Patients' characteristics
The demographic characteristics of the patients with SLE and SSc and their serological data are presented in Table 1. The mean age of the patients with SLE was 48 years, and that of the patients with SSc was 53 years. Among the patients with SLE, 14 (93%) were females, whereas all nine patients with SSc were females. The mean erythrocyte sedimentation rates were 18.0 ± 22.1 and 33.8 ± 29.3 mm/h in the patients with SLE and SSc, respectively. In total, 14 (93%) patients with SLE and 8 (89%) patients with SSc were positive for antinuclear antibodies. Further, six (40%) patients with SLE were anti-dsDNA positive. Three (20%) patients with SLE and four (44%) patients with SSc overexpressed the anti-Scl-70 autoantibody.
Table 1
Demographic and serological characteristics of patients with systemic lupus erythematosus and systemic sclerosis
Characteristics
|
Patients with SLE (n = 15)
|
Patients with SSc (n = 9)
|
Age (years)
|
47.73 ± 14.35
|
53.80 ± 15.30
|
Female sex
|
14 (93%)
|
9 (100%)
|
Serological characteristics
|
|
ESR (mm/h)
|
18.0 ± 22.1
|
33.8 ± 29.3
|
hCRP
|
0.32 ± 0.33
|
0.23 ± 0.21
|
IgG
|
1,112.0 ± 620.0
|
911.0 ± 1,257.2
|
C3
|
85.67 ± 29.18
|
76.97 ± 25.22
|
C4
|
16.62 ± 5.94
|
19.14 ± 8.99
|
ANA positivity
|
14 (93%)
|
8 (89%)
|
Coexisting autoantibodies
|
|
Anti-dsDNA
|
6 (40%)
|
0 (0%)
|
Anti-Scl-70
|
3 (20%)
|
4 (44%)
|
Anti-SS-A
|
8 (53%)
|
0 (0%)
|
Anti-SS-B
|
3 (20%)
|
0 (0%)
|
Anti-Sm
|
5 (33%)
|
0 (0%)
|
Anti-RNP
|
6 (40%)
|
0 (0%)
|
Continuous variables are expressed as the mean ± standard deviation. Categorical variables are expressed as n (%). |
Abbreviations: ESR erythrocyte sedimentation rate; SLE systemic lupus erythematosus; SSc systemic sclerosis |
TLR gene expression and housekeeping genes
Gene expression levels were determined in FASTQ format. The raw TLR gene expression data are presented in Table 2, which shows significant differences (p < 0.05) between the patients with SLE and SSc. At first, we compared TLR1, TLR2, TLR3, TLR4, and TLR5 gene expression normalized by ACTB for patients with SLE and online dataset (SLE) of NCBI in Table 3. Then, the normalized gene expression levels of TLRs of SLE and SSc were compared. The data of TLR1 and TLR3 are presented in Fig. 1.
Table 2
Raw TLR gene expression data for patients with systemic lupus erythematosus and systemic sclerosis
Gene
|
Patients with SSc
(n = 9)
|
Patients with SLE
(n = 15)
|
p-Value
|
TLR1
|
7,096.89 ± 6,153.97
|
1,414.80 ± 821.70
|
0.0001*
|
TLR2
|
6,550.89 ± 3,466.45
|
2,776.00 ± 1,694.09
|
0.0013*
|
TLR3
|
48.89 ± 36.13
|
16.20 ± 12.84
|
0.0024*
|
TLR4
|
7,429.78 ± 5,198.17
|
2,775.07 ± 1,432.09
|
0.0027*
|
TLR5
|
822.56 ± 861.45
|
313.67 ± 174.03
|
0.0297*
|
TLR6
|
2,522.22 ± 1,742.09
|
949.67 ± 564.37
|
0.0177*
|
TLR7
|
434.00 ± 270.27
|
197.93 ± 142.01
|
0.0133*
|
TLR8
|
3,900.67 ± 2,070.58
|
1,714.67 ± 805.28
|
0.0001*
|
TLR10
|
267.11 ± 139.92
|
147.73 ± 102.11
|
0.0095*
|
ACTB
|
32,554.67 ± 12,131.16
|
59,349.11 ± 70,677.27
|
0.2384
|
GAPDH
|
3,043.13 ± 1,321.43
|
5,051.33 ± 1,689.00
|
0.0021*
|
PGK1
|
6,520.67 ± 4,210.41
|
2,592.80 ± 890.90
|
0.0001*
|
PPIB
|
866.33 ± 712.27
|
316.40 ± 94.47
|
0.0008*
|
SDHA
|
1,004.56 ± 1,027.37
|
291.07 ± 97.20
|
0.0054*
|
TBP
|
161.00 ± 136.16
|
81.87 ± 32.73
|
0.2449
|
Data are presented as the mean (counts) ± standard deviation. * =“p < 0.05” |
Abbreviations: SLE systemic lupus erythematosus; SSc systemic sclerosis |
Table 3
Comparison of TLR gene expression data for patients with SLE and online data set (SLE) normalized by ACTB
Gene
|
Patients with SLE
(n = 15)
|
Data set with SLE
(n = 16)
|
p-Value
|
TLR1
|
426.70 ± 170.39
|
428.89 ± 135.24
|
0.8001
|
TLR2
|
828.02 ± 342.87
|
718.96 ± 276.48
|
0.4232
|
TLR3
|
4.85 ± 3.43
|
4.28 ± 2.57
|
0.6926
|
TLR4
|
837.51 ± 299.26
|
655.35 ± 170.21
|
0.0544
|
TLR5
|
93.98 ± 40.44
|
83.66 ± 40.48
|
0.3791
|
Data are presented as the mean (counts) ± standard deviation. * =“p < 0.05” |
Abbreviations: SLE systemic lupus erythematosus |
Post-normalization TLR gene expression levels
Normalization to ACTB expression
The TLR1 and TLR3 gene expression values normalized to those of ACTB are presented in Fig. 1a. The median relative expression levels of TLR1 and TLR3 in the patients with SSc were 12.88 (range: 9.51–35.08) and 0.13 (0.06–0.25), respectively. These levels were significantly higher than those in the patients with SLE [TLR1: 4.44 (3.08–5.28), p = 0.0001; TLR3: 0.040 (0.023–0.067), p = 0.0026]. The relative expression levels of TLR2, TLR4, TLR5, and TLR6 were also significantly different between the patients with SSc and SLE when normalized to those of ACTB.
Normalization to GAPDH expression
The TLR1 and TLR3 gene expression values normalized to those of GAPDH are shown in Fig. 1b. The median relative expression levels of TLR1 and TLR3 in the patients with SSc were 69.04 (47.61–370.96) and 0.88 (0.39–1.55), respectively. These levels were significantly higher than those in the patients with SLE [TLR1: 47.42 (9.97–98.81), p = 0.018; TLR3: 0.45 (0.02–0.10), p = 0.032].
Normalization to PGK1 expression
The TLR1 and TLR3 gene expression values normalized to those of PGK1 are shown in Fig. 1c. The only TLR gene that showed a significant difference in the expression levels between the patients with SLE and SSc was TLR1. The median relative expression level of TLR1 in the patients with SSc was 85.66 (47.44–187.28), which was significantly higher (p = 0.0013) than that in the patients with SLE [57.48 (21.02–94.98)].
Normalization to PPIB expression
The expression levels of the TLR1 and TLR3 genes normalized to those of PPIB are shown in Fig. 1d. The median relative expression level of TLR1 in the patients with SSc was 66.21 (51.44–158.67), which was significantly higher (p = 0.0067) than that in the patients with SLE [46.77 (10.12–89.76)].
Normalization to SDHA expression
The expression levels of the TLR1 and TLR3 genes normalized to those of SDHA are shown in Fig. 1e. The median relative expression level of TLR1 in the patients with SSc was 68.05 (41.22–152.40), which was significantly higher (p = 0.021) than that in the patients with SLE [42.59 (13.92–86.24)].
Normalization to TBP expression
The expression levels of the TLR1 and TLR3 genes normalized to those of TBP are shown in Fig. 1 f. The median relative expression levels of TLR1 and TLR3 in the patients with SSc were 40.68 (14.63–208.18) and 0.36 (0.13–0.80), respectively. These levels were significantly higher than those in the patients with SLE [TLR1: 15.06 (6.11–28.74), p = 0.001; TLR3: 0.21 (0.02–0.47), p = 0.034].
Thus, there were significant differences in the relative TLR gene expression levels between the patients with SSc and SLE. In particular, the TLR1 expression values were significantly different between the two groups when normalized to those of all of the housekeeping genes. By contrast, the TLR3 expression values were significantly different upon normalization to those of only some of the housekeeping genes. A summary of significant differences is shown in Table 4.
Table 4
Significant intergroup differences in TLRs gene expression levels after normalization to those of housekeeping genes
|
TLR1
|
TLR2
|
TLR3
|
TLR4
|
TLR5
|
TLR6
|
TLR7
|
TLR8
|
TLR10
|
ACTB
|
V
|
V
|
V
|
V
|
V
|
V
|
|
|
|
GADPH
|
V
|
|
V
|
|
|
|
|
|
|
PGK1
|
V
|
|
|
|
|
|
|
|
|
PPIB
|
V
|
|
|
|
|
|
|
|
|
SDHA
|
V
|
|
|
|
|
|
|
|
|
TBP
|
V
|
|
V
|
|
|
|
|
|
|
"V" indicates a significant difference in the relative expression levels of TLR genes between patients with systemic lupus erythematosus and systemic sclerosis