Changes in immunological parameters by ageing in rural healthy Indian adults and their associations with sex and lifestyle.

Several factors including sex and lifestyle have been reported to contribute to the age-related alteration of immune functions. The study was undertaken to determine age-related differences in the proportion of peripheral blood mononuclear lymphocytes in the Indian population using blood samples from 67 healthy adults (33 females and 34 males) aged between 20 and 80 years old. In the linear regression analysis to estimate the relationship with age categories, there was a significant increase in the frequency of natural killer cells with ageing, while their cytolytic activity significantly declined. The frequency of CD4+ T cells increased with age, whereas that of CD8+ T cells decreased, resulting in the age-associated increase of the CD4/CD8 ratio. The subsets of B cells did not show any significant relationship with age. Although there were variations between the male and female subgroups in effect size of ageing, the trends were in the same direction in all the parameters. Reduced fat intake was associated with a lower frequency of CD4+ T cells, and higher serum cotinine level was associated with a higher CD4/CD8 ratio. The results indicate that cellular immunity in the Indian population is affected by ageing, while humoral immunity is less susceptible to ageing.

IL-3 inhibits rat osteoclast differentiation induced by TNF-α and other pro-osteoclastogenic cytokines.

IL-3, a haematopoiesis regulatory factor, has previously been shown to inhibit both mouse and human osteoclast differentiation and bone resorption. Here, the role of rat IL-3 on rat osteoclast differentiation was evaluated to address whether the inhibitory action of IL-3 on osteoclastogenesis is conserved in various species. It was observed that IL-3 inhibited rat osteoclast differentiation induced by both TNF-α and receptor activator of NF-ĸB ligand (RANKL). TNF-α is known to induce bone loss in postmenopausal osteoporotic women and it also synergise with many pro-osteoclastogenic cytokines to cause huge pathological bone loss. Importantly, it was found that rat IL-3 inhibits the synergistic action of TNF-α with RANKL and IL-1ß, TGFß1 and TGF-ß3. IL-3 downregulates the TNF-α-induced nuclear translocation of NF-ĸB-p65 and c-fos without affecting c-jun. Interestingly, we observed that IL-3 also inhibits osteoclast differentiation in vivo in rats induced by TNF-α. All these results suggest that inhibitory action of IL-3 on osteoclastogenesis is conserved in various species including mice, rats and humans. Thus, our results clearly indicate that IL-3 has therapeutic potential to treat pathological bone loss in important skeletal diseases.

An optimized step-by-step protocol for isolation of nucleus pulposus, annulus fibrosus, and end plate cells from the mouse intervertebral discs and subsequent preparation of high-quality intact total RNA.

Intervertebral disc degeneration is the most significant, and least understood, cause of chronic back pain, affecting almost one in seven individuals at some point of time. Each intervertebral disc has three components; central nucleus pulposus (NP), concentric layers of annulus fibrosus (AF), and a pair of end plate (EP) that connects the disc to the vertebral bodies. Understanding the molecular and cellular basis of intervertebral disc growth, health, and aging will generate significant information for developing therapeutic approaches. Rapid and efficient preparations of homogeneous and pure cells are crucial for meaningful and rigorous downstream analysis at the cellular, molecular, and biochemical level. Cross-sample contamination may influence the interpretation of the results. In addition to altering gene expression, slow or delayed isolation procedures will also cause the degradation of cells and biomolecules that create a bias in the outcomes of the study. The mouse model system is extensively used to understand the intervertebral disc biology. Here we describe two protocols: (a) for efficient isolation of pure NP, AF, and EP cells from mouse lumbar intervertebral disc. We validated the purity of the NP and AF cells using Shh Cre/+ ; R26 mT/mG/+ dual-fluorescent reporter mice where all NP cells are GPF+ve, and by the sensitive approach of qPCR analysis using TaqMan probes for Shh, and Brachyury as NP-specific markers, Tenomodulin as AF-specific marker, and Osteocalcin as bone-specific marker. (b) For isolation of high-quality intact RNA with RIN of 9.3 to 10 from disc cells. These methods will be useful for the rigorous analysis of NP and AF cells, and improve our understanding of intervertebral disc biology.

IL-3 Differentially Regulates Membrane and Soluble RANKL in Osteoblasts through Metalloproteases and the JAK2/STAT5 Pathway and Improves the RANKL/OPG Ratio in Adult Mice.

Bone remodeling comprises balanced activities between osteoclasts and osteoblasts, which is regulated by various factors, including hormones and cytokines. We previously reported that IL-3 inhibits osteoclast differentiation and pathological bone loss. IL-3 also enhances osteoblast differentiation and bone formation from mesenchymal stem cells. However, the role of IL-3 in regulation of osteoblast-osteoclast interactions and underlying mechanisms is not yet delineated. In this study, we investigated the role of IL-3 on the regulation of osteoblast-specific molecules, receptor activator of NF-κB ligand (RANKL), and osteoprotegerin (OPG) that modulate bone homeostasis. We found that IL-3 increases RANKL expression at both the transcriptional and translational levels, and it showed no effect on OPG expression in calvarial osteoblasts. The increased RANKL expression by IL-3 induces mononuclear osteoclasts; however, it does not induce multinuclear osteoclasts. Interestingly, IL-3 decreases soluble RANKL by reducing ectodomain shedding of membrane RANKL through downregulation of metalloproteases mainly a disintegrin and metalloproteinase (ADAM)10, ADAM17, ADAM19, and MMP3. Moreover, IL-3 increases membrane RANKL by activating the JAK2/STAT5 pathway. Furthermore, IL-3 enhances RANKL expression in mesenchymal stem cells of wild-type mice but not in STAT5a knockout mice. Interestingly, IL-3 restores RANKL expression in adult mice by enhancing bone-specific RANKL and decreasing serum RANKL. Furthermore, IL-3 increases the serum OPG level in adult mice. Thus, our results reveal, to our knowledge for the first time, that IL-3 differentially regulates two functional forms of RANKL through metalloproteases and the JAK2/STAT5 pathway, and it helps in restoring the decreased RANKL/OPG ratio in adult mice. Notably, our studies indicate the novel role of IL-3 in regulating bone homeostasis in important skeletal disorders.

Adipose-Derived Mesenchymal Stem Cells Prevent Systemic Bone Loss in Collagen-Induced Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammatory synovitis leading to joint destruction and systemic bone loss. The inflammation-induced bone loss is mediated by increased osteoclast formation and function. Current antirheumatic therapies primarily target suppression of inflammatory cascade with limited or no success in controlling progression of bone destruction. Mesenchymal stem cells (MSCs) by virtue of their tissue repair and immunomodulatory properties have shown promising results in various autoimmune and degenerative diseases. However, the role of MSCs in prevention of bone destruction in RA is not yet understood. In this study, we investigated the effect of adipose-derived MSCs (ASCs) on in vitro formation of bone-resorbing osteoclasts and pathological bone loss in the mouse collagen-induced arthritis (CIA) model of RA. We observed that ASCs significantly inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis in both a contact-dependent and -independent manner. Additionally, ASCs inhibited RANKL-induced osteoclastogenesis in the presence of proinflammatory cytokines such as TNF-α, IL-17, and IL-1ß. Furthermore, treatment with ASCs at the onset of CIA significantly reduced clinical symptoms and joint pathology. Interestingly, ASCs protected periarticular and systemic bone loss in CIA mice by maintaining trabecular bone structure. We further observed that treatment with ASCs reduced osteoclast precursors in bone marrow, resulting in decreased osteoclastogenesis. Moreover, ASCs suppressed autoimmune T cell responses and increased the percentages of peripheral regulatory T and B cells. Thus, we provide strong evidence that ASCs ameliorate inflammation-induced systemic bone loss in CIA mice by reducing osteoclast precursors and promoting immune tolerance.

High fat diet promotes achievement of peak bone mass in young rats.

The relationship between obesity and bone is complex. Epidemiological studies demonstrate positive as well as negative correlation between obesity and bone health. In the present study, we investigated the impact of high fat diet-induced obesity on peak bone mass. After 9 months of feeding young rats with high fat diet, we observed obesity phenotype in rats with increased body weight, fat mass, serum triglycerides and cholesterol. There were significant increases in serum total alkaline phosphatase, bone mineral density and bone mineral content. By micro-computed tomography (µ-CT), we observed a trend of better trabecular bones with respect to their microarchitecture and geometry. This indicated that high fat diet helps in achieving peak bone mass and microstructure at younger age. We subsequently shifted rats from high fat diet to normal diet for 6 months and evaluated bone/obesity parameters. It was observed that after shifting rats from high fat diet to normal diet, fat mass, serum triglycerides and cholesterol were significantly decreased. Interestingly, the gain in bone mineral density, bone mineral content and trabecular bone parameters by HFD was retained even after body weight and obesity were normalized. These results suggest that fat rich diet during growth could accelerate achievement of peak bone mass that is sustainable even after withdrawal of high fat diet.