The expression profile in the mouse hepatitis B virus X (HBx)-transfected model was investigated in order to lay a foundation for further study on the implication of cytokines expression in hepatitis B virus (HBV) infection. Hydrodynamic injection method via the tail vein was used to establish the animal HBx-transfected model. By using microassay, the differential expression of gene in each group was analyzed, which was further confirmed by using real-time PCR and semi-quantitative PCR. Most of chemokine genes such as Ccl2, Ccl5, Ccl9, MIG and IP-10 were up-regulated in the HBx-transfected mouse model versus the control mice, which was coincided with the microarray results. Western blotting and immunohistochemistry were applied to detect the expression of MIG and IP-10 in the liver tissues. Simultaneously, ELISA was adopted to measure the content of IFN-γ in the liver tissues. DNA microassay revealed that the expression of 611 genes changed in HBx-transfected mice as compared with that in pCMV-tag2B-transfected mice, and most of the screened chemokines were up-regulated (including MIG and IP-10). Additionally, IFN-γ protein levels were increased by 20.7% (P<0.05) in pCMV-tag2B-HBx-transfected mice as compared with the untreated mice. IFN-γ protein levels were reduced by 53.9% (P<0.05) in pCMV-tag2B-transfected mice as compared with the untreated mice, which was consistent with the up-regulation of MIG and IP-10. It was suggested HBx transfection could induce the expression of MIG and IP-10 in the liver tissues, which might play the roles in HBV-related liver immunity and cytokines-mediated antiviral effect.

This study aimed to identify allergic rhinitis (AR)-related hub genes and functionally enriched pathways in a murine model. Dataset GSE52804 (including three normal controls and three AR mice) was downloaded from Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) were identified. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and protein-protein interaction (PPI) analyses of DEGs were performed to identify the hub genes in AR. The DEGs were classified into different modules by using the weighted gene co-expression network analysis (WGCNA). Moreover, to verify the potential hub genes, nasal mucosa tissues were obtained from murine AR models (n = 5) and controls (n = 5), and qRT-PCR and Western blot were performed. In this study, a total of 634 DEGs were identified. They were significantly enriched in 14 GO terms, such as integral component of membrane, plasma membrane, and G-protein-coupled receptor signaling pathway. Meanwhile, there were eight terms of KEGG pathways significantly enriched, such as Olfactory transduction, Cytokine-cytokine receptor interaction, and TNF signaling pathway. The top 10 hub genes (Rtp1, Rps27a, Penk, Cxcl2, Gng8, Gng3, Cxcl1, Cxcr2, Ccl9, and Anxa1) were identified by the PPI network. DEGs were classified into seven modules by WGCNA. According to qRT-PCR validation of the five genes of interest (Rtp1, Rps27a, Penk, Cxcl2, and Anxa1), the expression level of Rtp1 mRNA was significantly decreased in the AR group compared with the control group, while there are enhanced Rps27a, Penk, Cxcl2, and Anxa1 mRNA expressions in the AR mice group compared with the control group. Western blot was also performed to further explore the expression of Anxa1 in the protein level, and the results showed a similar expression trend.

Keywords: Anxa1; WGCNA; allergic rhinitis; bioinformatics analysis; hub genes.

Meningiomas are primary central nervous system (CNS) tumors that originate from the arachnoid cells of the meninges. Recurrence occurs in higher grade meningiomas and a small subset of Grade I meningiomas with benign histology. Currently, there are no established circulating tumor markers which can be used for diagnostic and prognostic purposes in a non-invasive way for meningiomas. Here, we aimed to identify potential biomarkers of meningioma in patient sera. For this purpose, we collected preoperative (n = 30) serum samples from the meningioma patients classified as Grade I (n = 23), Grade II (n = 4), or Grade III (n = 3). We used a high-throughput, multiplex immunoassay cancer panel comprising of 92 cancer-related protein biomarkers to explore the serum protein profiles of meningioma patients. We detected 14 differentially expressed proteins in the sera of the Grade I meningioma patients in comparison to the age- and gender-matched control subjects (n = 12). Compared to the control group, Grade I meningioma patients showed increased serum levels of amphiregulin (AREG), CCL24, CD69, prolactin, EGF, HB-EGF, caspase-3, and decreased levels of VEGFD, TGF-α, E-Selectin, BAFF, IL-12, CCL9, and GH. For validation studies, we utilized an independent set of meningioma tumor tissue samples (Grade I, n = 20; Grade II, n = 10; Grade III, n = 6), and found that the expressions of amphiregulin and Caspase3 are significantly increased in all grades of meningiomas either at the transcriptional or protein level, respectively. In contrast, the gene expression of VEGF-D was significantly lower in Grade I meningioma tissue samples. Taken together, our study identifies a meningioma-specific protein signature in blood circulation of meningioma patients and highlights the importance of equilibrium between tumor-promoting factors and anti-tumor immunity.

Regenerative medicine using umbilical cord blood (UCB) cells shows promise for the treatment of cerebral palsy. Although the efficacy of this therapy has been seen in the clinic, the mechanisms by which UCB cells interact and aid in the improvement of symptoms are not clear. We explored the chemokine expression profile in damaged brain tissue in the neonatal mouse ischemia-reperfusion (IR) brain injury model that was infused with human UCB (hUCB) cells. IR brain injury was induced in 9-day-old NOD/SCID mice. hUCB cells were administered 3 weeks post brain injury. Chemokine expression profiles in the brain extract were determined at various time points. Inflammatory chemokines such as CCL1, CCL17, and CXCL12 were transiently upregulated by 24 hours post brain injury. Upregulation of other chemokines, including CCL5, CCL9, and CXCL1 were prolonged up to 3 weeks post brain injury, but most chemokines dissipated over time. There were marked increases in levels of CCL2, CCL12, CCL20, and CX3CL1 in response to hUCB cell treatment, which might be related to the new recruitment and differentiation of neural stem cells, leading to the induction of tissue regeneration. We propose that the chemokine expression profile in the brain shifted from responding to tissue damage to inducing tissue regeneration. hUCB cell administration further enhanced the production of chemokines, and chemokine networks may play an active role in tissue regeneration in neonatal hypoxic-ischemic brain injury.

The incidence of thyroid cancer, the most frequent endocrine neoplasia, is rapidly increasing. Significant progress has recently been made in the identification of genetic lesions in thyroid cancer; however, whether inflammation contributes to thyroid cancer progression remains unknown. Using a mouse model of aggressive follicular thyroid cancer (FTC; Thrb^PV/PVPten^+/- mice), we aimed to elucidate a cause-effect relationship at the molecular level. The Thrb^PV/PVPten^+/- mouse expresses a dominantly negative thyroid hormone receptor β (denoted as PV) and a deletion of a single allele of the Pten gene. These two oncogenic signaling pathways synergistically activate PI3K-AKT signaling to drive cancer progression as in human FTC. At the age of 5-7 weeks, thyroids of Thrb^PV/PVPten^+/- mice exhibited extensive hyperplasia accompanied by 77.5-fold infiltration of inflammatory monocytes as compared with normal thyroids. Global gene expression profiling identified altered expression of 2387 genes, among which 1353 were upregulated and 1034 were down-regulated. Further analysis identified markedly elevated expression of inflammation mediators and cytokines such as, Csf1r, Csf1, SPP1, Aif1, IL6, Ccl9, Ccl3, Ccl12, and Ccr2 genes and decreased expression of Kit, Ephx2, Cd163, IL15, Ccl11, and Cxcl13 genes. These changes elicited the inflammatory responses in the hyperplastic thyroid of Thrb^PV/PVPten^+/- mice, reflecting early events in thyroid carcinogenesis. We next tested whether attenuating the inflammatory responses could mitigate thyroid cancer progression. We treated the mice with an inhibitor of colony-stimulating factor 1 receptor (CSF1R), pexidartinib (PLX-3397; PLX). CSF1R mediates the activity of the cytokine, colony stimulating factor 1 (CSF1), in the production, differentiation, and functions of monocytes and macrophages. Treatment with PLX decreased 94% and 62% of inflammatory monocytes in the thyroid and bone marrow, respectively, versus controls. Further, PLX suppressed the expression of critical cytokine and inflammation-regulating genes such as Csf1r, SPP1 (OPN), Aif1, IL6, Ccl9, Ccl3, Ccl12, and Ccr2 (25%-80%), resulting in inhibition of 89% tumor cell proliferation, evidenced by Ki-67 immunostaining. These preclinical findings suggest that inflammation occurs in the early stage of thyroid carcinogenesis and plays a critical in cancer progression. Importantly, attenuation of inflammation by inhibitors such as PLX would be beneficial in preventing thyroid cancer.

Keywords: CSF1R inhibitors thyroid cancer; Inflammation; colony-stimulating factor 1 receptor; monocytes; mouse model; pexidartinib (PLX-3397).

BACKGROUND

Increasing evidence has indicated parathyroid hormone type 1 receptor (PTHR1) plays important roles for the development and progression of osteosarcoma (OS). However, its function mechanisms remain unclear. The goal of this study was to further illuminate the roles of PTHR1 in OS using microarray data.

METHODS

Microarray data were available from the Gene Expression Omnibus database under the accession number GSE46861, including six tumors from mice with PTHR1 knockdown (PTHR1.358) and six tumors from mice with control knockdown (Ren.1309). Differentially expressed genes (DEGs) between PTHR1.358 and Ren.1309 were identified using the LIMMA method, and then, protein-protein interaction (PPI) network was constructed using data from STRING database to screen crucial genes associated with PTHR1. KEGG pathway enrichment analysis was performed to investigate the underlying functions of DEGs using DAVID tool.

RESULTS

A total of 1163 genes were identified as DEGs, including 617 downregulated (Lef1, lymphoid enhancer-binding factor 1) and 546 upregulated genes (Dkk1, Dickkopf-related protein 1). KEGG enrichment analysis indicated upregulated DEGs were involved in Renin-angiotensin system (e.g., Agt, angiotensinogen) and Wnt signaling pathway (e.g., Dkk1), while downregulated DEGs participated in Basal cell carcinoma (e.g., Lef1). A PPI network (534 nodes and 2830 edges) was constructed, in which Agt gene was demonstrated to be the hub gene and its interactive genes (e.g., CCR3, CC chemokine receptor 3; and CCL9, chemokine CC chemokine ligand 9) were inflammation related.

CONCLUSIONS

Our present study preliminarily reveals the pro-malignant effects of PTHR1 in OS cells may be mediated by activating Wnt, angiogenesis, and inflammation pathways via changing the expressions of the crucial enriched genes (Dkk1, Lef1, Agt-CCR3, and Agt-CCL9).

Oxidative stress is a key contributor to the pathogenesis of stroke-reperfusion injury. Neuroinflammatory peptides released after ischemic stroke mediate reperfusion injury. Previous studies, including ours, have shown that lipocalin-2 (LCN2) is secreted in response to cerebral ischemia to promote reperfusion injury. Genetic deletion of LCN2 significantly reduces brain injury after stroke, suggesting that LCN2 is a mediator of reperfusion injury and a potential therapeutic target. Immunotherapy has the potential to harness neuroinflammatory responses and provides neuroprotection against stroke. Here we report that LCN2 was induced on the inner surface of cerebral endothelial cells, neutrophils, and astrocytes that gatekeep the blood-brain barrier (BBB) after stroke. LCN2 monoclonal antibody (mAb) specifically targeted LCN2 in vitro and in vivo, attenuating the induction of LCN2 and pro-inflammatory mediators (iNOS, IL-6, CCL2, and CCL9) after stroke. Administration of LCN2 mAb at 4 h after stroke significantly reduced neurological deficits, cerebral infarction, edema, BBB leakage, and infiltration of neutrophils. The binding epitope of LCN2 mAb was mapped to the β3 and β4 strands, which are responsible for maintaining the integrity of LCN2 cup-shaped structure. These data indicate that LCN2 can be pharmacologically targeted using a specific mAb to reduce reperfusion injury after stroke.

Keywords: Lipocalin-2; immunotherapy; neutrophils; oxidative stress; reperfusion injury; stroke.

Interferon-β has therapeutic efficacy in Multiple Sclerosis by reducing disease exacerbations and delaying relapses. Previous studies have suggested that the effects of type I IFN in Experimental Autoimmune Encephalomyelitis (EAE) in mice were targeted to myeloid cells. We used mice with a conditional deletion (cKO) of the type I IFN receptor (IFNAR) in T regulatory (Treg) cells to dissect the role of IFN signaling on Tregs. cKO mice developed severe EAE with an earlier onset than control mice. Although Treg cells from cKO mice were more activated, the activation status and effector cytokine production of CD4⁺Foxp3^- T cells in the draining lymph nodes (dLN) was similar in WT and cKO mice during the priming phase. Production of chemokines (CCL8, CCL9, CCL22) by CD4⁺Foxp3^- T cells and LN resident cells from cKO mice was suppressed. Suppression of chemokine production was accompanied by a substantial reduction of myeloid derived suppressor cells (MDSCs) in the dLN of cKO mice, while generation of MDSCs and recruitment to peripheral organs was comparable. This study demonstrates that signaling by type I IFNs in Tregs reduces their capacity to suppress chemokine production, with resultant alteration of the entire microenvironment of draining lymph nodes leading to enhancement of MDSC homing, and beneficial effects on disease outcome.

Keywords: Chemokines; Experimental autoimmune encephalomyelitis; Myeloid derived suppressor cells; Regulatory T cells; Type 1 IFN Receptor-α.

Pathogen-specific memory T cells (T_M) contribute to enhanced immune protection under conditions of reinfection, and their effective recruitment into a recall response relies, in part, on cues imparted by chemokines that coordinate their spatiotemporal positioning. An integrated perspective, however, needs to consider T_M as a potentially relevant chemokine source themselves. In this study, we employed a comprehensive transcriptional/translational profiling strategy to delineate the identities, expression patterns, and dynamic regulation of chemokines produced by murine pathogen-specific T_M CD8⁺T_M, and to a lesser extent CD4⁺T_M, are a prodigious source for six select chemokines (CCL1/3/4/5, CCL9/10, and XCL1) that collectively constitute a prominent and largely invariant signature across acute and chronic infections. Notably, constitutive CCL5 expression by CD8⁺T_M serves as a unique functional imprint of prior antigenic experience; induced CCL1 production identifies highly polyfunctional CD8⁺ and CD4⁺T_M subsets; long-term CD8⁺T_M maintenance is associated with a pronounced increase of XCL1 production capacity; chemokines dominate the earliest stages of the CD8⁺T_M recall response because of expeditious synthesis/secretion kinetics (CCL3/4/5) and low activation thresholds (CCL1/3/4/5/XCL1); and T_M chemokine profiles modulated by persisting viral Ags exhibit both discrete functional deficits and a notable surplus. Nevertheless, recall responses and partial virus control in chronic infection appear little affected by the absence of major T_M chemokines. Although specific contributions of T_M-derived chemokines to enhanced immune protection therefore remain to be elucidated in other experimental scenarios, the ready visualization of T_M chemokine-expression patterns permits a detailed stratification of T_M functionalities that may be correlated with differentiation status, protective capacities, and potential fates.

Proteasomes are complex macromolecular structures existing in various forms to regulate a myriad of cellular processes. Besides degrading unwanted or misfolded proteins (proteostasis), distinct immune functions were ascribed for the immunoproteasome and thymoproteasome (TPr) complexes. For instance, antigen degradation during ongoing immune responses mainly relies on immunoproteasome activity, whereas intrathymic CD8 T-cell development requires peptide generation by the TPr complex. Despite these substantial differences, the functional contribution of the TPr to peripheral T-cell immunity remains ill-defined. We herein explored whether the use of mesenchymal stromal cells (MSCs) engineered to exhibit altered proteasomal activity through de novo expression of the TPr complex can be exploited as a novel anti-cancer vaccine capable of triggering potent CD8 T-cell activation. Phenotypic and molecular characterization of MSC-TPr revealed a substantial decrease in MHCI (H2-K^b and H2-D^d) expression along with elevated secretion of various chemokines (CCL2, CCL9, CXCL1, LIX, and CX3CL1). In parallel, transcriptomic analysis pinpointed the limited ability of MSC-TPr to present endogenous antigens, which is consistent with their low expression levels of the peptide-loading proteins TAP, CALR, and PDAI3. Nevertheless, MSC-TPr cross-presented peptides derived from captured soluble proteins. When tested for their protective capacity, MSC-TPr triggered modest anti-tumoral responses despite efficient generation of effector memory CD4 and CD8 T cells. In contrast, clodronate administration prior to vaccination dramatically enhanced the MSC-TPr-induced anti-tumoral immunity clearly highlighting a refractory role mediated by phagocytic cells. Thus, our data elute to a DC cross-priming-dependant pathway in mediating the therapeutic effect of MSC-TPr.

Keywords: antigen cross-presentation; cancer vaccine; clodronate; cross-priming; efferocytosis; mesenchymal stromal cell; thymoproteasome.

To examine the involvement of CD8alpha+ intraepithelial lymphocytes (IELs) in chemokine expression by villus epithelial cells, villus and crypt fractions were collected by mechanical isolation using a chelating buffer and specific antibodies in CD8alpha+ cell-depleted rats. A larger population of CD8alpha+ cells was observed by histochemical evaluation in villus epithelia than in crypt epithelia in rat small intestine, and CCL9 and CCL28 expression was higher in the crypt fraction than in the villus fraction. The mRNA expression of CCL28 in villus fractions isolated from rat small intestinal mucosa was significantly reduced compared to that of CCL9, and was accompanied by CD8alpha depletion. Using a combined histochemical and flow cytometric approach, CD8alphaalpha+ cells were detected in the intraepithelial region of the villus epithelium. Thus, CCL28 expression in villus epithelial cells is partially supported by CD8alphaalpha+ cells, and CD8alpha+ IELs are involved in CCL28 expression.

BACKGROUND

The purpose of this study is to identify key genes and microRNAs (miRNAs) involved in autoantibody-mediated arthritis (AMA).

METHODS

A time-course microarray data (ID: GSE27492) of peripheral blood leukocytes, ankle tissue, and synovial fluid from K/BxN mouse serum-transferred mice were downloaded from Gene Expression Omnibus. Those samples were collected at days 0, 1, 3, 7, 12, and 18 after serum injection. Limma of R was employed to identify differentially expressed genes (DEGs) in samples collected at days 1-18 compared with those collected at day 0. Consistent DEGs were obtained by taking the interaction of DEGs from different time points, followed by functional enrichment analysis. MiRNAs were screened out and constructed into regulatory network with DEGs using Cytoscape.

RESULTS

In total, 17 consistent DEGs were obtained, including downregulated Ephx1 and upregulated AF251705, Adam8, Arg1, Basp1, Ccl2, Ccl7, Ccl9, Ccr2, Clec4a2, Clec4d, Cxcl1, Fabp5, Fcgr1, Gp49a, Il1rn, and Saa3. Those DEGs were associated with biological processes of immune response, inflammatory response, and defense response; chemokine signaling pathway; cytokine-cytokine receptor interaction; and NOD-like receptor signaling pathway. Additionally, 202 miRNAs were identified to have a regulatory effect on 9 of the 17 DEGs. Notably, miR-944, miR-374a, and miR374b were found to regulate the expression of Cxcl1, Ccl7, and Ccl2. Clec4d was targeted by 78 miRNAs.

CONCLUSIONS

Our study reveals that 17 DEGs and 202 miRNAs may be associated with autoimmune disorder in the progression of AMA, which could guide future researches.

Allergic inflammatory diseases are a steadily growing health concern. Mast cells, a driving force behind allergic pathologies, modulate metabolic pathways to carry out various functions following IgE-FcεRI-mediated activation. Tafazzin (TAZ) is a cardiolipin transacylase that functions to remodel, and thereby mature, cardiolipin, which is important for efficient energy production through oxidative phosphorylation. In this study, we aimed to evaluate the contribution of TAZ in IgE-mediated mast cell activation. Fetal liver-derived mast cells (FLMCs) were differentiated from mice with a doxycycline (dox)-inducible TAZ short hairpin RNA (shRNA) cassette (TAZ shRNA^+/+) and littermate wild-types (WTs). TAZ knockdown in FLMCs following dox treatment was confirmed by Western blotting (99.1% by day 5), whereas flow cytometry confirmed FLMC phenotype (c-kit⁺ FcεRI⁺) and retention of receptor expression post-dox. Five-day dox-treated WT and TAZ shRNA^+/+ FLMCs were activated via allergen-bound IgE cross-linking of FcεRI under stem cell factor potentiation. With dox, and in response to allergen, TAZ shRNA^+/+ FLMCs displayed a 25% reduction in oxygen consumption and a significant 31% reduction in mast cell degranulation compared with dox-treated WT FLMCs. Secretion of TNF, CCL1, and CCL2 were significantly reduced, with CCL9 also impaired. Notably, gene expression was not impaired for any inflammatory mediator measured. Functionally, this suggests that TAZ is a contributor to mast cell degranulation and inflammatory mediator secretion. Given unimpacted induced gene expression for mediators measured, we propose that TAZ reduction impairs mast cell exocytosis mechanisms. We thus identify a potential new contributor to immunometabolism that enhances our understanding of mast cell signaling metabolic pathway interactions during allergic inflammation.

A deep knowledge of the profound immunological response induced by traumatic brain injury (TBI) raises the possibility of novel therapeutic interventions. Existing studies have highlighted the important roles of C-C motif ligands in the development of neuroinflammation after brain injury; however, the participation of macrophage inflammatory protein-1 (MIP-1) family members in this phenomenon is still undefined. Therefore, the goal of our study was to evaluate changes in macrophage inflammatory protein-1 (MIP-1) family members (CCL3, CCL4, and CCL9) and their receptors (CCR1 and CCR5) in a mouse model of TBI (induced by controlled cortical impact (CCI)). We also investigated the pattern of activation of immunological cells (such as neutrophils, microglia and astroglia), which on one hand express CCR1/CCR5, and on the other hand might be a source of the tested chemokines in the injured brain. We investigated changes in mRNA (RT-qPCR) and/or protein (ELISA and Western blot) expression in brain structures (the cortex, hippocampus, thalamus, and striatum) at different time points (24 h, 4 days, 7 days, 2 weeks, and/or 5 weeks) after trauma. Our time-course studies revealed the upregulation of the mRNA expression of all members of the MIP-1 family (CCL3, CCL4, and CCL9) in all tested brain structures, mainly in the early stages after injury. A similar pattern of activation was observed at the protein level in the cortex and thalamus, where the strongest activation was observed 1 day after CCI; however, we did not observe any change in CCL3 in the thalamus. Analyses of CCR1 and CCR5 demonstrated the upregulation of the mRNA expression of both receptors in all tested cerebral structures, mainly in the early phases post injury (24 h, 4 days and 7 days). Protein analysis showed the upregulation of CCR1 and CCR5 in the thalamus 24 h after TBI, but we did not detect any change in the cortex. We also observed the upregulation of neutrophil marker (MPO) at the early time points (24 h and 7 days) in the cortex, while the profound activation of microglia (IBA-1) and astroglia (GFAP) was observed mainly on day 7. Our findings highlight for the first time that CCL3, CCL4, CCL9 and their receptors offer promising targets for influencing secondary neuronal injury and improving TBI therapy. The results suggest that the MIP-1 family is an important target for pharmacological intervention for brain injury.

Activating transcription factor (ATF) 3 regulates chemokine expression in various cell types and tissues. Herein, we studied this regulation in contracting muscle cells in vitro, and in skeletal muscle after muscle-damaging exercise in vivo. C2C12 myotubes with normal or low ATF3 levels (atf3_siRNA) were electrically stimulated (EPS). Also, ATF3-knockout (ATF3-KO) and control mice ran downhill until exhaustion, and muscles were analyzed post-exercise. EPS increased ATF3 levels in myotubes (P < 0.01). Chemokine C-C motif ligand (ccl) 2 mRNA increased post-EPS, but atf3_siRNA attenuated the response (P < 0.05). Atf3_siRNA up-regulated ccl6 basal mRNA, and down-regulated ccl9 and chemokine C-X-C motif ligand (cxcl) 1 basal mRNAs. Post-exercise, ATF3-KO mice showed exacerbated mRNA levels of ccl6 and ccl9 in soleus (P < 0.05), and similar trends were observed for ccl2 and interleukin (il) 1β (P < 0.09). In quadriceps, il6 mRNA level increased only in ATF3-KO (P < 0.05), and cxcl1 mRNA showed a similar trend (P = 0.082). Cluster of differentiation-68 (cd68) mRNA, a macrophage marker, increased in quadriceps and soleus independently of genotype (P < 0.001). Our data demonstrate that ATF3 regulates chemokine expression in muscle cells in vitro and skeletal muscle in vivo, but the regulation differs in each model. Cells other than myofibers may thus participate in the response observed in skeletal muscle. Our results also indicate that ATF3-independent mechanisms would regulate macrophage infiltration upon muscle-damaging exercise. The implications of chemokine regulation in skeletal muscle remain to be determined.

Severe acute pancreatitis is a lethal inflammatory disease frequently accompanied by pancreatic necrosis. We aimed to identify a key regulator in the development of pancreatic necrosis. A cytokine/chemokine array using sera from patients with acute pancreatitis (AP) revealed that serum CXCL16 levels were elevated according to the severity of pancreatitis. In a mouse model of AP, Cxcl16 expression was induced in pancreatic acini in the late phase with the development of pancreatic necrosis. Cxcl16-/- mice revealed similar sensitivity as wild-type (WT) mice to the onset of pancreatitis, but better resisted development of acinar cell necrosis with attenuated neutrophil infiltration. A cytokine array and immunohistochemistry revealed lower expression of Ccl9, a neutrophil chemoattractant, in the pancreatic acini of Cxcl16-/- mice than WT mice. Ccl9 mRNA expression was induced by stimulation with Cxcl16 protein in pancreatic acinar cells in vitro, suggesting a Cxcl16/Ccl9 cascade. Neutralizing antibody against Cxcl16 ameliorated pancreatic injury in the mouse AP model with decreased Ccl9 expression and less neutrophil accumulation. In conclusion, Cxcl16 expressed in pancreatic acini contributes to the development of acinar cell necrosis through the induction of Ccl9 and subsequent neutrophil infiltration. CXCL16 could be a new therapeutic target in AP.

This study aims to investigate the global profiling of genes and miRNAs expression to explore the regulatory effects of eicosapentaenoic acid (EPA) in visceral adipose tissue (VAT) of obese mice. We used male mice, fed either a high-fat diet (HF) or HF supplemented with EPA (HF-EPA), for 11 weeks. RNA, and small RNA profiling, were performed by RNAseq analysis. We conducted analyses using Ingenuity Pathway Analysis software (IPA^®) and validated candidate genes and miRNAs related to lipid mediators and inflammatory pathways using qRT-PCR. We identified 153 genes differentially downregulated, and 62 microRNAs differentially expressed in VAT from HF-EPA compared to HF. Genes with a positive association with inflammation, chemotaxis, insulin resistance, and inflammatory cell death, such as Irf5, Alox5ap, Tlrs, Cd84, Ccr5, Ccl9, and Casp1, were downregulated by EPA. Moreover, EPA significantly reduced LTB4 levels, a lipid mediator with a central role in inflammation and insulin resistance in obesity. The pathways and mRNA/microRNA interactions identified in our study corroborated with data validated for inflammatory genes and miRNAs. Together, our results identified key VAT inflammatory targets and pathways, which are regulated by EPA. These targets merit further investigation to better understand the protective mechanisms of EPA in obesity-associated inflammation.

Keywords: adipose tissue; eicosapentaenoic acid; inflammation; leukotriene-B4; obesity.

Mouse Ccr1l1 (Ccr1-like 1) encodes an orphan G protein-coupled receptor (GPCR) with highest homology to the inflammatory and highly promiscuous chemokine receptors Ccr1 and Ccr3 (70 and 50% amino acid identity, respectively). Ccr1l1 was first cloned in 1995, yet current knowledge of this putative chemokine receptor is limited to its gene organization and chromosomal localization. Here we report that Ccr1l1 is a Rodentia-specific gene selectively expressed in eosinophils. However, eosinophil phenotypes, development and responsiveness to chemokines were all normal in naïve Ccr1l1 knockout mice. We demonstrate for the first time that recombinant Ccr1l1 is expressed on the plasma membrane of transfected cells and contains an extracellular N-terminus and an intracellular C-terminus, consistent with GPCR topology. Using receptor internalization, β-arrestin recruitment, calcium flux and chemotaxis assays, we excluded all 37 available mouse chemokines, including Ccr1 ligands, and two viral chemokines as Ccr1l1 ligands, and demonstrated that mouse Ccr1, but not Ccr1l1, exhibits constitutive signaling activity. However, sequence analysis and structural modeling revealed that Ccr1l1 is well-equipped to act as a classical signaling GPCR, with N-terminal sulfotyrosines as the only signaling and chemokine-binding determinant absent in Ccr1l1. Hereof, we show that a sulfatable N-terminal Ccr1 Y¹⁸ residue is essential for chemotaxis and calcium responses induced by Ccl3 and Ccl9/10 but substituting the corresponding Ccr1l1 F¹⁹ residue with tyrosine failed to confer responsiveness to Ccr1 ligands. Although Ccr1l1 remains an extreme outlier in the chemokine receptor family, our study supports it might respond to unidentified mouse chemokine ligands in eosinophil-driven immune responses.

Keywords: Ccr1; calcium flux; cell migration; chemotaxis; constitutive activity; eosinophil; receptor internalization; sulfotyrosine; tyrosine sulfation.

Ethnopharmacological relevance: Jatropha curcas L. (Euphorbiaceae), as a drought resistant shrub mainly cultivated in tropical and subtropical areas worldwide, is widely used as traditional medicine to cure arthritis, dysentery, abscess and pneumonia in Asian, African and South American folklores. The methanolic extracts of the roots have been revealed the anti-inflammatory activity in vivo and vitro.

Aim of study: This research aimed to provide promising anti-inflammatory candidates from the roots of J. curcas. In addition, RNA-Seq was conducted to give targeted genes involved in the anti-inflammatory action.

Materials and methods: The diterpenoids were isolated from the CH₂Cl₂ fraction of the methanolic extract from the roots of J. curcas by column chromatography (CC): silica gel, Sephadex LH-20, ODS, semi-preparative reversed-phase high-performance liquid chromatography (HPLC). The structures were identified based on HR-ESI-MS and 1D, 2D-NMR spectroscopic analysis. Their anti-inflammatory effects were tested on lipopolysaccharide (LPS, 500 ng/mL)-stimulated murine RAW264.7 macrophages. Furthermore, we conducted transcriptome-wide RNA sequencing to profile gene expression alterations in LPS-induced RAW264.7 cells upon treatment with jatrocurcasenone I (4) and analyzed the underlying genes targeted by this compound.

Results: Six diterpenoids were obtained from J. curcas, and four of them were identified to be new lathyrane diterpenoids: jatrocurcasenones F-I (1-4). Compounds 3 and 4 exhibited potent inhibitory activities against LPS-induced nitric oxide (NO) production in RAW264.7 cells with IC₅₀ values of 11.28 μM and 7.71 μM, respectively. Western blotting analysis showed that the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were suppressed with the supplementation of 3 and 4. The results of RNA-seq showed that 4 (20 μM) exhibited regulation on the 587 differentially expressed genes (DEGs) induced by LPS (500 ng/mL). Transcriptome-wide RNA sequencing indicated that the protective activity of 4 supplementation was most likely driven by modulating expression levels of IL-1α, IL-1β, IL-1f6, IL-6, IL-1rn, IL-27, Ccl2, Ccl5, Ccl7, Ccl9, Ccl22, Cxcl10, Tnfsf12, Tnfsf15, Lta, Trim25, Bcl2a1a, Dusp1, Dusp2, Ptgs2, Edn1 and Nr4a1.

Conclusions: This study offered four new lathyrane diterpenoids, of them, jatrocurcasenone I (4) showed significant anti-inflammatory activity. RNA-Seq suggested that Jatrocurcasenone I (4) could be a candidate drug for the prevention inflammation-mediated diseases by modulating 24 candidate DEGs.

Keywords: Anti-inflammatory; Diterpeniods; Jatropha curcas; Transcriptome sequencing.

Streptococcus suis serotype 2 (SS2) is a serious zoonotic pathogen; it can lead to symptoms of streptococcal toxic shock syndrome (STSS) in humans and sepsis in pigs, and poses a great threat to public health. The SS2 MetQ gene deletion strain has attenuated antiphagocytosis, although the mechanism of antiphagocytosis and pathogenesis of MetQ in SS2 has remained unclear. In this study, stable isotope labeling by amino acids in cell culture (SILAC) based liquid chromatography-mass spectrometry (LC-MS) and subsequent bioinformatics analysis was used to determine differentially expressed proteins of RAW264.7 cells infected with △MetQ and ZY05719. Proteomic results were verified by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting for selected proteins. Further research was focused mainly on immune system processes related to downregulated proteins, such as Src and Ccl9, and actin cytoskeleton and endocytosis related upregulated proteins, like Pstpip1 and Ppp1r9b. The proteomic results in this study shed light on the mechanism of antiphagocytosis and innate immunity of macrophages infected with △MetQ and ZY05719, which might provide novel targets to prevent or control the infection of SS2.

Keywords: Antiphagocytosis; Macrophages; MetQ; Quantitative proteomics; Streptococcus suis serotype 2.

A major green tea component, epigallocatechin-3-gallate (EGCG), has been proven protective against lethal sepsis in experimental setting, but its protective mechanisms remain incompletely understood. Here, we provide evidence to support EGCG's capacities in stimulating G-CSF production and neutrophilia in vivo. In an animal model of sepsis, EGCG significantly elevated peritoneal levels of G-CSF and several chemokines (e.g., MCP-1/CCL2 and MIP-1γ/CCL9), and consequently increased peritoneal neutrophil numbers (neutrophilia) at a late stage. In vitro, EGCG divergently affected HMGB1-mediated production of several chemokines: reducing CXCL15 and RANTES/CCL5, but elevating G-CSF and MIP-1α/CCL3 production by peritoneal macrophages. Similarly, it significantly induced the expression and secretion of G-CSF and MIP-1α/CCL3 in human peripheral blood mononuclear cells. Based on our preliminary data, it may be important to search for anti-inflammatory and G-CSF-stimulating agents for the clinical management of inflammatory diseases.

20-Hydroxyl-3-oxolupan-28-oic acid (HOA), a minor component from Mahonia bealei (Fort.) Carr. leaves, has been found to attenuate inflammatory responses. However, the underlying molecular mechanism is still unclear. In this study, we performed a comprehensive transcriptional study to investigate genetic changes. We used RNA sequencing technology to analyses the transcriptional changes in RAW 264.7 cells in a control group, lipopolysaccharide (LPS)-induced group, and HOA-treated group. We identified 1,313 and 388 differentially expressed genes (DEGs) in the control/LPS group and LPS/HOA group, respectively. Gene Ontology (GO) classification revealed that the DEGs were mainly enriched in a series of inflammatory and immune-related processes. The results of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that the DEGs were mainly enriched in inflammatory-related pathways such as the nuclear factor-kappa B (NF-κB) signaling pathway, cytokine-cytokine receptor interaction, chemokine signaling pathway, mitogen-activated protein kinase (MAPK) pathway, and Janus kinase-signal transducer and activator of transcription proteins (JAK-STAT) signaling pathway. The results of qPCR validation revealed that dynamic changes in immune-related mRNAs such as Saa3, Bcl2l1, Mapkapk2, Ccl9, Sdc4, Ddx3x, Socs3, Prdx5, Tlr4, Lif, IL15, Tnfaip3, Tet2, Tgf-β1, and Ccl20, which were significantly upregulated in the LPS group and downregulated in the HOA group. Taken together, these results suggest that HOA may be used as a source of anti-inflammatory agents as well as a dietary complement for health promotion.

Keywords: 20-hydroxy-3-oxolupan-28-oic acid; Mahonia bealei; NF-κB; anti-inflammatory; transcriptome analyses.

Whereas the prevalence of several cancer types is decreasing, skin malignancies are growing more common every year. Malignant melanoma is the most aggressive form of skin cancer with high metastatic capacity. In most cases, malignant melanoma shows acquired therapy resistance. We evaluated the ability of Ocoxin, a natural compound-based antioxidant and anti-inflammatory nutritional complement, to exert an antitumor effect in melanoma. To do so, the cytotoxicity of Ocoxin in a panel of BRAF-mutated murine and human melanoma cell lines was tested alone and in combination with BRAF inhibitor Vemurafenib. Our results revealed a potent cytotoxic effect of Ocoxin against melanoma cells and a synergic effect when combined with Vemurafenib, reducing viability and increasing apoptosis. Besides, Ocoxin interferes with the cell cycle, impairs the inherent and fibroblast-mediated melanoma cell migration, and reduces resistance to BRAF inhibition. Proteomic analysis revealed reduced tumor secretion of inflammatory factors Galectin-1, Osteopontin, CCL5, and CCL9 upon treatment with Ocoxin. Moreover, RNASeq showed that Ocoxin downregulated the cell cycle and proliferation-related genes. In vivo, Ocoxin reduced the number of lung metastasis of YUMM-1.7 melanoma cells. Therefore, Ocoxin arises as a good candidate for clinical trials analyzing the beneficial effects in patients suffering from this cutaneous malignancy.

Keywords: BRAF inhibition; adjuvant; cancer nutrition; chemoresistance; fibroblasts; melanoma; tumor microenvironment.

Although obesity has been associated with an increased risk and aggressiveness of many types of carcinoma, whether it promotes squamous cell carcinoma remains unclear. To reveal the role of obesity in oral squamous cell carcinoma (OSCC) initiation and development, we used 4NQO-induced OSCC model mice to examine the impact of dietary obesity on carcinogenesis. The results showed that high-fat diet (HFD)-induced obesity significantly promoted the incidence of OSCC and altered the local immune microenvironment with the expansion of CD11b⁺Gr1⁺ myeloid-derived suppressor cells (MDSCs). The underlying mechanism that induced an immunosuppressive local microenvironment in obesity was the recruitment of MDSCs through the CCL9/CCR1 axis and enhancement of MDSC immunosuppressive function via intracellular fatty acid uptake. Furthermore, clinical samples verified the increase in infiltrated CD33⁺ (a marker of human MDSCs) cells in obese OSCC patients, and data from the TCGA dataset confirmed that CD33 expression was positively correlated with local adipocytes in OSCC. Survival analysis showed that enrichment of adipocytes and high expression of CD33 were associated with poor prognosis in OSCC patients. Strikingly, depletion of MDSCs significantly ameliorated HFD-promoted carcinogenesis in 4NQO-induced model mice. These findings indicate that obesity is also an important risk factor for OSCC, and cancer immunotherapy, especially targeting MDSCs, may exhibit greater antitumor efficacy in obese patients.