Pro-inflammatory Cytokines Drive Deregulation of Potassium Channel Expression in Primary Synovial Fibroblasts

Figures

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  FIGURE 1. Experimental pipeline and verification. (A) Schematic of the next generation sequencing (NGS) pipeline. RNA was extracted from synovial tissue from eight animals, were split into control and test groups and the test samples were treated with 10 ng IL1β and TNFα as described in the section “Materials and Methods.” These eight samples sequenced with Illumina and raw data files were uploaded using a FTP client to a bioinformatics platform for analysis. Within the bioinformatics platform, reads were aligned/mapped with Hisat2 program using the reference genome Rat Jul. 2014 (RGSC 6.0/rn6) (rn6). The resulting BAM files were used to measure phred quality score for all samples. Also, BAM files along with an annotation file (Rat GFF3) to assemble mapped reads and quantify gene expression. Such assembly and quantification took place with StringTie and CuffDiff package/tool that generated gene abundance estimates and differentially expressed genes, respectively. After that, using several servers and a local custom-script in MATLAB software allowed us to filter the transcriptome for all known ion channel genes for further analysis (for example, see Tables 1, 2). (B) Expression of CD248 in…

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  FIGURE 2. Discriminant analyses of global IL-1β TNFα treatment effects. (A) Shows the kernel density plots of the discriminant component co-ordinates for the control and IL-1β/TNFα treatment groups; co-ordinates on the x-axis and density on the y-axis; Individual co-ordinate center points are illustrated by the vertical line and circle. There is clear separation between control and cytokine treated FLS samples. (B) A graphical confusion matrix showing the actual group membership (y-axis) and predicted cluster membership (on the y-axis). All groups are correctly clustered with greater than 0.9 probability. (C) The top 15 contributors to the linear discriminator function: Fn-1, fibronectin 1; Col1a1, collagen type I alpha 1 chain; Col3a1, collagen type III alpha 1 chain; Lcn2, lipocalin 2; PAI, serpine1; Grem1, gremlin 1; Mmp2, matrix metallopeptidase 2; Thbs2, thrombospondin 2; Cxcl12, C-X-C motif chemokine ligand 12; Mgp, matrix Gla protein; C3, complement C3; Col1a2, collagen type I alpha 2 chain; Sod2, superoxide dismutase 2; Lrp1, LDL receptor related protein 1; Col1a2, collagen type I alpha 2.

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  FIGURE 3. Enriched calcium regulation pathway. Genes mapped to the calcium signaling canonical pathway by IPA. Red, increased expression; green, decreased expression (for interpretation of the references to color in the figure legend, please refer to the online version of this article).

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  FIGURE 4. The correlation between control and NGS expression data. (A) We found a strong correlation between control and treated (72 h of IL1β + TNFα) channel expression data. Each point represents the intersection of four control and four test values. In (B), the correlation between qPCR (ΔΔCts) and NGS ratio (FPKM values) are shown. Data suggest that there only a weak linear correlation between qPCR and NGS. P-value = 0.3. R = 0.29. n = 14 (eight animals for NGS and six animals for PCR). Genes that were upregulated in both datasets are indicated in red. (C) Summary of NGS expression of all known (rat) splice variants of Kcnma1 and associated Kcnmb subunits.

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  FIGURE 5. Whole-cell voltage-gated currents from control and cytokine treated FLS. (A) Top panels show the voltage step protocols and the evoked currents are shown below for control (left) and IL1β + TNFα (right) conditions. Note that FLS exhibit both transient and sustained currents. These phases of current were then analyzed separately as indicated. (B) Current–voltage curves (left) from the transient currents recorded in a number or experiments such as that illustrated in (A). There was no significant difference between control and treated transient current density. Data points are shown as mean ± SEM (n = 18 for control and n = 11 for IL1β + TNFα). (C) Current–voltage curves from the sustained currents recorded in a number or experiments such as that illustrated in (A). The red line in the current–voltage curve is the difference current for control-cytokine treated. (D) Difference conductance–voltage curve for the cytokine difference current shown in (C). The line is fit with a Boltzmann, see text.

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  FIGURE 6. Effects of BK channel drugs on FLS whole-cell currents. (A) Representative examples of untreated (control) cells before (left) and in the presence of 1 μM of the BK channel opener NS1619 (left). Voltage protocols as shown in Figure 5. (B) Representative raw example current families of cytokine (10 ng/ml IL1β + TNFα) treated cells in the absence (left) and presence (right) of 1 μM NS1619. (C) Current–voltage curves from a number of control FLS cells such as that shown in (A). Current is significantly greater in the presence of NS1619, p < 0.05, n = 9 and 6. (D) Current–voltage curves for a number of treated FLS cells such as that shown in (B). These two curves are not significantly different from each other n = 6 and 6. (E) Current–voltage curves from a number of control FLS cells treated with and without paxilline; there is a significant decrease in paxilline current p < 0.05 n = 13,5), but (F) there was no significant different of current density in the presence of the BK inhibitor paxilline following cytokine treatment (n = 6, 12). (G) Numerical simulation of data from Clark et al. (2017) model. To quantify the degree of change of BK channel modulation apparently taking place…