Blocking JAM-C improves the Th1 cell response and favours healing in C57BL/6 mice.

<p>(<b>A–E</b>) <b>Mice were inoculated with L. major in the ear dermis and treated with H33 or isotype control antibody for 3 weeks, twice a week.</b> (A) The area of the lesion was monitored weekly and representative pictures of ear lesions are shown at 4 and 8 weeks p.i. Scale bars, 0.5 mm. Data represent the mean ± SEM of twenty mice per group pooled from two separate experiments for the time point 4 weeks; and fifteen mice per group pooled from two separate experiments for the time point 8 weeks. (B) The parasite burden in infected ears was measured by LDA 4 and 8 weeks p.i. Data are expressed as a percentage of the mean of the control group ± SEM of mice from panel A. (C–D) The number of draining lymph node CD4⁺ (C) and CD8⁺ (D) T cells analyzed by flow cytometry 4 and 8 weeks p.i. Data represent the mean ± SEM of mice from panel A. (E) Draining lymph node cells were restimulated for 72 hrs with UV-irradiated <i>L. major</i> and the secreted IFN-γ was measured. Data are expressed as a percentage of the mean of the control group ± SEM of mice from panel A. Data were analyzed by the unpaired Student's t test with *:p<0.05 and **: p<0.01. For panels B and E, raw data from one experiment are also provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004550#ppat.1004550.s008" target="_blank">S8 Figure</a>.</p

Blocking JAM-C boosts the Th2 cell response and worsens the disease in BALB/c mice.

<p>The number of emigrating neutrophils (A), mo-DCs (B), dermal DCs (C) and dermal mφ (D) was measured in the ears of H33-treated (H33, black bar) versus isotype control-treated mice (Ctr, white bars) 24 hours post <i>L. major</i> infection. Data represent the mean ± SEM of twelve mice per group pooled from 2 separate experiments, and were analyzed by the unpaired Student's t test with *: p<0.05. (E) Mice were inoculated with 2×10⁶ stationary phase <i>L. major</i> promastigotes in the ear dermis and treated with H33 or control antibody for 3 weeks. The area of the lesion was monitored weekly for 6 weeks. Representative ear pictures are shown. Scale bars, 1 mm. Data represent the mean ± SEM of twenty mice per group pooled from two separate experiments. (F–L) Mice were inoculated with 1×10⁴ stationary phase <i>L. major</i> promastigotes in the ear dermis and treated with H33 or control antibody for 3 weeks. The area of the lesion was monitored weekly for 4 weeks. Representative ear pictures are shown. Scale bars, 0.5 mm. Data represent the mean ± SEM of ten mice per group pooled from two separate experiments. (G–H) The parasite burden in infected ears (G) and draining lymph nodes (H) were measured by LDA. Data are expressed as a percentage of the mean of the control group ± SEM of mice from panel F. (I–J) The number of CD4⁺ (I) and CD8⁺ (J) T cells were measured. Data represent the mean ± SEM of mice from panel F. (K–L) Draining lymph nodes cells were restimulated with UV-irradiated <i>L. major</i> for 72 hours, and the IL-4 (K) and IFN-γ (L) produced were measured. Data are expressed as a percentage of the mean of the control group ± SEM of mice from panel F. Data were analyzed by the unpaired Student's t test with *:p<0.05 and **: p<0.01. For panels expressing results as a percentage of the mean of the control, raw data of one experiment are provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004550#ppat.1004550.s009" target="_blank">S9 Figure</a>.</p

The antibody H33 mimics JAM-C downregulation after L. major inoculation, and locally increases vascular permeability after infection.

<p>(A) JAM-C levels in endothelial cells populations of mouse ear. Ears were enzymatically digested and stained for FACS analysis. CD45⁻ CD31⁺ gp38⁻ cells represent blood endothelial cells (BECs), whereas CD45⁻ CD31⁺ gp38⁺ cells are lymphatic endothelial cells (LECs). For each population, a representative histogram overlay is shown with JAM-C in endothelial cells from naïve ears (black filled), JAM-C in endothelials cells from <i>L. major</i> infected ears (blank filled), and the isotype control staining (grey filled). (B) The median fluorescence intensity (MFI) of JAM-C in naïve mouse ears (white bars) versus <i>L. major</i> infected mouse ears (black bars) was measured in BECs and LECs. The Y-axis scale represents MFI normalized to the mean MFI of naïve ears. Data represent the mean ± SEM of ten individual mice pooled from two separate experiments, and were analyzed by the unpaired Student's t test with ***: p<0.001. (C) Mice were treated with H33 or control antibody 2 hours before Evans blue was injected i.v. and <i>L. major</i> inoculated i.d. in the ear dermis. Skin permeability was assessed by the absorbance of Evans blue extracted from the sample normalized to the weight of ear. Results are shown for naïve versus <i>L. major</i> infected animals treated with H33 (black bars) or control antibody (blank bars). Representative ear pictures are shown. Data represent the mean ± SEM of seventeen mice per group pooled from two separate experiments, and were analyzed by the unpaired Student's t test with ***: p<0.001. (D) Ear sections from control antibody-treated (top panel) or H33-treated mice (bottom panel) were stained for JAM-C (green) and CD31 (red). Nucleus was stained with DAPI (blue). Scale bars, 10 µm. Control staining for JAM-C is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004550#ppat.1004550.s003" target="_blank">Figure S3</a>. (E) The pixel intensity across 10 representative cells of similar size taken from three mice per group was measured and expressed as a percentage of the maximal pixel intensity. Data represent the average profile plot for the 10 cells per group analyzed.</p

Blocking JAM-C increases the number of DCs migrating to the draining lymph node.

<p>(A) The ear draining lymph node cells were harvested and stained for FACS analysis 18 hours after FITC-painting. Representative FACS dot plots are shown. (B) The number of IA^high CD11c⁺ FITC⁺ migratory DCs was counted. Data are expressed as a percentage of the mean of the control group ± SEM of eighteen mice per group pooled from 3 separate experiments, and were analyzed by the unpaired Student's t test with ***: p<0.001. Raw data from one representative experiment are provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004550#ppat.1004550.s007" target="_blank">S7 Figure</a>.</p

Blocking JAM-C enhances DC migration and boosts the immune responses to <i>L. major</i> infection.

<p>By removing JAM-C out of functions, H33 increases adhesion of leukocytes and potentiates vascular permeability and cell migration of leukocytes after <i>L. major</i> infection. Increased numbers of recruited neutrophils result in higher levels of the chemokine CCL3 attracting monocytes and mo-DCs in C57BL/6 mice. The number of migratory DCs to lymph nodes increases, and the subsequent T cell response is mounted more efficiently. Resistant C57BL/6 mice develop a higher IFN-γ-dominated Th1 response while susceptible BALB/c mice develop a stronger IL-4-dominated Th2 response. This has a significant healing effect in resistant animals whereas susceptible mice display an exacerbated disease.</p

Blocking JAM-C increases the number of leukocytes recruited to the site of <i>L. major</i> infection.

<p>(A) Representative dot plots of neutrophils (CD11b⁺ Ly6C⁺ Ly6G⁺); monocytes (CD11b⁺ Ly6C⁺ Ly6G⁻ CD11c⁻ IA⁻); mo-DCs (CD11b⁺ Ly6C⁺ Ly6G⁻ CD11c⁺ IA⁺); dermal mφ (CD11b⁺ Ly6C⁻ Ly6G⁻ CD11c^low IA⁺); dermal DCs (CD11b⁺ Ly6C⁻ Ly6G⁻ CD11c^high IA⁺) in control versus H33-treated animals. (B–F) The number of neutrophils (B), monocytes (C), mo-DCs (D), dermal mφ (E) and dermal DCs (F) was measured in the H33-treated (H33, black bar) versus isotype control-treated mice (Ctr, white bars) 24 hours p.i. Data represent the mean ± SEM of twenty mice per group pooled from 3 separate experiments, and were analyzed by the unpaired Student's t test with *: p<0.05 and **: p<0.01. (G) CCL3 protein levels normalized to the weight of ears were measured in H33-treated (H33, black bar) versus isotype control-treated mice (Ctr, white bars) 8 and 24 hours p.i. Data represent the mean ± SEM of ten mice per group pooled from 2 separate experiments, and were analyzed by the unpaired Student's t test with **: p<0.01. (H–I) The parasite burden in infected ears (H) and draining lymph nodes (LN) (I) were measured 48 hours p.i. by limiting dilution assay (LDA). Data are expressed as a percentage of the mean of the control group ± SEM of ten mice per group pooled from 2 separate experiments, and were analyzed by the unpaired Student's t test. For panel H and I, raw data of one representative experiment are provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004550#ppat.1004550.s006" target="_blank">S6 Figure</a>.</p

N1 and N2 alone can drive CD4⁺ Th1 differentiation.

<p>(A) N1^ΔCD4Cre, N2^ΔCD4Cre, N1N2^ΔCD4Cre, and control mice were infected with 3×10⁶<i>L. major</i> promastigotes and lesion size measured weekly. Data are represented as the mean of lesion size ± SEM with n≥3 mice per group. (B) Parasite load in the lesion was assessed by LDA 6 weeks after infection. Mean parasite number is given ± SEM (n≥3 mice per group) (C, D) Six weeks after infection, IFNγ (C), IL-4 and IL-13 (D) secretion was assessed in supernatant of dLN cells restimulated or not with UV-irradiated <i>L. major</i> for 72 h. Histograms show the mean cytokine secretion ± SEM (n≥3 mice per group). n.d. not-detectable, n.s. not significant. * p-value<0.05 versus control mice.</p

The impact of Notch signaling on Th1 differentiation is RBP-Jκ-independent.

<p>(A) RBP-Jκ^ΔCD4Cre and RBP-Jκ<i>^lox/lox</i> mice were infected s.c. with 3×10⁶<i>L. major</i> and lesion size measured weekly. Group mean of lesion size ± SEM for n≥3 mice per group is shown. (B) Parasite load in footpad was analyzed by LDA 5 weeks post infection. Data represent mean parasite number ± SEM for n≥3 mice per group. (C, D) IFNγ (C), IL-4, IL-13 and IL-5 (D) levels were measured in supernatant of dLN cells isolated from <i>L. major</i>-infected mice 5 weeks post infection and restimulated for 72 h with or without UV-treated <i>L. major</i>. Group mean of cytokine secretion ± SEM is given (n≥3 mice per group). n.s. not significant. * p-value<0.05 versus control mice.</p

N1N2^ΔCD4Cre mice transcribe T-bet and IFNγ in dLN CD4⁺ T cells but do not secrete it.

<p>(A) Proliferation of CD4⁺ T cells was assessed by FACS. Draining LN cells of <i>L. major</i>-infected mice were isolated 6 weeks after infection, stained with CFSE and restimulated with UV-treated <i>L. major</i> for 72 h. Representative flow cytometry plots gated on CD4⁺ T cells are shown. Numbers in plots represent the mean percentage of proliferating cells ± SEM for 5 mice. (B) Intracellular levels of IFNγ were analysed by FACS in <i>L. major</i>-infected dLN cells restimulated for 72 h with UV-treated <i>L. major</i>. Representative flow cytometry plots are given. Numbers in plots represent the mean percentage of IFNγ⁺ cells within CD4⁺ T cells ± SEM for 5 mice. (C) Draining LN CD4⁺ T cells from N1N2^ΔCD4Cre and N1N2<i>^lox/lox</i> mice were sorted by FACS 21 days post <i>L. major</i> infection, T-bet and IFNγ mRNA levels were analyzed by quantitative RT-PCR. Data are represented as the mean ± SEM mRNA transcript levels normalized to HPRT mRNA levels (n≥3 mice per group). (D) Phosphorylation of STAT1 was assessed by FACS on dLN cells of N1N2^ΔCD4Cre and N1N2<i>^lox/lox</i> mice 3 weeks post infection. Naive mice were used as control. Representative flow cytometry plots gated on CD4⁺ T cells are shown. Numbers in quadrants indicate the mean frequency of pSTAT1⁺CD4⁺ T cells ± SEM. pSTAT1 mean fluorescence intensity MFI ± SEM is shown (n≥3 mice per group). (E) Draining LN cells of <i>L. major</i>-infected mice were restimulated <i>ex vivo</i> with PMA/ionomcyin for 4 h and level of intracellular IFNγ was assessed by FACS. The frequency of CD4⁺IFNγ⁺ T cells is given ± SEM for n≥3 mice per group. (F) mRNA expression of IL-13 and IL-5 was analyzed by quantitative real-time PCR in dLN cells isolated from N1N2^ΔCD4Cre and N1N2<i>^lox/lox</i> mice 6 weeks post <i>L. major</i> infection. Results are given as mean mRNA expression relative to HPRT ± SEM for n≥3 mice per group. Data are representative of 2–3 individual experiments. * p-value<0.05 versus control mice.</p

N1N2^ΔCD4Cre mice on the C57BL/6 <i>L. major</i> resistant background are susceptible to infection.

<p>(A) N1N2^ΔCD4Cre and control N1N2<i>^lox/lox</i> mice were infected with 3×10⁶<i>L. major</i> promastigotes and lesion size measured weekly. Dots represent group mean of lesion size ± SEM. (B, C) Six weeks after infection, parasite load was assessed by LDA in footpads (B), dLN (C) and spleen (D). Histograms represent the mean number of parasite ± SEM (n≥3 mice per group). (E–G) IFNγ (E), IL-4 (F), IL-13 and IL-5 (G) secretion was quantified in supernatants of draining lymph node cells restimulated or not with UV-irradiated <i>L. major</i> 6 weeks after infection. Mean cytokine secretion ± SEM are given (n≥3 mice per group). Data are representative of at least 3 individual experiments. n.d. not-detectable. * p-value<0.05 versus control mice.</p