Acidification changes affect the inflammasome in human nucleus pulposus cells

Journal of Inflammation, Aug 2016

Background Interleukin (IL)-1β is involved in the pathology of intervertebral disc degeneration. Under normal conditions, IL-1β is present in cells in an inactive form (pro-IL-1β). However, under pathological conditions, pro-IL-1β is turned into its active form (IL-1β) by the inflammasome, a multi-protein complex of the innate immune response that activates caspase-1. Under conditions of degeneration, the disc experiences an environment of increased acidification. However, the implications of acidification on the innate immune response remain poorly explored. Methods Here we have studied how pH changes in human nucleus pulposus cells affect inflammasome activation by immunoblot analysis of protein lysates obtained from nucleus pulposus cells that were exposed to different pH levels in culture. Results In this study, we have found that in nucleus pulposus cells, with increased acidification, there was a decrease in inflammasome activation consistent with lower levels of active IL-1β. However, this effect at a pH of 6.5, the lowest pH level tested, was abrogated when cells were treated with IL-1β. Conclusions Taken together, these findings suggest that the inflammatory response through IL-1β experienced by the human disc is not initiated in nucleus pulposus cells when the stimulus is acidification.

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Acidification changes affect the inflammasome in human nucleus pulposus cells

Brand et al. Journal of Inflammation Acidification changes affect the inflammasome in human nucleus pulposus cells Frank J. Brand III 1 2 Mahtab Forouzandeh 1 2 Harmanpreet Kaur 1 2 Francesco Travascio 2 3 Juan Pablo de Rivero Vaccari 0 1 2 0 Department of Neurological Surgery, Lois Pope LIFE Center , 1095 NW 14th Terrace, 3-25JJ, Miami, FL 33136-1060 , USA 1 Department of Neurological Surgery, The Miami Project to Cure Paralysis, Miller School of Medicine, University of Miami , Miami, FL 33136 , USA 2 Abbreviations: ALR, AIM-2-like receptor; ASC, Apoptosis-associated speck-like protein containing a caspase recruitment domain; ATP , Adenosine tri-phosphate; ECM, Extracellular matrix; HNPC, Human nucleus pulposus cells; IDD, Intervertebral disc degeneration; IL, Interleukin; IVD, Intervertebral disc; LDH, Lactose dehydrogenase; LPS, Lipopolysaccharide; MMP, Matrix metalloproteinases; NLR, NOD-like receptor; TLR, Toll-like receptor; TNF, Tumor necrosis factor; XIAP, X-linked inhibitor of apoptosis protein 3 Biomechanics Research Laboratory, Department of Industrial Engineering, University of Miami , Coral Gables, FL 33146 , USA Background: Interleukin (IL)-1β is involved in the pathology of intervertebral disc degeneration. Under normal conditions, IL-1β is present in cells in an inactive form (pro-IL-1β). However, under pathological conditions, pro-IL-1β is turned into its active form (IL-1β) by the inflammasome, a multi-protein complex of the innate immune response that activates caspase-1. Under conditions of degeneration, the disc experiences an environment of increased acidification. However, the implications of acidification on the innate immune response remain poorly explored. Methods: Here we have studied how pH changes in human nucleus pulposus cells affect inflammasome activation by immunoblot analysis of protein lysates obtained from nucleus pulposus cells that were exposed to different pH levels in culture. Results: In this study, we have found that in nucleus pulposus cells, with increased acidification, there was a decrease in inflammasome activation consistent with lower levels of active IL-1β. However, this effect at a pH of 6.5, the lowest pH level tested, was abrogated when cells were treated with IL-1β. Conclusions: Taken together, these findings suggest that the inflammatory response through IL-1β experienced by the human disc is not initiated in nucleus pulposus cells when the stimulus is acidification. Innate Immunity; Intervertebral disc degeneration; Caspase-1; Inflammasome; Inflammation Background Low back pain is a major pathological condition that affects approximately 84 % of the population at some point in their life [ 1 ]. Estimates indicate that in the United States, 25 % of the population suffers from low back or neck pain [ 2 ]. Accordingly, the societal costs associated with low back and neck pain exceeds $100 billion per year in the United States alone [ 3 ]. Intervertebral disc (IVD) degeneration (IDD) is believed to be a major contributor to back pain [ 4–7 ]. The etiology of IDD has been linked to genetic factors, aging or excessive manual labor [8]. All these factors contribute to increase inflammation, which eventually leads to increased degradation of aggrecan and collagen in the extracellular matrix of the disc [ 9 ]. Historically most studies looking at inflammation have focused on looking at professional immune cells as neutrophils and macrophages. However, recent studies indicate that cells that are not professionally considered as immune cells do play an important role on inflammation. This concept applies to cells as diverse as neurons [ 10–13 ] sperm cells [ 14, 15 ] or keratinocytes [ 16, 17 ]. Importantly, targeting inflammation in these cells has been shown to offer an important therapeutic potential. Thus in the context of IDD, it is important to understand the contribution of cells like NP or annulus fibrosus in regards to the inflammatory response in order to gain a better understanding of how inflammation contributes to IDD. The inflammasome is a multi-protein complex in which caspase-1 is activated followed by processing of prointerleukin (IL)-1β and pro-IL-18 into their respective active forms. The inflammasome has been previously shown to play a role in infections [ 18–20 ], metabolic syndromes [ 21, 22 ], autoimmune diseases [ 23–25 ] and injury [ 11, 13, 26–28 ]. A positive correlation between the degree of IDD and inflammasome content in the disc has also been previously reported [29]. The pro-inflammatory cytokines IL-1β and tumor necrosis factor (TNF) are two key cytokines that are involved in the pathology of IDD [ 9, 30–32 ] and degradation of the IVD [ 33, 34 ]. IL-1β is present in the cell in an inactive form as pro-IL-1β, and it relies on the inflammasome for its maturation into active IL-1β [ 35 ]. The inflammasome is a multi-protein complex comprised of a nod-like receptor (NLR) such as NLRP1 or NLRP3, (...truncated)


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Frank Brand, Mahtab Forouzandeh, Harmanpreet Kaur, Francesco Travascio, Juan de Rivero Vaccari. Acidification changes affect the inflammasome in human nucleus pulposus cells, Journal of Inflammation, 2016, pp. 29, 13, DOI: 10.1186/s12950-016-0137-0