BACKGROUND

Tristetraprolin (TTP/ZFP36) family proteins have anti-inflammatory activity by binding to and destabilizing pro-inflammatory mRNAs such as Tnf mRNA, and represent a potential therapeutic target for inflammation-related diseases. Tea has anti-inflammatory properties but the molecular mechanisms have not been completely elucidated. We hypothesized that TTP and/or its homologues might contribute to the beneficial effects of tea as an anti-inflammatory product.

METHODS

Quantitative real-time PCR was used to investigate the effects of green tea (0, 1, and 2 g solid extract/kg diet) on the expression of Ttp family genes (Ttp/Tis11/Zfp36, Zfp36l1/Tis11b, Zfp36l2/Tis11d, Zfp36l3), pro-inflammatory genes (Tnf, Csf2/Gm-csf, Ptgs2/Cox2), and Elavl1/Hua/Hur and Vegf genes in liver and muscle of rats fed a high-fructose diet known to induce insulin resistance, oxidative stress, inflammation, and TNF-alpha levels.

RESULTS

Ttp and Zfp36l1 mRNAs were the major forms in both liver and skeletal muscle. Ttp, Zfp36l1, and Zfp36l2 mRNA levels were more abundant in the liver than those in the muscle. Csf2/Gm-csf and Zfp36l3 mRNAs were undetectable in both tissues. Tea (1 g solid extract/kg diet) increased Ttp mRNA levels by 50-140% but Tnf mRNA levels decreased by 30% in both tissues, and Ptgs2/Cox2 mRNA levels decreased by 40% in the muscle. Tea (2 g solid extract/kg diet) increased Elavl1/Hua/Hur mRNA levels by 40% in the liver but did not affect any of the other mRNA levels in liver or muscle.

CONCLUSIONS

These results show that tea can modulate Ttp mRNA levels in animals and suggest that a post-transcriptional mechanism through TTP could partially account for tea's anti-inflammatory properties. The results also suggest that drinking adequate amounts of green tea may play a role in the prevention of inflammation-related diseases.

BACKGROUND

Mesenchymal stem cells (MSCs) represent a heterogeneous cell population that is promising for regenerative medicine. The present study was designed to assess whether VCAM-1 can be used as a marker of MSC subpopulation with superior angiogenic potential.

METHODS

MSCs were isolated from placenta chorionic villi (CV). The VCAM-1(+/-) CV-MSCs population were separated by Flow Cytometry and subjected to a comparative analysis for their angiogenic properties including angiogenic genes expression, vasculo-angiogenic abilities on Matrigel in vitro and in vivo, angiogenic paracrine activities, cytokine array, and therapeutic angiogenesis in vascular ischemic diseases.

RESULTS

Angiogenic genes, including HGF, ANG, IL8, IL6, VEGF-A, TGFβ, MMP2 and bFGF, were up-regulated in VCAM-1(+)CV-MSCs. Consistently, angiogenic cytokines especially HGF, IL8, angiogenin, angiopoitin-2, μPAR, CXCL1, IL-1β, IL-1α, CSF2, CSF3, MCP-3, CTACK, and OPG were found to be significantly increased in VCAM-1(+) CV-MSCs. Moreover, VCAM-1(+)CV-MSCs showed remarkable vasculo-angiogenic abilities by angiogenesis analysis with Matrigel in vitro and in vivo and the conditioned medium of VCAM-1(+) CV-MSCs exerted markedly pro-proliferative and pro-migratory effects on endothelial cells compared to VCAM-1(-)CV-MSCs. Finally, transplantation of VCAM-1(+)CV-MSCs into the ischemic hind limb of BALB/c nude mice resulted in a significantly functional improvement in comparison with VCAM-1(-)CV-MSCs transplantation.

CONCLUSIONS

VCAM-1(+)CV-MSCs possessed a favorable angiogenic paracrine activity and displayed therapeutic efficacy on hindlimb ischemia. Our results suggested that VCAM-1(+)CV-MSCs may represent an important subpopulation of MSC for efficient therapeutic angiogenesis.

Mechanical loading of mammalian tissues is a potent promoter of tissue growth and regeneration, whilst unloading in microgravity can cause reduced tissue regeneration, possibly through effects on stem cell tissue progenitors. To test the specific hypothesis that mechanical unloading alters differentiation of bone marrow mesenchymal and hematopoietic stem cell lineages, we studied cellular and molecular aspects of how bone marrow in the mouse proximal femur responds to unloading in microgravity. Trabecular and cortical endosteal bone surfaces in the femoral head underwent significant bone resorption in microgravity, enlarging the marrow cavity. Cells isolated from the femoral head marrow compartment showed significant down-regulation of gene expression markers for early mesenchymal and hematopoietic differentiation, including FUT1(-6.72), CSF2(-3.30), CD90(-3.33), PTPRC(-2.79), and GDF15(-2.45), but not stem cell markers, such as SOX2. At the cellular level, in situ histological analysis revealed decreased megakaryocyte numbers whilst erythrocytes were increased 2.33 fold. Furthermore, erythrocytes displayed elevated fucosylation and clustering adjacent to sinuses forming the marrow-blood barrier, possibly providing a mechanistic basis for explaining spaceflight anemia. Culture of isolated bone marrow cells immediately after microgravity exposure increased the marrow progenitor's potential for mesenchymal differentiation into in-vitro mineralized bone nodules, and hematopoietic differentiation into osteoclasts, suggesting an accumulation of undifferentiated progenitors during exposure to microgravity. These results support the idea that mechanical unloading of mammalian tissues in microgravity is a strong inhibitor of tissue growth and regeneration mechanisms, acting at the level of early mesenchymal and hematopoietic stem cell differentiation.

CAPC, also known as LRRC26, is expressed in normal prostate and salivary gland. We developed a mAb to CAPC and used it to characterize the protein and study its function. CAPC protein was detected in normal prostate and salivary gland, in several human breast cancer cell lines and in the prostate cancer cell line LNCaP. Knockdown of CAPC by siRNA in LNCaP cells enhanced anchorage-independent growth in soft agar. Conversely, overexpression of CAPC in MDA-231 breast cancer cells and A431 epidermoid cancer cells inhibited growth in soft agar and tumorigenesis in nude mice, and suppressed the metastasis of MDA-231 cells to the lung. Overexpression of CAPC downregulated NF-κB activity and its target genes, including GM-CSF (CSF2), CXCL1, IL8 and LTB1. It also suppressed genes encoding the serine protease mesotrypsin (PRSS3) and cystatin SN (CST1). CAPC expressing tumors showed a decrease in the number of proliferating cells and a large increase in ECM. The role of CAPC in the suppression of tumor growth and metastasis may be through its alteration of the tumor microenvironment.

The vascular endothelium is integrally involved in the host response to infection and in organ failure during acute inflammatory disorders such as sepsis. Gram-negative and Gram-positive bacterial lipoproteins circulate in sepsis and can directly activate the endothelium by binding to endothelial cell (EC) TLR2. In this report, we perform the most comprehensive analysis to date of the immune-related genes regulated after activation of endothelial TLR2 by bacterial di- and triacylated lipopeptides. We found that TLR2 activation specifically induces the expression of the genes IL-6, IL-8, CSF2, CSF3, ICAM1 and SELE by human umbilical vein ECs and human lung microvascular ECs. These proteins participate in neutrophil recruitment, adherence and activation at sites of inflammation. Significantly, our studies demonstrate that TLR2-mediated EC responses are specifically geared towards recruitment, activation, and survival of neutrophils and not mononuclear leukocytes, that ECs do not require priming by other inflammatory stimuli to respond to bacterial lipopeptides and, unlike mononuclear leukocytes, TLR2 agonists do not induce ECs to secrete TNF-α. This study suggests that endothelial TLR2 may be an important regulator of neutrophil trafficking to sites of infection in general, and that direct activation of lung endothelial TLR2 may contribute to acute lung injury during sepsis.

There is a need for efficacious therapies for metastatic castration-resistant prostate cancer (mCRPC) after disease progression on docetaxel. The SRC tyrosine kinase and its related family members may be important drivers of prostate cancer and can be inhibited by dasatinib. mCRPC patients, after one previous chemotherapy, started dasatinib at 70 mg twice daily, amended to 100 mg daily. The primary endpoint was the disease control (DC) rate, defined as complete response (CR), partial response (PR), or stable disease (SD) in prostate specific antigen (PSA), RECIST, bone scan, and FACT-P score. Up to 41 patients were to be accrued (two-stage design, 21+20) to rule out a null-hypothesized effect of 5 versus 20% (α=0.05, β=0.1). Secondary endpoints included progression-free survival, toxicity, and pharmacokinetic and pharmacodynamic correlatives. Of 38 patients, 27 were evaluable for response or toxicity. The median duration of therapy was 55 days (6-284). Five patients showed DC after 8 weeks of therapy (18.5% DC, 95% CI: 6.3-38.1%). One PR (3.7% response rate, 95% CI: 0.1-19.0%) was observed in a patient treated for 284 days. Twelve patients (43%) discontinued treatment for toxicity. Dasatinib induced a decrease in phytohemagglutinin-stimulated CSF2, CD40L, GZMB, and IL-2 mRNAs in blood cells, indicating target engagement. Decreases in plasma IL-6 and bone alkaline phosphatase, and in urinary N-telopeptide, were associated with DC. Dasatinib has definite but limited activity in advanced mCRPC, and was poorly tolerated. The observation of a patient with prolonged, objective, clinically significant benefit warrants molecular profiling to select the appropriate patient population.

The human interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating-factor (GM-CSF, or CSF2) gene cluster arose by duplication of an ancestral gene. Although just 10 kb apart and responsive to the same signals, the IL-3 and GM-CSF genes are nevertheless regulated independently by separate, tissue-specific enhancers. To understand the differential regulation of the IL-3 and GM-CSF genes we have investigated a cluster of three ubiquitous DNase I-hypersensitive sites (DHSs) located between the two genes. We found that each site contains a conserved CTCF consensus sequence, binds CTCF, and recruits the cohesin subunit Rad21 in vivo. The positioning of these sites relative to the IL-3 and GM-CSF genes and their respective enhancers is conserved between human and mouse, suggesting a functional role in the organization of the locus. We found that these sites effectively block functional interactions between the GM-CSF enhancer and either the IL-3 or the GM-CSF promoter in reporter gene assays. These data argue that the regulation of the IL-3 and the GM-CSF promoters depends on the positions of their enhancers relative to the conserved CTCF/cohesin-binding sites. We suggest that one important role of these sites is to enable the independent regulation of the IL-3 and GM-CSF genes.

Molecular mapping techniques have defined the region of gene loss in two patients with the 5q- syndrome and uncharacteristically small 5q deletions (5q31-q33). The allelic loss of 10 genes localized to 5q23-qter (centromere-CSF2-EGR1-FGFA-GRL-ADRB2-CS F1R-SPARC-GLUH1-NKSF1-FLT4-telomere) was investigated in peripheral blood cell fractions. Gene dosage experiments demonstrated that CSF2, EGR1, NKSF1, and FLT4 were retained on the 5q- chromosome in both patients and that FGFA was retained in one patient, thus placing these genes outside the critical region. GRL, ADRB2, CSF1R, SPARC, and GLUH1 were shown to be deleted in both patients. The proximal breakpoint is localized between EGR1 and FGFA in one patient and between FGFA and ADRB2 in the other, and the distal breakpoint is localized between GLUH1 and NKSF1 in both patients. Pulsed-field gel electrophoresis was used to map the 5q deletion breakpoints, and breakpoint-specific fragments were detected with FGFA in the granulocyte but not the lymphocyte fraction of one patient. This study has established the critical region of gene loss of the 5q- chromosome in the 5q- syndrome, giving the location for a putative tumor-suppressor gene in the 5.6-Mb region between FGFA and NKSF1.

Successful embryonic development is dependent on factors secreted by the reproductive tract. Dickkopf-1 (DKK1), an antagonist of the wingless-related mouse mammary tumor virus (WNT) signaling pathway, is one endometrial secretory protein potentially involved in maternal-embryo communication. The purpose of this study was to investigate the roles of DKK1 in embryo cell fate decisions and competence to establish pregnancy. Using in vitro-produced bovine embryos, we demonstrate that exposure of embryos to DKK1 during the period of morula to blastocyst transition (between d 5 and 8 of development) promotes the first 2 cell fate decisions leading to increased differentiation of cells toward the trophectoderm and hypoblast lineages compared with that for control embryos treated with vehicle. Moreover, treatment of embryos with DKK1 or colony-stimulating factor 2 (CSF2; an endometrial cytokine known to improve embryo development and pregnancy establishment) between d 5 and 7 of development improves embryo survival after transfer to recipients. Pregnancy success at d 32 of gestation was 27% for cows receiving control embryos treated with vehicle, 41% for cows receiving embryos treated with DKK1, and 39% for cows receiving embryos treated with CSF2. These novel findings represent the first evidence of a role for maternally derived WNT regulators during this period and could lead to improvements in assisted reproductive technologies.

The regional specialization of intestinal immune cells is affected by the longitudinal heterogeneity of environmental factors. Although the distribution of group 3 innate lymphoid cells (ILC3s) is well characterized in the lamina propria, it is poorly defined in Peyer's patches (PPs) along the intestine. Given that PP ILC3s are closely associated with mucosal immune regulation, it is important to characterize the regulatory mechanism of ILC3s. Here, we found that terminal ileal PPs of specific pathogen-free (SPF) mice have fewer NKp46+ ILC3s than jejunal PPs, while there was no difference in NKp46+ ILC3 numbers between terminal ileal and jejunal PPs in antibiotics (ABX)-treated mice. We also found that butyrate levels in the terminal ileal PPs of SPF mice were higher than those in the jejunal PPs of SPF mice and terminal ileal PPs of ABX-treated mice. The reduced number of NKp46+ ILC3s in terminal ileal PPs resulted in a decrease in Csf2 expression and, in turn, resulted in reduced regulatory T cells and enhanced antigen-specific T-cell proliferation. Thus, we suggest that NKp46+ ILC3s are negatively regulated by microbiota-derived butyrate in terminal ileal PPs and the reduced ILC3 frequency is closely associated with antigen-specific immune induction in terminal ileal PPs.

BACKGROUND

Rheumatoid arthritis (RA) is a systemic, chronic inflammatory disease influenced by both genetic and environmental factors, leading to joint destruction and functional impairment. Recently, a large-scaled GWAS meta-analysis using more than 37,000 Japanese samples were conducted and 13 RA susceptibility loci were identified. However, it is not clear whether these loci have significant impact on joint destruction or not. This is the first study focused on the 13 loci to investigate independent genetic risk factors for radiographic progression in the first five years from onset of RA.

METHODS

Sharp/van der Heijde score of hands at 5-year disease duration, which represents joint damage, were measured retrospectively and used as an outcome variable in 865 Japanese RA patients. Genetic factors regarded as putative risk factors were RA-susceptible polymorphisms identified by the Japanese GWAS meta-analysis, including HLA-DRB1 (shared epitope, SE), rs2240340 (PADI4), rs2230926 (TNFAIP3), rs3093024 (CCR6), rs11900673 (B3GNT2), rs2867461 (ANXA3), rs657075 (CSF2), rs12529514 (CD83), rs2233434 (NFKBIE), rs10821944 (ARID5B), rs3781913 (PDE2A-ARAP1), rs2841277 (PLD4) and rs2847297 (PTPN2). These putative genetic risk factors were assessed by a stepwise multiple regression analysis adjusted for possible non-genetic risk factors: autoantibody positivity (anti-citrullinated peptide antibody [ACPA] and rheumatoid factor), history of smoking, gender and age at disease onset.

RESULTS

The number of SE alleles (P = 0.002) and risk alleles of peptidyl arginine deiminase type IV gene (PADI4, P = 0.04) had significant impact on progressive joint destruction, as well as following non-genetic factors: ACPA positive (P = 0.0006), female sex (P = 0.006) and younger age of onset (P = 0.02).

CONCLUSIONS

In the present study, we found that PADI4 risk allele and HLA-DRB1 shared epitope are independent genetic risks for radiographic progression in Japanese rheumatoid arthritis patients. The results of this study give important knowledge of the risks on progressive joint damage in RA patients.

Human mammary epithelial cells (HMECs) exhibit an increase in phosphocholine (PC) and total choline-containing compounds, as well as a switch from high glycerophosphocholine (GPC)/low PC to low GPC/high PC, with progression to malignant phenotype. The treatment of human breast cancer cells with a nonsteroidal anti-inflammatory agent, indomethacin, reverted the high PC/low GPC pattern to a low PC/high GPC pattern indicative of a less malignant phenotype, supported by decreased invasion. Here, we have characterized mechanisms underlying indomethacin-induced alterations in choline membrane metabolism in malignant breast cancer cells and nonmalignant HMECs labeled with [1,2-13C]choline using 1H and 13C magnetic resonance spectroscopy. Microarray gene expression analysis was performed to understand the molecular mechanisms underlying these changes. In breast cancer cells, indomethacin treatment activated phospholipases that, combined with an increased choline phospholipid biosynthesis, led to increased GPC and decreased PC levels. However, in nonmalignant HMECs, activation of the anabolic pathway alone was detected following indomethacin treatment. Following indomethacin treatment in breast cancer cells, several candidate genes, such as interleukin 8, NGFB, CSF2, RHOB, EDN1, and JUNB, were differentially expressed, which may have contributed to changes in choline metabolism through secondary effects or signaling cascades leading to changes in enzyme activity.

EBV has been reported to impair monocyte in vitro differentiation into dendritic cells (DCs) and reduce cell survival. In this study, we added another layer of knowledge to this topic and showed that these effects correlated with macroautophagy/autophagy, ROS and mitochondrial biogenesis reduction. Of note, autophagy and ROS, although strongly interconnected, have been separately reported to be induced by CSF2/GM-CSF (colony stimulating factor 2) and required for CSF2-IL4-driven monocyte in vitro differentiation into DCs. We show that EBV infects monocytes and initiates a feedback loop in which, by inhibiting autophagy, reduces ROS and through ROS reduction negatively influences autophagy. Mechanistically, autophagy reduction correlated with the downregulation of RAB7 and ATG5 expression and STAT3 activation, leading to the accumulation of SQSTM1/p62. The latter activated the SQSTM1-KEAP1- NFE2L2 axis and upregulated the anti-oxidant response, reducing ROS and further inhibiting autophagy. ROS decrease correlated also with the reduction of mitochondria, the main source of intracellular ROS, achieved by the downregulation of NRF1 and TFAM, mitochondrial biogenesis transcription factors. Interestingly, mitochondria supply membranes and ATP required for autophagy execution, thus their reduction may further reduce autophagy in EBV-infected monocytes. In conclusion, this study shows for the first time that the interconnected reduction of autophagy, intracellular ROS and mitochondria mediated by EBV switches monocyte differentiation into apoptosis, giving new insights into the mechanisms through which this virus reduces immune surveillance. Abbreviations: ACTB: actin beta; ATG5: autophagy related 5; BAF: bafilomycin A1; BECN1: beclin 1; CAT: catalase; CSF2: colony stimulating factor 2; CT: control; CYCS (cytochrome C: somatic); DCs: dendritic cells; EBV: Epstein-Barr virus; GSR: glutathione-disulfide reductase; KEAP1: kelch like ECH associated protein 1; IL4: interleukin 4; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MET: metformin; NAC: N-acetylcysteine; NFE2L2/NRF2 nuclear factor: erythroid 2 like 2; NRF1 (nuclear respiratory factor 1); clPARP1: cleaved poly(ADP-ribose) polymerase; Rapa: Rapamycin; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; TFAM: (transcription factor A: mitochondrial); TUBA1A: tubulin alpha 1a.

Seminal fluid interacts with epithelial cells lining the female reproductive tract to induce expression of proinflammatory cytokines and chemokines, initiating immune tolerance mechanisms to facilitate pregnancy. TGFB cytokines are key signaling agents in seminal plasma but do not fully account for the female response to seminal fluid. We hypothesized that additional molecular pathways are utilized in seminal fluid signaling. Affymetrix microarray was employed to compare gene expression in the endometrium of mice 8 h after mating with either intact males or seminal fluid deficient (SVX/VAS) males. Bioinformatics analysis revealed TLR4 signaling as a strongly predicted upstream regulator activated by the differentially expressed genes and implicated TGFB signaling as a second key pathway. Quantitative PCR and microbead data confirmed that seminal fluid induces endometrial synthesis of several TLR4-regulated cytokines and chemokines, including CSF3, CXCL1, CXCL2, IL1A, IL6, LIF, and TNF. In primary uterine epithelial cells, CSF3, CXCL1, and CXCL2 were strongly induced by the TLR4 ligand LPS but suppressed by TGFB, while IL1A, TNF, and CSF2 were induced by both ligands. TLR4 was confirmed as essential for the full endometrial cytokine response using mice with a null mutation in Tlr4, where seminal fluid failed to induce endometrial Csf3, Cxcl2, Il6, and Tnf expression. This study provides evidence that TLR4 contributes to seminal fluid modulation of the periconception immune environment. Activation of TLR4 signaling by microbial or endogenous components of seminal fluid is thus implicated as a key element of the female tract response to seminal fluid at the outset of pregnancy in mice.

Unstimulated monocytes of at-risk/type 1 diabetic humans and macrophages of the NOD mouse have markedly elevated autocrine GM-CSF production and persistent STAT5 phosphorylation. We analyzed the relationship between GM-CSF production and persistent STAT5 phosphorylation in NOD macrophages using reciprocal congenic mouse strains containing either diabetes-susceptible NOD (B6.NODC11), or diabetes-resistant C57L (NOD.LC11) loci on chromosome 11. These intervals contain the gene for GM-CSF (Csf2; 53.8 Mb) and those for STAT3, STAT5A, and STAT5B (Stat3, Stat5a, and Stat5b; 100.4-100.6 Mb). High GM-CSF production and persistent STAT5 phosphorylation in unactivated NOD macrophages can be linked to a region (44.9-55.7 Mb) containing the Csf2 gene, but not the Stat3/5a/5b genes. This locus, provisionally called Idd4.3, is upstream of the previously described Idd4.1 and Idd4.2 loci. Idd4.3 encodes an abundance of cytokine genes that use STAT5 in their macrophage activation signaling and contributes approximately 50% of the NOD.LC11 resistance to diabetes.

BACKGROUND

Infestation of ovine skin with the ectoparasitic mite Psoroptes ovis results in a rapid cutaneous immune response, leading to the crusted skin lesions characteristic of sheep scab. Little is known regarding the mechanisms by which such a profound inflammatory response is instigated and to identify novel vaccine and drug targets a better understanding of the host-parasite relationship is essential. The main objective of this study was to perform a combined network and pathway analysis of the in vivo skin response to infestation with P. ovis to gain a clearer understanding of the mechanisms and signalling pathways involved.

RESULTS

Infestation with P. ovis resulted in differential expression of 1,552 genes over a 24 hour time course. Clustering by peak gene expression enabled classification of genes into temporally related groupings. Network and pathway analysis of clusters identified key signalling pathways involved in the host response to infestation. The analysis implicated a number of genes with roles in allergy and inflammation, including pro-inflammatory cytokines (IL1A, IL1B, IL6, IL8 and TNF) and factors involved in immune cell activation and recruitment (SELE, SELL, SELP, ICAM1, CSF2, CSF3, CCL2 and CXCL2). The analysis also highlighted the influence of the transcription factors NF-kB and AP-1 in the early pro-inflammatory response, and demonstrated a bias towards a Th2 type immune response.

CONCLUSIONS

This study has provided novel insights into the signalling mechanisms leading to the development of a pro-inflammatory response in sheep scab, whilst providing crucial information regarding the nature of mite factors that may trigger this response. It has enabled the elucidation of the temporal patterns by which the immune system is regulated following exposure to P. ovis, providing novel insights into the mechanisms underlying lesion development. This study has improved our existing knowledge of the host response to P. ovis, including the identification of key parallels between sheep scab and other inflammatory skin disorders and the identification of potential targets for disease control.

Ionizing radiation induces cellular senescence to suppress cancer cell proliferation. However, it also induces deleterious bystander effects in the unirradiated neighboring cells through the release of senescence-associated secretory phenotypes (SASPs) that promote tumor progression. Although autophagy has been reported to promote senescence, its role is still unclear. We previously showed that radiation induces senescence in PTTG1-depleted cancer cells. In this study, we found that autophagy was required for the radiation-induced senescence in PTTG1-depleted breast cancer cells. Inhibition of autophagy caused the cells to switch from radiation-induced senescence to apoptosis. Senescent cancer cells exerted bystander effects by promoting the invasion and migration of unirradiated cells through the release of CSF2 and the subsequently activation of the JAK2-STAT3 and AKT pathways. However, the radiation-induced bystander effects were correlated with the inhibition of endogenous autophagy in bystander cells, which also resulted from the activation of the CSF2-JAK2 pathway. The induction of autophagy by rapamycin reduced the radiation-induced bystander effects. This study reveals, for the first time, the dual role of autophagy in radiation-induced senescence and bystander effects.

The contribution of mast cells in the microenvironment of solid malignancies remains controversial. Here we functionally assess the impact of tumor-adjacent, submucosal mast cell accumulation in murine and human intestinal-type gastric cancer. We find that genetic ablation or therapeutic inactivation of mast cells suppresses accumulation of tumor-associated macrophages, reduces tumor cell proliferation and angiogenesis, and diminishes tumor burden. Mast cells are activated by interleukin (IL)-33, an alarmin produced by the tumor epithelium in response to the inflammatory cytokine IL-11, which is required for the growth of gastric cancers in mice. Accordingly, ablation of the cognate IL-33 receptor St2 limits tumor growth, and reduces mast cell-dependent production and release of the macrophage-attracting factors Csf2, Ccl3, and Il6. Conversely, genetic or therapeutic macrophage depletion reduces tumor burden without affecting mast cell abundance. Therefore, tumor-derived IL-33 sustains a mast cell and macrophage-dependent signaling cascade that is amenable for the treatment of gastric cancer.

Our previous studies showed that the non-psychoactive cannabinoid, cannabidiol (CBD), ameliorates the clinical symptoms in mouse myelin oligodendrocyte glycoprotein (MOG)35-55-induced experimental autoimmune encephalomyelitis model of multiple sclerosis (MS) as well as decreases the memory MOG35-55-specific T cell (TMOG) proliferation and cytokine secretion including IL-17, a key autoimmune factor. The mechanisms of these activities are currently poorly understood.

Herein, using microarray-based gene expression profiling, we describe gene networks and intracellular pathways involved in CBD-induced suppression of these activated memory TMOG cells. Encephalitogenic TMOG cells were stimulated with MOG35-55 in the presence of spleen-derived antigen presenting cells (APC) with or without CBD. mRNA of purified TMOG was then subjected to Illumina microarray analysis followed by ingenuity pathway analysis (IPA), weighted gene co-expression network analysis (WGCNA) and gene ontology (GO) elucidation of gene interactions. Results were validated using qPCR and ELISA assays.

Gene profiling showed that the CBD treatment suppresses the transcription of a large number of proinflammatory genes in activated TMOG. These include cytokines (Xcl1, Il3, Il12a, Il1b), cytokine receptors (Cxcr1, Ifngr1), transcription factors (Ier3, Atf3, Nr4a3, Crem), and TNF superfamily signaling molecules (Tnfsf11, Tnfsf14, Tnfrsf9, Tnfrsf18). "IL-17 differentiation" and "IL-6 and IL-10-signaling" were identified among the top processes affected by CBD. CBD increases a number of IFN-dependent transcripts (Rgs16, Mx2, Rsad2, Irf4, Ifit2, Ephx1, Ets2) known to execute anti-proliferative activities in T cells. Interestingly, certain MOG35-55 up-regulated transcripts were maintained at high levels in the presence of CBD, including transcription factors (Egr2, Egr1, Tbx21), cytokines (Csf2, Tnf, Ifng), and chemokines (Ccl3, Ccl4, Cxcl10) suggesting that CBD may promote exhaustion of memory TMOG cells. In addition, CBD enhanced the transcription of T cell co-inhibitory molecules (Btla, Lag3, Trat1, and CD69) known to interfere with T/APC interactions. Furthermore, CBD enhanced the transcription of oxidative stress modulators with potent anti-inflammatory activity that are controlled by Nfe2l2/Nrf2 (Mt1, Mt2a, Slc30a1, Hmox1).

Microarray-based gene expression profiling demonstrated that CBD exerts its immunoregulatory effects in activated memory TMOG cells via (a) suppressing proinflammatory Th17-related transcription, (b) by promoting T cell exhaustion/tolerance, (c) enhancing IFN-dependent anti-proliferative program, (d) hampering antigen presentation, and (d) inducing antioxidant milieu resolving inflammation. These findings put forward mechanism by which CBD exerts its anti-inflammatory effects as well as explain the beneficial role of CBD in pathological memory T cells and in autoimmune diseases.

Hyperinflammation contributes to lung injury and subsequent acute respiratory distress syndrome (ARDS) with high mortality in patients with severe coronavirus disease 2019 (COVID-19). To understand the underlying mechanisms involved in lung pathology, we investigated the role of the lung-specific immune response. We profiled immune cells in bronchoalveolar lavage fluid and blood collected from COVID-19 patients with severe disease and bacterial pneumonia patients not associated with viral infection. By tracking T cell clones across tissues, we identified clonally expanded tissue-resident memory-like Th17 cells (Trm17 cells) in the lungs even after viral clearance. These Trm17 cells were characterized by a a potentially pathogenic cytokine expression profile of IL17A and CSF2 (GM-CSF). Interactome analysis suggests that Trm17 cells can interact with lung macrophages and cytotoxic CD8⁺ T cells, which have been associated with disease severity and lung damage. High IL-17A and GM-CSF protein levels in the serum of COVID-19 patients were associated with a more severe clinical course. Collectively, our study suggests that pulmonary Trm17 cells are one potential orchestrator of the hyperinflammation in severe COVID-19.

BACKGROUND

Allele frequencies reported from public databases or articles are mostly based on small sample sizes. Differences in genotype frequencies by age, race and sex have implications for studies designed to examine genetic susceptibility to disease. In a community-based cohort of 9,960 individuals, we compared the allele frequencies of 49 single nucleotide polymorphisms (SNPs) of genes involved in inflammatory pathways to the frequencies reported on public databases, and examined the genotypes frequencies by age and sex. The genes in which SNPs were analyzed include CCR2, CCR5, COX1, COX2, CRP, CSF1, CSF2, IFNG, IL1A, IL1B, IL2, IL4, IL6, IL8, IL10, IL13, IL18, LTA, MPO, NOS2A, NOS3, PPARD, PPARG, PPARGC1 and TNF.

RESULTS

Mean(SD) age was 53.2(15.5); 98% were Caucasians and 62% were women. Only 1 out of 33 SNPs differed from the SNP500Cancer database in allele frequency by >10% in Caucasians (n = 9,831), whereas 12 SNPs differed by >10% (up to 50%) in African Americans (n = 105). Two out of 15 SNPs differed from the dbSNP database in allele frequencies by >10% in Caucasians, and 5 out of 15 SNPs differed by >10% in African Americans. Age was similar across most genotype groups. Genotype frequencies did not differ by sex except for TNF(rs1799724), IL2(rs2069762), IL10(rs1800890), PPARG(rs1801282), and CRP(rs1800947) with differences of less than 4%.

CONCLUSIONS

When estimating the size of samples needed for a study, particularly if a reference sample is used, one should take into consideration the size and ethnicity of the reference sample. Larger sample size is needed for public databases that report allele frequencies in non-Caucasian populations.

BACKGROUND

In experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, mice genetically deficient in the transcription factor signal transducer and activator of transcription 4 (STAT4) are resistant to disease. In contrast, deletion or inhibition of the Th1-associated cytokines IL-12 or IFNγ which act upstream and downstream of STAT4, respectively, does not ameliorate disease. These discordant findings imply that STAT4 may act in a non-canonical role during EAE. Recently, STAT4 has been shown to regulate GM-CSF production by CD4 T cells and this cytokine is necessary for the induction of EAE. However, it is not known if STAT4 controls GM-CSF production by both Th1 and Th17 effector CD4 T cells.

METHODS

This study utilized the MOG(35-55) peptide immunization model of EAE. Intracellular cytokine staining and novel mixed bone marrow chimeric mice were used to study the CD4 T cell-intrinsic role of STAT4 during disease. STAT4 chromatin-immunoprecipitation (ChIP-PCR) experiments were performed to show STAT4 directly interacts with the Csf2 gene loci.

RESULTS

Herein, we demonstrate that STAT4 controls CD4 T cell-intrinsic GM-CSF production by both Th1 and Th17 CD4 T cells during EAE as well as in vitro. Importantly, we show that STAT4 interacts with the Csf2 locus in MOG(35-55)-activated effector CD4 T cells demonstrating direct modulation of GM-CSF.

CONCLUSIONS

Overall, these studies illustrate a previously unrecognized role of STAT4 to regulate GM-CSF production by not only Th1 cells, but also Th17 effector CD4 T cell subsets during EAE pathogenesis. Critically, these data highlight for the first time that STAT4 is able to modulate the effector profile of Th17 CD4 T cell subsets, which redefines our current understanding of STAT4 as a Th1-centric factor.

Mucosa-associated lymphoid tissue 1 (MALT1) controls antigen receptor-mediated signalling to nuclear factor κB (NF-κB) through both its adaptor and protease function. Upon antigen stimulation, MALT1 forms a complex with BCL10 and CARMA1, which is essential for initial IκBα phosphorylation and NF-κB nuclear translocation. Parallel induction of MALT1 protease activity serves to inactivate negative regulators of NF-κB signalling, such as A20 and RELB. Here we demonstrate a key role for auto-proteolytic MALT1 cleavage in B- and T-cell receptor signalling. MALT1 cleavage occurred after Arginine 149, between the N-terminal death domain and the first immunoglobulin-like region, and did not affect its proteolytic activity. Jurkat T cells expressing an un-cleavable MALT1-R149A mutant showed unaltered initial IκBα phosphorylation and normal nuclear accumulation of NF-κB subunits. Nevertheless, MALT1 cleavage was required for optimal activation of NF-κB reporter genes and expression of the NF-κB targets IL-2 and CSF2. Transcriptome analysis confirmed that MALT1 cleavage after R149 was required to induce NF-κB transcriptional activity in Jurkat T cells. Collectively, these data demonstrate that auto-proteolytic MALT1 cleavage controls antigen receptor-induced expression of NF-κB target genes downstream of nuclear NF-κB accumulation.

The mechanisms of inflammation in acne are currently subject of intense investigation. This study focused on the activation of adaptive and innate immunity in clinically early visible inflamed acne lesions and was performed in two independent patient populations. Biopsies were collected from lesional and non-lesional skin of acne patients. Using Affymetrix Genechips, we observed significant elevation of the signature cytokines of the Th17 lineage in acne lesions compared to non-lesional skin. The increased expression of IL-17 was confirmed at the RNA and also protein level with real-time PCR (RT-PCR) and Luminex technology. Cytokines involved in Th17 lineage differentiation (IL-1β, IL-6, TGF-β, IL23p19) were remarkably induced at the RNA level. In addition, proinflammatory cytokines and chemokines (TNF-α, IL-8, CSF2 and CCL20), Th1 markers (IL12p40, CXCR3, T-bet, IFN-γ), T regulatory cell markers (Foxp3, IL-10, TGF-β) and IL-17 related antimicrobial peptides (S100A7, S100A9, lipocalin, hBD2, hBD3, hCAP18) were induced. Importantly, immunohistochemistry revealed significantly increased numbers of IL-17A positive T cells and CD83 dendritic cells in the acne lesions. In summary our results demonstrate the presence of IL-17A positive T cells and the activation of Th17-related cytokines in acne lesions, indicating that the Th17 pathway is activated and may play a pivotal role in the disease process, possibly offering new targets of therapy.