Ubiquitin Specific Protease 21 Is Dispensable for Normal Development, Hematopoiesis and Lymphocyte Differentiation

<div><p>USP21 is a ubiquitin specific protease that catalyzes protein deubiquitination, however the identification of its physiological substrates remains challenging. USP21 is known to deubiquitinate transcription factor GATA3 and death-domain kinase RIPK1 <i>in vitro</i>, however the <i>in vivo</i> settings where this regulation plays a biologically significant role remain unknown. In order to determine whether USP21 is an essential and non-redundant regulator of GATA3 or RIPK1 activity <i>in vivo</i>, we characterized <i>Usp21</i>-deficient mice, focusing on mouse viability and development, hematopoietic stem cell function, and lymphocyte differentiation. The <i>Usp21</i>-knockout mice were found to be viable and fertile, with no significant dysmorphology, in contrast to the GATA3 and RIPK1 knockout lines that exhibit embryonic or perinatal lethality. Loss of USP21 also had no effect on hematopoietic stem cell function, lymphocyte development, or the responses of antigen presenting cells to TLR and TNFR stimulation. GATA3 levels in hematopoietic stem cells or T lymphocytes remained unchanged. We observed that aged <i>Usp21</i>-knockout mice exhibited spontaneous T cell activation, however this was not linked to altered GATA3 levels in the affected cells. The contrast in the phenotype of the <i>Usp21</i>-knockout line with the previously characterized GATA3 and RIPK1 knockout mice strongly indicates that USP21 is redundant for the regulation of GATA3 and RIPK1 activity during mouse development, in hematopoietic stem cells, and in lymphocyte differentiation. The <i>Usp21</i>-deficient mouse line characterized in this study may serve as a useful tool for the future characterization of USP21 physiological functions.</p></div

Transmission electron microscopy (TEM) images of <i>S.</i> Typhi and <i>S.</i> Typhimurium showing expression of Vi polysaccharide.

<p>S. Typhi (A), S. Typhimurium C5507 (B), <i>S.</i> Typhimurium SGB1 (C5507 D<i>tviB</i>::kan^r) (C) visualised using TEM. S. Typhi (D), S. Typhimurium C5507 (E), <i>S.</i> Typhimurium SGB1 (C5507 D<i>tviB</i>::kan^r) (F) visualised using TEM in conjunction with immunogold labelling using anti-Vi antibody.</p

Total number of cytokine producing cells after infection with Vi⁺ or Vi⁻<i>S.</i> Typhimurium.

<p>Total cell number for spleen (i.v. infection) or MLN (oral infection) after 24 h (1×10⁴) ± SEM.</p><p>*indicates significant values of p<0.05;</p><p>**, p<0.01;</p><p>***, p<0.001, as determined by one-way ANOVA followed by Bonferroni's multiple comparison test when compared to naïve mice.</p>†<p>indicates significant values of p<0.05;</p>††<p>, p<0.01;</p>†††<p>, p<0.001, as determined by one-way ANOVA followed by Bonferroni's multiple comparison test when Vi⁺ are compared to Vi⁻ infected mice.</p

Vi induction of IL-10 impacts on chemotaxis and activation of splenocytes <i>in vitro</i>.

<p>(A) Spleen cells were isolated from naïve mice and stimulated for 24 h with 1∶1 ratio of Vi⁺ or Vi⁻<i>Salmonella</i> strains and supernatants were analysed for levels of IL-10. Mean ± SEM are presented from six individual animals. Significance was determined using Mann-Whitney test, *** p<0.001. (B) Splenocytes were also stimulated with the various <i>S.</i> Typhimurium strains in the presence of anti-IL-10, isotype control antibody or rIL-10. Cells were then stained with surface antibodies and the percentage and expression/mean fluorescent intensity (MFI) of the activation marker CD69 on NK cells and PMN was analysed by flow cytometry. Columns represent the percentage of MFI ± SEM and significant differences were determined as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002131#ppat-1002131-g006" target="_blank">Figure 6</a>. (C) Chemotactic migration of splenocytes towards supernatants from <i>Salmonella</i> stimulated cultures (in the presence of anti-IL-10, control antibody or rIL-10). After 4 h incubation, the level of cell migration was determined by reading the relative fluorescent units (RFU) at 480/520 nm. Data represents mean ± SEM minus negative (media alone) controls. Significant differences in values of * p<0.05; **, p<0.01; ***, p<0.001, as determined by Kruskal-Wallis followed by Dunn's multiple comparison test or ^† indicating significant values when compared to r-IL-10.</p

Expression of the Vi capsule induces differential innate immune responses shortly after infection.

<p>Cells were isolated from the spleens after i.v. infection (A) or MLN after oral infection (B) of C57BL/6 mice 24 h after infection with C5507 (Vi⁺) or SGB1 (Vi⁻) <i>S.</i> Typhimurium and stained with flurochrome-labelled mAb and analysed by flow cytometry in which 20,000–200,000 events were recorded. Columns represent the percentage ± SEM. Significant differences in values of * p<0.05; **, p<0.01; ***, p<0.001, as determined by one-way ANOVA followed by Bonferroni's multiple comparison test.</p

Innate cell numbers after infection with Vi⁺ or Vi⁻<i>S.</i> Typhimurium.

<p>Total cell number for spleen (i.v. infection) or MLN (oral infection) after 24 h (1×10⁴) ± SEM.</p><p>*indicates significant values of p<0.05;</p><p>**, p<0.01;</p><p>***, p<0.001, as determined by one-way ANOVA followed by Bonferroni's multiple comparison test when compared to naïve mice.</p>†<p>indicates significant values of p<0.05;</p>††<p>, p<0.01;</p>†††<p>, p<0.001, as determined by one-way ANOVA followed by Bonferroni's multiple comparison test when Vi⁺ are compared to Vi⁻ infected mice.</p

Colonisation of C57BL/6 mice with <i>S.</i> Typhimurium C5, <i>S.</i> Typhimurium C5507, and <i>S.</i> Typhimurium SGB1 (C5507 D<i>tviB</i>::kan^r).

<p>(A) Groups of ten C57BL/6 genetically susceptible mice were inoculated orally with 1×10⁸ cfu of <i>S.</i> Typhimurium C5 (closed circles), <i>S.</i> Typhimurium C5507 (open circles), or <i>S.</i> Typhimurium SGB1 (C5507 D<i>tviB</i>::kan^r) (closed squares). Horizontal bar indicates the geometric mean. Mice were culled on day 5 post-inoculation and the cfu in mesenteric lymph nodes, cecum, ileum, spleen and liver homogenates determined. (B) A group of five 129/sv genetically resistant mice were inoculated orally with an equal mixture of 1×10⁹ CFU <i>S.</i> Typhimurium C5507 Vi⁺, and <i>S.</i> Typhimurium SGB1 Vi⁻ (C5507 D<i>tviB</i>::kan^r). The mean log10 ratio of these two strains in fresh fecal pellets on days 1, 4, 7 and 10 post inoculation are plotted (top), the CFU per 100 mg of <i>S.</i> Typhimurium C5507 Vi⁺ (open circles) and <i>S.</i> Typhimurium SGB1 Vi⁻ (filled circles) are plotted (below).</p

Vi expression also impacts on the cytokine profile of cells after <i>S.</i> Typhimurium infection.

<p>Isolated splenocytes after i.v. infection (A) or MLN after oral infection (B) from naïve, or 24 h infected C5507 (Vi⁺) and SGB1 (Vi⁻) mice were stimulated for 6 h with BD Leukocyte Activation Cocktail with BD GolgiPlug (BD Biosciences) <i>in vitro</i>, permeabilised and stained with anti-cytokine flurochrome-labelled mAb. (C) Cells were stained with mAb to specific surface markers F4/80⁺, CD11c⁺ and DX5⁺/CD3⁻ the identity of innate populations, permeabilised and stained with anti-IL-10. Data represent percent of cytokine positive cells out of total spleen populations ± SEM. Significant differences in values of * p<0.05; **, p<0.01; ***, p<0.001, as determined by one-way ANOVA followed by Bonferroni's multiple comparison test.</p

IFNλ can restrict mCMV replication <i>in vitro</i>.

<p>(A) <i>Ifnλr1</i> expression by 3T3 and BNLCL2 cells was determined by qPCR. (B) 3T3 (top) and BNLCL2 (bottom) cells were incubated with/without 50U/ml IFNα and/or IFNβ, or 50ng/ml IFNλ2 (IL-28A) for 24hrs and infected with mCMV at multiplicities of infection (MOI), as stated in the figure. After 4 days, infectious virions in supernatant were quantified by plaque assay. Statistical significance of PFU in IFNλ2-treated versus control cells is shown. Virus load in spleen (C), liver (D) and salivary glands (E) of WT and <i>Ifnλr1</i>^<i>-/-</i> mice was assessed 4 (D&E) and 33 (E) days p.i. (F) IFNλ2/3 protein in spleen (left), liver (middle) and salivary glands (right) was measured at day 0 and 2 days p.i (spleen and liver) or 0 and 26 days p.i (salivary glands). Results are shown as mean + SEM of 3–7 mice/group. (G) WT and <i>Ifnλr1</i>^<i>-/-</i> mice were infected (i.n) with mCMV in a volume of 25μl or 50μl and after 4 days, lung infectious viral load was quantified by plaque assay. Statistical significance was assessed using 1-way ANOVA (B) or Mann Whitney-U (C-E, G) or students T-Test (F) and is depicted where appropriate. Panel G represents merged data from two experiments whereas all other data represent at least two biological replicates performed separately.</p

Total number of PMN and NK cells after infection with Vi⁺ or Vi⁻<i>S.</i> Typhimurium in TLR4^−/− and MyD88^−/− mice.

<p>Total cell number of PMN and NK cells in the spleen 24 h post (×10⁴) ± SEM.</p><p>*indicates significant values of *, p<0.01;</p><p>**, p<0.001, as determined by one-way ANOVA followed by Bonferroni's multiple comparison test when compared to naïve mice.</p>†<p>indicates significant values of †, p<0.01;</p>††<p>, p<0.001, as determined by one-way ANOVA followed by Bonferroni's multiple comparison test when WT Vi⁻ are compared to KO Vi⁻ infected mice.</p