[Show abstract][Hide abstract] ABSTRACT: Tumors contain a heterogeneous myeloid fraction comprised of discrete MHC-IIhi and MHC-IIlo tumor-associated macrophage (TAM) subpopulations that originate from Ly6Chi monocytes. However, the mechanisms regulating the abundance and phenotype of distinct TAM subsets remain unknown. Here, we investigated the role of macrophage colony-stimulating factor (M-CSF) in TAM differentiation and polarization in different mouse tumor models. We demonstrate that treatment of tumor-bearing mice with a blocking anti-M-CSFR monoclonal antibody resulted in a reduction of mature TAMs due to impaired recruitment, extravasation, proliferation, and maturation of their Ly6Chi monocytic precursors. M-CSFR signaling blockade shifted the MHC-IIlo/MHC-IIhi TAM balance in favor of the latter as observed by the preferential differentiation of Ly6Chi monocytes into MHC-IIhi TAMs. In addition, the genetic and functional signatures of MHC-IIlo TAMs were downregulated upon M-CSFR blockade, indicating that M-CSFR signaling shapes the MHC-IIlo TAM phenotype. Conversely, granulocyte macrophage (GM)-CSFR had no effect on the mononuclear tumor infiltrate or relative abundance of TAM subsets. However, GM-CSFR signaling played an important role in fine-tuning the MHC-IIhi phenotype. Overall, our data uncover the multifaceted and opposing roles of M-CSFR and GM-CSFR signaling in governing the phenotype of macrophage subsets in tumors, and provide new insight into the mechanism of action underlying M-CSFR blockade.
Cancer Research 11/2015; DOI:10.1158/0008-5472.CAN-15-0869 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: IL-4/IL-13-induced alternatively activated macrophages (M(IL-4/IL-13), AAMs or M2) are known to express E-cadherin, enabling them to engage in heterotypic cellular interactions and IL-4-driven macrophage fusion in vitro. Here we show that E-cadherin overexpression in Raw 264.7 macrophages inhibits their inflammatory response to LPS stimulation, as demonstrated by a reduced secretion of inflammatory mediators like interleukin (IL)-6, tumor necrosis factor (TNF) and nitric oxide (NO). To study the function of E-cadherin in M(IL-4/IL-13) macrophages in vivo, we generated macrophage-specific E-cadherin-deficient C57BL/6 mice. Using this new tool, we analyzed immunological parameters during two typical AAM-associated Th2-driven diseases and assessed Th2-associated granuloma formation. Although E-cadherin is strongly induced in AAMs during Taenia crassiceps helminth infections and allergic airway inflammation, its deletion in macrophages does not affect the course of both Th2 cytokine-driven diseases. Moreover, macrophage E-cadherin expression is largely redundant for granuloma formation around Schistosoma mansoni ova. Overall, we conclude that E-cadherin is a valuable AAM marker which suppresses the inflammatory response when overexpressed. Yet E-cadherin deletion in macrophages does not affect M(LPS+IFNγ) and M(IL-4) polarization in vitro, nor in vivo macrophage function, at least in the conditions tested.
[Show abstract][Hide abstract] ABSTRACT: African trypanosomosis is a chronic debilitating disease affecting the health and economic well-being of developing countries. The immune response during African trypanosome infection consisting of a strong proinflammatory M1-type activation of the myeloid phagocyte system (MYPS) results in iron deprivation for these extracellular parasites. Yet, the persistence of M1-type MYPS activation causes the development of anemia (anemia of chronic disease, ACD) as a most prominent pathological parameter in the mammalian host, due to enhanced erythrophagocytosis and retention of iron within the MYPS thereby depriving iron for erythropoiesis. In this review we give an overview of how parasites acquire iron from the host and how iron modulation of the host MYPS affects trypanosomosis-associated anemia development. Finally, we also discuss different strategies at the level of both the host and the parasite that can/might be used to modulate iron availability during African trypanosome infections.
[Show abstract][Hide abstract] ABSTRACT: Monocytes consist of two well-defined subsets, the Ly6C+ and Ly6C- monocytes. Both CD11b+ myeloid cells populations have been proposed to infiltrate tissues during inflammation. While infiltration of Ly6C+ monocytes is an established pathogenic factor during hepatic inflammation, the role of Ly6C- monocytes remains elusive. Mice suffering experimental African trypanosome infection die from systemic inflammatory response syndrome (SIRS) that is initiated by phagocytosis of parasites by liver myeloid cells and culminates in apoptosis/necrosis of liver myeloid and parenchymal cells that reduces host survival. C57BL/6 mice are considered as trypanotolerant to Trypanosoma congolense infection. We have reported that in these animals, IL-10, produced among others by myeloid cells, limits the liver damage caused by pathogenic TNF-producing Ly6C+ monocytes, ensuring prolonged survival. Here, the heterogeneity and dynamics of liver myeloid cells in T. congolense-infected C57/BL6 mice was further dissected. Moreover, the contribution of Ly6C- monocytes to trypanotolerance was investigated. By using FACS analysis and adoptive transfer experiments, we found that the accumulation of Ly6C- monocytes and macrophages in the liver of infected mice coincided with a drop in the pool of Ly6C+ monocytes. Pathogenic TNF mainly originated from Ly6C+ monocytes while Ly6C- monocytes and macrophages were major and equipotent sources of IL-10 within myeloid cells. Moreover, Nr4a1 (Nur77) transcription factor-dependent Ly6C- monocytes exhibited IL-10-dependent and cell contact-dependent regulatory properties contributing to trypanotolerance by suppressing the production of TNF by Ly6C+ monocytes and by promoting the differentiation of the latter cells into macrophages. Thus, Ly6C- monocytes can dampen liver damage caused by an extensive Ly6C+ monocyte-associated inflammatory immune response in T. congolense trypanotolerant animals. In a more general context, Ly6C- or Ly6C+ monocyte targeting may represent a therapeutic approach in liver pathogenicity induced by chronic infection.
[Show abstract][Hide abstract] ABSTRACT: Author Summary
Extracellular trypanosomes, causative agents of sleeping sickness and Nagana, threaten human and animal health throughout the world. Anemia is a hallmark feature of virtually every type of trypanosome infection. During the early phase of experimental murine trypanosomosis, acute anemia occurs as witnessed by a 50% reduction in red blood cells within a 48 hour time span. The acute nature of this phenomenon suggests the implication of a consumptive process such as erythrophagocytosis. However, due to the multiple significant drawbacks of the presently used phagocytosis techniques, this has never been straightforwardly demonstrated. Here we developed a new erythrophagocytosis assay based on the labeling of red blood cells with the acid-sensitive dye pHrodo. This assay unequivocally distinguishes erythrophagocytozing cells in vivo and in vitro via flow cytometry and fluorescent microscopy. Using this new assay, we show that the acute anemia during experimental trypanosomosis is a result of enhanced erythrophagocytosis by activated liver monocytic cells and neutrophils as well as by activated splenic macrophages. Moreover, the red blood cell membrane composition and stability are altered during the infection, priming them for enhanced clearance by the myeloid phagocytic system.
[Show abstract][Hide abstract] ABSTRACT: Pancreas injury by partial duct ligation (PDL) activates a healing response, encompassing β-cell neogenesis and proliferation. Macrophages were recently shown to promote β-cell proliferation after PDL, but they remain poorly characterized. We assessed myeloid cell diversity and the factors driving myeloid cell dynamics following acute pancreas injury by PDL. In naive and sham-operated pancreas, the myeloid cell compartment consisted mainly of two distinct tissue-resident macrophage types, designated MHC-IIlo and MHC-IIhi macrophages, the latter being predominant. MHC-IIlo and MHC-IIhi pancreas macrophages differed at the molecular level, with MHC-IIlo macrophages being more M2-activated. After PDL, there was an early surge of Ly6Chi monocyte infiltration in the pancreas, followed by a transient MHC-IIlo macrophage peak and ultimately a restoration of the MHC-IIhi macrophage-dominated steady-state equilibrium. These intricate macrophage dynamics in PDL pancreas depended on monocyte recruitment by CCR2 and M-CSFR as well as on M-CSFR-dependent local macrophage proliferation. Functionally, MHC-IIlo macrophages were more angiogenic. We further demonstrated that, at least in CCR2-KO mice, tissue macrophages, rather than Ly6Chi monocyte-derived macrophages, contributed to β-cell proliferation. Together, our study fully characterizes the macrophage subsets in the pancreas and clarifies the complex dynamics of macrophages after PDL injury.This article is protected by copyright. All rights reserved
European Journal of Immunology 01/2015; 45(5). DOI:10.1002/eji.201445013 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The current review article describes the functional relationship between tumor-associated macrophages (TAM) as key cellular contributors to cancer malignancy on the one hand and macrophage-colony-stimulating factor (M-CSF or CSF-1) as an important molecular contributor on the other. We recapitulate the available data on expression of M-CSF and the M-CSF receptor (M-CSFR) in human tumor tissue as constituents of a stromal macrophage signature and on the limits of the predictive and prognostic value of plasma M-CSF levels. After providing an update on current insights into the nature of TAM heterogeneity at the level of M1/M2 phenotype and TAM subsets, we give an overview of experimental evidence, based on genetic, antibody-mediated, and pharmacological disruption of M-CSF/M-CSFR signaling, for the extent to which M-CSFR signaling can not only determine the TAM quantity, but can also contribute to shaping the phenotype and heterogeneity of TAM and other related tumor-infiltrating myeloid cells (TIM). Finally, we review the accumulating information on the - sometimes conflicting - effects blocking M-CSFR signaling may have on various aspects of cancer progression such as tumor growth, invasion, angiogenesis, metastasis, and resistance to therapy and we thereby discuss in how far these different effects actually reflect a contribution of TAM.
Frontiers in Immunology 10/2014; 5:489. DOI:10.3389/fimmu.2014.00489
[Show abstract][Hide abstract] ABSTRACT: Kupffer cells (KCs) are liver resident macrophages which are important for tissue homeostasis and have been implicated in immunogenic, tolerogenic and pathogenic immune reactions depending on the insult. These cells and the biomarkers they express thus represent interesting in vivo sensors for monitoring liver inflammation. In the current study, we explored whether KCs can be monitored non-invasively using single-photon-emission computed tomography (SPECT) with 99mTc labeled nanobodies (Nbs) targeting selected biomarkers. Nbs targeting V-set and immunoglobulin domain-containing 4 (Vsig4) or macrophage mannose receptor (MMR) accumulated in the liver of untreated mice. The liver targeting of anti-Vsig4 Nbs, but not anti-MMR Nbs, was blunted upon depletion of macrophages, highlighting specificity of anti-Vsig4 Nbs for liver macrophage imaging. Ex vivo flow cytometry and immunohistochemistry analysis confirmed that anti-Vsig4 Nbs specifically targeted KCs but no other cell types in the liver. Upon induction of acute hepatitis using concanavalin A (ConA), down-regulation of the in vivo imaging signal obtained using anti-Vsig4 Nbs reflected reduction in KC numbers and transient modulation of Vsig4 expression on KCs. Overall, these results indicate that Nbs targeting Vsig4 as molecular imaging biomarker enable non-invasive monitoring of KCs during hepatic inflammation.
[Show abstract][Hide abstract] ABSTRACT: The life cycle of African trypanosomes involves adaptations to the defense mechanisms of two completely different hosts, the insect vector Glossina and the mammalian host. This interplay ultimately determines host resistance and/or tolerance to parasite infection. In the tsetse fly, the immune deficiency (IMD)-regulated pathway, the scavenger receptor peptidoglycan-recognition protein LB (PGRP-LB), and the reactive oxygen species (ROS)-mediated response modulate the insect's capacity to transmit the parasite. In experimental mice, control of parasite burden and tissue pathogenicity relies on timely regulated interactions between myeloid cells exhibiting distinct activation states (M1 versus M2 type). Tsetse fly saliva and various trypanosome components including adenylate cyclases, DNA, a kinesin heavy chain, and variant surface glycoprotein (VSG) interfere with resistance and tolerance to infection.
Trends in Parasitology 09/2014; 30(11). DOI:10.1016/j.pt.2014.08.006 · 6.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Author Summary
Uncontrolled inflammation is a major contributor to pathogenicity development during many chronic parasitic infections, including African trypanosome infections. Hence, therapies should aim at re-establishing the balance between pro- and anti-inflammatory responses to reduce tissue damage. Our experiments uncovered that macrophage migration inhibitory factor (MIF) plays a pivotal role in trypanosomiasis-associated pathogenicity development. Hereby, MIF-deficient and neutralizing anti-MIF antibody-treated wild type (WT) T. brucei-infected mice exhibited decreased inflammatory responses, reduced liver damage and anemia (i.e. the most prominent pathogenicity features) compared to WT control mice. The reduced tissue damage coincided with reduced infiltration of pathogenic monocytic cells and neutrophils, whereby neutrophil-derived MIF contributed more significantly than monocyte-derived MIF to tissue damage. MIF also promoted anemia development by suppressing red blood cell production and enhancing their clearance. The clinical significance of these findings follows from human genetic data indicating that low-expression (protective) MIF alleles are enriched in Africans. The current findings therefore offer promise for human translation and open the possibility of assessing MIF levels or MIF genotype as an indication of an individual's risk for severe trypanosomiasis. Furthermore, given the unmet medical need of African trypanosomiasis affecting millions of people, these findings highlight MIF as a potential new therapeutic target for treatment of trypanosomiasis-associated pathogenicity.
[Show abstract][Hide abstract] ABSTRACT: An accurate and noninvasive tracer able to detect molecular events underlying the development of rheumatoid arthritis (RA) would be useful for RA diagnosis and drug efficacy assessment. A complement receptor of the Ig superfamily (CRIg) is expressed on synovial macrophages of RA patients, making it an interesting target for molecular imaging of RA. We aim to develop a radiotracer for the visualization of CRIg in a mouse model for RA using radiolabeled single-domain variable antibody VHH fragments (Nanobodies).
Quantitative polymerase chain reaction was used to locate CRIg expression in mice with collagen-induced arthritis (CIA). A Nanobody, NbV4m119, was generated to specifically target CRIg. Flow cytometry, phosphorimaging, and confocal microscopy were used to confirm NbVm119 binding to CRIg-positive cells. SPECT (SPECT/CT) was used to image arthritic lesions in the inflamed paws of 29 mice using (99m)Tc-NbV4m119 Nanobody.
CRIg is constitutively expressed in the liver and was found to be upregulated in synovial tissues of CIA mice. SPECT/CT imaging revealed that (99m)Tc-NbV4m119 specifically targeted CRIg-positive liver macrophages in naïve wild-type but not in CRIg(-/-) (CRIg knockout) mice. In CIA mice, (99m)Tc-NbV4m119 accumulation in arthritic lesions increased according to the severity of the inflammation. In the knees of mice with CIA, (99m)Tc-NbV4m119 was found to accumulate even before the onset of macroscopic clinical symptoms.
SPECT/CT imaging with (99m)Tc-NbV4m119 visualizes joint inflammation in CIA. Furthermore, imaging could predict which mice will develop clinical symptoms during CIA. Consequently, imaging of joint inflammation with CRIg-specific Nanobodies offers perspectives for clinical applications in RA patients.
Journal of Nuclear Medicine 03/2014; 55(5). DOI:10.2967/jnumed.113.130617 · 6.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To increase the safety and possibly efficacy of HIV-1 derived lentivectors (LVs) as an anti-cancer vaccine, we recently developed the Nanobody (Nb) display technology to target LVs to antigen presenting cells (APCs). In this study, we extend these data with exclusive targeting of LVs to conventional dendritic cells (DCs), which are believed to be the main cross-presenting APCs for the induction of a TH1-conducted antitumor immune response. The immunogenicity of these DC-subtype targeted LVs was compared to that of broad tropism, general APC-targeted and non-infectious LVs. Intranodal immunization with ovalbumin encoding LVs induced proliferation of antigen specific CD4+ T cells, irrespective of the LVs' targeting ability. However, the cytokine secretion profile of the restimulated CD4+ T cells demonstrated that general APC targeting induced a similar TH1-profile as the broad tropism LVs while transduction of conventional DCs alone induced a similar and less potent TH1 profile as the non-infectious LVs. This observation contradicts the hypothesis that conventional DCs are the most important APCs and suggests that the activation of other APCs is also meaningful. Despite these differences, all targeted LVs were able to stimulate cytotoxic T lymphocytes, be it to a lesser extent than broad tropism LVs. Furthermore this induction was shown to be dependent on type I interferon for the targeted and non-infectious LVs, but not for broad tropism LVs. Finally we demonstrated that the APC-targeted LVs were as potent in therapy as broad tropism LVs and as such deliver on their promise as safer and efficacious LV-based vaccines.
[Show abstract][Hide abstract] ABSTRACT: Background
Tuberculosis-associated Immune Reconstitution Inflammatory Syndrome (TB-IRIS) is a common complication of combined antiretroviral therapy (cART) in HIV-TB co-infected patients. However, the disease mechanism is poorly understood, prognosis of TB-IRIS is currently impossible, and diagnosis is highly challenging. We analyzed whether the gene expression of monocytes could be correlated with TB-IRIS pathogenesis and could be used to classify patients predisposed to TB-IRIS.
Monocyte gene expression was compared between patients who developed TB-IRIS and matched controls. We carried out whole-genome expression profiling using Affymetrix GeneChip® ST 1.1 arrays at two time-points: before cART initiation (baseline) and at week two post-cART initiation. For each time-point, we used different statistical approaches to identify molecular signatures which could be used as classifiers. We also functionally mapped the modulated cellular pathways using the software package Ingenuity Pathway Analysis.
At baseline, before introduction of cART and before onset of symptoms, monocyte gene expression was already perturbed in patients who subsequently developed TB-IRIS, indicating a possible involvement of monocytes in TB-IRIS predisposition. The differences in monocyte gene expression in TB-IRIS patients became even more clear after two weeks of cART (when TB-IRIS commonly occurs), with more than 100 genes for which expression showed a fold change greater than 1.5. Both at baseline and at week two post-cART initiation, a classifier of 8 and 9 genes, respectively could be built, which allowed discrimination of TB-IRIS cases and controls. Pathway analyses revealed that the majority of the dysregulated genes in TB-IRIS – at the time of the IRIS episode, but also already at baseline – are associated with infection and inflammation. Relevant biological functions which were perturbed before/during TB-IRIS included “Role of Pattern Recognition Receptors in Recognition of Bacteria and Viruses” and “Complement System”.
Our results indicate an involvement of monocytes in predisposition to/development of TB-IRIS, and suggest a number of functional pathways which may play a role in TB-IRIS development. This comprehensive study of gene regulation in monocytes provides baseline data for further studies into biomarkers for prognosis and diagnosis of TB-IRIS.
[Show abstract][Hide abstract] ABSTRACT: Tumor-associated macrophages (TAM) are exposed to multiple microenvironmental cues in tumors, which collaborate to endow these cells with protumoral activities. Hypoxia, caused by an imbalance in oxygen supply and demand due to a poorly organized vasculature, is often a prominent feature in solid tumors. However, to what extent tumor hypoxia regulates the TAM phenotype in vivo is unknown. Here, we show that the myeloid infiltrate in mouse lung carcinoma tumors encompasses two morphologically distinct CD11bhiF4/80hiLy6Clo TAM subsets, designated as MHC-II(lo) and MHC-II(hi) TAM, both of which were derived from tumor-infiltrating Ly6C(hi) monocytes. MHC-II(lo) TAM express higher levels of prototypical M2 markers and reside in more hypoxic regions. Consequently, MHC-II(lo) TAM contain higher mRNA levels for hypoxia-regulated genes than their MHC-II(hi) counterparts. To assess the in vivo role of hypoxia on these TAM features, cancer cells were inoculated in PHD2-haplodeficient mice, resulting in better oxygenated tumors. Interestingly, reduced tumor hypoxia did not alter the relative abundance of TAM subsets nor their M2 marker expression, but specifically lowers hypoxia-sensitive gene expression and angiogenic activity in the MHC-II(lo) TAM subset. The same observation in PHD2(+/+) → PHD2(+/-) bone marrow chimeras also suggests organization of a better-oxygenized microenvironment. Together, our results show that hypoxia is not a major driver of TAM subset differentiation, but rather specifically fine-tunes the phenotype of M2-like MHC-II(lo) TAM.
Cancer Research 11/2013; 74(1). DOI:10.1158/0008-5472.CAN-13-1196 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tumor growth coincides with an accumulation of myeloid-derived suppressor cells (MDSCs) which exert immune suppression and which consist of two main subpopulations, known as monocytic CD11b(+) CD115(+) Ly6G(-) Ly6C(high) MO-MDSCs and granulocytic CD11b(+) CD115(-) Ly6G(+) Ly6C(int) PMN-MDSCs. However, whether these distinct MDSC subsets hamper all aspects of early CD8(+) T-cell activation - including cytokine production, surface marker expression, survival and cytotoxicity - is currently unclear. Here, employing an in vitro co-culture system, we demonstrate that splenic MDSC subsets suppress antigen-driven CD8(+) T-cell proliferation, but differ in their dependency on IFN-γ, STAT-1, IRF-1 and NO to do so. Moreover, MO- and PMN-MDSCs diminish IL-2 levels, but only MO-MDSCs affect IL-2Rα (CD25) expression and STAT-5 signaling. Unexpectedly however, both MDSC populations stimulate IFN-γ production by CD8(+) T cells on a per-cell basis, illustrating that some T-cell activation characteristics are actually stimulated by MDSCs. Conversely, MO-MDSCs counteract the activation-induced change in CD44, CD62L, CD162 and granzyme B expression, while promoting CD69 and Fas upregulation. Together, these effects result in an altered CD8(+) T-cell adhesiveness to the extracellular matrix and selectins, sensitivity to FasL-mediated apoptosis and cytotoxicity. Hence, MDSCs intricately influence different CD8(+) T-cell activation events in vitro, whereby some parameters are suppressed while others are stimulated. This article is protected by copyright. All rights reserved.
European Journal of Immunology 11/2013; 43(11). DOI:10.1002/eji.201343349 · 4.03 Impact Factor