On-demand erythrocyte disposal and iron recycling requires transient macrophages in the liver

Iron is an essential component of the erythrocyte protein hemoglobin and is crucial to oxygen transport in vertebrates. In the steady state, erythrocyte production is in equilibrium with erythrocyte removal1. In various pathophysiological conditions, however, erythrocyte life span is severely compromised, which threatens the organism with anemia and iron toxicity2,3. Here we identify an on-demand mechanism that clears erythrocytes and recycles iron. We show that Ly-6Chigh monocytes ingest stressed and senescent erythrocytes, accumulate in the liver via coordinated chemotactic cues, and differentiate to ferroportin 1 (FPN1)-expressing macrophages that can deliver iron to hepatocytes. Monocyte-derived FPN1+ Tim-4neg macrophages are transient, reside alongside embryonically-derived Tim-4high Kupffer cells, and depend on Csf1 and Nrf2. The spleen likewise recruits iron-loaded Ly-6Chigh monocytes, but these do not differentiate into iron-recycling macrophages due to the suppressive action of Csf2. Inhibiting monocyte recruitment to the liver leads to kidney and liver damage. These observations identify the liver as the primary organ supporting rapid erythrocyte removal and iron recycling and uncover a mechanism by which the body adapts to fluctuations in erythrocyte integrity

Twenty Years of G-CSFClinical and Nonclinical Discoveries /

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Identification of Antibody and Small Molecule Antagonists of Ferroportin-Hepcidin Interaction

The iron exporter ferroportin and its ligand, the hormone hepcidin, control fluxes of stored and recycled iron for use in a variety of essential biochemical processes. Inflammatory disorders and malignancies are often associated with high hepcidin levels, leading to ferroportin down-regulation, iron sequestration in tissue macrophages and subsequent anemia. The objective of this research was to develop reagents to characterize the expression of ferroportin, the interaction between ferroportin and hepcidin, as well as to identify novel ferroportin antagonists capable of maintaining iron export in the presence of hepcidin. Development of investigative tools that enabled cell-based screening assays is described in detail, including specific and sensitive monoclonal antibodies that detect endogenously-expressed human and mouse ferroportin and fluorescently-labeled chemically-synthesized human hepcidin. Large and small molecule antagonists inhibiting hepcidin-mediated ferroportin internalization were identified, and unique insights into the requirements for interaction between these two key iron homeostasis molecules are provided

Structure of hepcidin-bound ferroportin reveals iron homeostatic mechanisms.

The serum level of iron in humans is tightly controlled by the action of the hormone hepcidin on the iron efflux transporter ferroportin. Hepcidin regulates iron absorption and recycling by inducing the internalization and degradation of ferroportin1. Aberrant ferroportin activity can lead to diseases of iron overload, such as haemochromatosis, or iron limitation anaemias2. Here we determine cryogenic electron microscopy structures of ferroportin in lipid nanodiscs, both in the apo state and in complex with hepcidin and the iron mimetic cobalt. These structures and accompanying molecular dynamics simulations identify two metal-binding sites within the N and C domains of ferroportin. Hepcidin binds ferroportin in an outward-open conformation and completely occludes the iron efflux pathway to inhibit transport. The carboxy terminus of hepcidin directly contacts the divalent metal in the ferroportin C domain. Hepcidin binding to ferroportin is coupled to iron binding, with an 80-fold increase in hepcidin affinity in the presence of iron. These results suggest a model for hepcidin regulation of ferroportin, in which only ferroportin molecules loaded with iron are targeted for degradation. More broadly, our structural and functional insights may enable more targeted manipulation of the hepcidin-ferroportin axis in disorders of iron homeostasis

Bispecific T cell engager (BiTE^®) antibody constructs can mediate bystander tumor cell killing

<div><p>For targets that are homogenously expressed, such as CD19 on cells of the B lymphocyte lineage, immunotherapies can be highly effective. Targeting CD19 with blinatumomab, a CD19/CD3 bispecific antibody construct (BiTE^®), or with chimeric antigen receptor T cells (CAR-T) has shown great promise for treating certain CD19-positive hematological malignancies. In contrast, solid tumors with heterogeneous expression of the tumor-associated antigen (TAA) may present a challenge for targeted therapies. To prevent escape of TAA-negative cancer cells, immunotherapies with a local bystander effect would be beneficial. As a model to investigate BiTE^®-mediated bystander killing in the solid tumor setting, we used epidermal growth factor receptor (EGFR) as a target. We measured lysis of EGFR-negative populations in vitro and in vivo when co-cultured with EGFR-positive cells, human T cells and an EGFR/CD3 BiTE^® antibody construct. Bystander EGFR-negative cells were efficiently lysed by BiTE^®-activated T cells only when proximal to EGFR-positive cells. Our mechanistic analysis suggests that cytokines released by BiTE^®-activated T-cells induced upregulation of ICAM-1 and FAS on EGFR-negative bystander cells, contributing to T cell-induced bystander cell lysis.</p></div

EGFR-negative bystander cells were lysed by BiTE^®-activated T cells when co-cultured with EGFR-positive cells.

<p>NUGC4 (EGFR-positive) and SW620 (EGFR-negative) cells were mixed in various ratios and incubated with T cells (E:T ratio 10:1) and EGFR BiTE^® in duplicate plates. Thousands of cells/well were analyzed, with good agreement between replicate plates. This result was reproducible (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183390#pone.0183390.s003" target="_blank">S2C Fig</a>). After 48 hours, cells were stained and analyzed as describe in Materials and Methods. Cytotoxicity of (A) EGFR-positive and (B) EGFR-negative cells was measured by nuclear count (N = 2, all data points shown). (C) IFNγ and (D) TNFα were measured using commercially available MSD assays (N = 3, mean +/- sd). (E) T cells from quadruplicate wells were combined and percent CD69+/CD25+ cells determined by flow cytometry.</p

EGFR-negative cells were sensitized to bystander killing by T cell cytokines.

<p>(A) EGFR BiTE^®, T cells and NUGC4 cells (E:T ratio 10:1) were incubated in 96-well plates for 48 hours; supernatants containing T cells were either transferred directly (medium + cells) or clarified by centrifugation prior to transfer (medium only) to 96-well plates containing SW620 cells, or SW620 cells were directly treated with T cells and EGFR BiTE^® (no transfer control); N = 3, mean +/- sd (B) T cells + EGFR BiTE^® + NUGC4 cells were added to the top chamber of Transwell^® assays with 1μm and 5μm membranes; SW620 (or NUGC4 as control) cells were added to the bottom chambers. Percent cytotoxicity in the bottom chambers was determined with CellTiter-Glo^®. (C) SW620 cells were pre-treated for 24 hours +/- cytokines (10ng/ml IFNγ + 5ng/ml TNFα), then incubated for 24 hours with either resting T cells or BiTE^®-activated T cells. Cells were enumerated by nuclear count with cellular imaging; N = 4, mean +/- sd. Significance values: ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.</p

Lysis of bystander EGFR-negative tumor cells in tumor xenografts.

<p>Luciferase-labeled EGFR-negative cells (SW620-LUC), EGFR-positive cells (HCT116) or equal numbers of each cell line were mixed with human T cells (E:T 1:1, where the number of T cells is equal to the number of total combined target cells in mixed implants) and implanted in immunocompromised mice. MEC14 negative control BiTE^® or EGFR BiTE^® was dosed once daily. (A) Tumor growth for 1:1 mixture implants was measured by luminescence on days 8 and 11 using an imaging system. (B) Tumor volume was measured with calipers on day 21. Data represent averages of 5 replicate animals +/- SEM. Significance values: ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.</p

Blockade of IFNγ R1, TNFR1, ICAM-1 or FAS provided partial protection from BiTE^®-mediated cytotoxicity.

<p>(A) SW620 cells were pretreated with either IFNγ R1- or TNFR1-blocking antibodies or mouse IgG1 control antibody at 2 μg/ml (final) for one hour prior to addition of resting T cells or BiTE^®-activated T cells (E:T ratio 10:1) for 48 hours; N = 6, mean +/- sd. SW620 cells were pretreated with cytokines (5ng/ml IFNγ + 10ng/ml TNFα) for 18 hours to induce ICAM-1 and FAS, then incubated with (B) 5 μg/ml anti-ICAM-1 (final) or (C) 2.5 μg/ml anti-FAS (final) neutralizing antibodies (+ cytokines + blocking Ab) or control antibody (no cytokines and + cytokines) for one hour followed by addition of BiTE^®-activated T cells (E:T ratio 10:1) for 24 hours. Cell count was determined by imaging; N = 4, mean +/- sd. Significance values: ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.</p