Figure 6. LL-37 upregulates the expression of IFN-a, but not TNF-a in thymic pDC. Sorted pDC were incubated with LL-37 plus or minus eukaryotic DNA/RNA or LL-37-DNA/RNA complex pretreated with DNAse and RNAse. CpG-A 2216 was used as a positive control. Q-PCR was performed for IFN-a and TNF-a mRNA. doi:10.1371/journal.pone.0024252.g006 trigger peripheral blood pDC to secrete IFN-a via TLR-9 [29]. The same group reported that eukaryotic RNA/LL-37 complexes trigger the secretion of IFN-a by pDC through TLR7 activation [30]. More recently it was demonstrated that IFN-a secretion in response to CpG-ODNs can also be mediated by the cytosolic sensors aspartate-glutamate-any amino acid-aspartate/histidine (DExD/H)-box helicase 36 (DHX36) and DHX9, which is TLR7 and 9 independent but MyD88 dependent [40]. CAMP1, the gene encoding LL-37, is expressed in many tissues with the highest expression levels in bone marrow and thymus [31]. Our results show that LL-37 is mainly expressed in the thymic medulla, although some expression was also observed in the cortex. The co- localization of pDC and LL-37 in the thymic medulla where nucleic acids may be present from thymocytes undergoing apoptosis as a result of negative selection [32], suggests that LL- 37 is a likely trigger for the constitutive secretion of IFN-a in the thymus. Although it is known that macrophages will rapidly phagocytose apoptotic cells, we previously showed that apoptotic CD4+ thymocytes can be identified in the human thymus [41]. We also found LL-37 expression in the fetal spleen, but as there is little apoptosis in this organ, it can be assumed that the lack of DNA accounts for the lack of IFN-a secretion and subsequent MxA expression in the fetal spleen. We previously reported that MxA is highly expressed in pDC and mature thymocytes in response to CpG or HIV-1 induced type I IFN production [17]. In the current study we show that pDC constitutively express IFN-a in the absence of microbial stimulation, and are likely the main source of IFN-a secretion in the thymus. The observation that pDC in normal thymus express high levels of MxA indicates that they also responded to secreted IFN-a in an autocrine fashion. This is consistent with the notion that pDC employ type I IFNs for their survival [36]. It is of note, that the mean fluorescence intensity of MxA is significantly higher in pDC than in other thymocyte subsets, although the explanation for this is currently unclear. It is possible that this can be attributed to increased responsiveness to type I IFNs due to higher expression levels of the type I IFN receptor or downstream signaling molecules. Alternatively, it can be envisioned that negative regulators of the JAK/STAT pathway, such as SOCS proteins, are differentially activated in pDC compared to thymocytes. Constitutive IFN-a Expression in the Human Thymus Our current data using immunofluorescence, and those of Meager et al [42], indicate that MxA expressing cells are mainly present in the thymic medulla. We confirmed these findings by flow cytometry as detailed characterization of MxA+ cells enriches for cells with a medullary phenotype, including CD3+/hiCD27+C- 2 + D1a CD45RA . Meager et al. [42], in a study of anti-interferon autoantibodies in autoimmunity, showed that resident macro- phages expressed IFN-a in the postnatal thymus, but did not express MxA and are therefore likely only storing IFN-a and not actively secreting it. The data presented here suggest that IFN-a is continuously produced in the thymic medulla by pDC resulting in MxA expression in medullary thymocytes and pDC. However it is still unclear what role this cytokine plays in thymopoiesis in vivo. We previously reported that IFN-a is a reversible inhibitor of human T cell development [16]. Murine studies found that exogenous IFN-a interfered with early T and B cell development at stages where IL-7 stimulation is necessary [13]. Utilizing the OP-9 culture system to mimic thymopoiesis in vitro, we showed that IFN-a inhibits early stages of thymocyte development as it delayed the acquisition of CD1a on developing CD34+ thymic progenitor cells [16]. In addition we found that IFN-a inhibited later stages of T cell development from CD3+hiCD1a+/hi to CD3+/hiCD1a-[16]. Cells of these developmental stages, which are present at the cortico-medullary junction, coincide with stages where thymocytes are responsive to IL-7 induced proliferation. Taken together, these data suggest that IFN-a may serve to control the rate of thymopoiesis. Another attractive consideration is that IFN-a secretion may play a role in negative selection. Autoreactive T cells are negatively selected in the medulla and are removed via apoptosis (reviewed in[43]) induced by several mechanisms including TRAIL-mediated cell death (reviewed in[44]). This is consistent with reports demonstrating that the expression of MxA increases a cell’s susceptibility to cell death [45,46]. Hence, it is possible that mature MxA+ thymocytes may be more sensitive to cell death as a result of MxA expression. The increased susceptibility to cell death may reduce the signal intensity necessary to delete autoreactive T cells. The expression of MxA in mature thymocytes may aid in reducing the threshold needed for negative selection. Finally, we believe that our data have implications in the pathogenesis of CCR5-tropic compared to CXCR4-tropic HIV-1 infection in the thymus. Previously we reported that the proportion of pDC expressing IFN-a increased after infection with either CXCR4 or CCR5-tropic HIV-1 [17]. We and others have shown that infection with CCR5-tropic viruses leads to infection of mature thymocytes within the thymic medulla [47,48,49,50]. We speculate that the constitutive expression of ISG in the medulla may make this region relatively more resistant to infection. This notion is in line with the reduced pathogenicity of CCR5-tropic strains when compared to CXCR4-tropic strains [50,51]. In contrast, CXCR4-tropic strains may be more pathogenic due to the fact that they initially infect cells located within the cortex where we did not observe constitutive ISG expression [47,49,51,52]. It is tempting to speculate that by infecting cells that are distantly located from pDC and the ISG, these virus strains are able to replicate much more efficiently. This suggests that constitutive IFN-a secretion, in addition to its potential role in T cell development, is essential in inhibiting acute infection with CCR5-tropic HIV-1. Supporting Information Figure S1 pressed in pSTAT1 and IRF-7 are preferentially ex- mature thymocytes. Total thymocytes were PLoS ONE | www.plosone.org 9 August 2011 | Volume 6 | Issue 8 | e24252