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

Regulatory B cells shape the development of Th2 immune responses in BALB/c mice infected with Leishmania major through IL-10 production.

Recent evidence indicates that B cells are required for susceptibility to infection with Leishmania major in BALB/c mice. In this study, we analyzed the role of the IL-10 produced by B cells in this process. We showed that B cells purified from the spleen of BALB/c mice produced IL-10 in response to stimulation with L. major in vitro. In vivo, early IL-10 mRNA expression is detected after L. major infection in B cells from draining lymph nodes of susceptible BALB/c, but not of resistant C57BL/6 mice. Although adoptive transfer of naive wild-type B cells prior to infection in B cell-deficient BALB/c mice restored Th2 cell development and susceptibility to infection with L. major of these otherwise resistant mice, adoptive transfer of IL-10(-/-) B cells mice did not. B cells stimulated by L. major, following in vitro or in vivo encounter, express the CD1d and CD5 molecules and the IL-10 produced by these cells downregulate IL-12 production by L. major-stimulated dendritic cells. These observations indicate that IL-10 secreting B cells are phenotypically and functionally regulatory B cells. Altogether these results demonstrate that the IL-10 produced by regulatory CD1d+ CD5+ B cells in response to L. major is critical for Th2 cell development in BALB/c mice

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

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

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

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

MyD88 and TLR9 are involved in IL-12 production following infection and their absence increases susceptibility to infection.

<p>At indicated time points RNA from draining popliteal lymph node cells of wild-type, TLR9^−/− and Myd88^−/− mice infected by <i>L.g.</i> LRV^high (Fig3A left panel) or <i>L.g.</i> LRV^low (Fig3A right panel) were reverse-transcribed into cDNA and relative transcript levels were determined using gene specific primers by quantitative real time PCR. Results are expressed as mean± SEM of the individual mice analyzed per group with the average value for the C57BL/6 mice given a value of 1. A.U refers to Arbitrary Units of fold change. (B) Mice (n≥4) were infected in the hind footpads and footpad swelling was measured weekly over 8 weeks using a Vernier caliper. (D) At 8 weeks post-infection mice were sacrificed and footpad parasite burden was determined using parasite specific Kmp11 primers. Results are expressed as the mean ± SEM. Significance determined at *p≤0.05, **p≤0.01, ***p≤0.005.</p