Thermolytic CpG-containing DNA oligonucleotides as potential immunotherapeutic prodrugs

A CpG-containing DNA oligonucleotide functionalized with the 2-(N-formyl-N-methyl)aminoethyl thiophosphate protecting group (CpG ODN fma1555) was prepared from phosphoramidites 1a–d using solid-phase techniques. The oligonucleotide behaved as a prodrug by virtue of its conversion to the well-studied immunomodulatory CpG ODN 1555 through thermolytic cleavage of the 2-(N-formyl-N-methyl)aminoethyl thiophosphate protecting group. Such a conversion occurred at 37°C with a half-time of 73 h. The immunostimulatory properties of CpG ODN fma1555 were evaluated in two in vivo assays, one of which consisted of mice challenged in the ear with live Leishmania major metacyclic promastigotes. Local intradermal administration of CpG ODN fma1555 was as effective as that of CpG ODN 1555 in reducing the size of Leishmania lesions over time. In a different infectious model, CpG ODN 1555 prevented the death of Tacaribe-infected mice (43% survival) when administered between day 0 and 3 post infection. Administration of CpG ODN fma1555 three days before infection resulted in improved immunoprotection (60–70% survival). Moreover, co-administration of CpG ODN fma1555 and CpG ODN 1555 in this model increased the window for therapeutic treatment against Tacaribe virus infection, and thus supports the use of thermolytic oligonucleotides as prodrugs in the effective treatment of infectious diseases.     

Dendritic cell-mediated vaccination relies on interleukin-4 receptor signaling to avoid tissue damage after Leishmania major infection of BALB/c mice

Prevention of tissue damages at the site of Leishmania major inoculation can be achieved if the BALB/c mice are systemically given L. major antigen (LmAg)-loaded bone marrow-derived dendritic cells (DC) that had been exposed to CpG-containing oligodeoxynucleotides (CpG ODN). As previous studies allowed establishing that interleukin-4 (IL-4) is involved in the redirection of the immune response towards a type 1 profile, we were interested in further exploring the role of IL-4. Thus, wild-type (wt) BALB/c mice or DC-specific IL-4 receptor alpha (IL-4Rα)-deficient (CD11c^creIL-4Rα^−/lox) BALB/c mice were given either wt or IL-4Rα-deficient LmAg-loaded bone marrow-derived DC exposed or not to CpG ODN prior to inoculation of 2×10⁵ stationary-phase L. major promastigotes into the BALB/c footpad. The results provide evidence that IL4/IL-4Rα-mediated signaling in the vaccinating DC is required to prevent tissue damage at the site of L. major inoculation, as properly conditioned wt DC but not IL-4Rα-deficient DC were able to confer resistance. Furthermore, uncontrolled L. major population size expansion was observed in the footpad and the footpad draining lymph nodes of CD11c^creIL-4Rα^−/lox mice immunized with CpG ODN-exposed LmAg-loaded IL-4Rα-deficient DC, indicating the influence of IL-4Rα-mediated signaling in host DC to control parasite replication. In addition, no footpad damage occurred in BALB/c mice that were systemically immunized with LmAg-loaded wt DC doubly exposed to CpG ODN and recombinant IL-4. We discuss these findings and suggest that the IL4/IL4Rα signaling pathway could be a key pathway to trigger when designing vaccines aimed to prevent damaging processes in tissues hosting intracellular microorganisms.     

PyNTTTTGT and CpG Immunostimulatory Oligonucleotides: Effect on Granulocyte/Monocyte Colony-Stimulating Factor (GM-CSF) Secretion by Human CD56+ (NK and NKT) Cells

CD56⁺ cells have been recognized as being involved in bridging the innate and acquired immune systems. Herein, we assessed the effect of two major classes of immunostimulatory oligonucleotides (ODNs), PyNTTTTGT and CpG, on CD56⁺ cells. Incubation of human peripheral blood mononuclear cells (hPBMC) with some of these ODNs led to secretion of significant amounts of interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α) and granulocyte/monocyte colony-stimulating factor (GM-CSF), but only if interleukin 2 (IL2) was present. IMT504, the prototype of the PyNTTTTGT ODN class, was the most active. GM-CSF secretion was very efficient when non-CpG ODNs with high T content and PyNTTTTGT motifs lacking CpGs were used. On the other hand, CpG ODNs and IFNα inhibited this GM-CSF secretion. Selective cell type removal from hPBMC indicated that CD56⁺ cells were responsible for GM-CSF secretion and that plasmacytoid dendritic cells (PDCs) regulate this process. In addition, PyNTTTTGT ODNs inhibited the IFNα secretion induced by CpG ODNs in PDCs by interference with the TLR9 signaling pathway. Since IFNα is essential for CD56⁺ stimulation by CpG ODNs, there is a reciprocal interference of CpG and PyNTTTTGT ODNs when acting on this cell population. This suggests that these synthetic ODNs mimic different natural alarm signals for activation of the immune system.     

Plasmacytoid Dendritic Cell Dynamics Tune Interferon-Alfa Production in SIV-Infected Cynomolgus Macaques

IFN-I production is a characteristic of HIV/SIV primary infections. However, acute IFN-I plasma concentrations rapidly decline thereafter. Plasmacytoid dendritic cells (pDC) are key players in this production but primary infection is associated with decreased responsiveness of pDC to TLR 7 and 9 triggering. IFNα production during primary SIV infection contrasts with increased pDC death, renewal and dysfunction. We investigated the contribution of pDC dynamics to both acute IFNα production and the rapid return of IFNα concentrations to pre-infection levels during acute-to-chronic transition. Nine cynomolgus macaques were infected with SIVmac251 and IFNα-producing cells were quantified and characterized. The plasma IFN-I peak was temporally associated with the presence of IFNα⁺ pDC in tissues but IFN-I production was not detectable during the acute-to-chronic transition despite persistent immune activation. No IFNα⁺ cells other than pDC were detected by intracellular staining. Blood-pDC and peripheral lymph node-pDC both lost IFNα⁻ production ability in parallel. In blood, this phenomenon correlated with an increase in the counts of Ki67⁺-pDC precursors with no IFNα production ability. In tissues, it was associated with increase of both activated pDC and KI67⁺-pDC precursors, none of these being IFNα⁺ in vivo. Our findings also indicate that activation/death-driven pDC renewal rapidly blunts acute IFNα production in vivo: pDC sub-populations with no IFNα-production ability rapidly increase and shrinkage of IFNα production thus involves both early pDC exhaustion, and increase of pDC precursors.     

Interferon-Gamma Promotes Infection of Astrocytes by Trypanosoma cruzi

The inflammatory cytokine interferon-gamma (IFNγ) is crucial for immunity against intracellular pathogens such as the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (CD). IFNγ is a pleiotropic cytokine which regulates activation of immune and non-immune cells; however, the effect of IFNγ in the central nervous system (CNS) and astrocytes during CD is unknown. Here we show that parasite persists in the CNS of C3H/He mice chronically infected with the Colombian T. cruzi strain despite the increased expression of IFNγ mRNA. Furthermore, most of the T. cruzi-bearing cells were astrocytes located near IFNγ⁺ cells. Surprisingly, in vitro experiments revealed that pretreatment with IFNγ promoted the infection of astrocytes by T. cruzi increasing uptake and proliferation of intracellular forms, despite inducing increased production of nitric oxide (NO). Importantly, the effect of IFNγ on T. cruzi uptake and growth is completely blocked by the anti-tumor necrosis factor (TNF) antibody Infliximab and partially blocked by the inhibitor of nitric oxide synthesis L-NAME. These data support that IFNγ fuels astrocyte infection by T. cruzi and critically implicate IFNγ-stimulated T. cruzi-infected astrocytes as sources of TNF and NO, which may contribute to parasite persistence and CNS pathology in CD.     

Leishmania Specific CD4 T Cells Release IFNγ That Limits Parasite Replication in Patients with Visceral Leishmaniasis

Visceral leishmaniasis (VL) is associated with increased circulating levels of multiple pro-inflammatory cytokines and chemokines, including IL-12, IFNγ, and TNFα, and elevated expression of IFNγ mRNA in lesional tissue such as the spleen and bone marrow. However, an immunological feature of VL patients is that their peripheral blood mononuclear cells (PBMCs) typically fail to respond to stimulation with leishmanial antigen. Unexpectedly, it was recently shown that Leishmania specific IFNγ, can readily be detected when a whole blood stimulation assay (WBA) is used. We sought to define the conditions that permit whole blood cells to respond to antigen stimulation, and clarify the biological role of the IFNγ found to be released by cells from VL patients. CD4+ T cells were found to be crucial for and the main source of the IFNγ production in Leishmania stimulated whole blood (WB) cultures. Complement, antibodies and red blood cells present in whole blood do not play a significant role in the IFNγ response. The IFNγ production was reduced by blockade of human leukocyte antigen (HLA)-DR, indicating that the response to leishmanial antigens observed in WB of active VL patients is a classical HLA- T cell receptor (TCR) driven reaction. Most importantly, blockade of IFNγ in ex-vivo splenic aspirate cultures demonstrated that despite the progressive nature of their disease, the endogenous IFNγ produced in patients with active VL serves to limit parasite growth.     

Effect of Arginine on Pre-nucleus Stage of Interferon Beta-1b Aggregation

Understanding the mechanism of aggregation of a therapeutic protein would not only ease the manufacturing processing but could also lead to a more stable finished product. Aggregation of recombinant interferon (IFNβ-1b) was studied by heating, oxidizing, or seeding of unformulated monomeric solution. The formation of aggregates was monitored by dynamic light scattering (DLS) and UV spectroscopy. The autocatalytic monomer loss model was used to fit the data on aggregation rates. The influence of pre-nucleation on aggregation step was demonstrated by inducing the liquid samples containing a monomer form of folded IFNβ-1b by heat and also an oxidizing agent. Results tend to suggest that the nucleus includes a single protein molecule which has been probably deformed. Seeding tests showed that aggregation of IFNβ-1b was probably initiated when 1.0% (w/w) of monomers converted to nucleus form. Chemiluminescence spectroscopy analysis of the sample indicated the generation of 3.0 μM of hydrogen peroxide (H₂O₂) during nucleation stage of IFNβ-1b aggregation. Arginine with a concentration of 200 mM was sufficient to suppress aggregation of IFNβ-1b by decreasing the rate of pre-nucleation step. We proposed the formation of pre-nucleus structures prior to nucleation as the mechanism of aggregation of IFNβ-1b. Furthermore, we have showed the positive anti-aggregation effect of arginine on pre-nucleation step.KEY WORDS: aggregation, arginine, interferon beta-1b, mechanism, pre-nucleus     

Interferon Alpha Signalling and Its Relevance for the Upregulatory Effect of Transporter Proteins Associated with Antigen Processing (TAP) in Patients with Malignant Melanoma

Introduction

Interferon alpha (IFNα) is routinely used in the clinical practice for adjuvant systemic melanoma therapy. Understanding the molecular mechanism of IFNα effects and prediction of response in the IFNα therapy regime allows initiation and continuation of IFNα treatment for responder and exclusion of non-responder to avoid therapy inefficacy and side-effects. The transporter protein associated with antigen processing-1 (TAP1) is part of the MHC class I peptide-loading complex, and important for antigen presentation in tumor and antigen presenting cells. In the context of personalized medicine, we address this potential biomarker TAP1 as a target of IFNα signalling.

Results

We could show that IFNα upregulates TAP1 expression in peripheral blood mononuclear cells (PBMCs) of patients with malignant melanoma receiving adjuvant high-dose immunotherapy. IFNα also induced expression of TAP1 in mouse blood and tumor tissue and suppressed the formation of melanoma metastasis in an in vivo B16 tumor model. Besides its expression, TAP binding affinity and transport activity is induced by IFNα in human monocytic THP1 cells. Furthermore, our data revealed that IFNα clearly activates phosphorylation of STAT1 and STAT3 in THP1 and A375 melanoma cells. Inhibition of Janus kinases abrogates the IFNα-induced TAP1 expression. These results suggest that the JAK/STAT pathway is a crucial mediator for TAP1 expression elicited by IFNα treatment.

Conclusion

We suppose that silencing of TAP1 expression provides tumor cells with a mechanism to escape cytotoxic T-lymphocyte recognition. The observed benefit of IFNα treatment could be mediated by the shown dual effect of TAP1 upregulation in antigen presenting cells on the one hand, and of TAP1 upregulation in ‘silent’ metastatic melanoma cells on the other hand. In conclusion, this work contributes to a better understanding of the mode of action of IFNα which is essential to identify markers to predict, assess and monitor therapeutic response of IFNα treatment in the future.     

A Fluorescence Reporter Model Defines “Tip-DCs” as the Cellular Source of Interferon β in Murine Listeriosis

Production of type I interferons, consisting mainly of multiple IFNα subtypes and IFNβ, represents an essential part of the innate immune defense against invading pathogens. While in most situations, namely viral infections, this class of cytokines is indispensable for host survival they mediate a detrimental effect during infection with L. monocytogenes by rendering macrophages insensitive towards IFNγ signalling which leads to a lethal bacterial pathology in mice. Due to a lack of suitable analytic tools the precise identity of the cell population responsible for type I IFN production remains ill-defined and so far these cells have been described to be macrophages. As in general IFNβ is the first type I interferon to be produced, we took advantage of an IFNβ fluorescence reporter-knockin mouse model in which YFP is expressed from a bicistronic mRNA linked by an IRES to the endogenous ifnb mRNA to assess the IFNβ production on a single cell level in situ. Our results showed highest frequencies and absolute numbers of IFNβ⁺ cells in the spleen 24 h after infection with L. monocytogenes where they were located predominately in the white pulp within the foci of infection. Detailed FACS surface marker analyses, intracellular cytokine stainings and T cell proliferation assays revealed that the IFNβ⁺ cells were a phenotypically and functionally further specialized subpopulation of TNF and iNOS producing DCs (Tip-DCs) which are known to be essential for the early containment of L. monocytogenes infection. We proved that the IFNβ⁺ cells exhibited the hallmark characteristics of Tip-DCs as they produced iNOS and TNF and possessed T cell priming abilities. These results point to a yet unappreciated ambiguous role for a multi-effector, IFNβ producing subpopulation of Tip-DCs in controlling the balance between containment of L. monocytogenes infection and effects detrimental to the host driven by IFNβ.     

Spontaneous formation of nucleic acid-based nanoparticles is responsible for high interferon-alpha induction by CpG-A in plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDC) represent a highly specialized immune cell subset that produces large quantities of the anti-viral cytokines type I interferons (IFN-alpha and IFN-beta) upon viral infection. PDC employ a member of the family of toll-like receptors, TLR9, to detect CpG motifs (unmethylated CG dinucleotides in certain base context) present in viral DNA. A certain group of CpG motif-containing oligodeoxynucleotides (CpG ODN), CpG-A, was the first synthetic stimulus available that induced large amounts of interferon-alpha (IFN-alpha) in PDC. However, the mechanism responsible for this activity remained elusive. CpG-A is characterized by a central palindrome and poly(G) at the 5' and 3' end. Here we demonstrate that CpG-A self-assembles to higher order tertiary structures via G-tetrad formation of their poly(G) motifs. Spontaneous G-tetrad formation of CpG-A required the palindrome sequence allowing structure formation in a physiological environment. Once formed, G-tetrad-linked structures were stable even under denaturing conditions. Atomic force microscopy revealed that the tertiary structures formed by CpG-A represent nucleic acid-based nanoparticles in the size range of viruses. Similarly sized preformed polystyrene nanoparticles loaded with a CpG ODN that is otherwise weak at inducing IFN-alpha (CpG-B) gained the potency of CpG-A to induce IFN-alpha. Higher ODN uptake in PDC was not responsible for the higher IFN-alpha-inducing activity of CpG-A or of CpG-B-coated nanoparticles as compared with CpG-B. Based on these results we propose a model in which the spatial configuration of CpG motifs as particle is responsible for the virus-like potency of CpG-A to induce IFN-alpha in PDC.     

An Early Reduction in Treg Cells Correlates with Enhanced Local Inflammation in Cutaneous Leishmaniasis in CCR6-Deficient Mice

Resistance to Leishmania major infection is dependent on the development of a cell-mediated Th1 immune response in resistant C57BL/6 mice whereas Th2-prone BALB/c mice develop non-healing lesions after infection. The chemokine receptor CCR6 is shared by anti-inflammatory regulatory T cells and pro-inflammatory Th17 cells. In a recent study we showed that C57BL/6 mice deficient in CCR6 exhibited enhanced footpad swelling and impaired T helper cell migration indicated by reduced recruitment of total T helper cells into the skin after infection and a reduced delayed type hypersensitivity reaction. Based on these findings we tested whether the lack of CCR6 alters Treg or Th17 cell responses during the course of Leishmania major infection. When we analyzed T cell subsets in the lymph nodes of CCR6-deficient mice, Th17 cell numbers were not different. However, reduced numbers of Treg cells paralleled with a stronger IFNγ response. Furthermore, the early increase in IFNγ-producing cells correlated with increased local tissue inflammation at later time points. Our data indicate an important role of CCR6 for Treg cells and a redundant role for Th17 cells in a Th1 cell-driven anti-parasitic immune response against Leishmania major parasites in resistant C57BL/6 mice.     

The BH3-only protein Bik/Blk/Nbk inhibits nuclear translocation of activated ERK1/2 to mediate IFNgamma-induced cell death

IFNγ induces cell death in epithelial cells, but the mediator for this death pathway has not been identified. In this study, we find that expression of Bik/Blk/Nbk is increased in human airway epithelial cells (AECs [HAECs]) in response to IFNγ. Expression of Bik but not mutant BikL61G induces and loss of Bik suppresses IFNγ-induced cell death in HAECs. IFNγ treatment and Bik expression increase cathepsin B and D messenger RNA levels and reduce levels of phospho–extracellular regulated kinase 1/2 (ERK1/2) in the nuclei of bik^+/+ compared with bik^−/− murine AECs. Bik but not BikL61G interacts with and suppresses nuclear translocation of phospho-ERK1/2, and suppression of ERK1/2 activation inhibits IFNγ- and Bik-induced cell death. Furthermore, after prolonged exposure to allergen, hyperplastic epithelial cells persist longer, and nuclear phospho-ERK is more prevalent in airways of IFNγ^−/− or bik^−/− compared with wild-type mice. These results demonstrate that IFNγ requires Bik to suppress nuclear localization of phospho-ERK1/2 to channel cell death in AECs.     

Tumor Progression Locus 2 Promotes Induction of IFNλ, Interferon Stimulated Genes and Antigen-Specific CD8+ T Cell Responses and Protects against Influenza Virus

Mitogen-activated protein kinase (MAP) cascades are important in antiviral immunity through their regulation of interferon (IFN) production as well as virus replication. Although the serine-threonine MAP kinase tumor progression locus 2 (Tpl2/MAP3K8) has been implicated as a key regulator of Type I (IFNα/β) and Type II (IFNγ) IFNs, remarkably little is known about how Tpl2 might contribute to host defense against viruses. Herein, we investigated the role of Tpl2 in antiviral immune responses against influenza virus. We demonstrate that Tpl2 is an integral component of multiple virus sensing pathways, differentially regulating the induction of IFNα/β and IFNλ in a cell-type specific manner. Although Tpl2 is important in the regulation of both IFNα/β and IFNλ, only IFNλ required Tpl2 for its induction during influenza virus infection both in vitro and in vivo. Further studies revealed an unanticipated function for Tpl2 in transducing Type I IFN signals and promoting expression of interferon-stimulated genes (ISGs). Importantly, Tpl2 signaling in nonhematopoietic cells is necessary to limit early virus replication. In addition to early innate alterations, impaired expansion of virus-specific CD8⁺ T cells accompanied delayed viral clearance in Tpl2^-/- mice at late time points. Consistent with its critical role in facilitating both innate and adaptive antiviral responses, Tpl2 is required for restricting morbidity and mortality associated with influenza virus infection. Collectively, these findings establish an essential role for Tpl2 in antiviral host defense mechanisms.     

The TrxG Complex Mediates Cytokine Induced De Novo Enhancer Formation in Islets

To better understand how β-cells respond to proinflammatory cytokines we mapped the locations of histone 3 lysine 4 monomethylation (H3K4me1), a post-translational histone modification enriched at active and poised cis-regulatory regions, in IFNγ, Il-1β, and TNFα treated pancreatic islets. We identified 96,721 putative cis-regulatory loci, of which 3,590 were generated de novo, 3,204 had increased H3K4me1, and 5,354 had decreased H3K4me1 in IFNγ, Il-1β, and TNFα exposed islets. Roughly 10% of the de novo and increased regions were enriched for the repressive histone modification histone 3 lysine 27 trimethylation (H3K27me3) in untreated cells, and these were frequently associated with chemokine genes. We show that IFNγ, Il-1β, and TNFα exposure overcomes this repression and induces chemokine gene activation in as little as three hours, and that this expression persists for days in absence of continued IFNγ, Il-1β, and TNFα exposure. We implicate trithorax group (TrxG) complexes as likely players in the conversion of these repressed loci to an active state. To block the activity of these complexes, we suppressed Wdr5, a core component of the TrxG complexes, and used the H3K27me3 demethylase inhibitor GSK-J4. We show that GSK-J4 is particularly effective in blunting IFNγ, Il-1β, and TNFα-induced chemokine gene expression in β-cells; however, it induced significant islet-cell apoptosis and β-cell dysfunction. Wdr5 suppression also reduced IFNγ, Il-1β, and TNFα induced chemokine gene expression in β-cells without affecting islet-cell survival or β-cell function after 48hrs, but did begin to increase islet-cell apoptosis and β-cell dysfunction after four days of treatment. Taken together these data suggest that the TrxG complex is potentially a viable target for preventing cytokine induced chemokine gene expression in β-cells.     

CpG oligodeoxynucleotides protect normal and SIV-infected macaques from Leishmania infection

Oligodeoxynucleotides containing CpG motifs (CpG ODNs) mimic microbial DNA and activate effectors of the innate immune response, which limits the spread of pathogens and promotes an adaptive immune response. CpG ODNs have been shown to protect mice from infection with intracellular pathogens. Unfortunately, CpG motifs that optimally stimulate humans are only weakly active in mice, mandating the use of nonhuman primates to monitor the activity and safety of "human" CpG ODNs in vivo. This study demonstrates that CpG ODN treatment of rhesus macaques significantly reduces the severity of the lesions caused by a challenge with Leishmania: Leishmania superinfection is common in immunocompromised hosts, particularly those infected with HIV. This study shows that PBMCs from HIV-infected subjects respond to stimulation with CpG ODNs. To determine whether CpG ODNs can protect retrovirus-infected primates, SIV-infected macaques were treated with CpG ODNs and then challenged with Leishmania: Both lesion size and parasite load were significantly reduced in the CpG-treated animals. These findings support the clinical development of CpG ODNs as immunoprotective agents in normal and HIV-infected patients.     

IL-4 Attenuates Th1-Associated Chemokine Expression and Th1 Trafficking to Inflamed Tissues and Limits Pathogen Clearance

Interleukin 4 (IL-4) plays a central role in the orchestration of Type 2 immunity. During T cell activation in the lymph node, IL-4 promotes Th2 differentiation and inhibits Th1 generation. In the inflamed tissue, IL-4 signals promote innate and adaptive Type-2 immune recruitment and effector function, positively amplifying the local Th2 response. In this study, we identify an additional negative regulatory role for IL-4 in limiting the recruitment of Th1 cells to inflamed tissues. To test IL-4 effects on inflammation subsequent to Th2 differentiation, we transiently blocked IL-4 during ongoing dermal inflammation (using anti-IL-4 mAb) and analyzed changes in gene expression. Neutralization of IL-4 led to the upregulation of a number of genes linked to Th1 trafficking, including CXCR3 chemokines, CCL5 and CCR5 and an associated increase in IFNγ, Tbet and TNFα genes. These gene expression changes correlated with increased numbers of IFNγ-producing CD4+ T cells in the inflamed dermis. Moreover, using an adoptive transfer approach to directly test the role of IL-4 in T cell trafficking to the inflamed tissues, we found IL-4 neutralization led to an early increase in Th1 cell recruitment to the inflamed dermis. These data support a model whereby IL-4 dampens Th1-chemokines at the site of inflammation limiting Th1 recruitment. To determine biological significance, we infected mice with Leishmania major, as pathogen clearance is highly dependent on IFNγ-producing CD4+ T cells at the infection site. Short-term IL-4 blockade in established L. major infection led to a significant increase in the number of IFNγ-producing CD4+ T cells in the infected ear dermis, with no change in the draining LN. Increased lymphocyte influx into the infected tissue correlated with a significant decrease in parasite number. Thus, independent of IL-4''s role in the generation of immune effectors, IL-4 attenuates lymphocyte recruitment to the inflamed/infected dermis and limits pathogen clearance.     

Pathogen- and Host-Directed Antileishmanial Effects Mediated by Polyhexanide (PHMB)

Background

Cutaneous leishmaniasis (CL) is a neglected tropical disease caused by protozoan parasites of the genus Leishmania. CL causes enormous suffering in many countries worldwide. There is no licensed vaccine against CL, and the chemotherapy options show limited efficacy and high toxicity. Localization of the parasites inside host cells is a barrier to most standard chemo- and immune-based interventions. Hence, novel drugs, which are safe, effective and readily accessible to third-world countries and/or drug delivery technologies for effective CL treatments are desperately needed.

Methodology/Principal Findings

Here we evaluated the antileishmanial properties and delivery potential of polyhexamethylene biguanide (PHMB; polyhexanide), a widely used antimicrobial and wound antiseptic, in the Leishmania model. PHMB showed an inherent antileishmanial activity at submicromolar concentrations. Our data revealed that PHMB kills Leishmania major (L. major) via a dual mechanism involving disruption of membrane integrity and selective chromosome condensation and damage. PHMB’s DNA binding and host cell entry properties were further exploited to improve the delivery and immunomodulatory activities of unmethylated cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODN). PHMB spontaneously bound CpG ODN, forming stable nanopolyplexes that enhanced uptake of CpG ODN, potentiated antimicrobial killing and reduced host cell toxicity of PHMB.

Conclusions

Given its low cost and long history of safe topical use, PHMB holds promise as a drug for CL therapy and delivery vehicle for nucleic acid immunomodulators.