Acadesine suppresses TNF-α induced complement component 3 (C3), in retinal pigment epithelial (RPE) cells

Abstract

Rationale Age-related macular degeneration (AMD) is the most prevalent form of irreversible blindness in the developed world. Aging, inflammation and complement dysregulation affecting the retinal pigment epithelium (RPE), are considered significant contributors in its pathogenesis and several evidences have linked tumor necrosis factor alpha (TNF-α) and complement component 3 (C3) with AMD. Acadesine, an analog of AMP and an AMP-activated protein kinase (AMPK) activator, has been shown to have cytoprotective effects in human clinical trials as well as having anti-inflammatory and anti-vascular exudative effects in animals. The purpose of this study was to evaluate if acadesine is able to suppress TNF-α induced C3 in RPE cells. Methods ARPE-19 and human primary RPE cells were cultured and allowed to grow to confluence. TNF-α was used for C3 induction in the presence or absence of acadesine. Small molecule inhibitors and siRNA were used to determine if acadesine exerts its effect via the extracellular or intracellular pathway and to evaluate the importance of AMPK for these effects. The expression level of C3 was determined by immunoblot analysis. Results Acadesine suppresses TNF-α induced C3 in a dose dependent manner. When we utilized the adenosine receptor inhibitor dipyridamole (DPY) along with acadesine, acadesine’s effects were abolished, indicating the necessity of acadesine to enter the cell in order to exert it’s action. However, pretreatment with 5-iodotubericidin (5-Iodo), an adenosine kinase (AK) inhibitor, didn’t prevent acadesine from decreasing TNF-α induced C3 expression suggesting that acadesine does not exert its effect through AMP conversion and subsequent activation of AMPK. Consistent with this, knockdown of AMPK α catalytic subunit did not affect the inhibitory effect of acadesine on TNF-α upregulation of C3. Conclusions Our results suggest that acadesine suppresses TNF-α induced C3, likely through an AMPK-independent pathway, and could have potential use in complement over activation diseases.

Investigation of the effect of TNF-α on damage to retinal pigment epithelial cells in age-related macular degeneration

“…Aging, inflammation, and dysregulation of the complement system affect the retinal pigment epithelium, and evidence has been found to involve tumor necrosis factor alpha and complement component 3 (C3) as one of the key factors in the development of age-related macular degeneration [6]. The primary effects of TNF are related to the cytoplasmic domain TNF-R1, which sequentially recruits death domains in a number of key signaling proteins: primarily the death domain associated with the TNF-α receptor (tumor necrosis factor receptor type 1associated via death domain protein, TRADD); Fas associated death domain (FADD); a starter of caspase-8 apoptosis (also known as FADD-like ICE, Fas-associated death domain-like IL-1β-converting enzyme, or FLICE), causing degeneration of numerous proteins [14].…”

Complement System and Alarmin HMGB1 Crosstalk: For Better or Worse

“…For example, Ab-mediated complement activation or HMGB1 can contribute to enhance IFNγ secretion ( 53 , 54 ), which in turn can enhance C1s secretion ( 55 , 56 ) or NLRP3 inflammasome signaling ( 53 ). TNFα, which is amplified by HMGB1 in response to LPS ( 57 ), may trigger C3 or C1s secretion ( 58 , 59 ). Of note, it was early shown that IFNγ and TNFα are both secreted by human T cells as a response to C1q-bearing immune complexes, but not by non-opsonized complexes ( 60 ).…”

Neuroprotection for Age-Related Macular Degeneration

“…Likewise, different upstream stimuli may lead to activation of different cellular death pathways 31,32,[85][86][87] . For example, when ROS are the predominant cell death stimulus, ferroptosis (iron-dependent death) is the predominant pathway [87][88][89][90][91][92] ; whereas when tumor necrosis factor alpha (TNF-alpha) or FASL are the predominant stimuli, apoptosis and/or RIPK-dependent necrosis are the predominant pathways 24,25,31,32,86,[93][94][95][96] . To make things more complex, infiltrating immune cells also contribute to neurodegeneration via the inflammasome and cross-talk with several other pathways 80,85,[97][98][99][100][101][102][103][104][105][106] .…”

“… 31 , 32 , 85 , 86 , 87 For example, when ROS are the predominant cell death stimulus, ferroptosis (iron-dependent death) is the predominant pathway, 87 , 88 , 89 , 90 , 91 , 92 whereas when tumor necrosis factor alpha and FAS/FAS ligand are the predominant stimuli, apoptosis and receptor interacting protein kinase–dependent necrosis are the predominant pathways. 24 , 25 , 31 , 32 , 86 , 93 , 94 , 95 , 96 To make things more complex, infiltrating immune cells also contribute to neurodegeneration via the inflammasome and cross-talk with several other pathways. 80 , 85 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 , 105 , 106 On the basis of these findings, we speculate that successful future approaches must target multiple pathways; optimal neuroprotection may require inhibition of upstream and downstream targets simultaneously because cellular death activation is stimulus dependent.…”