Antigen variation is a successful defense system adopted by several infectious agents to evade the host immune response. The principle of this defense strategy in the African trypanosome paradigm involves a dense packing of variant surface glycoproteins (VSG) exposing only highly variable and immuno-dominant epitopes to the immune system, whereas conserved epitopes become inaccessible for large molecules. Reducing the size of binders that target the conserved, less-immunogenic, cryptic VSG epitopes forms an obvious solution to combat these parasites. This goal was achieved by introducing dromedary Heavy-chain antibodies. We found that only these unique antibodies recognize epitopes common to multiple VSG classes. After phage display of their antigen-binding repertoire, we isolated a single domain antibody fragment with high specificity for the conserved Asn-linked carbohydrate of VSG. In sharp contrast to labeled concanavalin-A that stains only the flagellar pocket where carbohydrates are accessible because of less dense VSG packing, the single domain binder stains the entire surface of viable parasites, irrespective of the VSG type expressed. This corroborates the idea that small antibody fragments, but not larger lectins or conventional antibody fragments, are able to penetrate the dense VSG coat to target their epitope. The diagnostic potential of this fluorescently labeled binder was proven by the direct, selective, and sensitive detection of parasites in blood smears. The employment of this binder as a molecular recognition unit in immuno-toxins designed for trypanosomosis therapy becomes feasible as well. This was illustrated by the specific trypanolysis induced by an antibody::β-lactamase fusion activating a prodrug.

Tumors contain a heterogeneous myeloid fraction comprised of discrete MHC-II^hi and MHC-II^lo tumor-associated macrophage (TAM) subpopulations that originate from Ly6C^hi 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 Ly6C^hi monocytic precursors. M-CSFR signaling blockade shifted the MHC-II^lo/MHC-II^hi TAM balance in favor of the latter as observed by the preferential differentiation of Ly6C^hi monocytes into MHC-II^hi TAMs. In addition, the genetic and functional signatures of MHC-II^lo TAMs were downregulated upon M-CSFR blockade, indicating that M-CSFR signaling shapes the MHC-II^lo 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-II^hi 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 Res; 76(1); 35–42. ©2015 AACR.

The vitamin D receptor (VDR) is a hormone nuclear receptor regulating bone and calcium homeostasis. Studies revealing the expression of VDR on immune cells point toward a role for VDR-dependent signaling pathways in immunity. Here we verified the ability of the natural VDR ligand, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) to interfere in inflammatory and T cell stimulatory capacity of macrophages, in particular within a chronic inflammatory disease features of experimental type 1 diabetes (T1D). We demonstrated that VDR is constitutively expressed in macrophages and both the levels of VDR and its downstream targets, are clearly induced by 1,25(OH)(2)D(3). In control mice, macrophage programming with 1,25(OH)(2)D(3) partially abrogated the activation-provoked expression of IL-12p40, TNFα and iNOS as well as the effector T cell-recruiting chemokines, CXCL9, CXCL10 and CXCL11. Targeting VDR signaling in macrophages counteracted their T-cell stimulatory ability despite essentially unaltered expression of antigen-presenting and costimulatory molecules. Furthermore, even in non-obese diabetic (NOD) mice, where macrophages/monocytes featured a heightened responsiveness toward danger signals and a superior T cell stimulatory capacity, 1,25(OH)(2)D(3) successfully curtailed these basic macrophage-mediated functions. Interestingly, the inhibitory action of the active compound was associated with an IL-10-dependent mechanism since 1,25(OH)(2)D(3)-treatment of IL-10-deficient macrophages failed to reproduce the characteristic repression on inflammatory mediators or T cell proliferation. Combined, these results highlight the possible therapeutic applicability of this natural immunomodulator, due to its ability to counteract macrophage inflammatory and T cell-activating pathways.

The development of classically activated monocytic cells (M1) is a prerequisite for effective elimination of parasites, including African trypanosomes. However, persistent activation of M1 that produce pathogenic molecules such as TNF and NO contributes to the development of trypanosome infection-associated tissue injury including liver cell necrosis in experimental mouse models. Aiming to identify mechanisms involved in regulation of M1 activity, we have recently documented that during Trypanosoma brucei infection, CD11b⁺Ly6C⁺CD11c⁺ TNF and iNOS producing DCs (Tip-DCs) represent the major pathogenic M1 liver subpopulation. By using gene expression analyses, KO mice and cytokine neutralizing antibodies, we show here that the conversion of CD11b⁺Ly6C⁺ monocytic cells to pathogenic Tip-DCs in the liver of T. brucei infected mice consists of a three-step process including (i) a CCR2-dependent but CCR5- and Mif-independent step crucial for emigration of CD11b⁺Ly6C⁺ monocytic cells from the bone marrow but dispensable for their blood to liver migration; (ii) a differentiation step of liver CD11b⁺Ly6C⁺ monocytic cells to immature inflammatory DCs (CD11c⁺ but CD80/CD86/MHC-II^low) which is IFN-γ and MyD88 signaling independent; and (iii) a maturation step of inflammatory DCs to functional (CD80/CD86/MHC-II^high) TNF and NO producing Tip-DCs which is IFN-γ and MyD88 signaling dependent. Moreover, IL-10 could limit CCR2-mediated egression of CD11b⁺Ly6C⁺ monocytic cells from the bone marrow by limiting Ccl2 expression by liver monocytic cells, as well as their differentiation and maturation to Tip-DCs in the liver, showing that IL-10 works at multiple levels to dampen Tip-DC mediated pathogenicity during T. brucei infection. A wide spectrum of liver diseases associates with alteration of monocyte recruitment, phenotype or function, which could be modulated by IL-10. Therefore, investigating the contribution of recruited monocytes to African trypanosome induced liver injury could potentially identify new targets to treat hepatic inflammation in general, and during parasite infection in particular. Most infections are associated with host inflammatory responses that can result in multiple organ failure and death. It is therefore essential to understand the mechanisms balancing host immune response and tissue damage. Mouse models of African trypanosome infection represent valuable tools to study the mechanisms contributing to the inflammatory (pathogenic) or anti-inflammatory (anti-pathogenic) immune response. We recently identified TNF and NO producing DCs (Tip-DCs) as major contributors to liver pathogenicity in Trypanosoma brucei infected mice. Herein, the role of different chemokine and cytokines in the generation of Tip-DCs was investigated. Tip-DCs originated from bone marrow derived monocytes that egressed to the blood in a CCR2 chemokine receptor dependent manner. Then, monocytes extravasated to inflamed liver where IFN-γ and MyD88 signaling promoted their maturation to Tip-DCs. Both the egression of monocytes from bone marrow and their IFN-γ/MyD88 dependent maturation to Tip-DCs was counteracted by IL-10, hereby reducing liver pathogenicity. Liver injury, affecting millions of persons worldwide with often lethal consequences, frequently results from uncontrolled activation of recruited monocyte-derived cells that can be modulated by IL-10. Thus, the mechanisms regulating liver immunopathogenicity during parasitic infection identified herein could lead to new therapeutic policies in the field of hepatic inflammation.

Liver X receptors (LXRs) are key regulators of macrophage function, controlling transcriptional programs involved in lipid homeostasis and inflammation. However, exactly how LXRs modulate inflammation during infection remains unknown. To explore this, we used a mouse model of Mycobacterium tuberculosis infection. Upon intratracheal infection with M. tuberculosis, LXRs and LXR target genes were induced in CD11c⁺ lung and alveolar cells. Furthermore, mice deficient in both LXR isoforms, LXRα and LXRβ (Lxra^–/–Lxrb^–/– mice), were more susceptible to infection, developing higher bacterial burdens and an increase in the size and number of granulomatous lesions. Interestingly, mice solely deficient in LXRα, but not those lacking only LXRβ, mirrored the susceptibility of the Lxra^–/–Lxrb^–/– animals. Lxra^–/–Lxrb^–/– mice failed to mount an effective early neutrophilic airway response to infection and showed dysregulation of both pro- and antiinflammatory factors in CD11c⁺ lung cells. T cell responses were strongly affected in Lxra^–/–Lxrb^–/– mice, showing near-complete abrogation of the infection-induced Th1 function — and even more so Th17 function — in the lungs. Treatment of WT mice with the LXR agonists TO901317 and GW3965 resulted in a 10-fold decrease of the pulmonary bacterial burden and a comparable increase of Th1/Th17 function in the lungs. The dependence of LXR signaling on the neutrophil IL-17 axis represents what we believe to be a novel function for these nuclear receptors in resistance to M. tuberculosis infection and may provide a new target for therapeutics.