IRF-5 Promotes Cell Death in CD4 T Cells during Chronic Infection

International audienceThe transcription factor interferon regulatory factor 5 (IRF-5) plays an important function in innate immunity and in initiating pro-inflammatory responses against pathogens. IRF-5 is constitutively expressed in several cell types, including plasmacytoid dendritic cells, monocytes, and B cells. We have previously reported that IRF-5 is also expressed in T cells during infection. The role of IRF-5 in T cells is yet unknown. Here, we demonstrate that IRF-5 is increasingly expressed in interferon (IFN)-γ+ CD4 T cells over the course of L. donovani infection. This transcription factor is induced by apoptotic material via Toll-like receptor 7 (TLR7) and promotes the expression of death receptor 5 (DR5). IRF-5 activation sensitizes CD4 T cells to cell death. Because tissue disruption and chronic inflammation are common characteristics of persistent infections, activation of IRF-5 in CD4 T cells may represent a common pathway that leads to suppression of protective CD4 T cell responses, favoring the establishment of chronic infection

HIF-1α is a key regulator in potentiating suppressor activity and limiting the microbicidal capacity of MDSC-like cells during visceral leishmaniasis

<div><p><i>Leishmania donovani</i> is known to induce myelopoiesis and to dramatically increase extramedullary myelopoiesis. This results in splenomegaly, which is then accompanied by disruption of the splenic microarchitecture, a chronic inflammatory environment, and immunosuppression. Chronically inflamed tissues are typically hypoxic. The role of hypoxia on myeloid cell functions during visceral leishmaniasis has not yet been studied. Here we show that <i>L</i>. <i>donovani</i> promotes the output from the bone marrow of monocytes with a regulatory phenotype that function as safe targets for the parasite. We also demonstrate that splenic myeloid cells acquire MDSC-like function in a HIF-1α-dependent manner. HIF-1α is also involved in driving the polarization towards M2-like macrophages and rendering intermediate stage monocytes more susceptible to <i>L</i>. <i>donovani</i> infection. Our results suggest that HIF-1α is a major player in the establishment of chronic <i>Leishmania</i> infection and is crucial for enhancing immunosuppressive functions and lowering leishmanicidal capacity of myeloid cells.</p></div

Myeloid cells, particularly Ly6C^hi and Ly6C^lo/int monocytes, accumulate in the spleen of <i>L</i>. <i>donovani</i> infected mice over the course of infection.

<p>Mice were infected with <i>L</i>. <i>donovani</i> and sacrificed at various time points after infection. Neutrophils were excluded from all analysis involving monocytes. <b>(A)</b> Representative FACS plots depicting the gating strategy used to identify neutrophils (left) and percentage of neutrophils in the spleen of infected mice (right).<b>(B)</b> Gating strategy used to identify Ly6C⁺ monocytes (left) and percentage of splenic Ly6C^hi (upper graph) and Ly6C^lo/int (lower graph) monocytes. <b>(C)</b> Percentage of CD11c⁺ myeloid cells. <b>(D)</b> Percentage of CD11c⁺ Ly6C⁺ (left histogram raw and upper graph) and Ly6C^lo/int (right histogram raw and lower graph) monocytes. All data represent mean ± SEM of one of 4 independent experiments, n = 4.</p

HIF-1α deficient intermediate stage monocytes are more resistant to <i>L</i>. <i>donovani</i> infection under hypoxic conditions.

<p>Monocytes were derived for three days from the bone marrow of naïve <i>Hif</i>^{<i>flox/flox</i>}-<i>Cd11c-Cre</i>^<i>+</i> and <i>Cre</i>^<i>-</i> as described in the material and method section. M-CSF was then removed from the medium and cells were then infected with fluorescently-labelled <i>L</i>. <i>donovani</i> amastigotes prior to activation or not with IFNγ. The infection was monitored for 24h. <b>(A-B)</b> Percentage of infected Ly6C^hi <b>(A)</b> and Ly6C^lo/int <b>(B)</b> monocytes. <b>(C)</b> Examples of ImageStream analysis; pictures illustrate: nucleus (purple), parasites (green), Ly6C (red). <b>(D-E)</b> ImageStream analysis of numbers of parasites per cells in Ly6C^hi <b>(D)</b> and Ly6C^lo/int <b>(E)</b> monocytes at 1h (left graph), 12h (middle graph) and 24h (right graph). <b>(F-G)</b> Monocytes were treated as described above, but M-CSF was left in the medium. <b>(F)</b> Percentage of Ly6C^hi and <b>(G)</b> Ly6C^lo/int monocytes infected with <i>L</i>. <i>donovani</i>. All data represent mean ± SEM of one of 2 independent experiments, n = 3. * denotes <i>p</i><0.05, ** denotes <i>p</i><0.01, and *** denotes p<0.001</p

CD11c-specific HIF-1α-knockout mice produce more monocyte’s progenitors and display enhanced output of inflammatory monocytes in the bone marrow.

<p><i>Hif</i>^{<i>flox/flox</i>}-<i>Cd11c</i>-<i>Cre</i>^<i>+</i> and <i>Cre</i>^<i>-</i> mice were infected with <i>L</i>. <i>donovani</i> and sacrificed at various time point of infection. <b>(A)</b> Gating strategy to identify GMPs in the bone marrow of <i>L</i>.<i>donovani</i> infected mice. <b>(B)</b> Frequency (left graph) and absolute numbers (right graph) of GMPs in the bone marrow of mice over the course of infection. <b>(C-E)</b> Frequency (upper graph) and absolute numbers (lower graph) of granulocytes <b>(C)</b>, Ly6C^hi monocytes<b>(D),</b> Ly6C^lo/int monocytes <b>(E)</b> in the bone marrow of infected mice. <b>(F-G)</b> Percentage of CCR2⁺ <b>(F)</b> and CXCR4⁺ <b>(G)</b> Ly6C^hi monocytes in the bone marrow. <b>(H)</b> Percentage of Sca-1⁺ Ly6C^hi monocytes in the bone marrow. <b>(I)</b> Percentage of MHCII⁺ Ly6C^hi monocytes in the bone marrow. All data represent mean ± SEM of one of 4 independent experiments, n = 4. * denotes <i>p</i><0.05. </p

HIF-1α governs glucose metabolism in <i>L</i>. <i>donovani</i> infected splenocytes.

<p><i>Hif</i>^{<i>flox/flox</i>}-<i>Cd11c-Cre</i>^<i>+</i> and <i>Cre</i>^<i>-</i> mice were infected with <i>L</i>. <i>donovani</i> and sacrificed at various time point of infection. <b>(A)</b> Intracellular lactate concentration and <b>(B)</b> intracellular glucose concentration in splenocytes from infected mice at various time points of infection. <b>(C)</b> Mean fluorescence intensity of ROS expression in splenocytes from infected mice over the course of infection. <b>(D)</b> Percentage of ROS⁺ neutrophils. <b>(E)</b> Percentage of ROS⁺ Ly6C^hi monocytes. (<b>F</b>) Frequency of CD11b^hiLy6C⁺ splenocytes expressing IFNγR. All data represent mean ± SEM of one of 4 independent experiments, n = 4. * denotes <i>p</i><0.05, ** denotes <i>p</i><0.01, and *** denotes p<0.001 </p

HIF-1α expression in CD11c⁺ cells exacerbates infection in the bone marrow.

<p><i>Hif</i>^{<i>flox/flox</i>}-<i>Cd11c-Cre</i>^<i>+</i> and <i>Cre</i>^<i>-</i> mice were infected with <i>L</i>. <i>donovani</i> and sacrificed at various time point of infection. Graph shows the number of parasites present in one tibia and one femur of each infected mouse over the course of infection. All data represent mean ± SEM of one of 4 independent experiments, n = 4. * denotes <i>p</i><0.05 and ** denotes <i>p</i><0.01.</p

Primer sequences used in qPCR.

<p>Primer sequences used in qPCR.</p