Clodronate is not protective in lethal viral encephalitis despite substantially reducing inflammatory monocyte infiltration in the CNS.

Bone marrow (BM)-derived monocytes induce inflammation and tissue damage in a range of pathologies. In particular, in a mouse model of West Nile virus (WNV) encephalitis (WNE), nitric oxide-producing, Ly6Chi inflammatory monocytes from the BM are recruited to the central nervous system (CNS) and contribute to lethal immune pathology. Reducing the migration of these cells into the CNS using monoclonal antibody blockade, immune-modifying particles or CSF-1R inhibitors reduces neuroinflammation, improving survival and/or clinical outcomes. Macrophages can also be targeted more broadly by administration of clodronate-encapsulated liposomes, which induce apoptosis in phagocytes. In this study, clodronate reduced the inflammatory infiltrate by 70% in WNE, however, surprisingly, this had no effect on disease outcome. More detailed analysis demonstrated a compensatory increase in neutrophils and enhanced activation status of microglia in the brain. In addition, we observed increased numbers of Ly6Chi BM monocytes with an increased proliferative capacity and expression of SCA-1 and CD16/32, potentially indicating output of immature cells from the BM. Once in the brain, these cells were more phagocytic and had a reduced expression of antigen-presenting molecules. Lastly, we show that clodronate also reduces non-myeloid cells in the spleen and BM, as well as ablating red blood cells and their proliferation. These factors likely impeded the therapeutic potential of clodronate in WNE. Thus, while clodronate provides an excellent system to deplete macrophages in the body, it has larger and broader effects on the phagocytic and non-phagocytic system, which must be considered in the interpretation of data.

The cytokines interleukin-6 and interferon-α induce distinct microglia phenotypes.

BACKGROUND: Elevated production of the cytokines interleukin (IL)-6 or interferon (IFN)-α in the central nervous system (CNS) is implicated in the pathogenesis of neurological diseases such as neuromyelitis optica spectrum disorders or cerebral interferonopathies, respectively. Transgenic mice with CNS-targeted chronic production of IL-6 (GFAP-IL6) or IFN-α (GFAP-IFN) recapitulate important clinical and pathological features of these human diseases. The activation of microglia is a prominent manifestation found both in the human diseases and in the transgenic mice, yet little is known about how this contributes to disease pathology. METHODS: Here, we used a combination of ex vivo and in situ techniques to characterize the molecular, cellular and transcriptomic phenotypes of microglia in GFAP-IL6 versus GFAP-IFN mice. In addition, a transcriptomic meta-analysis was performed to compare the microglia response from GFAP-IL6 and GFAP-IFN mice to the response of microglia in a range of neurodegenerative and neuroinflammatory disorders. RESULTS: We demonstrated that microglia show stimulus-specific responses to IL-6 versus IFN-α in the brain resulting in unique and extensive molecular and cellular adaptations. In GFAP-IL6 mice, microglia proliferated, had shortened, less branched processes and elicited transcriptomic and molecular changes associated with phagocytosis and lipid processing. In comparison, microglia in the brain of GFAP-IFN mice exhibited increased proliferation and apoptosis, had larger, hyper-ramified processes and showed transcriptomic and surface marker changes associated with antigen presentation and antiviral response. Further, a transcriptomic meta-analysis revealed that IL-6 and IFN-α both contribute to the formation of a core microglia response in animal models of neurodegenerative and neuroinflammatory disorders, such as Alzheimer's disease, tauopathy, multiple sclerosis and lipopolysaccharide-induced endotoxemia. CONCLUSIONS: Our findings demonstrate that microglia responses to IL-6 and IFN-α are highly stimulus-specific, wide-ranging and give rise to divergent phenotypes that modulate microglia responses in neuroinflammatory and neurodegenerative diseases.

Integrated immune dynamics define correlates of COVID-19 severity and antibody responses.

SARS-CoV-2 causes a spectrum of COVID-19 disease, the immunological basis of which remains ill defined. We analyzed 85 SARS-CoV-2-infected individuals at acute and/or convalescent time points, up to 102 days after symptom onset, quantifying 184 immunological parameters. Acute COVID-19 presented with high levels of IL-6, IL-18, and IL-10 and broad activation marked by the upregulation of CD38 on innate and adaptive lymphocytes and myeloid cells. Importantly, activated CXCR3+cTFH1 cells in acute COVID-19 significantly correlate with and predict antibody levels and their avidity at convalescence as well as acute neutralization activity. Strikingly, intensive care unit (ICU) patients with severe COVID-19 display higher levels of soluble IL-6, IL-6R, and IL-18, and hyperactivation of innate, adaptive, and myeloid compartments than patients with moderate disease. Our analyses provide a comprehensive map of longitudinal immunological responses in COVID-19 patients and integrate key cellular pathways of complex immune networks underpinning severe COVID-19, providing important insights into potential biomarkers and immunotherapies.

Staining of Phosphorylated Signalling Markers Protocol for Mass Cytometry.

Mass cytometry is a multi-parametric technique that offers insight into functional and biological systems at a single cell level (Tanner et al., Cancer Immunol Immunother 62:955-965, 2013). One of the major advantages of mass cytometry is the ability to measure multiple intracellular markers, including phosphorylated proteins that are part of major signaling pathways, such as NF-κB, JAK/STAT, and ERK/MAPK. Here we describe an optimized mass cytometry protocol for staining human clinical blood samples with panels that include phosphorylated antibodies.

Analysis of the Murine Bone Marrow Hematopoietic System Using Mass and Flow Cytometry.

The hematopoietic system produces erythrocytes (red blood cells), leukocytes (white blood cells), and thrombocytes (platelets) throughout the life of an organism. Long-lived hematopoietic stem cells give rise to early progenitors with multi-lineage potential that progressively differentiate into lineage-specific progenitors. Following lineage commitment, these progenitors proliferate and expand, before eventually differentiating into their mature forms. This process drives the up- and downregulation of a wide variety of surface and intracellular markers throughout differentiation, making cytometric analysis of this interconnected system challenging. Moreover, during inflammation, the hematopoietic system can be mobilized to re-prioritize the production of various lineages, in order to match increased demand, often at the expense of other lineages. As such, the response of the hematopoietic system in the bone marrow (BM) is a critical component of both immunity and disease. Because of the complexity of the hematopoietic system in steady state and disease, high-dimensional cytometry technologies are well suited to the exploration of these complex systems. Here we describe a protocol for the extraction of murine bone marrow, and preparation for examination using high-dimensional flow or mass cytometry. Additionally, we describe methods for performing cell cycle assays using bromodeoxyuridine (BrdU) or iododeoxyuridine (IdU). Finally, we describe an analytical method that allows for a system-level analysis of the hematopoietic system in steady state or inflammatory scenarios.

Defective inflammatory monocyte development in IRF8-deficient mice abrogates migration to the West Nile virus-infected brain.

IRF8 (interferon-regulatory factor-8) plays a critical role in regulating myeloid cell differentiation. However, the role of this transcription factor in the development of Ly6C+ inflammatory monocytes and their migration to the infected brain has not been examined. We have previously shown that West Nile virus (WNV) infection of wild-type (WT) mice triggers a significant increase in numbers of Ly6C+ monocytes in the bone marrow. These cells traffic via the blood to the infected brain, where they give rise to proinflammatory macrophages. Here, we show that WNV-infected IRF8-deficient (IRF8-/-) mice had significantly reduced numbers of Ly6C+ monocytes in the periphery, with few of these cells found in the blood. Furthermore, low numbers of inflammatory monocyte-derived macrophages were observed in the brains of IRF8-/- mice throughout infection. Adoptive transfer of IRF8-/- Ly6C+ monocytes demonstrated that these cells were intrinsically unable to traffic to the inflamed brain. Low expression of the chemokine receptor CCR2 and integrin VLA-4 by IRF8-/- monocytes likely contributed to this defect, as the interactions between these proteins and their ligands are critical for monocyte egress and migration to inflammatory foci. These data highlight a critical role for IRF8 in inflammatory monocyte differentiation and migration during WNV infection.

Therapeutic inflammatory monocyte modulation using immune-modifying microparticles.

Inflammatory monocyte-derived effector cells play an important role in the pathogenesis of numerous inflammatory diseases. However, no treatment option exists that is capable of modulating these cells specifically. We show that infused negatively charged, immune-modifying microparticles (IMPs), derived from polystyrene, microdiamonds, or biodegradable poly(lactic-co-glycolic) acid, were taken up by inflammatory monocytes, in an opsonin-independent fashion, via the macrophage receptor with collagenous structure (MARCO). Subsequently, these monocytes no longer trafficked to sites of inflammation; rather, IMP infusion caused their sequestration in the spleen through apoptotic cell clearance mechanisms and, ultimately, caspase-3-mediated apoptosis. Administration of IMPs in mouse models of myocardial infarction, experimental autoimmune encephalomyelitis, dextran sodium sulfate-induced colitis, thioglycollate-induced peritonitis, and lethal flavivirus encephalitis markedly reduced monocyte accumulation at inflammatory foci, reduced disease symptoms, and promoted tissue repair. Together, these data highlight the intricate interplay between scavenger receptors, the spleen, and inflammatory monocyte function and support the translation of IMPs for therapeutic use in diseases caused or potentiated by inflammatory monocytes.