Following neuronal injury, microglia initiate repair by phagocytosing dead neurons without eliciting inflammation. Prior evidence indicates triggering receptor expressed by myeloid cells-2 (TREM2) ...promotes phagocytosis and retards inflammation. However, evidence that microglia and neurons directly interact through TREM2 to orchestrate microglial function is lacking. We here demonstrate that TREM2 interacts with endogenous ligands on neurons. Staining with TREM2-Fc identified TREM2 ligands (TREM2-L) on Neuro2A cells and on cultured cortical and dopamine neurons. Apoptosis greatly increased the expression of TREM2-L. Furthermore, apoptotic neurons stimulated TREM2 signaling, and an anti-TREM2 mAb blocked stimulation. To examine the interaction between TREM2 and TREM2-L in phagocytosis, we studied BV2 microglial cells and their engulfment of apoptotic Neuro2A. One of our anti-TREM2 mAb, but not others, reduced engulfment, suggesting the presence of a functional site on TREM2 interacting with neurons. Further, Chinese hamster ovary cells transfected with TREM2 conferred phagocytic activity of neuronal cells demonstrating that TREM2 is both required and sufficient for competent uptake of apoptotic neuronal cells. Finally, while TREM2-L are expressed on neurons, TREM2 is not; in the brain, it is found on microglia. TREM2 and TREM2-L form a receptor-ligand pair connecting microglia with apoptotic neurons, directing removal of damaged cells to allow repair.
Traumatic brain injury (TBI) provokes inflammatory responses, including a dramatic rise in brain macrophages in the area of injury. The pathway(s) responsible for macrophage infiltration of the ...traumatically injured brain and the effects of macrophages on functional outcomes are not well understood. C-C-chemokine receptor 2 (CCR2) is known for directing monocytes to inflamed tissues. To assess the role of macrophages and CCR2 in TBI, we determined outcomes in CCR2-deficient (Ccr2(-/-)) mice in a controlled cortical impact model. We quantified brain myeloid cell numbers post-TBI by flow cytometry and found that Ccr2(-/-) mice had greatly reduced macrophage numbers (∼80-90% reduction) early post-TBI, compared with wild-type mice. Motor, locomotor, and cognitive outcomes were assessed. Lack of Ccr2 improved locomotor activity with less hyperactivity in open field testing, but did not affect anxiety levels or motor coordination on the rotarod three weeks after TBI. Importantly, Ccr2(-/-) mice demonstrated greater spatial learning and memory, compared with wild-type mice eight weeks after TBI. Although there was no difference in the volume of tissue loss, Ccr2(-/-) mice had significantly increased neuronal density in the CA1-CA3 regions of the hippocampus after TBI, compared with wild-type mice. These data demonstrate that Ccr2 directs the majority of macrophage homing to the brain early after TBI and indicates that Ccr2 may facilitate harmful responses. Lack of Ccr2 improves functional recovery and neuronal survival. These results suggest that therapeutic blockade of CCR2-dependent responses may improve outcomes following TBI.
The elderly population suffers from higher rates of complications during fracture healing that result in increased morbidity and mortality. Inflammatory dysregulation is associated with increased age ...and is a contributing factor to the myriad of age‐related diseases. Therefore, we investigated age‐related changes to an important cellular regulator of inflammation, the macrophage, and the impact on fracture healing outcomes. We demonstrated that old mice (24 months) have delayed fracture healing with significantly less bone and more cartilage compared to young mice (3 months). The quantity of infiltrating macrophages into the fracture callus was similar in old and young mice. However, RNA‐seq analysis demonstrated distinct differences in the transcriptomes of macrophages derived from the fracture callus of old and young mice, with an up‐regulation of M1/pro‐inflammatory genes in macrophages from old mice as well as dysregulation of other immune‐related genes. Preventing infiltration of the fracture site by macrophages in old mice improved healing outcomes, with significantly more bone in the calluses of treated mice compared to age‐matched controls. After preventing infiltration by macrophages, the macrophages remaining within the fracture callus were collected and examined via RNA‐seq analysis, and their transcriptome resembled macrophages from young calluses. Taken together, infiltrating macrophages from old mice demonstrate detrimental age‐related changes, and depleting infiltrating macrophages can improve fracture healing in old mice.
Delayed fracture healing in old mice is associated with transcriptomic differences between old and young macrophages infiltrating the fracture callus. An aged macrophage phenotype was characterized by increased pro‐inflammatory gene expression compared to young. Inhibition of macrophage infiltration in old mice improved fracture healing outcomes and demonstrated a resident macrophages population with a transcriptome more similar to young mice.
Second-generation direct-acting antivirals (2G DAA) to cure HCV have led to dramatic clinical improvements. HCV-associated hepatocellular carcinoma (HCC), however, remains common. Impaired immune ...tumor surveillance may play a role in HCC development. Our cohort evaluated the effects of innate immune types and clinical variables on outcomes including HCC.
Participants underwent full HLA class I/KIR typing and long-term HCV follow-up.
A total of 353 HCV+ participants were followed for a mean of 7 years. Cirrhosis: 25% at baseline, developed in 12% during follow-up. 158 participants received 2G DAA therapy. HCC developed without HCV therapy in 20 subjects, 24 HCC after HCV therapy, and 10 of these after 2G DAA. Two predictors of HCC among 2G DAA-treated patients: cirrhosis (OR, 10.0, p = 0.002) and HLA/KIR profiles predicting weak natural killer (NK) cell-mediated immunity (NK cell complementation groups 6, 9, 11, 12, OR of 5.1, p = 0.02). Without 2G DAA therapy: cirrhosis was the main clinical predictor of HCC (OR, 30.8, p < 0.0001), and weak NK-cell-mediated immunity did not predict HCC.
Cirrhosis is the main risk state predisposing to HCC, but weak NK-cell-mediated immunity may predispose to post-2G DAA HCC more than intermediate or strong NK-cell-mediated immunity.
Increased osteoclastic bone resorption leads to periarticular erosions and systemic osteoporosis in RA patients. Although a great deal is known about how osteoclasts differentiate from precursors and ...resorb bone, the identity of an osteoclast precursor (OCP) population in vivo and its regulatory role in RA remains elusive. Here, we report the identification of a CD11b(-/lo)Ly6C(hi) BM population with OCP activity in vitro and in vivo. These cells, which can be distinguished from previously characterized precursors in the myeloid lineage, display features of both M1 and M2 monocytes and expand in inflammatory arthritis models. Surprisingly, in one mouse model of RA (adoptive transfer of SKG arthritis), cotransfer of OCP with SKG CD4+ T cells diminished inflammatory arthritis. Similar to monocytic myeloid-derived suppressor cells (M-MDSCs), OCPs suppressed CD4+ and CD8+ T cell proliferation in vitro through the production of NO. This study identifies a BM myeloid precursor population with osteoclastic and T cell-suppressive activity that is expanded in inflammatory arthritis. Therapeutic strategies that prevent the development of OCPs into mature bone-resorbing cells could simultaneously prevent bone resorption and generate an antiinflammatory milieu in the RA joint.
In traumatic brain injury (TBI), a diversity of brain resident and peripherally derived myeloid cells have the potential to worsen damage and/or to assist in healing. We define the heterogeneity of ...microglia and macrophage phenotypes during TBI in wild-type (WT) mice and Ccr2−/− mice, which lack macrophage influx following TBI and are resistant to brain damage. We use unbiased single-cell RNA sequencing methods to uncover 25 microglia, monocyte/macrophage, and dendritic cell subsets in acute TBI and normal brains. We find alterations in transcriptional profiles of microglia subsets in Ccr2−/− TBI mice compared to WT TBI mice indicating that infiltrating monocytes/macrophages influence microglia activation to promote a type I IFN response. Preclinical pharmacological blockade of hCCR2 after injury reduces expression of IFN-responsive gene, Irf7, and improves outcomes. These data extend our understanding of myeloid cell diversity and crosstalk in brain trauma and identify therapeutic targets in myeloid subsets.
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•TBI elevates distinct phenotypes of microglia, macrophages, and dendritic cells•Ccr2 deficiency alters cell proportions and reduces ISG expression in microglia•TBI induces crosstalk between microglia and circulating monocytes•Preclinical translational studies to target human CCR2 after TBI improves outcomes
By single-cell RNA sequencing of traumatically injured and normal brains from wild-type and Ccr2−/− mice, Somebang et al. define microglia, macrophage, and dendritic cell phenotypes in TBI. Targeting mouse and/or human CCR2 reduces specific TBI brain CNS myeloid compartments, dampens type I interferon responses, and improves cognition after TBI.
Traumatic brain injury (TBI) elicits innate inflammatory responses that can lead to secondary brain injury. To better understand the mechanisms involved in TBI‐induced inflammation, we examined the ...nature of macrophages responding to TBI in mice. In this model, brain macrophages were increased >20‐fold the day after injury and >77‐fold 4 days after injury in the ipsilateral hemisphere compared with sham controls. TBI macrophage subsets were identified by using a reporter mouse strain (YARG) that expresses eYFP from an internal ribosome entry site (IRES) inserted at the 3′ end of the gene for arginase‐1 (Arg1), a hallmark of alternatively activated (M2) macrophages. One day after TBI, 21 ± 1.5% of ipsilateral brain macrophages expressed relatively high levels of Arg1 as detected by yellow fluorescent protein, and this subpopulation declined thereafter. Arg1+ cells localized with macrophages near the TBI lesion. Gene expression analysis of sorted Arg1+ and Arg1− brain macrophages revealed that both populations had profiles that included features of conventional M2 macrophages and classically activated (M1) macrophages. The Arg1+ cells differed from Arg1− cells in multiple aspects, most notably in their chemokine repertoires. Thus, the macrophage response to TBI initially involves heterogeneous polarization toward at least two major subsets.
Lung myeloid cells are important in pulmonary immune homeostasis and in the pathogenesis of chronic obstructive pulmonary disease (COPD). Multiparameter immunophenotypic characterization of these ...cells is challenging because of their autofluorescence and diversity. We evaluated the immunophenotypic landscape of airway myeloid cells in COPD using time of flight mass cytometry. Cells from BAL, which were obtained from never-smokers (
= 8) and smokers with (
= 20) and without (
= 4) spirometric COPD, were examined using a 44-parameter time of flight mass cytometry panel. Unsupervised cluster analysis was used to identify cellular subtypes that were confirmed by manual gating. We identified major populations of CD68
and CD68
cells with 22 distinct phenotypic clusters, of which 18 were myeloid cells. We found a higher abundance of putative recruited myeloid cells (CD68
classical monocytes) in BAL from patients with COPD. CD68
classical monocyte population had distinct responses to smoking and COPD that were potentially related to their recruitment from the interstitium and vasculature. We demonstrate that BAL cells from smokers and subjects with COPD have lower AXL expression. Also, among subjects with COPD, we report significant differences in the abundance of PDL1
and PDL2
clusters and in the expression of PDL1 and PDL2 across several macrophage subtypes suggesting modulation of inflammatory responses. In addition, several phenotypic differences in BAL cells from subjects with history of COPD exacerbation were identified that could inform potential disease mechanisms. Overall, we report several changes to the immunophenotypic landscape that occur with smoking, COPD, and past exacerbations that are consistent with decreased regulation and increased activation of inflammatory pathways.
Ferritin is a spherical molecule composed of 24 subunits of two types, ferritin H chain (FHC) and ferritin L chain (FLC). Ferritin stores iron within cells, but it also circulates and binds ...specifically and saturably to a variety of cell types. For most cell types, this binding can be mediated by ferritin composed only of FHC (HFt) but not by ferritin composed only of FLC (LFt), indicating that binding of ferritin to cells is mediated by FHC but not FLC. By using expression cloning, we identified human transferrin receptor-1 (TfR1) as an important receptor for HFt with little or no binding to LFt. In vitro, HFt can be precipitated by soluble TfR1, showing that this interaction is not dependent on other proteins. Binding of HFt to TfR1 is partially inhibited by diferric transferrin, but it is hindered little, if at all, by HFE. After binding of HFt to TfR1 on the cell surface, HFt enters both endosomes and lysosomes. TfR1 accounts for most, if not all, of the binding of HFt to mitogen-activated T and B cells, circulating reticulocytes, and all cell lines that we have studied. The demonstration that TfR1 can bind HFt as well as Tf raises the possibility that this dual receptor function may coordinate the processing and use of iron by these iron-binding molecules.
T cell immunoglobulin-domain and mucin-domain (TIM) proteins constitute a receptor family that was identified first on kidney and liver cells; recently it was also shown to be expressed on T cells. ...TIM-1 and -3 receptors denote different subsets of T cells and have distinct regulatory effects on T cell function. Ferritin is a spherical protein complex that is formed by 24 subunits of H- and L-ferritin. Ferritin stores iron atoms intracellularly, but it also circulates. H-ferritin, but not L-ferritin, shows saturable binding to subsets of human T and B cells, and its expression is increased in response to inflammation. We demonstrate that mouse TIM-2 is expressed on all splenic B cells, with increased levels on germinal center B cells. TIM-2 also is expressed in the liver, especially in bile duct epithelial cells, and in renal tubule cells. We further demonstrate that TIM-2 is a receptor for H-ferritin, but not for L-ferritin, and expression of TIM-2 permits the cellular uptake of H-ferritin into endosomes. This is the first identification of a receptor for ferritin and reveals a new role for TIM-2.
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