Nicole J. Horwood

Alarmins: awaiting a clinical response.

Alarmins are endogenous molecules that are constitutively available and released upon tissue damage and activate the immune system. Current evidence indicates that uncontrolled and excessive release of alarmins contributes to the dysregulated processes seen in many inflammatory and autoimmune conditions, as well as tumorigenesis and cancer spread. Conversely, alarmins have also been found to play a major role in the orchestration of tissue homeostasis, including repair and remodeling in the heart, skin, and nervous system. Here, we provide an update and overview on alarmins, highlighting the areas that may benefit from this clinical translation.

The Antiproliferative Effect of Mesenchymal Stem Cells Is a Fundamental Property Shared by All Stromal Cells

Although it has been widely demonstrated that human mesenchymal stem cells exert potent immunosuppressive effects, there is little information as to whether more mature mesenchymal stromal cells (SC) share the same property. Accordingly, we set out to test the ability of SC from different human tissues to inhibit the proliferation of PBMC following polyclonal stimuli. Chondrocytes, as well as fibroblasts from synovial joints, lung, and skin, were used as a source of SC. Irrespective of their differentiation potential and/or content of progenitor cells, SC from all tissues exhibited antiproliferative functions. This was in marked contrast to parenchymal cells. Although SC did not interfere with early T lymphocyte activation, they arrested stimulated T cells in the G0/G1 phase of the cell cycle and rescued them from apoptosis. In addition, IFN-γ and TNF-α production were reduced. We observed that the inhibitory effect is ultimately mediated by soluble factors, the production of which requires SC to be licensed in an inflammatory environment by cell contact. We conclude that the immunosuppressive effect of mesenchymal cells is not confined to multipotent stem cells, but is a fundamental characteristic of all stroma. Our data suggest that SC, appropriately licensed, regulate T cell homeostasis.

Interleukin 18 inhibits osteoclast formation via T cell production of granulocyte macrophage colony-stimulating factor.

IL-18 inhibits osteoclast (OCL) formation in vitro independent of IFN-gamma production, and this was abolished by the addition of neutralizing antibodies to GM-CSF. We now establish that IL-18 was unable to inhibit OCL formation in cocultures using GM-CSF-deficient mice (GM-CSF -/-). Reciprocal cocultures using either wild-type osteoblasts with GM-CSF -/- spleen cells or GM-CSF -/- osteoblasts with wild-type spleen cells were examined. Wild-type spleen cells were required to elicit a response to IL-18 indicating that cells of splenic origin were the IL-18 target. As T cells comprise a large proportion of the spleen cell population, the role of T cells in osteoclastogenesis was examined. Total T cells were removed and repleted in various combinations. Addition of wild-type T cells to a GM-CSF -/- coculture restored IL-18 inhibition of osteoclastogenesis. Major subsets of T cells, CD4+ and CD8+, were also individually depleted. Addition of either CD4+ or CD8+ wild-type T cells restored IL-18 action in a GM-CSF -/- background, while IL-18 was ineffective when either CD4+ or CD8+ GM-CSF -/- T cells were added to a wild-type coculture. These results highlight the involvement of T cells in IL-18-induced OCL inhibition and provide evidence for a new OCL inhibitory pathway whereby IL-18 inhibits OCL formation due to action upon T cells promoting the release of GM-CSF, which in turn acts upon OCL precursors.

TNF-alpha promotes fracture repair by augmenting the recruitment and differentiation of muscle-derived stromal cells.

With an aging population, skeletal fractures are increasing in incidence, including the typical closed and the less common open fractures in normal bone, as well as fragility fractures in patients with osteoporosis. For the older age group, there is an urgent unmet need to induce predictable bone formation as well as improve implant fixation in situations such as hip joint replacement. Using a murine model of slow-healing fractures, we have previously shown that coverage of the fracture with muscle accelerated fracture healing and increased union strength. Here, we show that cells from muscle harvested after 3 d of exposure to an adjacent fracture differentiate into osteoblasts and form bone nodules in vitro. The osteogenic potential of these cells exceeds that of adipose and skin-derived stromal cells and is equivalent to bone marrow stromal cells. Supernatants from human fractured tibial bone fragments promote osteogenesis and migration of muscle-derived stromal cells (MDSC) in vitro. The main factor responsible for this is TNF-α, which promotes first MDSC migration, then osteogenic differentiation at low concentrations. However, TNF-α is inhibitory at high concentrations. In our murine model, addition of TNF-α at 1 ng/mL at the fracture site accelerated healing. These data indicate that manipulating the local inflammatory environment to recruit, then differentiate adjacent MDSC, may be a simple yet effective way to enhance bone formation and accelerate fracture repair. Our findings are based on a combination of human specimens and an in vivo murine model and may, therefore, translate to clinical care.

Bruton's tyrosine kinase is required for lipopolysaccharide-induced tumor necrosis factor alpha production.

Lipopolysaccharide (LPS), a product of Gram-negative bacteria, is potent mediator of tumor necrosis factor (TNF)α production by myeloid/macrophage cells. Inhibitors capable of blocking the signaling events that result in TNFα production could provide useful therapeutics for treating septic shock and other inflammatory diseases. Broad spectrum tyrosine inhibitors are known to inhibit TNFα production, however, no particular family of tyrosine kinases has been shown to be essential for this process. Here we show that the Brutons tyrosine kinase (Btk)-deficient mononuclear cells from X-linked agammaglobulinemia patients have impaired LPS-induced TNFα production and that LPS rapidly induces Btk kinase activity in normal monocytes. In addition, adenoviral overexpression of Btk in normal human monocytes enhanced TNFα production. We examined the role of Btk in TNFα production using luciferase reporter adenoviral constructs and have established that overexpression of Btk results in the stabilization of TNFα mRNA via the 3′ untranslated region. Stimulation with LPS also induced the activation of related tyrosine kinase, Tec, suggesting that the Tec family kinases are important components for LPS-induced TNFα production. This study provides the first clear evidence that tyrosine kinases of the Tec family, in particular Btk, are key elements of LPS-induced TNFα production and consequently may provide valuable therapeutic targets for intervention in inflammatory conditions.

Bruton’s Tyrosine Kinase Is Required for TLR2 and TLR4-Induced TNF, but Not IL-6, Production

Bruton’s tyrosine kinase (Btk), the gene mutated in the human immunodeficiency X-linked agammaglobulinemia, is activated by LPS and is required for LPS-induced TNF production. In this study, we have investigated the role of Btk both in signaling via another TLR (TLR2) and in the production of other proinflammatory cytokines such as IL-1β, IL-6, and IL-8. Our data show that in X-linked agammaglobulinemia PBMCs, stimulation with TLR4 (LPS) or TLR2 (N-palmitoyl-S-[2, 3-bis(palmitoyloxy)-(2R)-propyl]-(R)-cysteine) ligands produces significantly less TNF and IL-1β than in normal controls. In contrast, a lack of Btk has no impact on the production of IL-6, IL-8, or the anti-inflammatory cytokine, IL-10. Our previous data suggested that Btk lies within a p38-dependent pathway that stabilizes TNF mRNA. Accordingly, TaqMan quantitative PCR analysis of actinomycin D time courses presented in this work shows that overexpression of Btk is able to stabilize TNF, but not IL-6 mRNA. Furthermore, using the p38 inhibitor SB203580, we show that the TLR4-induced production of TNF, but not IL-6, requires the activity of p38 MAPK. These data provide evidence for a common requirement for Btk in TLR2- and TLR4-mediated induction of two important proinflammatory cytokines, TNF and IL-1β, and reveal important differences in the TLR-mediated signals required for the production of IL-6, IL-8, and IL-10.

A novel mechanism for TNF-alpha regulation by p38 MAPK: involvement of NF-kappa B with implications for therapy in rheumatoid arthritis.

TNF-α is a key factor in a variety of inflammatory diseases. This study examines the role of p38 MAPK in the regulation of TNF-α in primary human cells relevant to inflammation, e.g., macrophages and rheumatoid synovial cells. Using a dominant negative variant (D168A) of p38 MAPK and a kinase inhibitor, SB203580, we confirm in primary human macrophages that p38 MAPK regulates TNF-α production using a posttranscriptional mechanism requiring the 3′ untranslated region of the gene. However, in LPS-activated primary human macrophages we also detect a second previously unidentified mechanism, the p38 MAPK modulation of TNF-α transcription. This is mediated through p38 MAPK regulation of NF-κB. Interestingly this mechanism was not observed in rheumatoid synovial cells. Importantly however, the dominant negative mutant of p38 MAPK, but not SB203580 was effective at inhibiting spontaneous TNF-α production in these ex vivo rheumatoid synovial cell cultures. These data indicate there are potential major differences in the role of p38 MAPK in inflammatory signaling that have a bearing on the use of this kinase as a target for therapy. These results indicate despite disappointing results with p38 MAPK inhibitors in the clinic, this kinase is a valid target in rheumatoid disease.

Monocytes Induce STAT3 Activation in Human Mesenchymal Stem Cells to Promote Osteoblast Formation

A major therapeutic challenge is how to replace bone once it is lost. Bone loss is a characteristic of chronic inflammatory and degenerative diseases such as rheumatoid arthritis and osteoporosis. Cells and cytokines of the immune system are known to regulate bone turnover by controlling the differentiation and activity of osteoclasts, the bone resorbing cells. However, less is known about the regulation of osteoblasts (OB), the bone forming cells. This study aimed to investigate whether immune cells also regulate OB differentiation. Using in vitro cell cultures of human bone marrow-derived mesenchymal stem cells (MSC), it was shown that monocytes/macrophages potently induced MSC differentiation into OBs. This was evident by increased alkaline phosphatase (ALP) after 7 days and the formation of mineralised bone nodules at 21 days. This monocyte-induced osteogenic effect was mediated by cell contact with MSCs leading to the production of soluble factor(s) by the monocytes. As a consequence of these interactions we observed a rapid activation of STAT3 in the MSCs. Gene profiling of STAT3 constitutively active (STAT3C) infected MSCs using Illumina whole human genome arrays showed that Runx2 and ALP were up-regulated whilst DKK1 was down-regulated in response to STAT3 signalling. STAT3C also led to the up-regulation of the oncostatin M (OSM) and LIF receptors. In the co-cultures, OSM that was produced by monocytes activated STAT3 in MSCs, and neutralising antibodies to OSM reduced ALP by 50%. These data indicate that OSM, in conjunction with other mediators, can drive MSC differentiation into OB. This study establishes a role for monocyte/macrophages as critical regulators of osteogenic differentiation via OSM production and the induction of STAT3 signalling in MSCs. Inducing the local activation of STAT3 in bone cells may be a valuable tool to increase bone formation in osteoporosis and arthritis, and in localised bone remodelling during fracture repair.

Inhibition of osteoclast function reduces hematopoietic stem cell numbers in vivo.

Osteoblasts play a crucial role in the hematopoietic stem cell (HSC) niche; however, an overall increase in their number does not necessarily promote hematopoiesis. Because the activity of osteoblasts and osteoclasts is coordinately regulated, we hypothesized that active bone-resorbing osteoclasts would participate in HSC niche maintenance. Mice treated with bisphosphonates exhibited a decrease in proportion and absolute number of Lin(-)cKit(+)Sca1(+) Flk2(-) (LKS Flk2(-)) and long-term culture-initiating cells in bone marrow (BM). In competitive transplantation assays, the engraftment of treated BM cells was inferior to that of controls, confirming a decrease in HSC numbers. Accordingly, bisphosphonates abolished the HSC increment produced by parathyroid hormone. In contrast, the number of colony-forming-unit cells in BM was increased. Because a larger fraction of LKS in the BM of treated mice was found in the S/M phase of the cell cycle, osteoclast impairment makes a proportion of HSCs enter the cell cycle and differentiate. To prove that HSC impairment was a consequence of niche manipulation, a group of mice was treated with bisphosphonates and then subjected to BM transplantation from untreated donors. Treated recipient mice experienced a delayed hematopoietic recovery compared with untreated controls. Our findings demonstrate that osteoclast function is fundamental in the HSC niche.

Potential of mesenchymal stem cell therapy

Purpose of review Mesenchymal stem cells have the capacity to differentiate into several mesenchymal tissues, including the components of the hematopoietic stem cell niche. Mesenchymal stem cells also exhibit a powerful immunosuppressive activity. Here we review the most recent data to identify the properties of therapeutic significance. Recent findings Mesenchymal stem cells are attractive not only in regenerative medicine but also for the treatment of autoimmune diseases and graft-versus-host disease. Initial experience in animal models and the clinical setting have produced very encouraging results whereby mesenchymal stem cells have been shown to accelerate recovery after myocardial infarction, improve growth velocity in children with osteogenesis imperfecta, and ameliorate severe graft-versus-host disease as well as, in mouse models, rheumatoid arthritis and multiple sclerosis. Their use in the clinical setting, however, must be considered with caution because there is evidence that mesenchymal stem cells may also contribute to the maintenance of cancer stem cells. Summary The interest generated by mesenchymal stem cells has rapidly favored several initiatives to test their therapeutic potentials. There is still much to investigate to characterize their phenotype, understand their mechanisms of action, and optimize their in-vitro expansion for clinical use.