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NK cells are key innate immune cells that play critical roles in host defense. Although NK cells have been shown to regulate immunity to some infectious diseases, their role in immunity to Trypanosoma congolense has not been investigated. NK cells are vital sources of IFN-γ and TNF-α; two key cytokines that are known to play important roles in resistance to African trypanosomes. In this article, we show that infection with T. congolense leads to increased levels of activated and functional NK cells in multiple tissue compartments. Systemic depletion of NK cells with anti-NK1.1 mAb led to increased parasitemia, which was accompanied by significant reduction in IFN-γ production by immune cells in the spleens and liver of infected mice. Strikingly, infected NFIL3−/− mice (which genetically lack NK cell development and function) on the normally resistant background were highly susceptible to T. congolense infection. These mice developed fulminating and uncontrolled parasitemia and died significantly earlier (13 ± 1 d) than their wild-type control mice (106 ± 26 d). The enhanced susceptibility of NFIL3−/− mice to infection was accompanied by significantly impaired cytokine (IFN-γ and TNF-α) response by CD3+ T cells in the spleens and liver. Adoptive transfer of NK cells into NFIL3−/− mice before infection rescued them from acute death in a perforin-dependent manner. Collectively, these studies show that NK cells are critical for optimal resistance to T. congolense, and its deficiency leads to enhanced susceptibility in infected mice.

Breast cancer is the most common cancer in women worldwide. Hormone receptor breast cancers are the most common ones and, about 2 out of every 3 cases of breast cancer are estrogen receptor (ER) positive. Selective ER modulators, such as tamoxifen, are the first line of endocrine treatment of breast cancer. Despite the expression of hormone receptors some patients develop tamoxifen resistance and 50% present de novo tamoxifen resistance. Recently, we have demonstrated that activated mammalian target of rapamycin (mTOR) is positively associated with overall survival and recurrence free survival in ER positive breast cancer patients who were later treated with tamoxifen. Since altered expression of protein kinase B (PKB)/Akt in breast cancer cells affect N-myristoyltransferase 1 (NMT1) expression and activity, we investigated whether mTOR, a downstream target of PKB/Akt, regulates NMT1 in ER positive breast cancer cells (MCF7 cells). We inhibited mTOR by treating MCF7 cells with rapamycin and observed that the expression of NMT1 increased with rapamycin treatment over the period of time with a concomitant decrease in mTOR phosphorylation. We further employed mathematical modelling to investigate hitherto not known relationship of mTOR with NMT1. We report here for the first time a collection of models and data validating regulation of NMT1 by mTOR.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of bone marrow–derived myeloid cells that have immune-suppressive activities. These cells have been reported to suppress T cell immunity against tumors as well as in some parasitic and bacterial infections. However, their role during Trypanosoma congolense infection has not been studied. Given that immunosuppression is a hallmark of African trypanosomiasis, we investigated the role of MDSCs in immunity to T. congolense infection. We found increased numbers of MDSCs in the spleen and liver of infected mice, which correlated with increased parasitemia. Depletion of MDSCs significantly increased the percentage of proliferating and IFN-γ–producing CD4+ T cells from the spleen of T. congolense–infected mice. Furthermore, MDSCs from T. congolense–infected mice directly suppressed CD4+ T cell proliferation in a coculture setting. This suppressive effect was abolished by the arginase-1 inhibitor, Nω-hydroxy-nor-l-arginine (nor-NOHA), indicating that MDSCs suppress CD4+ T cell proliferation and function in an arginase-1–dependent manner. Indeed, depletion of MDSCs during infection led to control of the first wave of parasitemia and prolonged survival of infected mice. This was also associated with increased CD4+ T cell proliferation and IFN-γ production. Taken together, our findings identify an important role of MDSCs in the pathogenesis of experimental T. congolense infection via suppression of T cell proliferative and effector cytokine responses in an arginase-1–dependent manner.

Although it is known that Trypanosoma congolense infection in mice is associated with increased production of proinflammatory cytokines by macrophages and monocytes, the intracellular signaling pathways leading to the production of these cytokines remain unknown. Here, we investigated the innate receptors and intracellular signaling pathways that are involved in T. congolense-induced proinflammatory cytokine production in macrophages. We show that the production of IL-6, IL-12, and TNF-a by macrophages in vitro and in vivo following interaction with T. congolense is dependent on phosphorylation of mitogen-activated protein kinase (MAPK) including ERK, p38, JNK, and signal transducer and activation of transcription (STAT) proteins. Specific inhibition of MAPKs and STATs signaling pathways significantly inhibited T. congolense-induced production of proinflammatory cytokines in macrophages. We further show that T. congolense-induced proinflammatory cytokine production in macrophages is mediated via Toll-like receptor 2 (TLR2) and involves the adaptor molecule, MyD88. Deficiency of MYD88 and TLR2 leads to impaired cytokine production by macrophages in vitro and acute death of T. congolense-infected relatively resistant mice. Collectively, our findings provide a mechanistic insight into T. congolense-induced activation of the innate immune system that leads to the production of proinflammatory cytokines and resistance to the infection. Understanding the innate immune receptors and signaling molecules involved in T. congolense infection may help identify novel targets for immunomodulation aimed at regulating the disease.

NK cells are key innate immune cells that play critical roles in host defense. Although NK cells have been shown to regulate immunity to some infectious diseases, their role in immunity to

Diminazene aceturate (Berenil) is the most commonly used trypanolytic agent in livestock. We previously showed that Berenil downregulates Trypanosoma congolense (T. congolense)-induced cytokine production in macrophages both in vitro and in vivo. Here, we investigated the molecular mechanisms through which the drug alters T. congolense-induced cytokine production in macrophages. We show that pretreatment of macrophages with Berenil significantly downregulated T. congolense-induced phosphorylation of mitogen-activated protein kinase (p38), signal transducer and activator of transcription (STAT) proteins including STAT1 and STAT3, and NFκB activity both in vitro and in vivo. Collectively, our results reveal a mechanistic insight through which Berenil downregulates T. congolense-induced cytokine production in macrophages by inhibiting key signaling molecules and pathways associated with proinflammatory cytokine production.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of bone marrow-derived myeloid cells that have immune-suppressive activities. These cells have been reported to suppress T cell immunity against tumors as well as in some parasitic and bacterial infections. However, their role during

Diminazene aceturate or Berenil has been the drug of choice for treatment of animal trypanosomiasis. Although the compound has been in the market since 1955, its mechanisms of action have remained poorly understood. While some earlier reports show that Berenil possesses trypanolytic and trypanostatic properties, some studies show it may also indirectly affect the host immune system. Our recent extensive studies show that treatment with Berenil reduces pro-inflammatory cytokine (IL-6, IL-12 and TNF) production in macrophages in vivo and in vitro following stimulation with Trypanosoma congolense, lipopolysaccharide (LPS), unmethylated bacterial CpG motifs and Poly I:C. This global effect was not due to downregulation of Toll-like receptor (TLR) expression on innate immune cells. Instead, Berenil significantly downregulated phosphorylation of mitogen activated protein kinases (MAPKs, including ERK, p38 and JNK), signal transducer and activator of transcription (STAT) proteins (including STAT1 and STAT3) and NFκB p65 subunit, key signaling molecules and transcription factors involved in the production of proinflammatory cytokines. The ability of Berenil to downregulate major intracellular signaling pathways that lead to proinflammatory cytokine production suggests that it could be used to treat conditions caused by excessive production of inflammatory cytokines.

BALB/c mice are highly susceptible to experimental intraperitoneal Trypanosoma congolense infection. However, a recent report showed that these mice are relatively resistant to primary intradermal low-dose infection. Paradoxically, repeated low-dose intradermal infections predispose mice to enhanced susceptibility to an otherwise noninfectious dose challenge. Here, we explored the mechanisms responsible for this low-dose-induced susceptibility to subsequent low-dose challenge infection. We found that akin to intraperitoneal infection, low-dose intradermal infection led to production of interleukin-10 (IL-10), IL-6, IL-12, tumor necrosis factor alpha (TNF-α), transforming growth factor β (TGF-β), and gamma interferon (IFN-γ) by spleen and draining lymph node cells. Interestingly, despite the absence of parasitemia, low-dose intradermal infection led to expansion of CD4 + CD25 + Foxp3 + cells (T regulatory cells [Tregs]) in both the spleens and lymph nodes draining the infection site. Depletion of Tregs by anti-CD25 monoclonal antibody (MAb) treatment during primary infection or before challenge infection following repeated low-dose infection completely abolished the low-dose-induced enhanced susceptibility. In addition, Treg depletion was associated with dramatic reduction in serum levels of TGF-β and IL-10. Collectively, these findings show that low-dose intradermal infection leads to rapid expansion of Tregs, and these cells mediate enhanced susceptibility to subsequent infection.

The breakdown of L-arginine to ornithine and urea by host arginase supports Leishmania proliferation in macrophages. Studies using arginase-null mutants show that Leishmania-derived arginase plays an important role in disease pathogenesis. We investigated the role of parasite-derived arginase in secondary (memory) anti-Leishmania immunity in the resistant C57BL/6 mice. We found that C57BL/6 mice infected with arginase-deficient (arg−) L. major failed to completely resolve their lesion and maintained chronic pathology after 16 wk, a time when the lesion induced by wild-type L. major is completely resolved. This chronic disease was associated with impaired Ag-specific proliferation and IFN-γ production, a concomitant increase in programmed cell death-1 (PD-1) expression on CD4+ T cells, and failure to induce protection against secondary L. major challenge. Treatment with anti–PD-1 mAb restored T cell proliferation and IFN-γ production in vitro and led to complete resolution of chronic lesion in arg− L. major–infected mice. These results show that infection with arg− L. major results in chronic disease due in part to PD-1–mediated clonal exhaustion of T cells, suggesting that parasite-derived arginase contributes to the overall quality of the host immune response and subsequent disease outcome in L. major–infected mice. They also indicate that persistent parasites alone do not regulate the quality of secondary anti-Leishmania immunity in mice and that the quality of the primary immune response may be playing a hitherto unrecognized dominant role in this process.

Pathogens, such as bacteria, viruses, and parasites, possess specific molecules or proteins that are recognized by several host innate immune receptors, leading to the activation of several intracellular signaling molecules and pathways. The magnitude and quality of these events significantly affect the outcome of infection. African trypanosomes, including Trypanosoma congolense, are capable of manipulating the host immune response, including the activity of macrophages, which are the key immune cells that contribute to the immunopathogenesis of African trypanosomiasis. Although it is known that immune hyperactivation and excessive pro-inflammatory cytokine production are the hallmarks of African trypanosomiasis, the mechanisms through which these events are triggered are poorly defined. However, it is known that macrophages may play a significant role in these processes, because phagocytosis of trypanosomes by macrophages initiates intracellular signal transduction cascades that lead to the release of pro-inflammatory cytokines and alteration in cell function. This review highlights recent progress in our understanding of the innate immune receptors, signaling pathways, and transcription factors involved in T. congolense-induced pro-inflammatory cytokine production in macrophages. It will reveal the existence of complex signaling events through which the parasite modulates the host immune response, thus identifying novel targets that could aid in designing strategies to effectively control the disease.

BALB/c mice are highly susceptible to experimental Trypanosoma congolense infections, whereas C57BL/6 mice are relatively resistant. Infected highly susceptible BALB/c mice die of systemic inflammatory response syndrome. Because interleukin-17 (IL-17) and Th17 cells regulate inflammatory responses, we investigated their role in the pathogenesis of experimental African trypanosomiasis in mice. We show that the production of IL-17 by spleen and liver cells and the serum IL-17 level increased after T. congolense infection in mice. Interestingly, infected highly susceptible BALB/c mice produced more IL-17 and had more Th17 cells than infected relatively resistant C57BL/6 mice. Paradoxically, neutralization of IL-17 with anti-IL-17 monoclonal antibody in vivo induced higher parasitemia in both the susceptible and the relatively resistant mice. Interestingly, anti-IL-17 antibody-treated mice had higher serum levels of alanine aminotransferase and aspartate aminotransferase, and the production of IL-10 and nitric oxide by liver cells was markedly decreased. Moreover, recombinant IL-17-treated mice exhibited significantly faster parasite control and lower peak parasitemia compared to control mice. Collectively, these results suggest that the IL-17/Th17 axis plays a protective role in murine experimental African trypanosomiasis.

e15114 Background: Colorectal cancer (CRC) is the second most common cause of cancer related deaths in North America. Most CRCs arise from pre-malignant adenomatous polyps with some of these polyps developing into cancer over years providing an ideal opportunity for detection and intervention. However, the unpleasant nature of the current screening methods often leads to non-compliance. N-Myristoyltransferase (NMT) has been reported to be overexpressed in CRC tissues. In this study we show that NMT2, an isoform of NMT, is overexpressed in peripheral blood mononuclear cells (PBMC) of individuals with colorectal adenomatous polyps or cancers compared to healthy controls. Methods: PBMC’s were isolated from blood samples of CRC patients (n = 14), individuals with - non-adenomatous polyps (n = 8); adenomatous polyps (n = 18) and healthy controls (n = 24). Immunohistochemistry (IHC) was used to determine the NMT2 expression in these samples. IHC-scores were derived from assessment of both staining intensity (scale 0-3) and percentage of positive cells (0-100%), which were multiplied to generate an IHC -score between 0-300. Observers calculating the IHC scores were unaware of the polyp/CRC status. A receiver operating characteristic (ROC) analysis was performed to assess the overall performance of the NMT2 test via the area under the curve (AUC). Results: NMT2 was significantly overexpressed in PBMC of CRC patients (median IHC score- 196) compared to control subjects (median IHC score- 50). In addition, IHC scores were significantly higher for adenomatous polyps (median IHC score- 165) compared to those with non-adenomatous polyps (median IHC score- 54.8). We included non-adenomatous polyps and healthy subjects in “controls” and adenomatous polyps in “cases” along with CRC in the ROC analysis. The ROC curve of NMT2 expression displayed substantial separation between controls/non adenomatous polyps compared to CRC/adenomatous polyps [AUC = 0.913 (95% CI: 0.830-0.994)]. Conclusions: Our results show an increase in NMT2 expression in PBMC of CRC patients and individuals with adenomatous polyps suggesting that NMT2 could be used as a novel blood based biomarker for the screening and early detection of CRC. Validation studies are the next step.

BACKGROUND: Trypanosoma congolense are extracellular and intravascular blood parasites that cause debilitating acute or chronic disease in cattle and other domestic animals. Diminazene aceturate (Berenil) has been widely used as a chemotherapeutic agent for trypanosomiasis in livestock since 1955. As in livestock, treatment of infected highly susceptible BALB/c mice with Berenil leads to rapid control of parasitemia and survival from an otherwise lethal infection. The molecular and biochemical mechanisms of action of Berenil are still not very well defined and its effect on the host immune system has remained relatively unstudied. Here, we investigated whether Berenil has, in addition to its trypanolytic effect, a modulatory effect on the host immune response to Trypanosoma congolense. METHODOLOGY/PRINCIPAL FINDINGS: BALB/c and C57BL/6 mice were infected intraperitoneally with T. congolense, treated with Berenil and the expression of CD25 and FoxP3 on splenic cells was assessed directly ex vivo. In addition, serum levels and spontaneous and LPS-induced production of pro-inflammatory cytokines by splenic and hepatic CD11b⁺ cells were determined by ELISA. Berenil treatment significantly reduced the percentages of CD25⁺ cells, a concomitant reduction in the percentage of regulatory (CD4⁺Foxp3⁺) T cells and a striking reduction in serum levels of disease exacerbating pro-inflammatory cytokines including IL-6, IL-12, TNF and IFN-γ. Furthermore, Berenil treatment significantly suppressed spontaneous and LPS-induced production of inflammatory cytokines by splenic and liver macrophages and significantly ameliorated LPS-induced septic shock and the associated cytokine storm. CONCLUSIONS/SIGNIFICANCE: Collectively, these results provide evidence that in addition to its direct trypanolytic effect, Berenil also modulates the host immune response to the parasite in a manner that dampen excessive immune activation and production of pathology-promoting pro-inflammatory cytokines, suggesting that this drug may also be beneficial for treatment of disease conditions caused by excessive production of inflammatory cytokines.

Background BALB/c mice are highly susceptible while C57BL/6 are relatively resistant to experimental Trypanosoma congolense infection. Although regulatory T cells (Tregs) have been shown to regulate the pathogenesis of experimental T. congolense infection, their exact role remains controversial. We wished to determine whether Tregs contribute to distinct phenotypic outcomes in BALB/c and C57BL/6 mice and if so how they operate with respect to control of parasitemia and production of disease-exacerbating proinflammatory cytokines. Methodology/Findings BALB/c and C57BL/6 mice were infected intraperitoneally (i.p) with 103 T. congolense clone TC13 and both the kinetics of Tregs expansion and intracellular cytokine profiles in the spleens and livers were monitored directly ex vivo by flow cytometry. In some experiments, mice were injected with anti-CD25 mAb prior or post T. congolense infection or adoptively (by intravenous route) given highly enriched naïve CD25+ T lymphocytes prior to T. congolense infection and the inflammatory cytokine/chemokine levels and survival were monitored. In contrast to a transient and non significant increase in the percentages and absolute numbers of CD4+CD25+Foxp3+ T cells (Tregs) in C57BL/6 mouse spleens and livers, a significant increase in the percentage and absolute numbers of Tregs was observed in spleens of infected BALB/c mice. Ablation or increasing the number of CD25+ cells in the relatively resistant C57BL/6 mice by anti-CD25 mAb treatment or by adoptive transfer of CD25+ T cells, respectively, ameliorates or exacerbates parasitemia and production of proinflammatory cytokines. Conclusion Collectively, our results show that regulatory T cells contribute to susceptibility in experimental murine trypanosomiasis in both the highly susceptible BALB/c and relatively resistant C57BL/6 mice., Author Summary BALB/c mice are highly susceptible while C57BL/6 is relatively resistant to experimental Trypanosoma congolense infection. Acute death observed in infected BALB/c mice is usually associated with the excessive production of pro-inflammatory cytokines. Regulatory T cells (Tregs) have been shown to play a significant role in the pathogenesis of many diseases including those caused by parasites. However, the role of Tregs in the pathogenesis of T. congolense infection remains unclear. We were interested in addressing the following questions: Do Tregs contribute to the distinct phenotypic outcomes observed in T. congolense-infected BALB/c and C57BL/6 mice? If so, where and how do they operate with respect to parasitemia and cytokine response? By selectively altering the numbers of these cells either by targeted depletion with monoclonal antibody or adoptive transfer of highly enriched naïve CD25+ cells prior to infection, we show that Tregs impairs efficient parasite control and impacts on production of disease-exacerbating proinflammatory cytokines. Collectively, our findings suggest that Tregs contribute to enhanced susceptibility to experimental T. congolense infection in mice.

BALB/c mice are highly susceptible to experimental T. congolense infection. Interestingly, a recent report showed that these mice are relatively resistant to primary intradermal low dose infection. Paradoxically, repeated low dose intradermal infections predispose to enhanced susceptibility to an otherwise non-infectious dose challenge. Here, we explored the mechanisms responsible for this low-dose-induced susceptibility to subsequent low dose reinfection. We found that akin to intraperitoneal infection, low dose intradermal infection leads to production of IL-10, IL-6, IL-12, TGF-β and IFN-γ by spleen and draining lymph node cells. Interestingly, despite the absence of parasitemia, low dose intradermal infection led to expansion of CD4+CD24+Foxp3+ cells (Tregs) in both the spleens and lymph nodes draining the infection site. Depletion of Tregs by anti-CD25 mAb treatment during primary or before reinfection following repeated low dose infection completely abolished the low dose-induced enhanced susceptibility to rechallenge infection. In addition, Tregs depletion was associated with dramatic reduction in serum levels of TGF-β and IL-10. Collectively, these findings show that low dose intradermal infection leads to rapid expansion of Tregs and these cells mediate enhanced susceptibility to subsequent infection.