Lack of CCR3 leads to a skeletal phenotype only in male mice.

We recently showed that adult male mice that lacked the C-C-chemokine receptor 3 (CCR3) exhibited disturbed bone remodeling, which resulted in a cortical bone phenotype of thin femoral cortical bone. However, it remains unknown whether this phenotype would be present during bone modeling, or it affects female mice. Here, we analyzed juvenile and adolescent CCR3-deficient mice to determine when bone modeling was affected in the absence of CCR3 signaling. To investigate whether the CCR3 bone phenotype was sex-related, we analyzed both young female and male mice, and adult females. Micro-computed tomography (µCT) and histomorphometric analyses in adolescent CCR3-deficient male mice revealed reduced cortical bone volume and thickness, and an increase in periosteal mineralization. Interestingly, no skeletal phenotype was observed in adolescent or adult female CCR3-deficient mice. Among juvenile CCR3-deficient mice, neither males nor females showed a skeletal phenotype, which indicated that bone modeling was not affected by the CCR3 deficiency. In summary, adolescent and adult male mice that lacked CCR3 receptors exhibited a cortical phenotype that was not present in female mice, probably due to an estrogen protective mechanism. Based on these and our previous results, we suggest that the importance of CCR3 in cortical bone turnover is related to sex hormones. Because only a few molecules are known to control cortical bone turnover, our novel finding that CCR3 regulated cortical bone thickness only in males suggested that CCR3 is a novel target for controlling cortical bone morphology in male individuals, and perhaps, in post-menopausal women.

Engineering a 3D In Vitro Model of Human Gingival Tissue Equivalent with Genipin/Cytochalasin D.

Although three-dimensional (3D) co-culture of gingival keratinocytes and fibroblasts-populated collagen gel can mimic 3D structure of in vivo tissue, the uncontrolled contraction of collagen gel restricts its application in clinical and experimental practices. We here established a stable 3D gingival tissue equivalent (GTE) using hTERT-immortalized gingival fibroblasts (hGFBs)-populated collagen gel directly crosslinked with genipin/cytochalasin D and seeding hTERT-immortalized gingival keratinocytes (TIGKs) on the upper surface for a 2-week air-liquid interface co-culture. MTT assay was used to measure the cell viability of GTEs. GTE size was monitored following culture period, and the contraction was analyzed. Immunohistochemical assay was used to analyze GTE structure. qRT-PCR was conducted to examine the mRNA expression of keratinocyte-specific genes. Fifty µM genipin (G50) or combination (G + C) of G50 and 100 nM cytochalasin D significantly inhibited GTE contraction. Additionally, a higher cell viability appeared in GTEs crosslinked with G50 or G + C. GTEs crosslinked with genipin/cytochalasin D showed a distinct multilayered stratified epithelium that expressed keratinocyte-specific genes similar to native gingiva. Collagen directly crosslinked with G50 or G + C significantly reduced GTE contraction without damaging the epithelium. In summary, the TIGKs and hGFBs can successfully form organotypic multilayered cultures, which can be a valuable tool in the research regarding periodontal disease as well as oral mucosa disease. We conclude that genipin is a promising crosslinker with the ability to reduce collagen contraction while maintaining normal cell function in collagen-based oral tissue engineering.

CCR3 deficiency is associated with increased osteoclast activity and reduced cortical bone volume in adult male mice.

Increasing evidence emphasizes the importance of chemokines and chemokine receptors as regulators of bone remodeling. The C-C chemokine receptor 3 (CCR3) is dramatically upregulated during osteoclastogenesis, but the role of CCR3 in osteoclast formation and bone remodeling in adult mice is unknown. Herein, we used bone marrow macrophages derived from adult male CCR3-proficient and CCR3-deficient mice to study the role of CCR3 in osteoclast formation and activity. CCR3 deficiency was associated with formation of giant hypernucleated osteoclasts, enhanced bone resorption when cultured on bone slices, and altered mRNA expression of related chemokine receptors and ligands. In addition, primary mouse calvarial osteoblasts isolated from CCR3-deficient mice showed increased mRNA expression of the osteoclast activator-related gene, receptor activator of nuclear factor kappa-B ligand, and osteoblast differentiation-associated genes. Microcomputed tomography analyses of femurs from CCR3-deficient mice revealed a bone phenotype that entailed less cortical thickness and volume. Consistent with our in vitro studies, the total number of osteoclasts did not differ between the genotypes in vivo. Moreover, an increased endocortical osteoid mineralization rate and higher trabecular and cortical bone formation rate was displayed in CCR3-deficient mice. Collectively, our data show that CCR3 deficiency influences osteoblast and osteoclast differentiation and that it is associated with thinner cortical bone in adult male mice.

Innate lymphoid cells are present in gingivitis and periodontitis.

BACKGROUND: Innate lymphoid cells (ILCs) are the most recently identified leukocytes of the immune system and these cells are increasingly acknowledged to play important roles in host defence and tissue repair. ILCs are also contributors of inflammatory diseases such as asthma and colitis. We analyzed the presence and relative proportions of the different ILC subsets (ILC1, ILC2 and ILC3) in gingivitis and periodontitis. Further, we investigated if ILCs express receptor activator of nuclear factor kappa-B ligand (RANKL), a cytokine crucial for osteoclast differentiation and bone resorption. METHODS: We collected gingivitis and periodontitis soft tissue and characterized ILC subsets including RANKL expression in single-cell suspensions using flow cytometry. RESULTS: ILCs were detected both in gingivitis and periodontitis. The majority of ILCs, in both conditions, were ILC1s. Furthermore, RANKL expression was detected on a fraction of the ILC1s. CONCLUSIONS: Our discovery of the presence of ILCs both in gingivitis and periodontitis and concomitant expression of RANKL on a fraction of the ILC1 population suggest that these cells may be of importance in periodontal disease. In addition, our findings provide a new insight into the field of oral immunology.

Association Between Marginal Jawbone Loss and Onset of Rheumatoid Arthritis and Relationship to Plasma Levels of RANKL.

OBJECTIVE: To investigate whether periodontitis, characterized by marginal jawbone loss, precedes the onset of symptoms of rheumatoid arthritis (RA), and to analyze plasma levels of RANKL (a cytokine that is crucial for bone resorption) and anti-citrullinated peptide antibodies (ACPAs) in presymptomatic individuals compared with matched referent controls. METHODS: Marginal jawbone loss was measured on dental radiographs of the premolar/molar regions in the jaws in 176 subjects, 93 of whom subsequently developed RA. Among these participating subjects, 46 had documented radiographs predating symptom onset, and 45 cases could be matched to controls, according to sex, age, and smoking status. Plasma RANKL concentrations were analyzed using enzyme-linked immunosorbent assay. A receiver operating characteristic curve was used to define the cutoff value for RANKL positivity. RESULTS: Bone loss was significantly greater in presymptomatic subjects classified as never smokers compared with that in controls, and increasing levels of bone loss were associated with a higher risk of the subsequent development of RA (hazard ratio 1.03, 95% confidence interval 1.01-1.05). No association between jawbone loss and RA was observed in smokers. A significantly greater extent of marginal jawbone loss was detected in RANKL-positive presymptomatic subjects, and even more pronounced jawbone loss was observed in those who were positive for both RANKL and ACPA. CONCLUSION: Marginal jawbone loss preceded the clinical onset of RA symptoms, but this was observed only in nonsmokers. Moreover, marginal jawbone loss was significantly greater in RANKL-positive presymptomatic subjects compared with RANKL-negative presymptomatic subjects and was highest in presymptomatic subjects positive for both ACPA and RANKL.

Non-Hematopoietic and Hematopoietic SIRPα Signaling Differently Regulates Murine B Cell Maturation in Bone Marrow and Spleen.

B lymphocyte development occurs in the bone marrow, while final differentiation and maturation can occur in both the bone marrow and the spleen. Here we provide evidence that signal regulatory protein α (SIRPα), an Ig-superfamily ITIM-receptor expressed by myeloid but not by lymphoid cells, is involved in regulating B cell maturation. Lack of SIRPα signaling in adult SIRPα-mutant mice resulted in a reduced maturation of B cells in the bone marrow, evident by reduced numbers of semi-mature IgD+IgMhi follicular type-II (F-II) and mature IgD+IgMlo follicular type-I (F-I) B cells, as well as reduced blood B cell numbers. In addition, lack of SIRPα signaling also impaired follicular B cell maturation in the spleen. Maturing BM or splenic B cells of SIRPα-mutant mice were found to express higher levels of the pro-apoptotic protein BIM and apoptosis was increased among these B cells. Bone marrow reconstitution experiments revealed that the B cell maturation defect in bone marrow and blood was due to lack of SIRPα signaling in non-hematopoietic cells, while hematopoietic SIRPα signaling was important for follicular B cell maturation in the spleen. Adding on to our previous findings of a stromal cell defect in SIRPα-mutant mice was the finding that gene expression of receptor activator of nuclear factor-ĸB ligand (RANKL) was significantly lower in cultured bone marrow stromal cells of SIRPα mutant mice. These data suggest a novel and opposite contribution of SIRPα signaling within non-hematopoietic and hematopoietic cells, respectively, to maintain B cell maturation and to prevent apoptosis in the bone marrow and spleen of adult mice.