Evidence of necroptosis in osteoarthritic disease: investigation of blunt mechanical impact as possible trigger in regulated necrosis

Abstract

Joint injuries are highly associated with cell death and development of posttraumatic osteoarthritis (PTOA). The present study focused on necroptosis as a possible modality of chondrocyte death after cartilage trauma and its relevance in OA disease in general. For this purpose, apoptosis- and necroptosis-associated markers were determined in highly degenerated (ICRS ≥ 3) as well as macroscopically intact cartilage tissue (ICRS ≤ 1) by means of real-time PCR and immunohistochemistry (IHC). Moreover, influence of blunt trauma and/or stimulation with cycloheximide (CHX), TNF-a, and caspase-inhibitor zVAD were investigated in cartilage explants (ICRS ≤ 1). Further characterization of necroptosis was performed in isolated chondrocytes. We found that gene expression levels of RIPK3 (4.2-fold, P < 0.0001) and MLKL (2.7-fold, P < 0.0001) were elevated in highly degenerated cartilage tissue, which was confirmed by IHC staining. After ex vivo trauma and/or CHX/TNF stimulation, addition of zVAD further enhanced expression of necroptosis-related markers as well as release of PGE2 and nitric oxide, which was in line with increased cell death and subsequent release of intracellular HMGB1 and dsDNA in CHX/TNF stimulated chondrocytes. However, trauma and/or chemically induced cell death and subsequent release of pro-inflammatory mediators could be largely attenuated by RIPK1-inhibitor necrostatin 1 or antioxidant N-acetylcysteine. Overall, the study provided clear evidence of necroptotic cell death in OA disease. Moreover, a possible link between cartilage injury and necroptotic processes was found, depending on oxidative stress and cytokine release. These results contribute to further understanding of cell death in PTOA and development of novel therapeutic approaches.

Fig. 1. Expression of necroptosis- and apoptosis-related markers is elevated in OA cartilage.

Necroptosis- and apoptosis-related markers in highly degenerated cartilage tissue of OA patients (ICRS grade ≥ 3) were determined by a gene expression analysis of apoptosis and necroptosis markers as well as immunohistochemical analysis (IHC) of b cleaved CASP8, c RIPK3, and d p-MLKL. Macroscopically intact cartilage (ICRS grade ≤ 1) served as control. Bars in the IHC images represent 200 µm. Statistical analysis was performed by an unpaired multiple t test; error bars indicate median and range from min to max; n = 13. Significant differences between groups were depicted as: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 2. Treatment with NAC, Nec-1, and NSA, respectively, prevent from necroptotic cell death.

After 4 days, cell viability was evaluated by Live/Dead staining in the following experimental approaches: a effects of trauma in absence/presence of TNF and treatment with zVAD or Nec-1; b titration of appropriate duration and concentration for chemical induction of necroptosis by TNF/CHX stimulation in impacted and unimpacted cartilage explants, respectively; c evaluation of therapeutic effects of Nec-1 or NAC after TNF/CHX stimulation with/ without co-stimulation by zVAD; f exemplary comparison of necroptosis inhibitors NSA and Nec-1, respectively. Statistical analysis was performed by a, f 1-way and b, c 2-way ANOVA, respectively, including a Bonferroni posttest (a–c: n ≥ 5; F: n ≥ 4). d Counting of double positive cells and statistical analysis, performed by Kruskal–Wallis test, including a Dunn’s posttest (n ≥ 4). e Exemplary fluorescence images of the live/dead analysis. Living cells exhibit a green fluorescence, dead cells a red one. Double positive cells appear in orange/yellow, due to overlay of both colors (exemplarily indicated by white arrows). Significant differences between groups were depicted as: [vs C] ^cP < 0.05, ^ccP < 0.01, ^cccP < 0.001, ^ccccP < 0.0001; [vs T] ^tP < 0.05; [between delineated groups] *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Values are given as boxplots with median and whiskers (min to max); striped bars = impacted, blank bars = unimpacted; C (dotted line, green) = control level, T (dashed line, red) = trauma level

Fig. 3. Enhanced gene expression of necroptosis- and apoptosis-related markers after TNF/CHX stimulation w/ and w/o zVAD can be attenuated by Nec-1 and NAC, respectively, to some extent.

Gene expression analysis of a caspase 3, b caspase 8, c RIPK1, d RIPK3, and e MLKL necroptosis- and apoptosis-associated markers after trauma and/or stimulation with TNF/CHX w/ and w/o zVAD. Striped bars = impacted, blank bars = unimpacted. Statistical analysis was performed by one-way ANOVA, including a Bonferroni posttest (n ≥ 4). Significant differences between groups were depicted as: [vs C] ^cP < 0.05, ^ccP < 0.01, ^ccccP < 0.0001; [vs T] ^ttP < 0.01, ^ttttP < 0.0001; [between delineated groups] *P < 0.05, **P < 0.01, ****P < 0.0001. Values are given as boxplots with median and whiskers (min to max); striped bars = impacted, blank bars = unimpacted

Fig. 4. Release of inflammatory mediators is differently influenced by chemically induced necroptosis but can be attenuated by Nec-1 and NAC, respectively.

Necroptosis was chemically induced in impacted and unimpacted cartilage explants by means of TNF/CHX w/ and w/o zVAD for 4 days (n ≥ 4). In addition, therapeutic effects of Nec-1 and NAC were evaluated in this context. Amounts of inflammatory markers a nitrite and b PGE2, respectively, were determined by Griess assay and ELISA, respectively. Statistical analysis was performed by one-way ANOVA, including a Bonferroni posttest. Significant differences between groups were depicted as: [vs C] ^ccccP < 0.0001; [between delineated groups] **P < 0.01, ***P < 0.001, ****P < 0.0001. Values are given as boxplots with median and whiskers (min to max); striped bars = impacted, blank bars = unimpacted

Fig. 5. Necroptosis marker p-MLKL can be detected in OA cartilage and after TNF/CHX stimulation.

Exemplary images of impacted and differently stimulated cartilage explants after immunohistochemical staining of a cleaved CASP8, b RIPK3, and c p-MLKL. In addition, percentage of cleaved CASP8− (d) and p-MLKL− (e) positive cells was quantified (n = 3). Images were acquired using a 20× objective; the bars represent 200 µm

Fig. 6. Morphological alteration and DAMP release of chondrocytes undergoing regulated cell death.

Isolated chondrocytes were stimulated as follows: b trauma-conditioned medium (TCM), c 100 ng/mL TNF, d 100 ng/mL TNF + 10 µg/mL CHX, and e treated with Nec-1, or f 100 ng/mL TNF + 10 µg/mL CHX + 20 µM zVAD, and g treated with Nec-1 for 24 h. a Untreated cells served as control. Yellow arrows indicate cell debris/dead cells. Images were acquired using a 10× and 32× objective (magnification of d and f; illustrated in the middle columns); the bars represent 200 µm. h Cell viability of chondrocytes was evaluated by alamarBlue assay. Fluorescence intensity of treated cells was normalized to the untreated control. i Total release of dsDNA into the culture medium was quantified by Hoechst 33258. j Amounts of HMGB1 in the culture medium was determined by means of a specific ELISA and normalized to the results of the alamarBlue assay. Statistical analysis was performed by one-way ANOVA, including a Bonferroni posttest (n = 4); values are given as boxplots with median and whiskers (min to max). Significant differences between groups were depicted as: [vs C] ^cP < 0.05, ^ccP < 0.01, ^ccccP < 0.0001; [between delineated groups] *P < 0.05, **P < 0.01