Abnormal thymocyte maturation in LynΔN transgenic mice.

<p><b>A)</b> Representative thymi from 2-week old control and LynΔN mice. Bare scale : 0.5 cm. <b>B)</b> Total thymocyte numbers were determined. Lines indicate the mean, and each symbol represents one individual mouse. <b>C)</b> Total thymocytes from LynΔN and control mice were analyzed by flow cytometry for CD4 and CD8 expression, as shown in this representative flow cytometry profile. <b>D)</b> Proportion and <b>E)</b> number of all thymocyte subsets of 2-week old control and LynΔN mice were quantified; Lines indicate the mean, and each symbol represents one individual mouse<b>.</b> For these experiments, n = 15 for WT and n = 17 for LynΔN mice. P value, Student <i>t</i> test for unpaired data.</p

Splenic alterations in LynΔN mice are dependent on TNFR1 signaling.

<p><b>A)</b> Total splenocyte numbers from LynΔN and control mice in TNFR1+/− or TNFR1−/− background were determined. Lines indicate the mean, and each symbol represents one individual mouse. <b>B)</b> Splenic B cells from LynΔN and control mice in TNFR1+/− or TNFR1−/− background were analyzed by flow cytometry (CD19 and B220 expression), as shown in this representative flow cytometry profile. <b>C)</b> Proportion of CD19/B220 splenic B cell was quantified for LynΔN/TNFR1+/− and LynΔN/TNFR1−/− mice; each dot represents one individual mice. For these experiments, n = 5 for TNFR1+/−, TNFR1−/−, LynΔN/TNFR1+/− and n = 10 for LynΔN/TNFR1−/−. P value, Student <i>t</i> test for unpaired data.</p

TCR dependent proliferation and apoptosis are unaltered in total thymocytes of LynΔN mice.

<p><b>A)</b> Apoptosis index determined by TUNEL staining (green staining) was analyzed onto frozen thymi sections of 2-week old LynΔN and control mice. <b>B)</b> AnnexinV positive cells were analyzed onto freshly isolated thymocytes from control and LynΔN transgenic mice (n = 6 in each group) by flow cytometry. <b>C)</b> Total thymocytes from LynΔN and control mice were stimulated or not with 10 µg/ml of plate-coated anti-CD3 for the indicated time periods and AnnexinV+/PI+ dead cells were determined by flow cytometry. <b>D)</b> Total thymocytes from LynΔN and control mice were stimulated with 10 µg/ml of plate-coated anti-CD3 plus soluble anti-CD28 (2 µg/ml) mAbs for 72 h. 10 µM BrdU was added for 16 hours, and proliferation was measured by BrdU incorporation. Results are expressed as mean ± SD. Data are representative of at least 3 independent experiments.</p

Thymic alteration in LynΔN mice is dependent on TNFR1 signaling.

<p><b>A)</b> Total thymocyte numbers from LynΔN and control mice in TNFR1+/− or TNFR1−/− background were determined. Lines indicate the mean, and each symbol represents one individual mouse. <b>B)</b> Total thymocytes from LynΔN and control mice in TNFR1+/− or TNFR1−/− background were analyzed by flow cytometry for CD4 and CD8 expression, as shown in this representative flow cytometry profile. <b>C)</b> Proportion of all thymocyte subsets was quantified for LynΔN/TNFR1+/− and LynΔN/TNFR1−/− mice; each dot represents one individual mice. For these experiments, n = 5 for TNFR1+/−, TNFR1−/−, LynΔN/TNFR1+/− and n = 10 for LynΔN/TNFR1−/−. P value, Student <i>t</i> test for unpaired data.</p

Normal splenic T cell but abnormal B cell distribution in LynΔN transgenic mice.

<p><b>A)</b> Total splenocyte numbers were determined. Lines indicate the mean, and each symbol represents one individual mouse. <b>B)</b> Splenic T cells from LynΔN and control mice were analyzed by flow cytometry (CD4 and CD8 expression), as shown in this representative flow cytometry profile. <b>C)</b> Proportion and <b>D)</b> cell numbers of CD4+ and CD8+ splenic T cell of 2-week old control and LynΔN mice were quantified; Lines indicated the mean, and each dot represents one individual mice. For these experiments, n = 15 for WT and n = 17 for LynΔN mice. P value, Student <i>t</i> test for unpaired data. <b>E)</b> Splenic B cells from LynΔN and control mice were analyzed by flow cytometry (CD19 and B220 expression), as shown in this representative flow cytometry profile. <b>F)</b> Proportion and <b>G)</b> number of CD19+/B220+ splenic B cell of 2-week old control and LynΔN mice were quantified; Lines indicated the mean, and each dot represents one individual mice. For these experiments, n = 15 for WT and n = 17 for LynΔN mice. P value, Student <i>t</i> test for unpaired data.</p

TNF specific cell death is increased in LynΔN thymocytes.

<p><b>A)</b> Semi-quantitative RT-PCR analysis of TNFα and actin using RNA isolated from 2-week-old LynΔN and control thymi. <b>B)</b> Freshly isolated thymocytes from control or LynΔN mice were left untreated or incubated with either TNFα (50 ng/ml) for 18 h. Then, AnnexinV positive cells were determined by flow cytometry. Results are expressed as the percentage of TNF specific cell death (percentage of AnV+ cells in TNF stimulated condition – percentage of AnV+ cells in control condition). For this experiment, n = 3 for WT and n = 4 for LynΔN mice. P value, Student <i>t</i> test for unpaired data. <b>C)</b> Freshly isolated thymocytes from control or LynΔN mice were left untreated or incubated with either TNFα (50 ng/ml) for 10 and 30 minutes. Degradation and phosphorylation of IκBα was assayed by western blot analysis of total cell extracts. Hsp60 was used as a loading control.</p

CNF1 potentiates LPS-triggered immune responses.

<p>(A) Monocytes (5x10⁵ cells per condition) isolated from mouse blood were treated with PBS (control) or with 1 μg/ml CNF1 toxin for 10 h with or without 100 ng/ml LPS. Cell culture supernatants were analyzed using mouse ELISArray kits (n = 3). The data are shown as fold inductions compared with the control condition. (B, C and D) Monocytes (5x10⁵ cells per condition) purified from mouse blood were treated for 10 h. Cells were treated as indicated with 0.1, 1, or 10 μg/ml of CNF1 toxin or the CNF1 mutant C866S alone or in combination with ultrapure <i>E</i>. <i>coli</i> LPS at 1, 10 or 100 ng/ml (n = 3). (B) KC, (C) IL-6, and (D) IL-1β cytokine secretion was analyzed using ELISA (n = 4). (E) Female BALB/c mice were intravenously infected with 10⁷ CFUs of <i>E</i>. <i>coli</i> expressing CNF1 (<i>E</i>. <i>coli</i>^CNF1+) + PBS as a control or with an <i>E</i>. <i>coli</i>^CNF1+ + IL-1β antagonist (Kineret; 1.5 mg/kg) prior to the collection of peripheral blood at 3, 6, 24 and 48 h for the measurement of bacteremia (n = 10). P-values <0.05 (*); and P-values <0.01 (**) were considered statistically significant.</p

CNF1-triggered immunity requires inflammatory caspases.

<p>(A and B) IL-1β production and maturation/secretion after treatment with CNF1, LPS or CNF1 (1 μg/ml) + LPS (100 ng/ml) for 10 h. Actin and BSA were used as loading controls. (B) Graph showing the quantification of IL-1β secretion normalized to the control (n = 3). (C and D) Female C57BL/6 WT (C) or congenic C1^-/-C11^-/- mice (D) were intravenously infected with 10⁷ CFUs of <i>E</i>. <i>coli</i>^CNF1+ or with the isogenic CNF1-deleted mutant (<i>E</i>. <i>coli</i>^CNF1-) prior to the collection of peripheral blood at 3, 6, 24 and 48 h for bacteremia measurements. The data are expressed as the mean ± SEM (n = 7–8). (E and F) Analysis of the circulating levels of IL-1β (E) and KC (F) in the mouse sera by ELISA. Serum samples from mice infected with 10⁷<i>E</i>. <i>coli</i>^CNF1+ or with the isogenic mutant <i>E</i>. <i>coli</i>^CNF1- were collected at 3 h after intravenous infection and analyzed by ELISA (n = 3). P-values<0.05 (*); and P-values<0.01 (**) were considered statistically significant.</p

CNF1-triggered IL-1β maturation requires activated Rac, ASC and caspase-1.

<p>(A) Western blot analysis of the production and maturation/secretion of IL-1β by primary macrophages following treatment with CNF1, LPS or CNF1+LPS for 10 h. Actin and BSA were used as loading controls. (B) Quantification of caspase-1 activity in macrophages following treatment with CNF1+LPS for 6 h using YVAD-Fluorescent Labelled Inhibitor Caspase-1 Activity (FLICA). (C) Western blot analysis of macrophages IL-1β maturation/secretion upon transfection of HA-Rac2^Q61E and LPS treatment. (D) Co-immunoprecipitation of Myc-Rac2 and caspase-1 using an anti-Myc antibody following the treatment of HEK 293T cells with CNF1+LPS for 6 h.</p