OBJECTIVE

To compare the cyto- and chemokine profiles in the aqueous humor of PEXS eyes in the absence or presence of secondary glaucoma with or without luxation of the intraocular lens (IOL).

METHODS

Samples of aqueous humor were collected intraoperatively from 20 healthy controls and from 73 eyes with PEX-syndrome, which was manifested in the absence of any other local or systemic desease. The latter group was sub-devided into eyes with an early form of PEX-syndrome in the absence of complications (PEX, n = 33), those with a late form of PEX-syndrome and glaucoma (PEXG, n = 30), and those with a late form of PEX-syndrome with luxation of the IOL that required surgery (PEXL, n = 10). The samples were analyzed in parallel after storage at -80°C. The levels of 40 cytokines were simultaneously quantified using the Bio-Plex® multiplex beads system. The inter-group data were statistically compared using the Kruskal-Wallis test (p ≤ 0.01).

RESULTS

PEX and PEXG were comparable in their cytokine profiles for all 40 cytokines, whereas the cytokine profile in PEXL-eyes revealed higher levels of all but 5 cytokines (CXCL13, CCL27, IL-2, CCL3, CCL20; p ≤ 0.01). This latter finding is indicative of a non-specific inflammatory reaction in the context of IOL-luxation. The concentrations of 6 cytokines lay below the detection limit in all groups.

CONCLUSIONS

The local up-regulation of 85% of the detectable cytokines in the aqueous humor of PEXL-eyes may be linked either with a progression of the disease or a breakdown of the antero-posterior barrier in the context of IOL-luxation.

BACKGROUND

TNF inhibitors have been used as a treatment for moderate to severe RA patients. However, reliable biomarkers that predict therapeutic response to TNF inhibitors are lacking. In this study, we investigated whether chemokines may represent useful biomarkers to predict the response to TNF inhibitor therapy in RA.

METHODS

RA patients (n = 29) who were initiating adalimumab or etanercept were recruited from the rheumatology clinics at Cooper University Hospital. RA patients were evaluated at baseline and 14 weeks after TNF inhibitor therapy, and serum levels of CXCL10, CXCL13, and CCL20 were measured by ELISA. Responders (n = 16) were defined as patients who had good or moderate response at week 14 by EULAR response criteria, and nonresponders (n = 13) were defined as having no response.

RESULTS

Responders had higher levels of baseline CXCL10 and CXCL13 compared to nonresponders (p = 0.03 and 0.002 respectively). There was no difference in CCL20 levels. CXCL10 and CXCL13 were highly correlated with each other, and were higher in seropositive RA patients. CXCL10 and CXCL13 levels were decreased after TNF inhibitor therapy in responders. Baseline additive levels of CXCL10 + 13 were correlated with changes in DAS score at 14 weeks after TNF inhibitor therapy (r = 0.42, p = 0.03), and ROC curve analyses for predictive ability of CXCL10 + 13 showed an AUC of 0.83.

CONCLUSIONS

Elevated baseline levels of CXCL10 and CXCL13 were associated with favorable response to TNF inhibitor therapy in RA. Subjects with high CXCL10 and high CXCL13 may represent a subset of RA patients whose inflammatory reactions are primarily driven by TNF.

Chemokines, which regulate leukocyte trafficking and infiltration of local sites, are attractive candidates for improving the efficacy of cancer immunotherapy by enhancing the accumulation of immune cells in tumor tissue. Herein, we evaluated the antitumor effects of intratumoral injection of RGD fiber-mutant adenoviral vectors (AdRGDs) encoding the chemokines CCL17, CCL19, CCL20, CCL21, CCL22, CCL27, XCL1 or CX3CL1 in a murine model of preexisting CT26 colon carcinoma. Among these 8 chemokine-expressing AdRGDs, injection of AdRGD-CCL17 most effectively induced tumor regression and generated specific immunity in rechallenge experiments. Tumor elimination activity by intratumoral injection of AdRGD-CCL17 depended on both the vector dose and the number of injections, and mainly required CD8+ CTLs in an effector phase as confirmed by analysis using BALB/c nude mice and an in vivo depletion assay. In addition, CCL17 gene transduction induced significant increases in the number of infiltrating macrophages and CD8+ T cells in CT26 tumors, and changed the tumor microenvironment to an immunologic activation state in which there was enhanced expression of lymphocyte activation markers and cell adhesion molecules. Thus, our data provide evidence that CCL17 gene transduction of local tumor sites is a promising approach for the development of a cancer immunogene therapy that can recruit activated tumor-infiltrating immune effector cells.

The protein kinase D (PKD) family consists of three serine-threonine kinases involved in cellular proliferation, motility, and apoptosis. We previously reported that human toll-like receptor 5 (TLR5) contains a consensus PKD phosphorylation site. Flagellin stimulation of cells activated PKD1, and inhibition of PKD1 reduced flagellin-induced interleukin-8 (IL-8) production in epithelial cells. In the current work, we examined PKD1 and PKD2 involvement downstream of TLR5, TLR4 and TLR2. We found that inhibition of either kinase with shRNA reduced IL-8 and CCL20 release due to TLR4 and TLR2 agonists to a similar extent as previously reported for TLR5. PKD1 and PKD2 inhibition reduced NF-kappaB activity but not MAPK activation. These results demonstrate that both PKD1 and PKD2 are required for inflammatory responses following TLR2, TLR4, or TLR5 activation, although PKD1 is more strongly involved. These kinases likely act downstream of the TLRs themselves to facilitate NF-kappaB activation but not MAP kinase phosphorylation.

Chemokines direct cellular infiltration to tissues, and their receptors and signaling pathways represent targets for therapy in diseases such as multiple sclerosis (MS). The chemokine CCL20 is expressed in choroid plexus, a site of entry of T cells to the central nervous system (CNS). The CCL20 receptor CCR6 has been reported to be selectively expressed by CD4(+) T cells that produce the cytokine IL-17 (Th17 cells). Th17 cells and interferon-gamma (IFNγ)-producing Th1 cells are implicated in induction of MS and its animal model experimental autoimmune encephalomyelitis (EAE). We have assessed whether CCR6 identifies specific inflammatory T cell subsets in EAE. Our approach was to induce EAE, and then examine chemokine receptor expression by cytokine-producing T cells sorted from CNS at peak disease. About 7% of CNS-infiltrating CD4(+) T cells produced IFNγ in flow cytometric cytokine assays, whereas less than 1% produced IL-17. About 1% of CD4(+) T cells produced both cytokines. CCR6 was expressed by Th1, Th1+17 and by Th17 cells, but not by CD8(+) T cells. CD8(+) T cells expressed CXCR3, which was also expressed by CD4(+) T cells, with no correlation to cytokine profile. Messenger RNA for IFNγ, IL-17A, and the Th1 and Th17-associated transcription factors T-bet and RORγt was detected in both CCR6(+) and CXCR3(+) CD4(+) T cells. IFNγ, but not IL-17A mRNA expression was detected in CD8(+) T cells in CNS. CCR6 and CD4 were co-localized in spinal cord infiltrates by double immunofluorescence. Consistent with flow cytometry data some but not all CD4(+) T cells expressed CCR6 within infiltrates. CD4-negative CCR6(+) cells included macrophage/microglial cells. Thus we have for the first time directly studied CD4(+) and CD8(+) T cells in the CNS of mice with peak EAE, and determined IFNγ and IL17 expression by cells expressing CCR6 and CXCR3. We show that neither CCR6 or CXCR3 align with CD4 T cell subsets, and Th1 or mixed Th1+17 predominate in EAE.

Seminal plasma (SP) is considered as a vehicle to carry sperm into female reproductive tract, of which functions have not been completely understood. This study aimed to identify the function of seminal exosomes on porcine endometrium. Exosomes were isolated from the sperm-rich fraction of boar semen and were confirmed by the expression of exosome marker HSP70 and size distribution using nano-sight tracking analysis. Porcine endometrial epithelial cells (EECs) were then treated with seminal exosomes, and RNA extracted were subjected to global expression analysis. Transcripts related to "immune response", "inflammatory response" and their associated signaling pathways were up-regulated in EECs treated with seminal exosome, whereas those associated with "steroid biosynthesis", "metabolic pathways" and "T cell differentiation" were down-regulated. The decrease in PMVK, SC5D, INSIG1, HSD17B7, NSDHL, HMGCR, SQLE and FDFT1, and increase in CCL20, TNFSF15, AMCFII, CXCL2 and CXCL8 were also found in the endometrium from the naturally mated pigs. Moreover, changes in exosome-induced CYP24A1, EBP, CCL20, AMCFII and IL1A expression were not regulated by the exosome removed SP. These observations indicated that exosomes present in SP are involved in the immune-related gene regulation in the uterus, which could pave the passage for sperm and possibly fertilized eggs.

Sex differences in the incidence of respiratory diseases have been reported. Women are more susceptible to inflammatory lung disease induced by air pollution and show worse adverse pulmonary health outcomes than men. However, the mechanisms underlying these differences remain unknown. In the present study, we hypothesized that sex differences in the expression of lung inflammatory mediators affect sex-specific immune responses to environmental toxicants. We focused on the effects of ground-level ozone, a major air pollutant, in the expression and regulation of lung immunity genes. We exposed adult male and female mice to 2 ppm of ozone or filtered air (control) for 3 h. We compared mRNA levels of 84 inflammatory genes in lungs harvested 4 h postexposure using a PCR array. We also evaluated changes in lung histology and bronchoalveolar lavage fluid cell counts and protein content at 24 and 72 h postexposure. Our results revealed sex differences in lung inflammation triggered by ozone exposure and in the expression of genes involved in acute phase and inflammatory responses. Major sex differences were found in the expression of neutrophil-attracting chemokines (Ccl20, Cxcl5, and Cxcl2), the proinflammatory cytokine interleukin-6, and oxidative stress-related enzymes (Ptgs2, Nos2). In addition, the phosphorylation of STAT3, known to mediate IL-6-related immune responses, was significantly higher in ozone-exposed mice. Together, our observations suggest that a differential regulation of the lung immune response could be implicated in the observed increased susceptibility to adverse health effects from ozone observed in women vs. men.

Adjuvant chemotherapy offered to treat colon cancer is based on the TNM staging system, which often fails due to molecular heterogeneity and undefined molecular mechanisms independent of TNM. Therefore, identification of markers to better predict therapeutic option and outcome is needed. In this study we have characterised the clinical association of CCR6 with colon cancer and defined CCR6-mediated molecular pathway.

Immunohistochemistry, RT-qPCR, western blot and FACS were used to determine expression of CCR6 and/or EMT markers in colon tissues/cells. BrdU assay and trans-well system were used to determine cell proliferation, migration and invasion in response to CCL20.

CCR6 was higher in cancer cases compared to normal adjacent tissue and expression was associated with nodal status and distant metastasis. Similarly, CCR6 expression was higher in cells derived from node-positive cases and highest expression was in cells derived from metastatic cases. Significant changes in EMT markers, that is, E-cadherin, vimentin, β-catenin, N-cadherin, α-SMA, SNAILl and ZEB1 were observed in response to CCL20 along with decreased proliferation, increased migratory and invasive potential.

Results suggest CCR6 as a potential therapeutic target as well as biomarker in addition to nodal status for predicting therapeutic option.

OBJECTIVE

About 60-80 % of chronic hepatitis B virus (HBV) carriers are characterized with persistently normal alanine transaminase (ALT). Differences of cytokine expression are associated with the prognosis of HBV infection. We investigated the expression pattern of 30 cytokines associated with anti-HBV immunity in patients with normal ALT.

METHODS

Four patient groups (immune tolerance, inactive hepatitis B surface antigen carriers, resolved hepatitis B, and control; 10 subjects per group) were assigned. Thirty cytokines, including IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-9, IL-10, IL-12p40, IL-12p70, IL-15, IL-17A, IL-17C, IL-21, IL-22, IL-23p19, IL-28A, IL-29, CCL5, CCL16, CCL20, CCL22, CXCL9, CXCL10, CXCL11, TNFRSF8, TNFRSF18, IL-6R, gp130, and TGF-β1, were measured using a human cytokine antibody array. Signal intensities were obtained by laser scanner. Protein-protein interactions were analyzed by STRING (Search Tool for the Retrieval of Interacting Genes/Proteins).

RESULTS

Significant differences of signal intensities were observed for IL-2, IL-4, IL-6, IL-7, IL-9, IL-10, IL-12p40, IL-12p70, IL-15, IL-21, IL-23p19, IL-28A, and IL-29. The lowest intensity was in controls. Among three HBV infection groups, significant differences were observed in IL-2, IL-4, IL-12p70, IL-15, IL-21, IL-23p19, and IL-29. The highest intensity was in the inactive group. All cytokines with significant differences were involved JAK-STAT signaling that up-regulate FOXP3, SOCS3 and MX1.

CONCLUSIONS

Differential expression of cytokines in JAK-STAT signaling is an important factor associated with prognosis of HBV infection. The elevation of γC cytokines, IL-12p70, IL-23p19, and IL-29 may promote spontaneous HBeAg seroconversion and HBV clearance.

Moderate-to-severe atopic dermatitis (AD) has been associated with significant disease burden and systemic abnormalities and often requires systemic treatments. Currently, safe and effective oral systemic treatments for moderate-to-severe AD are not yet available. ASN002 is an oral inhibitor of the Janus kinase/spleen tyrosine kinase signaling pathways, targeting several cytokine axes (T_HHHHWe sought to evaluate the effect of ASN002 on the cellular and molecular biomarker profile of patients with moderate-to-severe AD and to correlate changes in biomarkers to improvements in clinical severity measures and pruritus.Thirty-six patients with moderate-to-severe AD were randomized to groups with dose escalation of ASN002 (20, 40, and 80 mg) and a placebo group. Skin biopsy specimens were performed at baseline, day 15, and day 29. Gene expression studies were conducted by using microarray and quantitative RT-PCR, and cellular infiltrates and protein expression were studied by using immunohistochemistry.

RESULTS

ASN002 reversed the lesional skin transcriptome toward a nonlesional phenotype. It also rapidly and significantly suppressed key inflammatory pathways implicated in AD pathogenesis, including T_HHHCCL20, S100A7/S100A8/S100A9, and IL36G/IL36RN), and T_HThe Janus kinase/spleen tyrosine kinase inhibitor ASN002 significantly suppressed key AD inflammatory pathways, corresponding to clinical response. ASN002 might be an effective novel therapeutic agent for moderate-to-severe AD.

Staphylococcus aureus is a leading cause of invasive infection. It also infects wet mucosal tissues including the cornea and conjunctiva. Conflicting evidence exists on the expression of Toll-like receptors by human corneal epithelial cells. It was therefore of interest to determine how epithelial cells from this immune privileged tissue respond to S. aureus. Further, it was of interest to determine whether cytolytic toxins, with the potential to cause ion flux or potentially permit effector molecule movement across the target cell membrane, alter the response. Microarrays were used to globally assess the response of human corneal epithelial cells to S. aureus. A large increase in abundance of transcripts encoding the antimicrobial dendritic cell chemokine, CCL20, was observed. CCL20 release into the medium was detected, and this response was found to be largely TLR2 and NOD2 independent. Corneal epithelial cells also respond to S. aureus by increasing the intracellular abundance of mRNA for inflammatory mediators, transcription factors, and genes related to MAP kinase pathways, in ways similar to other cell types. The corneal epithelial cell response was surprisingly unaffected by toxin exposure. Toxin exposure did, however, induce a stress response. Although model toxigenic and non-toxigenic strains of S. aureus were employed in the present study, the results obtained were strikingly similar to those reported for stimulation of vaginal epithelial cells by clinical toxic shock toxin expressing isolates, demonstrating that the initial epithelial cellular responses to S. aureus are largely independent of strain as well as epithelial cell tissue source.

CC chemokines (or β-chemokines) are 28 chemotactic cytokines with an N-terminal CC domain that play an important role in immune system cells, such as CD4⁺ and CD8⁺ lymphocytes, dendritic cells, eosinophils, macrophages, monocytes, and NK cells, as well in neoplasia. In this review, we discuss human CC motif chemokine ligands: CCL1, CCL3, CCL4, CCL5, CCL18, CCL19, CCL20, CCL21, CCL25, CCL27, and CCL28 (CC motif chemokine receptor CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10 ligands). We present their functioning in human physiology and in neoplasia, including their role in the proliferation, apoptosis resistance, drug resistance, migration, and invasion of cancer cells. We discuss the significance of chemokine receptors in organ-specific metastasis, as well as the influence of each chemokine on the recruitment of various cells to the tumor niche, such as cancer-associated fibroblasts (CAF), Kupffer cells, myeloid-derived suppressor cells (MDSC), osteoclasts, tumor-associated macrophages (TAM), tumor-infiltrating lymphocytes (TIL), and regulatory T cells (T_reg). Finally, we show how the effect of the chemokines on vascular endothelial cells and lymphatic endothelial cells leads to angiogenesis and lymphangiogenesis.

Keywords: CC chemokine; angiogenesis; cancer; chemokine; lymphangiogenesis; organ-specific metastasis; tumor; tumor microenvironment.

Although olfactory mucosa possesses long-lived horizontal basal stem cells (HBCs) and remarkable regenerative capacity, the function of human olfactory neuroepithelium is significantly impaired in chronic inflammatory rhinosinusitis. Here, we show that, while inflammation initially damages olfactory neurons and activates HBC-mediated regeneration, continued inflammation locks HBCs in an undifferentiated state. Global gene expression in mouse HBCs reveals broad upregulation of NF-κB-regulated cytokines and chemokines including CCL19, CCL20, and CXCL10, accompanied by enhancement of "stemness"-related transcription factors. Loss-of-function studies identify an NF-κB-dependent role of HBCs in amplifying inflammatory signaling, contributing to macrophage and T cell local proliferation. Chronically activated HBCs signal macrophages to maintain immune defense and prevent Treg development. In diseased human olfactory tissue, activated HBCs in a P63⁺ undifferentiated state similarly contribute to inflammation through chemokine production. These observations establish a mechanism of chronic rhinosinusitis-associated olfactory loss, caused by a functional switch of neuroepithelial stem cells from regeneration to immune defense.

Hirschsprung disease (HSCR) is a rare congenital anomaly characterized by the absence of enteric ganglia in the distal intestinal tract. While classified as a multigenic disorder, the altered function of the RET tyrosine kinase receptor is responsible for the majority of the pathogenesis of HSCR. Recent evidence demonstrate a strong association between RET and the homeostasis of immune system. Here, we utilize a unique cohort of fifty HSCR patients to fully characterize the expression of RET receptor on both innate (monocytes and Natural Killer lymphocytes) and adaptive (B and T lymphocytes) human peripheral blood mononuclear cells (PBMCs) and to explore the role of RET signaling in the immune system. We show that the increased expression of RET receptor on immune cell subsets from HSCR individuals correlates with the presence of loss-of-function RET mutations. Moreover, we demonstrate that the engagement of RET on PBMCs induces the modulation of several inflammatory genes. In particular, RET stimulation with glial-cell line derived neurotrophic factor family (GDNF) and glycosyl-phosphatidylinositol membrane anchored co-receptor α1 (GFRα1) trigger the up-modulation of genes encoding either for chemokines (CCL20, CCL2, CCL3, CCL4, CCL7, CXCL1) and cytokines (IL-1β, IL-6 and IL-8) and the down-regulation of chemokine/cytokine receptors (CCR2 and IL8-Rα). Although at different levels, the modulation of these "RET-dependent genes" occurs in both healthy donors and HSCR patients. We also describe another set of genes that, independently from RET stimulation, are differently regulated in healthy donors versus HSCR patients. Among these "RET-independent genes", there are CSF-1R, IL1-R1, IL1-R2 and TGFβ-1, whose levels of transcripts were lower in HSCR patients compared to healthy donors, thus suggesting aberrancies of inflammatory responses at mucosal level. Overall our results demonstrate that immune system actively participates in the physiopathology of HSCR disease by modulating inflammatory programs that are either dependent or independent from RET signaling.

A previous report by this laboratory demonstrated that bacterial iron chelator (siderophore) triggers inflammatory signals, including the production of CXC chemokine IL-8, in human intestinal epithelial cells (IECs). Microarray-based gene expression profiling revealed that iron chelator also induces macrophage inflammatory protein 3 alpha (MIP-3alpha)/CC chemokine-ligand 20 (CCL20). As CCL20 is chemotactic for the cells involved in host adaptive immunity, this suggests that iron chelator may stimulate IECs to have the capacity to link mucosal innate and adaptive immunity. The basal medium from iron chelator deferoxamine (DFO)-treated HT-29 monolayers was as chemotactic as recombinant human CCL20 at equivalent concentrations to attract CCR6(+) cells. The increase of CCL20 protein secretion appeared to correspond to that of CCL20 mRNA levels, as determined by real-time quantitative RT-PCR. The efficacy of DFO at inducing CCL20 mRNA was also observed in human PBMCs and in THP-1 cells, but not in human umbilical vein endothelial cells. Interestingly, unlike other proinflammatory cytokines, such as TNF-alpha and IL-1beta, a time-dependent experiment revealed that DFO slowly induces CCL20, suggesting a novel mechanism of action. A pharmacologic study also revealed that multiple signaling pathways are differentially involved in CCL20 production by DFO, while some of those pathways are not involved in TNF-alpha-induced CCL20 production. Collectively, these results demonstrate that, in addition to some bacterial products known to induce host adaptive immune responses, direct chelation of host iron by infected bacteria may also contribute to the initiation of host adaptive immunity in the intestinal mucosa.

The proinflammatory cytokines interleukin (IL)-17 and tumour necrosis factor (TNF)-α are targets for treatment in many chronic inflammatory diseases. Here, we examined their role in liver inflammatory response compared to that of IL-6. Human hepatoma cells (HepaRG, Huh7.5 and HepG2 cells) and primary human hepatocytes (PHH) were cultured with IL-6, IL-17 and/or TNF-α. To determine the contribution of the IL-6 pathway in the IL-17/TNF-α-mediated effect, an anti-IL-6 receptor antibody was used. IL-17 and TNF-α increased in synergy IL-6 secretion by HepaRG cells and PHH but not by Huh7.5 and HepG2 cells. This IL-17/TNF-α synergistic cooperation enhanced the levels of C-reactive protein (CRP) and aspartate aminotransferase (ASAT) in HepaRG cell and PHH cultures through the induction of IL-6. IL-17/TNF-α also up-regulated IL-8, monocyte chemoattractant protein (MCP)-1 and chemokine (C-C motif) ligand 20 (CCL20) chemokines in synergy through an IL-6-independent pathway. Interestingly, first exposure to IL-17, but not to TNF-α, was crucial for the initiation of the IL-17/TNF-α synergistic effect on IL-6 and IL-8 production. In HepaRG cells, IL-17 enhanced IL-6 mRNA stability resulting in increased IL-6 protein levels. The IL-17A/TNF-α synergistic effect on IL-6 and IL-8 induction was mediated through the activation of extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase, nuclear factor-κB and/or protein kinase B (Akt)-phosphatidylinositol 3-kinase signalling pathways. Therefore, the IL-17/TNF-α synergistic interaction mediates systemic inflammation and cell damage in hepatocytes mainly through IL-6 for CRP and ASAT induction. Independently of IL-6, the IL-17A/TNF-α combination may also induce immune cell recruitment by chemokine up-regulation. IL-17 and/or TNF-α neutralization can be a promising therapeutic strategy to control both systemic inflammation and liver cell attraction.

Previous studies have revealed that carcinoma-associated fibroblasts communicate microenvironment-derived signals through chemokine/chemokine receptor interaction, resulting in carcinogenesis. C-C motif chemokine ligand 20 (CCL20)/C-C motif chemokine receptor 6 (CCR6) interactions are involved in the pathogenesis of colonic malignancies. The present study aimed to characterize the roles of CCL20/CCR6 and the extracellular signal-regulated kinase (ERK) signaling pathway in lung adenocarcinoma growth. Lung adenocarcinoma samples obtained at surgery were assessed for the expression, tissue localization and production of CCL20/CCR6. In addition, colony formation, ERK signaling and chemokine production were measured to assess the responsiveness of the A549 cell line to CCL20 stimulation. CCL20 and CCR6 were found to be highly expressed in the majority of samples in the recurrence group (76 and 66%, respectively). The staining indexes of CCL20 and CCR6 in the recurrence group were 149.3 and 134.4, respectively, which were significantly higher than those in the non-recurrence group (57.2 and 58.0, respectively); the protein and mRNA expression levels determined by western blot and reverse transcription-quantitative polymerase chain reaction were also found to be high in the recurrence group For A549 cells, the colony-forming capacity was increased by CCL20 stimulation, and this effect was dependent in part on ERK phosphorylation. Collectively, the findings suggest that CCR6 and CCL20 may serve a role in lung adenocarcinoma, leading to proliferation and migration via autocrine or paracrine mechanisms. The disruption of CCL20/CCR6 interactions may be a promising strategy for the treatment of cancer.

Overdosing of the analgesic acetaminophen (APAP, paracetamol) is a major cause of acute liver injury. Whereas toxicity is initiated by hepatocyte necrosis, course of disease is regulated by mechanisms of innate immunity having the potential to serve in complex manner pathogenic or pro-regenerative functions. Interleukin (IL)-36γ has been identified as novel IL-1-like cytokine produced by and targeting epithelial (-like) tissues. Herein, we investigated IL-36γ in acute liver disease focusing on murine APAP-induced hepatotoxicity. Enhanced expression of hepatic IL-36γ and its prime downstream chemokine target CCL20 was detected upon liver injury. CCL20 expression coincided with the later regeneration phase of intoxication. Primary murine hepatocytes and human Huh7 hepatocellular carcinoma cells indeed displayed enhanced IL-36γ expression when exposed to inflammatory cytokines. Administration of IL-36 receptor antagonist (IL-36Ra) decreased hepatic CCL20 in APAP-treated mice. Unexpectedly, IL-36Ra likewise increased late phase hepatic injury as detected by augmented serum alanine aminotransferase activity and histological necrosis which suggests disturbed tissue recovery upon IL-36 blockage. Finally, we demonstrate induction of IL-36γ in inflamed livers of endotoxemic mice. Observations presented introduce IL-36γ as novel parameter in acute liver injury which may contribute to the decision between unleashed tissue damage and initiation of liver regeneration during late APAP toxicity.

Human milk oligosaccharides (HMO) have been shown to interact directly with immune cells. However, large quantities of HMO are required for intervention or clinical studies, but these are unavailable in most cases. In this respect, bovine milk is potentially an excellent source of commercially viable analogues of these unique molecules. In the present study, we compared the transcriptional response of colonic epithelial cells (HT-29) to the entire pool of HMO and bovine colostrum oligosaccharides (BCO) to determine whether the oligosaccharides from bovine milk had effects on gene expression that were similar to those of their human counterparts. Gene set enrichment analysis of the transcriptional data revealed that there were a number of similar biological processes that may be influenced by both treatments including a response to stimulus, signalling, locomotion, and multicellular, developmental and immune system processes. For a more detailed insight into the effects of milk oligosaccharides, the effect on the expression of immune system-associated glycogenes was chosen as a case study when performing validation studies. Glycogenes in the current context are genes that are directly or indirectly regulated in the presence of glycans and/or glycoconjugates. RT-PCR analysis revealed that HMO and BCO influenced the expression of cytokines (IL-1β, IL-8, colony-stimulating factor 2 (granulocyte-macrophage) (GM-CSF2), IL-17C and platelet factor 4 (PF4)), chemokines (chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (C-X-C motif) ligand 3 (CXCL3), chemokine (C-C motif) ligand 20 (CCL20), chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand 6 (CXCL6), chemokine (C-C motif) ligand 5 (CCL5), chemokine (C-X3-C motif) ligand 1 (CX3CL1) and CXCL2) and cell surface receptors (interferon γ receptor 1 (IFNGR1), intercellular adhesion molecule-1 (ICAM-1), intercellular adhesion molecule-2 (ICAM-2) and IL-10 receptor α (IL10RA)). The present study suggests that milk oligosaccharides contribute to the development and maturation of the intestinal immune response and that bovine milk may be an attractive commercially viable source of oligosaccharides for such applications.

Oral squamous cell carcinomas are associated with a poor prognosis, which may be partly due to functional impairment of the immune response. Lymphocyte recruitment to the tumor site is facilitated by high-endothelial venules, whereas expression of programmed-death ligand 1 (PD-L1) can impair T-cell function. Thus, we hypothesize that these factors are important in shaping the immune response in oral squamous cell carcinoma. In the present study, we characterized the immune infiltrate in formalin-fixed, paraffin-embedded tumor samples from 75 oral squamous cell carcinoma patients. We used immunohistochemistry to determine the distribution of immune cell subsets, high-endothelial venules, and PD-L1, as well as quantitative real-time polymerase chain reaction to assess the expression of inflammatory cytokines and chemokines associated with lymphocyte trafficking. Finally, we calculated correlations between the presence of immune cell subsets, the gene expression patterns, high-endothelial venules, PD-L1, and the clinicopathological parameters, including patient survival. The presence of high-endothelial venules correlated with increased number of CD3+ T cells and CD20+ B cells, higher levels of the chemokines CXCL12 and CCL21, and lower levels of CCL20, irrespective of the tumors' T stage. In univariate analysis, high levels of CD20+ B cells and CD68+ macrophages, positive high-endothelial venule status, and low T and N stages predicted longer patient survival. However, only the presence of high-endothelial venules and a low T stage were independent positive prognosticators. This indicates that high-endothelial venules are important mediators and a convenient marker of an antitumor immune response in oral squamous cell carcinoma. Our findings suggest that these vessels are a potential immunomodulatory target in this type of cancer. PD-L1 staining in tumor cells correlated with lower T stage, increased infiltration of CD4+ cells, and higher expression of several inflammation-related cytokines. Thus, oral squamous cell carcinomas rich in CD4+ cells may preferentially respond to PD-1/PD-L1 blockade therapy.

OBJECTIVE

Production of chemokines by intestinal epithelial cells is a key step in the amplification of the destructive immune-inflammatory response in patients with inflammatory bowel diseases [IBD]. In this study, we examined whether intestinal epithelial cells express macrophage colony-stimulating factor receptor 1 [M-CSFR-1], the functional receptor of interleukin-34 [IL-34], a cytokine that is over-produced in IBD and supposed to sustain inflammatory pathways.

METHODS

M-CSFR-1 expression was evaluated in intestinal samples of IBD patients, controls, and colon epithelial cell lines by real-time polymerase chain reaction [PCR], immunohistochemistry, and western blotting. DLD-1 cells were stimulated with IL-34 in the presence or absence of MAP kinase inhibitors, chemokine induction was assessed by real-time PCR and enzyme-linked immunosorbent assay [ELISA], and mitogen-activated protein (MAP) kinase activation was monitored by western blotting. The effect of a neutralising IL-34 antibody on CC chemokine ligand (CCL) 20 synthesis was tested in ex vivo organ cultures of IBD mucosal explants.

RESULTS

Enhanced expression of M-CSFR-1 RNA transcripts was seen in inflamed mucosa of IBD patients as compared with controls. Immunohistochemical analysis confirmed up-regulation of M-CSFR-1 in IBD and showed that both epithelial and lamina propria mononuclear cells expressed this receptor. Stimulation of DLD-1 with IL-34 increased CCL20 production through an ERK1/2-dependent mechanism. Consistently, treatment of IBD explants with anti-IL-34 reduced CCL20 production.

CONCLUSIONS

These data show that intestinal epithelial cells are a target of IL-34 and suggest that this cytokine contributes to mediating the cross-talk between epithelial cells and immune cells in IBD.

BACKGROUND

There has been a growing evidence for the role of chemokines in the pathology of multiple sclerosis. Recently, there has been great emphasis placed on humoral immunity and the T(H)-17 response, which has not yet been thoroughly described in MS. The aim of this study was to investigate the role of specific chemokines involved in B-cell migration (CXCL13) and in the T(H)-17 immune response (IL-17, CCL17, CCL20).

METHODS

Using ELISA, the chosen chemokine concentrations were measured in the serum and cerebrospinal fluid of relapsing-remitting MS patients with both active and stable disease, and the relapse prediction rate was calculated.

RESULTS

We found that the CSF concentrations of CXCL13 in patients with RRMS both, during relapse and remission, were significantly higher than in controls. CCL17 and CCL20 were not detected in CSF in either of the groups, whereas serum CCL20 level was significantly higher in remission than during relapse. Intravenous methylprednisolone treatment of patients with relapse did not influence serum CXCL13 and CCL20 levels. However, it did lower CCL17 and IL-17 concentrations.

CONCLUSIONS

CXCL13 is an important mediator in MS that is strongly linked to the neuroinflammatory activity of the disease. However, more studies are needed for elucidating the roles of CCL17, CCL20 and IL-17 in MS pathology.

Human antigen presenting cells (APC) found in peripheral blood are considered to be precursors that have been released from the bone marrow and are in transit to the peripheral tissues. These APC populations include myeloid dendritic cells (mDC), plasmacytoid DC (pDC) and monocytes (Mo). To assign specialized functional roles and stages of development for APCs, CD33 expressing APC subsets were examined for their capacity to respond to chemokines. Three major CD33(+) subsets including CD33(bright)CD14(bright) Mo, CD33(bright)CD14(-) CD11c(+) mDC and CD33(dim)CD14(-) pDC were present. Dendritic cells subsets and Mo expressed low levels of CC and CXC receptors, but distinctive chemokine receptor expression profiles were not observed. The percentage of cells expressing a particular chemokine receptor varied from donor to donor and over time in the same donor. Myeloid DC and Mo but not pDC migrated toward CXCL12 in a concentration dependent manner. Monocytes and pDC, but not myeloid DC, were attracted by high concentrations of CXCL10. All CD33(+) subsets migrated in a concentration dependent manner toward CCL19, but responded less robustly to CCL21. CCL20 was not chemoattractant for any population. Despite the finding that APC did not exhibit unique surface chemokine receptor expression patterns, they exhibited differential migration to CXCL12, CXCL10 and CCL21 but not to CCL20 or CCL19.