Leukocyte populations change profoundly in the human endometrium during the menstrual cycle. However the predominant cell, the uterine natural killer (uNK) cell does not contain steroid receptors. From gene array analysis we identified a transcript encoding chemokine (C-X-C motif) ligand 14 (CXCL14) which is markedly up-regulated in the secretory phase of the cycle. We confirm this data by northern blotting and quantitative PCR. Using in situ hybridization we localized CXCL14 mRNA to the glandular epithelial cells where it was detected only in the secretory phase of the cycle. Candidate progesterone response elements were identified at positions -2028/-2007 and -722/-697 (PRE1 and PRE2, respectively) relative to the translation start site. These were functionally tested using luciferase reporter deletion constructs, electrophoretic mobility shift assays and site-directed mutagenesis. The deletion/mutation of these sites reduced progesterone induction by 40 and 20%, respectively. Finally, we demonstrated that recombinant CXCL14 stimulated uNK cell chemotaxis in vitro. We therefore conclude that CXCL14 is likely to be regulated by progesterone in human endometrium and that it may exert a chemoattractive effect on uNK cells and in part be responsible for their clustering around the epithelial glands.

The chemokine BRAK/CXCL14 (BRAK) is expressed in normal squamous epithelium, but is not expressed or is expressed at negligible levels in head and neck squamous cell carcinoma. Malignant cells are known to be dedifferentiated compared with normal epithelial cells, suggesting a role for differentiation cues in the expression of BRAK. Thus, we examined the relationship between BRAK expression and stages of differentiation level in epithelial cells. Immunohistochemical analysis showed that BRAK protein was expressed in cells above the spinous cell layer in normal epithelia. In HSC-3 cells in culture, expression of BRAK mRNA was significantly upregulated by cell contact in a cell density-dependent manner, and mRNA expression of cell differentiation markers such as involucrin, cystatin-A, TGM1, TGM3, and TGM5 was concomitantly augmented. Furthermore, the upregulation of BRAK induced by cell contact was suppressed by chlorpromazine, a specific inhibitor of calmodulin. We previously reported that GC boxes and a TATA-like sequence in the BRAK promoter region are associated with the expression of BRAK. Using a promoter assay and ChIP, we demonstrated that binding of the stimulating protein-1 (SP1) transcription factor to a GC box upstream of the BRAK transcription start site was necessary for cell density-dependent upregulation of BRAK. These results indicated that upregulation of BRAK was accompanied by differentiation of epithelial cells induced by calcium/calmodulin signaling, and that SP1 binding to the BRAK promoter region played an important role in this signaling.

CXCL12 and CXCL14 are evolutionarily conserved members of the CXC-type chemokine family. CXCL12 binds specifically to the G-protein-coupled receptor CXCR4 to induce the migration of primordial germ cells, hematopoietic stem cells, and inflammation-associated immune cells. In addition, CXCL12-CXCR4 signaling is often enhanced in malignant tumor cells and facilitates increased proliferation as well as metastasis. Although macrophage migration inhibitory factor and extracellular ubiquitin interact with CXCR4 as agonistic factors, CXCL12 was believed to be the sole chemokine ligand for CXCR4. However, a very recent report revealed that CXCL14 binds to CXCR4 with high affinity and efficiently inhibits CXCL12-mediated chemotaxis of hematopoietic progenitor and leukemia-derived cells. CXCL14 does not directly cross-compete with CXCL12 for the CXCR4 binding but instead inactivates CXCR4 via receptor internalization. Because both CXCL12 and CXCL14 are expressed during embryogenesis and brain development in mice, these two chemokines could function in an interactive fashion. We propose that the CXCL14 gene has been conserved from fish to man due to its role in fine-tuning the strength of CXCL12-mediated signal transduction. In addition to its biological implications, the above finding will be important for designing anti-cancer compounds targeting the CXCL12-CXCR4 signaling axis. In fact, a stabilized dimeric peptide containing the C-terminal 51-77 amino acid residues of CXCL14 has been shown to have stronger CXCL12 antagonistic activity than full-length CXCL14.

The present study investigated the effects of oxidative stress induced by reactive oxygen species (ROS), such as hydrogen peroxide (H(2)O(2)) and hydroxyl radical (HO(*)), on the expression of both BRAK , which is also known as non-ELR motif angiostatic CXC chemokine ligand 14 (CXCL14), in head and neck squamous cell carcinoma (HNSCC) cells. When HNSCC cells were cultured in the presence of ROS, the expression of BRAK was significantly decreased whereas that of IL-8 was increased. Interestingly, the effects on the expression of both genes in HNSCC cells were much greater with HO(blacksquare, square, filled) than with H(2)O(2). The effects of ROS on both BRAK and IL-8 expression were attenuated by pre-treatment with N-acetyl-L-cysteine (NAC), epidermal growth factor receptor (EGFR), and mitogen-activated protein kinase (MAPK) inhibitors. These results indicate that oxidative stress induced by H(2)O(2) or HO(*) stimulates angiogenesis and tumuor progression by altering the gene expression of BRAK and IL-8 via the EGFR/MEK/ERK pathway in human HNSCC cells.

During Mycobacterium tuberculosis (M.tb) infection, the initial interactions between the pathogen and the host cell determines internalization and innate immune response events. It is established that detergents such as Tween alter the mycobacterial cell wall and solubilize various lipids and proteins. The implication of this is significant since induced changes on the cell wall affect macrophage uptake and the immune response to M.tb. Importantly, during transmission between hosts, aerosolized M.tb enters the host in its native form, i.e. in a detergent-free environment, thus in vitro and in vivo studies should mimic this as closely as possible. To this end, we have optimized a procedure for growing and processing detergent-free M.tb and assessed the response of murine macrophages (BMDM) infected with multi drug-resistant M.tb (R179 Beijing 220 clinical isolate) using RNAseq. We compared the effects of the host response to M.tb cultured under standard laboratory conditions (Tween 80 containing medium -R179T), or in detergent-free medium (R179NT). RNAseq comparisons reveal 2651 differentially expressed genes in BMDMs infected with R179T M.tb vs. BMDMs infected with R179NT M.tb. A range of differentially expressed genes involved in BMDM receptor interaction with M.tb (Mrc1, Ifngr1, Tlr9, Fpr1 and Itgax) and pro-inflammatory cytokines/chemokines (Il6, Il1b, Tnf, Ccl5 and Cxcl14) were selected for analysis through qPCR. BMDMs infected with R179NT stimulate a robust inflammatory response. Interestingly, R179NT M.tb induce transcription of Fpr1, a receptor which detects bacterial formyl peptides and initiates a myriad of immune responses. Additionally we show that the host components Cxcl14, with an unknown role in M.tb infection, and Tlr9, an emerging role player, are only stimulated by infection with R179NT M.tb. Taken together, our results suggest that the host response differs significantly in response to Tween 80 cultured M.tb and should therefore not be used in infection experiments.

BACKGROUND

Periodontitis is a chronic inflammatory disease triggered by the host immune response. Epigenetic modifications also affect the immune response. We assessed CpG methylation in 22 inflammatory candidate genes (ATF2, CCL25, CXCL14, CXCL3, CXCL5, CXCL6, FADD, GATA3, IL10RA, IL12A, IL12B, IL13, IL13RA1, IL15, IL17C, IL17RA, IL4R, IL6R, IL6ST, IL7, INHA, and TYK2) with respect to the occurrence of aggressive periodontitis (AgP).

METHODS

In this study 15 AgP patients (53.3% males, 41.4±10.5 years) and 10 controls (40.0% males, 36.9±17.5 years) were included. The methylation patterns of gingival biopsies were quantified using EpiTect® Methyl Signature PCR Array Human Inflammatory Response.

RESULTS

In gingival biopsies taken from patients with AgP, CpG methylation of CCL25 (1.73% vs. 2.59%, p=0.015) and IL17C (6.89% vs. 19.27%, p=0.002) was significantly reduced as compared with periodontally healthy tissues.

CONCLUSIONS

We showed for the first time a differential methylation pattern for CCL25 and IL17C in periodontitis. CCL25 plays an important role in T-cell development, whereas IL17C regulates innate epithelial immune responses. The decrease in CpG methylation is presumably accompanied by an increase in gene expression. This could lead to a greater availability of CCL25 and interleukin 17C and support periodontal loss of attachment.

Cardiac patch is considered a promising strategy for enhancing stem cell therapy of myocardial infarction (MI). However, the underlying mechanisms for cardiac patch repairing infarcted myocardium remain unclear. In this study, we investigated the mechanisms of PCL/gelatin patch loaded with MSCs on activating endogenous cardiac repair. PCL/gelatin patch was fabricated by electrospun. The patch enhanced the survival of the seeded MSCs and their HIF-1α, Tβ4, VEGF and SDF-1 expression and decreased CXCL14 expression in hypoxic and serum-deprived conditions. In murine MI models, the survival and distribution of the engrafted MSCs and the activation of the epicardium were examined, respectively. At 4 weeks after transplantation of the cell patch, the cardiac functions were significantly improved. The engrafted MSCs migrated across the epicardium and into the myocardium. Tendency of HIF-1α, Tβ4, VEGF, SDF-1 and CXCL14 expression in the infarcted myocardium was similar with expression in vitro. The epicardium was activated and epicardial-derived cells (EPDCs) migrated into deep tissue. The EPDCs differentiated into endothelial cells and smooth muscle cells, and some of EPDCs showed to have differentiated into cardiomyocytes. Density of blood and lymphatic capillaries increased significantly. More c-kit+ cells were recruited into the infarcted myocardium after transplantation of the cell patch. The results suggest that epicardial transplantation of the cell patch promotes repair of the infarcted myocardium and improves cardiac functions by enhancing the survival of the transplanted cells, accelerating locality paracrine, and then activating the epicardium and recruiting endogenous c-kit+ cells. Epicardial transplantation of the cell patch may be applied as a novel effective MI therapy.

The chemokine receptor, CXC chemokine receptor 4 (CXCR4), is selective for CXC chemokine ligand 12 (CXCL12), is broadly expressed in blood and tissue cells, and is essential during embryogenesis and hematopoiesis. CXCL14 is a homeostatic chemokine with unknown receptor selectivity and preferential expression in peripheral tissues. Here, we demonstrate that CXCL14 synergized with CXCL12 in the induction of chemokine responses in primary human lymphoid cells and cell lines that express CXCR4. Combining subactive concentrations of CXCL12 with 100-300 nM CXCL14 resulted in chemotaxis responses that exceeded maximal responses that were obtained with CXCL12 alone. CXCL14 did not activate CXCR4-expressing cells (i.e., failed to trigger chemotaxis and Ca2+ mobilization, as well as signaling via ERK1/2 and the small GTPase Rac1); however, CXCL14 bound to CXCR4 with high affinity, induced redistribution of cell-surface CXCR4, and enhanced HIV-1 infection by >3-fold. We postulate that CXCL14 is a positive allosteric modulator of CXCR4 that enhances the potency of CXCR4 ligands. Our findings provide new insights that will inform the development of novel therapeutics that target CXCR4 in a range of diseases, including cancer, autoimmunity, and HIV.-Collins, P. J., McCully, M. L., Martínez-Muñoz, L., Santiago, C., Wheeldon, J., Caucheteux, S., Thelen, S., Cecchinato, V., Laufer, J. M., Purvanov, V., Monneau, Y. R., Lortat-Jacob, H., Legler, D. F., Uguccioni, M., Thelen, M., Piguet, V., Mellado, M., Moser, B. Epithelial chemokine CXCL14 synergizes with CXCL12 via allosteric modulation of CXCR4.

Vertebrate eye development is a complex multistep process coordinated by signals from the lens, optic cup and periocular mesenchyme. Although chemokines are increasingly being recognized as key players in cell migration, proliferation, and differentiation during embryonic development, their potential role during eye development has not been examined. In this study, we demonstrate by section in situ hybridization that CXCL12 and CXCL14 are expressed during ocular development. CXCL12 is expressed in the periocular mesenchyme, ocular blood vessels, retina, and eyelid mesenchyme, and its expression pattern is conserved between chick and mouse in most tissues. Expression of CXCL14 is localized in the ocular ectoderm, limbal epithelium, scleral papillae, eyelid mesenchyme, corneal keratocytes, hair follicles, and retina, and it was only conserved in the upper eyelid ectoderm of chick and mouse. The unique and non-overlapping patterns of CXCL12 and CXCL14 expression in ocular tissues suggest that these two chemokines may interact and have important functions in cell proliferation, differentiation and migration during eye development.

Using cDNA microarray technology, the expression of chemokine genes in the elicitation site of 2,4,6-trinitrochlorobenzene-induced contact hypersensitivity (CHS) was examined in mice. Of the 33 genes analyzed, levels of 11 gene expressions changed, and these can be assigned to four groups based on their kinetic patterns; (1) LARC/CCL20 whose mRNA level increased rapidly at 3 h post-challenge and then gradually decreased, (2) JE/CCL2, MARC/CCL7, MIP-1gamma/CCL9, monocyte chemoattractant protein (MCP)-5/CCL12, ELC/CCL19 and BRAK/CXCL14 whose mRNA levels increased with time and reached the maximum at 6-9 h post-challenge, (3) LIX/CXCL5, Mig/CXCL9 and IP-10/CXCL10 whose mRNA levels increased gradually at least up to 12 h post challenge, and (4) SLC/CCL21 whose mRNA level decreased gradually with time after challenge. The findings suggest that sequential expression of chemokine genes is essential for orientating non-specific skin response to hapten-specific CHS response through the recruitment of inflammatory cells such as neutrophils, monocytes/macrophages and T-cells from the circulation into the tissue site.

Clear cell renal cell carcinoma (ccRCC) is often resistant to existing therapy. We found elevated S100A6 levels in ccRCC tissues, associated with higher grade pathological features and clinical stages in ccRCC patients. Knockdown of S100A6 inhibited cell proliferation in vitro and tumor growth in vivo. Gene expression profiling suggests a novel function of S100A6 in suppressing apoptosis, as well as a relationship between S100A6 and CXCL14, a pro-inflammatory chemokine. We suggest that the S100A6/CXCL14 signaling pathway is a potential therapeutic target in ccRCC.

Using mouse gene expression microarray analysis, we earlier obtained dynamic profiles of whole genome expression in the CCl(4)-induced liver injury mouse model. CXCL14 expression was increased in the liver injury phase and returned to normal after liver regeneration suggesting its involvement in the liver injury or regeneration regulation. The role of CXCL14 in liver injury was investigated. The dynamic of CXCL14 transcription was analyzed in CCl(4)-induced mouse liver damage by qRT-PCR. Plasmid mediated CXCL14 overexpression and antibody neutralization of endogenous CXCL14 were used to demonstrate its effects and mechanisms on CCl(4)-induced liver injury and acute liver failure. We showed that CXCL14 expression was immediately upregulated post CCl(4) injection with a dose-dependent response. CXCL14 over-expression aggravated CCl(4)-induced liver injuries, evidenced by enhanced acidophilic change and necrosis of hepatocyte, increased fat deposition in hepatocytes (P<0.01), and inhibited hepatocyte proliferation (P<0.01). On the contrary, anti-CXCL14 antibody treatment reduced the severity of CCL4-induced liver injuries Significant reductions in hepatic necrosis area (P<0.05), the liver fat deposition (P<0.01), and the lipid peroxidation measured by serum MDA (P<0.05) were observed. Importantly, the antibody treatment reduced the mouse mortality caused by CCl4-induced liver failure (P<0.05). The data suggest that CXCL14 and its receptor present potential targets for the treatment of liver diseases.

We immunohistochemically investigated the distribution of CXCL14, also called BRAK protein in the rat hypothalamus using anti-human CXCL14 serum. CXCL14-immunoreactive somata were localized in the periventricular area and paraventricular and supraoptic hypothalamic nuclei. In the former, immunoreactive neuronal somata, confirmed by double staining with a neuronal marker, NeuN, contained diffuse CXCL14-like immunoreactivity in their perikarya. In contrast, immunoreactive somata in the latter contained immunoreactive puncta within their perikarya. Very dense immunoreactive fibers and puncta were seen in the median eminence. Dense immunoreactive fibers were seen in the arcuate nucleus and ventromedial hypothalamic nucleus. Other hypothalamic areas contained a few immunoreactive fibers and puncta. These results demonstrated for the first time that CXCL14 protein is present in a subset of hypothalamic neurons and suggest that CXCL14 participates in hypothalamic functions such as control of autonomic nervous systems and/or participates in immune cell recruitment via the median eminence.

Staphylococcus aureus (S. aureus) is problematic for lactating mammals and public health. Understanding of mechanisms by which the hosts respond to severe invasion of S. aureus remains elusive. In this study, the genome-wide expression of mRNAs and miRNAs in bovine mammary gland cells were interrogated at 24 h after intra-mammary infection (IMI) with high or low concentrations of S. aureus. Compared to the negative control quarters, 194 highly-confident responsive genes were identified in the quarters with high concentration (109 cfu/mL) of S. aureus, which were predominantly implicated in pathways and biological processes pertaining to innate immune system, such as cytokine-cytokine receptor interaction and inflammatory response. In contrast, only 21 highly-confident genes were significantly differentially expressed in face of low concentration (106 cfu/mL) of S. aureus, which slightly perturbed the cell signaling and invoked corresponding responses like vasoconstriction, indicating limited perturbations and immunological evading. Additionally, the significant up-regulations of bta-mir-223 and bta-mir-21-3p were observed in the quarters infected by high concentration of S. aureus. Network analysis suggested that the two miRNAs' pivotal roles in defending hosts against bacterial infection probably through inhibiting CXCL14 and KIT. The significant down-regulation of CXCL14 was also observed in bovine mammary epithelial cells at 24 h post-infection of S. aureus (108 cfu/mL) in vitro. Integrated analysis with QTL database further suggested 28 genes (e.g., CXCL14, KIT, and SLC4A11) as candidates of bovine mastitis. This study first systematically revealed transcriptional and post-transcriptional responses of bovine mammary gland cells to invading S. aureus in a dosage-dependent pattern, and highlighted a complicated responsive mechanism in a network of miRNA-gene-pathway interplay.

Recurrent glioblastomas are frequently found near subventricular zone (SVZ) areas of the brain where neural stem cells (NSCs) reside, and glioblastoma-derived extracellular vesicles (EVs) are reported to play important roles in tumour micro-environment, but the details are not clear. Here, we investigated the possibility that NSCs are involved in glioblastoma relapse mediated by glioblastoma-derived EVs. We studied changes to NSCs by adding glioblastoma-derived EVs into a culture system of NSCs, and found that NSCs differentiated into a type of tumour-promoting cell. These transformed cells had distinguished proliferation activity, a high migration rate, and clone-forming ability revealed by CCK-8, wound healing and soft agar clone formation assays, respectively. In vivo assays indicated that these cells could accelerate tumour formation by Ln229 cells in nude mice. Moreover, to explore the mechanisms underlying NSC transformation, single cell transcriptome sequencing was performed; our results suggest that several key genes such as S100B, CXCL14, EFEMP1, SCRG1, GLIPR1, HMGA1 and CD44 and dysregulated signalling may be important for the transformation of NSCs. It is also indicated that NSCs may be involved in glioblastoma recurrence through EV release by glioblastoma in this work. This could help to illuminate the mechanism of glioblastoma relapse, which occurs in a brief period after surgical excision, and contribute to finding new ways to treat this disease.

C-X-C motif chemokine 12/C-X-C chemokine receptor type 4 (CXCL12/CXCR4) signaling is involved in ontogenesis, hematopoiesis, immune function and cancer. Recently, the orphan chemokine CXCL14 was reported to inhibit CXCL12-induced chemotaxis - probably by allosteric modulation of CXCR4. We thus examined the effects of CXCL14 on CXCR4 regulation and function using CXCR4-transfected human embryonic kidney (HEK293) cells and Jurkat T cells. CXCL14 did not affect dose-response profiles of CXCL12-induced CXCR4 phosphorylation, G protein-mediated calcium mobilization, dynamic mass redistribution, kinetics of extracellular signal-regulated kinase 1 (ERK1) and ERK2 phosphorylation or CXCR4 internalization. Hence, essential CXCL12-operated functions of CXCR4 are insensitive to CXCL14, suggesting that interactions of CXCL12 and CXCL14 pathways depend on a yet to be identified CXCL14 receptor.

Idiopathic Pulmonary Fibrosis (IPF) is a progressive disorder that is marked by an over accumulation of activated fibroblast populations. Despite the improved understanding of many mechanisms within this disease, global gene expression analysis has few focused studies on the fibroblast, the central effector cell of progressive fibrosis. We present a unique analysis of IPF pulmonary fibroblasts as they transition through cell culture and identify in vitro altered cellular processes. Fibroblasts were isolated from diseased (n = 8) and non-diseased (n = 4) lungs. Global gene expression analysis was carried out at the initial point of isolation and after 3 weeks of culture. We identify several genes that are altered by removal of the fibroblast from the IPF environment. Comparison of this subset of genes to four previously published whole lung analyses refined our list to a small subset of key fibroblast specific genes important in IPF. Application of STRING database analysis and confirmation via in-vitro and histological assay highlights the CXCL14/CXCR4 chemokine axis with a possible role in the progression and/or activation of fibroblasts within the IPF lung. Our findings, present a possible therapeutic target for IPF and a model for the study and discovery of novel protein and processes in this terrible disease.

CXCL14 (C-X-C motif chemokine ligand 14) is a conserved member of chemokine family and functions as a chemoattractant for multiplicate immunocytes. CXCL14 expression is constitutive in normal tissues, but absent in wide range of epithelial tumors. Many reports have claimed its important role in tumorigenesis and vascularization. An association between rs2237062 polymorphism and hepatocellular carcinoma (HCC) susceptibility was found in patients with chronic HCV infection in Japanese population. Here we analyzed, by using a polymerase chain reaction-ligation detection reaction (PCR-LDR), the polymorphism in 202 non-HCC patients with HBV infection, 361 HBV-related HCC patients and 407 healthy controls. The aim was to detect the possible association of this single-nucleotide polymorphism (SNP) with HBV-related HCC susceptibility and progression. However, no association was found between rs2237062 polymorphism and susceptibility to HBV infection or HBV-related HCC. Intriguingly, our stratification analysis revealed that HBV-related HCC patients in advanced phase (TNM-II-IV stage) had significantly higher C allele frequency at this polymorphism than patients at early stage (TNM-I stage) (33.5% vs. 25.7%), and its odds ratio reached 1.47 (95% CI 1.06-2.04, P = 0.021). These results suggest that the rs2237062 polymorphism in the CXCL14 gene might influence HBV-related HCC progression in Chinese population.

Bisphenol-A (BPA) is a ubiquitous estrogen-like endocrine disrupting compound (EDC). BPA exposure in utero has been linked to breast cancer and abnormal mammary gland development in mice. The recent rise in incidence of human breast cancer and decreased age of first detection suggests a possible environmental etiology. We hypothesized that developmental programming of carcinogenesis may involve an aberrant immune response. Both innate and adaptive immunity play a role in tumor suppression through cytolytic CD8, NK, and Th1 T-cells. We hypothesized that BPA exposure in utero would lead to dysregulation of both innate and adaptive immunity in the mammary gland. CD1 mice were exposed to BPA in utero during gestation (days 9-21) via osmotic minipump. At 6 weeks, the female offspring were ovariectomized and estradiol was given at 8 weeks. RNA and protein were extracted from the posterior mammary glands, and the mRNA and protein levels were measured by PCR array, qRT-PCR, and western blot. In mouse mammary tissue, BPA exposure in utero significantly decreased the expression of members of the chemokine CXC family (Cxcl2, Cxcl4, Cxcl14, and Ccl20), interleukin 1 (Il1) gene family (Il1β and Il1rn), interleukin 2 gene family (Il7 receptor), and interferon gene family (interferon regulatory factor 9 (Irf9), as well as immune response gene 1 (Irg1). Additionally, BPA exposure in utero decreased Esr1 receptor gene expression and increased Esr2 receptor gene expression. In utero exposure of BPA resulted in significant changes to inflammatory modulators within mammary tissue. We suggest that dysregulation of inflammatory cytokines, both pro-inflammatory and anti-inflammatory, leads to a microenvironment that may promote disordered cell growth through inhibition of the immune response that targets cancer cells.

Purpose: A subset of estrogen receptor-positive (ER-positive) breast cancer (BC) contains high levels of tumor-infiltrating lymphocytes (TILs), similar to triple-negative BC (TNBC). The majority of immuno-oncology trials target TNBCs because of the greater proportion of TIL-rich TNBCs. The extent to which the immune microenvironments of immune-rich ER-positive BC and TNBC differ is unknown.

Patients and methods: RNA sequencing data from The Cancer Genome Atlas (TCGA; n = 697 ER-positive BCs; n = 191 TNBCs) were used for discovery; microarray expression data from Molecular Taxonomy of Breast Cancer International Consortium (METABRIC; n = 1,186 ER-positive BCs; n = 297 TNBCs) was used for validation. Patients in the top 25th percentile of a previously published total TIL metagene score distribution were considered immune rich. We compared expression of immune cell markers, immune function metagenes, and immuno-oncology therapeutic targets among immune-rich subtypes.

Results: Relative fractions of resting mast cells (TCGA P _adj = .009; METABRIC P _adj = 4.09E-15), CD8⁺ T cells (TCGA P _adj = .015; METABRIC P _adj = 0.390), and M2-like macrophages (TCGA P _adj= 4.68E-05; METABRIC P _adj = .435) were higher in immune-rich ER-positive BCs, but M0-like macrophages (TCGA P _adj = 0.015; METABRIC P _adj = .004) and M1-like macrophages (TCGA P _adj = 9.39E-08; METABRIC P _adj = 6.24E-11) were higher in immune-rich TNBCs. Ninety-one immune-related genes (eg, CXCL14, CSF3R, TGF-B3, LRRC32/GARP, TGFB-R2) and a transforming growth factor β (TGF-β) response metagene were significantly overexpressed in immune-rich ER-positive BCs, whereas 41 immune-related genes (eg, IFNG, PD-L1, CTLA4, MAGEA4) were overexpressed in immune-rich TNBCs in both discovery and validation data sets. TGF-β pathway member genes correlated negatively with expression of immune activation markers (IFNG, granzyme-B, perforin) and positively with M2-like macrophages (IL4, IL10, and MMP9) and regulatory T-cell (FOXP3) markers in both subtypes.

Conclusion: Different immunotherapy strategies may be optimal in immune-rich ER-positive BC and TNBC. Drugs targeting the TGF-β pathway and M2-like macrophages are promising strategies in immune-rich ER-positive BCs to augment antitumor immunity.

Breast implant-associated anaplastic large cell lymphoma is a new provisional entity in the revised World Health Organization classification of lymphoid malignancies, the pathogenesis and cell of origin of which are still unknown. We performed gene expression profiling of microdissected breast implant-associated anaplastic large cell lymphoma samples and compared their transcriptional profiles with those previously obtained from normal T-cells and other peripheral T-cell lymphomas and validated expression of selected markers by immunohistochemistry. Our results indicate that most breast implant-associated anaplastic large cell lymphomas exhibit an activated CD4+ memory T-cell phenotype, which is associated with CD25 and FoxP3 expression. Gene ontology analyses revealed upregulation of genes involved in cell motility programs (e.g., CCR6, MET, HGF, CXCL14) in breast implant-associated anaplastic large cell lymphomas compared to normal CD4+ T-cells and upregulation of genes involved in myeloid cell differentiation (e.g., PPARg, JAK2, SPI-1, GAB2) and viral gene transcription (e.g., RPS10, RPL17, RPS29, RPL18A) compared to other types of peripheral T-cell lymphomas. Gene set enrichment analyses also revealed shared features between the molecular profiles of breast implant-associated anaplastic large cell lymphomas and other types of anaplastic large cell lymphomas, including downregulation of T-cell receptor signaling and STAT3 activation. Our findings provide novel insights into the biology of this rare disease and further evidence that breast implant-associated anaplastic large cell lymphoma represents a distinct peripheral T-cell lymphoma entity.

The pathogenic drivers of sporadic and familial motor neuron disease (MND), such amyotrophic lateral sclerosis (ALS), are unknown. MND impairs the Ran GTPase cycle, which controls nucleocytoplasmic transport, ribostasis and proteostasis; however, cause-effect mechanisms of Ran GTPase modulators in motoneuron pathobiology have remained elusive. The cytosolic and peripheral nucleoporin Ranbp2 is a crucial regulator of the Ran GTPase cycle and of the proteostasis of neurological disease-prone substrates, but the roles of Ranbp2 in motoneuron biology and disease remain unknown. This study shows that conditional ablation of Ranbp2 in mouse Thy1 motoneurons causes ALS syndromes with hypoactivity followed by hindlimb paralysis, respiratory distress and, ultimately, death. These phenotypes are accompanied by: a decline in the nerve conduction velocity, free fatty acids and phophatidylcholine of the sciatic nerve; a reduction in the g-ratios of sciatic and phrenic nerves; and hypertrophy of motoneurons. Furthermore, Ranbp2 loss disrupts the nucleocytoplasmic partitioning of the import and export nuclear receptors importin β and exportin 1, respectively, Ran GTPase and histone deacetylase 4. Whole-transcriptome, proteomic and cellular analyses uncovered that the chemokine receptor Cxcr4, its antagonizing ligands Cxcl12 and Cxcl14, and effector, latent and activated Stat3 all undergo early autocrine and proteostatic deregulation, and intracellular sequestration and aggregation as a result of Ranbp2 loss in motoneurons. These effects were accompanied by paracrine and autocrine neuroglial deregulation of hnRNPH3 proteostasis in sciatic nerve and motoneurons, respectively, and post-transcriptional downregulation of metalloproteinase 28 in the sciatic nerve. Mechanistically, our results demonstrate that Ranbp2 controls nucleocytoplasmic, chemokine and metalloproteinase 28 signaling, and proteostasis of substrates that are crucial to motoneuronal homeostasis and whose impairments by loss of Ranbp2 drive ALS-like syndromes.

BACKGROUND

It is well known that iodide exacerbates thyroid function in subclinical hypothyroid patients with autoimmune thyroiditis. To investigate the immunological mechanism of iodine-induced thyroid dysfunction, we studied the effect of iodide in cultured human thyroid follicles, which respond to physiological concentrations of human thyrotropin (TSH) (0.3-10 microU/mL) and maintain the Wolff-Chaikoff effect.

METHODS

Thyroid follicles obtained from Graves' patients at subtotal thyroidectomy were precultured in medium containing 0.5% fetal calf serum and 10(-8) M iodide for 5 days, and then cultured with the medium containing bovine TSH (30 microU/mL) and low (10(-8)M) or high (10(-5)M) concentrations of iodide. After 3-72 hours of culture, the effect of iodide on thyroid cell mRNA expression was analyzed by microarray and reverse transcriptase-polymerase chain reaction.

RESULTS

After 48 hours of culture, iodide nearly doubled the mRNA expression levels of the immunity-associated genes (intercellular adhesion molecule-1, transforming growth factor beta 1-induced protein, early growth response gene 1, guanylate-binding protein 1, and annexin A1) and decreased the mRNA expression of sodium-iodide symporter to less than 20%. Further, the mRNA expression levels of chemokines (CCL2, CXCL8, and CXCL14) increased nearly twofold, whereas their receptors did not show any significant response. Real-time polymerase chain reaction analyses confirmed that iodide increased the mRNA expression levels of these genes in a time- and concentration-dependent manner. Immunohistochemical studies revealed that the chemokines were expressed mainly in the thyroid follicular cells in addition to the immune cells. The iodide-induced increase in CCL2 was greater in thyroid follicles obtained from thyroid gland that had been moderately infiltrated with the immunocompetent cells.

CONCLUSIONS

We have demonstrated that iodide stimulates thyroid follicular cells to produce chemokines, particularly CCL2, CXCL8, and CXCL14. These chemokines and intercellular adhesion molecule-1 would attract immunocompetent cells into thyroid gland. These in vitro findings suggest that iodide at high concentrations may induce thyroid dysfunction through not only biochemical but also immunological mechanisms, particularly in patients with autoimmune thyroid disorders.

The uterine natural killer cells (uNK cells) are the major immune cells in pregnant uterus and the number of uNK cells is dramatically increased during placentation and embryo development. The uNK cells are necessary for the immune tolerance, cytokine secretion and angiogenesis of placenta. Former studies indicated that the population expansion of uNK cells was accomplished through recruitment of NK cell precursors from the spleen and bone marrow, but not proliferation of NK cells. However, the necessary molecules within this process were little understood. Here in our study, we found the co-localized expression of Cxcl14 protein with uNK cells in E13.5 pregnant uterus. Moreover, we used Cxcl14 knockout mice to examine uNK cells in mesometrial lymphoid aggregate of pregnancy (MLAp) and decidua basalis (DB) of E13.5 pregnant uterus and found significantly decreased uNK cells in Cxcl14(-/-) pregnant uteri compared with Cxcl14(+/-) pregnant uteri. To further explorer the molecular change in MLAp and DB after Cxcl14 knockout, we isolated the MLAp and DB from Cxcl14(+/+) and Cxcl14(-/-) pregnant uteri and performed microarray analysis. We found many genes were up and down regulated after Cxcl14 knockout. In conclusion, our results suggested the important function of Cxcl14 in uNK cells and the proper level of Cxcl14 protein were required to recruit NK cells to pregnant uterus.