Notch regulates Th17 differentiation and controls trafficking of IL-17 and metabolic regulators within Th17 cells in a context-dependent manner

Th17 cells play critical roles in host defense and autoimmunity. Emerging data support a role for Notch signaling in Th17 cell differentiation but whether it is a positive or negative regulator remains unclear. We report here that T cell-specific deletion of Notch receptors enhances Th17 cell differentiation in the gut, with a corresponding increase in IL-17 secretion. An increase in Th17 cell frequency was similarly observed following immunization of T cell specific Notch mutant mice with OVA/CFA. However, in this setting, Th17 cytokine secretion was impaired, and increased intracellular retention of IL-17 was observed. Intracellular IL-17 co-localized with the CD71 iron transporter in the draining lymph node of both control and Notch-deficient Th17 cells. Immunization induced CD71 surface expression in control, but not in Notch-deficient Th17 cells, revealing defective CD71 intracellular transport in absence of Notch signaling. Moreover, Notch receptor deficient Th17 cells had impaired mTORC2 activity. These data reveal a context-dependent impact of Notch on vesicular transport during high metabolic demand suggesting a role for Notch signaling in the bridging of T cell metabolic demands and effector functions. Collectively, our findings indicate a prominent regulatory role for Notch signaling in the fine-tuning of Th17 cell differentiation and effector function

Redundant Notch1 and Notch2 Signaling Is Necessary for IFNγ Secretion by T Helper 1 Cells During Infection with Leishmania major

The protective immune response to intracellular parasites involves in most cases the differentiation of IFNγ-secreting CD4+ T helper (Th) 1 cells. Notch receptors regulate cell differentiation during development but their implication in the polarization of peripheral CD4+ T helper 1 cells is not well understood. Of the four Notch receptors, only Notch1 (N1) and Notch2 (N2) are expressed on activated CD4+ T cells. To investigate the role of Notch in Th1 cell differentiation following parasite infection, mice with T cell-specific gene ablation of N1, N2 or both (N1N2ΔCD4Cre) were infected with the protozoan parasite Leishmania major. N1N2ΔCD4Cre mice, on the C57BL/6 L. major-resistant genetic background, developed unhealing lesions and uncontrolled parasitemia. Susceptibility correlated with impaired secretion of IFNγ by draining lymph node CD4+ T cells and increased secretion of the IL-5 and IL-13 Th2 cytokines. Mice with single inactivation of N1 or N2 in their T cells were resistant to infection and developed a protective Th1 immune response, showing that CD4+ T cell expression of N1 or N2 is redundant in driving Th1 differentiation. Furthermore, we show that Notch signaling is required for the secretion of IFNγ by Th1 cells. This effect is independent of CSL/RBP-Jκ, the major effector of Notch receptors, since L. major-infected mice with a RBP-Jκ deletion in their T cells were able to develop IFNγ-secreting Th1 cells, kill parasites and heal their lesions. Collectively, we demonstrate here a crucial role for RBP-Jκ-independent Notch signaling in the differentiation of a functional Th1 immune response following L. major infection

Çédille, revista de estudios franceses

Presentació

Rapid Sequestration of Leishmania mexicana by Neutrophils Contributes to the Development of Chronic Lesion.

The protozoan Leishmania mexicana parasite causes chronic non-healing cutaneous lesions in humans and mice with poor parasite control. The mechanisms preventing the development of a protective immune response against this parasite are unclear. Here we provide data demonstrating that parasite sequestration by neutrophils is responsible for disease progression in mice. Within hours of infection L. mexicana induced the local recruitment of neutrophils, which ingested parasites and formed extracellular traps without markedly impairing parasite survival. We further showed that the L. mexicana-induced recruitment of neutrophils impaired the early recruitment of dendritic cells at the site of infection as observed by intravital 2-photon microscopy and flow cytometry analysis. Indeed, infection of neutropenic Genista mice and of mice depleted of neutrophils at the onset of infection demonstrated a prominent role for neutrophils in this process. Furthermore, an increase in monocyte-derived dendritic cells was also observed in draining lymph nodes of neutropenic mice, correlating with subsequent increased frequency of IFNγ-secreting T helper cells, and better parasite control leading ultimately to complete healing of the lesion. Altogether, these findings show that L. mexicana exploits neutrophils to block the induction of a protective immune response and impairs the control of lesion development. Our data thus demonstrate an unanticipated negative role for these innate immune cells in host defense, suggesting that in certain forms of cutaneous leishmaniasis, regulating neutrophil recruitment could be a strategy to promote lesion healing

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

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

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 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

Treatment with anti-IL-13 does not restore resistance of N1N2^ΔCD4Cre mice to <i>L. major</i>.

<p>(A) N1N2^ΔCD4Cre and control N1N2<i>^lox/lox</i> mice were infected s.c. with 3×10⁶<i>L. major</i> promastigotes. At day 21 of infection, N1N2^ΔCD4Cre mice were treated i.p. with either anti-IL-13 mAb or PBS as control. Treatment was repeated once a week until the end of the experiment, and lesion development was monitored. Group means of lesion size ± SEM (n≥3 mice per group) are represented. The parasite load at the site of infection was assessed by LDA 47 days post infection. Group means of parasite number are given ± SEM (n≥3 mice per group). (B) CD4⁺ T cells were isolated by MACS from dLN of <i>L. major</i>-infected mice 47 days post infection and restimulated with or without UV-treated <i>L. major</i> in presence of irradiated syngenic splenocytes. IFNγ level was measured in supernatant after 72 h of stimulation. Data are expressed as the group mean ± SEM of cytokine measurement of n≥3 draining lymph nodes (C) Ym1 and Fizz1 mRNA expression was analyzed in dLN cells by quantitative real-time PCR and normalized to HPRT mRNA expression. Results are represented as fold-increase in mRNA levels relative to levels measured in control mice ± SEM (n≥3 mice per group). Data are representative of three independent experiments. Similar results were obtained when anti-IL-13 was administrated 7 days post infection (data not shown). * p-value<0.05 versus control mice.</p