Piotr Tymoszuk

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 removal. In various pathophysiological conditions, however, erythrocyte life span is compromised severely, which threatens the organism with anemia and iron toxicity. Here we identify an on-demand mechanism that clears erythrocytes and recycles iron. We show that monocytes that express high levels of lymphocyte antigen 6 complex, locus C1 (LY6C1, also known as Ly-6C) ingest stressed and senescent erythrocytes, accumulate in the liver via coordinated chemotactic cues, and differentiate into ferroportin 1 (FPN1, encoded by SLC40A1)-expressing macrophages that can deliver iron to hepatocytes. Monocyte-derived FPN1+Tim-4neg macrophages are transient, reside alongside embryonically derived T cell immunoglobulin and mucin domain containing 4 (Timd4, also known as Tim-4)high Kupffer cells (KCs), and depend on the growth factor Csf1 and the transcription factor Nrf2 (encoded by Nfe2l2). The spleen, likewise, recruits iron-loaded Ly-6Chigh monocytes, but these do not differentiate into iron-recycling macrophages, owing to the suppressive action of Csf2. The accumulation of a transient macrophage population in the liver also occurs in mouse models of hemolytic anemia, anemia of inflammation, and sickle cell disease. Inhibition of monocyte recruitment to the liver during stressed erythrocyte delivery leads to kidney and liver damage. These observations identify the liver as the primary organ that supports rapid erythrocyte removal and iron recycling, and uncover a mechanism by which the body adapts to fluctuations in erythrocyte integrity.

Lapatinib and doxorubicin enhance the Stat1‐dependent antitumor immune response

The dual erbB1/2 tyrosine kinase inhibitor lapatinib as well as the anthracycline doxorubicin are both used in the therapy of HER2‐positive breast cancer. Using MMTV‐neu mice as an animal model for HER2‐positive breast cancer, we observed enhanced tumor infiltration by IFN‐γ‐secreting T cells after treatment with doxorubicin and/or lapatinib. Antibody depletion experiments revealed a contribution of CD8+ but not CD4+ T cells to the antitumor effect of these drugs. Doxorubicin treatment additionally decreased the content of immunosuppressive tumor‐associated macrophages (TAMs) in the tumor bed. In contrast, Stat1‐deficient mice were resistant to tumor growth inhibition by lapatinib and/or doxorubicin and exhibited impaired T‐cell activation and reduced T‐cell infiltration of the tumor in response to drug treatment. Furthermore, Stat1‐deficiency resulted in reduced expression of the T‐cell chemotactic factors CXCL9, CXCL10, and CXCL11 in the tumor epithelium. The inhibition of TAM infiltration of the tumor by doxorubicin and the immunosuppressive function of TAMs were found to be Stat1 independent. Taken together, the results point to an important contribution toward enhancing T‐cell and IFN‐γ‐based immunity by lapatinib as well as doxorubicin and emphasize the role of Stat1 in building an effective antitumor immune response.

In situ proliferation contributes to accumulation of tumor-associated macrophages in spontaneous mammary tumors

Infiltration of a neoplasm with tumor‐associated macrophages (TAMs) is considered an important negative prognostic factor and is functionally associated with tumor vascularization, accelerated growth, and dissemination. However, the ontogeny and differentiation pathways of TAMs are only incompletely characterized. Here, we report that intense local proliferation of fully differentiated macrophages rather than low‐pace recruitment of blood‐borne precursors drives TAM accumulation in a mouse model of spontaneous mammary carcinogenesis, the MMTVneu strain. TAM differentiation and expansion is regulated by CSF1, whose expression is directly controlled by STAT1 at the gene promoter level. These findings appear to be also relevant for human breast cancer, in which an interrelationship between STAT1, CSF1, and macrophage marker expression was identified. We propose that, akin to various MU subtypes in nonmalignant tissues, local proliferation and CSF1 play a vital role in the homeostasis of TAMs.

High STAT1 mRNA levels but not its tyrosine phosphorylation are associated with macrophage infiltration and bad prognosis in breast cancer

BackgroundSTAT1 has been attributed a function as tumor suppressor. However, in breast cancer data from microarray analysis indicated a predictive value of high mRNA expression levels of STAT1 and STAT1 target genes belonging to the interferon-related signature for a poor response to therapy. To clarify this issue we have determined STAT1 expression levels and activation by different methods, and investigated their association with tumor infiltration by immune cells. Additionally, we evaluated the interrelationship of these parameters and their significance for predicting disease outcome.MethodsExpression of STAT1, its target genes SOCS1, IRF1, CXCL9, CXCL10, CXCL11, IFIT1, IFITM1, MX1 and genes characteristic for immune cell infiltration (CD68, CD163, PD-L1, PD-L2, PD-1, CD45, IFN-γ, FOXP3) was determined by RT-PCR in two independent cohorts comprising 132 breast cancer patients. For a subset of patients, protein levels of total as well as serine and tyrosine-phosphorylated STAT1 were ascertained by immunohistochemistry or immunoblotting and protein levels of CXCL10 by ELISA.ResultsmRNA expression levels of STAT1 and STAT1 target genes, as well as protein levels of total and serine-phosphorylated STAT1 correlated with each other in neoplastic tissue. However, there was no association between tumor levels of STAT1 mRNA and tyrosine-phosphorylated STAT1 and between CXCL10 serum levels and CXCL10 expression in the tumor. Tumors with increased STAT1 mRNA amounts exhibited elevated expression of genes characteristic for tumor-associated macrophages and immunosuppressive T lymphocytes. Survival analysis revealed an association of high STAT1 mRNA levels and bad prognosis in both cohorts. A similar prognostically relevant correlation with unfavorable outcome was evident for CXCL10, MX1, CD68, CD163, IFN-γ, and PD-L2 expression in at least one collective. By contrast, activation of STAT1 as assessed by the level of STAT1-Y701 phosphorylation was linked to positive outcome. In multivariate Cox regression, the predictive power of STAT1 mRNA expression was lost when including expression of CXCL10, MX1 and CD68 as confounders.ConclusionsOur study confirms distinct prognostic relevance of STAT1 expression levels and STAT1 tyrosine phosphorylation in breast cancer patients and identifies an association of high STAT1 levels with elevated expression of STAT1 target genes and markers for infiltrating immune cells.

Momelotinib inhibits ACVR1/ALK2, decreases hepcidin production and ameliorates anemia of chronic disease in rodents

Patients with myelofibrosis (MF) often develop anemia and frequently become dependent on red blood cell transfusions. Results from a phase 2 study for the treatment of MF with the Janus kinase 1/2 (JAK1/2) inhibitor momelotinib (MMB) demonstrated that MMB treatment ameliorated anemia, which was unexpected for a JAK1/2 inhibitor, because erythropoietin-mediated JAK2 signaling is essential for erythropoiesis. Using a rat model of anemia of chronic disease, we demonstrated that MMB treatment can normalize hemoglobin and red blood cell numbers. We found that this positive effect is driven by direct inhibition of the bone morphogenic protein receptor kinase activin A receptor, type I (ACVR1), and the subsequent reduction of hepatocyte hepcidin production. Of note, ruxolitinib, a JAK1/2 inhibitor approved for the treatment of MF, had no inhibitory activity on this pathway. Further, we demonstrated the effect of MMB is not mediated by direct inhibition of JAK2-mediated ferroportin (FPN1) degradation, because neither MMB treatment nor myeloid-specific deletion of JAK2 affected FPN1 expression. Our data support the hypothesis that the improvement of inflammatory anemia by MMB results from inhibition of ACVR1-mediated hepcidin expression in the liver, which leads to increased mobilization of sequestered iron from cellular stores and subsequent stimulation of erythropoiesis.

Lipocalin 2 drives neutrophilic inflammation in alcoholic liver disease

BACKGROUND & AIMS Alcoholic steatohepatitis (ASH) is characterised by neutrophil infiltration that contributes to hepatic injury and disease. Lipocalin-2 (LCN2) was originally identified as siderophore binding peptide in neutrophils, which exerted tissue protective effects in several disease models. Here we investigate the role of LCN2 in the pathogenesis of alcohol-induced liver injury. METHODS We compared hepatic LCN2 expression in ASH patients, alcoholic cirrhosis patients without evidence of ASH and patients with non-alcoholic fatty liver disease (NAFLD; i.e. simple steatosis). To mechanistically dissect LCN2 function in alcohol-induced liver injury, we subjected wild-type (WT) and Lcn2-deficient (Lcn2(-/-)) mice to the Lieber-DeCarli diet containing 5% ethanol (EtOH) or isocaloric maltose. Adoptive transfer experiments were performed to track neutrophil migration. Furthermore, we tested the effect of antibody-mediated LCN2 neutralisation in an acute model of ethanol-induced hepatic injury. RESULTS Patients with ASH exhibited increased hepatic LCN2 immunoreactivity compared to patients with alcoholic cirrhosis or simple steatosis, which mainly localised to neutrophils. Similarly, ethanol-fed mice exhibited increased LCN2 expression that mainly localised to leukocytes and especially neutrophils. Lcn2(-/-) mice were protected from alcoholic liver disease (ALD) as demonstrated by reduced neutrophil infiltration, liver injury and hepatic steatosis compared to WT controls. Adoptive transfers revealed that neutrophil-derived LCN2 critically determines hepatic neutrophil immigration and persistence during chronic alcohol exposure. Antibody-mediated neutralisation of LCN2 protected from hepatic injury and neutrophilic infiltration after acute alcohol challenge. CONCLUSIONS LCN2 drives ethanol-induced neutrophilic inflammation and propagates the development of ALD. Despite a critical role for LCN2 in immunity and infection, pharmacological neutralisation of LCN2 might be of promise in ALD.

MMTV-neu mice deficient in STAT1 are susceptible to develop ovarian teratomas.

Signal transducer and activator of transcription 1 (STAT1) serves in the protection of the organism against pathogens and other harmful insults. It is implicated in innate immune response, immunosurveillance, tumor-suppression, and the response to genotoxic as well as oxidative stress. We report here that 9 of 140 examined STAT1 deficient mouse mammary tumor virus-neu (MMTV-neu) mice developed differentiated ovarian teratomas, which histologically resemble benign dermatoid cysts. Conventional karyotyping revealed diploidy without structural rearrangements of the chromosomes. STAT1 proficient MMTV-neu mice with the same genetic background (FVB/N), and STAT1 deficient C57BL/6 mice failed to develop this type of tumor. This indicates that STAT1 deficiency promotes teratoma formation and this depends on MMTV-neu expression and/or the genetic background. Since ovarian teratomas are considered to develop as a consequence of alterations in the maturation of oocytes and follicular cells, we compared the ovaries from non-tumor bearing STAT1 deficient and proficient MMTV-neu mice. No detectable alterations in the number and proportion of the different follicular developmental stages were detected, implying the absence of non-redundant functions of STAT1 in normal folliculogenesis, as well as in follicular atresia. However, strong staining for STAT1 was detectable in granulosa and theca cells. These results point to a role for STAT1 in protecting from teratoma formation in a later step of tumorigenesis, e.g. by inducing apoptosis and eliminating premature or aberrantly formed follicles which have the potential to transform into teratomas.

Genetic and Dietary Iron Overload Differentially Affect the Course of Salmonella Typhimurium Infection

Genetic and dietary forms of iron overload have distinctive clinical and pathophysiological features. HFE-associated hereditary hemochromatosis is characterized by overwhelming intestinal iron absorption, parenchymal iron deposition, and macrophage iron depletion. In contrast, excessive dietary iron intake results in iron deposition in macrophages. However, the functional consequences of genetic and dietary iron overload for the control of microbes are incompletely understood. Using Hfe+/+ and Hfe−/− mice in combination with oral iron overload in a model of Salmonella enterica serovar Typhimurium infection, we found animals of either genotype to induce hepcidin antimicrobial peptide expression and hypoferremia following systemic infection in an Hfe-independent manner. As predicted, Hfe−/− mice, a model of hereditary hemochromatosis, displayed reduced spleen iron content, which translated into improved control of Salmonella replication. Salmonella adapted to the iron-poor microenvironment in the spleens of Hfe−/− mice by inducing the expression of its siderophore iron-uptake machinery. Dietary iron loading resulted in higher bacterial numbers in both WT and Hfe−/− mice, although Hfe deficiency still resulted in better pathogen control and improved survival. This suggests that Hfe deficiency may exert protective effects in addition to the control of iron availability for intracellular bacteria. Our data show that a dynamic adaptation of iron metabolism in both immune cells and microbes shapes the host-pathogen interaction in the setting of systemic Salmonella infection. Moreover, Hfe-associated iron overload and dietary iron excess result in different outcomes in infection, indicating that tissue and cellular iron distribution determines the susceptibility to infection with specific pathogens.

The BH3-only protein BIM contributes to late-stage involution in the mouse mammary gland

After cessation of lactation, involution of the mouse mammary gland proceeds in two distinct phases, a reversible and an irreversible one, which leads to the death and removal of alveolar cells. Cell death is preceded by the loss of STAT5 activity, which abrogates cell differentiation and gain of STAT3 activity. Despite early observations implicating BCL2 (B cell lymphoma 2) family proteins in this process, recent evidence suggests that STAT3-controlled cathepsin activity is most critical for cell death at the early stage of involution. Somewhat surprisingly, this cell death associates with but does not depend on the activation of pro-apoptotic effector caspases. However, transgenic overexpression of BCL2, that blocks caspase activation, delays involution while conditional deletion of BclX accelerates this process, suggesting that BCL2 family proteins are needed for the effective execution of involution. Here, we report on the transcriptional induction of multiple pro-apoptotic BCL2 family proteins of the ‘BH3-only’ subgroup during involution and the rate-limiting role of BIM in this process. Loss of Bim delayed epithelial cell clearance during involution after forced weaning in mice, whereas the absence of related Bmf had minor and loss of Bad or Noxa no impact on this process. Consistent with a contribution of BCL2 family proteins to the second wave of cell death during involution, loss of Bim reduced the number of apoptotic cells in this irreversible phase. Notably, the expression changes observed within the BCL2 family did not depend on STAT3 signalling, in line with its initiating role early in the process, but rather appear to result from relief of repression by STAT5. Our findings support the existence of a signalling circuitry regulating the irreversible phase of involution in mice by engaging BH3-only protein-driven mitochondrial apoptosis.

Secretoneurin gene therapy improves hind limb and cardiac ischaemia in Apo E−/− mice without influencing systemic atherosclerosis

AIMS Hypercholesterolaemia is a major risk factor for cardiovascular diseases and has been shown to influence angiogenesis in the hind limb ischaemia (HLI) model. The impaired up-regulation of angiogenic factors seems to be one of the underlying mechanisms for reduced vessel formation. Since we found that secretoneurin (SN) is up-regulated in hypoxic skeletal muscle cells and exerts beneficial effects in myocardial and HLI, we hypothesized that SN therapy might improve neovascularization in hypercholesterolaemic Apo E(-/-) (Apo E knockout) mice suffering from an impaired vascular response. METHODS AND RESULTS For in vitro experiments, endothelial cells (ECs) were incubated with oxidized low-density lipoprotein (oxLDL) to mimic hypercholesterolaemia. EC function was impaired by oxLDL, but SN induced EC proliferation and in vitro tube formation under these conditions. In the HLI model, injection of SN plasmid resulted in a significant better outcome regarding blood flow recovery, amputation rate, and vessel density. In the myocardial infarction (MI) model, the SN group showed improvement in cardiac parameters. Aortic plaque area was not influenced by local SN injection. Interestingly, SN-induced recruitment of angiogenic monocytic cells was abolished under hypercholesterolaemia. CONCLUSIONS SN gene therapy exerts beneficial effects in cardiovascular animal models in Apo E(-/-) mice without influencing atherosclerosis and might qualify as a promising therapy for cardiovascular disorders.