocytes, CD68⁺ macrophages, dendritic/Langerhans cells and CD79α⁺ plasma cells. In primary cultures of monocytes or inflammatory macrophages (M1), IL-36ß and IL-36Ra were produced constitutively, but IL-36α, γ and IL-38 were produced after lipopolysaccharide stimulation. These distinct expression profiles may help to explain why only subgroups of RA and CD patients have a potentially elevated IL-36 agonists/antagonists ratio.

Inhibition of osteolysis and increase of bone formation after local administration of siRNA-targeting RANK in a polyethylene particle-induced osteolysis model.

Receptor activator of nuclear factor kappa-B (RANK) and RANK-ligand are relevant targets for the treatment of polyethylene particle-induced osteolysis. This study assessed the local administration of siRNA, targeting both human RANK and mouse Rank transcripts in a mouse model. Four groups of mice were implanted with polyethylene (PE) particles in the calvaria and treated locally with 2.5, 5 and 10 µg of RANK siRNA or a control siRNA delivered by the cationic liposome DMAPAP/DOPE. The tissues were harvested at day 9 after surgery and evaluated by micro-computed tomography, tartrate-resistant acid phosphatase (TRAP) immunohistochemistry for macrophages and osteoblasts, and gene relative expression of inflammatory and osteolytic markers. 10 µg of RANK siRNA exerted a protective effect against PE particle-induced osteolysis, decreasing the bone loss and the osteoclastogenesis, demonstrated by the significant increase in the bone volume (P<0.001) and by the reduction in both the number of TRAP(+) cells and osteoclast activity (P<0.01). A bone anabolic effect demonstrated by the formation of new trabecular bone was confirmed by the increased immunopositive staining for osteoblast-specific proteins. In addition, 5 and 10 µg of RANK siRNA downregulated the expression of pro-inflammatory cytokines (P<0.01) without depletion of macrophages. Our findings show that RANK siRNA delivered locally by a synthetic vector may be an effective approach for reducing osteolysis and may even stimulate bone formation in aseptic loosening of prosthetic implants.

Interleukin 34 expression is associated with synovitis severity in rheumatoid arthritis patients.

OBJECTIVES: Interleukin (IL) 34 is a new cytokine implicated in macrophage differentiation and osteoclastogenesis. This study assessed IL-34 expression in the tissue of patients with rheumatoid arthritis (RA). METHODS: Immunohistochemistry was performed in synovial biopsies from patients with RA (n=20), osteoarthritis (n=3) or other inflammatory arthritis (n=4). IL-34 was detected in the synovial fluid by ELISA and its messenger RNA expression was studied by quantitative PCR in rheumatoid synovial fibroblasts after stimulation by tumour necrosis factor α (TNFα) and IL-1ß. Wild-type, jnk1(-/-)-jnk2(-/-) and nemo(-/-) murine fibroblasts and pharmacological inhibition were used to determine the involvement of nuclear factor kappa B (NF-κB) and JNK in that effect. RESULTS: IL-34 was expressed in 24/27 biopsies, with three samples from RA patients being negative. A significant association was found between IL-34 expression and synovitis severity. Levels of IL-34 and the total leucocyte count in synovial fluid were correlated. TNFα and IL-1ß stimulated IL-34 expression by synovial fibroblasts in a dose/time-dependent manner through the NF-κB and JNK pathway. CONCLUSION: This work for the first time identifies IL-34 expression in the synovial tissue of patients with arthritis. This cytokine, as a downstream effector of TNFα and IL-1ß, may contribute to inflammation and bone erosions in RA.

Histopathologic changes of the ear in canine models of mucopolysaccharidosis types I and VII.

Mucopolysaccharidosis (MPS) types I and VII are inborn errors of metabolism caused by mutation of enzymes involved in glycosaminoglycan catabolism, which leads to intralysosomal accumulation of glycosaminoglycans. In children, severe forms of MPS I and VII are characterized by somatic and neurologic manifestations, including a poorly understood hearing loss. The purpose of this study is to describe the age-related histopathologic changes of the ear in spontaneous canine models of MPS I and VII. Pathologic changes in the ear were assessed in MPS I and VII dogs ranging from 1.6 to 9.3 months of age. Paraffin-embedded sections of the whole ear and Epon-embedded semithin sections of the cochlea were examined. The following lesions were blindly scored in the middle and inner ear: inflammation, cells vacuolization, thickening of osseous and membranous structures, perivascular vacuolated macrophages infiltration, and bone resorption. All dogs had lysosomal storage within cells of tympanic membrane, ossicles, tympanic bone and mucosa, cochlear bone, spiral ligament, limbus, and stria vascularis. The MPS I dogs mainly had progressive cochlear lesions. The MPS VII dogs had severe and early middle ear lesions, including chronic otitis media and bone resorption. The MPS I dog only partially recapitulates the pathology seen in humans; specifically, the dog model lacks inflammatory middle ear disease. In contrast, the MPS VII dog has severe inflammatory middle ear disease similar to that reported in the human. In conclusion, the canine MPS VII model appears to be a good model to study MPS VII-related deafness.