Fate of swallowed interleukin 8 and TNFα, and effect on the Wistar rat gut

To evaluate the passage of cytokines through the gastrointestinal tract, we investigated the digestion of interleukin-8 (IL-8) and tumour necrosis factor α (TNFα), in vitro and in vivo, and their propensity to induce intestinal inflammation. We serially immuno-assayed IL-8 and TNFα solutions co-incubated with each of three pancreatin preparations at pH 4.5 and pH 8. We gavaged IL-8, TNFα and marker into 15 Wistar rats, and measured their faecal cytokine concentrations by ELISA and histologically examined their guts. IL-8 immunoreactivity was extinguished by all pancreatin preparations after 1 h of incubation at 37 °C. TNFα concentration progressively fell from 1 to 4 h with all enzyme preparations. Buffer control samples maintained their cytokine concentrations throughout incubation. No IL-8 or TNFα was detected in any rat faecal pellets. There was no significant proinflammatory effect of the gavaged cytokines on rat intestine. IL-8 and TNFα in aqueous solution could well be fully digested in the CF gut when transit time is normal and exogenous enzymes are provided, although cytokines swallowed in viscous sputum may be protected from such digestion

GEF-H1-RhoA signaling pathway mediates LPS-induced NF-κB transactivation and IL-8 synthesis in endothelial cells

Secretion of proinflammatory cytokines by LPS activated endothelial cells contributes substantially to the pathogenesis of sepsis. However, the mechanism involved in this process is not well understood. In the present study, we determined the roles of GEF-H1 (Guanine-nucleotide exchange factor-H1)-RhoA signalling in LPS-induced interleukin-8 (IL-8, CXCL8) production in endothelial cells. First, we observed that GEF-H1 expression was upregulated in a dose- and time-dependent manner as consistent with TLR4 (Toll-like receptor 4) expression after LPS stimulation. Afterwards, Clostridium difficile toxin B-10463 (TcdB-10463), an inhibitor of Rho activities, reduced LPS-induced NF-κB phosphorylation. Inhibition of GEF-H1 and RhoA expression reduced LPS-induced NF-κB and p38 phosphorylation. TLR4 knockout blocked LPS-induced activity of RhoA, however, MyD88 knockout did not impair the LPS-induced activity of RhoA. Nevertheless, TLR4 and MyD88 knockout both significantly inhibited transactivation of NF-κB. GEF-H1-RhoA and MyD88 both induced significant changes in NF-κB transactivation and IL-8 synthesis. Co-inhibition of GEF-H1-RhoA and p38 expression produced similar inhibitory effects on LPS-induced NF-κB transactivation and IL-8 synthesis as inhibition of p38 expression alone, thus confirming that activation of p38 was essential for the GEF-H1-RhoA signalling pathway to induce NF-κB transactivation and IL-8 synthesis. Taken together, these results demonstrate that LPS-induced NF-κB activation and IL-8 synthesis in endothelial cells are regulated by the MyD88 pathway and GEF-H1-RhoA pathway.

Molecular dynamics simulations of CXCL-8 and its interactions with a receptor peptide, heparin fragments, and sulfated linked cyclitols

CXCL-8 (Interleukin 8) is a CXC chemokine with a central role in the human immune response. We have undertaken extensive in silico analyses to elucidate the interactions of CXCL-8 with its various binding partners, which are crucial for its biological function. Sequence and structure analyses showed that residues in the thirdq β-sheet and basic residues in the heparin binding site are highly variable, while residues in the second β-sheet are highly conserved. Molecular dynamics simulations in aqueous solution of dimeric CXCL-8 have been performed with starting geometries from both X-ray and NMR structures showed shearing movements between the two antiparallel C-terminal helices. Dynamic conservation analyses of these simulations agreed with experimental data indicating that structural differences between the two structures at quaternary level arise from changes in the secondary structure of the N-terminal loop, the 310-helix, the 30s, 40s, and 50s loops and the third β-sheet, resulting in a different interhelical separation. Nevertheless, the observation of these different states indicates that CXCL-8 has the potential to undergo conformational changes, and it seems likely that this feature is relevant to the mode of binding of glycosaminoglycan (GAG) mimetics such as cyclitols. Simulations of the receptor peptide fragment−CXCL-8 complex identified several specific interactions of the receptor peptide with CXCL-8 that could be exploited in the structure-based design of competitive peptides and nonpeptidic molecules targeting CXCL-8 for combating inflammatory diseases. Simulations of the CXCL-8 dimer complexed with a 24-mer heparin fragment and of the CXCL-8−receptor peptide complex revealed that Arg60, Lys64, and Arg68 in the dimer bind to cyclitols in a horseshoe pattern, defining a region which is spatially distinct from the receptor binding site. There appears to be an optimum number of sulfates and an optimum length of alkyl spacers required for the interaction of cyclitol inhibitors with the dimeric form of CXCL-8. Calculation of the binding affinities of cyclitol inhibitors reflected satisfactorily the ranking of experimentally determined inhibitory potencies. The findings of these molecular modeling studies will help in the search for inhibitors which can modulate various CXCL-8 biological activities and serve as an excellent model system to study CXC-inhibitor interactions.

Epigenetics underpinning the regulation of the CXC (ELR+) chemokines in non-small cell lung cancer

Background: Angiogenesis may play a role in the pathogenesis of Non-Small Cell Lung cancer (NSCLC). The CXC (ELR+) chemokine family are powerful promoters of the angiogenic response. Methods: The expression of the CXC (ELR+) family members (CXCL1-3/GROα-γ, CXCL8/IL-8, CXCR1/2) was examined in a series of resected fresh frozen NSCLC tumours. Additionally, the expression and epigenetic regulation of these chemokines was examined in normal bronchial epithelial and NSCLC cell lines. Results: Overall, expression of the chemokine ligands (CXCL1, 2, 8) and their receptors (CXCR1/2) were down regulated in tumour samples compared with normal, with the exception of CXCL3. CXCL8 and CXCR1/2 were found to be epigenetically regulated by histone post-translational modifications. Recombinant CXCL8 did not stimulate cell growth in either a normal bronchial epithelial or a squamous carcinoma cell line (SKMES-1). However, an increase was observed at 72 hours post treatment in an adenocarcinoma cell line. Conclusions: CXC (ELR+) chemokines are dysregulated in NSCLC. The balance of these chemokines may be critical in the tumour microenvironment and requires further elucidation. It remains to be seen if epigenetic targeting of these pathways is a viable therapeutic option in lung cancer treatment. © 2011 Baird et al.

A concise approach to the polycyclic scaffold of frondosin D

Frondosins A−E, 1−5 (Figure 1), are a family of related marine sesquiterpenoids first isolated in their dextro-rotatory form from the sponge Dysidea frondosa.(1a) Additionally, levo-rotatory frondosins A and D were isolated from an unidentified Eurospongia species.(1b) Frondosins A−E are compounds of interest due to their promising interleukin-8 (IL-8) affinity and protein kinase C inhibition.(1a) IL-8 antagonists are of particular interest in view of their antiinflammatory,(2a) anti-HIV,(1b, 2b) and antitumor(2c-2f) properties. To date, frondosins A, B, and C have been synthesized.(3) Notwithstanding these successes, the frondosins have proved quite a formidable synthetic challenge, and as of yet, there has been no synthesis of frondosin D or E. In this report, we describe our approaches to the molecular scaffold of frondosins D. This work has culminated in a very effective means of producing the trimethylbicyclo[5.4.0]undecane ring system common to all frondosins (shown in bold, Figure 1).

Acute resistance exercise increases the expression of chemotactic factors within skeletal muscle

Intense resistance exercise causes mechanical loading of skeletal muscle, followed by muscle adaptation. Chemotactic factors likely play an important role in these processes. Purpose We investigated the time course of changes in the expression and tissue localization of several key chemotactic factors in skeletal muscle during the early phase of recovery following resistance exercise. Methods Muscle biopsy samples were obtained from vastus lateralis of eight untrained men (22+-0.5 yrs) before and 2, 4 and 24 h after three sets of leg press, squat and leg extension at 80% 1 RM. Results Monocyte chemotactic protein-1 (95×), interleukin-8 (2,300×), IL-6 (317×), urokinase-type plasminogen activator (15×), vascular endothelial growth factor (2×) and fractalkine (2.5×) mRNA was significantly elevated 2 h post-exercise. Interleukin-8 (38×) and interleukin-6 (58×) protein was also significantly elevated 2 h post-exercise, while monocyte chemotactic protein-1 protein was significantly elevated at 2 h (22×) and 4 h (21×) post-exercise. Monocyte chemotactic protein-1 and interleukin-8 were expressed by cells residing in the interstitial space between muscle fibers and, in some cases, were co-localized with CD68+ macrophages, PAX7+ satellite cells and blood vessels. However, the patterns of staining were inconclusive and not consistent. Conclusion In conclusion, resistance exercise stimulated a marked increase in the mRNA and protein expression of various chemotactic factors in skeletal muscle. Myofibers were not the dominant source of these factors. These findings suggest that chemotactic factors regulate remodeling/adaptation of skeletal muscle during the early phase of recovery following resistance exercise.

The p38 mitogen-activated protein kinase regulates interleukin-1beta-induced IL-8 expression via an effect on the IL-8 promoter in intestinal epithelial cells

Several lines of evidence implicate the p38 mitogen-activated protein kinase (p38 MAPK) in the proinflammatory response to bacterial agents and cytokines. Equally, the transcription factor, nuclear factor (NF)-kappaB, is recognized to be a critical determinant of the inflammatory response in intestinal epithelial cells (IECs). However, the precise inter-relationship between the activation of p38 MAPK and activation of the transcription factor NF-kappaB in the intestinal epithelial cell (IEC) system, remains unknown. Here we show that interleukin (IL)-1beta activates all three MAPKs in Caco-2 cells. The production of IL-8 and monocyte chemotactic protein 1 (MCP-1) was attenuated by 50% when these cells were preincubated with the p38 MAPK inhibitor, SB 203580. Further investigation of the NF-kappaB signalling system revealed that the inhibitory effect was independent of the phosphorylation and degradation of IkappaBalpha, the binding partner of NF-kappaB. This effect was also independent of the DNA binding of the p65 Rel A subunit, as well as transactivation, determined by an NF-kappaB luciferase construct, using both SB 203580 and dominant-negative p38 MAPK. Evaluation of IL-8 and MCP-1 RNA messages by reverse transcription-polymerase chain reaction (RT-PCR) revealed that the inhibitory effect of SB 203580 was associated with a reduction in this parameter. Using an IL-8-luciferase promoter construct, an effect of p38 upon its activation by both pharmacological and dominant-negative p38 construct co-transfection was demonstrated. It is concluded that p38 MAPK influences the expression of chemokines in intestinal epithelial cells, through an effect upon the activation of the chemokine promoter, and does not directly involve the activation of the transcription factor NF-kappaB

Serum YKL-40 and Interleukin 6 Levels in Hodgkin Lymphoma

Purpose Serum levels of the inflammatory markers YKL-40 and IL-6 are increased in many conditions, including cancers. We examined serum YKL-40 and IL-6 levels in patients with Hodgkin lymphoma (HL), a tumor with strong immunologic reaction to relatively few tumor cells, especially in nodular sclerosis HL. Experimental Design We analyzed Danish and Swedish patients with incident HL (N=470) and population controls from Denmark (N= 245 for YKL-40; N= 348 for IL-6). Serum YKL-40 and IL-6 levels were determined by ELISA, and log-transformed data were analysed by linear regression, adjusting for age and sex. Results Serum levels of YKL-40 and IL-6 were increased in HL patients compared to controls (YKL-40: 3.6-fold, IL-6: 8.3-fold; both p<0.0001). In samples from pre-treatment HL patients (N=176), levels were correlated with more advanced stages (ptrend 0.0001 for YKL-40 and 0.013 for IL-6) and in those with B symptoms, but levels were similar in nodular sclerosis and mixed cellularity subtypes, by EBV status, and in younger (<45 years old) and older patients. Patients tested soon after treatment onset had significantly lower levels than pre-treatment patients, but even >6 months after treatment onset, serum YKL-40 and IL-6 levels remained significantly increased, compared to controls. In patients who died (N=12), pre-treatment levels for both YKL-40 and IL-6 were higher than in survivors, although not statistically significantly. Conclusions Serum YKL-40 and IL-6 levels were increased in untreated HL patients and those with more advanced stages but did not differ significantly by HL histology. Following treatment, serum levels were significantly lower.

A novel immunodeficiency disorder characterized by genetic amplification of interleukin 25

Many primary immunodeficiency disorders of differing etiologies have been well characterized, and much understanding of immunological processes has been gained by investigating the mechanisms of disease. Here, we have used a whole-genome approach, employing single-nucleotide polymorphism and gene expression microarrays, to provide insight into the molecular etiology of a novel immunodeficiency disorder. Using DNA copy number profiling, we define a hyperploid region on 14q11.2 in the immunodeficiency case associated with the interleukin (IL)-25 locus. This alteration was associated with significantly heightened expression of IL25 following T-cell activation. An associated dominant type 2 helper T cell bias in the immunodeficiency case provides a mechanistic explanation for recurrence of infections by pathogens met by Th1-driven responses. Furthermore, this highlights the capacity of IL25 to alter normal human immune responses.

Interleukin-1β stimulates human renal fibroblast proliferation and matrix protein production by means of a transforming growth factor-β-dependent mechanism

One of the hallmarks of progressive renal disease is the development of tubulointerstitial fibrosis. This is frequently preceded by macrophage infiltration, raising the possibility that macrophages relay fibrogenic signals to resident tubulointerstitial cells. The aim of this study was to investigate the potentially fibrogenic role of interleukin-1beta (IL-1beta), a macrophage-derived inflammatory cytokine, on cortical fibroblasts (CFs). Primary cultures of human renal CFs were established and incubated for 24 hours in the presence or absence of IL-1beta. We found that IL-1beta significantly stimulated DNA synthesis (356.7% +/- 39% of control, P <.003), fibronectin secretion (261.8 +/- 11% of control, P <.005), collagen type 1 production, (release of procollagen type 1 C-terminal-peptide, 152.4% +/- 26% of control, P <.005), transforming growth factor-beta (TGF-beta) secretion (211% +/- 37% of control, P <.01), and nitric oxide (NO) production (342.8% +/- 69% of control, P <.002). TGF-beta (1 ng/mL) and the phorbol ester phorbol 12-myristate 13-acetate (PMA, 25 nmol/L) produced fibrogenic effects similar to those of IL-1beta. Neither a NO synthase inhibitor (N(G)-methyl-l-arginine, 1 mmol/L) nor a protein kinase C (PKC) inhibitor (bis-indolylmaleimide 1, 1 micromol/L) altered the enhanced level of fibronectin secretion or DNA synthesis seen in response to IL-1beta treatment. However, addition of a TGF-beta-neutralizing antibody significantly reduced IL-1beta-induced fibronectin secretion (IL-1beta + IgG, 262% +/- 72% vs IL-1beta + alphaTGF-beta 156% +/- 14%, P <.02), collagen type 1 production (IL-1beta + IgG, 176% +/- 28% vs IL-1beta + alphaTGF-beta, 120% +/- 14%, P <.005) and abrogated IL-1beta-induced DNA synthesis (245% +/- 49% vs 105% +/- 21%, P <.005). IL-1beta significantly stimulated CF DNA synthesis and production of fibronectin, collagen type 1, TGFbeta, and NO. The fibrogenic and proliferative action of IL-1beta on CF appears not to involve activation of PKC or production of NO but is at least partly TGFbeta-dependent.

Preliminary characterisation of tumor necrosis factor alpha and interleukin-10 responses to chlamydia pecorum infection in the koala (Phascolarctos cinereus)

Debilitating infectious diseases caused by Chlamydia are major contributors to the decline of Australia's iconic native marsupial species, the koala (Phascolarctos cinereus). An understanding of koala chlamydial disease pathogenesis and the development of effective strategies to control infections continue to be hindered by an almost complete lack of species-specific immunological reagents. The cell-mediated immune response has been shown to play an influential role in the response to chlamydial infection in other hosts. The objective of this study, hence, was to provide preliminary data on the role of two key cytokines, pro-inflammatory tumour necrosis factor alpha (TNFα) and anti-inflammatory interleukin 10 (IL10), in the koala Chlamydia pecorum response. Utilising sequence homology between the cytokine sequences obtained from several recently sequenced marsupial genomes, this report describes the first mRNA sequences of any koala cytokine and the development of koala specific TNFα and IL10 real-time PCR assays to measure the expression of these genes from koala samples. In preliminary studies comparing wild koalas with overt chlamydial disease, previous evidence of C. pecorum infection or no signs of C. pecorum infection, we revealed strong but variable expression of TNFα and IL10 in wild koalas with current signs of chlamydiosis. The description of these assays and the preliminary data on the cell-mediated immune response of koalas to chlamydial infection paves the way for future studies characterising the koala immune response to a range of its pathogens while providing reagents to assist with measuring the efficacy of ongoing attempts to develop a koala chlamydial vaccine.

Enrichment of circulating interleukin-17-secreting interleukin-23 receptor-positive γ/δ T cells in patients with active ankylosing spondylitis

Objective Ankylosing spondylitis (AS) is a common inflammatory arthritis affecting primarily the axial skeleton. IL23R is genetically associated with AS. This study was undertaken to investigate and characterize the role of interleukin-23 (IL-23) signaling in AS pathogenesis. Methods The study population consisted of patients with active AS (n = 17), patients with psoriatic arthritis (n = 8), patients with rheumatoid arthritis, (n = 9), and healthy subjects (n = 20). IL-23 receptor (IL-23R) expression in T cells was determined in each subject group, and expression levels were compared. Results The proportion of IL-23R-expressing T cells in the periphery was 2-fold higher in AS patients than in healthy controls, specifically driven by a 3-fold increase in IL-23R-positive γ/δ T cells in AS patients. The proportions of CD4+ and CD8+ cells that were positive for IL-17 were unchanged. This increased IL-23R expression on γ/δ T cells was also associated with enhanced IL-17 secretion, with no observable IL-17 production from IL-23R-negative γ/δ T cells in AS patients. Furthermore, γ/δ T cells from AS patients were heavily skewed toward IL-17 production in response to stimulation with IL-23 and/or anti-CD3/CD28. Conclusion Recently, mouse models have shown IL-17-secreting γ/δ T cells to be pathogenic in infection and autoimmunity. Our data provide the first description of a potentially pathogenic role of these cells in a human autoimmune disease. Since IL-23 is a maturation and growth factor for IL-17-producing cells, increased IL-23R expression may regulate the function of this putative pathogenic γ/δ T cell population.

Association between endometriosis and the interleukin 1A (IL1A) locus

STUDY QUESTION Are single-nucleotide polymorphisms (SNPs) at the interleukin 1A (IL1A) gene locus associated with endometriosis risk? SUMMARY ANSWER We found evidence for strong association between IL1A SNPs and endometriosis risk. WHAT IS KNOWN ALREADY Genetic factors contribute substantially to the complex aetiology of endometriosis and the disease has an estimated heritability of ∼51%. We, and others, have conducted genome-wide association (GWA) studies for endometriosis, which identified a total of nine independent risk loci. Recently, two small Japanese studies reported eight SNPs (rs6542095, rs11677416, rs3783550, rs3783525, rs3783553, rs2856836, rs1304037 and rs17561) at the IL1A gene locus as suggestively associated with endometriosis risk. There is also evidence of a link between inflammation and endometriosis. STUDY DESIGN, SIZE, DURATION We sought to further investigate the eight IL1A SNPs for association with endometriosis using an independent sample of 3908 endometriosis cases and 8568 controls of European and Japanese ancestry. The study was conducted between October 2013 and July 2014. PARTICIPANTS/MATERIALS, SETTING, METHODS By leveraging GWA data from our previous multi-ethnic GWA meta-analysis for endometriosis, we imputed variants in the IL1A region, using a recent 1000 Genomes reference panel. After combining summary statistics for the eight SNPs from our European and Japanese imputed data with the published results, a fixed-effect meta-analysis was performed. An additional meta-analysis restricted to endometriosis cases with moderate-to-severe (revised American Fertility Society stage 3 or 4) disease versus controls was also performed. MAIN RESULTS AND THE ROLE OF CHANCE All eight IL1A SNPs successfully replicated at P < 0.014 in the European imputed data with concordant direction and similar size to the effects reported in the original Japanese studies. Of these, three SNPs (rs6542095, rs3783550 and rs3783525) also showed association with endometriosis at a nominal P < 0.05 in our independent Japanese sample. Fixed-effect meta-analysis of the eight SNPs for moderate-to-severe endometriosis produced a genome-wide significant association for rs6542095 (odds ratio = 1.21; 95% confidence interval = 1.13–1.29; P = 3.43 × 10−8). LIMITATIONS, REASONS FOR CAUTION The meta-analysis for moderate-to-severe endometriosis included results of moderate-to-severe endometriosis cases from our European data sets and all endometriosis cases from the Japanese data sets, as disease stage information was not available for endometriosis cases in the Japanese data sets. WIDER IMPLICATIONS OF THE FINDINGS SNP rs6542095 is located ∼2.3 kb downstream of the IL1A gene and ∼6.9 kb upstream of cytoskeleton-associated protein 2-like (CKAP2L) gene. The IL1A gene encodes the IL1a protein, a member of the interleukin 1 cytokine family which is involved in various immune responses and inflammatory processes. These results provide important replication in an independent Japanese sample and, for the first time, association of the IL1A locus in endometriosis patients of European ancestry. SNPs within the IL1A locus may regulate other genes, but if IL1A is the target, our results provide supporting evidence for a link between inflammatory responses and the pathogenesis of endometriosis. STUDY FUNDING/COMPETING INTEREST(S) The research was funded by grants from the Australian National Health and Medical Research Council and Wellcome Trust. None of the authors has competing interests for the study.

Influence of interleukin-1 beta on platelet-poor plasma clot formation: A potential impact on early bone healing

Objectives Hematoma quality (especially the fibrin matrix) plays an important role in the bone healing process. Here, we investigated the effect of interleukin-1 beta (IL-1β) on fibrin clot formation from platelet-poor plasma (PPP). Methods Five-milliliter of rat whole-blood samples were collected from the hepatic portal vein. All blood samples were firstly standardized via a thrombelastograph (TEG), blood cell count, and the measurement of fibrinogen concentration. PPP was prepared by collecting the top two-fifths of the plasma after centrifugation under 400 × g for 10min at 20°C. The effects of IL-1β cytokines on artificial fibrin clot formation from PPP solutions were determined by scanning electronic microscopy (SEM), confocal microscopy (CM), turbidity, and clot lysis assays. Results The lag time for protofibril formation was markedly shortened in the IL-1β treatment groups (243.8 ± 76.85 in the 50 pg/mL of IL-1β and 97.5 ± 19.36 in the 500 pg/mL of IL-1β) compared to the control group without IL-1β (543.8 ± 205.8). Maximal turbidity was observed in the control group. IL-1β (500 pg/mL) treatment significantly decreased fiber diameters resulting in smaller pore sizes and increased density of the fibrin clot structure formed from PPP (P < 0.05). The clot lysis assay revealed that 500 pg/mL IL-1β induced a lower susceptibility to dissolution due to the formation of thinner and denser fibers. Conclusion IL-1β can significantly influence PPP fibrin clot structure, which may affect the early bone healing process.