In contrast to chronic TLR activation and inflammation

In glycosylase to chronic TLR activation and inflammation, short time stimulation of the innate immune system may have completely different tasks with respect to tissue maturation and repair. It is well known that bone healing during fracture repair is induced by platelet derived growth factor stimulation after injury, followed by an inflammatory phase that precedes the osteogenic and the remodeling phases (Kolar et al., 2010; Ai-Aql et al., 2008). Osteogenic non-canonical wnt-signaling through WNT5A secretion is stimulated by proinflammatory stimuli (Rauner et al., 2012). We therefore hypothesized that both the endogenous production of SAA and exogenous SAA exposure during injury and infection may modulate the osteogenic differentiation process. During in vitro osteogenic differentiation using established osteogenic media SAA1 and 2 are both stimulated and this is associated with a very similar proinflammatory phenotype observed in our aging model. The peak of the expression is however early in the course of osteogenic differentiation and seems to cease at later time points. When we on top added rhSAA1 to these routine osteogenic differentiation experiments we observed a marked acceleration of the induction of osteogenic differentiation and the pro-inflammatory phenotype (Fig. 4). We also realized that WNT5A and ROR2, two key players in non-canonical wnt signaling and stimulation of osteogenic differentiation, are direct targets of TLR4 activation during osteogenic differentiation. Mechanistically this involved p38 and p65 phosphorylation and NFκB activation. We conclude from these data that in the early phase of bone regeneration and fracture healing the process of osteogenic differentiation is directly enhanced by TLR4 activation (confirmed by using the TLR4 inhibitor CLI-095) via autocrine and paracrine SAA1 and 2 production and their downstream targets, which again amplify the WNT5A induction demonstrated earlier (Rauner et al., 2012). Mineralization is a hallmark of osteogenic differentiation, which is orchestrated by a set of genes and their substrates/products such as alkaline phosphatase and other ectophosphatases, phospho 1, ENPP and the calcification inhibitors osteopontin, FGF23 and osteocalcin, which propagate or inhibit crystallization to guarantee a coordinated mineralization process (Harmey et al., 2004). In order to characterize early and late osteogenic readouts under the influence of SAA we analyzed in vitro ALP activity and mineralization and we were able to demonstrate that early ALP activity and early and late mineralization are markedly enhanced in the presence of rhSAA1. This identical process could also be induced in osseous and extraosseous pathological calcification processes where SAA expression has been described. Hence SAA could also be tightly involved in calcifying inflammatory reactions such as we can see in atherosclerosis or in sclerosing bone metastases as frequently seen in prostate and breast cancer bone metastases, where also WNT5A is an important marker (Thiele et al., 2011; Zhang et al., 2012; Hansen et al., 2015; Le et al., 2005).
Conclusion The following are the supplementary related to this article.
Acknowledgments This work was supported by the German Research Foundation (FOR793 JA506/9-1 and in part by FOR1586 SKELMET). We thank Martina Regensburger for technical assistance and the orthopedic surgeons who supplied us with human femoral head material.
Introduction Over the last two decades, cell-based therapy has been evaluated in cardiovascular, oncologic, autoimmune and neurodegenerative diseases. In the neurological field preclinical studies have demonstrated the potential of stem cell injection (Lindvall and Kokaia, 2006). On the other hand, their clinical application is still limited and controversial (Viswanathan and Keating, 2011). Thanks to their self-renewal and differentiation abilities, stem cells had been originally considered as a possible tool to replace damaged cells by an initial selective migration to the injured area and a subsequent differentiation into the affected neurons (Bjorklund and Lindvall, 2000). Unfortunately, this fascinating mechanism of repair rapidly faded away and the hope of an effective care for neurodegenerative disorders by topic transplantation of highly committed neural stem cells and/or embryonic totipotential cells was more and more neglected. On the contrary, there are growing evidences of an alternative endocrine-like mechanism of action of stem cells in different murine models of acute and chronic neurological disorders that does not involve any specific cell differentiation toward the neural lineage (Silani et al., 2010; Uccelli et al., 2011, 2012). This mechanism, commonly referred as bystander effect, is mainly based on the secretion of trophic factors, anti-inflammatory cytokines, immunomodulatory agents and soluble molecules even far from the diseased area by transplanted cells. This experimental evidence greatly enlarged the spectrum of potential cell candidates, in particular stromal cells derived from extraembryonic tissues, and paved the way to alternative systemic routes of administrations, in addition to the intraparenchyma implantation (Corti et al., 2004; Zhao et al., 2007; Knippenberg et al., 2012a; Mitrecic et al., 2010; Willing et al., 2008). The possibility to perform preclinical studies by systemically injecting cells in the bloodstream (IV) or, more locally intracerebroventricularly (ICV), has been adopted in many models of neurodegenerative disorders. All these premises prompted the scientific community to focus the attention on the effect of systemically injected fetal stem cells in different models of neurodegenerative disorders. In particular a large number of studies have been recently carried out in mouse models of Amyotrophic Lateral Sclerosis (ALS).