Anti-oxidant inhibition of hyaluronan fragment-induced inflammatory gene expression

<p>Abstract</p> <p>Background</p> <p>The balance between reactive oxygen species (ROS) and endogenous anti-oxidants is important in maintaining healthy tissues. Excessive ROS states occur in diseases such as ARDS and Idiopathic Pulmonary Fibrosis. Redox imbalance breaks down the extracellular matrix component hyaluronan (HA) into fragments that activate innate immune responses and perpetuate tissue injury. HA fragments, via a TLR and NF-κB pathway, induce inflammatory gene expression in macrophages and epithelial cells. NAC and DMSO are potent anti-oxidants which may help balance excess ROS states.</p> <p>Methods</p> <p>We evaluated the effect of H₂O₂, NAC and DMSO on HA fragment induced inflammatory gene expression in alveolar macrophages and epithelial cells.</p> <p>Results</p> <p>NAC and DMSO inhibit HA fragment-induced expression of TNF-α and KC protein in alveolar and peritoneal macrophages. NAC and DMSO also show a dose dependent inhibition of IP-10 protein expression, but not IL-8 protein, in alveolar epithelial cells. In addition, H₂O₂synergizes with HA fragments to induce inflammatory genes, which are inhibited by NAC. Mechanistically, NAC and DMSO inhibit HA induced gene expression by inhibiting NF-κB activation, but NAC had no influence on HA-fragment-AP-1 mediated gene expression.</p> <p>Conclusion</p> <p>ROS play a central role in a pathophysiologic "vicious cycle" of inflammation: tissue injury generates ROS, which fragment the extracellular matrix HA, which in turn synergize with ROS to activate the innate immune system and further promote ROS, HA fragment generation, inflammation, tissue injury and ultimately fibrosis. The anti-oxidants NAC and DMSO, by inhibiting the HA induced inflammatory gene expression, may help re-balance excessive ROS induced inflammation.</p

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.

The Adenosine A2a Receptor Inhibits Matrix-Induced Inflammation in a Novel Fashion

Endogenous mediators within the inflammatory milieu play a critical role in directing the scope, duration, and resolution of inflammation. High-molecular-weight extracellular matrix hyaluronan (HA) helps to maintain homeostasis. During inflammation, hyaluronan is broken down into fragments that induce chemokines and cytokines, thereby augmenting the inflammatory response. Tissue-derived adenosine, released during inflammation, inhibits inflammation via the anti-inflammatory A2 adenosine receptor (A2aR). We demonstrate that adenosine modulates HA-induced gene expression via the A2aR. A2aR stimulation inhibits HA fragment–induced pro-fibrotic genes TNF-α, keratinocyte chemoattractant (KC), macrophage inflammatory protein (MIP)-2, and MIP-1α while simultaneously synergizing with hyaluronan fragments to up-regulate the TH1 cytokine IL-12. Interestingly, A2aR stimulation mediates these affects via the novel cAMP-activated guanine nucleotide exchange factor EPAC. In addition, A2aR-null mice are more susceptible to bleomycin-induced lung injury, consistent with a role for endogenous adenosine in inhibiting the inflammation that may lead to fibrosis. Indeed, the bleomycin treated A2aR-null mice demonstrate increased lung inflammation, HA accumulation, and histologic damage. Overall, our data elucidate the opposing roles of tissue-derived HA fragments and adenosine in regulating noninfectious lung inflammation and support the pursuit of A2aR agonists as a means of pharmacologically inhibiting inflammation that may lead to fibrosis

MiR-27a Functions as a Tumor Suppressor in Acute Leukemia by Regulating 14-3-3θ

<div><p>MicroRNAs (miRs) play major roles in normal hematopoietic differentiation and hematopoietic malignancies. In this work, we report that miR-27a, and its coordinately expressed cluster (miR-23a∼miR-27a∼miR-24-2), was down-regulated in acute leukemia cell lines and primary samples compared to hematopoietic stem-progenitor cells (HSPCs). Decreased miR-23a cluster expression in some acute leukemia cell lines was mediated by c-MYC. Replacement of miR-27a in acute leukemia cell lines inhibited cell growth due, at least in part, to increased cellular apoptosis. We identified a member of the anti-apoptotic 14-3-3 family of proteins, which support cell survival by interacting with and negatively regulating pro-apoptotic proteins such as Bax and Bad, as a target of miR-27a. Specifically, miR-27a regulated 14-3-3θ at both the mRNA and protein levels. These data indicate that miR-27a contributes a tumor suppressor-like activity in acute leukemia cells via regulation of apoptosis, and that miR-27a and 14-3-3θ may be potential therapeutic targets.</p> </div

The miR-23a cluster does not regulate 14-3-3ζ, or other 14-3-3 isotypes.

<p>(A) and (B) The effect of the miR-23a cluster on (A) 14-3-3ζ and (B) 14-3-3γ protein expression was measured via Western blot. The top panels are representative blots from one experiment, and the bottom panels are graphs of normalized protein expression from 3 independent experiments (±SEM), determined by densitometry analysis (p<0.05*). (C) The schematics show predicted miR-27a and miR-24 binding sites in the CDS and 3′UTR of 14-3-3ζ (top panel). HEK293T cells were co-transfected with 14-3-3ζL-157, 14-3-3ζL-627, or 14-3-3ζL-1075 (500 ng, white bars) alone and with 25 nM of miR-23a (light grey bars) or miR-24 (dark grey bars); HEK293 cells were also co-transfected with 14-3-3ζL-1266 (500 ng, white bars) alone and with 25 nM of miR-23a (light grey bars) or miR-27a (black bars) (bottom panel). Normalized mean Luc expression (±SEM, n≥3) is represented on the graphs.</p

Effects of c-MYC on expression of the miR-23a cluster.

<p>(A) Knock down of c-MYC expression in P493B cells by doxycycline-treatment. Cells were treated with 0 µg/ml (white bar), 0.1 µg/ml (grey bar) or 0.3 µg/ml (black bar) doxycycline for 48 hr, harvested, and total RNA isolated. Levels of miR-23a cluster member expression were measured via qRT-PCR, using U18 as an endogenous control, and normalized to untreated (0 µg/ml Dox) cells (2^−ΔΔCt = 1). (B) and (C) The effects of pharmacologic inhibition of c-MYC on miR-23a (B) and miR-27a (C) in REH, KOPN8, SUPB15, and K562 cell lines were determined by treatment with 2 doses of 10058-F4 (25 µM [grey bar] and 50 µM [black bar]) or vehicle (white bar) for 48 hr. Fold-expression of mature miR-23a and miR-27a were assessed by qRT-PCR and analyzed as above. Effect of the c-MYC inhibitor on miR-23a and miR-27a expression was determined by comparison of treated cells to the mean expression (±SEM) of vehicle-treated cells Significance was determined by a Student's t-test where p<0.05 (*) indicated significance (n = 2–6 independent experiments). (D) and (E) The effect of siRNA knock-down of endogenous c-MYC on expression of miR-23a (D) and miR-27a (E) in K562, Karpas45, and Molt16 cells were assessed by transfection with 50 nM (grey bar) or 100 nM (black bar) of an siRNA-c-MYC pool or 100 nM of control siRNA (white bar). Expression levels of miR-23a and miR-27a and significance of results were assessed as in (B) and (C). n≥3 independent experiments for all samples.</p