Dermal exposure to cobalt studied in vitro in keratinocytes – effects of cobalt exposure on inflammasome activated cytokines, and mRNA response

Figures & data

Figure 1. (A) IL-1β secretion by HaCaT cells after exposure to cobalt chloride concentrations 0, 100, 500 µM and 1 mM. Exposure time is 30 minutes (control), 24 hours, and 48 hours. Calcium incubation of HaCaT cells (high: 1.8 mM and low: 0.15 mM) calcium concentrations are shown. (B) IL-18 secretion production by HaCaT-cells after CoCl₂ exposure 0, 100, 500 µM and 1 mM, at time points 30 minutes, 24 hours, and 48 hours. Results of high (1.8 mM) and low (0.15 mM) calcium culturing conditions are shown.

Figure 2. Gene expression in HaCaT cells after CoCl₂ exposure of 0, 500 µM and 1 mM for 24 hours and 48 hours, respectively. High (1.8 mM) and low (0.15 mM) calcium culturing conditions are shown. Results are presented as the normalized gene expression for each of IL1B (A), IL18 (B), NLRP3 (C), CASP1 (D), and PYCARD (E). Results are shown as median value and range (min-max).

Figure 3. Caspase-1 activity in HaCaT cells determined with FLICA staining, analysed by flow cytometry. The cells were exposed to 500 µM and 1 mM CoCl₂, respectively, for 24 hours under high and low calcium incubation. The results are expressed in percentage as fluorescence signal of the activated caspase-1 in exposed cells, compared to unexposed, and shown as mean value and standard deviation (*p ≤ 0.05, **p ≤ 0.005).

Figure 4. Caspase-1 activity in HaCaT cells determined by flow cytometry. Gating strategy for the cell population (A). By moving forward with the same population, gating strategy is shown using unstimulated (0 µg) stained cells (B), and the gating of FLICA positive cells (C).

Table 1. ANOVA analyses for dose- and time trends for IL-1β and IL-18 secretion from the HaCaT cells^a,b.

Cytokine	N	Exposure time/CoCl2 concentration/ High-low calcium incubation	Mean	95% CI	p-value
IL-1β	40	30 minutes	0.55	0.06–1.03
	40	24 hours	0.24	0.05–0.43	0.586
	40	48 hours	0.42	0.15–0.69	1.000
	30	0 µM	0.22	–0.00 to 0.44
	30	100 µM	0.27	0.01–0.52	1.000
	30	500 µM	0.32	–0.04 to 0.69	1.000
	30	1 mM	0.81	0.21–1.41	0.183
	60	Low calcium	0.26	0.14–0.39
	60	High calcium	0.55	0.18–0.91	0.145
IL-18	38	30 minutes	0.79	0.25–1.34
	40	24 hours	0.93	0.49–1.36	1.000
	40	48 hours	1.30	0.79–1.82	0.439
	30	0 µM	0.39	0.04–0.74
	30	100 µM	0.64	0.18–1.10	1.000
	29	500 µM	0.81	0.27–1.35	1.000
	29	1 mM	2.23	1.54–2.93	0.000
	60	Low calcium	1.04	0.65–1.42
	58	High calcium	0.99	0.56–1.41	0.862

AM: Arithmetic mean, 95% CI: 95% confidence interval of the mean, bold values: statistically significant values (p < 0.05).

^aAll data ln-transformed. ^bn = 120.

Table 2. Linear regression mixed model analyses for dose- and time trends for IL-1β and IL-18 secretion from the HaCaT cells^a,b.

Cytokine	Exposure time/CoCl2 concentration/ High-low calcium incubation	OR	95% CI
IL-1β	30 minutes	1.00
	24hours	0.73	0.46–1.16
	48 hours	0.88	0.56–1.40
	0 µM	1.00
	100 µM	1.05	0.62–1.79
	500 µM	1.11	0.65–1.89
	1 mM	1.80	1.06–3.07
	Low calcium	1.00
	High calcium	0.75	0.52–1.10
IL-18	30 minutes	1.00
	24 hours	1.12	0.60–2.08
	48 hours	1.63	0.87–3.04
	0 µM	1.00
	100 µM	1.29	0.63–2.62
	500 µM	1.51	0.73–3.09
	1 mM	6.23	3.04–12.77
	Low calcium	1.00
	High calcium	1.04	0.63–1.73

OR: Odds ratio, CI 95%: 95% confidence interval, bold values: statistically significant values (p < 0.05).

^aAll data ln-transformed. ^bn = 120.

Table 3. Linear regression mixed model analyses for caspase-1 activation in HaCaT cells, dose trends, high (1.8 mM) and low (0.15 mM) calcium incubation^a,b.

Exposed and unexposed CoCl2 concentration/ High-low calcium incubation	OR	95% CI
Exposed	5.96	3.07–11.56
Unexposed	1.00	–
1 mM CoCl2	2.81	1.45–5.45
500 µM CoCl2	1.00	–
High calcium	1.20	0.62–2.33
Low calcium	1.00	–

OR: Odds ratio; 95% CI: 95% confidence interval.

^aAll data ln-transformed. ^bn = 24.

Table 4. ANOVA analyses showing dose- and time relationships for gene expression of IL1B, IL18, NLRP3, CASP1 and PYCARD at high (1.8 mM) and low (0.15 mM) calcium incubation of HaCaT cells exposed to CoCl₂^a,b.

Genes	N	Exposure time/CoCl2 concentration/ High-low calcium incubation	Mean	95% CI	p-value
IL1B	35	24 hours	–6.58	–7.04–(–6.11)
	30	48 hours	–7.27	–7.75–(–6.80)	0.038
	24	0 µM	–7.91	–8.06–(–7.76)
	24	500 µM	–7.32	–7.66–(–6.98)	0.001
	17	1 mM	–4.87	–5.00–(–4.74)	0.000
	31	Low calcium	–7.06	–7.53–(–6.59)
	34	High calcium	–6.75	–7.25–(–6.25)	0.365
IL18	35	24 hours	2.40	2.22–2.58
	30	48 hours	2.52	2.36–2.67	0.335
	24	0 µM	2.45	2.37–2.53
	24	500 µM	2.87	2.80–2.94	0.000
	17	1 mM	1.88	1.64–2.11	0.000
	31	Low calcium	2.33	2.13–2.52
	34	High calcium	2.57	2.43–2.71	0.041
NLRP3	35	24 hours	–7.47	–8.07–(–6.88)
	30	48 hours	–7.64	–8.17–(–7.10)	0.688
	24	0 µM	–8.53	–8.77–(–8.29)
	24	500 µM	–8.33	–8.58–(–8.08)	0.700
	17	1 mM	–5.06	–5.34–(–4.78)	0.000
	31	Low calcium	–7.78	–8.36–(–7.20)
	34	High calcium	–7.34	–7.90–(–6.78)	0.269
CASP1	34	24hours	–4.53	–4.94–(–4.12)
	30	48 hours	–4.99	–5.61–(–4.37)	0.199
	24	0 µM	–3.04	–3.13–(–2.96)
	24	500 µM	–6.03	–6.33–(–5.73)	0.000
	16	1 mM	–5.36	–5.51–(–5.22)	0.000
	30	Low calcium	–4.78	–5.36–(–4.21)
	34	High calcium	–4.71	–5.18–(–4.24)	0.847
PYCARD	35	24hours	–0.48	–0.78–(–0.20)
	30	48 hours	–0.49	–0.78–(–0.19)	0.989
	24	0 µM	0.37	0.30–0.45
	24	500 µM	–0.55	–0.65–(–0.46)	0.000
	17	1 mM	–1.60	–1.78–(–1.42)	0.000
	31	Low calcium	–0.46	–0.75–(–0.17)
	34	High calcium	–0.51	–0.81–(–0.21)	0.787

AM: Arithmetic mean, 95% CI: 95% confidence interval of the mean, bold values: statistically significant values (p < 0.05).

^aAll data ln-transformed. ^bn = 65.

Table 5. Linear regression mixed model analyses showing dose- and time relationships for gene expression of IL1B, IL18, NLRP3, CASP1, and PYCARD at high (1.8 mM) and low (0.15 mM) calcium incubation of HaCaT cells exposed to CoCl₂^a,b.

Genes	Exposure time/CoCl2 concentration/ High-low calcium incubation	OR	95% CI
IL1B	24hours	1.00
	48 hours	0.67	0.52–0.87
	0 µM	1.00
	500 µM	1.80	1.33–2.42
	1 mM	19.46	13.98–27.09
	Low calcium	1.00
	High calcium	1.17	0.90–1.52
IL18	24hours	1.00
	48 hours	0.98	0.87–1.09
	0 µM	1.00
	500 µM	1.52	0.69–3.36
	1 mM	0.54	0.25–1.14
	Low calcium	1.00
	High calcium	1.38	0.74–2.58
NLRP3	24hours	1.00
	48 hours	1.25	0.94–1.65
	0 µM	1.00
	500 µM	1.22	0.39–3.78
	1 mM	33.03	12.58–86.70
	Low calcium	1.00
	High calcium	1.20	0.51–2.78
CASP1	24hours	1.00
	48 hours	0.57	0.47–0.69
	0 µM	1.00
	500 µM	0.05	0.03–0.09
	1 mM	0.09	0.06–0.14
	Low calcium	1.00
	High calcium	1.21	0.82–1.80
PYCARD	24hours	1.00
	48 hours	0.83	0.74–0.93
	0 µM	1.00
	500 µM	0.40	0.35–0.45
	1 mM	0.13	0.12–0.16
	Low calcium	1.00
	High calcium	1.38	0.74–2.58

OR: Odds ratio, 95% CI: 95% confidence interval, bold values: statistically significant values (p < 0.05).

^aAll data ln-transformed. ^bn = 65.

Data availability statement

All data are within the manuscript.