Interleukin-23 instructs protective multifunctional CD4 T cell responses after immunization with the Mycobacterium tuberculosis subunit vaccine H1 DDA/TDB independently of interleukin-17A.

Interleukin (IL)-17A-producing T helper (Th)17 cells are increasingly being acknowledged to be associated with protective immunity to Mycobacterium tuberculosis (Mtb). Subunit vaccines potently promote protective immune responses against Mtb infection that correlate with an expansion of IL-23-dependent Th17 cells. Previous studies revealed that after vaccination, IL-23 is required for protection against challenge with Mtb but the underlying IL-23-dependent-and possibly IL-17A-mediated-mechanisms remain elusive. Therefore, we here analyzed the early outcome of Mtb infection in C57BL/6, IL-23p19-deficient (-/-), and IL-17A-/- mice after vaccination with the subunit vaccine H1-DDA/TDB to investigate the role of the IL-23-Th17 immune axis for the instruction of vaccine-induced protection. While in IL-23p19-/- mice the protective effect was reduced, protection after vaccination was maintained in IL-17A-/- animals for the course of infection of 6 weeks, indicating that after vaccination with H1-DDA/TDB early protection against Mtb is-although dependent on IL-23-not mediated by IL-17A. In contrast, IL-17A deficiency appears to have an impact on maintaining long-term protection. In fact, IL-23 instructed the vaccine-induced memory immunity in the lung, in particular the sustained expansion of tumor necrosis factor (TNF)+IL-2+ multifunctional T cells, independently of IL-17A. Altogether, a targeted induction of IL-23 during vaccination against Mtb might improve the magnitude and quality of vaccine-induced memory immune responses. KEY MESSAGES: After subunit Mtb vaccination with H1-DDA/TDB, IL-23 but not IL-17A contributes to vaccine-induced early protection against infection with Mtb. IL-17F does not compensate for IL-17A deficiency in terms of H1-DDA/TDB-induced protection against Mtb infection. IL 23 promotes the H1-DDA/TDB-induced accumulation of effector memory T cells independently of IL 17A. IL-23 arbitrates the induction of H1-specific IFN-γ-TNF+IL-2+ double-positive multifunctional CD4 T cells after subunit Mtb vaccination in an IL-17A-independent manner.

The increased protection and pathology in Mycobacterium tuberculosis-infected IL-27R-alpha-deficient mice is supported by IL-17A and is associated with the IL-17A-induced expansion of multifunctional T cells.

During Mycobacterium tuberculosis (Mtb) infection, mice lacking the IL-27R exhibit lower bacterial burdens but develop an immunopathological sequelae in comparison to wild-type mice. We here show that this phenotype correlates with an enhanced recruitment of antigen-specific CCR6+ CD4+ T cells and an increased frequency of IL-17A-producing CD4+ T cells. By comparing the outcome of Mtb infection in C57BL/6, IL-27R-deficient and IL-27R/IL-17A-double deficient mice, we observed that both the increased protection and elevated immunopathology are supported by IL-17A. Whereas IL-17A neither impacts the development of Tr1 cells nor the expression of PD1 and KLRG1 on T cells in IL-27R-deficient mice during infection, it regulates the presence of multifunctional T-cells in the lungs, co-expressing IFN-γ, IL-2 and TNF. Eventually, IL-17A supports Cxcl9, Cxcl10 and Cxcl13 expression and the granulomatous response in the lungs of infected IL-27R-deficient mice. Taken together, IL-17A contributes to protection in Mtb-infected IL-27R-deficient mice probably through a chemokine-mediated recruitment and strategic positioning of multifunctional T cells in granulomas. As IL-27 limits optimal antimycobacterial protection by inhibiting IL-17A production, blocking of IL-27R-mediated signaling may represent a strategy for improving vaccination and host-directed therapy in tuberculosis. However, because IL-27 also prevents IL-17A-mediated immunopathology, such intervention has to be tightly controlled.

De Novo Fatty Acid Synthesis During Mycobacterial Infection Is a Prerequisite for the Function of Highly Proliferative T Cells, But Not for Dendritic Cells or Macrophages.

Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, is able to efficiently manipulate the host immune system establishing chronic infection, yet the underlying mechanisms of immune evasion are not fully understood. Evidence suggests that this pathogen interferes with host cell lipid metabolism to ensure its persistence. Fatty acid metabolism is regulated by acetyl-CoA carboxylase (ACC) 1 and 2; both isoforms catalyze the conversion of acetyl-CoA into malonyl-CoA, but have distinct roles. ACC1 is located in the cytosol, where it regulates de novo fatty acid synthesis (FAS), while ACC2 is associated with the outer mitochondrial membrane, regulating fatty acid oxidation (FAO). In macrophages, mycobacteria induce metabolic changes that lead to the cytosolic accumulation of lipids. This reprogramming impairs macrophage activation and contributes to chronic infection. In dendritic cells (DCs), FAS has been suggested to underlie optimal cytokine production and antigen presentation, but little is known about the metabolic changes occurring in DCs upon mycobacterial infection and how they affect the outcome of the immune response. We therefore determined the role of fatty acid metabolism in myeloid cells and T cells during Mycobacterium bovis BCG or Mtb infection, using novel genetic mouse models that allow cell-specific deletion of ACC1 and ACC2 in DCs, macrophages, or T cells. Our results demonstrate that de novo FAS is induced in DCs and macrophages upon M. bovis BCG infection. However, ACC1 expression in DCs and macrophages is not required to control mycobacteria. Similarly, absence of ACC2 did not influence the ability of DCs and macrophages to cope with infection. Furthermore, deletion of ACC1 in DCs or macrophages had no effect on systemic pro-inflammatory cytokine production or T cell priming, suggesting that FAS is dispensable for an intact innate response against mycobacteria. In contrast, mice with a deletion of ACC1 specifically in T cells fail to generate efficient T helper 1 responses and succumb early to Mtb infection. In summary, our results reveal ACC1-dependent FAS as a crucial mechanism in T cells, but not DCs or macrophages, to fight against mycobacterial infection.

Suppressor of Cytokine Signaling 3 in Macrophages Prevents Exacerbated Interleukin-6-Dependent Arginase-1 Activity and Early Permissiveness to Experimental Tuberculosis.

Suppressor of cytokine signaling 3 (SOCS3) is a feedback inhibitor of interleukin (IL)-6 signaling in macrophages. In the absence of this molecule, macrophages become extremely prone to an IL-6-dependent expression of arginase-1 (Arg1) and nitric oxide synthase (NOS)2, the prototype markers for alternative or classical macrophage activation, respectively. Because both enzymes are antipodean macrophage effector molecules in Mycobacterium tuberculosis (Mtb) infection, we assessed the relevance of SOCS3 for macrophage activation during experimental tuberculosis using macrophage-specific SOCS3-deficient (LysMcreSOCS3loxP/loxP) mice. Aerosol infection of LysMcreSOCS3loxP/loxP mice resulted in remarkably higher bacterial loads in infected lungs and exacerbated pulmonary inflammation. This increased susceptibility to Mtb infection was accompanied by enhanced levels of both classical and alternative macrophage activation. However, high Arg1 expression preceded the increased induction of NOS2 and at early time points of infection mycobacteria were mostly found in cells positive for Arg1. This sequential activation of Arg1 and NOS2 expression in LysMcreSOCS3loxP/loxP mice appears to favor the initial replication of Mtb particularly in Arg1-positive cells. Neutralization of IL-6 in Mtb-infected LysMcreSOCS3loxP/loxP mice reduced arginase activity and restored control of mycobacterial replication in LysMcreSOCS3loxP/loxP mice. Our data reveal an unexpected role of SOCS3 during experimental TB: macrophage SOCS3 restrains early expression of Arg1 and helps limit Mtb replication in resident lung macrophages, thereby limiting the growth of mycobacteria. Together, SOCS3 keeps IL-6-dependent divergent macrophage responses such as Nos2 and Arg1 expression under control and safeguard protective macrophage effector mechanisms.

During acute experimental infection with the reticulotropic Trypanosoma cruzi strain Tulahuen IL-22 is induced IL-23-dependently but is dispensable for protection.

Protective immunity against Trypanosoma cruzi, the causative agent of Chagas disease, depends on the activation of macrophages by IFN-γ and IL-17A. In contrast, IL-10 prevents immunopathology. IL-22 belongs to the IL-10 cytokine family and has pleiotropic effects during host defense and immunopathology, however its role in protection and pathology during T. cruzi infection has not been analyzed yet. Therefore, we examined the role of IL-22 in experimental Chagas disease using the reticulotropic Tulahuen strain of T. cruzi. During infection, IL-22 is secreted by CD4-positive cells in an IL-23-dependent fashion. Infected IL-22(-/-) mice exhibited an increased production of IFN-γ and TNF and displayed enhanced numbers of activated IFN-γ-producing T cells in their spleens. Additionally, the production of IL-10 was increased in IL-22(-/-) mice upon infection. Macrophage activation and by association the parasitemia was not affected in the absence of IL-22. Apart from a transient increase in the body weight loss, infected IL-22(-/-) mice did not show any signs for an altered immunopathology during the first fourteen days of infection. Taken together, although IL-22 is expressed, it seems to play a minor role in protection and pathology during the acute systemic infection with the reticulotropic Tulahuen strain of T. cruzi.

Epstein-Barr virus-induced gene 3 suppresses T helper type 1, type 17 and type 2 immune responses after Trypanosoma cruzi infection and inhibits parasite replication by interfering with alternative macrophage activation.

The Epstein-Barr virus-induced gene 3 (EBI3) is a member of the interleukin-12 (IL)-12) family structurally related to the subunit p40 of IL-12 and forms a heterodimer either with the p28 subunit to build IL-27 or with p35 to form IL-35. Interleukin-27 is secreted by antigen-presenting cells whereas IL-35 appears to be produced mainly by regulatory T cells and regulatory B cells but both cytokines negatively regulate inflammatory immune responses. We here analysed the function of EBI3 during infection with the intracellular parasite Trypanosoma cruzi. Compared with C57BL/6 wild-type mice, EBI3-deficient (EBI3(-/-) ) mice showed a higher parasitaemia associated with an increased mortality rate. The EBI3(-/-) mice displayed an elevated inflammatory immune response with an increased production of T helper type 1 (Th1-), Th2- and Th17-derived cytokines. The increased Th2 immune response appears to have over-ridden the otherwise protective Th1 and Th17 immune responses by the induction of arginase-1-expressing alternatively activated macrophages in these mice. Hence, neutralization of IL-4 and arginase-1 activity partially restored protective immune responses in EBI3(-/-) mice. So far, our results demonstrate that EBI3 is an essential general regulator of inflammatory immune responses in experimental Chagas disease and is required for control of T. cruzi infection by inhibiting Th2-dependent alternative macrophage activation. Further studies are needed to dissect the underlying mechanisms and clarify whether EBI3 association with IL-27 or/and IL-35 accounts for its anti-inflammatory character in parasitic disease.

The IL-13/IL-4Rα axis is involved in tuberculosis-associated pathology.

Human tuberculosis (TB) is a leading global health threat and still constitutes a major medical challenge. However, mechanisms governing tissue pathology during post-primary TB remain elusive, partly because genetically or immunologically tractable animal models are lacking. In human TB, the demonstration of a large relative increase in interleukin (IL)-4 and IL-13 expression, which correlates with lung damage, indicates that a subversive T helper (TH)2 component in the response to Mycobacterium tuberculosis (Mtb) may undermine protective immunity and contribute to reactivation and tissue pathology. Up to now, there has been no clear evidence regarding whether IL-4/IL-13-IL-4 receptor-α (Rα)-mediated mechanisms may in fact cause reactivation and pathology. Unfortunately, the virtual absence of centrally necrotizing granulomas in experimental murine TB is associated with a poor induction of a TH2 immune response. We therefore hypothesize that, in mice, an increased production of IL-13 may lead to a pathology similar to human post-primary TB. In our study, aerosol Mtb infection of IL-13-over-expressing mice in fact resulted in pulmonary centrally necrotizing granulomas with multinucleated giant cells, a hypoxic rim and a perinecrotic collagen capsule, with an adjacent zone of lipid-rich, acid-fast bacilli-containing foamy macrophages, thus strongly resembling the pathology in human post-primary TB. Granuloma necrosis (GN) in Mtb-infected IL-13-over-expressing mice was associated with the induction of arginase-1-expressing macrophages. Indirect blockade of the endogenous arginase inhibitor l-hydroxyarginine in Mtb-infected wild-type mice resulted in a strong arginase expression and precipitated a similar pathology of GN. Together, we here introduce an experimental TB model that displays many features of centrally necrotizing granulomas in human post-primary TB and demonstrate that IL-13/IL-4Rα-dependent mechanisms leading to arginase-1 expression are involved in TB-associated tissue pathology.

9- and 11-Substituted 4-azapaullones are potent and selective inhibitors of African trypanosoma.

Trypanosomes from the "brucei" complex are pathogenic parasites endemic in sub-Saharan Africa and causative agents of severe diseases in humans and livestock. In order to identify new antitrypanosomal chemotypes against African trypanosomes, 4-azapaullones carrying α,ß-unsaturated carbonyl chains in 9- or 11-position were synthesized employing a procedure with a Heck reaction as key step. Among the so prepared compounds, 5a and 5e proved to be potent antiparasitic agents with antitrypanosomal activity in the submicromolar range.

IL-17A promotes macrophage effector mechanisms against Trypanosoma cruzi by trapping parasites in the endolysosomal compartment.

The contribution of the IL-23-IL-17A pathway to resistance against extracellular bacterial infections is well established, whereas its role in immunity to intracellular pathogens is much less clear. To analyze the contribution of the IL-23-IL-17A-axis to resistance against Trypanosoma cruzi infection, we infected IL-23p19(-/-) mice and IL-17A(-/-) mice with T. cruzi. Both mouse strains were susceptible to T. cruzi infection despite strong Th1 immune responses. In vitro experiments revealed that IL-17A, but not IL-23, directly stimulates macrophages to internalize T. cruzi parasites by phagocytosis, which is in contrast to the active invasion process normally used by T. cruzi. In contrast to the active entry of parasites into macrophages, the IL-17A-driven phagocytosis prolonged residency of parasites in the endosomal/lysosomal compartment of the macrophage, which subsequently led to eradication of parasites. This IL-17A-dependent mechanism represents a novel function of IL-17A trapping pathogens in endosomal/lysosomal compartments and enhancing exposure time to antimicrobial effectors of the macrophage.

Molecular interaction of Siglecs (sialic acid-binding Ig-like lectins) with sialylated ligands on Trypanosoma cruzi.

The protozoan parasite Trypanosoma cruzi (T. cruzi) is transmitted by blood-sucking insect vectors. After transmission, parasites circulate in the blood as trypomastigotes and invade a variety of cells to multiply intracellularly as amastigotes. The acute phase triggers an immune response that restricts the dissemination and proliferation of parasites. However, parasites are able to persist in different tissues for decades causing the pathology of Chagas' disease. T. cruzi expresses a trans-sialidase (TS). This unique enzyme transfers sialic acid from host glycoconjugates to mucin-like molecules on the parasite and is supposed to be a major virulence factor. TS and sialylated structures were implicated in the persistence of parasites. We discuss here the recent findings on the function of sialylated structures on the surface of T. cruzi with a special emphasis on their property to interact with sialic acid-binding Ig-like lectins, which may allow the parasite to modulate the immune system of the host.

Sialylated ligands on pathogenic Trypanosoma cruzi interact with Siglec-E (sialic acid-binding Ig-like lectin-E).

Trypanosoma cruzi causes a suppression of the immune system leading to persistence in host cells. The trans-sialidase expressed by T. cruzi is a major virulence factor and transfers sialic acid from host glycoconjugates to mucin-like molecules on the parasite. Here we demonstrate that these sialylated structures play a role in the immunosuppression. We used two T. cruzi strains, whose TS activity correlated with their pathogenicity. The Tulahuen strain, characterized by a high TS activity efficiently infected mice, whereas the Tehuantepec strain showing a reduced TS activity could not establish a patent parasitemia. In vitro analysis revealed that these two strains invaded phagocytic and non-phagocytic host cells at a comparable rate, but they exhibited different potentials to modulate dendritic cell function. In contrast to Tehuantepec, the Tulahuen strain suppressed the production of the proinflammatory cytokine IL-12 and subsequent T-cell activation. This inhibitory effect was absent upon desialylation of the parasite. Therefore, we analysed whether sialylated structures of T. cruzi interact with the inhibitory sialic acid-binding protein Siglec-E on DC. Indeed, Siglec-E interacted with the pathogenic Tulahuen strain, but showed a diminished binding to the Tehuantepec strain. Ligation of Siglec-E on DC using antibodies confirmed this inhibitory effect on DC function.