Investigation of Association of Complement 5 Genetic Polymorphisms with Sepsis and Sepsis-Induced Inflammatory Responses

Figures & data

Table 1 Clinical Characteristics of Sepsis Patients and Healthy Controls

Variable	Sepsis (n=636)	Control (n=753)	P value
Demographics
Age, years, mean ± SEM	61.90±0.70	60.39±0.61	0.102
Male/female, number	421/215	472/281	0.174
Sepsis status, n (%)
Sepsis subtype	351 (55.2)	N.A
Septic shock	285 (44.8)	N.A
Source of infection, n (%)
Respiratory tract infection	410 (64.5)	N.A
Primary bloodstream infection	88 (13.8)	N.A
Abdominal infection	172 (27.0)	N.A
Urinary tract infection	69 (10.8)	N.A
Catheter-associated infection	48 (7.5)	N.A
Brain	47 (7.4)	N.A
Trauma	46 (7.2)
Others	47 (7.4)	N.A
Infection types, n (%)
Gram-positive	96 (15.1)	N.A
Gram-negative	333 (52.4)	N.A
Mixed Gram-negative and -positive	200 (31.4)	N.A
Fungus	143 (22.5)	N.A
Negative blood culture	32 (5.0)	N.A
Pathogenic bacteria, n (%)
Acinetobacter baumannii	161 (25.3)	N.A
Pseudomonas aeruginosa	79 (12.4)	N.A
Escherichia coli	74 (11.6)	N.A
Monilia albicans	59 (9.3)	N.A
Staphylococcus aureus	53 (8.3)	N.A
Klebsiella pneumoniae	45 (7.1)	N.A
Yeast sample sporophyte	39 (6.1)	N.A
Aspergillus	25 (3.9)	N.A
Others	110 (17.3)	N.A
APACHE II score, mean ± SD	26.7±4.5	N.A
qSOFA score, mean ± SD	2.51±0.61	N.A
SOFA score, mean ± SD	8.26±4.41	N.A
28-day mortality, n (%)	166 (26.1)	N.A

Abbreviations: N.A, not applicable; APACHE II, Acute Physiology and Chronic Health Evaluation II; qSOFA, quick sepsis-related organ dysfunction assessment; SOFA, sepsis-related organ dysfunction assessment.

Table 2 Genotype and Allele Frequencies Distribution of the C5 Polymorphisms in the Sepsis Patients and Healthy Controls

SNP	Sepsis n (%)	Control n (%)	P	Adjusted P ^a	OR (95% CI)	Adjusted P ^b	Adjusted OR (95% CI) ^b
rs17611
CC	118 (18.6)	162 (21.5)	0.368	0.507	–	–	–
CT	313 (49.2)	351 (46.6)	–	–	–	–	–
TT	205 (32.2)	240 (31.9)	–	–	–	–	–
(CC+CT) vs TT	431 (67.8)	513 (68.1)	0.886	0.886	0.984 (0.785, 1.235)	0.934	0.991 (0.790, 1.242)
(CT+TT) vs CC	518 (81.4)	591 (78.5)	0.171	0.507	1.203 (0.926, 1.575)	0.170	1.204 (0.923, 1.570)
C allele	549 (43.2)	675 (44.8)	0.380	0.507	0.935 (0.804, 1.086)	–	–
T allele	723 (56.8)	831 (55.2)	–	–	1.000 (reference)	–	–
rs2269067
GG	325 (63.5)	394 (56.1)	0.035	0.046	–	–	–
GC	160 (31.2)	266 (37.9)	–	–	–	–	–
CC	27 (5.3)	42 (6.0)	–	–	–	–	–
(GG+GC) vs CC	485 (94.7)	660 (94.0)	0.598	0.598	1.143 (0.6938, 1.850)	0.610	1.139 (0.692, 1.874)
(GC+CC) vs GG	187 (36.5)	308 (43.9)	0.010	0.040	0.736 (0.581, 0.929)	0.011	0.737 (0.583, 0.932)
G allele	810 (79.1)	1054 (75.1)	–	–	1.000 (reference)	–	–
C allele	214 (20.9)	350 (24.9)	0.020	0.040	0.796 (0.656, 0.964)	–	–

Notes: ^aFalse discovery rate-adjusted P-value for multiple hypotheses testing by using the Benjamini–Hochberg method; ^badjusted for age and gender by using multivariable logistic regression analysis.

Abbreviations: OR, odds ratio; 95% CI, 95% confidence interval.

Table 3 Genotype and Allele Frequencies Distribution of the C5 Polymorphisms in the Different Sepsis Status

SNP	Sepsis Subtype n (%)	Septic Shock n (%)	P	Adjusted P ^a	OR (95% CI)	Adjusted P ^b	Adjusted OR (95% CI) ^b
rs17611
CC	58 (16.5)	60 (21.1)	0.066	0.088	–	–	–
CT	167 (47.6)	146 (51.2)	–	–	–	–	–
TT	126 (35.9)	79 (27.7)	–	–	–	–	–
(CC+CT) vs TT	225 (64.1)	206 (72.3)	0.028	0.056	1.460 (1.041, 2.042)	0.018	1.513 (1.075, 2.130)
(CT+TT) vs CC	293 (83.5)	225 (78.9)	0.144	0.144	0.742 (0.497, 1.106)	0.129	0.731 (0.488, 1.095)
C allele	283 (40.3)	266 (46.7)	0.023	0.056	1.295 (1.039, 1.616)	–	–
T allele	419 (59.7)	304 (53.3)	–	–	1.000 (reference)	–	–
rs2269067
GG	186 (62.4)	139 (65.0)	0.839	0.909	–	–	–
GC	96 (32.2)	64 (29.9)	–	–	–	–	–
CC	16 (5.4)	11 (5.1)	–	–	–	–	–
(GG+GC) vs CC	282 (94.6)	203 (94.9)	0.909	0.909	1.047 (0.473, 2.409)	0.909	1.047 (0.473, 2.320)
(GC+CC) vs GG	112 (37.6)	75 (35.0)	0.557	0.909	0.896 (0.618, 1.287)	0.595	0.905 (0.626, 1.308)
G allele	468 (78.5)	342 (79.9)	–	–	1.000 (reference)	–	–
C allele	128 (21.5)	86 (20.1)	0.591	0.909	0.919 (0.68, 1.243)	–	–

Notes: ^aFalse discovery rate-adjusted P-value for multiple hypotheses testing by using the Benjamini–Hochberg method; ^badjusted for age, gender, and APACHE II score by using multivariable logistic regression analysis.

Abbreviations: OR, odds ratio; 95% CI, 95% confidence interval.

Table 4 Genotype and Allele Frequencies Distribution of the C5 Polymorphisms in the 28-Day Surviving and Non-Surviving Sepsis Patients

SNP	Survivors n (%)	Non-Survivors n (%)	P	Adjusted P ^a	OR (95% CI)	Adjusted P ^b	Adjusted OR (95% CI) ^b
rs17611
CC	80 (17.0)	38 (22.9)	0.023	0.031	–	–	–
CT	225 (47.9)	88 (53.0)	–	–	–	–	–
TT	165 (35.1)	40 (24.1)	–	–	–	–	–
(CC+CT) vs TT	305 (64.9)	126 (75.9)	0.009	0.018	1.704 (1.143, 2.564)	0.003	1.878 (1.238, 2.850)
(CT+TT) vs CC	390 (83.0)	128 (77.1)	0.094	0.094	0.691 (0.451, 1.076)	0.146	0.718 (0.459, 1.123)
C allele	385 (41.0)	164 (49.4)	0.008	0.018	1.407 (1.090, 1.802)	–	–
T allele	555 (59.0)	168 (50.6)	–	–	1.000 (reference)	–	–
rs2269067
GG	252 (65.8)	73 (56.6)	0.171	0.228	–	–	–
GC	112 (29.2)	48 (37.2)	–	–	–	–	–
CC	19 (5.0)	8 (6.2)	–	–	–	–	–
(GG+GC) vs CC	364 (95.0)	121 (93.8)	0.586	0.586	1.267 (0.574, 2.944)	0.503	0.743 (0.311, 1.775)
(GC+CC) vs GG	131 (34.2)	56 (43.4)	0.060	0.148	0.678 (0.451, 1.029)	0.053	1.510 (0.995, 2.290)
G allele	616 (80.4)	194 (75.2)	–	–	1.000 (reference)	–	–
C allele	150 (19.6)	64 (24.8)	0.074	0.148	1.355 (0.970, 1.898)	–	–

Notes: ^aFalse discovery rate-adjusted P-value for multiple hypotheses testing by using the Benjamini–Hochberg method; ^badjusted for age, gender, APACHE II score and SOFA score by using multivariable logistic regression analysis.

Abbreviations: OR, odds ratio; 95% CI, 95% confidence interval.

Figure 1 The location of rs2269067 and rs17611 polymorphisms in C5 gene and their effect on 28-day survival of sepsis patients. (A) The human C5 gene is located on chromosome 9q34.1 (120,952,335–121,050,275) and contains 41 exons according to the GRCh38.p13 primary assembly. The green bar represents the exons. In the visual, the miss-sense rs17611 C>T polymorphism is located within the exon 19 of C5 gene and causes a V802I change in C5, while rs2269067 G>C polymorphism is located within the intron near to exon 30; (B and C) the effect of rs2269067 and rs17611 on the 28-day survival of sepsis patients was evaluated by Kaplan–Meier survival analysis; (D and E) the sepsis-related organ dysfunction assessment (SOFA) score in sepsis patients; (F and G) the correlation of C5 genetic polymorphisms with SOFA score in sepsis patients. Values of relative expression levels are shown as mean ± SD; ***P<0.001.

Figure 2 The influence of C5 genetic polymorphisms on C5 gene expression and C5a production in sepsis patients and healthy controls. (A and B) The C5 gene expression and C5a production levels in sepsis patients and healthy controls; (C and D) the C5 gene expression and C5a production levels in sepsis subtype and septic shock subgroups; (E and F) the C5 gene expression and C5a production levels in survivor and non-survivor subgroups; (G and H) the distribution of C5 and C5a expression in individuals with different rs2269067 genotypes; (I and J) the distribution of C5 and C5a expression in individuals with different rs17611 genotypes. Values of relative expression levels are shown as mean ± SD; *P<0.05, **P<0.01, ***P<0.001.

Figure 3 The influence of C5 genetic SNPs on inflammatory cytokine production in sepsis patients and healthy controls. (A–C) The IL-1β, TNF-α and IL-6 plasma levels in sepsis patients and healthy controls; (D–F) the IL-1β, TNF-α and IL-6 plasma levels in sepsis subtype and septic shock subgroups; (G–I) the IL-1β, TNF-α and IL-6 plasma levels in survivor and non-survivor subgroups; (J–L) the distribution of IL-1β, TNF-α and IL-6 plasma levels in individuals with different rs2269067 genotypes; (M–O) the distribution of IL-1β, TNF-α and IL-6 plasma levels in individuals with different rs17611 genotypes. Values of relative expression levels are shown as mean ± SD; *P<0.05, **P<0.01, ***P<0.001.

Figure 4 C5a enhances LPS-induced inflammatory cytokine production and cell apoptosis. THP-1 monocytes were stimulated with C5a (100 ng/mL), LPS (500 ng/mL), or C5a plus LPS for 16 h in vitro. (A–C) The supernatant concentrations of inflammatory cytokines (IL-1β, TNF-α and IL-6) were measured by ELISA; (D and E) the apoptosis of THP-1 monocytes stimulated with C5a, LPS, or C5a plus LPS was also measured by using annexin-V-FITC/PI staining on a flow cytometer. Values of relative expression levels are shown as mean ± SD; *P<0.05, **P<0.01.