Inhibition of T cell-dependent and RANKL-dependent osteoclastogenic processes associated with high levels of bone mass in interleukin-15 receptor-deficient mice.

OBJECTIVE: T cell production of RANKL, interferon-γ (IFNγ), and other cytokines in inflammatory processes such as rheumatoid arthritis or secondary to conditions such as estrogen deficiency stimulates osteoclast activity, which leads to bone resorption and bone loss. The purpose of this study was to characterize the effects of interleukin-15 (IL-15), a master T cell growth factor whose role in bone remodeling remains unknown. METHODS: We used mice lacking the IL-15 receptor (IL-15Rα(-/-) ) to investigate the effects of IL-15 on osteoclast development, T cell and dendritic cell activation in vitro and in vivo, bone mass, and microarchitecture in intact and ovariectomized (OVX) mice. RESULTS: In wild-type (WT) animals, IL-15 and RANKL provided a costimulatory signal for osteoclast development. Spleens from IL-15Rα(-/-) mice contained few c-Kit+ osteoclast precursors, and the expression of NF-ATc1 and the osteoclastogenic response to RANKL were impaired. In addition, dendritic cell-dependent and T cell-dependent mechanisms of osteoclast activation, including RANKL and IFNγ production, were impaired in IL-15Rα(-/-) mice. In turn, IL-15Rα(-/-) T cells failed to stimulate WT osteoclasts, whereas WT T cells failed to stimulate IL-15Rα(-/-) osteoclasts. Compared with WT mice, both intact and OVX IL-15Rα(-/-) mice had significantly greater bone mineral density and microarchitecture, including a higher trabecular bone volume fraction and cortical thickness. The numbers of osteoclasts on the bone surface as well as markers of bone turnover were significantly decreased in IL-15Rα(-/-) mice. CONCLUSION: In the absence of IL-15 signaling, several converging mechanisms of osteoclastogenesis are inhibited, both directly and indirectly, through T cells, which leads to a high bone mass phenotype. Targeting the IL-15 pathway may represent a novel therapeutic approach to treating primary and secondary osteoporosis.

Metataxonomic and Metabolic Impact of Fecal Microbiota Transplantation From Patients With Pancreatic Cancer Into Germ-Free Mice: A Pilot Study.

Background: Body weight (BW) loss is prevalent in patients with pancreatic cancer (PC). Gut microbiota affects BW and is known to directly shape the host immune responses and antitumor immunity. This pilot study evaluated the link between gut microbiota, metabolic parameters and inflammatory/immune parameters, through the fecal material transplantation (FMT) of PC patients and healthy volunteers into germ-free (GF) mice. Methods: We transplanted the feces from five PC patients and five age- and gender-matched healthy volunteers into two GF mice each. Mouse BW and energy intake were measured every 1-5 days, oral glucose on day 21, insulin tolerance on day 26, fecal bacterial taxonomic profile by 16S rRNA gene sequencing on day 5, 10, 15 and 30, and gut-associated lymphoid tissue T cells, plasma cytokines and weights of fat and muscle mass at sacrifice (day 34). Results are presented as mean ± SD. The continuous parameters of mice groups were compared by linear univariate regressions, and their bacterial communities by Principal Coordinates Analysis (PCoA), Bray-Curtis similarity and ANCOM test. Results: Recipients of feces from PC patients and healthy volunteers had similar BW gain and food intake. Visceral fat was lower in recipients of feces from PC patients than from healthy individuals (0.72 ± 0.17 vs. 0.92 ± 0.14 g; coeff -0.19, 95% CI -0.38, -0.02, p=0.035). The other non-metataxonomic parameters did not differ between groups. In PCoA, microbiota from PC patients clustered apart from those of healthy volunteers and the same pattern was observed in transplanted mice. The proportions of Clostridium bolteae, Clostridium scindens, Clostridium_g24_unclassified and Phascolarctobacterium faecium were higher, while those of Alistipes obesi, Lachnospiraceae PAC000196_s and Coriobacteriaceae_unclassified species were lower in PC patients and in mice transplanted with the feces from these patients. Conclusion: In this pilot study, FMT from PC patients was associated with a decrease in visceral fat as compared to FMT from healthy individuals. Some of the differences in fecal microbiota between PC and control samples are common to humans and mice. Further research is required to confirm that feces contain elements involved in metabolic and immune alterations.

In vivo investigations on anti-fibrotic potential of proteasome inhibition in lung and skin fibrosis.

In systemic sclerosis (SSc), a disease characterized by fibrosis of the skin and internal organs, the occurrence of interstitial lung disease is responsible for high morbidity and mortality. We previously demonstrated that proteasome inhibitors (PI) show anti-fibrotic properties in vitro by reducing collagen production and favoring collagen degradation in a c-jun N-terminal kinase (JNK)-dependent manner in human fibroblasts. Therefore, we tested whether PI could control fibrosis development in bleomycin-induced lung injury, which is preceded by massive inflammation. We extended the study to test PI in TSK-1/+ mice, where skin fibrosis develops in the absence of overt inflammation. C57Bl/6 mice received bleomycin intratracheally and were treated or not with PI. Lung inflammation and fibrosis were assessed by histology and quantification of hydroxyproline content, type I collagen mRNA, and TGF-beta at Days 7, 15, and 21, respectively. Histology was used to detect skin fibrosis in TSK-1/+mice. The chymotryptic activity of 20S proteasome was assessed in mice blood. JNK and Smad2 phosphorylation were evaluated by Western blot on lung protein extracts. PI reduced collagen mRNA levels in murine lung fibroblasts, without affecting their viability in vitro. In addition, PI inhibited the chymotryptic activity of proteasome and enhanced JNK and TGF-beta signaling in vivo. PI failed to prevent bleomycin-induced lung inflammation and fibrosis and to attenuate skin fibrosis in TSK-1/+mice. In conclusion, our results provide direct evidence that, despite promising in vitro results, proteasome blockade may not be a strategy easily applicable to control fibrosis development in diseases such as lung fibrosis and scleroderma.

Enoxaparin Attenuates Mouse Colon Cancer Liver Metastases by Inhibiting Heparanase and Interferon-γ-inducible Chemokines.

BACKGROUND/AIM: Low-molecular-weight heparin (LMWH) has been suggested to reduce the risk of cancer progression in both preclinical and clinical studies but the underlying mechanisms remain poorly explored. The aim of the study was to investigate the anti-metastatic role of enoxaparin, a clinically-used LMWH, in a murine model of colon cancer and to explore its underlying mechanisms. MATERIALS AND METHODS: Using a reproducible mouse model of colon carcinomas, we assessed the capacity of enoxaparin, a LMWH, to affect tumor metastasis of colon carcinoma cell lines in mice. RESULTS: The hepatic growth of colon carcinoma metastases was strongly inhibited by enoxaparin compared to (Ctrl) group (p=0.001). This effect was associated to an inhibition of heparanase mRNA expression and protein production both in vivo and in vitro. In addition, enoxaparin inhibited the liver and serum production of interferon gamma (Ifnγ)-inducible chemokine receptor ligands. Overexpression of heparanase prompted proliferation, migration and growth of colon carcinoma in vitro and in vivo to a point that was not affected by enoxaparin in vivo anymore. CONCLUSION: Enoxaparin decreased liver metastases in a mouse model of colon carcinoma. These results suggest that enoxaparin may benefit patients with cancer and deserves further laboratory and clinical investigations.