[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.
[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; · 6.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: African trypanosomiasis is a chronic debilitating disease affecting the health and economic well-being of many people in developing countries. The pathogenicity associated with this disease involves a persistent inflammatory response, whereby M1-type myeloid cells, including Ly6Chigh inflammatory monocytes, are centrally implicated. A comparative gene analysis between trypanosusceptible and trypanotolerant animals identified MIF (macrophage migrating inhibitory factor) as an important pathogenic candidate molecule. Using MIF-deficient mice and anti-MIF antibody treated mice, we show that MIF mediates the pathogenic inflammatory immune response and increases the recruitment of inflammatory monocytes and neutrophils to contribute to liver injury in Trypanosoma brucei infected mice. Moreover, neutrophil-derived MIF contributed more significantly than monocyte-derived MIF to increased pathogenic liver TNF production and liver injury during trypanosome infection. MIF deficient animals also featured limited anemia, coinciding with increased iron bio-availability, improved erythropoiesis and reduced RBC clearance during the chronic phase of infection. Our data suggest that MIF promotes the most prominent pathological features of experimental trypanosome infections (i.e. anemia and liver injury), and prompt considering MIF as a novel target for treatment of trypanosomiasis-associated immunopathogenicity.
[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; · 5.56 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.
[Show abstract][Hide abstract] ABSTRACT: In order to promote infection, the blood-borne parasite Trypanosoma brucei releases factors that upregulate arginase expression and activity in myeloid cells.
By screening a cDNA library of T. brucei with an antibody neutralizing the arginase-inducing activity of parasite released factors, we identified a Kinesin Heavy Chain isoform, termed TbKHC1, as responsible for this effect. Following interaction with mouse myeloid cells, natural or recombinant TbKHC1 triggered SIGN-R1 receptor-dependent induction of IL-10 production, resulting in arginase-1 activation concomitant with reduction of nitric oxide (NO) synthase activity. This TbKHC1 activity was IL-4Rα-independent and did not mirror M2 activation of myeloid cells. As compared to wild-type T. brucei, infection by TbKHC1 KO parasites was characterized by strongly reduced parasitaemia and prolonged host survival time. By treating infected mice with ornithine or with NO synthase inhibitor, we observed that during the first wave of parasitaemia the parasite growth-promoting effect of TbKHC1-mediated arginase activation resulted more from increased polyamine production than from reduction of NO synthesis. In late stage infection, TbKHC1-mediated reduction of NO synthesis appeared to contribute to liver damage linked to shortening of host survival time.
A kinesin heavy chain released by T. brucei induces IL-10 and arginase-1 through SIGN-R1 signaling in myeloid cells, which promotes early trypanosome growth and favors parasite settlement in the host. Moreover, in the late stage of infection, the inhibition of NO synthesis by TbKHC1 contributes to liver pathogenicity.
[Show abstract][Hide abstract] ABSTRACT: Background: Anemia of Inflammation (AI), or Anemia of Chronic Disease (ACD), is the most prominent form of anemia in hospitalized patients, affecting quality of life and resulting from an imbalance between erythrophagocytosis and erythropoiesis. Multiple mechanisms contribute to its pathogenesis, including iron restriction, direct erythropoietic suppression and shortened erythrocyte survival. A common parameter in AI is a persistent pro-inflammatory immune response of the host, whereby macrophages and their activation state play a key role. In the regulation of erythropoiesis, the enigmatic central macrophages are thought to be essential as part of erythroblastic islands.
Results: By scrutinizing African trypanosomiasis in mice as a model of infectious disease associated with AI, we could discriminate within the F4/80+ER-HR3+ macrophage population 4 distinct subsets (CD11b-/lowLy6C-/low, CD11b-/lowLy6C+, CD11b+Ly6C+Fo-SGL+ and CD11b+Ly6C-/low). Using an in vitro erythropoiesis assay we could establish that CD11b+Ly6C+Fo-SGL+ cells represent the best candidates to function as central macrophages, since these cells are able to aid in the maturation of erythroblasts. Furthermore, they express crucial adhesion molecules (VCAM-1, CD49d, CD169 and CD36) favoring erythroblastic island formation. Moreover, the gene expression levels of crucial iron-homeostasis-associated genes such as CD71, hmox-1, fpn-1 and fhc suggest that this population rather exports iron during infection in contrast to other myeloid cells. Interestingly, by comparing Trypanosome-infected wild-type and mice exhibiting different degrees of anemia, we observed that there was an inverse correlation between the CD11b+Ly6C+Fo-SGL+ population size and anemia development. Therefore, this population requires further investigation to unravel its exact role in AI development.
I would like to acknowledge all the technical staff that contributed to this work or gave logistic support: Ella Omasta, Marie-Thérèse Detobel, Maria Slazak, Yvon Elkrim, Lea Brys, Nadia Abou, Eddy Vercauteren
Weiss G (Iron metabolism in the anemia of chronic disease. Biochim Biophys Acta 1790:682-693.2009).
Stijlemans B, Vankrunkelsven A, Caljon G, Bockstal V, Guilliams M, Bosschaerts T, Beschin A, Raes G, Magez S, De Baetselier P (The central role of macrophages in trypanosomiasis-associated anemia: rationale for therapeutical approaches. Endocr Metab Immune Disord Drug Targets 10:71-82.2010).
15th International Congress of Immunology (ICI) 22 Aug - 27 Aug, 2013., Milan, Italy; 08/2013
[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 07/2013; · 4.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Antigen-presenting cells are a heterogeneous group of cells that are characterized by their functional specialization. Consequently, targeting specific antigen-presenting cell subsets offers opportunities to induce distinct T cell responses. Here we report on the generation and use of Nanobodies (Nbs) to target lentivectors specifically to human lymph node-resident myeloid dendritic cells, demonstrating that Nbs represent a powerful tool to redirect lentivectors to human antigen-presenting cell subsets.
[Show abstract][Hide abstract] ABSTRACT: Objective: Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is a common complication in HIV-TB co-infected patients receiving combined antiretroviral therapy (cART). This study investigated a putative contribution of mono-cytes to the development of TB-IRIS. Design: A prospective study was designed to compare gene expression between patients who developed TB-IRIS with matched non-TB-IRIS controls. Methods: We performed a hypothesis-generating transcriptome analysis on monocytes of HIV-TB co-infected patients. Identified pathways were subsequently analysed in patients' monocytes before and shortly after cART initiation, in a technically indepen-dent set-up (nCounter). Additionally, protein expression and enzymatic activities of specific factors were assessed at the systemic level. Results: Pathway analysis of microarray datasets and focused gene expression study revealed that, even before initiation of cART, the complement system is dysregulated in HIV–TB co-infected patients who are predisposed to developing TB-IRIS. Detailed analysis revealed differences between TB-IRIS patients and matched non-TB-IRIS cases, at the level of the balance between the effector C1Q and the inhibitor C1-INH, both before and 2 weeks after cART initiation. These differences were mirrored by increases in the downstream pro-inflammatory complement factor C5 over the course of 2 weeks of cART. Our results suggest that inappropriate control of complement activation could be associated with the 'flaring up' of inflammation observed during TB-IRIS.
[Show abstract][Hide abstract] ABSTRACT: Rheumatoid arthritis (RA) is a chronic autoimmune disease occurring in approximately 1% of the worldwide population. The disease primarily affects the joints, where inflammatory cells, such as macrophages, invade the synovium and cause cartilage and bone destruction. Currently, it is difficult to efficiently diagnose and monitor early-stage RA. In this study, we investigated whether SPECT/micro-CT imaging with 99mTc-labeled Nanobodies directed against the macrophage mannose receptor (MMR) is a useful tool for monitoring and quantifying joint inflammation in collagen-induced arthritis (CIA), a mouse model for RA. The expression of MMR was analyzed on macrophages and osteoclasts generated in vitro and in cells obtained from various organs from mice with CIA. METHODS: CIA was induced in DBA/1 mice by injection of collagen type II in complete Freund adjuvant, and cell suspensions from the inflamed joints and other organs were obtained. Macrophages and osteoclasts were generated in vitro from bone marrow cells. Expression of MMR was quantified by quantitative polymerase chain reaction and flow cytometry with specific Nanobodies and conventional antibodies. SPECT/micro-CT imaging was performed with 99mTc-labeled MMR and control Nanobodies. RESULTS: MMR was highly expressed on macrophages and to a lesser extent on osteoclasts generated in vitro. In mice with CIA, MMR expression was detected on cells from the bone marrow, lymph nodes, and spleen. In synovial fluid of arthritic joints, MMR was expressed on CD11b+F4/80+ macrophages. On in vivo SPECT/micro-CT imaging with consecutive injections of MMR and control Nanobodies, a strong MMR signal was seen in the knees, ankles, and toes of arthritic mice. Quantification of the SPECT imaging confirmed the specificity of the MMR signal in inflamed joints as compared with the control Nanobody. Dissection of the paws revealed an additional significant MMR signal in nonarthritic paws of affected mice (i.e., mice displaying symptoms of arthritis in other paws). CONCLUSION: Our data show that MMR is expressed on macrophages in vitro and in vivo in synovial fluid of inflamed paws, whereas expression is relatively low in other tissues. The use of Nanobodies against MMR in SPECT/micro-CT imaging generates the possibility to track inflammatory cells in vivo in arthritic joints.
Journal of Nuclear Medicine 02/2013; · 5.56 Impact Factor