Erythropoietin enhances Kupffer cell number and activity in the challenged liver.
ABSTRACT: Erythropoietin (EPO) is the main hormone driving mammalian erythropoiesis, with activity mediated via the surface receptor, EPO-R, on erythroid progenitor cells. Recombinant human EPO is currently used clinically for the treatment of anemia in patients with end-stage renal disease, and in certain cancer patients suffering from anemia induced either by the tumor itself or by chemotherapy. EPO-R expression is also detected in non-erythroid cells, including macrophages present in the peritoneum, spleen, and bone marrow (BM). Here we demonstrate that Kupffer cells (KCs) - the liver-resident macrophages - are EPO targets. We show that, in vitro, EPO initiated intracellular signalling and enhanced phagocytosis in a rat KC line (RKC-2) and in sorted KCs. Moreover, continuous EPO administration in mice, resulted in an increased number of KCs, up-regulation of liver EPO-R expression and ???elevated? production of the monocyte chemoattractant CCL2, with corresponding egress of Ly6Chi monocytes from the BM. In a model of acute acetaminophen-induced liver injury, EPO administration increased the recruitment of Ly6Chi monocytes and neutrophils to the liver. Taken together, our results reveal a new role for EPO in stimulating KC proliferation and phagocytosis, and in recruiting Ly6Chi monocytes in response to liver injury.
Project description:Kupffer cells (KCs), the resident tissue macrophages of the liver, play a crucial role in the clearance of pathogens and other particulate materials that reach the systemic circulation. Recent studies have identified KCs as a yolk sac-derived resident macrophage population that is replenished independently of monocytes in the steady state. Although it is now established that following local tissue injury, bone marrow derived monocytes may infiltrate the tissue and differentiate into macrophages, the extent to which newly differentiated macrophages functionally resemble the KCs they have replaced has not been extensively studied.We studied the two populations of KCs using intravital microscopy, morphometric analysis and gene expression profiling. An ion homeostasis gene signature, including genes associated with scavenger receptor function and extracellular matrix deposition, allowed discrimination between these two KC sub-types.Bone marrow derived "KCs" accumulating as a result of genotoxic injury, resemble but are not identical to their yolk sac counterparts. Reflecting the differential expression of scavenger receptors, yolk sac-derived KCs were more effective at accumulating acetylated low density lipoprotein, whereas surprisingly, they were poorer than bone marrow-derived KCs when assessed for uptake of a range of bacterial pathogens. The two KC populations were almost indistinguishable in regard to i) response to lipopolysaccharide challenge, ii) phagocytosis of effete red blood cells and iii) their ability to contain infection and direct granuloma formation against Leishmania donovani, a KC-tropic intracellular parasite.Bone marrow-derived KCs differentiate locally to resemble yolk sac-derived KC in most but not all respects, with implications for models of infectious diseases, liver injury and bone marrow transplantation. In addition, the gene signature we describe adds to the tools available for distinguishing KC subpopulations based on their ontology.Liver macrophages play a major role in the control of infections in the liver and in the pathology associated with chronic liver diseases. It was recently shown that liver macrophages can have two different origins, however, the extent to which these populations are functionally distinct remains to be fully addressed. Our study demonstrates that whilst liver macrophages share many features in common, regardless of their origin, some subtle differences in function exist.Gene expression data are available from the European Bioinformatics Institute ArrayExpress data repository (accession number E-MTAB-4954).
Project description:Rationale: Kupffer cells (KCs) play a crucial role in liver immune homeostasis through interacting with other immune cells and liver sinusoidal endothelial cells (LSECs). However, how KCs exactly interact with these cells for maintaining the homeostasis still require the further investigation. CXCL10 is a chemokine that has been implicated in chemoattraction of monocytes, T cells, NK cells, and dendritic cells, and promotion of T cell adhesion to endothelial cells. Although CXCL10 is also known to participate in the pathogenesis of hepatic inflammation, the degree to which it is functionally involved in the crosstalk between immune cells and regulation of immune response is still unclear.Methods: To dynamically investigate the function of KCs, we used our recently developed rapid cell ablation model, intermedilysin (ILY)/human CD59 (hCD59)-mediated cell ablation tool, to selectively ablate KC pool under normal condition or concanavalin A (Con A)- induced hepatitis. At certain time points after KCs ablation, we performed flow cytometry to monitor the amount of hepatic infiltrating immune cells. mRNA array was used to detect the change of hepatic cytokines and chemokines levels. Cytokines and chemokines in the serum were further measured by LEGENDplexTM mouse proinflammatory chemokine panel and inflammation panel. Evans blue staining and transmission electron microscopy were used to investigate the interaction between KCs and LSECs in steady condition. CXCL10 neutralizing antibody and CXCL10 deficient mouse were used to study the role of CXCL10 in immune cell migration and pathogenesis of Con A-induced hepatitis.Results: At steady state, elimination of KCs results in a reduction of hepatic infiltrating monocytes, T, B, and NK cells and a list of cytokines and chemokines at transcriptional level. In the meantime, the depletion of KCs resulted in increased sinusoidal vascular permeability. In the pathological condition, the KCs elimination rescues Con A-induced acute hepatitis through suppressing proinflammatory immune responses by down-regulation of hepatitis-associated cytokines/chemokines in serum such as CXCL10, and recruitment of infiltrating immune cells (monocytes, T, B, and NK cells). We further documented that deficiency or blockade of CXCL10 attenuated the development of Con A-induced hepatitis associated with reduction of the infiltrating monocytes, especially inflammatory Ly6Chi monocytes.Conclusions: This study supports the notion that KCs actively interact with immune cells and LSECs for maintaining immune response and liver homeostasis. Our data indicate that the interplay between KCs and infiltrated monocytes via CXCL10 contribute to Con A-induced hepatitis.
Project description:Many patients with anemia fail to respond to treatment with erythropoietin (Epo), a commonly used hormone that stimulates erythroid progenitor production and maturation by human BM or by murine spleen. The protein product of growth arrest-specific gene 6 (Gas6) is important for cell survival across several cell types, but its precise physiological role remains largely enigmatic. Here, we report that murine erythroblasts released Gas6 in response to Epo and that Gas6 enhanced Epo receptor signaling by activating the serine-threonine kinase Akt in these cells. In the absence of Gas6, erythroid progenitors and erythroblasts were hyporesponsive to the survival activity of Epo and failed to restore hematocrit levels in response to anemia. In addition, Gas6 may influence erythropoiesis via paracrine erythroblast-independent mechanisms involving macrophages. When mice with acute anemia were treated with Gas6, the protein normalized hematocrit levels without causing undesired erythrocytosis. In a transgenic mouse model of chronic anemia caused by insufficient Epo production, Gas6 synergized with Epo in restoring hematocrit levels. These findings may have implications for the treatment of patients with anemia who fail to adequately respond to Epo.
Project description:Multiple myeloma (MM) is a plasma cell malignancy, characterized by osteolytic lesions and monoclonal immunoglobulins. The anemia, accompanying the disease is often treated with recombinant human EPO. Diverse non-erythropoietic effects of EPO have led us to question its combined action on the immune system and bone in the 5T33MM mouse model. EPO administration to MM mice attenuated disease progression as demonstrated by a decrease in serum MM IgG2b, splenic CD138 expressing cells, IL-6 and ROR?? transcripts in bone marrow (BM). IFN-? transcript levels and macrophages (F4/80(+)CD11b(+)) in the BM both increased ~1.5 fold in the EPO-treated MM mice. In-vitro, EPO stimulated phagocytosis of 5T33MM cells (+30%) by BM-derived macrophages. In contrast, high-resolution microCT analysis of distal femurs revealed EPO-associated bone loss in both healthy and 5T33MM mice. EPO significantly increased expression of the osteoclastogenic nuclear factor-kappa B ligand (RANKL) in healthy mice, but not in MM mice, likely due to antagonizing effects on MM progression. Thus, in MM, EPO may act as a double-edged-sword stimulating immune response, while accelerating bone resorption, possibly via direct action on BM macrophages. This study supports a prudent approach of treating anemia in MM patients, aiming to maintain EPO-associated anti-MM effects, while considering bone damage.
Project description:Anemia is a known driver for hypoxia inducible factor (HIF) which leads to increased renal erythropoietin (EPO) synthesis. Bone marrow (BM) EPO receptor (EPOR) signals are transduced through a JAK2-STAT5 pathway. The origins of anemia of chronic kidney disease (CKD) are multifactorial, including impairment of both renal EPO synthesis as well as intestinal iron absorption. We investigated the HIF- EPO- EPOR axis in kidney, BM and proximal tibia in anemic juvenile CKD rats.CKD was induced by 5/6 nephrectomy in young (20 days old) male Sprague-Dawley rats while C group was sham operated. Rats were sacrificed 4 weeks after CKD induction and 5 minutes after a single bolus of IV recombinant human EPO. An additional control anemic (C-A) group was daily bled for 7 days.Hemoglobin levels were similarly reduced in CKD and C-A (11.4 ± 0.3 and 10.8±0.2 Vs 13.5±0.3 g/dL in C, p<0.0001). Liver hepcidin mRNA was decreased in CA but increased in CKD. Serum iron was unchanged while transferrin levels were mildly decreased in CKD. Kidney HIF2? protein was elevated in C-A but unchanged in CKD. Kidney EPO protein and mRNA levels were unchanged between groups. However, BM EPO protein (which reflects circulating EPO) was increased in C-A but remained unchanged in CKD. BM and proximal tibia EPOR were unchanged in C-A but decreased in CKD. Proximal tibial phospho-STAT5 increased after the EPO bolus in C but not in CKD.Compared to blood loss, anemia in young CKD rats is associated with inappropriate responses in the HIF-EPO-EPO-R axis: kidney HIF2? and renal EPO are not increased, BM and bone EPOR levels, as well as bone pSTAT5 response to EPO are reduced. Thus, anemia of CKD may be treated with additional therapeutic avenues beyond iron and EPO supplementation.
Project description:Fungal dissemination into the bloodstream is a critical step leading to invasive fungal infections. Here, using intravital imaging, we show that Kupffer cells (KCs) in the liver have a prominent function in the capture of circulating Cryptococcus neoformans and Candida albicans, thereby reducing fungal dissemination to target organs. Complement C3 but not C5, and complement receptor CRIg but not CR3, are involved in capture of C. neoformans. Internalization of C. neoformans by KCs is subsequently mediated by multiple receptors, including CR3, CRIg, and scavenger receptors, which work synergistically along with C5aR signaling. Following phagocytosis, the growth of C. neoformans is inhibited by KCs in an IFN-γ independent manner. Thus, the liver filters disseminating fungi from circulation via KCs, providing a mechanistic explanation for the enhanced risk of cryptococcosis among individuals with liver diseases, and suggesting a therapeutic strategy to prevent fungal dissemination through enhancing KC functions.
Project description:Intestinal mononuclear phagocytes (MPs) comprise dendritic cells (DCs) and macrophages (M?s) that play different roles in response to Salmonella infection. After phagocytosis, DCs expressing CD103 transport Salmonella from the intestinal tract to the mesenteric lymph nodes (MLN) and induce adaptive immune responses whereas resident M?s expressing CX3CR1 capture bacteria in the lumen and reside in the lamina propria (LP) where they induce a local immune response. CX3CR1+ M?s are generated from Ly6Chi monocytes that enter the colonic mucosa and differentiate locally. We previously demonstrated that the probiotic yeast Saccharomyces boulardii CNCM I-745 (S.b) prevents infection by Salmonella enterica serovar Typhimurium (ST), decreases ST translocation to the peripheral organs and modifies the pro-and anti-inflammatory cytokine profiles in the gut. In the present study, we investigated the effect of S.b on the migratory CD103+ DCs and the resident CX3CR1+ M?s. MPs were isolated from the LP of streptomycin-treated mice infected by ST with or without S.b treatment before or during the infection. In S.b-pretreated mice, we observed a decrease of the CD103+ DCs in the LP that was associated with the drop of ST recovery from MLN. Interestingly, S.b induced an infiltration of LP by classical Ly6Chi monocytes, and S.b modified the monocyte-M? maturation process in ST-infected mice. Our results showed that S.b treatment induced the expansion of Ly6Chi monocytes in the blood as well as in the bone marrow (BM) of mice, thus contributing to the M? replenishment in LP from blood monocytes. In vitro experiments conducted on BM cells confirmed that S.b induced the expansion of CX3CR1+ M?s and concomitantly ST phagocytosis. Altogether, these data demonstrate that Saccharomyces boulardii CNCM I-745 modulates the innate immune response. Although here, we cannot explicitly delineate direct effects on ST from innate immunity, S. b-amplified innate immunity correlated with partial protection from ST infection. This study shows that S.b can induce the expansion of classical monocytes that are precursors of resident M?s in the LP.
Project description:It is well established that Ly6Chi monocytes develop from common monocyte progenitors (cMoPs) and reside in the bone marrow (BM) until they are mobilized into the circulation. In our study, we found that BM Ly6Chi monocytes are not a homogenous population, as current data would suggest. Using computational analysis approaches to interpret multidimensional datasets, we demonstrate that BM Ly6Chi monocytes consist of two distinct subpopulations (CXCR4hi and CXCR4lo subpopulations) in both mice and humans. Transcriptome studies and in vivo assays revealed functional differences between the two subpopulations. Notably, the CXCR4hi subset proliferates and is immobilized in the BM for the replenishment of functionally mature CXCR4lo monocytes. We propose that the CXCR4hi subset represents a transitional premonocyte population, and that this sequential step of maturation from cMoPs serves to maintain a stable pool of BM monocytes. Additionally, reduced CXCR4 expression on monocytes, upon their exit into the circulation, does not reflect its diminished role in monocyte biology. Specifically, CXCR4 regulates monocyte peripheral cellular activities by governing their circadian oscillations and pulmonary margination, which contributes toward lung injury and sepsis mortality. Together, our study demonstrates the multifaceted role of CXCR4 in defining BM monocyte heterogeneity and in regulating their function in peripheral tissues.
Project description:We investigated serial changes of the Kupffer cell (KC) function and hepatic oxygen saturation (sO2) using contrast-enhanced ultrasound imaging (CEUS) and photoacoustic imaging (PAI) in preneoplastic changes during cholangiocarcinogenesis induced by obstructive cholangitis and N-nitrosodimethylamine in a mouse model. The CEUS and PAI were performed to assess Sonazoid contrast agent uptake by KC and changes in the sO2 of liver parenchyma. An extensive bile ductular reaction, cystic dilatation, and epithelial hyperplasia with dysplastic changes were noted in the experimental group. During the preneoplastic changes, the parenchymal echogenicity on the Kupffer-phase of CEUS was continuously decreased in the experimental group, and which means that the Sonazoid phagocytosis by KC was decreased. The number of KCs was increased in the CD68 analysis, indicating functionally impaired KCs. There was a simultaneous serial decrease in sO2 on PAI measurement of the experimental group during the preneoplastic changes. The experimental group also showed significantly higher expression of hypoxia-inducible factor-1? and vascular endothelial growth factor protein. Our study demonstrated that KC dysfunction and hypoxic environmental changes were the factors influencing preneoplastic change during cholangiocarcinogenesis, and we could non-invasively monitor these changes using CEUS and PAI.
Project description:Purpose:Kupffer cells (KCs) present dysfunctional immunity capacity among the diabetes mellitus patients. This study aims to investigate whether Lidocaine could reverse dysfunctions of KCs, in terms of phagocytosis, granulocyte recruitment and inflammatory mediator secretion. Methods:db/db and C57BL/6 mice were employed to establish diabetic and nondiabetic models. Upon intravenous injection of Lidocaine, KCs were isolated and cultured ex vivo. The functions of phagocytosis, recruiting granulocytes and inflammatory mediator secretion in KCs were compared between Lidocaine-treated and untreated (control) groups. Results:Comparing with nondiabetic mice, KCs in diabetic mice presented reduced phagocytosis, activated granulocyte recruitment, increased expression of intercellular cell adhesion molecule-1 (ICAM-1) and activated levels of inflammatory mediators. With Lidocaine injection, phagocytic functions of KCs in diabetic mice were improved significantly; in contrast, recruitment of granulocytes, expression of ICAM-1 and secretion of inflammatory mediators were reduced markedly. However, Lidocaine intervention did not alter KC functions in phagocytosis, granulocyte recruitment, ICAM-1 expression or inflammatory mediator secretion among nondiabetic mice. Conclusion:Lidocaine reversed diabetes-related dysfunctions of KCs in terms of phagocytosis, granulocyte recruitment, ICAM-1 expression or inflammatory mediator secretion.