mTORC1 links pathology in experimental models of Still's disease and macrophage activation syndrome.

Still's disease is a severe inflammatory syndrome characterized by fever, skin rash and arthritis affecting children and adults. Patients with Still's disease may also develop macrophage activation syndrome, a potentially fatal complication of immune dysregulation resulting in cytokine storm. Here we show that mTORC1 (mechanistic target of rapamycin complex 1) underpins the pathology of Still's disease and macrophage activation syndrome. Single-cell RNA sequencing in a murine model of Still's disease shows preferential activation of mTORC1 in monocytes; both mTOR inhibition and monocyte depletion attenuate disease severity. Transcriptomic data from patients with Still's disease suggest decreased expression of the mTORC1 inhibitors TSC1/TSC2 and an mTORC1 gene signature that strongly correlates with disease activity and treatment response. Unrestricted activation of mTORC1 by Tsc2 deletion in mice is sufficient to trigger a Still's disease-like syndrome, including both inflammatory arthritis and macrophage activation syndrome with hemophagocytosis, a cellular manifestation that is reproduced in human monocytes by CRISPR/Cas-mediated deletion of TSC2. Consistent with this observation, hemophagocytic histiocytes from patients with macrophage activation syndrome display prominent mTORC1 activity. Our study suggests a mechanistic link of mTORC1 to inflammation that connects the pathogenesis of Still's disease and macrophage activation syndrome.

TET2-mutant clonal hematopoiesis and risk of gout.

Gout is a common inflammatory arthritis caused by precipitation of monosodium urate (MSU) crystals in individuals with hyperuricemia. Acute flares are accompanied by secretion of proinflammatory cytokines, including interleukin-1ß (IL-1ß). Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related condition predisposing to hematologic cancers and cardiovascular disease. CHIP is associated with elevated IL-1ß, thus we investigated CHIP as a risk factor for gout. To test the clinical association between CHIP and gout, we analyzed whole exome sequencing data from 177 824 individuals in the MGB Biobank (MGBB) and UK Biobank (UKB). In both cohorts, the frequency of gout was higher among individuals with CHIP than without CHIP (MGBB, CHIP with variant allele fraction [VAF] ≥2%: odds ratio [OR], 1.69; 95% CI, 1.09-2.61; P = .0189; UKB, CHIP with VAF ≥10%: OR, 1.25; 95% CI, 1.05-1.50; P = .0133). Moreover, individuals with CHIP and a VAF ≥10% had an increased risk of incident gout (UKB: hazard ratio [HR], 1.28; 95% CI, 1.06-1.55; P = .0107). In murine models of gout pathogenesis, animals with Tet2 knockout hematopoietic cells had exaggerated IL-1ß secretion and paw edema upon administration of MSU crystals. Tet2 knockout macrophages elaborated higher levels of IL-1ß in response to MSU crystals in vitro, which was ameliorated through genetic and pharmacologic Nlrp3 inflammasome inhibition. These studies show that TET2-mutant CHIP is associated with an increased risk of gout in humans and that MSU crystals lead to elevated IL-1ß levels in Tet2 knockout murine models. We identify CHIP as an amplifier of NLRP3-dependent inflammatory responses to MSU crystals in patients with gout.

The neutrotime transcriptional signature defines a single continuum of neutrophils across biological compartments.

Neutrophils are implicated in multiple homeostatic and pathological processes, but whether functional diversity requires discrete neutrophil subsets is not known. Here, we apply single-cell RNA sequencing to neutrophils from normal and inflamed mouse tissues. Whereas conventional clustering yields multiple alternative organizational structures, diffusion mapping plus RNA velocity discloses a single developmental spectrum, ordered chronologically. Termed here neutrotime, this spectrum extends from immature pre-neutrophils, largely in bone marrow, to mature neutrophils predominantly in blood and spleen. The sharpest increments in neutrotime occur during the transitions from pre-neutrophils to immature neutrophils and from mature marrow neutrophils to those in blood. Human neutrophils exhibit a similar transcriptomic pattern. Neutrophils migrating into inflamed mouse lung, peritoneum and joint maintain the core mature neutrotime signature together with new transcriptional activity that varies with site and stimulus. Together, these data identify a single developmental spectrum as the dominant organizational theme of neutrophil heterogeneity.

Megakaryocyte emperipolesis: a new frontier in cell-in-cell interaction.

Histology of bone marrow routinely identifies megakaryocytes that enclose neutrophils and other hematopoietic cells, a phenomenon termed emperipolesis. Preserved across mammalian species and enhanced with systemic inflammation and platelet demand, the nature and significance of emperipolesis remain largely unexplored. Recent advances demonstrate that emperipolesis is in fact a distinct form of cell-in-cell interaction. Following integrin-mediated attachment, megakaryocytes and neutrophils both actively drive entry via cytoskeletal rearrangement. Neutrophils enter a vacuole termed the emperisome which then releases them directly into the megakaryocyte cytoplasm. From this surprising location, neutrophils fuse with the demarcation membrane system to pass membrane to circulating platelets, enhancing the efficiency of thrombocytogenesis. Neutrophils then egress intact, carrying megakaryocyte membrane and potentially other cell components along with them. In this review, we summarize what is known about this intriguing cell-in-cell interaction and discuss potential roles for emperipolesis in megakaryocyte, platelet and neutrophil biology.

High-dimensional analysis reveals a pathogenic role of inflammatory monocytes in experimental diffuse alveolar hemorrhage.

Diffuse alveolar hemorrhage (DAH) is a life-threatening pulmonary complication associated with systemic lupus erythematosus, vasculitis, and stem cell transplant. Little is known about the pathophysiology of DAH, and no targeted therapy is currently available. Pristane treatment in mice induces systemic autoimmunity and lung hemorrhage that recapitulates hallmark pathologic features of human DAH. Using this experimental model, we performed high-dimensional analysis of lung immune cells in DAH by mass cytometry and single-cell RNA sequencing. We found a large influx of myeloid cells to the lungs in DAH and defined the gene expression profile of infiltrating monocytes. Bone marrow-derived inflammatory monocytes actively migrated to the lungs and homed adjacent to blood vessels. Using 3 models of monocyte deficiency and complementary transfer studies, we established a central role of inflammatory monocytes in the development of DAH. We further found that the myeloid transcription factor interferon regulatory factor 8 is essential to the development of both DAH and type I interferon-dependent autoimmunity. These findings collectively reveal monocytes as a potential treatment target in DAH.

Megakaryocyte emperipolesis mediates membrane transfer from intracytoplasmic neutrophils to platelets.

Bone marrow megakaryocytes engulf neutrophils in a phenomenon termed emperipolesis. We show here that emperipolesis is a dynamic process mediated actively by both lineages, in part through the ß2-integrin/ICAM-1/ezrin pathway. Tethered neutrophils enter in membrane-bound vesicles before penetrating into the megakaryocyte cytoplasm. Intracytoplasmic neutrophils develop membrane contiguity with the demarcation membrane system, thereby transferring membrane to the megakaryocyte and to daughter platelets. This phenomenon occurs in otherwise unmanipulated murine marrow in vivo, resulting in circulating platelets that bear membrane from non-megakaryocytic hematopoietic donors. Transit through megakaryocytes can be completed as rapidly as minutes, after which neutrophils egress intact. Emperipolesis is amplified in models of murine inflammation associated with platelet overproduction, contributing to platelet production in vitro and in vivo. These findings identify emperipolesis as a new cell-in-cell interaction that enables neutrophils and potentially other cells passing through the megakaryocyte cytoplasm to modulate the production and membrane content of platelets.

Megakaryocytes as immune cells.

Platelets play well-recognized roles in inflammation, but their cell of origin-the megakaryocyte-is not typically considered an immune lineage. Megakaryocytes are large polyploid cells most commonly identified in bone marrow. Egress via sinusoids enables migration to the pulmonary capillary bed, where elaboration of platelets can continue. Beyond receptors involved in hemostasis and thrombosis, megakaryocytes express receptors that confer immune sensing capacity, including TLRs and Fc-γ receptors. They control the proliferation of hematopoietic cells, facilitate neutrophil egress from marrow, possess the capacity to cross-present antigen, and can promote systemic inflammation through microparticles rich in IL-1. Megakaryocytes internalize other hematopoietic lineages, especially neutrophils, in an intriguing cell-in-cell interaction termed emperipolesis. Together, these observations implicate megakaryocytes as direct participants in inflammation and immunity.

High-throughput identification of noncoding functional SNPs via type IIS enzyme restriction.

Genome-wide association studies (GWAS) have identified many disease-associated noncoding variants, but cannot distinguish functional single-nucleotide polymorphisms (fSNPs) from others that reside incidentally within risk loci. To address this challenge, we developed an unbiased high-throughput screen that employs type IIS enzymatic restriction to identify fSNPs that allelically modulate the binding of regulatory proteins. We coupled this approach, termed SNP-seq, with flanking restriction enhanced pulldown (FREP) to identify regulation of CD40 by three disease-associated fSNPs via four regulatory proteins, RBPJ, RSRC2 and FUBP-1/TRAP150. Applying this approach across 27 loci associated with juvenile idiopathic arthritis, we identified 148 candidate fSNPs, including two that regulate STAT4 via the regulatory proteins SATB2 and H1.2. Together, these findings establish the utility of tandem SNP-seq/FREP to bridge the gap between GWAS and disease mechanism.

The metabolic regulator mTORC1 controls terminal myeloid differentiation.

Monocytes are derived from hematopoietic stem cells through a series of intermediate progenitor stages, but the factors that regulate this process are incompletely defined. Using a Ccr2/Cx3cr1 dual-reporter system to model murine monocyte ontogeny, we conducted a small-molecule screen that identified an essential role of mechanistic target of rapamycin complex 1 (mTORC1) in the development of monocytes and other myeloid cells. Confirmatory studies using mice with inducible deletion of the mTORC1 component Raptor demonstrated absence of mature circulating monocytes, as well as disruption in neutrophil and dendritic cell development, reflecting arrest of terminal differentiation at the granulocyte-monocyte progenitor stage. Conversely, excess activation of mTORC1 through deletion of the mTORC1 inhibitor tuberous sclerosis complex 2 promoted spontaneous myeloid cell development and maturation. Inhibitor studies and stage-specific expression profiling identified failure to down-regulate the transcription factor Myc by the mTORC1 target ribosomal S6 kinase 1 (S6K1) as the mechanistic basis for disrupted myelopoiesis. Together, these findings define the mTORC1-S6K1-Myc pathway as a key checkpoint in terminal myeloid development.

Megakaryocytes compensate for Kit insufficiency in murine arthritis.

The growth factor receptor Kit is involved in hematopoietic and nonhematopoietic development. Mice bearing Kit defects lack mast cells; however, strains bearing different Kit alleles exhibit diverse phenotypes. Herein, we investigated factors underlying differential sensitivity to IgG-mediated arthritis in 2 mast cell-deficient murine lines: KitWsh/Wsh, which develops robust arthritis, and KitW/Wv, which does not. Reciprocal bone marrow transplantation between KitW/Wv and KitWsh/Wsh mice revealed that arthritis resistance reflects a hematopoietic defect in addition to mast cell deficiency. In KitW/Wv mice, restoration of susceptibility to IgG-mediated arthritis was neutrophil independent but required IL-1 and the platelet/megakaryocyte markers NF-E2 and glycoprotein VI. In KitW/Wv mice, platelets were present in numbers similar to those in WT animals and functionally intact, and transfer of WT platelets did not restore arthritis susceptibility. These data implicated a platelet-independent role for the megakaryocyte, a Kit-dependent lineage that is selectively deficient in KitW/Wv mice. Megakaryocytes secreted IL-1 directly and as a component of circulating microparticles, which activated synovial fibroblasts in an IL-1-dependent manner. Transfer of WT but not IL-1-deficient megakaryocytes restored arthritis susceptibility to KitW/Wv mice. These findings identify functional redundancy among Kit-dependent hematopoietic lineages and establish an unanticipated capacity of megakaryocytes to mediate IL-1-driven systemic inflammatory disease.

The tachykinins substance P and hemokinin-1 favor the generation of human memory Th17 cells by inducing IL-1ß, IL-23, and TNF-like 1A expression by monocytes.

The nervous system influences immune responses through the release of neural factors such as neuropeptides. Among them, the tachykinin substance P (SP) signals via the neurokinin 1 receptor (NK-1R), which is expressed by various immune cells. We thereby analyzed in this paper whether tachykinins may participate in human CD4(+) Th cell polarization. We report that SP and hemokinin-1 (HK-1) upregulate IL-17A and IFN-γ production by human memory CD4(+) T cells without affecting IL-4 and IL-10 production. SP and HK-1 switch non-Th17-committed CD4(+) memory T cells into bona fide Th17 cells and Th1/Th17 cells. In contrast, SP and HK-1 do not modulate the polarization of naive CD4(+) T cells. SP- and HK-1-induced Th17 cell generation is mediated through NK-1R and requires the presence of monocytes. SP and HK-1 trigger IL-1ß, IL-6, and TNF-α production, upregulate IL-23 production, and enhance TNF-like 1A expression on monocyte surface. Neutralization experiments demonstrated that IL-1ß, IL-23, and TNF-like 1A are involved in the SP- and HK-1-induced Th17 cell. The other members of the tachykinin family, neurokinins A and B, have no effect on the differentiation of naive and memory T cells. These results thereby show that SP and HK-1 are novel Th17 cell-inducing factors that may act locally on memory T cells to amplify inflammatory responses.