Diminazene aceturate (Berenil) modulates LPS induced pro-inflammatory cytokine production by inhibiting phosphorylation of MAPKs and STAT proteins.

Although diminazene aceturate (Berenil) is widely used as a trypanolytic agent in livestock, its mechanisms of action remain poorly understood. We previously showed that Berenil treatment suppresses pro-inflammatory cytokine production by splenic and liver macrophages leading to a concomitant reduction in serum cytokine levels in mice infected with Trypanosoma congolense or challenged with LPS. Here, we investigated the molecular mechanisms through which Berenil alters pro-inflammatory cytokine production by macrophages. We show that pre-treatment of macrophages with Berenil dramatically suppressed IL-6, IL-12 and TNF-α production following LPS, CpG and Poly I:C stimulation without altering the expression of TLRs. Instead, it significantly down-regulated phosphorylation of mitogen-activated protein kinases (p38, extracellular signal-regulated kinase and c-Jun N-terminal kinases), signal transducer and activator of transcription (STAT) proteins (STAT1 and STAT3) and NF-кB p65 activity both in vitro and in vivo. Interestingly, Berenil treatment up-regulated the phosphorylation of STAT5 and the expression of suppressor of cytokine signaling 1 (SOCS1) and SOCS3, which are negative regulators of innate immune responses, including MAPKs and STATs. Collectively, these results show that Berenil down-regulates macrophage pro-inflammatory cytokine production by inhibiting key signaling pathways associated with cytokine production and suggest that this drug may be used to treat conditions caused by excessive production of inflammatory cytokines.

Parasite-derived arginase influences secondary anti-Leishmania immunity by regulating programmed cell death-1-mediated CD4+ T cell exhaustion.

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.

Diminazene aceturate (Berenil) modulates the host cellular and inflammatory responses to Trypanosoma congolense infection.

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.

Infection with arginase-deficient Leishmania major reveals a parasite number-dependent and cytokine-independent regulation of host cellular arginase activity and disease pathogenesis.

The balance between the products of L-arginine metabolism in macrophages regulates the outcome of Leishmania major infection. L-arginine can be oxidized by host inducible NO synthase to produce NO, which contributes to parasite killing. In contrast, L-arginine hydrolysis by host arginase blocks NO generation and provides polyamines, which can support parasite proliferation. Additionally, Leishmania encode their own arginase which has considerable potential to modulate infectivity and disease pathogenesis. In this study, we compared the infectivity and impact on host cellular immune response in vitro and in vivo of wild-type (WT) L. major with that of a parasite arginase null mutant (arg(-)) L. major. We found that arg(-) L. major are impaired in their macrophage infectivity in vitro independent of host inducible NO synthase activities. As with in vitro results, the proliferation of arg(-) L. major in animal infections was also significantly impaired in vivo, resulting in delayed onset of lesion development, attenuated pathology, and low parasite burden. Despite this attenuated pathology, the production of cytokines by cells from the draining lymph node of mice infected with WT and arg(-) L. major was similar at all times tested. Interestingly, in vitro and in vivo arginase levels were significantly lower in arg(-) than in WT-infected cases and were directly correlated with parasite numbers inside infected cells. These results suggest that Leishmania-encoded arginase enhances disease pathogenesis by augmenting host cellular arginase activities and that contrary to previous in vitro studies, the host cytokine response does not influence host arginase activity.

Altered proinflammatory cytokine production and enhanced resistance to Trypanosoma congolense infection in lymphotoxin beta-deficient mice.

BALB/c mice are highly susceptible to Trypanosoma congolense infection, whereas C57BL/6 mice are relatively resistant. Overproduction of interferon-gamma (IFN-gamma) and other proinflammatory cytokines contribute to death in susceptible mice. Here, we show that lymphotoxin beta-deficient (LTbeta(-/-)) mice are more resistant than wild-type (WT) mice to T. congolense infection, as shown by a lower parasitemia level and a longer survival duration. The enhanced resistance of LTbeta(-/-) mice was associated with undetectable or low serum levels of proinflammatory cytokines (i.e., tumor necrosis factor-alpha, interleukin [IL]-6, IL-12, and monocyte chemotactic protein-1). Although infected LTbeta(-/-) mice had high numbers of CD4(+)CD25(+)Foxp3(+) cells and high serum IL-10 levels, these cells were not the major producers of IL-10. Treatment of LTbeta(-/-) mice with anti-IL-10R monoclonal antibody abolished their enhanced resistance, whereas depletion of CD25(+) cells further enhanced resistance among infected WT and LTbeta(-/-) mice. These results suggest that LTbeta plays critical role in regulating the outcome of T. congolense infection in mice.

Mitochondrial metabolism in hibernation: metabolic suppression, temperature effects, and substrate preferences.

We compared liver and skeletal muscle mitochondrial function among activity states to characterize regulated reversible metabolic suppression in the mammalian hibernator Spermophilus tridecemlineatus. At 37 degrees C, succinate oxidation was 70% lower in the liver mitochondria from torpid animals than in those from summer-active animals or in animals arousing from torpor. Respiration was very sensitive to temperature (Q(10) 5.8-9.8), and when measured at 25 degrees or 5 degrees C there was no difference among the three states. Liver mitochondria from summer-active animals oxidized pyruvate and beta -hydroxybutyrate at higher rates than those from torpid animals, and flux through complex 4 of the electron transport chain was about three- and fivefold higher than flux through complexes 2-4 and complexes 1-4, respectively. In the hibernating and arousing animals there was no difference in flux through complexes 2-4 and complex 4, suggesting a downregulation of cytochrome c oxidase in liver mitochondria during the hibernation season. Muscle mitochondrial respiration did not differ between the torpid and summer-active states in any of the parameters measured. The data support a regulated, reversible decrease of liver (but not muscle) mitochondrial oxidative phosphorylation in hibernating ground squirrels.