Cinnamic Acid Bornyl Ester Derivatives from Valeriana wallichii Exhibit Antileishmanial In Vivo Activity in Leishmania major-Infected BALB/c Mice

Human leishmaniasis covers a broad spectrum of clinical manifestations ranging from selfhealing cutaneous leishmaniasis to severe and lethal visceral leishmaniasis caused among other species by Leishmania major or Leishmania donovani, respectively. Some drug candidates are in clinical trials to substitute current therapies, which are facing emerging drugresistance accompanied with serious side effects. Here, two cinnamic acid bornyl ester derivatives (1 and 2) were assessed for their antileishmanial activity. Good selectivity and antileishmanial activity of bornyl 3-phenylpropanoate (2) in vitro prompted the antileishmanial assessment in vivo. For this purpose, BALB/c mice were infected with Leishmania major promastigotes and treated with three doses of 50 mg/kg/day of compound 2. The treatment prevented the characteristic swelling at the site of infection and correlated with reduced parasite burden. Transmitted light microscopy and transmission electron microscopy of Leishmania major promastigotes revealed that compounds 1 and 2 induce mitochondrial swelling. Subsequent studies on Leishmania major promastigotes showed the loss of mitochondrial transmembrane potential (ΔΨm) as a putative mode of action. As the cinnamic acid bornyl ester derivatives 1 and 2 had exhibited antileishmanial activity in vitro, and compound 2 in Leishmania major-infected BALB/c mice in vivo, they can be regarded as possible lead structures for the development of new antileishmanial therapeutic approaches

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 (CD11ccreIL-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 CD11ccreIL-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

Die auf dendritischen Zellen basierende Immunisierungsstrategie gegen Leishmania major in BALB/c Mäusen ist abhängig von der Stimulation der Interleukin-4 Rezeptor alpha Kette

Cutaneous leishmaniasis is endemic in tropical and subtropical regions of the world. Effective vaccination strategies are urgently needed because of the emergence of drug-resistant parasites and severe side effects of chemotherapy. The research group of Heidrun Moll previously established a DC-based vaccination strategy to induce complete and long-lasting immunity to experimental leishmaniasis using LmAg-loaded and CpG ODN-activated DC as a vaccine carrier. Prevention of tissue damages at the site of L. major inoculation can be achieved if the BALB/c mice were systemically given LmAg-loaded BMDC that had been exposed to CpG ODN. The interest in further exploring the role of IL-4 aroused as previous studies allowed establishing that IL-4 was involved in the redirection of the immune response towards a type 1 profile. Thus, wt BALB/c mice or DC-specific CD11ccreIL-4Rα-/lox BALB/c mice were given either wt or IL-4Rα-deficient LmAg-loaded BMDC exposed or not to CpG ODN prior to inoculation of 2 x 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 damages 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 LN in CD11ccreIL-4Rα-/lox mice immunized with CpG ODN-exposed LmAg-loaded IL-4Rα-deficient DC, indicating the influence of IL-4R-mediated signaling in host DC to control parasite replication. In addition, no footpad damage was observed in BALB/c mice that were systemically immunized with LmAg-loaded wt DC doubly exposed to CpG ODN and recombinant IL-4. Discussing these findings allow the assumption that triggering the IL4/IL4Rα signaling pathway could be a precondition when designing vaccines aimed to prevent damaging processes in tissues hosting intracellular microorganisms.Die kutane Leishmaniose ist vor allem in den tropischen und subtropischen Regionen endemisch. Die Notwendigkeit der Erforschung und Etablierung einer Impfstoffstrategie basiert auf dem Auftreten von starken Nebenwirkungen während einer medikamentösen Behandlung, als auch auf die Entwicklung von Resistenzen des Parasiten gegenüber herkömmlichen Behandlungsmethoden. Die Arbeitsgruppe um Heidrun Moll etablierte eine auf dendritischen Zellen (DZ) basierende Immunisierungsstrategie, welche langlebige Immunität gegen experimentelle Leishmaniose vermittelt. Dabei dienen CpG ODN-stimulierte DZ als Adjuvans für L.-major–Antigene (LmAg). Die durch Infektion mit Leishmania-Parasiten hervorgerufene Gewebeschädigung kann in BALB/c-Mäusen verhindert werden, vorausgesetzt eine systemische Verabreichung von LmAg-beladenen und CpG ODN-aktivierten DZ erfolgte eine Woche vor der Infektion. Es konnte gezeigt werden, dass der Schutz durch die Induktion einer von Interleukin (IL)-12 und Interferon (IFN)-gamma dominierten T-Helfer (Th)1-Immunantwort herbeigeführt wurde und kranke Kontrollmäuse eine IL-4-dominierte Th2 Immunantwort aufwiesen. Mittlerweile zeigen zahlreiche Studien, dass IL-4 nicht ausschließlich eine krankheitsfördernde Funktion innehat, sondern auch die Fähigkeit zur Einleitung eine Typ-1-Immunantwort besitzt. Auf Grund dieser Studien wurde das Augenmerk auf die Rolle von IL-4 in der DZ-basierten Immunisierung gegen Leishmaniose in BALB/c Mäusen gelegt. In der vorliegenden Arbeit wurde die Notwendigkeit der Stimulation der IL-4 Rezeptor alpha (IL-4Rα) Kette auf DZ, während einer DZ-basierten Immunisierung gegen Leishmaniose in BALB/c Mäusen gezeigt. Um dies zu erreichen, wurden Wildtyp (wt)-BALB/c-Mäuse oder DZ-spezifische CD11ccreIL-4Rα-/lox BALB/c Mäuse entweder mit wt oder IL-4Rα-defizienten LmAg-beladenen DZ mit oder ohne Aktivierung durch CpG ODN, eine Woche vor der Infektion mit 2x105 L. major Promastigoten in den Hinterfuß, immunisiert. Die in dieser Doktorarbeit gezeigten Ergebnisse lassen den Schluss zu, dass die Stimulation der IL-4Rα-Kette auf den als Adjuvans eingesetzten DZ erforderlich ist, um eine Gewebsschädigung an der Infektionsstelle zu verhindern, da konditionierte wt DZ, nicht aber IL-4Rα-defiziente DZ in der Lage sind, Schutz gegen Leishmaniose zu vermitteln. Des Weiteren konnte eine unkontrollierte Ausdehnung von Leishmania-Parasiten im infizierten Fuß und in den angrenzenden Lymphknoten von CD11ccreIL-4Rα-/lox Mäusen beobachtet werden, welche mit CpG ODN-aktivierten und LmAg-beladenen IL-4Rα-defizienten DZ immunisiert wurden. Dieser Befund zeigt den Einfluss der Stimulation der IL-4Rα-Kette auf wirtsansässigen DZ im Hinblick auf die Eindämmung der Parasitenreplikation und Parasitenverbreitung. Zusätzliche Analysen in BALB/c-Mäusen, welche mit LmAg-beladenen, CpG ODN- und rekombinanten IL-4-stimulierten DZ immunisiert wurden, zeigten einen resistenten klinischen Verlauf der Infektion. Die hier gezeigten Ergebnisse lassen die Vermutung zu, dass die durch die IL-4/IL-4Rα-Kette ausgelösten Signale in den DZ eine Grundvoraussetzung für eine erfolgreiche Immunisierung sind und sollten deswegen unbedingt bei der Entwicklung eines Impfstoffes gegen die gewebsschädigenden Folgen einer Leishmaniose oder anderer durch intrazelluläre Mikroorganismen verursachten Infektionen berücksichtigt werden

T Cell-Derived IL-10 Determines Leishmaniasis Disease Outcome and Is Suppressed by a Dendritic Cell Based Vaccine

Abstract In the murine model of Leishmania major infection, resistance or susceptibility to the parasite has been associated with the development of a Th1 or Th2 type of immune response. Recently, however, the immunosuppressive effects of IL-10 have been ascribed a crucial role in the development of the different clinical correlates of Leishmania infection in humans. Since T cells and professional APC are important cellular sources of IL-10, we compared leishmaniasis disease progression in T cell-specific, macrophage/neutrophil-specific and complete IL-10-deficient C57BL/6 as well as T cell-specific and complete IL-10-deficient BALB/c mice. As early as two weeks after infection of these mice with L. major, T cell-specific and complete IL-10-deficient animals showed significantly increased lesion development accompanied by a markedly elevated secretion of IFN-γ or IFN-γ and IL-4 in the lymph nodes draining the lesions of the C57BL/6 or BALB/c mutants, respectively. In contrast, macrophage/neutrophil-specific IL-10-deficient C57BL/6 mice did not show any altered phenotype. During the further course of disease, the T cell-specific as well as the complete IL-10-deficient BALB/c mice were able to control the infection. Furthermore, a dendritic cell-based vaccination against leishmaniasis efficiently suppresses the early secretion of IL-10, thus contributing to the control of parasite spread. Taken together, IL-10 secretion by T cells has an influence on immune activation early after infection and is sufficient to render BALB/c mice susceptible to an uncontrolled Leishmania major infection. Author Summary The clinical symptoms caused by infections with Leishmania parasites range from self-healing cutaneous to uncontrolled visceral disease and depend not only on the parasite species but also on the type of the host's immune response. It is estimated that 350 million people worldwide are at risk, with a global incidence of 1–1.5 million cases of cutaneous and 500,000 cases of visceral leishmaniasis. Murine leishmaniasis is the best-characterized model to elucidate the mechanisms underlying resistance or susceptibility to Leishmania major parasites in vivo. Using T cell-specific and macrophage-specific mutant mice, we demonstrate that abrogating the secretion of the immunosuppressive cytokine IL-10 by T cells is sufficient to render otherwise susceptible mice resistant to an infection with the pathogen. The healing phenotype is accompanied by an elevated specific inflammatory immune response very early after infection. We further show that dendritic cell-based vaccination against leishmaniasis suppresses the early secretion of IL-10 following challenge infection. Thus, our study unravels a molecular mechanism critical for host immune defense, aiding in the development of an effective vaccine against leishmaniasis

Decreased levels of activated DC in the infected popliteal lymph nodes in mice vaccinated with IL-4Rα^−/− BMDC.

<p>The lymphocytes of the draining popliteal lymph nodes of 5 BALB/c or 5 CD11c^creIL-4Rα^−/lox mice, treated as indicated, were collected six weeks post infection, surface-stained for CD11c, MHC class II and CD80 expression to determine the proportion of activated and mature DC in the lymph nodes and analysed using FACSCalibur. The x-axis of the dot blots label CD11c and the y-axis MHC class II or CD80, as indicated. The numbers indicate % of gated cells within the distinct quadrant. The mean ± SD of 5 mice each is shown as bar graphs (white column: PBS-treated group, grey column: wt DC/CpG/LmAg immunized group, black column: IL-4Rα^−/− DC/CpG/LmAg immunized group, lined column: wt DC/rIL-4/CpG/LmAg immunized group). **, p<0.005 compared to the respective PBS- treated control group.</p

IL-4Rα triggering of BMDC increases <i>L. major</i>-stimulated IL-12 secretion <i>in vivo</i>.

<p>BALB/c (<b>A</b>) or CD11c^creIL-4Rα^−/lox mice (<b>B</b>) were immunized with 5×10⁵ BMDC prepared as indicated and infected one week later with 2×10⁵<i>L. major</i> promastigotes. Total lymphocytes of the draining popliteal lymph nodes were collected six weeks post infection and incubated for 72 hours in the absence (white bars) or presence (black bars) of LmAg. The levels of IL-4, IFN-γ and IL-12 were measured by ELISA in the collected supernatants. The results of 5 mice are shown. *, p<0.05, **, p<0.005 compared to the respective PBS-treated control group.</p

IL-4Rα triggering of vaccine carrier leads to the control of IL-4 production by host lymphocytes.

<p>Lymphocytes of the draining popliteal lymph nodes of 5 BALB/c or CD11c^creIL-4Rα^−/lox mice, treated as indicated, were collected six weeks post infection and activated for 2 hours with PMA-ionomycin before adding monensin for the final 4 hours of culture. The cells were stained for CD4 and IFN-γ (<b>A</b>) or IL-4 (<b>B</b>) and for CD11c and IL-12 (<b>C</b>) or IL-4 (<b>D</b>) and analysed using FACSCalibur. The x-axis of the dot blots label CD11c or CD4 and the y-axis IL-4, IL-12 or IFN-γ as indicated. The numbers indicate % of gated cells within the distinct quadrant. The bar graphs show the percentage of gated cytokine-secreting lymphocytes as the mean ± SD of 5 BALB/c (black column) or CD11c^creIL-4Rα^−/lox mice (white column). *, p<0.05, **, p<0.005, compared to the respective PBS-treated control group.</p

Cinnamic Acid Bornyl Ester Derivatives from <i>Valeriana wallichii</i> Exhibit Antileishmanial <i>In Vivo</i> Activity in <i>Leishmania major</i>-Infected BALB/c Mice

<div><p>Human leishmaniasis covers a broad spectrum of clinical manifestations ranging from self-healing cutaneous leishmaniasis to severe and lethal visceral leishmaniasis caused among other species by <i>Leishmania major</i> or <i>Leishmania donovani</i>, respectively. Some drug candidates are in clinical trials to substitute current therapies, which are facing emerging drug-resistance accompanied with serious side effects. Here, two cinnamic acid bornyl ester derivatives (<b>1</b> and <b>2</b>) were assessed for their antileishmanial activity. Good selectivity and antileishmanial activity of bornyl 3-phenylpropanoate (<b>2</b>) <i>in vitro</i> prompted the antileishmanial assessment <i>in vivo</i>. For this purpose, BALB/c mice were infected with <i>Leishmania major</i> promastigotes and treated with three doses of 50 mg/kg/day of compound <b>2</b>. The treatment prevented the characteristic swelling at the site of infection and correlated with reduced parasite burden. Transmitted light microscopy and transmission electron microscopy of <i>Leishmania major</i> promastigotes revealed that compounds <b>1</b> and <b>2</b> induce mitochondrial swelling. Subsequent studies on <i>Leishmania major</i> promastigotes showed the loss of mitochondrial transmembrane potential (ΔΨ_m) as a putative mode of action. As the cinnamic acid bornyl ester derivatives <b>1</b> and <b>2</b> had exhibited antileishmanial activity <i>in vitro</i>, and compound <b>2</b> in <i>Leishmania major</i>-infected BALB/c mice <i>in vivo</i>, they can be regarded as possible lead structures for the development of new antileishmanial therapeutic approaches.</p></div

Compound-associated cell death is time-dependent.

<p><i>L</i>. <i>major</i> promastigotes were treated with solvent control (1% DMSO), apoptosis inducer miltefosine (122.7 μM), compound 1 (100 μM), and 2 (100 μM) in a time course experiment for 6 h, 10 h, and 24 h. Cells were harvested, stained with AV and PI and subsequently analyzed by flow cytometry. Analysis for compound 2 was performed in an independent experiment. Percentage for each quadrant is written below the corresponding dot blot figure. Lower-left (LL): live cells (AV^-/PI^-); upper-left (UL): early necrotic cells (AV^-/PI⁺); upper-right (UR): necrotic/late apoptotic cells (AV⁺/PI⁺); and lower-right (LR): early apoptotic cells (AV⁺/PI^-).</p

Compound 2 treatment of <i>L</i>. <i>major</i>-infected BALB/c mice is associated with reduced parasite burden in infected tissues and organs.

<p>Female BALB/c mice (n = 33) were infected with 5 × 10⁶<i>L</i>. <i>major</i> promastigotes into the right hind footpad. Treatment regimen started on d 21 post infection either using 50% DMSO/PBS (□) (n = 11), 50 mg/kg compound 2 (■) (n = 11) or mice were left untreated (▲) (n = 11). A. At the end of experiment on d 35 all mice (n = 33) were sacrificed and the parasite burden of the infected footpads (iFP) (n = 9) was determined using Limited Dilution Assays (LDA). B. Photographic documentation of the infected footpads of three exemplary mice per group shall reveal the severity of disease progression in non-healing mice and the control of clinical manifestation in compound 2-treated BALB/c mice. C. The parasite burden of the infection site-draining popliteal lymph nodes (pLN) of individual mice (n = 9) or D. spleens of individual mice (n = 6) was determined using LDA. n = number of animals; ** p<0.005; differences in parasite load between 50% DMSO/PBS-treated and compound 2-treated animals did not reach statistical significance for D.</p