Indoleamine 2,3-dioxygenase 1 (Ido1) is involved in the control of mouse caput epididymis immune environment

The epididymis maintains a state of immune tolerance towards spermatozoa while also protecting them and itself against infection and acute inflammation. The immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (Ido1) participates in this delicate local equilibrium. Using the mouse Ido1−/− model, we show here that the absence of IDO1 expression leads in the epididymis but not in serum to (1) an increase in the inflammatory state as evidenced by changes in the content of cytokines and chemokines, (2) the engagement of a Th1-driven inflammatory response as evidenced by changes in the Th17/Treg as well as Th1/Th2 equilibria, as well as (3) differences in the content of lipid intermediates classically involved in inflammation. Despite this more pronounced inflammatory state, Ido1−/− animals succeed in preserving the local epididymal immune situation due to the activation of compensatory mechanisms that are discussed

Chemokine profiles of the epididymis and plasma.

<p>Histograms show the levels of the inflammatory chemokines CCL1 (TCA-3, T-cell activation 3), CCL2 (MCP1, Monocyte chemoattractant protein 1), CCL3 (MIP-1α, Macrophage inflammatory protein 1), CCL5 (RANTES, regulated on activation normal T cell expressed and secreted), CCL11 (Eotaxin 1), CCL24 (Eotaxin 2), CCL25 (TECK, Thymus-Expressed Chemokine), CXCL5 (LIX, Lipopolysaccharide-induced CXC-chemokine), CXCL9 (MIG, Monokine induced by gamma interferon), CXCL11 (I-TAC, Interferon-inducible T-cell alpha chemoattractant), CXCL13 (BCA-1, B-cell attracting chemokine 1) and CX3CL1 (Neurotactin) in caput epididymidis extracts (<b>A</b>) and plasma (<b>B</b>) from 6 month-old <i>wt</i> and <i>IDO1−/−</i> animals. *<i>P</i>≤0.05; **<i>P</i>≤0.01.</p

Interleukin profile of the epididymis.

<p>Histograms show the levels of the interleukins IL-1ß, IL-2, IL-3, IL-4, IL-6, IL-9, IL-10, IL-12p70, IL-12p40p70, IL-13, and Il-17 in caput epididymidis extracts (<b>A</b>) and plasma (<b>B</b>) from 6 month-old <i>wt</i> and <i>IDO1−/−</i> animals. *<i>P</i>≤0.05; **<i>P</i>≤0.01.</p

Monitoring of the activation of the TGFß/BMP pathway in caput epididymis extracts.

<p>Bar graphs showing the levels of Smad3 and Smad1-5 proteins and their phosphorylated counterparts phosphoSmad3 and phosphoSmad1-5 upon activation in caput epididymidis extracts from <i>wt</i> and <i>IDO1^−/−</i> mice at 6 months of age. Bar graphs display means ± SEM using GAPDH as an internal standard for quantification (n = 4 for Smad3, and n = 8 for Smad1-5; *<i>P</i>≤0.05; **p<0.01).</p

Th17/Treg equilibrium in the caput epididymidis.

<p><b>A:</b> Differentiation of the Th17 and Treg lineages from naïve T cells are mutually exclusive <i>via</i> their respective cytokines. STAT3/RORc drive the differentiation of Th17 cells, while STAT5/FOXP3 drive the differentiation of Treg. In inflammatory or/and tolerogenic situations, IDO1 activity and the resulting KYNs promotes the differentiation of Treg cells and their immunosuppressive actions. <b>B:</b> Representative western blots showing the levels of STAT3 and STAT5 proteins and their phosphorylated counterparts phosphoSTAT3 and phosphoSTAT5 upon activation in caput epididymidis extracts from <i>wt</i> and <i>IDO1^−/−</i> mice at 6 months of age. Bar graphs display means ± SEM using GAPDH as an internal standard for quantification. (n = 7 for Stat3, and n = 3 for Stat5; **p<0.01). <b>C:</b> Quantitative RT-PCR estimations of <i>RORc</i> and <i>FOXP3</i> mRNA accumulations in caput epididymidis samples from <i>wt</i> (black bars) and <i>IDO1^−/−</i> (grey bars) male mice. For quantification of transcripts, the relative method was used to calculate mRNA relative level to <i>Cyclophilin B</i> standard. WT levels were set as 1 in the Y axis. Mean +/− SEM; n = 12. **<i>P</i>≤0.01.</p

Other inflammatory markers of the IDO1^−/− caput epididymidis.

<p><b>A:</b> Quantitative RT-PCR estimations of <i>COX1</i> and <i>COX2</i> mRNA accumulation in caput epididymidis samples from <i>wt</i> (black bars) and <i>IDO1^−/−</i> (grey bars) male mice. For quantification of transcripts, the relative method was used to calculate mRNA levels relative to <i>Cyclophilin B</i> standard. WT levels were set as 1 in the Y-axis. Mean +/− SEM; n = 12. *<i>P</i>≤0.05; **<i>P</i>≤0.01. <b>B:</b> Schematic representation of the omega 6- (ω6) and omega 3-derived (ω3) fatty acid (FA) intermediates (DGLA, Dihomo-gamma-linolenic acid; AA, Arachidonic acid; EPA, Eicosapentaenoic acid and DHA, Docosahexaenoic acid) precursors of the eicosanoids derivatives including prostaglandins, leucotrienes and thromboxanes. Bracketed numbers given above each FA species indicate difference recorded in the representation of these FA in caput epididymidis extracts of <i>IDO1^−/−</i> animals versus <i>wt</i> (n = 6). <b>C:</b> Histograms illustrate the recorded differences in sphingolipid intermediates (sphingomyelin and ceramides) concentration in caput epididymidis extracts of <i>wt</i> (black bars) and <i>IDO1^−/−</i> (grey bars) from 6 month-old animals. Cholesterol level was taken as a reference to show that the transgenic animals do not present a general disruption in their epididymal lipidic profile.</p

Analysis of the inflammatory status of the epididymis.

<p><b>A:</b> KYN:TRP ratios in caput epididymis and blood plasma, respectively, of 6 month-old <i>wt</i> and <i>IDO1−/−</i> animals. The Y-axis is a semi logarithmic representation in µmole/mmole. <b>B:</b> Histograms show the levels of INF-γ and TNF-α in caput epididymidis extracts and plasma from 6 month-old <i>wt</i> and <i>IDO1−/−</i> animals. <b>C:</b> Histogram shows the levels of the soluble TNF receptors (sTnfRI and sTnfRII) in caput epididymidis extracts from 6 month-old <i>wt</i> and <i>IDO1−/−</i> animals. *<i>P</i>≤0.05, **<i>P</i>≤0.01, ***<i>P</i>≤0.001.</p

Th1/Th2 equilibrium in the caput epididymidis.

<p><b>A:</b> Differentiation of the Th1 and Th2 lineages from naïve T cells are mutually exclusive <i>via</i> their respective cytokines. T-bet drives the differentiation of Th1 cells while Gata-3 drives the differentiation of Th2 cells. <b>B:</b> Quantitative RT-PCR estimations of T-bet and Gata-3 mRNA accumulation in caput epididymidis samples from <i>wt</i> (black bars) and <i>IDO1^−/−</i> (grey bars) male mice. For quantification of transcripts, the relative method was used to calculate mRNA levels relative to <i>Cyclophilin B</i> standard. WT levels were set as 1 in the Y-axis. Mean +/− SEM; n = 12. **<i>P</i>≤0.01. <b>C:</b> Histograms show the levels of leptin in caput epididymidis extracts and plasma from 6 month-old <i>wt</i> and <i>IDO1−/−</i> animals. **<i>P</i>≤0.01.</p

Flow cytometry analysis of lymphocytes populations amongst <i>wt</i> and <i>Ido1^−/−</i> caput epididymal cells.

<p>Cytogrammes show the gating strategy used to identify the different lymphocytes subpopulations. The lymphocyte gate was defined according to cell size (FSC) and granularity (SSC) criteria. The two tables show the number of cells (events) for each population gate in <i>wt</i> (left) and <i>Ido1^−/−</i> (right) animals. They also show the proportion of each population among all acquired cells (% total).</p