From Dead Cells to Healing Tissue
Removal of apoptotic and necrotic cells (efferocytosis) by macrophages is critical to inflammation resolution and is postulated to initiate tissue remodeling and regeneration. Limited understanding of the mechanisms regulating efferocytosis currently prohibits therapeutic targeting of this process. In this issue, Campana et al. (p. 1169) demonstrated that IL-6 plays a role in regulating the late cargo-digestion stage of efferocytosis. In vitro and in vivo analysis of glycoprotein nonmetastatic melanoma B–null (Gpnmb−) macrophages (which can engulf, but not process, apoptotic cargo) showed that recombinant murine IL-6 (rmIL-6) restored the phagocytic abilities of Gpnmb− macrophages in a phosphoSTAT3 (pSTAT3)-dependent manner. Blockade of pSTAT3 impaired phagocytosis and reduced production of IL-6 protein and mRNA 2 h after the start of phagocytosis in wild-type (Gpnmb+) macrophages in vitro, and decreased the infiltration of inflammatory macrophages in a model of sterile acute liver damage. Addition to macrophages of IL-10, which is induced by pSTAT3 and has been demonstrated to regulate phagocytosis, also rescued phagocytosis by Gpnmb− macrophages. In contrast to IL-6, addition of recombinant IL-10 to Gpnmb+ macrophages in which pSTAT3 was blocked dramatically decreased IL-10 production at 15 and 30 min and had no effect on IL-10 mRNA levels, indicating that pSTAT3 induces IL-10 secretion at the early stages of efferocytosis in a transcriptionally independent manner. Sterile liver injury was significantly increased in Gpnmb− mice when compared with Gpnmb+ controls. Furthermore, impairment of phagocytic cargo processing prevented, whereas administration of rmIL-6 improved a phenotypic switch from inflammatory (Ly6Chigh) to restorative (Ly6Clow) macrophages, the latter of which are necessary for tissue remodeling and regeneration. Thus, this study demonstrates that the STAT3–IL-6–IL-10 axis positively regulates macrophage efferocytosis and phenotypic conversion, providing a direct link between tissue repair and removal of cellular debris. This work defines a potential therapeutic target for clinical management of acute liver disease or chronic liver fibrosis.
NR4A1 Restricts Lineage Specification
Multipotent progenitors (MPP) have a limited capacity for self-renewal but can differentiate into all hematopoietic cells. Although MPP subsets are developmentally biased toward different lineages, their fate is not fixed and can change depending on the environmental signals, whose identity is unknown. The orphan nuclear receptor NR4A1 is a transcription factor that integrates external environmental signals and is expressed by myeloid-biased long-term hematopoietic stem cells. Mumau et al. (p. 1078) demonstrated that NR4A1 guides lineage specification of MPP of the spleen (MPPS), specifically regulating the potential of MPPS to differentiate into erythroid cells. The authors identified the novel MPPS population as Lineage−Sca1−cKit+CD41−CD16/32−CD71lowCD24high cells. Adoptive transfer of wild-type (WT) MPPS and GFP+ helper bone marrow cells into lethally irradiated UBC-GFP mice demonstrated that MPPS-derived cells contribute to neutrophil and monocyte populations in the spleen, but not in the bone marrow. Using NR4A1GFP reporter mice, the authors compared NR4A1 expression in splenic versus bone marrow stem cells and progenitors and found that splenic Lineage−Sca1+cKit+ cells express significantly higher levels of NR4A1 than their bone marrow counterparts. Moreover, NR4A1 expression in the spleen was highest within MPPS. NR4A1GFP+ and NR4A1GFP− MPPS from NR4A1GFP mice were cultured for 12 d; the colonies generated from NR4A1GFP+ MPPS were mostly monocytic and granulocytic and rarely erythroid or megakaryocytic, whereas NR4A1GFP− cells produced significantly fewer granulocytic/monocytic but more erythroid and megakaryocytic colonies. These data demonstrate that NR4A1 expression specifically identifies multipotent MPPS that are biased toward monocyte and granulocyte development. In vivo, Nr4a1−/− MPPS generated 5–10-fold more erythrocytes expressing the erythroid-specific Ag Ter119+ in the peripheral blood than did WT MPPS, demonstrating that, in the absence of NR4A1, MPPS are biased toward erythroid and megakaryocyte lineage commitment. Taken together, these data show that NR4A1 restricts the erythroid and megakaryocytic differentiation potential of progenitors in the spleen.
TRAILshort Confers Resistance to TRAIL Killing
Binding of TNF-related apoptosis-inducing ligand (TRAIL) to a family of cognate receptors (TRAIL-R1 to TRAIL-R4) induces apoptosis. TRAILshort is a novel splice variant of TRAIL that lacks apoptosis-inducing activity and antagonizes full-length TRAIL (TRAILFL). Given that HIV-infected cells expressing TRAIL-R are resistant to apoptosis, Nie et al. (p. 1110) examined the role of TRAILshort in human CD4+ T cell resistance to TRAIL-induced apoptosis. Resting CD4+ T cells produce TRAILshort in response to IFNα14 and IFNβ treatment. Moreover, treatment of uninfected donor PBMCs with TLR 7, 8, or 9 agonists induced TRAILshort mRNA expression by CD4+ T cells. Using three approaches, the authors confirmed that TRAILshort traffics to the plasma membrane and preferentially binds to TRAIL-R1 and -R2. Expression of hemagglutinin (HA)-tagged TRAILshort in 293T cells, followed by anti-HA pulldown and immunoblotting for ubiquitin, demonstrated that TRAILshort is ubiquinated and targeted for degradation, giving this splice variant a short half-life. TRAILshort not only localizes to the plasma membrane, but is also present in microvesicles, as demonstrated by isolation of extracellular vesicles from PHA-stimulated and unstimulated CD4+ T cells and immunoblotting for TRAILshort. SuperKiller TRAIL (sk-TRAIL) treatment in the presence of increasing doses of anti-TRAILshort induced a dose-dependent increase in Jurkat cell killing. Incubation of Jurkat cells with TRAILFL-expressing 293T cells induced apoptosis, which was decreased in the presence of 293T cells expressing TRAILshort. Thus, TRAILshort coexpression with TRAILFL antagonizes the proapoptotic effects of TRAILFL in a dose-dependent manner. TRAILshort-producing cells can transfer resistance to bystander cells, as demonstrated by the uptake of TRAILshort via microvesicles from Ruby-TRAILshort–transfected cells and subsequent protection from sk-TRAIL–induced killing. These data reveal that TRAILshort is sufficient to endow resistance to TRAIL-mediated killing and that its protective effects can be conferred on bystander cells via microvesicle transfer. Therefore, reversal of TRAIL resistance to apoptosis through TRAILshort-specific Ab treatment may have implications for the treatment of HIV.
Predicting Hepatitis C Viral Clearance
Although combination therapy with pegylated–IFN-α (PEG–IFN-α) and ribavirin (RBV) has been the standard treatment for chronic hepatitis C virus (HCV) infection for more than a decade, the contribution of the patient’s immune system to achieving sustained virologic response (SVR) is unclear. In this issue, Méndez-Lagares et al. (p. 1124) undertook a longitudinal, prospective study of 33 individuals with chronic HCV who were treated with PEG–IFN-α, RBV, and telaprevir/boceprevir (a direct-acting antiviral) and correlated SVR status with immune cell dynamics at baseline and after treatment. Consistent with previous observations, subjects achieving SVR showed minimal monocyte activation after the initiation of treatment, whereas non-SVR patients demonstrated significantly increased monocyte activation in the first wk of treatment. Baseline expression of the proliferation marker Ki-67 in both CD4+ and CD8+ memory T cells positively correlated to SVR. Increased expression levels of T-bet, a driver of Th1 differentiation and cytotoxic effector cell maturation, was also observed in central memory (CM) T cells of individuals achieving SVR. As the differences in Ki-67 and T-bet expression suggested possible variation in virus-specific effector T cell functions, the authors assessed the responses of CD4+ and CD8+ T cells to three HCV peptide pools and surprisingly found no disparity in the magnitude of Th1 responses in SVR versus non-SVR individuals 12 wk posttreatment, whereas SVR patients showed higher CM CD4+ and CD8+ T cell responses to all HCV Ags. Collectively, these data indicate that the status of the patient’s immune system prior to treatment may predict outcome; patients achieving SVR have a proliferating memory T cell compartment prior to treatment, ultimately manifesting as a higher frequency of circulating HCV-specific T cells by 12 wk posttreatment. Understanding the immune factors that facilitate viral clearance upon treatment may enable more efficient selection of viral therapies and inform efforts to develop HCV vaccine candidates.