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Linear ubiquitin chain assembly complex coordinates late thymic T-cell differentiation and regulatory T-cell homeostasis

View Article: PubMed Central - PubMed

ABSTRACT

The linear ubiquitin chain assembly complex (LUBAC) is essential for innate immunity in mice and humans, yet its role in adaptive immunity is unclear. Here we show that the LUBAC components HOIP, HOIL-1 and SHARPIN have essential roles in late thymocyte differentiation, FOXP3+ regulatory T (Treg)-cell development and Treg cell homeostasis. LUBAC activity is not required to prevent TNF-induced apoptosis or necroptosis but is necessary for the transcriptional programme of the penultimate stage of thymocyte differentiation. Treg cell-specific ablation of HOIP causes severe Treg cell deficiency and lethal immune pathology, revealing an ongoing requirement of LUBAC activity for Treg cell homeostasis. These data reveal stage-specific requirements for LUBAC in coordinating the signals required for T-cell differentiation.

No MeSH data available.


Inhibition of cell death in LUBAC-deficient thymocytes.(a) Cell death determined by PI uptake in thymocytes from control, HoipΔCd4, HoilΔCd4 and Sharpincpdm mice cultured for 24 h with combinations of agonists (T: 100 ng ml−1 TNF; S: 500 nM SMAC mimetic; Q:10 μM QVD-OPh;N: 10 μM necrostatin). Percentages (b) and absolute numbers (c) of CD4+FOXP3+ cells in the thymus of WT (n=18), Sharpincpdm (n=11), Mlkl−/− (n=4), Mlkl−/−Casp8−/− (n=4), SharpincpdmCasp8−/−Mlkl−/− (n=4), Rip3k−/−Casp8+/− (n=4), SharpincpdmRip3k−/−Casp8+/− (n=4), Tnf−/− (n=4) and SharpincpdmTnf−/− (n=5) mice. Shpncpdm refers to Sharpincpdm mice. (d) Surface expression of CD4 and CD8 on whole thymocytes from control, Casp8ΔCd4Mlkl−/−, HoilΔCd4 and HoipΔCd4Casp8Δcd4Mlkl−/−mice. Data are representative of two independent experiments with one mouse per genotype (a,d). For b and c, each symbol represents an individual mouse, with 4–14 mice per group; small horizontal lines indicate mean±s.d.; *P<0.05, **P<0.01, ***P<0.005 and ****P<0.001, respectively. One-way analsyis of variance with a Tukey's post-hoc test for multiple comparisons was used for statistical analysis.
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f5: Inhibition of cell death in LUBAC-deficient thymocytes.(a) Cell death determined by PI uptake in thymocytes from control, HoipΔCd4, HoilΔCd4 and Sharpincpdm mice cultured for 24 h with combinations of agonists (T: 100 ng ml−1 TNF; S: 500 nM SMAC mimetic; Q:10 μM QVD-OPh;N: 10 μM necrostatin). Percentages (b) and absolute numbers (c) of CD4+FOXP3+ cells in the thymus of WT (n=18), Sharpincpdm (n=11), Mlkl−/− (n=4), Mlkl−/−Casp8−/− (n=4), SharpincpdmCasp8−/−Mlkl−/− (n=4), Rip3k−/−Casp8+/− (n=4), SharpincpdmRip3k−/−Casp8+/− (n=4), Tnf−/− (n=4) and SharpincpdmTnf−/− (n=5) mice. Shpncpdm refers to Sharpincpdm mice. (d) Surface expression of CD4 and CD8 on whole thymocytes from control, Casp8ΔCd4Mlkl−/−, HoilΔCd4 and HoipΔCd4Casp8Δcd4Mlkl−/−mice. Data are representative of two independent experiments with one mouse per genotype (a,d). For b and c, each symbol represents an individual mouse, with 4–14 mice per group; small horizontal lines indicate mean±s.d.; *P<0.05, **P<0.01, ***P<0.005 and ****P<0.001, respectively. One-way analsyis of variance with a Tukey's post-hoc test for multiple comparisons was used for statistical analysis.

Mentions: Impaired LUBAC function can switch pro-survival TNFR1 signalling into caspase-8-dependent apoptosis or caspase-independent, RIPK1/RIPK3/MLKL-mediated necroptosis173839. TNF is produced constitutively in the thymic medulla by epithelial cells and dendritic cells8, prompting the hypothesis that LUBAC deficiency might lead to the death of medullary SP and Treg cells. Surprisingly, we found that TNF treatment of thymocytes from HoipΔCd4, HoilΔCd4 and Sharpincpdm mice did not induce greater cell death than observed in control thymocytes (Fig. 5a). Moreover, treatment of cells with TNF plus a small molecular mimetic of second mitochondria-derived activator of caspases (SMACs) did not induce additional death of HoipΔCd4, HoilΔCd4 and Sharpincpdm thymocytes. Similarly, when TNF/SMAC mimetic-induced cell death was blocked by the caspase inhibitor, QVD-OPh, to engage the alternative cell death mechanism, necroptosis, the viability of thymocytes from HoipΔCd4, HoilΔCd4 and Sharpincpdm mice was comparable to WT thymocytes (Fig. 5a). Necroptosis is dependent on the activities of RIPK1, RIPK3 and the pseudo-kinase MLKL. Blocking necroptosis using the RIPK1 inhibitor necrostatin-1 did not alter the survival of thymocytes from HoipΔCd4, HoilΔCd4 or Sharpincpdm mice in vitro. These data do not support the notion that LUBAC deficiency sensitizes thymocytes to TNF-induced cell death.


Linear ubiquitin chain assembly complex coordinates late thymic T-cell differentiation and regulatory T-cell homeostasis
Inhibition of cell death in LUBAC-deficient thymocytes.(a) Cell death determined by PI uptake in thymocytes from control, HoipΔCd4, HoilΔCd4 and Sharpincpdm mice cultured for 24 h with combinations of agonists (T: 100 ng ml−1 TNF; S: 500 nM SMAC mimetic; Q:10 μM QVD-OPh;N: 10 μM necrostatin). Percentages (b) and absolute numbers (c) of CD4+FOXP3+ cells in the thymus of WT (n=18), Sharpincpdm (n=11), Mlkl−/− (n=4), Mlkl−/−Casp8−/− (n=4), SharpincpdmCasp8−/−Mlkl−/− (n=4), Rip3k−/−Casp8+/− (n=4), SharpincpdmRip3k−/−Casp8+/− (n=4), Tnf−/− (n=4) and SharpincpdmTnf−/− (n=5) mice. Shpncpdm refers to Sharpincpdm mice. (d) Surface expression of CD4 and CD8 on whole thymocytes from control, Casp8ΔCd4Mlkl−/−, HoilΔCd4 and HoipΔCd4Casp8Δcd4Mlkl−/−mice. Data are representative of two independent experiments with one mouse per genotype (a,d). For b and c, each symbol represents an individual mouse, with 4–14 mice per group; small horizontal lines indicate mean±s.d.; *P<0.05, **P<0.01, ***P<0.005 and ****P<0.001, respectively. One-way analsyis of variance with a Tukey's post-hoc test for multiple comparisons was used for statistical analysis.
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f5: Inhibition of cell death in LUBAC-deficient thymocytes.(a) Cell death determined by PI uptake in thymocytes from control, HoipΔCd4, HoilΔCd4 and Sharpincpdm mice cultured for 24 h with combinations of agonists (T: 100 ng ml−1 TNF; S: 500 nM SMAC mimetic; Q:10 μM QVD-OPh;N: 10 μM necrostatin). Percentages (b) and absolute numbers (c) of CD4+FOXP3+ cells in the thymus of WT (n=18), Sharpincpdm (n=11), Mlkl−/− (n=4), Mlkl−/−Casp8−/− (n=4), SharpincpdmCasp8−/−Mlkl−/− (n=4), Rip3k−/−Casp8+/− (n=4), SharpincpdmRip3k−/−Casp8+/− (n=4), Tnf−/− (n=4) and SharpincpdmTnf−/− (n=5) mice. Shpncpdm refers to Sharpincpdm mice. (d) Surface expression of CD4 and CD8 on whole thymocytes from control, Casp8ΔCd4Mlkl−/−, HoilΔCd4 and HoipΔCd4Casp8Δcd4Mlkl−/−mice. Data are representative of two independent experiments with one mouse per genotype (a,d). For b and c, each symbol represents an individual mouse, with 4–14 mice per group; small horizontal lines indicate mean±s.d.; *P<0.05, **P<0.01, ***P<0.005 and ****P<0.001, respectively. One-way analsyis of variance with a Tukey's post-hoc test for multiple comparisons was used for statistical analysis.
Mentions: Impaired LUBAC function can switch pro-survival TNFR1 signalling into caspase-8-dependent apoptosis or caspase-independent, RIPK1/RIPK3/MLKL-mediated necroptosis173839. TNF is produced constitutively in the thymic medulla by epithelial cells and dendritic cells8, prompting the hypothesis that LUBAC deficiency might lead to the death of medullary SP and Treg cells. Surprisingly, we found that TNF treatment of thymocytes from HoipΔCd4, HoilΔCd4 and Sharpincpdm mice did not induce greater cell death than observed in control thymocytes (Fig. 5a). Moreover, treatment of cells with TNF plus a small molecular mimetic of second mitochondria-derived activator of caspases (SMACs) did not induce additional death of HoipΔCd4, HoilΔCd4 and Sharpincpdm thymocytes. Similarly, when TNF/SMAC mimetic-induced cell death was blocked by the caspase inhibitor, QVD-OPh, to engage the alternative cell death mechanism, necroptosis, the viability of thymocytes from HoipΔCd4, HoilΔCd4 and Sharpincpdm mice was comparable to WT thymocytes (Fig. 5a). Necroptosis is dependent on the activities of RIPK1, RIPK3 and the pseudo-kinase MLKL. Blocking necroptosis using the RIPK1 inhibitor necrostatin-1 did not alter the survival of thymocytes from HoipΔCd4, HoilΔCd4 or Sharpincpdm mice in vitro. These data do not support the notion that LUBAC deficiency sensitizes thymocytes to TNF-induced cell death.

View Article: PubMed Central - PubMed

ABSTRACT

The linear ubiquitin chain assembly complex (LUBAC) is essential for innate immunity in mice and humans, yet its role in adaptive immunity is unclear. Here we show that the LUBAC components HOIP, HOIL-1 and SHARPIN have essential roles in late thymocyte differentiation, FOXP3+ regulatory T (Treg)-cell development and Treg cell homeostasis. LUBAC activity is not required to prevent TNF-induced apoptosis or necroptosis but is necessary for the transcriptional programme of the penultimate stage of thymocyte differentiation. Treg cell-specific ablation of HOIP causes severe Treg cell deficiency and lethal immune pathology, revealing an ongoing requirement of LUBAC activity for Treg cell homeostasis. These data reveal stage-specific requirements for LUBAC in coordinating the signals required for T-cell differentiation.

No MeSH data available.