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Antibodies for flow cytometry (S1); List of gene specific primer sequences for qRT-PCR (S2).

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 Res; 76(1); 35–42. ©2015 AACR.

Effects of α-M-CSFR Ab treatment on Ly6Chi monocyte function (S1); Effect of M-CSFR blockade on the survival of Ly6Chi monocytes and TAM (S2); Effect of M-CSFR blockade on Ly6Chi monocyte percentages in peripheral blood, spleen and bone marrow (S3); Absolute numbers of myeloid cell subsets in tumors from isotype or α-M-CSFR Ab-treated animals (S4); Production of M-CSF, IL-34 and GM-CSF by 3LL-R tumors (S5); Effect of M-CSFR blockade on monocyte differentiation in TS/A breast carcinoma tumors (S6); M-CSFR-mediated regulation of gene expression is IL-4Rα- independent (S7); Mixed Leukocyte Reaction (MLR) by TAM subsets from isotype or α-M-CSFR Ab treated animals (S8); M-CSFR blockade enhanced the M1-like MHC-IIhi TAM gene signature independently of GM-CSFR signaling (S9).

Description of additional methods and procedures used in the study.

Uncontrolled growth of solid tumors will result in a hallmark hypoxic condition, whereby the key transcriptional regulator of hypoxia inducible factor-1α (HIF-1α) will be stabilized to activate the transcription of target genes that are responsible for the metabolism, proliferation, and metastasis of tumor cells. Targeting and inhibiting the transcriptional activity of HIF-1 may provide an interesting strategy for cancer therapy. In the present study, an immune library and a synthetic library were constructed for the phage display selection of Nbs against recombinant PAS B domain protein (rPasB) of HIF-1α. After panning and screening, seven different nanobodies (Nbs) were selected, of which five were confirmed via immunoprecipitation to target the native HIF-1α subunit. The inhibitory effect of the selected Nbs on HIF-1 induced activation of target genes has been evaluated after intracellular expression of these Nbs in HeLa cells. The dramatic inhibition of both intrabody formats on the expression of HIF-1-related target genes has been confirmed, which indicated the inhibitory efficacy of selected Nbs on the transcriptional activity of HIF-1.

Bovine African Trypanosomosis is an infectious parasitic disease affecting livestock productivity and thereby impairing the economic development of Sub-Saharan Africa. The most important trypanosome species implicated is T. congolense, causing anemia as most important pathological feature. Using murine models, it was shown that due to the parasite’s efficient immune evasion mechanisms, including (i) antigenic variation of the variable surface glycoprotein (VSG) coat, (ii) induction of polyclonal B cell activation, (iii) loss of B cell memory and (iv) T cell mediated immunosuppression, disease prevention through vaccination has so far been impossible. In trypanotolerant models a strong, early pro-inflammatory immune response involving IFN-γ, TNF and NO, combined with a strong humoral anti-VSG response, ensures early parasitemia control. This potent protective inflammatory response is counterbalanced by the production of the anti-inflammatory cytokine IL-10, which in turn prevents early death of the host from uncontrolled hyper-inflammation-mediated immunopathologies. Though at this stage different hematopoietic cells, such as NK cells, T cells and B cells as well as myeloid cells (i.e. alternatively activated myeloid cells (M2) or Ly6c- monocytes), were found to produce IL-10, the contribution of non-hematopoietic cells as potential IL-10 source during experimental T. congolense infection has not been addressed. Here, we report for the first time that during the chronic stage of T. congolense infection non-hematopoietic cells constitute an important source of IL-10. Our data shows that hepatocyte-derived IL-10 is mandatory for host survival and is crucial for the control of trypanosomosis-induced inflammation and associated immunopathologies such as anemia, hepatosplenomegaly and excessive tissue injury., Author summary Bovine African Trypanosomosis is a parasitic disease of veterinary importance that adversely affects the public health and economic development of sub-Saharan Africa. The most important trypanosome species implicated is T. congolense, causing anemia as most important pathological feature and major cause of death. Using murine models, it was shown that the disease is characterized by a well-timed and balanced production of pro-inflammatory cytokine promoting factors followed by an anti-inflammatory response, involving IL-10. The latter is required to attenuate infection-associated pathogenicity and to prevent early host death from uncontrolled hyper-inflammation mediated immunopathologies. However, the cellular source of IL-10 in vivo and the window within which these cells exert their function during the course of African trypanosomiasis remain poorly understood, which hampers the design of effective therapeutic strategies. Using a T. congolense infection mouse model, relevant for bovine trypanosomosis, we demonstrate that during the chronic stage of infection hepatocyte-derived IL-10, but not myeloid cell-derived IL-10, regulates the main infection-associated immunopathologies and ultimately mediates host survival. Hence, strategies that tilt the balance of hepatocyte cytokine production in favor of IL-10 could majorly impact the wellbeing and survival of T. congolense-infected animals. Given the unmet medical need for this parasite infection, our findings offer promise for improved treatment protocols in the field.

Sepsis-leading to septic shock-is the leading cause of death in intensive care units. The systemic inflammatory response to infection, which is initiated by activated myeloid cells, plays a key role in the lethal outcome. Macrophage migration inhibitory factor (MIF) is an upstream immunoregulatory mediator, released by myeloid cells, that underlies a common genetic susceptibility to different infections and septic shock. Accordingly, strategies that are aimed at inhibiting the action of MIF have therapeutic potential. Here, we report the isolation and characterization of tailorable, small, affinity-matured nanobodies (Nbs; single-domain antigen-binding fragments derived from camelid heavy-chain Abs) directed against MIF. Of importance, these bioengineered Nbs bind both human and mouse MIFs with nanomolar affinity. NbE5 and NbE10 inhibit key MIF functions that can exacerbate septic shock, such as the tautomerase activity of MIF (by blocking catalytic pocket residues that are critical for MIF's conformation and receptor binding), the TNF-inducing potential, and the ability of MIF to antagonize glucocorticoid action. A lead NbE10, tailored to be a multivalent, half-life extended construct (NbE10-NbAlb8-NbE10), attenuated lethality in murine endotoxemia when administered via single injection, either prophylactically or therapeutically. Hence, Nbs, with their structural and pharmacologic advantages over currently available inhibitors, may be an effective, novel approach to interfere with the action of MIF in septic shock and other conditions of inflammatory end-organ damage.-Sparkes, A., De Baetselier, P., Brys, L., Cabrito, I., Sterckx, Y. G.-J., Schoonooghe, S., Muyldermans, S., Raes, G., Bucala, R., Vanlandschoot, P., Van Ginderachter, J. A., Stijlemans, B. Novel half-life extended anti-MIF nanobodies protect against endotoxic shock.

The African trypanosome Trypanosoma brucei, which persists within the bloodstream of the mammalian host, has evolved potent mechanisms for immune evasion. Specifically, antigenic variation of the variant-specific surface glycoprotein (VSG) and a highly active endocytosis and recycling of the surface coat efficiently delay killing mediated by anti-VSG antibodies. Consequently, conventional VSG-specific intact immunoglobulins are non-trypanocidal in the absence of complement. In sharp contrast, monovalent antigen-binding fragments, including 15 kDa nanobodies (Nb) derived from camelid heavy-chain antibodies (HCAbs) recognizing variant-specific VSG epitopes, efficiently lyse trypanosomes both in vitro and in vivo. This Nb-mediated lysis is preceded by very rapid immobilisation of the parasites, massive enlargement of the flagellar pocket and major blockade of endocytosis. This is accompanied by severe metabolic perturbations reflected by reduced intracellular ATP-levels and loss of mitochondrial membrane potential, culminating in cell death. Modification of anti-VSG Nbs through site-directed mutagenesis and by reconstitution into HCAbs, combined with unveiling of trypanolytic activity from intact immunoglobulins by papain proteolysis, demonstrates that the trypanolytic activity of Nbs and Fabs requires low molecular weight, monovalency and high affinity. We propose that the generation of low molecular weight VSG-specific trypanolytic nanobodies that impede endocytosis offers a new opportunity for developing novel trypanosomiasis therapeutics. In addition, these data suggest that the antigen-binding domain of an anti-microbial antibody harbours biological functionality that is latent in the intact immunoglobulin and is revealed only upon release of the antigen-binding fragment., Evaluation Studies, Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

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 Res; 76(1); 35–42. ©2015 AACR.

Animal African trypanosomosis is a major threat to the economic development and human health in sub-Saharan Africa. Trypanosoma congolense infections represent the major constraint in livestock production, with anemia as the major pathogenic lethal feature. The mechanisms underlying anemia development are ill defined, which hampers the development of an effective therapy. Here, the contribution of the erythropoietic and erythrophagocytic potential as well as of hemodilution to the development of T. congolense-induced anemia were addressed in a mouse model of low virulence relevant for bovine trypanosomosis. We show that in infected mice, splenic extramedullary erythropoiesis could compensate for the chronic low-grade type I inflammation-induced phagocytosis of senescent red blood cells (RBCs) in spleen and liver myeloid cells, as well as for the impaired maturation of RBCs occurring in the bone marrow and spleen. Rather, anemia resulted from hemodilution. Our data also suggest that the heme catabolism subsequent to sustained erythrophagocytosis resulted in iron accumulation in tissue and hyperbilirubinemia. Moreover, hypoalbuminemia, potentially resulting from hemodilution and liver injury in infected mice, impaired the elimination of toxic circulating molecules like bilirubin. Hemodilutional thrombocytopenia also coincided with impaired coagulation. Combined, these effects could elicit multiple organ failure and uncontrolled bleeding thus reduce the survival of infected mice. MIF (macrophage migrating inhibitory factor), a potential pathogenic molecule in African trypanosomosis, was found herein to promote erythrophagocytosis, to block extramedullary erythropoiesis and RBC maturation, and to trigger hemodilution. Hence, these data prompt considering MIF as a potential target for treatment of natural bovine trypanosomosis., Author Summary Bovine African trypanosomosis is a parasitic disease of veterinary importance that adversely affects the public health and economic development of sub-Saharan Africa. Anemia is a major cause of death associated with this disease. Yet, the mechanisms underlying anemia development are not elucidated, which hampers the design of effective therapeutic strategies. We show here that in a Trypanosoma congolense infection mouse model relevant for bovine trypanosomosis, red blood cells (RBCs) are generated in the spleen. This compensates for the impaired maturation of RBCs occurring in the bone marrow, the normal site of RBC generation, and for the destruction of RBCs taking place in the liver and the spleen. Instead, anemia results from an increase in blood volume (hemodilution). The immune molecule Macrophage Migration Inhibitory Factor (MIF) was found to drive RBC destruction, to block RBC maturation, as well as to trigger hemodilution. Iron accumulation in tissue due to sustained RBC destruction and hemodilution causes tissue damage, which culminates in the release of toxic molecules like bilirubin, in impaired production of blood detoxifying molecules like albumin, and in defective coagulation. Combined, these effects initiate multiple organ failure that can reduce the survival of infected mice. Given the unmet medical need for this parasite infection, our findings offer promise for improved treatment protocols in the field.

Antiparasite responses are associated with the recruitment of monocytes that differentiate to macrophages and dendritic cells at the site of infection. Although classically activated monocytic cells are assumed to be the major source of TNF and NO during Trypanosoma brucei brucei infection, their cellular origin remains unclear. In this study, we show that bone marrow-derived monocytes accumulate and differentiate to TNF/inducible NO synthase-producing dendritic cells (TIP-DCs) in the spleen, liver, and lymph nodes of T. brucei brucei-infected mice. Although TIP-DCs have been shown to play a beneficial role in the elimination of several intracellular pathogens, we report that TIP-DCs, as a major source of TNF and NO in inflamed organs, could contribute actively to tissue damage during the chronic stage of T. brucei brucei infection. In addition, the absence of IL-10 leads to enhanced differentiation of monocytes to TIP-DCs, resulting in exacerbated pathogenicity and early death of the host. Finally, we demonstrate that sustained production of IL-10 following IL-10 gene delivery treatment with an adeno-associated viral vector to chronically infected mice limits the differentiation of monocytes to TIP-DCs and protects the host from tissue damage.

Anemia is a well-established infection-associated immunopathological feature of trypanosomiasis and the degree of the anemia is a reliable indicator of the severity of infection. Since infections with trypanosomes triggers a strong cytokine production and a type I immune response, the trypanosome-elicited anemia may be type I cytokine driven. This type of anemia termed anemia of chronic disease is characterized by an imbalance between erythrophagocytosis and erythropoiesis that is linked to a perturbed iron homeostasis including altered iron recycling by macrophages and iron sequestration. To further unravel the mechanisms underlying trypanosome-elicited anemia the expression profile of genes involved in erythrophagocytosis, uptake of iron-containing complexes and iron homeostasis was performed during the acute and chronic phase of experimental Trypanosoma brucei infections in a murine model. The results suggest that liver-associated erythrophagocytosis mediated by cytokine-activated macrophages (M1 cells) is the most likely main initiating event of aggressive anemia during the acute phase of infection. Persistence of strong type I cytokine production during the chronic phase of infection leads to hyper-activated M1 cells and a more progressive anemia. RT-PCR analysis of liver tissue demonstrates a strong increase of cell surface receptors involved in uptake of RBC and iron-containing compounds. For genes involved in iron processing we found an increase of ferroportin-1 (FPN-1), transferrin (Tf) and ceruloplasmin (CP) only in the acute phase, suggesting that export of iron is hampered in the chronic phase of infection. Our results suggest that in the chronic phase of trypanosomiasis, the iron-processing pathway is skewed towards iron sequestration, as evidenced by increased ferritin expression, while enhanced uptake of RBC/iron-containing compounds is maintained.

Uncontrolled inflammation is a major cause of tissue injury/pathogenicity often resulting in death of a host infected with African trypanosomes. Thus, comparing the immune response in hosts that develop different degrees of disease severity represents a promising approach to discover processes contributing to trypanosomiasis control. It is known that limitation of pathogenicity requires a transition in the course of infection, from an IFN-γ-dependent response resulting in the development of classically activated myeloid cells (M1), to a counterbalancing IL-10-dependent response associated with alternatively activated myeloid cells (M2). Herein, mechanisms and downstream effectors by which M2 contribute to lower the pathogenicity and the associated susceptibility to African trypanosomiasis have been explored. Gene expression analysis in IL-10 knockout and wild-type mice, that are susceptible and relatively resistant to Trypanosoma congolense infection, respectively, revealed a number of IL-10-inducible genes expressed by M2, including Sepp1 coding for selenoprotein P. Functional analyses confirm that selenoprotein P contributes to limit disease severity through anti-oxidant activity. Indeed, Sepp1 knockout mice, but not Sepp1Δ240-361 mice retaining the anti-oxidant motif but lacking the selenium transporter domain of selenoprotein P, exhibited increased tissue injury that associated with increased production of reactive oxygen species and increased apoptosis in the liver immune cells, reduced parasite clearance capacity of myeloid cells, and decreased survival. These data validate M2-associated molecules as functioning in reducing the impact of parasite infection on the host.

Tolerance to African trypanosomes requires the production of IFN-γ in the early stage of infection that triggers the development of classically activated macrophages controlling parasite growth. However, once the first peak of parasitemia has been controlled, down-regulation of the type 1 immune response has been described. In this study, we have evaluated whether regulatory T cells (Tregs) contribute to the limitation of the immune response occurring during Trypanosoma congolense infection and hereby influence the outcome of the disease in trypanotolerant C57BL/6 host. Our data show that Foxp3+ Tregs originating from the naturally occurring Treg pool expanded in the spleen and the liver of infected mice. These cells produced IL-10 and limited the production of IFN-γ by CD4+ and CD8+ effector T cells. Tregs also down-regulated classical activation of macrophages resulting in reduced TNF-α production. The Treg-mediated suppression of the type 1 inflammatory immune response did not hamper parasite clearance, but was beneficial for the host survival by limiting the tissue damages, including liver injury. Collectively, these data suggest a cardinal role for naturally occurring Tregs in the development of a trypanotolerant phenotype during African trypanosomiasis.

FedF is the two-domain tip adhesin of F18 fimbriae from enterotoxigenic Escherichia coli. Bacterial adherence, mediated by the N-terminal receptor-binding domain of FedF to carbohydrate receptors on intestinal microvilli, causes diarrhoea and oedema disease in newly weaned piglets and induces the secretion of Shiga toxins. A truncate containing only the receptor-binding domain of FedF was found to be further cleaved at its N-terminus. Reconstruction of this N-terminal truncate rendered FedF amenable to crystallization, resulting in crystals with space group P2(1)2(1)2(1) and unit-cell parameters a = 36.20, b = 74.64, c = 99.03 A that diffracted to beyond 2 A resolution. The binding specificity of FedF was screened for on a glycan array, exposing 264 glycoconjugates, to identify specific receptors for cocrystallization with FedF.

Shiga toxin Stx2e is the major known agent that causes edema disease in newly weaned pigs. This severe disease is characterized by neurological disorders, hemorrhagic lesions, and frequent fatal outcomes. Stx2e consists of an enzymatically active A subunit and five B subunits that bind to a specific glycolipid receptor on host cells. It is evident that antibodies binding to the A subunit or the B subunits of Shiga toxin variants may have the capability to inhibit their cytotoxicity. Here, we report the discovery and characterization of a VHH single domain antibody (nanobody) isolated from a llama phage display library that confers potent neutralizing capacity against Stx2e toxin. We further present the crystal structure of the complex formed between the nanobody (NbStx2e1) and the Stx2e toxoid, determined at 2.8 Å resolution. Structural analysis revealed that for each B subunit of Stx2e, one NbStx2e1 is interacting in a head-to-head orientation and directly competing with the glycolipid receptor binding site on the surface of the B subunit. The neutralizing NbStx2e1 can in the future be used to prevent or treat edema disease.

Resistance to Trypanosoma brucei brucei has been correlated with the ability of infected animals to produce interferon (IFN)-gamma and tumor necrosis factor (TNF) in an early phase of infection, followed by interleukin (IL)-4 and IL-10 in late and chronic stages of the disease. Contributions of IFN-gamma and IL-10 in the control of parasitemia and survival of mice infected with T. brucei brucei were investigated by using IFN-gamma(-/-) and IL-10(-/-) mice. Results suggest that IFN-gamma, mainly secreted by CD8(+) T cells, is essential for parasite control via macrophage activation, which results in TNF and nitric oxide secretions. IL-10, partially secreted by CD4(+) T cells, seems to be important for the survival of infected mice. Its absence resulted in the sustained secretion of inflammatory mediators, which indicated the role of IL-10 in maintaining the balance between pathogenic and protective immune responses during African trypanosomosis.

African trypanosomes are extracellular parasites causing sleeping sickness to human or nagana to livestock in sub-Saharan Africa. To gain insight into factors governing resistance/susceptibility to these parasites, the immune responses in mice infected with a Trypanosoma brucei phospholipase C null mutant (PLC(-/-)) or its wild type counterpart (WT) were compared. We found that the T. b. brucei mutant inducing a chronic infection triggers the production of type I cytokines during the early stage of infection, followed by the secretion of type II cytokines in the late/chronic phase of the disease. In contrast, WT-infected mice are killed within 5 weeks and remain locked in a type I cytokine response. The type I/type II cytokine balance may influence the development of different subsets of suppressive macrophages, i.e. classically activated macrophages (type I) versus alternatively activated macrophages (type II) that are antagonistically regulated. Therefore, the phenotype and accessory cell function of macrophages elicited during WT and PLC(-/-) T. b. brucei infections were addressed. Results indicate that classically activated macrophages develop in a type I cytokine environment in the early phase of both WT and PLC(-/-) trypanosome infections. In the late stage of infection, only PLC(-/-)-infected mice resisting the infection develop type II cytokine-associated alternative macrophages. In parallel, we found that mice susceptible to Trypanosoma congolense infection, showing an exponential parasite growth until they die, have a higher level of type II cytokines in the early stage of infection than resistant animals controlling the first peak of parasitaemia. The levels of type I cytokines were comparable in both T. congolense-resistant and -susceptible mice. On the basis of these results, we propose that survival to African trypanosome infection requires a type I cytokine environment and classical macrophage activation in the early stage of infection, enabling mice to control the first peak of parasitaemia. Thereafter, a switch to type II cytokine environment triggering alternative macrophage activation is required to enable progression of the disease into the chronic phase. The possible role of the sequential activation of alternative macrophages in the late/chronic stage of infection in the increased resistance of mice to PLC(-/-) T. b. brucei will be discussed.

The type I/type II cytokine balance may influence the development of different subsets of suppressive macrophages, i.e., classically activated macrophages (caMφ, type I) versus alternatively activated macrophages (aaMφ, type II). Recently, we showed that although mice infected with phospholipase C-deficient (PLC−/−) Trypanosoma brucei brucei exhibit a clear shift from type I to the type II cytokine production, wild type (WT)-infected mice remain locked in a type I cytokine response. In the present study, phenotype and accessory cell function of macrophages elicited during WT and PLC−/−T. b. brucei infection were compared. Results indicate that caMφ develop in a type I cytokine environment in the early phase of WT and PLC−/− trypanosome infection, correlating with inhibition of T cell activation triggered by a mitogen, a superantigen, or an antigen. In the late stage of infection, only PLC−/−-infected mice resisting the infection develop type II cytokine-associated aaMφ correlating with impaired antigen- but not mitogen- or superantigen-induced T cell activation.

During African trypanosomiasis, macrophages play a central role in T cell hyporesponsiveness to parasite-related and unrelated antigens. In this study, the ability of macrophages from Trypanosoma b. brucei-infected mice to present exogenous antigens to a major histocompatibility complex (MHC) class II-restricted CD4+ T cell hybridoma was analysed. We demonstrate that the antigen presentation capacity of macrophages from infected mice is markedly reduced as a result of a lower expression of [MHC class II-peptide] complexes on their plasma membrane. This defect did not result from a decreased antigen uptake/catabolism, a reduced MHC class II and intercellular adhesion molecule 1 expression on the surface of macrophages, a decreased affinity of MHC class II molecules for antigenic peptides, a competition between exogenous and parasite antigens, or the generation of inhibitory peptides. Our data indicate that the step resulting in coexpression of processed antigens and MHC class II molecules is affected in T. b. brucei-infected mice. Additionally, macrophages from infected mice secreted IL-10 that in turn contributes to the impairment of T cell activation.

Infection-associated immunoincompetence during malaria might result from macrophage dysfunction. In the present study, we investigated the role of macrophages as target for immunosuppression during infection, using the murine Plasmodium c. chabaudi model. Special attention has been paid to the analysis of processing/presentation of protein antigens and presentation of peptides, using cocultures of peritoneal exudate cells (PECs) from infected mice and antigen-specific T-cell hybridomas. The results obtained indicate a defective processing of protein antigens that becomes maximal at acute parasitemias. In addition, macrophages from acutely infected mice suppress the interleukin-2 production by the antigen-activated T-cell hybridomas. This effect was independent of prostaglandin and nitric oxide production by the macrophage. The possible role of parasite components in the impaired accessory cell function of PECs was investigated and hemozoin, the end-product of the hemoglobin catabolism by intraerythrocytic malaria parasites, was found to induce similar infection-associated deficiencies in vitro. Moreover, hemozoin, was shown to mimic the immunosuppressive effects induced in PECs during in-vivo infections with P. chabaudi. In conclusion, we propose that hemozoin is a key factor in the malaria-associated immunosuppression, affecting both the antigen processing and immunomodulatory functions of macrophages.

Coelomic fluid of earthworms Eisenia foetida (Oligochaeta, Annelida) exerts a mitogenic activity on murine splenocytes. Total coelomic fluid was subjected to size-exclusion chromatography and a semi-purified mitogenic fraction (fraction 5) was isolated and further characterized. Both coelomic fluid and the semi-purified fraction 5 block concanavalin A (ConA)-induced spleen cell proliferation but exert a synergistic effect on LPS-triggered spleen cell proliferation. Using a polyclonal antiserum neutralizing the mitogenic activity of the semi-purified fraction 5, a 60-kDa component was identified and named CMF (coelomic mitogenic factor). CMF was found to bind ConA which could account for its ability to inhibit ConA-induced spleen cell proliferation. CMF is present in the coelomic fluid as a trimer of a 20-kDa protein. N-terminal amino acid sequence of monomeric CMF reveals partial sequence homology with phospholipase A2 (PLA2). Moreover, CMF-enriched coelomic fluid fraction 5 exerts phospholipase activity comparable with that of bovine pancreatic PLA2. Our results suggest that coelomic fluid of E. foetida contains a ubiquitous PLA2-like enzyme which might be involved in immune reactions in earthworms such as anti-bacterial mechanisms.

The skin secretion of many amphibians contains an arsenal of bioactive molecules, including hormone-like peptides (HLPs) acting as defense toxins against predators, and antimicrobial peptides (AMPs) providing protection against infectious microorganisms. Several amphibian taxa seem to have independently acquired the genes to produce skin-secreted peptide arsenals, but it remains unknown how these originated from a non-defensive ancestral gene and evolved diverse defense functions against predators and pathogens. We conducted transcriptome, genome, peptidome and phylogenetic analyses to chart the full gene repertoire underlying the defense peptide arsenal of the frog Silurana tropicalis and reconstruct its evolutionary history. Our study uncovers a cluster of 13 transcriptionally active genes, together encoding up to 19 peptides, including diverse HLP homologues and AMPs. This gene cluster arose from a duplicated gastrointestinal hormone gene that attained a HLP-like defense function after major remodeling of its promoter region. Instead, new defense functions, including antimicrobial activity, arose by mutation of the precursor proteins, resulting in the proteolytic processing of secondary peptides alongside the original ones. Although gene duplication did not trigger functional innovation, it may have subsequently facilitated the convergent loss of the original function in multiple gene lineages (subfunctionalization), completing their transformation from HLP gene to AMP gene. The processing of multiple peptides from a single precursor entails a mechanism through which peptide-encoding genes may establish new functions without the need for gene duplication to avoid adaptive conflicts with older ones., Author Summary Many amphibians defend themselves against predation and infections by secreting a mixture of gene-encoded toxins and antimicrobials. How does such an integrated defense weapon arise and how does it diversify to gain distinct antipredatory and antimicrobial functions? We took advantage of the availability of a sequenced genome for the African clawed frog Silurana tropicalis to provide the first comprehensive overview of an amphibian peptide defense arsenal, from its underlying genes to its bioactive components. A reconstruction of the evolutionary history of this gene repertoire allows us to elucidate the timing and mode of evolution of distinct defense functions. Our study shows that the basal transition from a gastrointestinal hormone function to a skin-secretory defense function was accompanied by major restructuring of regulatory sequences in the ancestral gene. Instead, subsequently diversifying defense genes underwent functional shifts by entering a bifunctional stage (by cleavage of two distinct defense peptides from a single precursor protein) and occasionally losing the original defense function (by loss of the original defense peptide). This pattern provides an evolutionary explanation for the processing of structurally or functionally unrelated toxins from the same or closely related precursor proteins in other poisonous and venomous animals.

In macrophages, basal polyamine (putrescine, spermidine, and spermine) levels are relatively low but are increased upon IL-4 stimulation. This Th2 cytokine induces Arg1 activity, which converts arginine into ornithine, and ornithine can be decarboxylated by ODC to produce putrescine, which is further converted into spermidine and spermine. Recently, we proposed polyamines as novel agents in IL-4-dependent E-cadherin regulation in AAMs. Here, we demonstrate for the first time that several, but not all, AAM markers depend on polyamines for their IL-4-induced gene and protein expression and that polyamine dependency of genes relies on the macrophage type. Remarkably, Arg1-deficient macrophages display rather enhanced IL-4-induced polyamine production, suggesting that an Arg1-independent polyamine synthesis pathway may operate in macrophages. On the other side of the macrophage activation spectrum, LPS-induced expression of several proinflammatory genes was increased significantly in polyamine-depleted CAMs. Overall, we propose Arg1 independently produced polyamines as novel regulators of the inflammatory status of the macrophage. Indeed, whereas polyamines are needed for IL-4-induced expression of several AAM mediators, they inhibit the LPS-mediated expression of proinflammatory genes in CAMs.

Development of classically activated macrophages (M1 cells) is a prerequisite to controlling parasite growth and therefore resistance to African trypanosomiasis. However, if activation of M1 cells is uncontrolled, including their production of tumor necrosis factor (TNF) and nitric oxide (NO), collateral pathogenic damage to tissues ensues. We report the identification of a novel putative Trypanosoma brucei M1 cell-triggering protein. The recombinant trypanosome-suppressive immunomodulating factor (rTSIF) induced TNF and NO secretion by macrophages. Moreover, M1 cells triggered by rTSIF block T cell proliferation in a manner dependent on NO, interferon gamma, and cell contact. Furthermore, rTSIF could down-regulate type 2-oriented immune responses. Therefore, trypanosome-suppressive immunomodulating factor (TSIF) may represent a new parasite molecule with the potential to modulate the host immune network, whereby it could contribute to the inflammatory response required to control parasite growth and to the pathogenicity of African trypanosomiasis, including immunosuppression. TSIF knock-down trypanosomes died within 2 days, indicating that TSIF may be essential for parasite biology.

In animal trypanosomiasis the severity of infection is reflected by the degree of anemia which resembles anemia of inflammation, involving a skewed iron homeostasis leading to iron accumulation within the reticuloendothelial system. Myeloid cells (M cells) have been implicated in the induction and maintenance of this type of anemia and modulation of M cells through the main trypanosome-derived glycosylphosphatidylinositol (GPI)-anchor could attenuate both anemia and trypano-susceptibility in Trypanosoma brucei-infected mice. Herein the GPI-based treatment, allowing a straightforward comparison between trypanotolerance and susceptibility in T. brucei-infected C57Bl/6 mice, was further adopted to scrutinize mechanisms/pathways underlying trypanosome-elicited anemia. Hereby, the following interlinkable observations were made in GPI-based treated (GBT) T. brucei-infected mice: (i) a reduced inflammatory cytokine production and increased IL-10 production associated with alleviation of anemia and restoration of serum iron levels, (ii) a shift in increased liver expression of iron storage towards iron export genes, (iii) increased erythropoiesis in the bone marrow and extramedullar sites (spleen) probably reflecting a normalized iron homeostasis and availability. Collectively, our results demonstrate that reprogramming macrophages towards an anti-inflammatory state alleviates anemia of inflammation by normalizing iron homeostasis and restoring erythropoiesis.

Cells of the monocyte-macrophage lineage play a central role in the orchestration and resolution of inflammation. Plasticity is a hallmark of mononuclear phagocytes, and in response to environmental signals these cells undergo different forms of polarized activation, the extremes of which are called classic or M1 and alternative or M2. NF-kappaB is a key regulator of inflammation and resolution, and its activation is subject to multiple levels of regulation, including inhibitory, which finely tune macrophage functions. Here we identify the p50 subunit of NF-kappaB as a key regulator of M2-driven inflammatory reactions in vitro and in vivo. p50 NF-kappaB inhibits NF-kappaB-driven, M1-polarizing, IFN-beta production. Accordingly, p50-deficient mice show exacerbated M1-driven inflammation and defective capacity to mount allergy and helminth-driven M2-polarized inflammatory reactions. Thus, NF-kappaB p50 is a key component in the orchestration of M2-driven inflammatory reactions.

Cells of the monocyte-macrophage lineage play a central role in the orchestration and resolution of inflammation. Plasticity is a hallmark of mononuclear phagocytes, and in response to environmental signals these cells undergo different forms of polarized activation, the extremes of which are called classic or M1 and alternative or M2. NF-kappaB is a key regulator of inflammation and resolution, and its activation is subject to multiple levels of regulation, including inhibitory, which finely tune macrophage functions. Here we identify the p50 subunit of NF-kappaB as a key regulator of M2-driven inflammatory reactions in vitro and in vivo. p50 NF-kappaB inhibits NF-kappaB-driven, M1-polarizing, IFN-beta production. Accordingly, p50-deficient mice show exacerbated M1-driven inflammation and defective capacity to mount allergy and helminth-driven M2-polarized inflammatory reactions. Thus, NF-kappaB p50 is a key component in the orchestration of M2-driven inflammatory reactions.

Using variants of the murine BW5147 lymphoma cell-line, we have previously identified 3 monoclonal antibodies (MAbs) that discriminate between metastatic and nonmetastatic BW5147-derived T-cell hybridomas and lymphomas, as well as BW5147-unrelated T-lymphomas. These MAbs were reported to recognize an identical membrane-associated sialoglycoprotein, termed "metastatic T-cell hybridoma antigen" (MTH-Ag). Here, we document that the expression pattern of the MTH-Ag on metastatic and nonmetastatic BW5147 variants correlates with that of the P-selectin glycoprotein ligand 1 (PSGL-1), a sialomucin involved in leukocyte recruitment to sites of inflammation. Moreover, the MAbs against the MTH-Ag recognize PSGL-1 when it is transfected in MTH-Ag-negative BW5147 variants, suggesting that the MTH-Ag is PSGL-1. Overexpression of MTH-Ag/PSGL-1 in MTH-Ag-negative BW5147 variants did not affect their in vivo malignancy. Yet, down-regulation of MTH-Ag/PSGL-1 expression on metastatic, MTH-Ag-positive BW5147 variants, using an RNA interference (RNAi) approach, resulted, in a dose-dependent manner, in a significant reduction of liver and spleen colonization and a delay in mortality of the recipient mice upon intravenous inoculation. Collectively, these results demonstrate that, although MTH-Ag/PSGL-1 overexpression alone may not be sufficient for successful dissemination and organ colonization, MTH-Ag/PSGL-1 plays a critical role in hematogenous metastasis of lymphoid cancer cells.

Tumors may escape from immune control by the induction of CD11b(+)Gr-1(+) myeloid suppressor cells in the spleen. In this study, we demonstrate that this cell population can be subdivided into a CD11b(hi)Gr-1(int)SSC(lo)Ly6G(neg)M-CSFR(int) immature monocytic fraction and a CD11b(hi+)Gr-1(hi)SSC(hi)Ly6G(hi)M-CSFR(neg) granulocytic fraction. Upon in vitro culture, the monocytic CD11b(+)Gr-1(+) cell fraction is sufficient for cytotoxic T lymphocyte (CTL) suppression, which is linked to the gradual differentiation of these monocytic cells into mature F4/80(+) CD68(+) macrophages. These CTL-suppressive macrophages are alternatively activated (M2), as demonstrated by the expression of known and novel M2 signature genes. In search of M2-associated genes involved in the suppressive activity, it is shown that stimulation of peroxisome proliferator-activated receptor gamma (PPARgamma) and inhibition of phospholipase A(2) (PLA(2)) activity cooperate to alleviate CTL suppression. Of importance, purified tumor-associated macrophages display a similar M2 phenotype and are suppressive for antitumor CTLs, via a mechanism that can be almost completely reversed by PPARgamma ligands. Overall, our data identify PLA(2) and especially PPARgamma as new potential therapeutic targets to subvert macrophage-mediated CTL suppression in cancer.

Tumors may escape from immune control by the induction of CD11b(+)Gr-1(+) myeloid suppressor cells in the spleen. In this study, we demonstrate that this cell population can be subdivided into a CD11b(hi)Gr-1(int)SSC(lo)Ly6G(neg)M-CSFR(int) immature monocytic fraction and a CD11b(hi+)Gr-1(hi)SSC(hi)Ly6G(hi)M-CSFR(neg) granulocytic fraction. Upon in vitro culture, the monocytic CD11b(+)Gr-1(+) cell fraction is sufficient for cytotoxic T lymphocyte (CTL) suppression, which is linked to the gradual differentiation of these monocytic cells into mature F4/80(+) CD68(+) macrophages. These CTL-suppressive macrophages are alternatively activated (M2), as demonstrated by the expression of known and novel M2 signature genes. In search of M2-associated genes involved in the suppressive activity, it is shown that stimulation of peroxisome proliferator-activated receptor gamma (PPARgamma) and inhibition of phospholipase A(2) (PLA(2)) activity cooperate to alleviate CTL suppression. Of importance, purified tumor-associatedmacrophages display a similar M2 phenotype and are suppressive for antitumor CTLs, via a mechanism that can be almost completely reversed by PPARgamma ligands. Overall, our data identify PLA(2) and especially PPARgamma as new potential therapeutic targets to subvert macrophage-mediated CTL suppression in cancer.

The specificity and affinity characteristics of antibodies make them excellent probes in biosensor applications. Unfortunately, their large size, unstable behavior, and random immobilization properties create numerous problems. The single-domain antigen-binding fragment derived from heavy-chain antibodies of camelids (termed VHH) offers special advantages in terms of size, stability, and ease of generating different antibody constructs. In this study, we show the potential of those VHHs in sensing human prostate-specific antigen (hPSA) by SPR technology. Different VHH constructs were immobilized onto commercial and custom-built sensor surfaces by metal chelation, biotin-streptavidin interaction, or covalent coupling. The detection of subnanogram per milliliter hPSA concentrations could be attained on a covalently coupled three-dimensional dextran surface. Moreover, the ratio of different hPSA isoform concentrations could be assessed via a sandwich assay and resulted in the detection of clinically significant antigen concentrations within 15 min. In addition, for the first time, the intrinsic protein stability is presented as an important probe design factor, since our results reveal that higher intrinsic stability offers higher resistance to harsh regeneration conditions. In conclusion, we present VHHs as a novel class of biosensor probes rivaling conventional antibodies and their derived antibody fragments.

International audience; Processing of fibrillar collagens is required to generate collagen monomers able to self-assemble into elongated and cylindrical collagen fibrils. ADAMTS-2 belongs to the "A disintegrin and metalloproteinase with thrombospondin type 1 motifs" (ADAMTS) family. It is responsible for most of the processing of the aminopropeptide of type I procollagen in the skin, and it also cleaves type II and type III procollagens. ADAMTS are complex secreted enzymes that are implicated in various physiological and pathological processes. Despite accumulating evidence indicating that their activity is regulated by ancillary domains, additional information is required for a better understanding of the specific function of each domain. We have generated 17 different recombinant forms of bovine ADAMTS-2 and characterized their processing, activity, and cleavage specificity. The results indicated the following: (i) activation of the ADAMTS-2 zymogen involves several cleavages, by proprotein convertases and C-terminal processing, and generates at least seven distinct processed forms; (ii) the C-terminal domain negatively regulates enzyme activity, whereas two thrombospondin type 1 repeats are enhancer regulators; (iii) the 104-kDa form displays the highest aminoprocollagen peptidase activity on procollagen type I; (iv) ADAMTS-2 processes the aminopropeptide of alpha1 type V procollagen homotrimer at the end of the variable domain; and (v) the cleaved sequence (PA) is different from the previously described sites ((P/A)Q) for ADAMTS-2, redefining its cleavage specificity. This finding and the existence of multiple processed forms of ADAMTS-2 strongly suggest that ADAMTS-2 may be involved in function(s) other than processing of fibrillar procollagen types I-III.Processing of fibrillar collagens is required to generate collagen monomers able to self-assemble into elongated and cylindrical collagen fibrils. ADAMTS-2 belongs to the "A disintegrin and metalloproteinase with thrombospondin type 1 motifs" (ADAMTS) family. It is responsible for most of the processing of the aminopropeptide of type I procollagen in the skin, and it also cleaves type II and type III procollagens. ADAMTS are complex secreted enzymes that are implicated in various physiological and pathological processes. Despite accumulating evidence indicating that their activity is regulated by ancillary domains, additional information is required for a better understanding of the specific function of each domain. We have generated 17 different recombinant forms of bovine ADAMTS-2 and characterized their processing, activity, and cleavage specificity. The results indicated the following: (i) activation of the ADAMTS-2 zymogen involves several cleavages, by proprotein convertases and C-terminal processing, and generates at least seven distinct processed forms; (ii) the C-terminal domain negatively regulates enzyme activity, whereas two thrombospondin type 1 repeats are enhancer regulators; (iii) the 104-kDa form displays the highest aminoprocollagen peptidase activity on procollagen type I; (iv) ADAMTS-2 processes the aminopropeptide of alpha1 type V procollagen homotrimer at the end of the variable domain; and (v) the cleaved sequence (PA) is different from the previously described sites ((P/A)Q) for ADAMTS-2, redefining its cleavage specificity. This finding and the existence of multiple processed forms of ADAMTS-2 strongly suggest that ADAMTS-2 may be involved in function(s) other than processing of fibrillar procollagen types I-III.

Molecular markers, especially surface markers associated with type II, cytokine-dependent, alternatively activated macrophages (aaMF), remain scarce. Besides the earlier documented markers, macrophage mannose receptor and arginase 1, we demonstrated recently that murine aaMF are characterized by increased expression of found in inflammatory zone 1 (FIZZ1) and the secretory lectin Ym. We now document that expression of the two members of the mouse macrophage galactose-type C-type lectin gene family (mMGL1 and mMGL2) is induced in diverse populations of aaMF, including peritoneal macrophages elicited during infection with the protozoan Trypanosoma brucei brucei or the Helminth Taenia crassiceps and alveolar macrophages elicited in a mouse model of allergic asthma. In addition, we demonstrate that in vitro, interleukin-4 (IL-4) and IL-13 up-regulate mMGL1 and mMGL2 expression and that in vivo, induction of mMGL1 and mMGL2 is dependent on IL-4 receptor signaling. Moreover, we show that expression of MGL on human monocytes is also up-regulated by IL-4. Hence, macrophage galactose-type C-type lectins represent novel surface markers for murine and human aaMF.

Although it is well-established that macrophages can occur in distinct activation states, the molecular characteristics of differentially activated macrophages, and particularly those of alternatively activated macrophages (aaMφ), are still poorly unraveled. Recently, we demonstrated that the expression of FIZZ1 and Ym is induced in aaMφ as compared with classically activated macrophages (caMφ), elicitedin vitroor developedin vivoduring infection withTrypanosoma brucei brucei. In the present study, we analyzed the expression of FIZZ1 and Ym in caMφ and aaMφ elicited duringTrypanosoma congolenseinfection and show that the use of FIZZ1 and Ym for the identification of aaMφ is not limited toT. b. bruceiinfection and is independent of the organ sources from which macrophages are obtained. We also demonstrate that FIZZ1 can be used to discriminate between different populations of aaMφ. Furthermore, we studied the effects of various stimuli, and combinations thereof, on the expression of FIZZ1 and Ym in macrophages from different mouse strains and demonstrate that regulation of the expression of FIZZ1 and Ym in macrophages is not dependent on the mouse strain. Finally, we show that these genes can be used to monitor the macrophage activation status without the need to obtain pure macrophage populations.

Whereas functional heavy (H)-chain antibodies devoid of light (L)- chains account for about half of the circulating immunoglobulins in Camelidae, H-chain only antibodies (HCAbs) are not produced in other healthy mammals including rodents and humans. To test the feasibility of expressing single chain antibodies in the mouse, which on account of their small size and antigen-recognition properties would have a major impact on antibody engineering strategies, we constructed a rearranged dromedary H-chain gene encoding the immunoglobulin G2a (IgG2a) isotype with specificity for hen-egg lysozyme (HEL). This IgG2a H-chain gene was introduced into mouse myeloma cells not expressing endogenous immunoglobulin H- or L-chains. Unexpectedly the mouse cells processed and expressed the introduced H-chain as naturally occurring dromedary antibody. For this the first constant (C) region exon was proficiently removed from the recombinant H-chain transcript. This resulted in specific H-chain antibodies of the correct molecular weight (2 x 50 000 MW) secreted as disulfide-linked homodimers and displayed on the mouse cell surface as glycosyl-phosphatidyl-inositol-linked B-cell receptor. The results indicate that antibody expression and maturation without immunoglobulin L-chain is feasible and paves the way for the generation of transgenic single chain antibody repertoires.

Most of the mice bearing a s.c. BW-Sp3 lymphoma tumor mount a CD8+ T cell-mediated response resulting in tumor regression. Nonetheless, tumor progression occurs in some of the recipients and is associated with CTL inactivity. We demonstrated that T cell-activating APC were induced in regressors whereas T cell suppressive myeloid cells predominated in the spleen of progressors. Indeed, in vitro depletion of either the adherent or the CD11b+ populations restored T cell cytotoxicity and proliferation in these mice. This CTL inhibition was cell-to-cell contact-dependent but not mediated by NO. However, the same progressor suppressive cells prevented the activity of in vitro-restimulated CTLs derived from regressors in a cell-to-cell contact and NO-dependent fashion. Thus, either the NO-dependent or -independent suppressive pathway prevailed, depending on the target CTL population. In addition, the suppressive population expressed a high arginase activity, suggesting an association of the suppressive phenotype with alternatively activated (M2) myeloid cells. However, the high arginase activity is not directly involved in the suppressive process. Our results provide new insights for myeloid cell-mediated CTL inhibition during cancer progression.

The contribution of cytokines and chemokines to resistance and susceptibility to African trypanosomiasis remains controversial. In the present study, the levels of type I and type II cytokines and of the MCP-1 chemokine were compared during the early and late stages ofTrypanosoma congolenseinfection in susceptible BALB/c and resistant C57BL/6 mice. Moreover, the status of macrophage activation was compared in these animals by analyzing the inducible nitric oxide synthase-arginase balance, tumor necrosis factor secretion, and expression of the FIZZ1 and YM genes. Data show that changing from a predominant type I cytokine environment in the early stage of infection to a predominant type II cytokine environment and an enhanced MCP-1 secretion in the late stage of infection correlates with resistance toT. congolense. Concomitantly, macrophage activation evolves from a classical to a predominant alternative phenotype. We further confirmed that the simultaneous occurrence of type I/type II cytokines in the early stage of infection in susceptible BALB/c mice, reflected by the presence of macrophages exhibiting a mixed classical/alternative activation phenotype, is associated with uncontrolled parasite growth and early death. Interleukin-4 (IL-4) and IL-13 signaling did not influence the susceptibility of BALB/c mice toT. congolenseinfection and interestingly were not the main trigger to alternative macrophage activation. InT. congolense-resistant C57BL/6 mice, our results corroborated the induction of FIZZ1 and YM gene expressions with the alternative pathway of macrophage activation. In susceptible BALB/c mice, however, YM but not FIZZ1 induction reflected the emergence of alternatively activated macrophages. Hence, the FIZZ1 and YM genes may be useful markers to discriminate between distinct populations of alternatively activated macrophages.

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., 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.

We previously reported on a murine T lymphoma cell line, BW-Sp3, with inherent immunogenicity. BW-Sp3 tumors can elicit an anti-tumor CD8(+) CTL response capable of mediating a regression of subcutaneous tumors. However, this immune response is inadequate to eliminate cancer cells completely in a significant percentage of the recipients, resulting in progressing tumors. In this tumor model, tumor progression correlated with a tolerization of tumor-reactive T cells and cellular immunotherapy of tumor bearing animals, with or without B7-mediated costimulation, even increased tumor progression (Raes et al, 1998). In the present study, we investigated whether the co-expression of IFN gamma, together with B7-1, could have beneficial effects on immunotherapy. Although immunotherapy with IFN gamma and B7-1 single transfectants tended to tolerize anti-tumor T-cells and consequently increased tumor growth, the B7-1/IFN gamma double transfectants resulted in a more beneficial outcome. This phenomenon correlated with anincreased CTL-inducing potential of the double transfectants. Secondly, we wondered whether CTLA-4 signalling was involved in the down-regulation of the anti-tumor response. Indeed, when immunotherapy was provided along with anti-CTLA-4, the protection by B71/IFN gamma double transfectants was further improved and the tumor-promoting effect of BW-Sp3(B7-1) was compensated for. Our results indicate that B7-1, IFN gamma and the blockade of CTLA-4 cooperate to tilt the balance in favour of tumor elimination, while either factor alone fails to do so or even promotes tumor growth.