Dock10 regulates CD23 expression and sustains B-cell lymphopoiesis in secondary lymphoid tissue
Introduction
Dedicator of cytokinesis 10 (Dock10, Zizimin3) belongs to the Dock family of guanosine nucleotide exchange factors (GEFs) for small Rho GTPases (Yelo et al., 2008, Gadea and Blangy, 2014). Dock10 targets and activates Rac1 and Cdc42 (Ruiz-Lafuente et al., 2015, Jaudon et al., 2015, Gadea et al., 2008). Rho GTPases play essential roles in actin cytoskeleton dynamics and cell motility (Wennerberg and Der, 2004, Heasman and Ridley, 2008). Rac1 stimulates lamellipodia and membrane ruffles formation, and Cdc42 regulates cell polarity and induces filopodia (Aspenström et al., 2004, Chhabra and Higgs, 2007). Rac1 and Cdc42 regulate the actin cytoskeleton through interactions with Wiskott–Aldrich syndrome proteins, diaphanous-related formins, and p21 protein activated kinases. Dock10 is expressed as two forms with divergent amino termini, named Dock10.1 and Dock10.2, expressed preferentially by T cells and B cells, respectively (Fig. 1A). Dock10 expression, especially that of Dock10.2, is strongly upregulated in B cells by IL-4 (Yelo et al., 2008; Alcaraz-García et al., 2011; Ruiz-Lafuente et al., 2014). IL-4 is a pleiotropic cytokine that induces maturation of B cell precursors into Ig-secreting cells, isotype switching toward IgE, and has a potent antiapoptotic effect in vitro (Okada et al., 2003).
Rho GTPases are required for normal lymphopoiesis (Tybulewicz and Henderson, 2009, Mulloy et al., 2010). B-cell lymphopoiesis begins in the bone marrow (BM), where the immunoglobulin (Ig) genes are rearranged. B cells expressing surface IgM migrate to the spleen, where they maturate into two main subpopulations, the follicular (FO) B cells and the marginal zone (MZ) B cells, which make up the two separate areas of the lymphoid follicles (LFs), i.e., the germinal center (GC) and the MZ, respectively. The FO B cells, characterized by membrane CD23 expression, recirculate between secondary lymphoid organs. T-cell lymphopoiesis begins in the thymus, where CD4−CD8− double negative (DN) thymocytes differentiate into CD4+CD8+ double positive (DP) cells. DP thymocytes go through positive selection into either CD4+CD8− or CD4−CD8+ single positive (SP) thymocytes. SP thymocytes recirculate between secondary lymphoid organs. Rac1 and Cdc42 global KO mice are embryonic lethal. B cell-specific double deletion of Rac1 and Rac2 results in reduced numbers of both FO B cells and MZ B cells (Walmsley et al., 2003). T cell-specific double deletion of Rac1 and Rac2 blocks thymic differentiation resulting in decreased numbers of DP and SP thymocytes, and of T cells in peripheral blood (PB) and spleen (Guo et al., 2008, Dumont et al., 2009). B cell-specific deletion of Cdc42 impairs B-cell receptor (BCR) signaling and compromises B-cell development in BM and spleen, resulting in fewer and smaller LFs (Guo et al., 2009, Burbage et al., 2015). Specific deletion of Cdc42 in mature B cells impairs their adhesion capacity, ability to activate T cells, antibody affinity maturation and homing to the B cell follicles of the spleen (Gerasimcik et al., 2015). T cell-specific deletion of Cdc42 blocks thymic differentiation resulting in a reduction in the number of SP thymocytes (Guo et al., 2011).
Other Dock proteins significantly expressed in lymphoid tissue, such as Dock2, Dock8, and Dock11, have diverse effects in lymphocyte development (Gadea and Blangy, 2014). Dock2 is a Rac specific GEF that regulates lymphocyte trafficking in and out of the secondary lymphoid organs. In the Dock2 KO mice, there is an impaired homing of T and B lymphocytes in spleen and lymph nodes, and GCs are poorly structured (Fukui et al., 2001). Dock8 is a Cdc42 specific GEF whose mutations in humans cause a rare immune deficiency called autosomal recessive hyper IgE syndrome (Engelhardt et al., 2009). In mice, DOCK8 mutant B cells are unable to form MZ B cells or to persist in GCs and undergo affinity maturation (Randall et al., 2009). Dock11 is a Cdc42 specific GEF required for MZ but not FO B-cell development (Matsuda et al., 2015). Dock10 KO mice were also studied in the latter report and, like the Dock11 KO mice, displayed narrowed MZs, suggesting that Dock10 could also be involved in MZ development.
In this paper, we aimed to investigate Dock10 function by studying hematopoietic development in a global Dock10 KO mice model. Our results show that Dock10-deficient mice have decreased numbers of B cells in secondary lymphoid organs, and FO B cells display membrane CD23 overexpression. These results suggest that Dock10 plays a role in B-cell lymphopoiesis in secondary lymphoid tissue.
Section snippets
Mice
Our study model consisted of two cohorts of C57BL/6N mice, WT mice and Dock10 KO, respectively, established from two heterozygous breeding pairs kindly sent from the Helmholtz Zentrum München (Munich, Germany). The mutant allele, named Dock10tm1a(EUCOMM)Hmgu/Ieg (GenBank accession no. JN946611.1), designates a KO first allele, i.e., a reporter-tagged insertion allele with conditional potential. It bears the L1L2_Bact_P cassette just upstream of exon 4, and loxP sequences flanking exon 4 (Fig. 1
Dock10 protein and mRNA expression in mice
By western blot analysis of spleen protein extracts using two different antibodies, one that recognizes total Dock10 and another Dock10.1, it was shown that homozygous mice bearing the transgene had no expression of the Dock10 protein, and heterozygous mice had half the levels as those of homozygous WT mice (Fig. 1D). Therefore, though the mutant allele was created as ‘conditional ready’, mice were indeed KO without the need to cross with Cre mice to excise the critical exon 4. This was
Discussion
This study explores Dock10 function using global Dock10 KO mice. Since Dock10 expression is most prominent in PB lymphocytes, followed by spleen lymphocytes, we investigated whether Dock10 suppression affected hematopoietic development. The main findings were reductions of B cells in PB and spleen, but normal primary hematopoiesis in central organs. These results suggest that Dock10 plays a role in late stages of B lymphoid maturation. The B cell blockade involving FO and MZ B cells and not
Conclusions
The study of Dock10 KO mice suggests that Dock10 plays a role in B-cell development. Specifically, Dock10 sustains B-cell lymphopoiesis in secondary lymphoid tissue. In addition, CD23 was overexpressed on the membrane of FO B cells in Dock10 KO mice. Negative regulation of CD23 expression by Dock10 could play a role in B-cell development.
Conflict of interest
The authors declare that they have no conflicts of interest with the contents of this article.
Funding
This work was supported by Instituto de Salud Carlos III (grant numbers PI07/0135 and PI10/01226) (co-financed by the European Regional Development Fund, “Una manera de hacer Europa”), and Fundación Séneca, Agencia de Ciencia y Tecnología de la Región de Murcia (grant number 08721/PI/08).
Acknowledgements
We thank the European Mouse Mutant Archive (EMMA) network for granting Transnational Access to the Dock10 null mice to us.
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Both authors contributed equally to this work.