Fig 2: Prox1+/- mice are protected from DSS-induced colitis. (A) Mouse body weight changes during DSS treatment. Prox1+/- and NMRI littermate controls (WT), were treated with 3% (w/v) DSS in the drinking water for 8 days and the weight was measured daily for 12 days. Body weight changes were calculated as a percentage of weight prior to DSS treatment (Day 0) Data are plotted as means ± SEM from male and female mice of at least 10 mice per genotype. Results are representative of 2 independent experiments. * P < 0.05, 1-way ANOVA followed by Tukey´s multiple comparison test. (B) Macroscopic appearance (left) and quantification of the length of colons (right) at day 12 from WT and Prox1+/- mice treated with DSS or vehicle. Data are plotted as means ± SEM. (C, D) mRNA levels of IL-6 (C) and IL-1ß (D) in colon samples of WT and Prox1+/- mice treated with DSS. (E, F) Time course of cytokine levels in in colonic tissue explants showing the effect of DSS treatment in WT and Prox1+/- mice. Tissue levels of IL-16 (E) and IL-1ß (F) in colon were quantified by ELISA assay. Data expressed as mean ± SD and statistical significance evaluated by One-way ANOVA followed by Tukey’s post-test where * P < 0.05; *** P < 0.001; **** P < 0.0001. (G–J) Serum levels of IL-6 and IL-1ß from the different groups evaluated at day 5 (C, D) and day 12 (E, F) after DSS or vehicle treatment was determined by ELISA. Data are plotted as means ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; 1-way ANOVA followed by Tukey´s multiple comparison test. (K) Lacteal length shrinking is reduced in DSS-treated Prox1+/- mice compared to DSS-treated WT animals. (Left) Representative images of lacteals (LYVE-1, green; Prox1-tdTomato, red) on thick sections of the small intestine (proximal duodenum) from WT and Prox1+/- mice treated with vehicle or DSS for 12 days are shown. Scale bar is 200 µm (Right). Quantification of relative lacteal length from the different groups is shown. Dots indicate values of 113–225 villi/group in n = 5 mice/group pooled from two independent experiments. Data are plotted as means ± SD. ** P < 0.01; **** P < 0.0001; (L) Reduced colonic lymphatic vasculature area in Prox1+/- mice treated with DSS. The colon lymphatics were stained using LYVE-1 antibody (green) and using the transgenic Prox1-tdTomato mice (red). LYVE-1+ Prox1+ indicates the lymphatic vessels (left). Scale bar is 200 µm. Quantification of the area of colon lymphatics vessels (right). Dots indicate values of 34–103 villi/group in n = 5 mice/group pooled from two independent experiments. Error bars indicate SD. **** P < 0.0001 by 1-way ANOVA with post hoc Tukey’s test.

Fig 3: LRH-1 regulates the pro-inflammatory cytokine production in LPS-stimulated bone marrow-derived macrophages and Kupffer cells.a TNF, b IL-6, and c IL-1ß secreted protein levels in the supernatant of bone marrow-derived macrophages (BMDMs) pre-treated for 2 h with vehicle (PBS) or 3d2 (40 µM) and subsequently stimulated with control buffer (CTL) or LPS at indicated concentrations for 18 h. Mean values of triplicates ± SD of a representative experiment (n = 3) are shown (one-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001). d TNF protein levels in the supernatant of isolated Kupffer cells from wild-type C57BL/6 mice after 2 h pre-treatment with vehicle (PBS) or 3d2 (40 µM) and subsequently stimulated for 18 h with LPS at indicated concentrations. Mean values of triplicates ± SD of one representative experiment (n = 3) are shown (t test; *p < 0.05, ***p < 0.001). e TNF protein levels in the supernatant of BMDMs generated from wild-type C57BL/6 (WT) or SHP-deficient (SHP-/-) mice and stimulated for 18 h with LPS at indicated concentrations. Mean values of triplicates ± SD of a representative experiment (n = 3) are shown (t test; *p < 0.05, ***p < 0.001).

Fig 4: Proinflammatory gene expression in immortalized astrocytes upon LPS stimulation. A) The immortalized cells (WT5 or KO9) under the non-permissive condition were cultured with EMEM/1% FCS for 24 h and treated with 1 µg/ml of LPS for 12 h. The gene expression of Il6, Tnfa, Il1ß, Ccl2, Cxcl10 and Nos2 was analyzed by real-time PCR. Values are normalized to the expression of cyclophilin A. The expression level of WT5 after LPS stimulation in the respective genes was adjusted to 1. n = 5 for all groups. (*, p < 0.05). ND, not detected. B) The level of IL-6 and IL-1ß in culture medium was analyzed by ELISA. The immortalized cells were incubated as in A. n = 5 for all groups. (*, p < 0.05). ND, not detected.

Fig 5: Screen for genes that modulate dendritic cell phenotype and functionCAS9 expressing-inducible immortalized dendritic cells (iniDC CAS9) were transfected with specific CRISPR RNAs (crRNA) to perform gene knockouts. Dexamethasone and doxycycline (DEX/DOX) were removed from the culture system during transfection and recovery of cells to differentiate iniDC CAS9 cells into primary de-iniDC CAS9 which were then used for assessing the phagocytosis of dying cancer cells (Phago), quantification of IL-1ß, IL-6, and TNFa production upon stimulation with LPS, as well as the assessment of antigen cross-presentation capacity upon ovalbumin (OVA) treatment by interleukin-2 (IL-2) quantification in B3Z hybridoma co-culture supernatants. For each parameter, the readouts of crRNA-transfected wells were normalized to non-target control crRNA. Averaged ratios are summarized as a heatmap (A), in which Fpr1 is highlighted as a candidate gene whose KO leads to a loss of function phenotype in DC. Fpr1 KO de-iniDC CAS9 cells (Fpr1-/-) were analyzed for cytokine production (B–D) upon stimulation with LPS or MCA205 cells lysate, cross-presentation of OVA antigen or SINFKEL peptide (SL8) (E), and phagocytosis of MCA205 cells that were either untreated (UT) or crizotinib (CRIZO)-treated (F) Cytokine concentration and phagocytic events (CD11C+ CellTracker+ cells) are reported as bar charts (means ± SD of triplicates). Statistical differences were calculated by means of the ANOVA test (Dunnett's multiple comparisons test), with **p < 0.01, ***p < 0.001 as compared to UT, ###p<0.001 or $$$p<0.001 as compared between Fpr1-/- and wild type (wt) de-iniDC CAS9 cells with the same stimuli. Differentiated iniDC and iniDC CAS9 cells (de-iniDC) were pulsed with tumor cell lysate and intratumorally (i.t.) injected into subcutaneous MCA205 tumors grafted on immunocompetent mice to test their (immuno)therapeutic potential. Tumor growth curves are reported in (G) as mean ± SEM (n = 6). Statistical significance was calculated by means of the ANOVA Type 2 (Wald test) and significant results are indicated as *P<0.05 compared to the PBS group.