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 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

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

Cathepsin B in Antigen-Presenting Cells Controls Mediators of the Th1 Immune Response during <i>Leishmania major</i> Infection

<div><p>Resistance and susceptibility to <i>Leishmania major</i> infection in the murine model is determined by the capacity of the host to mount either a protective Th1 response or a Th2 response associated with disease progression. Previous reports involving the use of cysteine cathepsin inhibitors indicated that cathepsins B (Ctsb) and L (Ctsl) play important roles in Th1/Th2 polarization during <i>L. major</i> infection in both susceptible and resistant mouse strains. Although it was hypothesized that these effects are a consequence of differential patterns of antigen processing, the mechanisms underlying these differences were not further investigated. Given the pivotal roles that dendritic cells and macrophages play during <i>Leishmania</i> infection, we generated bone-marrow derived dendritic cells (BMDC) and macrophages (BMM) from <i>Ctsb</i>^−/− and <i>Ctsl</i>^−/− mice, and studied the effects of Ctsb and Ctsl deficiency on the survival of <i>L. major</i> in infected cells. Furthermore, the signals used by dendritic cells to instruct Th cell polarization were addressed: the expression of MHC class II and co-stimulatory molecules, and cytokine production. We found that <i>Ctsb</i>^−/− BMDC express higher levels of MHC class II molecules than wild-type (WT) and <i>Ctsl</i>^−/− BMDC, while there were no significant differences in the expression of co-stimulatory molecules between cathepsin-deficient and WT cells. Moreover, both BMDC and BMM from <i>Ctsb</i>^−/− mice significantly up-regulated the levels of interleukin 12 (IL-12) expression, a key Th1-inducing cytokine. These findings indicate that <i>Ctsb</i>^−/− BMDC display more pro-Th1 properties than their WT and <i>Ctsl</i>^−/− counterparts, and therefore suggest that Ctsb down-regulates the Th1 response to <i>L. major</i>. Moreover, they propose a novel role for Ctsb as a regulator of cytokine expression.</p></div

Comparison of <i>L. major</i> promastigote uptake and processing by BMDC from WT and cathepsin-deficient mice.

<p>(A) Representative histograms for WT BMDC (CD11c⁺-gated) infected for 2 hours with eGFP-tg <i>L. major</i> promastigotes, and further incubated for 4 and 24 hours in fresh medium. The percentage of eGFP⁺ cells was considered as percentage of remaining infected cells. (B) No significant differences between BMDC from WT and cathepsin-deficient mice were found in the uptake and processing of eGFP-tg promastigotes over the course of 24 hours. The results are expressed as mean ± SD of 3 independent experiments.</p

BMDC from cathepsin B-deficient mice express higher levels of MHC class II molecules in comparison with BMDC from cathepsin L-deficient and WT mice, but no differences were observed in the expression of co-stimulatory molecules.

<p>The expression of MHC class II molecules, CD40, CD86 and CD80 was measured by flow cytometry in BMDC in response to infection with <i>L. major</i> promastigotes (Inf), LmAg, heat-killed parasites (HK), or LPS. (A) Average MFI of MHC class II molecules, normalized to non-treated (NT) WT BMDC. (B) Average MFI of CD40, normalized WT NT BMDC. (C) Average MFI of CD80 and CD86, normalized WT NT BMDC. MFI values are expressed as mean ± SD of 4 independent experiments. Statistical significance was assessed between WT BMDC and Ctsb^−/− BMDC, and between WT BMDC and Ctsl^−/− BMDC for every single treatment, * p<0.05, *** p<0.005.</p

Characterization of the early inflammation in <i>L. major</i>-infected T cell-specific IL-10-deficient BALB/c mice.

<p>Enhanced inflammation in T cell-specific IL-10-deficient BALB/c mice 2 weeks after infection is associated with a mixed Th1/Th2 immune response. T cell-specific IL-10 mutant and IL-10-competent BALB/c mice were infected with <i>L. major</i> promastigotes into the right hind footpad. Increase in size of the infected footpad 2 weeks after infection (<b>A</b>). Frequency of <i>L. major</i>-infected cells in the draining lymph nodes (<b>B</b>). Cytokine secretion upon restimulation of lymph node cells with <i>Leishmania</i> antigen <i>in vitro</i> (<b>C, D and E</b>). Each symbol represents an individual mouse (<b>A and B</b>); the results are representative of three independent experiments. Data show the mean ± SEM of three independent experiments with 5–6 mice per group and experiment (<b>C, D and E</b>). ** <i>p</i><0.01.</p

BMDC and BMM from cathepsin B-deficient mice express higher levels of IL-12 in response to <i>L. major</i> than cells derived from WT and cathepsin L-deficient mice.

<p>(A) IL-12p70 in supernatants from non-treated BMDC(NT), BMDC infected (Inf) with <i>L. major</i> promastigotes at 48 hours p.i., BMDC stimulated with parasite lysate (LmAg), or with heat-killed parasites (HK), for 48 hours. (B) IL-12p40 and (C) IL-10 concentration in supernatants of BMDC at 48 hours p.i., or stimulation with LmAg or HK parasites. (D) IL-12p70 production in supernatants from non-treated BMM (NT), BMM infected (Inf) with <i>L. major</i> promastigotes at 48 hours p.i., and BMM stimulated for 48 hours with LmAg or HK parasites. (E) IL-12p40 and (F) IL-10 concentration in supernatants of BMM at 48 hours p.i., or stimulation with either LmAg or HK parasites. The results are expressed as mean ± SD of 5 independent experiments. For each experimental group (NT, Inf, LmAg and HK), statistical significance was estimated between WT and Ctsb^−/− cells, and between WT and Ctsl^−/− cells, * p<0.05, **p<0.01, *** p<0.005.</p

IL-12 is up-regulated at the transcriptional level in BMM from cathepsin B-deficient mice in response to <i>L. major</i> infection and LPS stimulation.

<p>Relative expression levels of (A) IL-12p40 and (B) IL-12p35 transcripts in mRNA from BMM at 6 hours or 24 hours p.i. with <i>L. major</i> promastigotes or LPS stimulation. Non-treated (NT) BMM from each mouse line were used as negative controls. The expression levels were estimated using the 2^{− ΔΔC}_T method, using WT NT BMM at t = 6 hours as a reference. The results are shown as mean ± SD of 4 independent experiments, * p<0.05.</p

<i>L. major</i> promastigotes survive comparably in BMM from cathepsin B- and cathepsin L-deficient mice, and these BMM show similar levels of NO production in response to <i>L. major</i> and LPS.

<p>WT, Ctsb^−/− and Ctsl^−/− BMM were infected with eGFP-tg <i>L. major</i> promastigotes, and the percentage of infected cells 24 hours post infection (p.i.) and 48 hours p.i. was determined by fluorescence microscopy (A). No statistically significant differences were found in the amounts of infected cells between WT and cathepsin-deficient BMM. (B) As in (A) the number of parasites per infected cells was determined by fluorescence microscopy, and used as an indicator for parasite proliferation. Although no significant differences were found between WT and cathepsin-deficient BMM, each line showed significant differences in the counts of parasites per infected cell between 24 hours and 48 hours p.i. (C) Parasite proliferation determined by luminescence of Luc-tg <i>L. major</i> promastigotes within BMM at 48 hours p.i. Data are shown as counts per second (CPS). (D) NO production in supernatants from BMM 48 hours after infection with <i>L. major</i> and stimulation with LPS. Results are expressed as mean ± SD from 3 independent experiments.</p