Efficacy of oleylphosphocholine (OlPC) in vitro and in a mouse model of invasive aspergillosis.

Invasive aspergillosis (IA) has become increasingly common and is characterised by high morbidity and mortality. Upcoming resistance threatens treatment with azoles and highlights the continuous need for novel therapeutics. This laboratory study investigated the in vitro and in vivo potential of the alkylphospholipid oleylphosphocholine (OlPC) against Aspergillus. In vitro activities of OlPC, miltefosine, posaconazole and voriconazole were determined for Aspergillus fumigatus, A. niger, A. terreus and A. flavus. In vivo efficacy of OlPC was evaluated in a systemic A. fumigatus mouse model, adopting a short-term and long-term oral or intraperitoneal dosing regimen. OlPC showed good in vitro activity against A. fumigatus (IC50 = 1.04 µmol l(-1)). Intraperitoneal administration of 50 mg kg(-1) day(-1) OlPC significantly reduced the fungal organ burdens at 4 days post-infection (dpi). Although 5- and 10-day OlPC treatment improved survival, organ burdens were not affected at 10 and 15 dpi. While this study showed excellent in vitro activity of OlPC against Aspergillus spp., its therapeutic efficacy in an acute mouse model for IA was less convincing. Given the limited therapeutic options in the current antifungal market for invasive infections, OlPC activity should be assessed in a less stringent in vivo model, potentially in combination treatment with other already marketed antifungal drugs.

Direct comparison of the efficacy and safety of oral treatments with oleylphosphocholine (OlPC) and miltefosine in a mouse model of L. major cutaneous leishmaniasis.

BACKGROUND: Cutaneous leishmaniasis (CL) represents a range of skin diseases caused by infection with Leishmania parasites and associated with tissue inflammation and skin ulceration. CL is clinically widespread in both the Old and New World but lacks treatments that are well tolerated, effective and inexpensive. Oleylphosphocholine (OlPC) is a new orally bioavailable drug of the alkylphosphocholine family with potent antileishmanial activity against a broad range of Leishmania species/strains. METHODOLOGY/PRINCIPAL FINDINGS: The potential of OlPC against Old World CL was evaluated in a mouse model of Leishmania (L.) major infection in BALB/c mice. Initial dose-response experiments showed that an oral daily dose of 40 mg/kg of OlPC was needed to impact time to cure and lesion sizes. This dose was then used to directly compare the efficacy of OlPC to the efficacy of the antileishmanial drugs miltefosine (40 mg/kg/day), fluconazole (160 mg/kg/day) and amphotericin B (25 mg/kg/day). OlPC, miltefosine and fluconazole were given orally for 21 days while amphotericin B was administered intraperitoneally for 10 days. Ulcer sizes and animal weights were followed up on a weekly basis and parasitemia was determined by means of a real-time in vivo imaging system which detects luminescence emitted from luciferase-expressing infecting L. major parasites. Amphotericin B and OlPC showed excellent efficacy against L. major lesions in terms of reduction of parasitic loads and by inducing complete healing of established lesions. In contrast, treatment with miltefosine did not significantly affect parasitemia and lesion sizes, while fluconazole was completely ineffective at the dose regimen tested. CONCLUSIONS/SIGNIFICANCE: Given the data showing the outstanding efficacy and tolerability of OlPC, our results suggest that OlPC is a promising new drug candidate to improve and simplify current clinical management of L. major CL.

Drug delivery by tattooing to treat cutaneous leishmaniasis.

This study establishes a proof-of-concept that a tattoo device can target intra-dermal drug delivery against cutaneous leishmaniasis (CL). The selected drug is oleylphosphocholine (OlPC) formulated as liposomes, particles known to be prone to macrophage ingestion. We first show that treatment of cultured Leishmania-infected macrophages with OlPC-liposomes results in a direct dose-dependent killing of intracellular parasites. Based on this, in vivo efficacy is demonstrated using a 10 day tattooing-mediated treatment in mice infected with L. major and L. mexicana. In both models this regimen results in rapid clinical recovery with complete regression of skin lesions by Day 28. Parasite counts and histopathology examination confirm high treatment efficacy at the parasitic level. Low amount of drug required for tattooing combined with fast clinical recovery may have a positive impact on CL patient management. This first example of tattoo-mediated drug delivery could open to new therapeutic interventions in the treatment of skin diseases.

First study on efficacy and tolerability of a new alkylphosphocholine molecule (oleylphosphocholine-OlPC) in the treatment of canine leishmaniosis due to Leishmania infantum.

The alkylphosphocholine oleylphosphocholine (OlPC) represents a potential new therapy for the treatment of canine leishmaniosis caused by Leishmania infantum. The aim of the present study was to evaluate the efficacy and safety of OlPC in a small cohort of dogs naturally infected with L. infantum and defined as clinically sick (LeishVet stages II and III). A total of eight dogs were included in the study and were treated orally with 4 mg/kg OlPC for 14 days. Dogs were assessed at the clinical and parasitological level at four time points during a total follow-up period of 90 days (before treatment and at 15, 30, and 90 days post-treatment onset). Ln-PCR, real-time quantitative PCR, antibody testing (IFAT), and culture of bone marrow aspirates were evaluated at the four time points. OlPC treatment induced a rapid and satisfactory clinical recovery in terms of clinical score reduction and weight gain, and treatment efficacy was found to be associated with a decrease in bone marrow parasitic load. Serological titers measured by IFAT were stable in any of the treated dogs at any time point after treatment. OlPC was well tolerated and no severe adverse events were noted in any of the treated dogs; even some dogs showed slight intestinal disorders. This proof-of-principle study is the first to show that short oral treatment with OlPC improves clinical signs of canine L. infantum leishmaniosis, highlighting the need to perform additional studies to optimize the dosing regimen and to assess long-term treatment efficacy of this drug.

The CD20 homolog Ms4a8a integrates pro- and anti-inflammatory signals in novel M2-like macrophages and is expressed in parasite infection.

Recently, we identified the CD20 homolog Ms4a8a as a novel molecule expressed by tumor-associated macrophages that directly enhances tumor growth. Here, we analyzed Ms4a8a(+) macrophages in M2-associated infectious pathologies. In late-stage Trypanosoma congolense and Taenia crassiceps infections, Ms4a8a expression was detected in hepatic and peritoneal macrophages respectively. Innate immunity in these infections is modulated by Toll-like receptor (TLR) signaling and TLR2/4/7 agonists strongly induced Ms4a8a expression in bone marrow derived macrophages (BMDMs) treated with M2 mediators (glucocorticoids/IL-4). LPS/dexamethasone/IL-4-induced Ms4a8a(+) BMDMs were characterized by strong expression of mRNA of mannose receptor (Mmr), arginase 1, and CD163, and by decreased iNOS expression. Coinduction of Ms4a8a by M2 mediators and TLR agonists involved the classical TLR signaling cascade via activation of MyD88/TRIF and NF-κB. Forced overexpression of Ms4a8a modulated the TLR4 response of RAW264.7 cells as shown by gene expression profiling. Upregulation of Hdc, Tcfec, and Sla was confirmed both in primary LPS/dexamethasone/IL-4-stimulated Ms4a8a(+) BMDMs and in peritoneal macrophages from late-stage Taenia crassiceps infection. In conclusion, we show that TLR signaling skews the typical alternative macrophage activation program to induce a special M2-like macrophage subset in vitro that also occurs in immunomodulatory immune reactions in vivo, a process directly involving the CD20 homolog Ms4a8a.

IL-10 limits production of pathogenic TNF by M1 myeloid cells through induction of nuclear NF-κB p50 member in Trypanosoma congolense infection-resistant C57BL/6 mice.

A balance between parasite elimination and control of infection-associated pathogenicity is crucial for resistance to African trypanosomiasis. By producing TNF and NO, CD11b(+) myeloid cells with a classical activation status (M1) contribute to parasitemia control in experimental Trypanosoma congolense infection in resistant C57BL/6 mice. However, in these mice, IL-10 is required to regulate M1-associated inflammation, avoiding tissue/liver damage and ensuring prolonged survival. In an effort to dissect the mechanisms behind the anti-inflammatory activity of IL-10 in T. congolense-infected C57BL/6 mice, we show, using an antibody blocking the IL-10 receptor, that IL-10 impairs the accumulation and M1 activation of TNF/iNOS-producing CD11b(+) Ly6C(+) cells in the liver. Using infected IL-10(flox/flox) LysM-Cre(+/+) mice, we show that myeloid cell-derived IL-10 limits M1 activation of CD11b(+) Ly6C(+) cells specifically at the level of TNF production. Moreover, higher production of TNF in infected IL-10(flox/flox) LysM-Cre(+/+) mice is associated with reduced nuclear accumulation of the NF-κB p50 subunit in CD11b(+) M1 cells. Furthermore, in infected p50(-/-) mice, TNF production by CD11b(+) Ly6C(+) cells and liver injury increases. These data suggest that preferential nuclear accumulation of p50 represents an IL-10-dependent anti-inflammatory mechanism in M1-type CD11b(+) myeloid cells that regulates the production of pathogenic TNF during T. congolense infection in resistant C57BL/6 mice.

Tip-DC development during parasitic infection is regulated by IL-10 and requires CCL2/CCR2, IFN-gamma and MyD88 signaling.

The development of classically activated monocytic cells (M1) is a prerequisite for effective elimination of parasites, including African trypanosomes. However, persistent activation of M1 that produce pathogenic molecules such as TNF and NO contributes to the development of trypanosome infection-associated tissue injury including liver cell necrosis in experimental mouse models. Aiming to identify mechanisms involved in regulation of M1 activity, we have recently documented that during Trypanosoma brucei infection, CD11b(+)Ly6C(+)CD11c(+) TNF and iNOS producing DCs (Tip-DCs) represent the major pathogenic M1 liver subpopulation. By using gene expression analyses, KO mice and cytokine neutralizing antibodies, we show here that the conversion of CD11b(+)Ly6C(+) monocytic cells to pathogenic Tip-DCs in the liver of T. brucei infected mice consists of a three-step process including (i) a CCR2-dependent but CCR5- and Mif-independent step crucial for emigration of CD11b(+)Ly6C(+) monocytic cells from the bone marrow but dispensable for their blood to liver migration; (ii) a differentiation step of liver CD11b(+)Ly6C(+) monocytic cells to immature inflammatory DCs (CD11c(+) but CD80/CD86/MHC-II(low)) which is IFN-gamma and MyD88 signaling independent; and (iii) a maturation step of inflammatory DCs to functional (CD80/CD86/MHC-II(high)) TNF and NO producing Tip-DCs which is IFN-gamma and MyD88 signaling dependent. Moreover, IL-10 could limit CCR2-mediated egression of CD11b(+)Ly6C(+) monocytic cells from the bone marrow by limiting Ccl2 expression by liver monocytic cells, as well as their differentiation and maturation to Tip-DCs in the liver, showing that IL-10 works at multiple levels to dampen Tip-DC mediated pathogenicity during T. brucei infection. A wide spectrum of liver diseases associates with alteration of monocyte recruitment, phenotype or function, which could be modulated by IL-10. Therefore, investigating the contribution of recruited monocytes to African trypanosome induced liver injury could potentially identify new targets to treat hepatic inflammation in general, and during parasite infection in particular.

The central role of macrophages in trypanosomiasis-associated anemia: rationale for therapeutical approaches.

Bovine African trypanosomiasis causes severe economical problems on the African continent and one of the most prominent immunopathological parameters associated with this parasitic infection is anemia. In this report we review the current knowledge of the mechanisms underlying trypanosomiasis-associated anemia. In first instance, the central role of macrophages and particularly their activation state in determining the outcome of the disease (i.e. trypanosusceptibility versus trypanotolerance) will be discussed. In essence, while persistence of classically activated macrophages (M1) contributes to anemia development, switching towards alternatively activated macrophages (M2) alleviates pathology including anemia. Secondly, while parasite-derived glycolipids such as the glycosylphosphatidylinositol (GPI) induce M1, host-derived IL-10 blocks M1-mediated inflammation, promotes M2 development and prevents anemia development. In this context, strategies aimed at inducing the M1 to M2 switch, such as GPI-based treatment, adenoviral delivery of IL-10 and induction of IL-10 producing regulatory T cells will be discussed. Finally, the crucial role of iron-homeostasis in trypanosomiasis-associated anemia development will be documented to stress the analogy with anemia of chronic disease (ACD), hereby providing new insight that might contribute to the treatment of ACD.

Understanding the role of monocytic cells in liver inflammation using parasite infection as a model.

Uncontrolled inflammation is a major cause of pathogenicity during chronic parasite infections. Novel therapies should therefore aim at re-establishing the balance between pro- and anti-inflammatory signals during disease to avoid tissue damage and ensure survival of the host. In this context, we are intending to identify strategies capable of inducing counter-inflammatory activity in injured liver and thereby increasing the resistance of the host to African trypanosomiasis as a model for parasite infection. Here, recent evidence is summarized revealing how monocytic cells recruited to the liver of African trypanosome-infected mice develop an M1 or M2 activation status, thereby maintaining the capacity of the host to control parasite growth while avoiding the development of liver damage, which otherwise culminates in early death of the host.

IL-10 dampens TNF/inducible nitric oxide synthase-producing dendritic cell-mediated pathogenicity during parasitic infection.

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.

Experimental expansion of the regulatory T cell population increases resistance to African trypanosomiasis.

Inflammatory responses mounted to eliminate parasites can be lethal if not counterbalanced by regulatory responses protecting the host from collateral tissue damage. Here, we show that the maintained inflammation associated with tissue damage, anemia, and reduced survival of Trypanosoma brucei-infected mice correlates with the absence of the expansion of the regulatory T (T(reg)) cell population. Induction of T(reg) cell expansion via CD28 superagonist antibody treatment in these mice down-regulated interferon-gamma production by T cells and tumor necrosis factor-alpha and reactive oxygen species production by classically activated macrophages, triggered the development of alternatively activated macrophages, delayed the onset of liver injury, diminished the anemia burden, and prolonged the survival of infected animals. Thus, triggering the expansion of the T(reg) cell population coupled with the induction of alternatively activated macrophages can restore the balance between pro- and anti-inflammatory signals and thereby limit the pathogenicity of African trypanosomiasis.

Alternatively activated myeloid cells limit pathogenicity associated with African trypanosomiasis through the IL-10 inducible gene selenoprotein P.

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-gamma-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 Sepp1(Delta)(240-361) 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.