Type I IFN is a major player in innate and adaptive immune responses. Besides, it is involved in organogenesis and tumor development. Generally, IFN responses are amplified by an autocrine loop with IFN-β as the priming cytokine. However, due to the lack of sensitive detection systems, where and how type I IFN is produced in vivo is still poorly understood. In this study, we describe a luciferase reporter mouse, which allows tracking of IFN-β gene induction in vivo. Using this reporter mouse, we reveal strong tissue-specific induction of IFN-β following infection with influenza or La Crosse virus. Importantly, this reporter mouse also allowed us to visualize that IFN-β is expressed constitutively in several tissues. As suggested before, low amounts of constitutively produced IFN might maintain immune cells in an activated state ready for a timely response to pathogens. Interestingly, thymic epithelial cells were the major source of IFN-β under noninflammatory conditions. This relatively high constitutive expression was controlled by the NF Aire and might influence induction of tolerance or T cell development.

BackgroundSeveral facultative anaerobic bacteria with potential therapeutic abilities are known to preferentially colonize solid tumors after systemic administration. How they efficiently find and invade the tumors is still unclear. However, this is an important issue to be clarified when bacteria should be tailored for application in cancer therapy.Methodology/Principal FindingsWe describe the initial events of colonization of an ectopic transplantable tumor by Salmonella enterica serovar Typhimurium. Initially, after intravenous administration, bacteria were found in blood, spleen, and liver. Low numbers were also detected in tumors associated with blood vessels as could be observed by immunohistochemistry. A rapid increase of TNF-α in blood was observed at that time, in addition to other pro-inflammatory cytokines. This induced a tremendous influx of blood into the tumors by vascular disruption that could be visualized in H&E stainings and quantified by hemoglobin measurements of tumor homogenate. Most likely, together with the blood, bacteria were flushed into the tumor. In addition, blood influx was followed by necrosis formation, bacterial growth, and infiltration of neutrophilic granulocytes. Depletion of TNF-α retarded blood influx and delayed bacterial tumor-colonization.ConclusionOur findings emphasize similarities between Gram-negative tumor-colonizing bacteria and tumor vascular disrupting agents and show the involvement of TNF-α in the initial phase of tumor-colonization by bacteria.

T-cell development depends on recruitment of bone marrow-derived precursor cells to the thymus via a multistep adhesion cascade involving the chemokine receptor CCR9. However, CCR9 deficiency does not result in complete abrogation of progenitor entry into the adult thymus. Therefore, we tested the hypothesis that additional chemokine/ chemokine receptor systems might play a role in this process. To this end, we generated mice deficient in both CCR9 and CCR7. Deficiency in both chemokine receptors resulted in severely reduced numbers of early T-cell progenitors and in near-complete abrogation of thymus reconstitution. Progenitors in bone marrow and peripheral blood remained largely unaffected in CCR7 ؊/؊ CCR9 ؊/؊ mice, and direct intrathymic transfer of precursors from CCR7 ؊/؊ CCR9 ؊/؊ mice as well as single-mutant mice showed that intrathymic differentiation of these precursors remained functional. Thus, our data reveal a previously unrecognized role of CCR7 in progenitor seeding of the adult thymus, which is largely masked by compensatory effects of CCR9 signals. In turn, CCR7 signals can partially compensate for CCR9 signals, thus explaining the rather mild phenotype of CCR9 ؊/؊ mice with respect to progenitor seeding. (Blood. 2010;115:1906-1912

T-cell receptor (TCR) signal strength determines selection and lineage fate at the CD4 + CD8+ double-positive stage of intrathymic T-cell development. Members of the miR-181 family constitute the most abundantly expressed microRNA at this stage of T-cell development. Here we show that deletion of miR-181a/b-1 reduced the responsiveness of double-positive thymocytes to TCR signals and virtually abrogated early invariant natural killer T (iNKT) cell development, resulting in a dramatic reduction in iNKT cell numbers in thymus as well as in the periphery. Increased concentrations of agonist ligand rescued iNKT cell development in miR-181a/b-1 −/− mice. Our results define a critical role of miR-181a/b-1 in early iNKT cell development and show that miR-181a/b-1 sets a TCR signaling threshold for agonist selection.

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MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by sequence-specific targeting of multiple mRNAs. Although lineage-, maturation-, and disease-specific miRNA expression has been described, miRNAdependent phenotypes and miRNAregulated signaling in hematopoietic cells are largely unknown. Combining functional genomics, biochemical analysis, and unbiased and hypothesis-driven miRNA target prediction, we show that lentivirally over-expressed miR-125b blocks G-CSF-induced granulocytic differentiation and enables G-CSF-dependent proliferation of murine 32D cells. In primary lineage-negative cells, miR-125b over-expression enhances colonyformation in vitro and promotes myelopoiesis in mouse bone marrow chimeras. We identified Stat3 and confirmed Bak1 as miR-125b target genes with approximately 30% and 50% reduction in protein expression, respectively. However, genespecific RNAi reveals that this reduction, alone and in combination, is not sufficient to block G-CSF-dependent differentiation. STAT3 protein expression, DNAbinding, and transcriptional activity but not induction of tyrosine-phosphorylation and nuclear translocation are reduced upon enforced miR-125b expression, indicating miR-125b-mediated reduction of one or more STAT3 cofactors. Indeed, we identified c-Jun and Jund as potential miR-125b targets and demonstrated reduced protein expression in 32D/ miR-125b cells. Interestingly, gene-specific silencing of JUND but not c-JUN partially mimics the miR-125b over-expression phenotype. These data demonstrate coordinated regulation of several signaling pathways by miR-125b linked to distinct phenotypes in myeloid cells. IntroductionMicroRNAs (miRNAs) represent an emerging class of noncoding single-stranded RNAs of approximately 22 nucleotides 1,2 that play an important role in posttranscriptional regulation of gene expression. miRNAs are processed from primary primiRNA transcripts to pre-miRNAs and mature miRNAs in a multistep process. Mature miRNAs are incorporated into and subsequently recruit a multi-protein effector complex RISC (RNA-induced silencing complex) to complementary miRNAbinding sites located preferentially within the 3ЈUTR of target mRNAs. Sequence-specific binding of RISC results in reduced mRNA translation and/or degradation through RNA interference (RNAi). 3 The interaction between a miRNA and its target mRNAs usually requires complementarity only within so-called seed sequence (miRNA nucleotides 2-8). Hence, a single miRNA has the potential to regulate hundreds of proteins 4-6 but resulting target protein repression is typically relatively mild. 5,6 Thereby, the ratio of regulatory RNAs to target mRNAs may modulate the silencing activity with a negative correlation between target abundance and target down-regulation. 7 To identify miRNA target genes, several prediction programs based on the hybridization energy of complementary miRNA/mRNAs sequences have been described. [8][9][10][11][12] However, the use of these programs is error-prone and identification of miRNA tar...

T-cell development in the thymus depends on continuous supply of T-cell progenitors from bone marrow (BM). Several extrathymic candidate progenitors have been described that range from multipotent cells to lymphoid cell committed progenitors and even largely T-lineage committed precursors. However, the nature of precursors seeding the thymus under physiologic conditions has remained largely elusive and it is not known whether there is only one physiologic T-cell precursor population or many. Here, we used a competitive in vivo assay based on depletion rather than enrichment of classes of BM-derived precursor populations, thereby only minimally altering physiologic precursor ratios to assess the contribution of various extrathymic precursors to T-lineage differentiation. We found that under these conditions multiple precursors, belonging to both multipotent progenitor (MPP) and common lymphoid progenitor (CLP) subsets have robust T-lineage potential. However, differentiation kinetics of different precursors varied considerably, which might ensure continuous thymic output despite gated importation of extrathymic precursors. In conclusion, our data suggest that the thymus functions to impose T-cell fate on any precursor capable of filling the limited number of progenitor niches. (Blood. 2010;115:1137-1144) IntroductionT-cell development in the thymus depends on continuous supply of T-cell progenitors from bone marrow (BM) via the circulation. In the thymus T-cell precursors pass through a series of defined developmental stages, with the most immature thymocytes residing in the double-negative (DN) subset, characterized by the absence of the surface markers CD4 and CD8. Thymocyte differentiation then proceeds through the CD4 ϩ CD8 ϩ double-positive (DP) stage, after which thymocytes become either CD4 or CD8 single positive (SP) and leave the thymus to enter the mature T-cell pool. The most immature T-cell progenitors in the thymus are lineage negative (lin Ϫ ), CD44 ϩ , CD25 Ϫ , Sca-1 high , CD117 (c-kit) high (LSK), and CD127 Ϫ/lo (IL-7R␣) early T-lineage progenitors (ETPs), 1 which constitute a subfraction of the CD44 ϩ CD25 Ϫ DN1 population. These cells were shown to have high T but only limited B and some myeloid potential. 2,3 ETPs could be further subdivided according to their expression levels of CD135 (Fms-like tyrosine kinase receptor 3 [Flt3]) 4 and loss of CD135 expression correlated with loss of B-cell potential.Like all hematopoietic lineages T cells are ultimately derived from hematopoietic stem cells (HSCs) residing in BM. HSCs can generate CD135 ϩ multipotent progenitors (MPPs), which are likewise of the LSK phenotype, 5 as well as more committed precursors such as RAG-1-positive early lymphoid progenitors (ELPs) 6 and L-selectin-positive progenitors (LSPs), 7 both of which constitute subsets of MPPs. Common lymphoid progenitors (CLPs), 8 which are lin Ϫ Sca-1 ϩ CD117 ϩ/lo CD127 ϩ CD135 ϩ , and lin Ϫ Sca-1 ϩ CD117 Ϫ CD127 ϩ CD135 ϩ B220 ϩ CLP-2 9 differ from MPP subsets in their lack of myeloid pot...

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