Issues with the Specificity of Immunological Reagents for NLRP3: Implications for Age-related Macular Degeneration

Abstract

Contradictory data have been presented regarding the implication of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome in age-related macular degeneration (AMD), the leading cause of vision loss in the Western world. Recognizing that antibody specificity may explain this discrepancy and in line with recent National Institutes of Health (NIH) guidelines requiring authentication of key biological resources, the specificity of anti-NLRP3 antibodies was assessed to elucidate whether non-immune RPE cells express NLRP3. Using validated resources, NLRP3 was not detected in human primary or human established RPE cell lines under multiple inflammasome-priming conditions, including purported NLRP3 stimuli in RPE such as DICER1 deletion and Alu RNA transfection. Furthermore, NLRP3 was below detection limits in ex vivo macular RPE from AMD patients, as well as in human induced pluripotent stem cell (hiPSC)-derived RPE from patients with overactive NLRP3 syndrome (Chronic infantile neurologic cutaneous and articulate, CINCA syndrome). Evidence presented in this study provides new data regarding the interpretation of published results reporting NLRP3 expression and upregulation in RPE and addresses the role that this inflammasome plays in AMD pathogenesis.

Asian age-related macular degeneration: from basic science research perspective

“…This was followed by numerous studies that linked inflammasome activation in RPE and AMD [57]. However, a recent study pointed out the problems related to the use of non-specific and unreliable commercially available antibodies in these experiments and clearly demonstrated no significant NLRP3 (a pivotal cytosolic innate immune sensor and regulator of inflammasome) expression in RPE cells [58]. Their results suggest that RPE cells do not contain significant amounts of NLRP3 that contribute to AMD pathogenesis and call into question the notion that inflammasome activation is involved in the pathogenesis of AMD.…”

Caspase-1-dependent inflammasomes mediate photoreceptor cell death in photo-oxidative damage-induced retinal degeneration

“…To date, much of the research on uncovering the role of NLRP3 in AMD pathogenesis has investigated the mode of activation of NLRP3 in cell culture-based studies, primarily focusing on the RPE 19,21,22,27,28,31,32 . However, a recent review by Kosmidou et al 30 calls into question the specificity of cited NLRP3 antibodies, demonstrating that NLRP3 localisation in the retina could not be replicated 30 . In situ hybridisation results from this study detected Nlrp3 labelling in the both the INL and GCL of mouse retinas at 5 days photo-oxidative damage and in human AMD retinas, a finding which is supported in another study using RNAscope in situ hybridization 37 .…”

“…Microglia were primed with 20 ng/mL LPS from Escherichia coli 0111:B4 (N4391, Sigma Aldrich, MO, USA) for 4 hours and stimulated with either 5 mM ATP (A6419, Sigma Aldrich, MO, USA) for 0.5 hours or 10 μM Nigericin sodium salt from Streptomyces hygroscopicus (N7143, Sigma Aldrich, MO, USA) for 1 hour (C8B4 only) 86 . Immortalised Müller cells (MIO-M1) and RPE cells (aRPE- 19) were primed with 20 ng/mL LPS for 4 hours, or either 10 ng/mL TNF-α (210-TA, R&D Systems, MN, USA) for 24 hours for MIO-M1 cells 29,87 , or 50 ng/mL Recombinant Human Interleukin-1α (IL-1α) Protein (ab9615, Abcam, Cambridge, UK) for 24 hours for aRPE19 cells 29,30,87 . MIO-M1 and aRPE-19 cells were then stimulated with 5 mM ATP for 0.5 hours following all priming agents.…”

“…In addition, NLRP3 can be activated by stimuli implicated in disease progression including complement components C1q 21 , C3a, and membrane attack complex 17,26 , or lipofuscin component bisretinoid A2E and amyloid beta both of which have been isolated in drusen deposits of patients with AMD [27][28][29] . For these reasons NLRP3 could be a prime candidate for propagating inflammation in AMD, however, extensive research into the role of NLRP3 to date has not shown a definitive role for this inflammasome in disease development 17,30 .…”

“…Although they demonstrate that the NLRP3 inflammasome can be activated in RPE by various stimulations such as oxidative stress 19 , accumulation of repetitive transposable elements of non-coding RNA (Alu RNA) 22,32 , or inflammatory pathway stimulants including drusen components lipofuscin 31 , C1q 21 , or Aβ-peptide 1-40 27,28 ; they do not provide any conclusive evidence to show NLRP3 involvement in AMD disease progression. In addition, a recent review of commercially available NLRP3 antibodies used in these studies has demonstrated the poor specificity and reliability of these products, finding that no studies showing evidence of NLRP3 involvement in AMD, or its presence in primary or established RPE cell lines, could be replicated 30 . This review, along with evidence implicating a negative role for NLRP3 in Alu-RNA induced retinal degeneration 22,32 , and a positive role in laser-induced choroidal neovascularisation (CNV) 21 , highlights the need for in vivo investigations into the role of the NLRP3 inflammasome, specifically avoiding the primary use of cell culture-based systems.…”