A DOCK8-WIP-WASp complex links T cell receptors to the actin cytoskeleton

Wiskott-Aldrich syndrome (WAS) is associated with mutations in the WAS protein (WASp), which plays a critical role in the initiation of T cell receptor-driven (TCR-driven) actin polymerization. The clinical phenotype of WAS includes susceptibility to infection, allergy, autoimmunity, and malignancy and overlaps with the symptoms of dedicator of cytokinesis 8 (DOCK8) deficiency, suggesting that the 2 syndromes share common pathogenic mechanisms. Here, we demonstrated that the WASpinteracting protein (WIP) bridges DOCK8 to WASp and actin in T cells. We determined that the guanine nucleotide exchange factor activity of DOCK8 is essential for the integrity of the subcortical actin cytoskeleton as well as for TCR-driven WASp activation, F-actin assembly, immune synapse formation, actin foci formation, mechanotransduction, T cell transendothelial migration, and homing to lymph nodes, all of which also depend on WASp. These results indicate that DOCK8 and WASp are in the same signaling pathway that links TCRs to the actin cytoskeleton in TCR-driven actin assembly. Further, they provide an explanation for similarities in the clinical phenotypes of WAS and DOCK8 deficiency

A DOCK8-WIP-WASp complex links T cell receptors to the actin cytoskeleton

Wiskott-Aldrich syndrome (WAS) is associated with mutations in the WAS protein (WASp), which plays a critical role in the initiation of T cell receptor–driven (TCR-driven) actin polymerization. The clinical phenotype of WAS includes susceptibility to infection, allergy, autoimmunity, and malignancy and overlaps with the symptoms of dedicator of cytokinesis 8 (DOCK8) deficiency, suggesting that the 2 syndromes share common pathogenic mechanisms. Here, we demonstrated that the WASp-interacting protein (WIP) bridges DOCK8 to WASp and actin in T cells. We determined that the guanine nucleotide exchange factor activity of DOCK8 is essential for the integrity of the subcortical actin cytoskeleton as well as for TCR-driven WASp activation, F-actin assembly, immune synapse formation, actin foci formation, mechanotransduction, T cell transendothelial migration, and homing to lymph nodes, all of which also depend on WASp. These results indicate that DOCK8 and WASp are in the same signaling pathway that links TCRs to the actin cytoskeleton in TCR-driven actin assembly. Further, they provide an explanation for similarities in the clinical phenotypes of WAS and DOCK8 deficiency.United States. Public Health Service (RO1AI114588)United States. Public Health Service (K08AI114968

TRIF signaling is essential for TLR4-driven IgE class switching

The TLR4 ligand LPS causes mouse B cells to undergo IgE and IgG1 isotype switching in the presence of IL-4. TLR4 activates two signaling pathways mediated by the adaptor molecules MyD88 and Toll/IL-IR domain-containing adapter-inducing IFN-beta (TRIF)-related adaptor molecule (TRAM), which recruits TRIF. Following stimulation with LPS plus IL-4, Tram(-/-) and Trif(-/-) B cells completely failed to express Cepsilon germline transcripts (GLT) and secrete IgE. In contrast, Myd88(-/-) B cells had normal expression of Cepsilon GLT but reduced IgE secretion in response to LPS plus IL-4. Following LPS plus IL-4 stimulation, Cgamma1 GLT expression was modestly reduced in Tram(-/-) and Trif(-/-) B cells, whereas Aicda expression and IgG1 secretion were reduced in Tram(-/-), Trif(-/-), and Myd88(-/-) B cells. B cells from all strains secreted normal amounts of IgE and IgG1 in response to anti-CD40 plus IL-4. Following stimulation with LPS plus IL-4, Trif(-/-) B cells failed to sustain NF-kappaB p65 nuclear translocation beyond 3 h and had reduced binding of p65 to the Iepsilon promoter. Addition of the NF-kappaB inhibitor, JSH-23, to wild-type B cells 15 h after LPS plus IL-4 stimulation selectively blocked Cepsilon GLT expression and IgE secretion but had little effect on Cgamma1 GLT expression and IgG secretion. These results indicate that sustained activation of NF-kappaB driven by TRIF is essential for LPS plus IL-4-driven activation of the Cepsilon locus and class switching to IgE

The Thoc1 encoded ribonucleoprotein is required for myeloid progenitor cell homeostasis in the adult mouse.

Co-transcriptionally assembled ribonucleoprotein (RNP) complexes are critical for RNA processing and nuclear export. RNPs have been hypothesized to contribute to the regulation of coordinated gene expression, and defects in RNP biogenesis contribute to genome instability and disease. Despite the large number of RNPs and the importance of the molecular processes they mediate, the requirements for individual RNP complexes in mammalian development and tissue homeostasis are not well characterized. THO is an evolutionarily conserved, nuclear RNP complex that physically links nascent transcripts with the nuclear export apparatus. THO is essential for early mouse embryonic development, limiting characterization of the requirements for THO in adult tissues. To address this shortcoming, a mouse strain has been generated allowing inducible deletion of the Thoc1 gene which encodes an essential protein subunit of THO. Bone marrow reconstitution was used to generate mice in which Thoc1 deletion could be induced specifically in the hematopoietic system. We find that granulocyte macrophage progenitors have a cell autonomous requirement for Thoc1 to maintain cell growth and viability. Lymphoid lineages are not detectably affected by Thoc1 loss under the homeostatic conditions tested. Myeloid lineages may be more sensitive to Thoc1 loss due to their relatively high rate of proliferation and turnover

Effects of Thoc1 deficiency on myeloid progenitor cell cycle and apoptosis.

<p>A) The cell cycle phase distribution of bone marrow GMPs from tamoxifen treated mice of the indicated genotype was measured by Hoechst 33343 staining and flow cytometry. The graph shows the mean and standard error for 7 different mice of each genotype. Asterisks mark statistically significant differences between genotypes (t-test P<0.01). B) The cell cycle distribution of HSC cells from tamoxifen treated mice was determined. Asterisks mark statistically significant differences between genotypes (t-test P<0.01). C) The cell cycle distribution of MPP cells from tamoxifen treated mice was determined. Asterisks mark statistically significant differences between genotypes (t-test P<0.01). D) The graph shows the percentage of in vitro cultured GMPs of the indicated genotype that stain positive for the apoptotic marker Annexin V and negative for DAPI subsequent to tamoxifen treatment. The data show the mean and standard error for a total of 7 different mice for each genotype analyzed in 2 independent pools of samples. Asterisks mark statistically significant differences between genotypes (t-test P<0.01). E) GMPs in D) were monitored for Cre mediated deletion of the floxed <i>Thoc1</i> allele by PCR and agarose gel electrophoresis. GMPs of the indicated genotype were treated with increasing doses of tamoxifen. The upper panel shows results from PCR using primers specific for the wild type or unrecombined floxed <i>Thoc1</i> alleles. The lower panel shows results using primers specific for the Cre deleted floxed <i>Thoc1</i> allele.</p

Thoc1 deficiency in the bone marrow causes a decline in myeloid cells, but not lymphocytes.

<p>A) CD45.1 wild type mice were irradiated and transplanted with bone marrow from Cd45.2 <i>Thoc1^F/F</i>:<i>Rosa26^CreERT2</i> (<i>Thoc1^F/F</i>) or <i>Thoc1^+/+</i>:<i>Rosa26^CreERT2</i> (<i>Thoc1^+/+</i>) mice. 9 weeks later, CD45.1 and CD45.2 positive peripheral white blood cells were counted by flow cytometry. The percentage of CD45.1 (host, blue) and CD45.2 (donor, red) cells is shown. Each data point is from a different mouse with bars representing the mean and standard error. B) Mice in A) were treated with tamoxifen and bone marrow isolated 10 days later. RNA was extracted and <i>Thoc1</i> RNA levels measured by real time RT-PCR. Each data point is from a different <i>Thoc1^F/F</i>:<i>Rosa26^CreERT2</i> (F/F) or <i>Thoc1^+/+</i>:<i>Rosa26^CreERT2</i> (+/+) mouse, and the data are normalized to one of the control mice. The difference in <i>Thoc1</i> RNA levels between genotypes is significant (t-test P = 0.003). C) Protein was extracted from the thymus (1–6) and spleen (7–9) of tamoxifen treated <i>Thoc1^F/F</i>:<i>Rosa26^CreERT2</i> (red) and <i>Thoc1^+/+</i>:<i>Rosa26^CreERT2</i> (blue) mice in B). Extracts were analyzed for the indicated proteins with actin serving as a loading control. D) The small intestine was harvested from mice in B) and tissue sections stained with H&E. Representative images from mice of the indicated genotype are shown. Scale bars represent 200 microns. E) Peripheral blood was isolated from tamoxifen treated <i>Thoc1^F/F</i>:<i>Rosa26^CreERT2</i> (F/F) or <i>Thoc1^+/+</i>:<i>Rosa26^CreERT2</i> (+/+) mice in B) and the percentage of the indicated cell types counted. Each data point is from a different mouse with bars representing the mean. Significant differences (t-test P<0.02) are noted by *. F) Spleen cells were isolated from the tamoxifen treated mice in B) and the percentage of the indicated cell types counted as in E). Significant differences (t-test P<0.01) are noted by ∧. F) Lymph node cells were isolated from the tamoxifen treated mice in B) and the percentage of the indicated cell types counted as in E).</p

Myeloid progenitor cells are affected by Thoc1 deficiency.

<p>A) Bone marrow was recovered from tamoxifen treated <i>Thoc1^F/F</i>:<i>Rosa26^CreERT2</i> (F/F) or <i>Thoc1^+/+</i>:<i>Rosa26^CreERT2</i> (+/+) mice and the number of viable cells counted by flow cytometry. Each data point is from a different mouse with bars representing the genotype mean. Differences between genotypes are significant (t-test P = 0.01). B) An equal number of viable bone marrow cells isolated in A) were cultured in methylcellulose to assess colony forming potential. Each data point shows the number of colonies generated with samples from a different mouse with bars representing the mean. Differences between genotypes are significant (t-test P = 0.0006). C) Pre-granulocyte macrophage progenitors (Pre-GMP), granulocyte macrophage progenitors (GMP), pre-megakaryocyte erythroid progenitors (Pre-MegE), and erythroid progenitors (Pre-CFU-E) were counted in the bone marrow from A) using immunophenotyping and flow cytometry. Each data point is from a different mouse with bars representing the mean. Significant differences (t-test P<0.01) between genotypes are noted by *. D) Data from C) is plotted as a percentage of total viable bone marrow cells analyzed. Significant differences (t-test P<0.02) between genotypes are noted by *. E) A schematic outlining a simplified view of hematopoiesis highlighting the bifurcation of multi-potent progenitor cells (MPP) cells into common lymphoid progenitor cells (CLP) or common myeloid progenitor cells (CMP). HSC indicates hematopoietic stem cells. F) HSC and MPP cells from bone marrow in A) were counted as in C). Results from the different genotypes are not significantly different (t-test P>0.18). G) Data from E) is plotted as a percentage of total viable bone marrow cells analyzed. Significant differences (t-test P<0.05) between genotypes are marked by *.</p