Delta-Aminolevulinate-Induced Host-Parasite Porphyric Disparity for Selective Photolysis of Transgenic Leishmania in the Phagolysosomes of Mononuclear Phagocytes: a Potential Novel Platform for Vaccine Delivery

Chromatographic separation of porphyrins extracted from DT-infected and uninfected macrophages showing the persistent presence of URO originating from uroporphyric DT Leishmania. J + DT + ALA, J774 cells infected with DT for 3 days, treated with 1 mM ALA daily after 3 days, and chased for 3 more days. J + DT − ALA, omission of the ALA treatments; J + 0 + ALA, omission of DT infection; J + 0 − ALA, J774 macrophages with neither DT infection nor ALA treatment. Lanes 1 to 3, days 1 to 3 of chase after the ALA treatments. Porphyrins extracted from ∼5 × 10⁶ macrophages were loaded in each lane. Copro (C), Proto (P), and Uro (U), carboxymethylated porphyrin markers corresponding to coproporphyrins, protoporphyrin IX, and uroporphyrins.

Depression of parasite loads in DT-infected, but not ST-infected, host cells after ALA treatments under incipient light. Shown are the kinetics of parasite loads (A) and host cell replication (A′) per culture of J774 cells infected with uroporphyrinogenic (DT) and nonporphyrinogenic (ST) mutants after exposure to ALA (+ ALA). The arrows indicate daily ALA treatments during medium renewals for 3 consecutive days from day 3 to day 5 after infection for a total period of 9 days. Statistical significance, indicated by the asterisks: P < 0.01, calculated using the Student t test. The error bars indicate standard errors.

Significant clearance of parasite loads from DT-infected host cells after ALA treatment followed by experimental illumination. J774 macrophages (A and A′) and fetal dendritic cells (B and B′) were infected with DTs, followed by exposure to 1 mM ALA and white-light illumination for 15 min (12.15 J/cm²). The parasite loads (A and B) and the total numbers of host cells (A′ and B′) were estimated per infected culture at the time points indicated under various conditions, as shown (± ALA ± light). The red and black arrows indicate the time points of 1× ALA treatment and light exposure, respectively. Statistical significance, denoted by the asterisks: P < 0.001, calculated using one-way ANOVA. The error bars indicate standard errors.

Selective photolysis of intra-PV uroporphyric DTs after experimental illumination of DT-infected host cells. Shown are phase-contrast (left) and porphyrin fluorescence (right) microscopy of uroporphyrinogenic-DT-infected J774 macrophages (J + DT) and fetal skin dendritic cells (FSDC + DT) with and without 1 mM ALA exposure and/or white-light illumination for 15 min (12.15 J/cm²), as indicated (± ALA ± light). The cells were examined 2 h after illumination. Scale bar = 10 μm.

Recruitment of MHC class II molecules to LAMP-1-positive PVs after infection of mouse peritoneal macrophages with DTs and their return to normal distribution after photolysis of intra-PV DTs. Mouse peritoneal macrophages (Phase contrast) infected with uroporphyrinogenic mutants (+ DT) (A and B) and uninfected controls (C and D) were exposed to IFN-γ to induce MHC class II expression. One set, consisting of infected (B) and uninfected (C) cells, was exposed to the conditions for selective photolysis of intra-PV uroporphyric DTs, i.e., treatment with 1 mM ALA overnight (+ ALA) followed by illumination (400 nm) at 7.5 J/cm² (+ light). The remaining set of infected (A) and uninfected (D) cells served as porphyrin-free and thus nonphotolytic controls. A monolayer of all cell samples (A to D) was further incubated for 2 h after photolytic treatments before processing for localization of MHC class II (green), LAMP-1 (red), and the nucleus (blue) by immunofluorescence microscopy. See Materials and Methods for experimental details. Scale bar = 10 μm.

Macrophage and Leishmania expression profiles of DT-infected cells after ALA-induced porphyrinogenesis and selective photolysis of intra-PV uroporphyric DTs. J, J774 macrophages; J + DT, J774 cells infected with DTs; J + DT + light, illumination of DT-infected cells with a long-wave UV lamp (λ_max = 366 nm) for 10 min; J + DT + ALA, treatment of J + DT with 1 mM ALA for 16 h; J + DT + ALA + light, exposure of J + DT to both ALA and light, followed by chase in ALA-free medium for 3 days. All samples were subjected to microarray analysis (∼21,000 mouse genes plus 4 Leishmania genes) (see Materials and Methods for details). (A) Hierarchical clustering of genes showing their expression patterns in DT-infected cells versus uninfected cells and subsequent changes that occur upon induction of Leishmania photolysis. A total of 3,102 macrophage genes showed altered expression (P < 0.05) compared to uninfected (J) and infected (J + DT) macrophages. The 318 clustered genes shown here had >2-fold changes in the expression levels in at least one of the treatments (J + DT + ALA, J + DT + light, and/or J + DT + ALA + light) in comparison to J + DT. The expression levels of these 318 genes in uninfected J cells (J) are included for reference to the preinfection expression patterns for these genes. The color scale indicates the change for each gene based on median intensity (pale green) from downregulated (dark blue to light blue) to upregulated (yellow to red) for each condition using units of Z (standard deviations). Gene clustering is indicated at left, with two principal families: red, those which start low, increase, and then return to low initial values; and green, those which start high, decrease, and then rise to initial values. (B) Progressive decrease of Leishmania DT-specific gene expression from ALA-induced uroporphyrinogenesis of DTs and their photolysis in infected host cells. The four Leishmania genes spotted in triplicate on the arrays were used to assess the effects of various treatments on the protozoan transcription as a measure of their persistence. The average normalized medium densities (nMD) for three individual spots for each of the four genes in the four replicate experiments were used to plot the expression levels. Leishmania ndk, nagt, and dhfr genes are plotted on the left-hand scale, and beta-tubulin is on the right-hand scale (1st bar of the 4 for each treatment group). The beta-tubulin value is higher, presumably resulting from cross-hybridization of the Leishmania probe with sequence-conserved murine beta-tubulin messages inherently abundant in macrophages. The error bars indicate standard errors.