To examine this possibility, CFSE splenocytes from LPS-treated mice were transferred into recipient mice treated with Dex after the inflammatory process was triggered by LPS (Fig. 2F). Interestingly, in this experimental condition the entrance of peripheral cells into the thymus occurred. Similar data were observed when T. cruzi infected mice were used instead of LPS-treated mice click here (data not shown). Overall, these data indicate that space is necessary but not sufficient for the entrance of cells into the thymus and we hypothesize that specific signals that recruit peripheral cells into the organ are also required. To characterize the phenotype of cells that

enter the thymus during Th1-inflammatory/infectious processes, we analyzed the expression of markers that discriminate between naïve, recently act-ivated or memory T cells (CD44, CD62L, R428 in vitro and CD69). Data shown in Fig. 3A demonstrate that cells that enter the thymus exhibited high expression of CD44 and CD62L but low expression of CD69. Together, cells migrating to the thymus exhibited surface expression markers compatible with a central memory phenotype. It has been demonstrated that traffic of peripheral B and T cells to the thymus in AKR mouse is mediated by the expression of L-selectin

on immigrating lymphocytes [11]. Thus, we analyzed CD62L expression in all the cell types recruited to the thymus in LPS-treated and T. cruzi infected mice. As shown in Fig. 3B, CD62L was expressed by most immigrating B and CD4+ T cells and about 70% of CD8+ lymphocytes,

suggesting that the integrin could represent an important pathway for cells to extravasate into the thymus. However, data presented in Fig. 3C demonstrate that CD62L is not involved AZD9291 cell line in cell migration to the thymus since splenocytes from LPS-treated mice incubated with an anti-CD62L neutralizing Ab before the adoptive transfer did not affect migration of either mature T or B cells to the thymus (Fig. 3C), but highly diminished the entrance of transferred cells to popliteal LNs (data not shown) [28]. Similar results were found in the LPS model (data not shown). did not participate in the entry of mature lymphocytes into the thymus, we focused our attention on other integrin/chemokines candidates. We found that the expression of the chemokine MCP-1 was highly upregulated in the thymi of LPS-treated, C. albicans, or T. cruzi infected mice compared with that of controls (Fig. 4A). Ex vivo treatment of thymocytes from T. cruzi infected mice with Brefeldin A for 4 h and then intracellular staining with an anti-MCP-1 Ab demonstrated a low but consistent detection of MCP-1+ cells (Supporting Information Fig. 1). The expression of MCP-1 was mainly restricted to B and CD4+ and CD8+ CD44lo resident thymocytes, but not to CD44hi peripheral T-cell counterparts or CD11b+ and CD11c+ subsets (Supporting Information Fig. 1).

, 1994) in conjunction with anti-APH_1387, and examined the cells using confocal microscopy. By comparing the staining patterns obtained with FK1 and FK2, we can infer whether the AVM is mono- or polyubiquitinated (Haglund et al., 2003; Collins et al., 2009; Ivanov & Roy, 2009). AVM staining by both FK2 and FK1 would indicate that the AVM is polyubiquitinated or poly- and monoubiquitinated, whereas staining with FK2 but not FK1 would suggest that the AVM is only monoubiquitinated. LBH589 FK1 staining yielded punctate patterns throughout infected and uninfected control cells (Fig. 1g and

k) similar to those obtained using FK2 (Fig. 1a,d and j). However, in contrast to that observed for FK2, neither an intense ring-like nor an aggregate FK1 staining pattern surrounding APH_1387- Seliciclib solubility dmso or APH_0032-positive A. phagocytophilum organisms was observed (Fig. 1i and data not shown). Moreover, FK1 staining did not colocalize with AVM-associated APH_1387 or APH_0032 signal. Similar results were obtained for A. phagocytophilum-infected RF/6A cells (data not shown). Because tetracycline treatment of A. phagocytophilum-infected host

cells results in dissociation of Rab GTPases from the AVM and delivery of the bacterium to lysosomes (Gokce et al., 1999; Huang et al., 2010a), we rationalized that bacterial protein synthesis is critical for the AVM to accumulate ubiquitinated conjugates. To test our hypothesis, we treated A. phagocytophilum-infected HL-60 cells with tetracycline or vehicle control for 60 min. The cells were screened with anti-Msp2 (P44) and FK2 followed by confocal microscopic examination. Whereas 39.9% ± 9.4% of AVMs

in control cells were FK2-positive, Cyclin-dependent kinase 3 tetracycline treated cells exhibited a significant reduction in ubiquitination, as only 16.0% ± 3.7% of AVMs were stained with FK2 (Fig. 5). This effect was reversible, as removal of the antibiotic restored AVM ubiquitination by 4 h. Thus, de novo bacterial protein synthesis is requisite for maintaining the association of ubiquitinated proteins with the AVM. This study demonstrates that A. phagocytophilum co-opts monoubiquitin conjugation machinery in a bacterial protein synthesis-dependent manner. Monoubiquitination of the AVM is important early during A. phagocytophilum development, as monoubiquitinated proteins rapidly associate with the ApV upon bacterial entry to produce a sparse punctate distribution pattern on the membranes of nascent ApVs. Monoubiquitination of the AVM is coincident with bacterial replication, as monoubiquitinated proteins continually accumulate on the AVM until 24 h, a time point at which we have documented that A. phagocytophilum begins to transition from the replicative and metabolically active reticulate cell form to the infectious dense-cored cell form (Troese & Carlyon, 2009).