Severe thymic atrophy in a mouse model of skin inflammation accounts for impaired TNFR1 signaling

Abstract

Transgenic mice expressing the caspase-cleaved form of the tyrosine kinase Lyn (LynΔN) develop a TNFα-dependent skin disease that accurately recapitulates human psoriasis. Participation of lymphocytes in this disease was confirmed by backcrossing LynΔN mice on a Rag-1 deficient background. The present study was therefore conducted to analyze whether modification of lymphocyte homeostasis does occur and participate in the phenotype of LynΔN mice. We show here that LynΔN mice consistently exhibit thymic atrophy that correlates with both a net decrease in the CD4+/CD8+ Double Positive (DP) and an increase in Single Positive (SP) thymocyte sub-populations, but also display an increase of splenic mature B cell. Interestingly, a normal immune phenotype was rescued in a TNFR1 deficient background. Finally, none of these immune alterations was detected in newborn mice before the onset of inflammation. Therefore, we conclude that chronic inflammation can induce thymic atrophy and perturb spleen homeostasis in LynΔN mice through the increased production of TNFα, LTß and TNFR1 signaling.

Figure 1. Abnormal thymocyte maturation in LynΔN transgenic mice.

A) Representative thymi from 2-week old control and LynΔN mice. Bare scale : 0.5 cm. B) Total thymocyte numbers were determined. Lines indicate the mean, and each symbol represents one individual mouse. C) 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. D) Proportion and E) 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. For these experiments, n = 15 for WT and n = 17 for LynΔN mice. P value, Student t test for unpaired data.

Figure 2. Normal splenic T cell but abnormal B cell distribution in LynΔN transgenic mice.

A) Total splenocyte numbers were determined. Lines indicate the mean, and each symbol represents one individual mouse. 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. C) Proportion and D) 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 t test for unpaired data. E) 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. F) Proportion and G) 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 t test for unpaired data.

Figure 3. TCR dependent proliferation and apoptosis are unaltered in total thymocytes of LynΔN mice.

A) Apoptosis index determined by TUNEL staining (green staining) was analyzed onto frozen thymi sections of 2-week old LynΔN and control mice. B) AnnexinV positive cells were analyzed onto freshly isolated thymocytes from control and LynΔN transgenic mice (n = 6 in each group) by flow cytometry. C) 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. D) 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.

Figure 4. TNF specific cell death is increased in LynΔN thymocytes.

A) Semi-quantitative RT-PCR analysis of TNFα and actin using RNA isolated from 2-week-old LynΔN and control thymi. 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 t test for unpaired data. C) 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.

Figure 5. Thymic alteration in LynΔN mice is dependent on TNFR1 signaling.

A) 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) 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. C) 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 t test for unpaired data.

Figure 6. Splenic alterations in LynΔN mice are dependent on TNFR1 signaling.

A) 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) 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. C) 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 t test for unpaired data.

Figure 7. Newborn LynΔN mice displayed normal thymocyte and splenic B cell populations.

A) Total thymocytes from LynΔN and control mice were analyzed by flow cytometry for CD4 and CD8 expression. B) Splenic B cells from LynΔN and control mice were analyzed by flow cytometry (CD19 and B220 expression). Data are the mean ± SD; n = 4 for WT and n = 6 for LynΔN mice.