Differential Impact of LPG-and PG-Deficient \u3cem\u3eLeishmania major\u3c/em\u3e Mutants on the Immune Response of Human Dendritic Cells

BACKGROUND: Leishmania major infection induces robust interleukin-12 (IL12) production in human dendritic cells (hDC), ultimately resulting in Th1-mediated immunity and clinical resolution. The surface of Leishmania parasites is covered in a dense glycocalyx consisting of primarily lipophosphoglycan (LPG) and other phosphoglycan-containing molecules (PGs), making these glycoconjugates the likely pathogen-associated molecular patterns (PAMPS) responsible for IL12 induction. METHODOLOGY/PRINCIPAL FINDINGS: Here we explored the role of parasite glycoconjugates on the hDC IL12 response by generating L. major Friedlin V1 mutants defective in LPG alone, (FV1 lpg1-), or generally deficient for all PGs, (FV1 lpg2-). Infection with metacyclic, infective stage, L. major or purified LPG induced high levels of IL12B subunit gene transcripts in hDCs, which was abrogated with FV1 lpg1- infections. In contrast, hDC infections with FV1 lpg2- displayed increased IL12B expression, suggesting other PG-related/LPG2 dependent molecules may act to dampen the immune response. Global transcriptional profiling comparing WT, FV1 lpg1-, FV1 lpg2- infections revealed that FV1 lpg1- mutants entered hDCs in a silent fashion as indicated by repression of gene expression. Transcription factor binding site analysis suggests that LPG recognition by hDCs induces IL-12 in a signaling cascade resulting in Nuclear Factor κ B (NFκB) and Interferon Regulatory Factor (IRF) mediated transcription. CONCLUSIONS/SIGNIFICANCE: These data suggest that L. major LPG is a major PAMP recognized by hDC to induce IL12-mediated protective immunity and that there is a complex interplay between PG-baring Leishmania surface glycoconjugates that result in modulation of host cellular IL12

Differential impact of LPG-and PG-deficient Leishmania major mutants on the immune response of human dendritic cells

<div><p>Background</p><p><i>Leishmania major</i> infection induces robust interleukin-12 (IL12) production in human dendritic cells (hDC), ultimately resulting in Th1-mediated immunity and clinical resolution. The surface of <i>Leishmania</i> parasites is covered in a dense glycocalyx consisting of primarily lipophosphoglycan (LPG) and other phosphoglycan-containing molecules (PGs), making these glycoconjugates the likely pathogen-associated molecular patterns (PAMPS) responsible for IL12 induction.</p><p>Methodology/Principal Findings</p><p>Here we explored the role of parasite glycoconjugates on the hDC IL12 response by generating <i>L</i>. <i>major</i> Friedlin V1 mutants defective in LPG alone, (FV1 <i>lpg1-</i>), or generally deficient for all PGs, (FV1 <i>lpg2-</i>). Infection with metacyclic, infective stage, <i>L</i>. <i>major</i> or purified LPG induced high levels of <i>IL12B</i> subunit gene transcripts in hDCs, which was abrogated with FV1 <i>lpg1-</i> infections. In contrast, hDC infections with FV1 <i>lpg2-</i> displayed increased <i>IL12B</i> expression, suggesting other PG-related/<i>LPG2</i> dependent molecules may act to dampen the immune response. Global transcriptional profiling comparing WT, FV1 <i>lpg1-</i>, FV1 <i>lpg2-</i> infections revealed that FV1 <i>lpg1-</i> mutants entered hDCs in a silent fashion as indicated by repression of gene expression. Transcription factor binding site analysis suggests that LPG recognition by hDCs induces IL-12 in a signaling cascade resulting in Nuclear Factor κ B (NFκB) and Interferon Regulatory Factor (IRF) mediated transcription.</p><p>Conclusions/Significance</p><p>These data suggest that <i>L</i>. <i>major</i> LPG is a major PAMP recognized by hDC to induce IL12-mediated protective immunity and that there is a complex interplay between PG-baring <i>Leishmania</i> surface glycoconjugates that result in modulation of host cellular IL12.</p></div

Enriched immunologically relevant pathways for genes expressed in <i>IL12B</i>-like patterns.

<p>Gene transcripts lists from <i>L</i>. <i>major</i> FV1 WT, FV1 <i>lpg1</i>⁻, and FV1 <i>lpg2</i>⁻ mutant <i>in vitro</i> infected monocyte-derived hDCs microarray analysis, with log₂ ratio over uninfected value patterns between samples that clustered with the <i>IL12B</i>, were analyzed to identify significantly enriched (Benjamini and Hochberg adjusted p<0.01) KEGG pathways. Three immunologically relevant pathways, which also contained <i>IL12B</i>, were enriched among that gene list: cytokine-cytokine receptor interactions (A), Jak-STAT signaling pathway (B), and toll-like receptor signaling pathway (C). All graphs display average log₂ ratio over uninfected values for genes present in the expression datasets of FV1 WT, FV1 <i>lpg1</i>⁻, and FV1 <i>lpg2</i>⁻ mutant infected samples which were members of the corresponding enriched pathways.</p

Formal names for <i>L</i>. <i>major</i> FV1 LPG and PG null mutants and add back lines.

<p>^aA ^ denotes gene disruption and a ^Δ denotes gene replacement</p><p>Formal names for <i>L</i>. <i>major</i> FV1 LPG and PG null mutants and add back lines.</p

Relative <i>TNF</i> and <i>IL10</i> levels in <i>L</i>. <i>major</i> Friedlin V1 infected DCs.

<p>Human DCs were infected with <i>L</i>. <i>major</i> FV1 parasites: <i>L</i>. <i>major</i> FV1 <i>(WT)</i>, LPG null mutant (FV1 <i>lpg1</i>⁻), LPG add back (FV1 <i>lpg1</i>⁻/+<i>LPG1</i>), PG null mutant (FV1 <i>lpg2</i>⁻), or PG add back (FV1 <i>lpg2</i>⁻/+<i>LPG2</i>). At 8 hrs post infection, (<b>A</b>) <i>TNF</i> (n = 5 donors) and (<b>B</b>) <i>IL10</i> (n = 5 donors) expression was measured by qRT-PCR. Fold changes were calculated using the ΔΔC_T method and are represented as fold change over uninfected samples. Box plots display the median value (line), the interquartile range (box), and Tukey whiskers encompassing data within 1.5 fold of the interquartile range; data outside this range are presented as individual data points (open circles). *Statistical significance (p<0.05). All values were significantly greater than uninfected.</p

<i>L</i>. <i>major</i> strain FV1 metacyclic promastigotes and LPG stimulate <i>IL12B</i> expression.

<p>(<b>A</b>) Human DCs were infected with <i>L</i>. <i>major</i> FV1 or <i>L</i>. <i>major</i> LV39c5 parasite strains (n = 4 donors). After 8 hours, RNA was extracted from infected hDCs for cDNA generation and analyzed for <i>IL12B</i> expression by qRT-PCR. All values were significantly greater than uninfected. (<b>B</b>) Human DCs were infected with <i>L</i>. <i>major</i> FV1 metacyclic promastigotes (metacyclic), procyclic promastigotes (procyclic) or amastigotes (n = 3 donors). After 8 hours, RNA was extracted from infected hDCs for cDNA generation and analyzed for <i>IL12B</i> expression by qRT-PCR. All values were significantly greater than uninfected, except for amastigote infections. (<b>C</b>) Human DCs were exposed to different concentrations of LPG (0.5, 1, and 10 μg), derived from <i>L</i>. <i>major</i> FV1 metacyclic promastigotes (n = 4 donors). After 8 hours, RNA was extracted from infected hDCs for cDNA generation and analyzed for <i>IL12B</i> expression by qRT-PCR. Fold change was calculated utilizing the ΔΔC_T method and depicted as fold change over uninfected samples. Box plots display the median value (line), the interquartile range (box), and Tukey whiskers encompassing data within 1.5 fold of the interquartile range. *Statistical significance as compared to uninfected control, (p<0.05). All values were significantly greater than uninfected.</p

Kinetic analysis of <i>IL12</i> and <i>IL23</i> expression modulation by <i>L</i>. <i>major</i> LPG and PG null mutants.

<p>Human DCs (n = 3 donors) were infected with <i>L</i>. <i>major</i> FV1 parasites: <i>L</i>. <i>major</i> FV1 (WT), LPG null mutant (FV1 <i>lpg1</i>⁻), LPG add back (FV1 <i>lpg1</i>⁻/+<i>LPG1</i>), PG null mutant (FV1 <i>lpg2</i>⁻), or PG add back (FV1 <i>lpg2</i>⁻/+<i>LPG2</i>). At 2, 4, 8, and 24 hours post infection, <i>IL12B</i> (<b>A</b>), <i>IL12A</i> (B), and <i>IL23A</i> (C) expression was measured by qRT-PCR. Fold changes were calculated using the ΔΔC_T method and are represented as fold change over uninfected samples. Mean values of individual donors ± SD are presented. Statistical significance p<0.05, ANOVA with Bonferroni multiple comparisons test.</p

Significantly enriched pathways for genes regulated similarly to <i>IL12B</i> by LPG.

<p>Significantly enriched pathways for genes regulated similarly to <i>IL12B</i> by LPG.</p

<i>L</i>. <i>major</i> FV1 <i>lpg1</i>⁻ and FV1 <i>lpg2</i>⁻ modulate the <i>IL12B</i> response in hDCs.

<p>Human DCs (n = 9 donors) were infected with <i>L</i>. <i>major</i> FV1 parasites: <i>L</i>. <i>major</i> FV1 (WT), LPG null mutant (FV1 <i>lpg1</i>⁻), LPG add back (FV1 <i>lpg1</i>⁻/+<i>LPG1</i>), PG null mutant (FV1 <i>lpg2</i>⁻), or PG add back (FV1 <i>lpg2</i>⁻/+<i>LPG2</i>). (<b>A</b>) At 8 hours post infection, <i>IL12B</i> expression was measured by qRT-PCR. Fold changes were calculated using the ΔΔC_T method and are represented as fold change over uninfected samples. Box plots display the median value (line), the interquartile range (box), and Tukey whiskers encompassing data within 1.5 fold of the interquartile range. All values were significantly greater than uninfected. Aliquots from the infected hDC samples were prepared by Diff-Quick staining and visualized by light microscopy. (<b>B</b>) The parasite index (#parasites/100 cells) and (<b>C</b>) the percentage of infected cells (%DC infected) is displayed. Mean values of individual donors ± SD are presented. *Statistical significance (p<0.05).</p