Inflammation and functional iron deficiency regulate fibroblast growth factor 23 production.

Circulating levels of fibroblast growth factor 23 (FGF23) are elevated in patients with chronic kidney disease (CKD), but the mechanisms are poorly understood. Here we tested whether inflammation and iron deficiency regulate FGF23. In wild-type mice, acute inflammation induced by single injections of heat-killed Brucella abortus or interleukin-1ß (IL-1ß) decreased serum iron within 6 h, and was accompanied by significant increases in osseous Fgf23 mRNA expression and serum levels of C-terminal FGF23, but no changes in intact FGF23. Chronic inflammation induced by repeated bacteria or IL-1ß injections decreased serum iron, increased osseous Fgf23 mRNA, and serum C-terminal FGF23, but modestly increased biologically active, intact FGF23 serum levels. Chronic iron deficiency mimicked chronic inflammation. Increased osseous FGF23 cleavage rather than a prolonged half-life of C-terminal FGF23 fragments accounted for the elevated C-terminal FGF23 but near-normal intact FGF23 levels in inflammation. IL-1ß injection increased Fgf23 mRNA and C-terminal FGF23 levels similarly in wildtype and Col4a3(ko) mice with CKD but markedly increased intact FGF23 levels only in the CKD mice. Inflammation increased Fgf23 transcription by activating Hif1α signaling. Thus, inflammation and iron deficiency stimulate FGF23 production. Simultaneous upregulation of FGF23 cleavage in osteocytes maintains near-normal levels of biologically active, intact circulating FGF23, whereas downregulated or impaired FGF23 cleavage may contribute to elevated intact serum FGF23 in CKD.

TLR7/8 Activation in Immune Cells and Muscle by RNA-Containing Immune Complexes: Role in Inflammation and the Pathogenesis of Myositis.

OBJECTIVE: Activation of endosomal toll-like receptors (TLRs) is one possible driver of inflammation in idiopathic inflammatory myopathies (IIM). We investigated the potential contribution of TLR7 and TLR8 to IIM pathogenesis. METHODS: Activation of TLR7/8 in healthy donor peripheral blood mononuclear cells (PBMCs) by immune complexes from patients with IIM and lupus was tested. Autoantibody profiling of patient IgG samples was performed using a 1581-antigen array. TLR7 and/or TLR8 activation by RNA molecules associated with autoantibodies was assessed. Gene expression in human myoblasts and satellite cells following treatment with supernatants from TLR7/8-activated PBMCs was evaluated by NanoString. C57BL/6 mice were dosed intramuscularly with the TLR7/8 agonist R848 and single-cell RNA-sequencing was performed on the muscle to ascertain the cell types responding to TLR7/8 activation and the downstream effects. RESULTS: Overall, 69 patients with IIM were included with representation of dermatomyositis (DM), polymyositis (PM), and inclusion body myositis (IBM) subsets. Immune complexes from patients with IIM, as well as autoantibody-associated RNAs His-tRNA, Y1, Y4 and U1, activated PBMCs to produce IFN-α and IL-6 via TLR7/8. Several canonical (Ro60, Ro52, HIST1H4A) and novel (IL-36RN) autoreactivities correlated highly with TLR7/8 activation. Supernatants from TLR7/8-activated PBMCs had a negative impact on human myoblasts and satellite cells. Endothelial cells were activated by R848 in mouse muscle in vivo, in addition to immune cells such as monocytes and macrophages. CONCLUSION: Our results suggest that patients with IIM have autoantibodies in their blood causing TLR7/8 activation, which leads to inflammation in muscles with potential deleterious effects.

Regulation of TMPRSS6 by BMP6 and iron in human cells and mice.

Mutations in transmembrane protease, serine 6 (TMPRSS6), encoding matriptase-2, are responsible for the familial anemia disorder iron-refractory iron deficiency anemia (IRIDA). Patients with IRIDA have inappropriately elevated levels of the iron regulatory hormone hepcidin, suggesting that TMPRSS6 is involved in negatively regulating hepcidin expression. Hepcidin is positively regulated by iron via the bone morphogenetic protein (BMP)-SMAD signaling pathway. In this study, we investigated whether BMP6 and iron also regulate TMPRSS6 expression. Here we demonstrate that, in vitro, treatment with BMP6 stimulates TMPRSS6 expression at the mRNA and protein levels and leads to an increase in matriptase-2 activity. Moreover, we identify that inhibitor of DNA binding 1 is the key element of the BMP-SMAD pathway to regulate TMPRSS6 expression in response to BMP6 treatment. Finally, we show that, in mice, Tmprss6 mRNA expression is stimulated by chronic iron treatment or BMP6 injection and is blocked by injection of neutralizing antibody against BMP6. Our results indicate that BMP6 and iron not only induce hepcidin expression but also induce TMPRSS6, a negative regulator of hepcidin expression. Modulation of TMPRSS6 expression could serve as a negative feedback inhibitor to avoid excessive hepcidin increases by iron to help maintain tight homeostatic balance of systemic iron levels.

Pharmacologic inhibition of hepcidin expression reverses anemia of chronic inflammation in rats.

Anemia of chronic inflammation (ACI) is the most frequent anemia in hospitalized patients and is associated with significant morbidity. A major underlying mechanism of ACI is the retention of iron within cells of the reticuloendothelial system (RES), thus making the metal unavailable for efficient erythropoiesis. This reticuloendothelial iron sequestration is primarily mediated by excess levels of the iron regulatory peptide hepcidin down-regulating the functional expression of the only known cellular iron export protein ferroportin resulting in blockade of iron egress from these cells. Using a well-established rat model of ACI, we herein provide novel evidence for effective treatment of ACI by blocking endogenous hepcidin production using the small molecule dorsomorphin derivative LDN-193189 or the protein soluble hemojuvelin-Fc (HJV.Fc) to inhibit bone morphogenetic protein-Smad mediated signaling required for effective hepcidin transcription. Pharmacologic inhibition of hepcidin expression results in mobilization of iron from the RES, stimulation of erythropoiesis and correction of anemia. Thus, hepcidin lowering agents are a promising new class of pharmacologic drugs to effectively combat ACI.

A novel validated enzyme-linked immunosorbent assay to quantify soluble hemojuvelin in mouse serum.

Hemojuvelin is a critical regulator of hepcidin expression and can be cleaved by proteases to form soluble hemojuvelin. Soluble hemojuvelin has been recently identified in human serum but the presence and quantity of soluble hemojuvelin in mouse serum is unknown. We developed a two-site enzyme-linked immunosorbent assay using a monoclonal anti-hemojuvelin as the capture antibody and a biotinylated polyclonal anti-hemojuvelin as the detection antibody to quantify the levels of soluble hemojuvelin in mouse serum. We validated this assay using cell-conditioned media and serum from Hemojuvelin-null and Bone morphogenetic protein 6-null mice. We also used this validated assay to measure serum soluble hemojuvelin concentrations in mice receiving an acute low iron or high iron treatment. This two-site enzyme-linked immunosorbent assay was highly specific for mouse hemojuvelin, with a lower limit of detection at 13.2-26.8 ng/mL of soluble hemojuvelin in mouse serum. The median serum soluble hemojuvelin concentration in wild-type C57BL/6J mice was 57.9 ± 22 ng/mL, which is 4- to 20-fold less than that reported in healthy human volunteers. After acute low iron diet treatment in these mice, serum soluble hemojuvelin levels were increased and correlated with lowered serum iron levels and decreased hepatic hepcidin expression. An acute high iron diet in wild-type mice or chronically iron-overloaded Bone morphogenetic protein 6-null mice did not significantly lower serum soluble hemojuvelin concentrations. Here we report reliable quantitation of mouse serum soluble hemojuvelin using a novel and validated enzyme-linked immunosorbent assay. This assay may provide a useful tool to elucidate the source and physiological role of serum soluble hemojuvelin in hepcidin regulation and iron metabolism using well-established mouse models of iron-related disorders.

A hepcidin lowering agent mobilizes iron for incorporation into red blood cells in an adenine-induced kidney disease model of anemia in rats.

BACKGROUND: Anemia is a common complication of chronic kidney disease (CKD) that negatively impacts the quality of life and is associated with numerous adverse outcomes. Excess levels of the iron regulatory hormone hepcidin are thought to contribute to anemia in CKD patients by decreasing iron availability from the diet and from body stores. Adenine treatment in rats has been proposed as an animal model of anemia of CKD with high hepcidin levels that mirrors the condition in human patients. METHODS: We developed a modified adenine-induced kidney disease model with a higher survival rate than previously reported models, while maintaining persistent kidney disease and anemia. We then tested whether the small molecule bone morphogenetic protein (BMP) inhibitor LDN-193189, which was previously shown to lower hepcidin levels in rodents, mobilized iron into the plasma and improved iron-restricted erythropoiesis in this model. RESULTS: Adenine-treated rats exhibited increased hepatic hepcidin mRNA, decreased serum iron, increased spleen iron content, low hemoglobin (Hb) and inappropriately low erythropoietin (EPO) levels relative to the degree of anemia. LDN-193189 administration to adenine-treated rats lowered hepatic hepcidin mRNA, mobilized stored iron into plasma and increased Hb content of reticulocytes. CONCLUSIONS: Our data suggest that hepcidin lowering agents may provide a new therapeutic strategy to improve iron availability for erythropoiesis in CKD.

The BMP coreceptor RGMb promotes while the endogenous BMP antagonist noggin reduces neurite outgrowth and peripheral nerve regeneration by modulating BMP signaling.

Repulsive guidance molecule b (RGMb) is a bone morphogenetic protein (BMP) coreceptor and sensitizer of BMP signaling, highly expressed in adult dorsal root ganglion (DRG) sensory neurons. We used a murine RGMb knock-out to gain insight into the physiological role of RGMb in the DRG, and address whether RGMb-mediated modulation of BMP signaling influences sensory axon regeneration. No evidence for altered development of the PNS and CNS was detected in RGMb(-/-) mice. However, both cultured neonatal whole DRG explants and dissociated DRG neurons from RGMb(-/-) mice exhibited significantly fewer and shorter neurites than those from wild-type littermates, a phenomenon that could be fully rescued by BMP-2. Moreover, Noggin, an endogenous BMP signaling antagonist, inhibited neurite outgrowth in wild-type DRG explants from naive as well as nerve injury-preconditioned mice. Noggin is downregulated in the DRG after nerve injury, and its expression is highly correlated and inversely associated with the known regeneration-associated genes, which are induced in the DRG by peripheral axonal injury. We show that diminished BMP signaling in vivo, achieved either through RGMb deletion or BMP inhibition with Noggin, retarded early axonal regeneration after sciatic nerve crush injury. Our data suggest a positive modulatory contribution of RGMb and BMP signaling to neurite extension in vitro and early axonal regrowth after nerve injury in vivo and a negative effect of Noggin.

Targeting the hepcidin-ferroportin axis to develop new treatment strategies for anemia of chronic disease and anemia of inflammation.

Anemia of chronic disease (ACD) or anemia of inflammation is prevalent in patients with chronic infection, autoimmune disease, cancer, and chronic kidney disease. ACD is associated with poor prognosis and lower quality of life. Management of ACD using intravenous iron and erythropoiesis stimulating agents are ineffective for some patients and are not without adverse effects, driving the need for new alternative therapies. Recent advances in our understanding of the molecular mechanisms of iron regulation reveal that increased hepcidin, the iron regulatory hormone, is a key factor in the development of ACD. In this review, we will summarize the role of hepcidin in iron homeostasis, its contribution to the pathophysiology of ACD, and novel strategies that modulate hepcidin and its target ferroportin for the treatment of ACD.

The dynamics of EBV shedding implicate a central role for epithelial cells in amplifying viral output.

To develop more detailed models of EBV persistence we have studied the dynamics of virus shedding in healthy carriers. We demonstrate that EBV shedding into saliva is continuous and rapid such that the virus level is replaced in < or =2 minutes, the average time that a normal individual swallows. Thus, the mouth is not a reservoir of virus but a conduit through which a continuous flow stream of virus passes in saliva. Consequently, virus is being shed at a much higher rate than previously thought, a level too high to be accounted for by replication in B cells in Waldeyer's ring alone. Virus shedding is relatively stable over short periods (hours-days) but varies through 3.5 to 5.5 logs over longer periods, a degree of variation that also cannot be accounted for solely by replication in B cells. This variation means, contrary to what is generally believed, that the definition of high and low shedder is not so much a function of variation between individuals but within individuals over time. The dynamics of shedding describe a process governing virus production that is occurring independently < or =3 times at any moment. This process grows exponentially and is then randomly terminated. We propose that these dynamics are best explained by a model where single B cells sporadically release virus that infects anywhere from 1 to 5 epithelial cells. This infection spreads at a constant exponential rate and is terminated randomly, resulting in infected plaques of epithelial cells ranging in size from 1 to 10(5) cells. At any one time there are a very small number (< or =3) of plaques. We suggest that the final size of these plaques is a function of the rate of infectious spread within the lymphoepithelium which may be governed by the structural complexity of the tissue but is ultimately limited by the immune response.

Inhibition of IRF5 cellular activity with cell-penetrating peptides that target homodimerization.

The transcription factor interferon regulatory factor 5 (IRF5) plays essential roles in pathogen-induced immunity downstream of Toll-, nucleotide-binding oligomerization domain-, and retinoic acid-inducible gene I-like receptors and is an autoimmune susceptibility gene. Normally, inactive in the cytoplasm, upon stimulation, IRF5 undergoes posttranslational modification(s), homodimerization, and nuclear translocation, where dimers mediate proinflammatory gene transcription. Here, we report the rational design of cell-penetrating peptides (CPPs) that disrupt IRF5 homodimerization. Biochemical and imaging analysis shows that IRF5-CPPs are cell permeable, noncytotoxic, and directly bind to endogenous IRF5. IRF5-CPPs were selective and afforded cell type- and species-specific inhibition. In plasmacytoid dendritic cells, inhibition of IRF5-mediated interferon-α production corresponded to a dose-dependent reduction in nuclear phosphorylated IRF5 [p(Ser462)IRF5], with no effect on pIRF5 levels. These data support that IRF5-CPPs function downstream of phosphorylation. Together, data support the utility of IRF5-CPPs as novel tools to probe IRF5 activation and function in disease.

Antiviral activity of bone morphogenetic proteins and activins.

Understanding the control of viral infections is of broad importance. Chronic hepatitis C virus (HCV) infection causes decreased expression of the iron hormone hepcidin, which is regulated by hepatic bone morphogenetic protein (BMP)/SMAD signalling. We found that HCV infection and the BMP/SMAD pathway are mutually antagonistic. HCV blunted induction of hepcidin expression by BMP6, probably via tumour necrosis factor (TNF)-mediated downregulation of the BMP co-receptor haemojuvelin. In HCV-infected patients, disruption of the BMP6/hepcidin axis and genetic variation associated with the BMP/SMAD pathway predicted the outcome of infection, suggesting that BMP/SMAD activity influences antiviral immunity. Correspondingly, BMP6 regulated a gene repertoire reminiscent of type I interferon (IFN) signalling, including upregulating interferon regulatory factors (IRFs) and downregulating an inhibitor of IFN signalling, USP18. Moreover, in BMP-stimulated cells, SMAD1 occupied loci across the genome, similar to those bound by IRF1 in IFN-stimulated cells. Functionally, BMP6 enhanced the transcriptional and antiviral response to IFN, but BMP6 and related activin proteins also potently blocked HCV replication independently of IFN. Furthermore, BMP6 and activin A suppressed growth of HBV in cell culture, and activin A inhibited Zika virus replication alone and in combination with IFN. The data establish an unappreciated important role for BMPs and activins in cellular antiviral immunity, which acts independently of, and modulates, IFN.

Plasma cell-specific transcription factor XBP-1s binds to and transactivates the Epstein-Barr virus BZLF1 promoter.

Epstein-Barr virus (EBV) in vivo is known to establish persistent infection in resting, circulating memory B cells and to productively replicate in plasma cells. Until now, the molecular mechanism of how EBV switches from latency to lytic replication in vivo was not known. Here, we report that the plasma cell differentiation factor, XBP-1s, activates the expression of the master regulator of EBV lytic activation, BZLF1. Using reporter assays, we observed that XBP-1s was able to transactivate the BZLF1 promoter, Zp, in a plasma cell line and other lymphoid cell lines but, interestingly, not in epithelial cell lines. We have identified an XBP-1s binding site on the ZID/ZII region of Zp, which when abolished by site-directed mutagenesis led to abrogation of XBP-1s binding and promoter activation. Using the chromatin immunoprecipitation assay, we observed direct binding of XBP-1s to endogenous Zp in an EBV-infected plasma cell line. Finally, in the same cell line, we observed that overexpression of XBP-1s resulted in increased expression of BZLF1, while knockdown of XBP-1s with short hairpin RNA drastically reduces BZLF1 expression. We suggest that EBV harnesses the B-cell terminal differentiation pathway via XBP-1s as a physiological signal to reactivate and begin viral replication. We are currently investigating other signals, such as the endoplasmic reticulum stress response proteins, which act upstream of XBP-1s, to identify other interacting factors that initiate and/or amplify the lytic switch.

Inflammation regulates TMPRSS6 expression via STAT5.

TMPRSS6 is a regulated gene, with a crucial role in the regulation of iron homeostasis by inhibiting hepcidin expression. The main regulator of iron homeostasis, the antimicrobial peptide hepcidin, which also has a role in immunity, is directly upregulated by inflammation. In this study, we analyzed whether inflammation is also a modulator of TMPRSS6 expression in vitro and in vivo and we determined the mechanism of this regulation A Human Hepatoma cell line was treated with interleukin-6 and mice were injected with lipopolysaccharide and TMPRSS6 expression and the regulatory mechanism were addressed. In this study, we demonstrate that inflammation downregulates TMPRSS6 expression in vitro and in vivo. The downregulation of Tmprss6 by inflammation in mice is not dependent on the Bmp-Smad pathway but occurs through a decrease in Stat5 phosphorylation. Moreover, Stat5 positively regulates Tmprss6 expression directly by binding to a Stat5 element located on the Tmprss6 promoter. Importantly, our results highlight the functional role of inflammatory modulation of TMPRSS6 expression in the regulation of hepcidin. TMPRSS6 inhibition via decreased STAT5 phosphorylation may be an additional mechanism by which inflammation stimulates hepcidin expression to regulate iron homeostasis and immunity.

Differential regulation of myeloid leukemias by the bone marrow microenvironment.

Like their normal hematopoietic stem cell counterparts, leukemia stem cells (LSCs) in chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML) are presumed to reside in specific niches in the bone marrow microenvironment (BMM) and may be the cause of relapse following chemotherapy. Targeting the niche is a new strategy to eliminate persistent and drug-resistant LSCs. CD44 (refs. 3,4) and interleukin-6 (ref. 5) have been implicated previously in the LSC niche. Transforming growth factor-ß1 (TGF-ß1) is released during bone remodeling and plays a part in maintenance of CML LSCs, but a role for TGF-ß1 from the BMM has not been defined. Here, we show that alteration of the BMM by osteoblastic cell-specific activation of the parathyroid hormone (PTH) receptor attenuates BCR-ABL1 oncogene-induced CML-like myeloproliferative neoplasia (MPN) but enhances MLL-AF9 oncogene-induced AML in mouse transplantation models, possibly through opposing effects of increased TGF-ß1 on the respective LSCs. PTH treatment caused a 15-fold decrease in LSCs in wild-type mice with CML-like MPN and reduced engraftment of immune-deficient mice with primary human CML cells. These results demonstrate that LSC niches in CML and AML are distinct and suggest that modulation of the BMM by PTH may be a feasible strategy to reduce LSCs, a prerequisite for the cure of CML.

Repulsive guidance molecule (RGM) family proteins exhibit differential binding kinetics for bone morphogenetic proteins (BMPs).

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta superfamily that exert their effects via type I and type II serine threonine kinase receptors and the SMAD intracellular signaling pathway to regulate diverse biologic processes. Recently, we discovered that the repulsive guidance molecule (RGM) family, including RGMA, RGMB, and RGMC/hemojuvelin (HJV), function as co-receptors that enhance cellular responses to BMP ligands. Here, we use surface plasmon resonance to quantitate the binding kinetics of RGM proteins for BMP ligands. We show that among the RGMs, HJV exhibits the highest affinity for BMP6, BMP5, and BMP7 with K(D) 8.1, 17, and 20 nM respectively, versus 28, 33, and 166 nM for RGMB, and 55, 83, and 63 nM for RGMA. Conversely, RGMB exhibits preferential binding to BMP4 and BMP2 with K(D) 2.6 and 5.5 nM respectively, versus 4.5 and 9.4 nM for HJV, and 14 and 22 nM for RGMA, while RGMA exhibits the lowest binding affinity for most BMPs tested. Among the BMP ligands, RGMs exhibit the highest relative affinity for BMP4 and the lowest relative affinity for BMP7, while none of the RGMs bind to BMP9. Thus, RGMs exhibit preferential binding for distinct subsets of BMP ligands. The preferential binding of HJV for BMP6 is consistent with the functional role of HJV and BMP6 in regulating systemic iron homeostasis. Our data may help explain the mechanism by which BMPs exert cell-context specific effects via a limited number of type I and type II receptors.