Sabotage and exploitation in macrophages parasitized by intracellular protozoans

Macrophages are crucial in immunity to infection. They possess potent antimicrobial function, and efficiently process and present peptide antigens for T-cell activation. Despite this, the intracellular protozoan parasites Toxoplasma gondii, Trypanosoma cruzi and Leishmania spp. target macrophages for infection. Each has adopted unique strategies to subvert macrophage antimicrobial functions. The parasites sabotage killing activities through sophisticated manipulation of intracellular macrophage signaling pathways. These subversive activities are probably dictated by the need to evade microbicidal effector function, as well as to avoid proinflammatory pathology that can destabilize the host-parasite interaction. The molecular details of how intracellular protozoans manipulate macrophage signal transduction pathways for their own ends are beginning to emerge.     

Trefoil Factor 2 Negatively Regulates Type 1 Immunity against Toxoplasma gondii

IL-12-mediated type 1 inflammation confers host protection against the parasitic protozoan Toxoplasma gondii. However, production of IFN-γ, another type 1 inflammatory cytokine, also drives lethality from excessive injury to the intestinal epithelium. As mechanisms that restore epithelial barrier function following infection remain poorly understood, this study investigated the role of trefoil factor 2 (TFF2), a well-established regulator of mucosal tissue repair. Paradoxically, TFF2 antagonized IL-12 release from dendritic cells (DCs) and macrophages, which protected TFF2-deficient (TFF2(-/-)) mice from T. gondii pathogenesis. Dysregulated intestinal homeostasis in naive TFF2(-/-) mice correlated with increased IL-12/23p40 levels and enhanced T cell recruitment at baseline. Infected TFF2(-/-) mice displayed low rates of parasite replication and reduced gut immunopathology, whereas wild-type (WT) mice experienced disseminated infection and lethal ileitis. p38 MAPK activation and IL-12p70 production was more robust from TFF2(-/-)CD8(+) DC compared with WT CD8(+) DC and treatment of WT DC with rTFF2 suppressed TLR-induced IL-12/23p40 production. Neutralization of IFN-γ and IL-12 in TFF2(-/-) animals abrogated resistance shown by enhanced parasite replication and infection-induced morbidity. Hence, TFF2 regulated intestinal barrier function and type 1 cytokine release from myeloid phagocytes, which dictated the outcome of oral T. gondii infection in mice.     

Inflammation drives dysbiosis and bacterial invasion in murine models of ileal Crohn's disease

Background and Aims

Understanding the interplay between genetic susceptibility, the microbiome, the environment and the immune system in Crohn’s Disease (CD) is essential for developing optimal therapeutic strategies. We sought to examine the dynamics of the relationship between inflammation, the ileal microbiome, and host genetics in murine models of ileitis.

Methods

We induced ileal inflammation of graded severity in C57BL6 mice by gavage with Toxoplasma gondii, Giardia muris, low dose indomethacin (LDI;0.1 mg/mouse), or high dose indomethacin (HDI;1 mg/mouse). The composition and spatial distribution of the mucosal microbiome was evaluated by 16S rDNA pyrosequencing and fluorescence in situ hybridization. Mucosal E. coli were enumerated by quantitative PCR, and characterized by phylogroup, genotype and pathotype.

Results

Moderate to severe ileitis induced by T. gondii (day 8) and HDI caused a consistent shift from >95% Gram + Firmicutes to >95% Gram - Proteobacteria. This was accompanied by reduced microbial diversity and mucosal invasion by adherent and invasive E. coli, mirroring the dysbiosis of ileal CD. In contrast, dysbiosis and bacterial invasion did not develop in mice with mild ileitis induced by Giardia muris. Superimposition of genetic susceptibility and T. Gondii infection revealed greatest dysbiosis and bacterial invasion in the CD-susceptible genotype, NOD2^−/−, and reduced dysbiosis in ileitis-resistant CCR2^−/− mice. Abrogating inflammation with the CD therapeutic anti-TNF-α-mAb tempered dysbiosis and bacterial invasion.

Conclusions

Acute ileitis induces dysbiosis and proliferation of mucosally invasive E. coli, irrespective of trigger and genotype. The identification of CCR2 as a target for therapeutic intervention, and discovery that host genotype and therapeutic blockade of inflammation impact the threshold and extent of ileal dysbiosis are of high relevance to developing effective therapies for CD.     

Deploying parasite profilin on a mission of invasion and danger

Interleukin-12 (IL-12) is important in inducing Th1 responses during infection with microbial pathogens such as the protozoan Toxoplasma gondii. In this issue of Cell Host & Microbe, Plattner and colleagues describe an engineered Toxoplasma strain that lacks profilin, an actin-binding molecule previously implicated in Toll-like receptor-11-dependent IL-12 induction and now shown to be important in parasite motility and host cell invasion.     

IFN-gamma overproduction and high level apoptosis are associated with high but not low virulence Toxoplasma gondii infection.

Toxoplasma gondii is an opportunistic intracellular parasite which induces a highly strong type 1 cytokine response. The present study focuses on defining the factors influencing the outcome of infection with tachyzoites of the type I, highly lethal RH strain, relative to the type II, low virulence strain ME49. Infection with the RH strain led to widespread parasite dissemination and rapid death of mice; in contrast, mice survived low virulence strain ME49 infection, and tachyzoite dissemination was much less extensive. Furthermore, massive apoptosis and disintegration of the splenic architecture was characteristic of RH, but not ME49, infection. In addition, hyperinduction of IFN-gamma and lack of NO production were found during RH, in contrast to ME49 infection. These data demonstrate that Toxoplasma strain characteristics exert a profound effect on the host immune response and that the latter itself is a crucial determinant in parasite virulence.     

T lymphocyte-dependent effector mechanisms of immunity to Toxoplasma gondii

Immunity to the opportunistic pathogen, Toxoplasma gondii, is highly dependent upon the effector activity of IFN-gamma-producing T lymphocytes. While IFN-gamma is required to survive infection, an understanding of its function remains incomplete. During infection, T. gondii simultaneously induces downregulatory antiinflammatory cytokines, thereby avoiding major host pathology mediated by proinflammatory cytokines such as IFN-gamma. The ability to induce the correct balance between these two opposing host responses likely accounts for the success of this organism as a parasite.     

IGFBP7 induces apoptosis of acute myeloid leukemia cells and synergizes with chemotherapy in suppression of leukemia cell survival

Despite high remission rates after chemotherapy, only 30–40% of acute myeloid leukemia (AML) patients survive 5 years after diagnosis. This extremely poor prognosis of AML is mainly caused by treatment failure due to chemotherapy resistance. Chemotherapy resistance can be caused by various features including activation of alternative signaling pathways, evasion of cell death or activation of receptor tyrosine kinases such as the insulin growth factor-1 receptor (IGF-1R). Here we have studied the role of the insulin-like growth factor-binding protein-7 (IGFBP7), a tumor suppressor and part of the IGF-1R axis, in AML. We report that IGFBP7 sensitizes AML cells to chemotherapy-induced cell death. Moreover, overexpression of IGFBP7 as well as addition of recombinant human IGFBP7 is able to reduce the survival of AML cells by the induction of a G2 cell cycle arrest and apoptosis. This effect is mainly independent from IGF-1R activation, activated Akt and activated Erk. Importantly, AML patients with high IGFBP7 expression have a better outcome than patients with low IGFBP7 expression, indicating a positive role for IGFBP7 in treatment and outcome of AML. Together, this suggests that the combination of IGFBP7 and chemotherapy might potentially overcome conventional AML drug resistance and thus might improve AML patient survival.Only 30–40% of acute myeloid leukemia (AML) patients survive 5 years after diagnosis.¹ This extremely poor prognosis is mainly caused by treatment failure due to chemotherapy resistance. This resistance is often a multifactorial phenomenon that can include enhanced expression or activation of receptor tyrosine kinases such as the insulin growth factor-1 receptor (IGF-1R).^{2, 3} The IGF-1R stimulates proliferation, protects cells from apoptosis and has been implicated in the development and maintenance of various cancers.^{4, 5} Several oncogenes require an intact IGF-1R pathway for their transforming activity⁶ and moreover, disruption or inhibition of IGF-1R activity has been shown to inhibit the growth and motility of a wide range of cancer cells in vitro and in mouse models.^{4, 5} IGF-1Rs are membrane receptors and binding of their ligand, the insulin-like growth factor-1 (IGF-1), results in receptor phosphorylation and activation of MAPK and PI3K/Akt signaling.⁴ Importantly, IGF-1, normally produced by the liver and bone marrow stromal cells, can stimulate the proliferation of cancer cells in vitro and genetic manipulations that reduce IGF-1 signaling can lead to decreased tumor growth.^{7, 8}In hematological malignancies, a role for IGF-1 signaling has been demonstrated in multiple myeloma (MM) where it stimulates growth and potently mediates survival.⁹ Several anti-IGF-1R strategies have been shown to inhibit MM growth.^{10, 11} In AML, expression of the IGF-1R and IGF-1 was detected in AML cell lines and primary AML blasts and stimulation with IGF-1 can promote the growth of AML cells.^{12, 13, 14} In addition, neutralizing IGF-1R antibodies and the tyrosine kinase inhibitors (TKIs) NVP-AEW541 and NVP-ADW742, have been shown to inhibit proliferation and to induce apoptosis.^{15, 16}In addition to its mitogenic and anti-apoptotic roles, directly influencing tumor development, IGF-1R appears to be a critical determinant of response to numerous anti-cancer therapies, including TKIs and chemotherapy.^{2, 3, 17, 18, 19, 20, 21, 22} In AML, activated IGF-1R signaling has been linked to cytarabine resistance, a drug included in every AML treatment schedule.¹⁷ Notably, in several cancer cell lines, a small subpopulation of drug-tolerant cancer cells exists that maintains their viability, after treatment with a lethal drug dose, via engagement of the IGF-1R.¹⁸The activity of the IGF-1R is tightly controlled at multiple levels, including their processing, endocytosis, trafficking and availability of its ligands.⁴ Ligand bioavailability is partly controlled by the family of secreted insulin-like growth factor-binding protein (IGFBP1 to IGFBP6), which can bind to IGFs therewith regulating the interaction of these ligands to their receptors. However, as IGFBPs are able to induce IGF-dependent and IGF-independent effects, the results of several studies on their role in cancer cell survival appeared to be controversial and complex.^{23, 24} In addition to IGFBPs, various IGFBP-related proteins have been identified.^{23, 25} One of these is the IGFB-related protein 1, also known as insulin-like growth factor-binding protein-7 (IGFBP7). IGFBP7 has 30% homology to IGFBP1 to IGFBP6 in its N-terminal domain and functions predominantly as a tumor suppressor.^{23, 24, 25, 26} In contrast to IGFBP1 to IGFBP6, which bind to the IGFs,²³ IGFBP7 is a secreted protein that can directly bind to the IGF-1R and thereby inhibits its activity.²⁷ The abundance of IGFBP7 is inversely correlated with tumor progression in hepatocellular carcinoma.²⁸ Importantly, decreased expression of IGFBP7 has been associated with therapy resistance^{29, 30} and increasing IGFBP7 levels can inhibit melanoma and breast cancer growth.^{31, 32} IGFBP7 was originally identified as being involved in Raf-mediated apoptosis and senescence³³ and also has been shown to induce senescence in mesenchymal stromal cells.³⁴We established that IGFBP7 induces a cell cycle block and apoptosis in AML cells and cooperates with chemotherapy in the induction of leukemia cell death. AML patients with low IGFBP7 expression have a worse outcome than patients with high IGFBP7 expression, indicating that AML patients might benefit from a combination therapy consisting of chemotherapy and IGFBP7. Our results define IGFBP7 as a focus to enhance chemotherapy efficacy and improve AML patient survival.     

Trichinella spiralis: influence of an immunodominant, carbohydrate-associated determinant on the host antibody response repertoire.

Host antibody responses to the G2.1 epitope, a carbohydrate-associated determinant shared by several Trichinella spiralis glycoproteins, were examined by competitive inhibition enzyme-linked immunosorbent assay (ELISA). The G2.1 epitope dominated the AKR/J mouse antibody response whether the antigens were injected or introduced through infection, as determined by the serum blocking ability of a G2.1 epitope-specific monoclonal antibody (mAb). Serum T. spiralis-binding activity from several other infected mouse strains was blocked 22 to 86% by the G2.1 epitope-specific mAb. In addition to mice, the G2.1 epitope evoked powerful antibody responses in four other species. The binding activity of Trichinella-reactive antibodies from infected rats and pigs was inhibited 56 and 34%, respectively, by the mAb. Greater than 48% of the T. spiralis serum-binding activity from 4/5 infected humans was G2.1-specific. Most of the rabbit antibody response induced by injection of a previously characterized 43-kDa antigen was also directed to the G2.1 determinant. The specificities of 10 T. spiralis-reactive mAb were examined, and 7 reacted with the immunodominant epitope. Finally, of nine helminth species examined, only T. spiralis and T. pseudospiralis extracts efficiently blocked G2.1-specific antibody binding to solid-phase antigens. These results suggest that the responses to G2.1 epitope may play an important role during infection.     

Protozoan encounters with Toll-like receptor signalling pathways: implications for host parasitism

Toll-like receptors (TLRs) have emerged as a major receptor family involved in non-self recognition. They have a vital role in triggering innate immunity and orchestrate the acquired immune response during bacterial and viral infection. However, the role of TLRs during infection with protozoan pathogens is less clear. Nevertheless, our understanding of how these parasitic microorganisms engage the host TLR signalling system has now entered a phase of rapid expansion. This Review describes recent insights into how parasitic protozoans are sensed by TLR molecules, and how the TLR system itself can be targeted by these microbial pathogens for their own survival.