Human Chondrocyte Activation by Toxins From Premolis semirufa, an Amazon Rainforest Moth Caterpillar: Identifying an Osteoarthritis Signature

Abstract

Pararamosis is a disease that occurs due to contact with the hairs of the larval stage of the Brazilian moth Premolis semirufa. Envenomation induces osteoarticular alterations with cartilage impairment that resembles joint synovitis. Thus, the toxic venom present in the caterpillar hairs interferes with the phenotype of the cells present in the joints, resulting in inflammation and promoting tissue injury. Therefore, to address the inflammatory mechanisms triggered by envenomation, we studied the effects of P. semirufa hair extract on human chondrocytes. We have selected for the investigation, cytokines, chemokines, matrix metalloproteinases (MMPs), complement components, eicosanoids, and extracellular matrix (ECM) components related to OA and RA. In addition, for measuring protein-coding mRNAs of some molecules associated with osteoarthritis (OA) and rheumatoid arthritis (RA), reverse transcription (RT) was performed followed by quantitative real-time PCR (RT-qPCR) and we performed the RNA-sequencing (RNA-seq) analysis of the chondrocytes transcriptome. In the supernatant of cell cultures treated with the extract, we observed increased IL-6, IL-8, MCP-1, prostaglandin E2, metalloproteinases (MMP-1, MMP-2, MMP-3 and MMP-13), and complement system components (C3, C4, and C5). We noticed a significant decrease in both aggrecan and type II collagen and an increase in HMGB1 protein in chondrocytes after extract treatment. RNA-seq analysis of the chondrocyte transcriptome allowed us to identify important pathways related to the inflammatory process of the disease, such as the inflammatory response, chemotaxis of immune cells and extracellular matrix (ECM) remodeling. Thus, these results suggest that components of Premolis semirufa hair have strong inflammatory potential and are able to induce cartilage degradation and ECM remodeling, promoting a disease with an osteoarthritis signature. Modulation of the signaling pathways that were identified as being involved in this pathology may be a promising approach to develop new therapeutic strategies for the control of pararamosis and other inflammatory joint diseases.

Keywords: caterpillar; cell signaling; chondrocyte; mediators; osteoarthritis; toxins.

Figure 1

Assessment of cytokines, chemokines, and eicosanoids in chondrocytes treated with the P. semirufa hair extract. Chondrocytes were cultured in 96-well plates at a density of 5 × 10⁴ cells/mL and treated with buffer, IL-1β (10 ng/mL) or pararama hair extract (15, 30, or 60 μg/mL) for 24, 48, and 72 h. After each treatment period, we measured the cell viability by an MTT assay. The supernatants were collected from cells treated with buffer , IL-1β [-x-], 15 μg/mL or 60 μg/mL pararama hair extract for 24, 48, and 72 h by centrifugation at 400 ×g at 4°C for 20 min to assess the concentration of cytokines, chemokines, and eicosanoids. The results represent two separate experiments performed in duplicate and are expressed as the mean of the concentrations of the molecules ± SEM. The data were analyzed using two-way ANOVA and Dunnett's post hoc test. **p < 0.01; ***p < 0.001 vs. the control (buffer treatment).

Figure 2

Complement components analysis in the supernatant of chondrocytes treated with the P. semirufa hair extract. Chondrocytes were cultured in 96-well plates at a density of 5 × 10⁴ cells/mL and treated with buffer , IL-1β [-x-], 15 μg/mL or 60 μg/mL pararama hair extract for 24, 48, and 72 h. After each treatment period, we removed the supernatants, centrifuged them at 400 ×g at 4°C for 20 min, and assessed the concentrations of complement components by ELISA. The results represent two separate experiments performed in duplicate and are expressed as the mean of the concentrations of the complement components ± SEM. The data were analyzed using two-way ANOVA and Dunnett's post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001 vs. the control (buffer treatment).

Figure 3

Matrix metalloproteinases analysis in the supernatant of chondrocytes treated with the P. semirufa hair extract. Chondrocytes were cultured in 96-well plates at a density of 5 × 10⁴ cells/mL and treated with buffer , IL-1β [-x-], 15 μg/mL or 60 μg/mL pararama hair extract for 24, 48, and 72 h. After each treatment period, we removed the supernatants, centrifuged them at 400 ×g at 4°C for 20 min, and assessed the concentration of matrix metalloproteinases by ELISA. The results represent two separate experiments performed in duplicate and are expressed as the mean of the concentrations of the metalloproteinases ± SEM. The data were analyzed using two-way ANOVA and Dunnett's post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001 vs. the control (buffer treatment).

Figure 4

Evaluation of aggrecan and type II collagen production and HMGB1 expression by high-content screening (HCS). Chondrocytes were cultured in 96-well plates at a density of 4 × 10⁴ cells/mL and treated with buffer, IL-1β or pararama hair extract (12 and 49 μg/mL) for 24, 48, and 72 h. After each treatment period, the cells were fixed and blocked. Then, the cells were incubated with anti-Aggrecan, anti-collagen type II or anti-HMGB1 antibodies. In parallel, the cell counts were assessed using Hoechst 33342 staining. The image acquisition and fluorescence intensity measurements were conducted by automatic scanning by using MetaXpress software and a 10× objective, with the Custom Module to calculate the stained area and Multi Wave Scoring Module. For each condition and channel, nine images per well in triplicate were acquired and analyzed. Representative fluorescence microscopy images correspond to the cells obtained after 24 h of treatment. The results were normalized and represent two independent experiments performed in triplicate and are expressed as the mean of the stained area ± SEM or the mean of the median fluorescence intensity ± SEM. The data were analyzed using Student's t-test. *p < 0.05; **p < 0.01; ***p < 0.001 vs. the control (buffer treatment).

Figure 5

Gene expression profile in chondrocytes treated with the P. semirufa hair extract. Chondrocytes (1 × 10⁵ cells/well) treated with buffer, IL-1β or extract (60 μg/mL) were collected, and total RNA was extracted using TRIzol. Relative mRNA quantification was carried out by RT-qPCR. All experiments were performed in triplicate, and the values are presented as the mean ± SEM normalized to GAPDH as an endogenous control. The data were analyzed using Student's t-test. *p < 0.05; **p < 0.01; ***p < 0.001 vs. the control, which was arbitrarily set to 1 (buffer treatment).

Figure 6

Possible intracellular signaling pathways activated in chondrocytes upon treatment with pararama hair extract. (A) Activation of cells by the complement system or by the interaction between low molecular weight hyaluronan and TLR2/TLR4. (B) Activation of cells by the interaction between low molecular weight hyaluronan and CD44 or by interaction between TGF-β receptors and TGF-β1. Illustration based on the data obtained with the use of the MetaCore pathway analysis tool (GeneGO/Thomson Reuters) and enriched with DEGs, which are highlighted in red boxes. Red boxes with black borders are the molecules that were validated in this study. Studies that assessed proteases that are able to cleave the C3 component and hyaluronidase that is able to cleave HA in the extract: (**7, *9).