Stromal cells as trend-setters for cells migrating into the lymph node.

Lymph node stromal cells are known to be immunorelevant during inflammation and tolerance. Differences between peripheral lymph nodes and mesenteric lymph nodes are important for an efficient and effective immune defense. Stromal cells were considered to be perfectly adapted to their draining area and not changeable concerning their expression pattern. Here we show that stromal cells can change their profile after isolation and transplantation into a different draining area. Subsequently, these newly organized lymph nodes are able to induce not only a region-specific but also an antigen-specific immune response. Thus, stromal cells are trend-setters for immune cells in producing a microenvironment that allows an optimized immune defense.

Transcriptional regulation of immediate-early gene response by THOC5, a member of mRNA export complex, contributes to the M-CSF-induced macrophage differentiation.

Hematopoiesis and commitment to a restricted lineage are guided by a timely expressed set of cytokine receptors and their downstream transcription factors. A member of the mRNA export complex, THOC5 (suppressors of the transcriptional defects of hpr1 delta by overexpression complex 5) is a substrate for several tyrosine kinases such as macrophage colony-stimulating factor (M-CSF) receptor and various leukemogenic tyrosine kinases, such as Bcr-Abl, or NPM-ALK. THOC5 tyrosine phosphorylation is elevated in stem cells from patients with chronic myeloid leukemia, suggesting that THOC5 may be involved in leukemia development. THOC5 is also an essential element in the maintenance of hematopoiesis in adult mice. In this report, we show that THOC5 is located in the nuclear speckles, and that it is translocated from the nucleus to cytoplasm during M-CSF-induced bone marrow-derived macrophage differentiation. Furthermore, we have identified THOC5 target genes by trancriptome analysis, using tamoxifen-inducible THOC5 knockout macrophages. Although only 99 genes were downregulated in THOC5-depleted macrophages, half of the genes are involved in differentiation and/or migration. These include well-known regulators of myeloid differentiation inhibitor of DNA binding (Id)1, Id3, Smad family member 6 (Smad6) and Homeobox (Hox)A1. In addition, a subset of M-CSF-inducible genes, such as Ets family mRNAs are THOC5 target mRNAs. Upon depletion of THOC5, unspliced v-ets erythroblastosis virus E26 oncogene homolog (Ets1) mRNA was accumulated in the nucleus. Furthermore, THOC5 was recruited to chromatin where Ets1 was transcribed and bound to unspliced and spliced Ets1 transcripts, indicating that THOC5 has a role in processing/export of M-CSF-inducible genes. In conclusion, regulation of immediate-early gene response by THOC5, a member of mRNA export complex contributes to the M-CSF-induced macrophage differentiation.

Macrophages from patients with atopic dermatitis show a reduced CXCL10 expression in response to staphylococcal α-toxin.

BACKGROUND: Patients with atopic dermatitis (AD) are frequently colonized with Staphylococcus aureus (S. aureus), one-third of them producing α-toxin, which is correlated with the severity of eczema in AD. Staphylococcus aureus colonizes in patients with psoriasis as well. Distinct expression of chemokine (C-C motif) ligand (CCL) and chemokine (C-X-C motif) ligand (CXCL) chemokines has been documented in both diseases. In this study, we investigated the effects of sublytic α-toxin concentrations on human macrophages that accumulate in the skin of patients with AD and psoriasis. METHODS: IFN-γ-induced protein of 10-kDa (IP-10)/CXCL10 and macrophage-derived chemokine (MDC)/CCL22 production were evaluated at the mRNA or at the protein level using qRT-PCR or ELISA, respectively. Cell surface markers' expression and chemotaxis were determined by flow cytometry and Boyden chamber technique, respectively. RESULTS: Sublytic concentrations of α-toxin strongly induced CXCL10 in macrophages at both the mRNA and the protein levels and significantly up-regulated MHC class II expression. Supernatants of α-toxin-stimulated macrophages induced the migration of human CD4+ lymphocytes via the CXCL10 receptor (CXCR3). Macrophages from patients with AD produced lower levels of CXCL10 compared to cells from patients with psoriasis as well as healthy controls in response to α-toxin. α-Toxin did not lead to a large variation in CCL22 production in macrophages from all three groups. CONCLUSIONS: Staphylococcal α-toxin contributes to Th1 polarization by induction of CXCL10 in macrophages. Macrophages from patients with AD and psoriasis responded to α-toxin in the induction of Th1-related chemokine CXCL10 diversely, which could favour the recruitment of distinct leucocyte subsets into the skin.

Mutant PIK3CA licenses TRAIL and CD95L to induce non-apoptotic caspase-8-mediated ROCK activation.

Constitutively active PI3K catalytic subunit alpha (PIK3CA) interfered with apoptosis induction downstream of death receptor-signaling complex formation allowing robust caspase-8 activation without triggering the execution steps of apoptosis. In mutant PIK3CA-expressing cells, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and CD95L stimulated nuclear factor kappaB (NFkappaB) activation, invasion, and transition to an amoeboid-like morphology. NFkappaB activation and adoption of amoeboid shape were inhibited by caspase-8 knockdown or FLIP-S expression, but only the cell morphology alterations required caspase-8 activity. Furthermore, we identified caspase-8-mediated, caspase-3-independent cleavage of the protein kinase rho-associated, coiled-coil containing protein kinase 1 as a novel mechanism for acquiring amoeboid shape and enhanced invasiveness in response to TRAIL and CD95L. Taken together, we provide evidence that mutated PIK3CA converts the 'tumor surveillance' activity of cancer cell-expressed death receptors and caspase-8 toward tumor promotion.

The mitogen-activated protein kinase (MAPK)-activated protein kinases MK2 and MK3 cooperate in stimulation of tumor necrosis factor biosynthesis and stabilization of p38 MAPK.

MK2 and MK3 represent protein kinases downstream of p38 mitogen-activated protein kinase (MAPK). Deletion of the MK2 gene in mice resulted in an impaired inflammatory response although MK3, which displays extensive structural similarities and identical functional properties in vitro, is still present. Here, we analyze tumor necrosis factor (TNF) production and expression of p38 MAPK and tristetraprolin (TTP) in MK3-deficient mice and demonstrate that there are no significant differences with wild-type animals. We show that in vivo MK2 and MK3 are expressed and activated in parallel. However, the level of activity of MK2 is always significantly higher than that of MK3. Accordingly, we hypothesized that MK3 could have significant effects only in an MK2-free background and generated MK2/MK3 double-knockout mice. Unexpectedly, these mice are viable and show no obvious defects due to loss of compensation between MK2 and MK3. However, there is a further reduction of TNF production and expression of p38 and TTP in double-knockout mice compared to MK2-deficient mice. This finding, together with the observation that ectopically expressed MK3 can rescue MK2 deficiency similarly to MK2, indicates that both kinases share the same physiological function in vivo but are expressed to different levels.

Alpha-toxin is produced by skin colonizing Staphylococcus aureus and induces a T helper type 1 response in atopic dermatitis.

BACKGROUND: Staphylococcus aureus is a well known trigger factor of atopic dermatitis (AD). Besides the superantigens, further exotoxins are produced by S. aureus and may have an influence on the eczema. OBJECTIVE: To explore the impact of staphylococcal alpha-toxin on human T cells, as those represent the majority of skin infiltrating cells in AD. METHODS: Adult patients with AD were screened for cutaneous colonization with alpha-toxin producing S. aureus. As alpha-toxin may induce necrosis, CD4(+) T cells were incubated with sublytic alpha-toxin concentrations. Proliferation and up-regulation of IFN-gamma on the mRNA and the protein level were assessed. The induction of t-bet translocation in CD4(+) T cells was detected with the Electrophoretic Mobility Shift Assay. RESULTS: Thirty-four percent of the patients were colonized with alpha-toxin producing S. aureus and alpha-toxin was detected in lesional skin of these patients by immunohistochemistry. Sublytic alpha-toxin concentrations induced a marked proliferation of isolated CD4(+) T cells. Microarray analysis indicated that alpha-toxin induced particularly high amounts of IFN-gamma transcripts. Up-regulation of IFN-gamma was confirmed both on the mRNA and the protein level. Stimulation of CD4(+) T cells with alpha-toxin resulted in DNA binding of t-bet, known as a key transcription factor involved into primary T helper type 1 (Th1) commitment. CONCLUSION: alpha-toxin is produced by S. aureus isolated from patients with AD. We show here for the first time that sublytic alpha-toxin concentrations activate T cells in the absence of antigen-presenting cells. Our results indicate that alpha-toxin is relevant for the induction of a Th1 like cytokine response. In AD, this facilitates the development of Th1 cell dominated chronic eczema.