OPN-a induces muscle inflammation by increasing recruitment and activation of pro-inflammatory macrophages.

NEW FINDINGS: What is the central question of this study? What is the functional relevance of OPN isoform expression in muscle pathology? What is the main finding and its importance? The full-length human OPN-a isoform is the most pro-inflammatory isoform in the muscle microenvironment, acting on macrophages and myoblasts in an RGD-integrin-dependent manner. OPN-a upregulates expression of tenascin-C (TNC), a known Toll-like receptor 4 (TLR4) agonist. Blocking TLR4 signalling inhibits the pro-inflammatory effects of OPN-a, suggesting that a potential mechanism of OPN action is by promoting TNC-TLR4 signalling. Although osteopontin (OPN) is an important mediator of muscle remodelling in health and disease, functional differences in human spliced OPN variants in the muscle microenvironment have not been characterized. We thus sought to define the pro-inflammatory activities of human OPN isoforms (OPN-a, OPN-b and OPN-c) on cells present in regenerating muscle. OPN transcripts were quantified in normal and dystrophic human and dog muscle. Human macrophages and myoblasts were stimulated with recombinant human OPN protein isoforms, and cytokine mRNA and protein induction was assayed. OPN isoforms were greatly increased in dystrophic human (OPN-a > OPN-b > OPN-c) and dog muscle (OPN-a = OPN-c). In healthy human muscle, mechanical loading also upregulated OPN-a expression (eightfold; P < 0.01), but did not significantly upregulate OPN-c expression (twofold; P > 0.05). In vitro, OPN-a displayed the most pronounced pro-inflammatory activity among isoforms, acting on both macrophages and myoblasts. In vitro and in vivo data revealed that OPN-a upregulated tenascin-C (TNC), a known Toll-like receptor 4 (TLR4) agonist. Inhibition of TLR4 signalling attenuated OPN-mediated macrophage cytokine production. In summary, OPN-a is the most abundant and functionally active human spliced isoform in the skeletal muscle microenvironment. Here, OPN-a promotes pro-inflammatory signalling in both macrophages and myoblasts, possibly through induction of TNC-TLR4 signalling. Together, our findings suggest that specific targeting of OPN-a and/or TNC signalling in the damaged muscle microenvironment may be of therapeutic relevance.

IFN-γ Prevents Adenosine Receptor (A2bR) Upregulation To Sustain the Macrophage Activation Response.

The priming of macrophages with IFN-γ prior to TLR stimulation results in enhanced and prolonged inflammatory cytokine production. In this study, we demonstrate that, following TLR stimulation, macrophages upregulate the adenosine 2b receptor (A2bR) to enhance their sensitivity to immunosuppressive extracellular adenosine. This upregulation of A2bR leads to the induction of macrophages with an immunoregulatory phenotype and the downregulation of inflammation. IFN-γ priming of macrophages selectively prevents the induction of the A2bR in macrophages to mitigate sensitivity to adenosine and to prevent this regulatory transition. IFN-γ-mediated A2bR blockade leads to a prolonged production of TNF-α and IL-12 in response to TLR ligation. The pharmacologic inhibition or the genetic deletion of the A2bR results in a hyperinflammatory response to TLR ligation, similar to IFN-γ treatment of macrophages. Conversely, the overexpression of A2bR on macrophages blunts the IFN-γ effects and promotes the development of immunoregulatory macrophages. Thus, we propose a novel mechanism whereby IFN-γ contributes to host defense by desensitizing macrophages to the immunoregulatory effects of adenosine. This mechanism overcomes the transient nature of TLR activation, and prolongs the antimicrobial state of the classically activated macrophage. This study may offer promising new targets to improve the clinical outcome of inflammatory diseases in which macrophage activation is dysregulated.

Cardiac macrophages: how to mend a broken heart.

A study by Epelman et al. (2014) in this issue of Immunity demonstrates that diverse subpopulations of macrophages reside in the adult heart and can be maintained by multiple mechanisms involving both local proliferation and contributions from monocytes.

Extrinsic and intrinsic control of macrophage inflammatory responses.

Macrophages make major contributions to inflammatory immunopathology. In this work, we examine three disease scenarios, in which M1s play a major role early in the disease but eventually transitions into a population of cells with immunoregulatory activity. We propose that the transition from an inflammatory to a regulatory phenotype is a natural progression that regularly occurs in stimulated macrophages and that the timing of this transition is critical to maintaining homeostasis. In the first section of this review, we discuss the exogenous microenvironmental cues that may induce macrophages to enter a regulatory state. In the second half of this review, we discuss a novel mechanism, whereby TLR-stimulated macrophages can intrinsically induce their own regulatory activation state. They do so by secreting and synthesizing endogenous "reprogramming" signals that work in an autocrine fashion to promote a regulatory phenotype. We propose that these endogenous regulatory mechanisms exist to prevent macrophage-mediated immunopathology. Thus, macrophages can respond to endogenous and exogenous cues to regulate their activation state, and without these controlled regulatory responses, M1 would persist to the detriment of the host.

TLR stimulation initiates a CD39-based autoregulatory mechanism that limits macrophage inflammatory responses.

Sepsis is a highly fatal disease caused by an initial hyperinflammatory response followed by a state of profound immunosuppression. Although it is well appreciated that the initial production of proinflammatory cytokines by macrophages accompanies the onset of sepsis, it remains unclear what causes the transition to an immunosuppressive state. In this study, we reveal that macrophages themselves are key regulators of this transition and that the surface enzyme CD39 plays a critical role in self-limiting the activation process. We demonstrate that Toll-like receptor (TLR)-stimulated macrophages modulate their activation state by increasing the synthesis and secretion of adenosine triphosphate (ATP). This endogenous ATP is paradoxically immunosuppressive due to its rapid catabolism into adenosine by CD39. Macrophages lacking CD39 are unable to transition to a regulatory state and consequently continue to produce inflammatory cytokines. The importance of this transition is demonstrated in a mouse model of sepsis, where small numbers of CD39-deficient macrophages were sufficient to induce lethal endotoxic shock. Thus, these data implicate CD39 as a key "molecular switch" that allows macrophages to self-limit their activation state. We propose that therapeutics targeting the release and hydrolysis of ATP by macrophages may represent new ways to treat inflammatory diseases.