The role of CD4+T cells in host protective responses against cutaneous Leishmaniasis using genome-wide transcriptomics.

Includes abstract.Includes bibliographical references.Leishmania major is a protozoan parasite and infection in the human host causes severe cutaneous Leishmaniasis. The study aims to determine how signaling via the IL-4Rα on CD4+T cells causes susceptibility to L. major. We compared gene expression patterns early during infection in CD4+ T cells in the absence or presence of IL-4Rα signaling. Non-healer BALB/c mice with a deletion of the IL-4Rα on all cells (IL-4Rα-/-) or CD4+ T cells only (iLCKcreIL-4Rαlox/-) and their controls (wild-type (WT) C57BL/6, WT BALB/c and littermate IL-4Rαlox/-) were subcutaneously infected with L. major. As expected, the C57BL/6 “healer” mice produced a predominant TH1 response, whereas the iLCKcreIL-4Rα-/lox mice and susceptible BALB/c mice produced a TH2 response

Deletion of IL-4 receptor alpha on dendritic cells renders BALB/c mice hypersusceptible to Leishmania major infection

In BALB/c mice, susceptibility to infection with the intracellular parasite Leishmania major is driven largely by the development of T helper 2 (Th2) responses and the production of interleukin (IL)-4 and IL-13, which share a common receptor subunit, the IL-4 receptor alpha chain (IL-4Rα). While IL-4 is the main inducer of Th2 responses, paradoxically, it has been shown that exogenously administered IL-4 can promote dendritic cell (DC) IL-12 production and enhance Th1 development if given early during infection. To further investigate the relevance of biological quantities of IL-4 acting on DCs during in vivo infection, DC specific IL-4Rα deficient (CD11ccreIL-4Rα-/lox) BALB/c mice were generated by gene targeting and site-specific recombination using the cre/loxP system under control of the cd11c locus. DNA, protein, and functional characterization showed abrogated IL-4Rα expression on dendritic cells and alveolar macrophages in CD11ccreIL-4Rα-/lox mice. Following infection with L. major, CD11ccreIL-4Rα-/lox mice became hypersusceptible to disease, presenting earlier and increased footpad swelling, necrosis and parasite burdens, upregulated Th2 cytokine responses and increased type 2 antibody production as well as impaired classical activation of macrophages. Hypersusceptibility in CD11ccreIL-4Rα-/lox mice was accompanied by a striking increase in parasite burdens in peripheral organs such as the spleen, liver, and even the brain. DCs showed increased parasite loads in CD11ccreIL-4Rα-/lox mice and reduced iNOS production. IL-4Rα-deficient DCs produced reduced IL-12 but increased IL-10 due to impaired DC instruction, with increased mRNA expression of IL-23p19 and activin A, cytokines previously implicated in promoting Th2 responses. Together, these data demonstrate that abrogation of IL-4Rα signaling on DCs is severely detrimental to the host, leading to rapid disease progression, and increased survival of parasites in infected DCs due to reduced killing effector functions

Deletion of IL-4 Receptor Alpha on Dendritic Cells Renders BALB/c Mice Hypersusceptible to Leishmania major Infection

In BALB/c mice, susceptibility to infection with the intracellular parasite Leishmania major is driven largely by the development of T helper 2 (Th2) responses and the production of interleukin (IL)-4 and IL-13, which share a common receptor subunit, the IL-4 receptor alpha chain (IL-4Rα). While IL-4 is the main inducer of Th2 responses, paradoxically, it has been shown that exogenously administered IL-4 can promote dendritic cell (DC) IL-12 production and enhance Th1 development if given early during infection. To further investigate the relevance of biological quantities of IL-4 acting on DCs during in vivo infection, DC specific IL-4Rα deficient (CD11ccreIL-4Rα-/lox) BALB/c mice were generated by gene targeting and site-specific recombination using the cre/loxP system under control of the cd11c locus. DNA, protein, and functional characterization showed abrogated IL-4Rα expression on dendritic cells and alveolar macrophages in CD11ccreIL-4Rα-/lox mice. Following infection with L. major, CD11ccreIL-4Rα-/lox mice became hypersusceptible to disease, presenting earlier and increased footpad swelling, necrosis and parasite burdens, upregulated Th2 cytokine responses and increased type 2 antibody production as well as impaired classical activation of macrophages. Hypersusceptibility in CD11ccreIL-4Rα-/lox mice was accompanied by a striking increase in parasite burdens in peripheral organs such as the spleen, liver, and even the brain. DCs showed increased parasite loads in CD11ccreIL-4Rα-/lox mice and reduced iNOS production. IL-4Rα-deficient DCs produced reduced IL-12 but increased IL-10 due to impaired DC instruction, with increased mRNA expression of IL-23p19 and activin A, cytokines previously implicated in promoting Th2 responses. Together, these data demonstrate that abrogation of IL-4Rα signaling on DCs is severely detrimental to the host, leading to rapid disease progression, and increased survival of parasites in infected DCs due to reduced killing effector functions

Cell populations infected with <i>L. major</i> parasites.

<p>CD11c^creIL-4Rα^-/lox and littermate mice were infected subcutaneously with 2×10⁶ stationary phase metacyclic GFP-expressing <i>L. major</i> IL81 promastigotes into the hind footpad. (A–B) Total cells from footpads were analysed for different cell populations at day 3 (A) and week 4 (B) after infection. (C–F) Number of GFP⁺<i>L. major</i> parasites was identified within indicated cell populations derived from footpad (C, E) and lymph node (D, F) at day 3 and week 4 after infection, respectively. Cell populations were differentiated based on the following markers; conventional dendritic cells (cDCs; CD11c^highMHCII^high), plasmacytoid DCs (pDCs; CD11c⁺PDCA⁺SiglecH⁺), macrophages (Mph; CD11b^highMHCII^highCD11c⁻), Eosinophils (Eos; SiglecF⁺CD11c⁻), Neutrophils (Neut; GR-1^highSSC^highFSC^high CD11c⁻). Data is expressed as mean ± SEM. Statistical analysis was performed defining differences to IL-4Rα^-/lox mice (*, <i>p</i>≤0.05, **, <i>p</i>≤0.01, ***, <i>p</i>≤0.001). FP = Footpad and LN = Lymph node.</p

Generation and characterization of CD11c^creIL-4Rα^-/lox BALB/c mice.

<p>(A) IL-4Rα^-/- BALB/c mice were intercrossed with CD11c^cre expressing and IL-4Rα^lox/lox mice to generate CD11c^creIL-4Rα^-/lox BALB/c mice. (B) Genotyping of CD11c^creIL-4Rα^-/lox mice. The deleted IL-4Rα PCR is 471 base pairs, loxP is 450 base pairs (floxed) or 356 base pairs (wildtype) and CD11c^cre specific is 517 base pairs. (C) IL-4Rα surface expression was analyzed by flow cytometry from naïve IL-4Rα^-/lox (solid line), IL-4Rα^-/- (dashed line) and CD11c^creIL-4Rα^-/lox (grey tinted) mice. DCs were CD11c⁺MHCII⁺ (SiglecF⁻ in lungs), alveolar macrophages were CD11c⁺SiglecF⁺, peritoneal macrophages were F480⁺CD11b⁺, B cells were CD19⁺CD3⁻ and T cells were CD3⁺CD19⁻. GM = geometric mean. (D) Genomic DNA was extracted from spleen DCs and IL-4Rα exon 8 (deleted in IL-4Rα deficient cells) was determined by RT-PCR and normalized to exon 5 (present in all cells). (E) Bone marrow-derived DCs were stimulated with LPS in the presence of absence of IL-4 or IL-13 and IL-12p40 was measured in the supernatants 48 hours later. (*, p<0.05, **, <i>p</i>≤0.01.</p

Abrogation of IL-4Rα expression in dendritic cells impairs dendritic cell instruction and alters DC phenotype <i>in vivo</i>.

<p>Mice were infected subcutaneously with 2×10⁶ stationary phase metacyclic GFP-<i>L. major</i> IL81 promastigotes into the hind footpad. (A) After 4 weeks of infection, total lymph node cells were restimulated with SLA and production of IL-12p40 and IL-10 was determined by ELISA. (B) Intracellular staining of IL-12p40 and IL-10 in lymph node dendritic cells following incubation with PMA/Ionomycin/Monensin for 4 h at 37°C. Percent cytokine producing cells are shown. mRNA expression of IL-12p40 (C), IL-18 (D), IL-10 (E), IL-23p19 (F) and Activin A (G) was determined by real-time RT-PCR from sorted LN dendritic cells. Expression was normalised against the housekeeping gene <i>HPRT</i>. (H) IL-12p70 production from BMDCs infected with <i>L. major</i> in the presence or absence of rIL-4 or rIL-13. Culture supernatants were collected after 48 hours to determine IL-12p70 levels by ELISA. Data is expressed as mean ±SEM. Statistical analysis was performed defining differences to IL-4Rα^-/lox mice (*, <i>p</i>≤0.05, **, <i>p</i>≤0.01, ***, <i>p</i>≤0.001).</p

Impairment of IL-4Rα signaling <i>in vivo</i> results in increased <i>L. major</i> parasite loads in peripheral organs.

<p>CD11c^creIL-4Rα^-/lox and littermate mice were infected subcutaneously with 2×10⁶ stationary phase metacyclic <i>L. major</i> (<i>L. m</i> LV39) promastigotes into the hind footpad. Parasite load was determined by limiting dilution assay (LDA) of single-cell suspensions from homogenized footpad, lymph node, spleen, liver and brain at week 3 (A) and week 8 (B) after infection. Similarly, organs were harvested from mice infected with GFP-expressing <i>L. major</i> (<i>L. m</i> IL81) at week 4 after infection for limiting dilution assay (C). At the same time point, histopathology was analysed using formalin-fixed spleen and liver (D) stained with H&E and Giemsa, respectively (original magnification ×100; asterisks indicate inflammatory foci and insets, arrows indicate amastigote parasites ×800). Frozen brain sections (E) were stained with Hoechst nuclear stain (blue) and visualized by confocal microscopy for the presence of GFP-<i>L. major</i> amastigote parasites (original magnification ×400). A representative of two individual experiments is shown with mean values ±SEM. Statistical analysis was performed defining differences to IL-4Rα^-/lox mice (*, <i>p</i>≤0.05, **, <i>p</i>≤0.01, ***, <i>p</i>≤0.001).</p

Infected DCs in CD11c^creIL-4Rα^-/lox mice have reduced iNOS production.

<p>CD11c^creIL-4Rα^-/lox and littermate mice were infected subcutaneously with 2×10⁶ stationary phase metacyclic GFP-expressing <i>L. major</i> IL81 promastigotes into the hind footpad. (A) At week 4 after infection, spleens were harvested to analyse number of GFP⁺<i>L. major</i> parasites within the indicated cell populations. Cell populations were differentiated based on the following markers; conventional dendritic cells (cDCs; CD11c^highMHCII^high), plasmacytoid DCs (pDCs; CD11c⁺PDCA⁺SiglecH⁺), macrophages (Mph; CD11b^highMHCII^highCD11c⁻), Eosinophils (Eos; SiglecF⁺CD11c⁻), Neutrophils (Neut; GR-1^highSSC^highFSC^high CD11c⁻). (B) Percentage of DCs producing iNOS. Total lymph node cells were surface-stained for CD11c^highMHCII^high DCs followed by intracellular staining for percent iNOS-producing DCs. (C) Histogram plots showing intracellular iNOS expression in CD11c^highMHCII^high DCs. Data is expressed as mean ± SEM. Statistical analysis was performed defining differences to IL-4Rα^-/lox mice (*, <i>p</i>≤0.05, **, <i>p</i>≤0.01).</p

T helper 2 immunity is enhanced in hypersusceptible CD11c^creIL-4Rα^-/lox mice in response to acute <i>L. major</i> IL81 infection.

<p>Experimental mice were infected subcutaneously with 2×10⁶ stationary phase metacyclic GFP-expressing <i>L. major</i> IL81 promastigotes into the hind footpad (A–D). At week 4 post infection, total CD4⁺ T cells from the draining lymph node were restimulated for 72 hrs with fixed APCs and soluble <i>Leishmania</i> antigen (SLA). The production of IFN-γ (A), IL-4 (B), IL-13 (C) and IL-10 (D) was determined by ELISA. (E–G) Antigen-specific IgG2a (E), IgG1 (F) and total IgE (G) antibody production was quantified from infected sera by ELISA. (H–I) Expression of iNOS and arginase 1 in total footpad cells. Total cells were isolated from footpads at week 4 after infection, surface-stained for CD11b^highMHCII^highCD11c⁻ macrophages followed by intracellular staining for iNOS (H) and arginase 1 (I). GM = geometric means. (J–K) Production of NO and arginase 1 in total footpad cells. Total cells were isolated from footpads at week 4 after infection and stimulated with 10 ng/ml LPS for 72 h. Production of NO was determined in cell supernatants (J) and cell lysates were assayed for arginase 1 production (K). A representative of two individual experiments is shown with mean values ±SEM. Statistical analysis was performed defining differences to IL-4Rα^-/lox mice as significant (*, <i>p</i>≤0.05, **, <i>p</i>≤0.01; ***, <i>p</i>≤0.001).</p

CD11c^creIL-4Rα^-/lox mice are hypersusceptible to cutaneous <i>L. major</i> infection.

<p>Mice were infected with <i>L. major</i> LV39 (MRHO/SV/59/P) (A to C) or with the more virulent GFP-expressing <i>L. major</i> IL81 (MHOM/IL/81/FEBNI) parasite strain (D to F). Footpad swelling was measured at weekly intervals in mice (5 per group) infected subcutaneously with 2×10⁶ stationary phase metacyclic <i>L. major</i> promastigotes into the hind footpad (A and D). “N” indicates necrosis or ulceration/mouse. Parasite burden was determined by limiting dilution of single-cell suspensions from homogenized footpads at 8 (B) or 4 week (E) after infection as well as from draining lymph nodes at 8 (C) or 4 week (F) after infection. At week 4 after infection (IL81), formalin-fixed footpads (G) were stained with Giemsa for histopathology (original magnification ×40; asterisks indicate inflammatory foci and insets, arrows indicate amastigote parasites ×800). A representative of two individual experiments is shown with mean values ±SEM. Statistical analysis was performed defining differences to IL-4Rα^-/lox mice as significant (*, <i>p</i>≤0.05, **, <i>p</i>≤0.01; ***, <i>p</i>≤0.001).</p