Hepatocellular carcinoma after direct-acting antivirals for hepatitis C is associated with KIR-HLA types predicting weak NK cell-mediated immunity.

BACKGROUND AND AIMS: 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. METHODS: Participants underwent full HLA class I/KIR typing and long-term HCV follow-up. RESULTS: 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. CONCLUSION: 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.

Lower PDL1, PDL2, and AXL Expression on Lung Myeloid Cells Suggests Inflammatory Bias in Smoking and Chronic Obstructive Pulmonary Disease.

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 (n = 8) and smokers with (n = 20) and without (n = 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 PDL1high and PDL2high 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.

Traumatic brain injury induces macrophage subsets in the brain.

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.

Single-cell RNA-seq analysis reveals cell subsets and gene signatures associated with Rheumatoid Arthritis Disease Activity.

Rheumatoid arthritis (RA) management lean toward achieving remission or low-disease activity. In this study, we conducted single-cell RNA sequencing (scRNAseq) of peripheral blood mononuclear cells (PBMCs) from 36 individuals (18 RA patients and 18 matched controls, accounting for age, sex, race, and ethnicity), to identify disease-relevant cell subsets and cell type-specific signatures associated with disease activity. Our analysis revealed 18 distinct PBMC subsets, including an IFITM3 overexpressing Interferon-activated (IFN-activated) monocyte subset. We observed an increase in CD4+ T effector memory cells in patients with moderate to high disease activity (DAS28-CRP ≥ 3.2), and a decrease in non-classical monocytes in patients with low disease activity or remission (DAS28-CRP < 3.2). Pseudobulk analysis by cell type identified 168 differentially expressed genes between RA and matched controls, with a downregulation of pro-inflammatory genes in the gamma-delta T cells subset, alteration of genes associated with RA predisposition in the IFN-activated subset, and non-classical monocytes. Additionally, we identified a gene signature associated with moderate-high disease activity, characterized by upregulation of pro-inflammatory genes such as TNF, JUN, EGR1, IFIT2, MAFB, G0S2, and downregulation of genes including HLA-DQB1, HLA-DRB5, TNFSF13B. Notably, cell-cell communication analysis revealed an upregulation of signaling pathways, including VISTA, in both moderate-high and remission-low disease activity contexts. Our findings provide valuable insights into the systemic cellular and molecular mechanisms underlying RA disease activity.

Binding and uptake of H-ferritin are mediated by human transferrin receptor-1.

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.

TIM-2 is expressed on B cells and in liver and kidney and is a receptor for H-ferritin endocytosis.

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.

CCR2 deficiency alters activation of microglia subsets in traumatic brain injury.

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.

Age-related changes to macrophages are detrimental to fracture healing in mice.

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.

A role for TREM2 ligands in the phagocytosis of apoptotic neuronal cells by microglia.

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.

TREM2, a DAP12-associated receptor, regulates osteoclast differentiation and function.

UNLABELLED: Deficiency of the signaling adapter protein DAP12 or its associated receptor TREM2 is associated with abnormal OC development in humans. Here we examine the role of TREM2 in mouse OC development and function, including migration and resorption in vitro. These results provide new evidence that TREM2 regulates OC function independent of its effects on multinucleated OC differentiation. INTRODUCTION: TREM2 (triggering receptor expressed in myeloid cells-2) associates with the signaling adapter DAP12 in osteoclasts (OCs). Genetic mutation or deletion of either the TYROBP (DAP12) or TREM2 gene is associated with the human disorder of brain and bone, Nasu-Hakola disease. We and others recently showed the critical requirement for immunoreceptor tyrosine-based activation motif (ITAM) signals through DAP12 and the Fc Receptor gamma chain (FcRgamma) during OC development. Here, we further define the role of TREM2 in OC differentiation and describe a role for TREM2 in OC migration and bone resorption. MATERIALS AND METHODS: We generated monoclonal anti-mouse TREM2 antibodies (mAb), analyzed pre-osteoclasts and mature OCs for TREM2 surface expression, and determined the effect of antibody ligation on in vitro OC differentiation, resorption, and migration. TREM2 RNA interference (RNAi) was used to disrupt expression of TREM2 in pre-osteoclasts. RESULTS: Using flow cytometry, our studies reveal that TREM2 is weakly expressed on C57BL/6 bone marrow macrophages (BMMs) and is upregulated during culture with RANKL and macrophage-colony stimulating factor (M-CSF). The expression of TREM2 is unaltered in DAP12-deficient OCs. Using C57BL/6 BMMs or RAW264.7 precursors, anti-TREM2 mAb treatment with RANKL and M-CSF enhances the formation of multinuclear TRACP+ OCs compared with control mAb treatment. In contrast, these agents have no effect on DAP12-deficient precursors. Monoclonal Ab blockade of TREM2 on OCs generated from C57BL/6 BMMs results in decreased resorption of artificial calcium-phosphate substrate and dentine. Reduction of TREM2 expression in RAW264.7 cells by RNAi results in loss of OC formation in response to RANKL and M-CSF. Anti-TREM2 cross-linking enhances migration of C57BL/6 OCs and RAW246.7 OCs in response to M-CSF. CONCLUSIONS: Our studies indicate that the TREM2 receptor regulates OC multinucleation as well as resorption and migration of mature OCs. Thus, TREM2-DAP12 signals regulate both OC formation and function.

CCR2 deficiency impairs macrophage infiltration and improves cognitive function after traumatic brain injury.

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.

Inflammatory arthritis increases mouse osteoclast precursors with myeloid suppressor function.

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.

Two structurally related rat Ly49 receptors with opposing functions (Ly49 stimulatory receptor 5 and Ly49 inhibitory receptor 5) recognize nonclassical MHC class Ib-encoded target ligands.

The Ly49 family of lectin-like receptors in rodents includes both stimulatory and inhibitory members. Although NK alloreactivity in mice is regulated primarily by inhibitory Ly49 receptors, in rats activating Ly49 receptors are equally important. Previous studies have suggested that activating rat Ly49 receptors are triggered by polymorphic ligands encoded within the nonclassical class Ib region of the rat MHC, RT1-CE/N/M, while inhibitory Ly49 receptors bind to widely expressed classical class Ia molecules encoded from the RT1-A region. To further investigate rat Ly49-mediated regulation of NK alloreactivity, we report in this study the identification and characterization of two novel paired Ly49 receptors that we have termed Ly49 inhibitory receptor 5 (Ly49i5) and Ly49 stimulatory receptor 5 (Ly49s5). Using a new mAb (mAb Fly5), we showed that Ly49i5 is an inhibitory receptor that recognizes ligands encoded within the class Ib region of the u and l haplotypes, while the structurally related Ly49s5 is an activating receptor that recognizes class Ib ligands of the u haplotype. Ly49s5 is functionally expressed in the high NK-alloresponder PVG strain, but not in the low alloresponder BN strain, in which it is a pseudogene. Ly49s5 is hence not responsible for the striking anti-u NK alloresponse previously described in BN rats (haplotype n), which results from repeated alloimmunizations with u haplotype cells. The present studies support the notion of a complex regulation of rat NK alloreactivity by activating and inhibitory Ly49 members, which may be highly homologous in the extracellular region and bind similar class Ib-encoded target ligands.

Pattern recognition by TREM-2: binding of anionic ligands.

We recently described the cloning of murine triggering receptor expressed by myeloid cells (TREM) 2, a single Ig domain DNAX adaptor protein 12-associated receptor expressed by cells of the myeloid lineage. In this study, we describe the identification of ligands for TREM-2 on both bacteria and mammalian cells. First, by using a TREM-2A/IgG1-Fc fusion protein, we demonstrate specific binding to a number of Gram-negative and Gram-positive bacteria and to yeast. Furthermore, we show that fluorescently labeled Escherichia coli and Staphylococcus aureus bind specifically to TREM-2-transfected cells. The binding of TREM-2A/Ig fusion protein to E. coli can be inhibited by the bacterial products LPS, lipoteichoic acid, and peptidoglycan. Additionally, binding can be inhibited by a number of other anionic carbohydrate molecules, including dextran sulfate, suggesting that ligand recognition is based partly on charge. Using a sensitive reporter assay, we demonstrate activation of a TREM-2A/CD3zeta chimeric receptor by both bacteria and dextran sulfate. Finally, we demonstrate binding of TREM-2A/Ig fusion to a series of human astrocytoma lines but not to a variety of other cell lines. The binding to astrocytomas, like binding to bacteria, is inhibited by anionic bacterial products, suggesting either a similar charge-based ligand recognition method or overlapping binding sites for recognition of self- and pathogen-expressed ligands.

Ly-49s3 is a promiscuous activating rat NK cell receptor for nonclassical MHC class I-encoded target ligands.

Previous studies of the rapid rejection of MHC-disparate lymphocytes in rats, named allogeneic lymphocyte cytotoxicity, have indicated that rat NK cells express activating receptors for nonclassical MHC class I allodeterminants from the RT1-C/E/M region. Using an expression cloning system that identifies activating receptors associated with the transmembrane adapter molecule DAP12, we have cloned a novel rat Ly-49 receptor that we have termed Ly-49 stimulatory receptor 3 (Ly-49s3). A newly generated anti-Ly-49s3 Ab, mAb DAR13, identified subpopulations of resting and IL-2-activated NK cells, but not T or B lymphocytes. Depletion of Ly-49s3-expressing NK cells drastically reduced alloreactivity in vitro, indicating that this subpopulation is responsible for a major part of the observed NK alloreactivity. DAR13-mediated blockade of Ly-49s3 inhibited killing of MHC-congenic target cells from the av1, n, lv1, and c haplotypes, but not from the u or b haplotypes. A putative ligand was mapped to the nonclassical MHC class I region (RT1-C/E/M) using intra-MHC recombinant strains. Relative numbers of Ly-49s3(+) NK cells were reduced, and surface levels of Ly-49s3 were lower, in MHC congenic strains expressing the putative Ly-49s3 ligand(s). In conclusion, we have identified a novel Ly-49 receptor that triggers rat NK cell-mediated responses.

Characterization of a novel killer cell lectin-like receptor (KLRH1) expressed by alloreactive rat NK cells.

NK cells have the ability to recognize and kill MHC-mismatched hemopoietic cells. In the present study, strain-specific differences in the rat NK allorecognition repertoire were exploited to generate Abs against receptors that may be involved in allogeneic responses. A mAb termed STOK9 was selected, and it reacted with subsets of NK cells and NKR-P1(+) T cells from certain rat strains possessing highly alloreactive NK cells. The STOK9(+) NK subset was broadly alloreactive and lysed Con A lymphoblast targets from a range of MHC-mismatched strains. The mAb STOK9 precipitated a 75-kDa dimeric glycoprotein from NK lysates. Expression cloning revealed that each monomer consisted of 231 aa with limited homology to other previously characterized killer cell lectin-like receptors (KLRs). This glycoprotein therefore constitutes a novel KLR branch, and it has been termed KLRH1. A gene in the central region of the natural killer gene complex on rat chromosome 4 encodes KLRH1. A mouse homolog appears to be present as deduced from analyses of genomic trace sequences. The function of KLRH1 is unknown, but it contains an immunoreceptor tyrosine-based inhibitory motif, suggesting an inhibitory function. The MHC haplotype of the host appears to influence KLRH1 expression, suggesting that it may function as an MHC-binding receptor on subsets of NK cells and T lymphocytes.