A functional genomics approach reveals suggestive quantitative trait loci associated with combined TLR4 and BCP crystal-induced inflammation and osteoarthritis

Objective: Basic calcium phosphate (BCP) crystals can activate the NLRP3 inflammasome and are potentially involved in the pathogenesis of osteoarthritis (OA). In order to elucidate relevant inflammatory mechanisms in OA, we used a functional genomics approach to assess genetic variation influencing BCP crystal-induced cytokine production. Method: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers who were previously genotyped and stimulated with BCP crystals and/or lipopolysaccharide (LPS) after which cytokines release was assessed. Cytokine quantitative trait locus (cQTL) mapping was performed. For in vitro validation of the cQTL located in anoctamin 3 (ANO3), PBMCs were incubated with Tamoxifen and Benzbromarone prior to stimulation. Additionally, we performed co-localisation analysis of our top cQTLs with the most recent OA meta-analysis of genome-wide association studies (GWAS). Results: We observed that BCP crystals and LPS synergistically induce IL-1β in human PBMCs. cQTL analysis revealed several suggestive loci influencing cytokine release upon stimulation, among which are quantitative trait locus annotated to ANO3 and GLIS3. As functional validation, anoctamin inhibitors reduced IL-1β release in PBMCs after stimulation. Co-localisation analysis showed that the GLIS3 locus was shared between LPS/BCP crystal-induced IL-1β and genetic association with Knee OA. Conclusions: We identified and functionally validated a new locus, ANO3, associated with LPS/BCP crystal-induced inflammation in PBMCs. Moreover, the cQTL in the GLIS3 locus co-localises with the previously found locus associated with Knee OA, suggesting that this Knee OA locus might be explained through an inflammatory mechanism. These results form a basis for further exploration of inflammatory mechanisms in OA.</p

Detection of Novel Biomarkers in Pediatric Autoimmune Hepatitis by Proteomic Profiling

Autoimmune hepatitis (AIH) is characterized by immune-mediated hepatocyte injury resulting in the destruction of liver cells, causing inflammation, liver failure, and fibrosis. Pediatric (AIH) is an autoimmune inflammatory disease that usually requires immunosuppression for an extended period. Frequent relapses after treatment discontinuation demonstrate that current therapies do not control intrahepatic immune processes. This study describes targeted proteomic profiling data in patients with AIH and controls. A total of 92 inflammatory and 92 cardiometabolic plasma markers were assessed for (i) pediatric AIH versus controls, (ii) AIH type 1 versus type 2, (iii) AIH and AIH–autoimmune sclerosing cholangitis overlapping syndrome and (iv) correlations with circulating vitamin D levels in AIH. A total of 16 proteins showed a nominally significant differential abundance in pediatric patients with AIH compared to controls. No clustering of AIH subphenotypes based on all protein data was observed, and no significant correlation of vitamin D levels was observed for the identified proteins. The proteins that showed variable expression include CA1, CA3, GAS6, FCGR2A, 4E-BP1 and CCL19, which may serve as potential biomarkers for patients with AIH. CX3CL1, CXCL10, CCL23, CSF1 and CCL19 showed homology to one another and may be coexpressed in AIH. CXCL10 seems to be the central intermediary link for the listed proteins. These proteins were involved in relevant mechanistic pathways for liver diseases and immune processes in AIH pathogenesis. This is the first report on the proteomic profile of pediatric AIH. The identified markers could potentially lead to new diagnostic and therapeutic tools. Nevertheless, considering the complex pathogenesis of AIH, more extensive studies are warranted to replicate and validate the present study’s findings

Assessment of LC3 I and II levels in PBMCs under starving conditions or pharmacological treatment with 3MA.

<p>Human PBMCs were pre-incubated for 1 hour at 37°C in either RPMI, starvation medium (EBSS) or EBSS with 3MA (10 mM) in which inhibitors of lysosomal fusion have been added: Ammonium chloride 20 mM and Leupeptine 100 µM. This was followed by 3 hours stimulation with culture medium, LPS (10 ng/ml) or Pam3Cys (10 µg/ml) prepared in the corresponding media (RPMI or EBSS). Cells were lysed and western blot of LC3 fractions I and II has been performed.</p

The influence of 3MA on the ATP-dependent release of IL-1β.

<p>PBMCs were pre-incubated for 1 hour in the presence or absence of 3MA and were stimulated for 4 hours with LPS (10 ng/ml). After the stimulation, supernatants were discarded and refreshed with RPMI or with RPMI containing 1 mM ATP and cells were incubated for another 15 min. Data are shown as mean ± SEM of supernatant IL-1β levels obtained in 8 volunteers, **p<0.01.</p

The effects of starvation on mRNA levels of inflammatory cytokines.

<p>Cells pre-incubated for 2 hours in either starvation medium (Earle's Balanced Salt Solution) or RPMI were stimulated for 4 hours with medium, LPS (10 ng/ml) or Pam3Cys (10 µg/ml) prepared respectively in starvation medium or RPMI. Subsequently, RT-PCR was performed and IL-1β mRNA levels are depicted as mean ± SEM of cells harvested from 6 volunteers, *p<0.05 (A). RT-PCR results of IL-1β (B) and TNFα (C) mRNA levels in cells pre-incubated for 2 hours with RPMI, starvation medium or starvation medium and 3MA (10 mM) followed by 4 hours stimulation with RPMI or LPS (10 ng/ml). Results are shown as mean ± SEM of data obtained in 4 volunteers.</p

MAPK influence on the 3MA-dependent modulation of IL-1β and TNFα production.

<p>PBMC samples conditioned for 1 hour in either medium or in the presence of MAPK inhibitors: MEK inhibitor (5 µM), JNK inhibitor (20 µM) or p38 inhibitor (1 µM) were subjected to 1 hour treatment with RPMI or 3MA (10 mM), followed by stimulation with RPMI, LPS (10 ng/ml) or Pam3Cys (10 µg/ml). After 24 hours incubation, specific ELISA was performed to determine the level of IL-1β in response to LPS (A) and Pam3Cys (B) stimulations, same as for TNFα (panels C and D, respectively). Data from 6 volunteers are shown as mean ± SEM. (E) Western blot of total and phosphorylated p38 in cells pre-treated for 1 hour with RPMI or 3MA (10 mM) and subjected to 1 hour stimulation with RPMI, LPS (10 ng/ml) or Pam3Cys (10 µg/ml). (F) Quantification of the effect of 3MA on the phosphorylation of p38.</p

Modulation of inflammatory cytokine production by autophagy inhibition.

<p>Freshly isolated human PBMCs were pre-incubated for 1 hour at 37°C in culture medium in the presence or absence of 3MA (10 mM) and afterwards stimulated with culture medium, LPS (10 ng/ml) or Pam3Cys (10 µg/ml). After 24 hours incubation, IL-1β (A), TNFα (B) and IL-10 (C) were measured in the supernatant by specific ELISA. Data are presented as means ± SEM of cells harvested from 15 volunteers, **p<0.01, ***p<0.001.</p

The effects of 3MA on transcription and processing of inflammatory cytokines.

<p>Cells pre-treated for 1 hour with culture medium with or without 3MA (10 mM) were stimulated for 4 hours with RPMI, LPS (10 ng/ml) or Pam3Cys (10 µg/ml). RT-PCR was performed and relative levels of IL-1β and TNFα (B) mRNA were determined in 4 volunteers. (C) Western blot of p35 caspase-1 after 1 hour pre-incubation with or without 3MA (10 mM in RPMI), followed by 2 hours stimulation with LPS (10 ng/ml). The picture is representative for results obtained from 6 volunteers.</p

Inflammasome-independent modulation of cytokine response by autophagy in human cells

Contains fulltext : 98007.pdf (publisher's version ) (Open Access)Autophagy is a cell housekeeping mechanism that has recently received attention in relation to its effects on the immune response. Genetic studies have identified candidate loci for Crohn's disease susceptibility among autophagy genes, while experiments in murine macrophages from ATG16L1 deficient mice have shown that disruption of autophagy increases processing of IL-1beta and IL-18 through an inflammasome-dependent manner. Using complementary approaches either inducing or inhibiting autophagy, we describe modulatory effects of autophagy on proinflammatory cytokine production in human cells. Inhibition of basal autophagy in human peripheral blood mononuclear cells (PBMCs) significantly enhances IL-1beta after stimulation with TLR2 or TLR4 ligands, while at the same time reducing the production of TNFalpha. In line with this, induction of autophagy by starvation inhibited IL-1beta production. These effects of autophagy were not exerted at the processing step, as inflammasome activation was not influenced. In contrast, the effect of autophagy on cytokine production was on transcription level, and possibly involving the inhibition of p38 mitogen activated protein kinase (MAPK) phosphorylation. In conclusion, autophagy modulates the secretion of proinflammatory cytokines in human cells through an inflammasome-independent pathway, and this is a novel mechanism that may be targeted in inflammatory diseases