Constitutive IFN-a Expression in the Human Thymus Figure 4. Thymic pDC constitutively express IFN-a, MxA and pSTAT1. A, pDC from post-natal thymus express IFN-a. Total thymocytes were stained for surface expression of CD123, CD45RA, CD4, CD8, CD27, CD1a and CD3 and intracellular expression of IFN-a. IFN-a expression of pDC was determined by gating on CD45RA + CD123+/hi cells, that lacked CD3 and CD1a. B, pDC from post-natal thymus express MxA and pSTAT1. Total thymocytes were stained for surface expression of CD45RA, CD123, and CD3 and intracellular expression of MxA or pSTAT1. MxA and pSTAT1 expression in pDC was determined by gating on CD45RA + CD123+/hi cells that lacked CD3. C, pDC express the highest level of MxA. Total thymocytes were stained for surface expression of CD45RA, CD123, and CD3 and intracellular expression of MxA. Mean fluorescence intensity (MFI) of MxA expression in pDC was determined by gating on CD3 2 CD45RA+CD123+/hi cells. MxA MFI expression in mature and immature thymocyte subsets was determined by gating on CD45RA+CD3+/hi, CD45RA2CD3+ and CD3low/2 cells. D, pDC are located in the medulla of normal thymus. Thymus tissue was formalin fixed and paraffin embedded. 4 um sections were mounted on slides. Thymus tissue was stained for CD123 followed by Rabbit anti- mouse IgG1 and Goat anti-Rabbit Alexa Fluor 594. MxA was conjugated with zenon anti-IgG2a Alexa 488. doi:10.1371/journal.pone.0024252.g004 compared to LL37/eukaryotic RNA/DNA complexes. In addi- tion, we found that autologous DNA from apoptotic thymocytes complexed with LL-37 induced MxA mRNA expression and IFN- a production in the supernatant of cultured pDC (data not shown). Collectively, these results provide evidence for the notion that LL- 37 when complexed with DNA/RNA is able to increase IFN-a production, which may subsequently induce MxA expression in the medulla of the thymus. Notably, while stimulation of pDC with CpG-A also resulted in the production of TNF-a, stimulation with LL-37 complexed with DNA/RNA did not (Fig. 6). This result suggests that thymic pDC do not promote a pro-inflammatory state in the thymic medulla. Discussion Here we show that the interferon induced protein MxA is constitutively expressed in post-natal and fetal thymus, but not in peripheral lymphoid tissues including fetal spleen and lymph nodes (from the same donor as the thymus). In addition, we show that the majority of thymic pDC express high levels of IFN-a. This together with their medullar localization strongly suggests that thymic pDC are responsible for inducing MxA expression exclusively in the medulla. Finally, we observed that the anti- microbial peptide LL-37 is expressed in the thymus, and that this in complex with eukaryotic DNA/RNA can stimulate thymic pDC to express IFN-a in vitro. Collectively, our findings imply that type I IFNs, which are constitutively expressed in the medulla, may have a direct role on the development of T cells in the thymus. MxA has been used as a reliable marker to detect type I IFN in clinical settings, including SLE and hepatitis C infection [37,38], as it is exclusively expressed after engagement of type I IFNs to its receptor [9]. Hence, we conclude that type I IFNs are constitutively expressed in the thymus, but not in the spleen or lymph node. This is consistent with our previous observations that IFN-a expressing pDC are present in the human fetal thymus/ liver implant in the SCID-hu mouse [17]. Our conclusion is further enforced by the findings that pSTAT1 and IRF-7, which are both activated upon type I IFN signaling, are expressed in the normal thymus [33]. The fact that not only post-natal but also fetal thymus tissue harbors a type I IFN stimulated gene signature endorses the notion that this phenomenon occurs in the absence of viral or bacterial infection and is further supported by the presence of ‘‘immunoreactive’’ IFN-a in fetal thymus tissues described many years ago [39]. A notable finding in our study is that MxA expressing thymocytes (Fig.1) and pDC (Fig. S3), were only present in fetal and postnatal thymus tissues, but not in fetal spleen or lymph node or in adult peripheral blood. This prompted us to search for a stimulus which was only present in the thymus, but not in the peripheral lymphoid organs. We considered the antimicrobial peptide LL-37 as a potential candidate based on a recent report that LL-37 isolated from psoriasis skin lesions can bind DNA and Figure 5. LL-37 is located in the cortex and medulla of the thymus and in non-B cell zones of the fetal spleen. Fetal thymus, spleen, and lymph nodes were formalin fixed and paraffin embedded. 4 um sections were mounted on slides and stained with a monoclonal antibody to LL-37 or CD19 followed by biotinylated rabbit anti-mouse antibody and streptavidin-biotin-peroxidase complex. IgG2a was used as control antibody. (C) cortex; (M) medulla. The fetal tissues were obtained from the same donor. doi:10.1371/journal.pone.0024252.g005 PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e24252