Predilection to Dermato-Lymphangio-Adenitis in Obstructive Lymphedema of Lower Limbs Depending on Genetic Polymorphisms at TNFα -308G>A,CCR2 -190G>A,CD14 -159C>T,TLR2 2029C>T,TLR4 1063A>G,TLR4 1363C>T,TGFβ 74G>C,and TGFβ 29T>C

Abstract

Background:

Infection is the most common type of complication observed in lymphedema and is promoted by lymphatic system dysfunction, which causes locoregional immune disorders. Infectious complications are primarily bacterial and most commonly cellulitis (dermato-lymphangio-adenitis, DLA) caused by patients' own skin Staphylococci epidermidis and aureus. The clinical course and outcomes in the immune response to infection have been shown to be associated with genetic polymorphisms.

Aim:

To investigate polymorphism of TNFα-308G>A, CCR2-190G>A, CD14-159C>T, TLR2 2029C>T, TLR4 1063A>G, TLR4 1363C>T, TGFβ 74G>C, and TGFβ 29T>C. The second part of study was the correlation of levels of TNFα and TGFβ with their genes polymorphism in one hundred patients with lower limb postdermatitis lymphedema.

Results:

(a) High percentage of TNFα homozygotes, no differences in genotypes of CD14-159C>T, CCR2-190G>A, TGFβ 74G>C, TGFβ 29T>C, and TLR4 1063A>G, low percentage of TLR2 2029C>T heterozygotes and homozygotes TT, and a high percentage of TLR4 1363C>T homozygotes TT, (b) low serum levels of TGFβ and TNFα in 19% and 43% of patients, respectively, however, lack of correlation between low levels of these cytokines and frequency of homozygotes CC and AA, respectively.

Conclusions:

The practical implications of finding high frequency of genotype TT of TLR4 1363C>T are indications for testing this gene in patients with obstructive lymphedema of lower limbs and early antibiotic prophylaxis of recurrent attacks of DLA, and during elective surgery of lymphedema. The obtained data are also important as a contribution to mapping of genetic variations in acquired lymphedema of lower limbs.

Introduction

The lymphatic system plays a prominent role in antigen presentation and defense against infection. Infection is the most common type of complication observed in lymphedema and is promoted by lymphatic system dysfunction, which causes locoregional immune disorders. Infectious complications are primarily bacterial and most commonly cellulitis (dermato-lymphangio-adenitis, DLA) caused by patients' own skin Staphylococci epidermidis and aureus.^1,2 DLA is a common and serious complication of obstructive lymphedema of limbs. The clinical characteristics of acute DLA are local tenderness and erythema of the skin, sometimes red streaks along the distribution of the superficial lymphatics and enlarged inguinal lymph nodes. Systemic symptoms include malaise, fever and chills. In its subacute or latent form, only skin involvement is observed.³ Each episode of DLA is commonly followed by worsening of leg swelling. Repeated episodes can lead to extensive fibrosis and further blockage of lymphatic ducts, which results in gross enlargement of limbs. Numerous clinical studies suggest that administration of antibiotic drugs interrupt the acute episodes and prevent their recurrence.^4–6 Many forms of lymphedema are secondary to malignancies, particularly following surgical removal of lymphatic tissue and after radiotherapy. Between 25% and 33% patients who undergo surgery for breast cancer are expected to develop lymphedema in the arm or trunk and the risk factors have been identified.⁷ In lower limbs, postinflammatory, posttraumatic, and post-iliac lymphadenectomy lymphedema is most common. DLA attacks frequency may reach 50% in advanced cases.⁸

The clinical course and outcomes in the immune response to infection have been shown to be associated with genetic polymorphisms.^9,10 Functional and association studies involving genetic polymorphisms in essential genes, including Toll-like receptors, cytokines, and coagulation factors, have provided important insights into the mechanisms involved in the pathogenesis of infection-induced organ dysfunction.^11–13

The advancement of high-throughput single-nucleotide polymorphism (SNP) genotyping provides valuable information on the interaction of multiple allelic variants and clinical outcome not only of sepsis but also local infective processes. More precise categorization of patients based on genetic background might lead to individualized targeted treatment.

The usually reported studies give an insight into the correlation between the genotype and clinical symptoms. This prompted us to study a complex of nine variation points in a randomly selected group of patients with present and at risk infections in obstructive lymphedema, admitted to our surgical department. In this study, we present a genetic association study of DLA with focus on seven single-nuclear polymorphisms linked to local and systemic septic conditions. We investigated frequencies of promoter's SNPs in TNFα-c.-308G>A, (rs1800629), CCR2 c.-190G>A (rs1799864), CD14 c.-159C>T (rs2569190), TLR2 c.2029C>T (rs121917864), TLR4 c.1063A>G (rs4986790), TLR4 c.1363C>T (rs4986791), TGFβ c.74G>C (+915G>C, rs1800471, and Arg25Pro), and TGFβ c.29T>C (+869T>C, rs1800470, and Leu10Pro) genes. The second part of our study was the correlation of levels of TNFα and TGFβ with their gene polymorphism.

Patients and Methods

One hundred fifty patients were, in an order as they were showing up, consecutively admitted to our Department of Surgery in the period 2011–2015 because of inflammatory processes of soft tissues in swollen lower limbs. They underwent screening for identification of lymphatic obstruction. The population consisted of patients originating from the Mazovia (mid-Poland), ethnically homogenous demographic region; there were 80 males and 70 females, aged 21–62 (median 44) years, mean BMI 24.1 (range 21.3–28.6).

Inclusion criteria

Lower limbs swelling after foot or calf dermatitis with at least one episode of acute DLA and lymphographic confirmation of lymphatic collectors' obstruction.

Exclusion criteria

Venous thrombosis on ultrasound investigation, previous limb trauma, cancer surgery and/or radiotherapy afflicting limbs, cardiac insufficiency, lipedema, and diabetes.

Out of the 150 patients with limb edema only those 100 who were shown to have lymphatic blockage on lymphographies, were included into genetic studies.

The study was carried out in two groups: (1) 100 patients with multiple episodes of DLA in diagnosed lymphedema of lower limbs stage II–IV and (2) control group of 129 blood donors, ethnically matched to the study group and randomly selected for comparing the studied group data.

The study protocol was approved by the Institute's ethics committee. Informed consent was obtained from patients.

Diagnosis of lymphedema

Clinical evaluation

Briefly, in stage II, pitting edema affected foot and lower half of the calf; in stage III, foot and calf were involved, with hard foot and ankle area skin; in stage IV, the whole limb was edematous with foot and calf skin hyperkeratosis and papillomatosis of toes.¹⁴

Lymphoscintigraphic evaluation

Evaluation of main lymphatic pathways was done on lymphoscintigraphic pictures. The 99mTc Nanocoll was injected into toeweb and sole and imaging was done 10 and 60 minutes later after standard walking at 3 km/h. In stage II, the delayed outline of calf lymphatics and small inguinal node appeared 30 minutes after injection. In stage III, small fragments of draining lymphatics were seen in the calf and thigh with some few inguinal nodes of irregular outline after 1–2 hours of walking. Stage IV was characterized by epifascial spread of tracer in the foot and entire calf without visualization of collecting lymphatics and nodes. In none of the investigated patients did the tracer pass on the affected side above the inguinal crease level.¹⁵

Fluorescent lymphography evaluation

Evaluation of superficial foot and calf lymphatic pathways with indocyanine green (ICG) lymphography confirmed lymph stasis at the subepidermal level. It was performed as follows: 0.2 mL of 0.25% ICG (Pulsion, Munich, Germany) was injected subcutaneously into both lower extremities at the first and fourth web space of the foot. Imaging was performed using a Photodynamic Eye (Hamamatsu Photonics, Hamamatsu, Japan) immediately, 2 and 24 hours after the injection. In lymphedema limbs, the dye did not visualize lymphatic collectors, but spread under the foot and calf skin. After 24 hours, it was found under the skin of the whole limb up to the inguinal ligament.

Combination of isotopic and fluorescent lymphographies provided evidence for obstruction of the superficial and deep anatomical lymphatic systems, and formation of collateral subepidermal lymph flow pathway, necessary for diagnosis of lymphedema in contrast to other types of limb edema.

Bacteriological confirmation of inflammatory reaction

Subcutis biopsy specimens and aspirates of edema fluid were cultured on differential bacteriological media to confirm the presence of bacteria in subcutis and tissue fluid during DLA episodes.^4,5 Staphylococci epidermidis and aureus (methicillin sensitive) were identified in 60% of specimens. All strains were sensitive to most antibiotics. This indicated that the defined strains were inhabiting in the patient and did not originate from the hospital.

Genetic polymorphism studies

Detection of genetic polymorphisms of TNFα-308G>A, CCR2-190G>A, CD14-159C>T, TLR2 2029C>T, TLR4 1063A>G, TLR4 1363C>T, TGFβ 74G>C, and TGFβ 29T>C was performed in 100 tests using polymerase chain reaction–restriction fragment length polymorphism. Levels of TNFα and TGFβ were measured using the commercially available Quantikine Human TGFβ-1 kit and Quantikine Human TNFα kit according to the manufacturer's protocol (R&D Systems, Minneapolis) with ELISA method.

Genomic deoxyribonucleic acid (DNA) was isolated from whole blood using the Blood Mini Kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer's instructions. Quantification of isolated DNA was performed spectrophotometrically using the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA). Polymerase chain reaction (PCR) amplification was performed using a Thermal Cycler (MJ Research Watertown, MA). The amplification products were digested with appropriate restriction enzymes and separated, and silver stained on ultrathin 12.5% polyacrylamide gels (Multiphore II System and Silver Staining Kit, Amersham Pharmacia Biotech, Uppsala, Sweden).

Polymerase chain reaction–restriction fragment length polymorphism analysis of TNFα-308G>A polymorphism

Polymerase chain reaction was performed using RedTaq polymerase (Sigma-Aldrich, St. Louis, MO). Approximately, 50 ng of sample DNA was added to a final reaction volume of 25 μL containing 2.5 μL 10 × buffer with MgCl₂, 0.5 μL deoxyribonucleoside triphosphate mix (Sigma-Aldrich), and 30 pmol of each primer (Genomed, Warsaw, Poland). The primer sequences for TNFα-308G>A gene were as follows: 5′AGG CAA TAG GTT TTG AGG GCC AT3′ (forward) and 5′TCC TCC CTG CTC CGA TTC CG 3′ (reverse). The temperature profile of PCR was as follows: 95°C for 5 min, followed by 30 cycles of 94°C for 30 s, 64°C for 30 s, and 72°C for 45 s, followed by one elongation step at 72°C for 10 min. Polymerase chain reaction products (10 μL) were incubated for 2 h with 1 U NcoI (Roche Applied Science, Basel, Switzerland) in a total volume of 20 μL at 37°C.

Polymerase chain reaction–restriction fragment length polymorphism analysis of CCR2-190G>A, CD14-159C>T polymorphism

Polymerase chain reactions were performed using Expand Long Template (Roche Applied Science, Basel, Switzerland). Approximately, 50 ng of sample DNA was added to a final reaction volume of 25 μL containing 2.5 μL 10 × buffer with MgCl₂, 0.5 μL deoxyribonucleoside triphosphate mix (Sigma-Aldrich), and 30 pmol of each primer (Genomed). The primer sequences for CCR2-190G>A gene were as follows: 5′ GAA AGT GGA TTG AAC AAG GAC 3′ (forward) and 5′ CAG GTT GAG CAG GTA AAT GT 3′ (reverse); the primer sequences for CD14-159C>T gene were as follows: 5′ GTG CCA ACA GAT GAG GTT CA 3′ (forward) and 5′ CGC AGC GGA AAT CTT CAT C 3′ (reverse). Temperature profile of PCR both for CCR2-190G>A and CD14-159C>T polymorphisms was as follows: 95°C for 5 min, followed by 30 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s, followed by one elongation step at 72°C for 10 min.

CCR2-190G>A

Polymerase chain reaction products (10 μL) were incubated for 3 h with 0.2 U FokI (Roche Applied Science, Basel, Switzerland) in a total volume of 20 μL at 37°C.

CD14-159C>T

Polymerase chain reaction products (10 μL) were incubated for 2 h with 1 U HaeIII (Roche Applied Science, Basel, Switzerland) in a total volume of 20 μL at 37°C.

Polymerase chain reaction–restriction fragment length polymorphism analysis of TLR2 2029C>T, TLR4 1063A>G, and TLR4 1363C>T polymorphisms

Polymerase chain reactions were performed using FastStart Taq (Roche Applied Science, Basel, Switzerland). Approximately, 50 ng of sample DNA was added to a final reaction volume of 25 μL containing 2.5 μL 10 × buffer with MgCl₂, 0.5 μL deoxyribonucleoside triphosphate mix (Sigma-Aldrich), and 30 pmol of each primer (Genomed). The primer sequences for TLR2 2029C>T gene were as follows: 5′ GCC TAC TGG GTG GAG AAC CT 3′ (forward) and 5′ GGC CAC TCC AGG TAG GTC TT 3′ (reverse). Temperature profile of PCR was as follows: 95°C for 10 min, followed by 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, followed by one elongation step at 72°C for 10 min. Polymerase chain reaction products (10 μL) were incubated for 2 h with 1 U AciI (Fermentas, Burlington, Canada) in a total volume of 20 μL at 37°C. The primer sequences for TLR4 1063A>G gene were as follows: 5′ GAT TAG CAT ACT TAG ACT ACT ACC TCC ATG 3′ (forward) and 5′ GAT CAA CTT CTG AAA AAG CAT TCC CAC 3′ (reverse); the primer sequences for TLR4 1363C>T gene were as follows: 5′ GGT TGC TGT TCT CAA AGT GAT TTT GGG AGA A 3′ (forward) and 5′ CCT GAA GAC TGG AGA GTG AGT TAA ATG CT 3′ (reverse). The temperature profile of PCR both for TLR4 1063A>G and TLR4 1363C>T polymorphisms was as follows: 95°C for 4 min, followed by 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, followed by one elongation step at 72°C for 10 min.

TLR4 1063A>G.

Polymerase chain reaction products (10 μL) were incubated for 1.5 h with 1 U NcoI (Fermentas, Burlington, Canada) in a total volume of 20 μL at 37°C.

TLR4 1363C>T.

Polymerase chain reaction products (5 μL) were incubated for 3 h with 10 U HinfI (Fermentas, Burlington, Canada) in a total volume of 10 μL at 37°C.

Polymerase chain reaction–restriction fragment length polymorphism analysis of TGFβ 74G>C and 29T>C polymorphisms

Polymerase chain reactions were performed using FastStart Taq (Roche Applied Science, Basel, Switzerland). Approximately, 50 ng of sample DNA was added to a final reaction volume of 25 μL containing 2.5 μL 10 × buffer with MgCl₂, 0.5 μL deoxyribonucleoside triphosphate mix (Sigma-Aldrich), and 30 pmol of each primer (Genomed). The primer sequences for TGFβ 74G>C gene were as follows: 5′ CGC TGC TGT GGG TAC TGG T 3′ (forward) and 5′ CTC CGG TTC TGC ACT CTC C 3′ (reverse); the primer sequences for TGFβ 29T>C gene were as follows: 5′ ACC ACA CCA GCC CTG TTC GCG C 3′ (forward) and 5′ AGC CAC AGC AGC GGT AGC AGG A 3′ (reverse). The temperature profile of PCR of TGFβ 74G>C polymorphism was as follows: 95°C for 5 min, followed by 35 cycles of 95°C for 60 s, 56°C for 60 s, and 72°C for 60 s, followed by one elongation step at 72°C for 10 min. Polymerase chain reaction products (10 μL) were incubated for 2 h with 1 U FseI (New England Biolabs Ltd., Ipswich, MA) in a total volume of 20 μL at 37°C. The temperature profile of PCR of TGFβ 29T>C polymorphism was as follows: 95°C for 5 min, followed by 35 cycles of 95°C for 60 s, 66°C for 60 s, and 74°C for 60 s, followed by one elongation step at 72°C for 10 min. Polymerase chain reaction products (10 μL) were incubated for 2 h with 1 U BsrBI (Promega, Madison, WI) in a total volume of 20 μL at 37°C.

Serum TNFα and TGFβ protein measurement

Concentration of cytokines was measured on ELISA plates (R&D, Abingdon, United Kingdom).

Statistical analysis

Statistical analysis was performed using the StatSoft Statistica version 10.0 program (StatSoft, Inc., Tulsa, OK). Differences at p < 0.05 were considered to be statistically significant. Comparison of allele and genotype frequencies for each polymorphism was analyzed by Fisher's exact test.

There was no statistically significant deviation in Hardy–Weinberg equilibrium between the local and investigated population according to the Genetic Lab, State Institute of Health (data provided to authors).

Results

TNFα-308G>A, CCR2-190G>A, CD14-159C>T, TLR2 2029C>T, TLR4 1063A>G, TLR4 1363C>T, TGFβ 74G>C, and TGFβ 29T>C polymorphism analysis.

The obtained data are presented in Table 1 and in Figures 1 –8.

Table 1.

The Frequency of Genotypes in the Investigated Groups

Gene/SNP	Genotypes	Controls (n, %)	p/OR (95% CI)	Study group (n, %)	p/OR (95% CI)
TNFα-308G>A rs1800629	GG	40 (31.0)	<0.0001 16.565 (3.570–76.873)	72 (73.5)	1.0000 (NS) 1.323 (0.1172–14.936)
	GA	87 (67.4)		24 (24.5)
	AA	2 (1.6)		2(2.0)
CCR2-190G>A rs1799864	GG	96 (74.4)	1.0000 (NS) 0.7322 (0.02936–18.258)	80 (81.6)	1.0000 (NS) 0.8653 (0.03464–21.617)
	GA	32 (24.8)		18(18.4)
	AA	1 (0.8)		0 (0.0)
CD14-159C>T rs2569190	CC	37 (30.3)	0.3667 (NS) 1.486 (0.6227–3.547)	42 (42.9)	0.3334 (NS) 1.605 (0.6506–3.959)
	CT	70 (57.4)		38 (38.8)
	TT	15 (12.3)		18 (18.3)
TLR2 2029C>T rs121917864	CC	72 (61.5)	1.0000 (NS) 0.9474 (0.3992–2.248)	86 (87.8)	0.0010 0.05102 (0.003015–0.8633)
	CT	26 (22.2)		12 (12.2)
	TT	19 (16.3)		0 (0.0)
TLR4 1063A>G rs4986790	AA	123 (95.3)	0.0171 3.533 (1.227–10.173)	92 (93.9)	0.7108 (NS) 1.326 (0.3250–5.412)
	AG	6 (4.7)		3 (6.1)
	GG	0 (0.0)		0 (0.0)
TLR4 1363C>T rs4986791	CC	84 (69.4)	<0.0001 2079.0 (115.05–37569)	0 (0.0)	<0.0001 450.42 (87.697–2313.4)
	CT	31 (25.6)		4 (4.1)
	TT	6 (5.0)		94 (95.9)
TGFβ 74G>C rs1800471	GG	57 (44.9)	0.0001 3.938 (1.984–7.819)	40 (40.8)	0.1007 (NS) 1.995 (0.9356–4.254)
	GC	47 (37.0)		28 (28.6)
	CC	23 (18.1)		30 (30.6)
TGFβ 29T>C rs1800470	TT	53 (41.4)	0.0249 3.078 (1.244–7.616)	92 (93.9)	0.0638 (NS) 0.1140 (0.006548–1.985)
	TC	65 (50.8)		6 (6.1)
	CC	10 (7.8)		0 (0.0)

There were differences in genotypes of TNFα-308G>A in patients and control subjects. Patients showed higher frequency of TNFα gene −308G>A polymorphism GG (74%) and lower of GA (24%) genotypes than in controls (31% and 67%, respectively), but statistically nonsignificant (NS). The genotype AA was at a similar level to the reference group (Fig. 1).

FIG. 1.

Percentage of genotypes of TNFα G-308A polymorphism in the study group compared with controls.

No statistically significant difference of CCR2-190G>A and CD14-159C>T genes in patients compared with healthy subjects was found (Figs. 2 and 3).

FIG. 2.

Percentage of genotypes of CCR2 G-190A polymorphism in the study group compared with controls.

FIG. 3.

Percentage of genotypes of CD14 C-159T polymorphism in the study group compared with controls. TLR2 2029 CC genotype was represented in 88% and CT in 12% in the study group compared to controls (62% and 22%, respectively). The mutated homozygote TT reached 16% in the reference group, whereas this genotype was not detected in patients (p = 0.001) (Fig. 4).

TLR2 2029 CC genotype was represented in 88% and CT in 12% in the study group compared to controls (62% and 22%, respectively). The homozygote TT reached 16% in reference group, whereas this genotype was not detected in patients (p = 0.001) (Fig. 4).

FIG. 4.

Percentage of genotypes of TLR2 C2029T polymorphism in the study group compared with controls, *p = 0.0010.

There were no differences in genotypes of TLR4 1063A>G polymorphism in patients and control subjects, p = NS (Fig. 5).

FIG. 5.

Percentage of genotypes of TLR4 A1063G polymorphism in the study group compared with controls.

Interestingly, there were differences of TLR4 1363C>T in patients and control subjects. Genotype TT was detected in around 100% of study group, whereas in the control group, this genotype was found only in 5%, p < 0.0001 (Fig. 6).

FIG. 6.

Percentage of genotypes of TLR4 C1363T polymorphism in the study group compared with controls, *p < 0.0001.

TGFβ 74GG and GC genotypes remained at the similar level in study group and in controls. There were more of TGFβ 74CC patients (31%) than healthy subjects (18%), but statistically nonsignificant (NS) (Fig. 7).

FIG. 7.

Percentage of genotypes of TGFβ G74C polymorphism in the study group compared with controls.

TGFβ 29TT genotype was represented at higher values in the study group (94%) than in controls (41%). TC genotype was detected in patients, only in 6% compared with controls (51%). Interestingly, the homozygote CC was not found in patients, p = NS (Fig. 8).

FIG. 8.

Percentage of genotypes of TGFβ T29C polymorphism in the study group compared with controls.

Serum levels of TNFα and TGFβ

The mean serum level of TNFα of controls was 3 ± 0.5 pg/mL, whereas it was 6.03 ± 3.68 pg/mL in 12% (p < 0.05) and lower 0.12 ± in 43% (p < 0.005) of patients. The mean level of TGFβ in controls was 70.0 ± 1.1 ng/mL, whereas it was 64.0 ± 12.7 ng/mL, in 15% (p < 0.05) and 23.3 ± 1.2 ng/mL, in 19% (p < 0.0%) of patients.

Genotypes of TNFα and TGFβ and their cytokine serum levels

TNFα: one homozygote AA (3%) and 5 heterozygotes GA (17%) were found in 29 patients with low serum level. TGFβ: one homozygote CC (7%) was found in 13 patients with low serum level. There was no difference between the group with low cytokine levels and the genetic variant frequency of the whole investigated group.

Discussion

Our studies provided the following information:

(a) High percentage of TNFα homozygotes, no difference in genotypes of CD14-159C>T, CCR2-190G>A, TGFβ 74G>C, TGFβ 29T>C, and TLR4 1063A>G, low percentage of TLR2 2029C>T heterozygotes, homozygotes TT, and a high percentage of TLR4 1363C>T homozygotes TT,

(b) Low serum levels of TGFβ and TNFα in 19% and 43% of patients, respectively; however, lack of correlation between low levels of these cytokines and frequency of homozygotes.

The TNFα promoter gene is in linkage disequilibrium with several HLA alleles that may be involved with the control of TNFα secretion or may be an independent risk factor for the development of various forms of infections. We found an increased frequency of TNFα-308GG genotype in 74% of patients, higher than in controls. In the pertinent literature, the elispot analysis demonstrated that existence of A allele was associated with higher TNF production compared with G allele.¹⁶ This was not observed in our studies as we found low TNFα levels both in patients with A and G allele. Altogether conflicting results have been reported for TNFα-308G>A, which was associated with disease severity and outcome in some, but not other studies.^17,18

There was no difference in CD14-159C>T and CCR2-190G>A polymorphisms.

A number of SNPs have been identified in the promoter region of the CD14 gene. One polymorphism at −159 in the promoter region has been associated with an increased susceptibility to inflammatory diseases.¹⁹ In the case of CD14 gene for the Caucasian population, in the SNP database, only two results that can be used for comparison with the above data were found. One of the described populations is “E-0,” which consisted of 20 people. Percentage of genotypes was as follows: 30% of nonpolymorphic homozygotes, 45% of heterozygotes, and 25% of polymorphic homozygotes. The second population is “CAUC1” (29 people). In this group, the percentage of genotypes was as follows: 34.5% of nonpolymorphic homozygotes, 55.2% of heterozygotes, and 10.3% of polymorphic homozygotes. The results for the “CAUC1” population are similar to the results obtained for the control group (31%, 57%, and 12%, respectively) [dbSNP].²⁰

TLR-ligand complexes activate signal transduction pathways in both the innate and adaptive immune systems, leading to the release of inflammatory mediators.^21,22 Toll-like receptor 2 (TLR2) is a member of the TLR family, which plays a central role in the innate immune response to a wide variety of microorganisms.¹² We observed a high percentage of CC and zero homozygotes TT. The clinical implications may be more frequent expression of this receptor in skin infections. TLR4 operates in synergy with CD14, a leucine-rich 55 kDa glycoprotein. Our studies did not reveal differences in TLR4 1063A>G and controls. There is not much information connected with that polymorphism for Caucasian population in the SNP database. Only two results can be used as a comparative material. First of the described population is “E-0” (20 people). Percentage of genotypes was as follows: 90% of nonpolymorphic homozygotes, 10% of heterozygotes, and no homozygotes GG was found. The second population is “CAUC1” (31 people) and the percentage of genotypes was as follows: 93.5% of nonpolymorphic homozygotes, 6.5% of heterozygotes, and polymorphic homozygotes were also not found. These results are similar to those obtained in this article. In the other populations, the occurrence of the GG genotype was described, but usually below 1%. Only in two populations (South African Americans and Indian Gujarati from Texas) the polymorphic genotype exceeded 1%.²⁰ However, there were strong differences of TLR4 1363C>T between patients and control subjects. Genotype TT was detected in around 100% of study group, whereas in the control group, this genotype was found only in 5% (p < 0.0001). This finding remains in concert with our previous data obtained from a group of patients with nonhealing wounds and sepsis.²³ However, our conclusions are restricted to the selected 1063A>G, whereas a number of other noninvestigated SNPs may play a role.

There are now a great number of published studies of polymorphisms in TLR4 and their association with many infectious diseases, including sepsis, gram-negative infections, and other bacterial diseases, including tuberculosis, malaria, and infections with respiratory syncytial virus and Candida spp.²⁴ Studies in knockout mice have indicated a role for TLR4 in protection against endotoxemi, but an increased susceptibility of TLR4-mutant mice to systemic gram-negative infections, such as Salmonella typhimurium.^25,26 This is because activation of TLR4 is required for protective immunity against infections, but also mediates the effects of systemic endotoxin/infections. Toll-like receptors (TLRs) present in innate immune cells recognize pathogen molecular patterns and influence immunity to control the host–parasite interaction. TLR4-defective mice were not able to clear their diminished fungal burdens.²⁷

Genetic variants of TLRs and specifically TLR4, and low levels of serum of TNF and TGF are now reported. TLR4 mutant mice had a reduced capacity to eliminate mycobacteria from the lungs, spread the infection to spleen and liver, with 10–100 times higher CFU organ levels than the wild-type mice, and succumbed within 5–7 months, whereas most of the wild-type mice controlled infection and survived the duration of the experiment.²⁸ Diminished expression and function of TLR4 were observed in infection with filarial parasite.²⁹ TLR4 knockout mice demonstrated markedly increased tail inflammatory lymphatic edema compared with wild-type mice.³⁰

TGFβ is a multifunctional cytokine that plays an important role in modulating cell growth. TGFβ1 inhibits the growth of normal epithelial cells. We investigated the polymorphism at TGFβ 29T>C. The TT homozygote percentage was evidently higher than in controls and the CC homozygote was absent. Higher frequency of TT homozygotes could result in a more advanced inflammatory reaction; however, this remains in the realm of speculation. No difference was observed in TGFβ 74G>C.

Serum level of TNFα was higher in 12% and lower in 43% of patients compared with controls. The level of TGFβ was higher in 15% and lower in 19% of patients compared with controls. Most important from the clinical point of view were low levels of these cytokines. There was no correlation with gene polymorphism. The possible explanation might be intensive consumption by the inflammatory infiltrating cells.

Previously, we studied the polymorphisms of selected allele of cytokines and TLRs at nine polymorphic sites in randomly selected groups of patients displaying symptoms of sepsis, acute tissue infections, and prolonged wound suppuration. The conclusion was that polymorphisms of TNFβ, CD14, TLR2, CCR2, and TGFβ genes at certain variation points could be predisposing to this type of infections.²³

This study showed that patients with lymphedema predisposing to episodes of DLA presented high percentage of TNFα homozygotes, no difference in genotypes of CD14, CCR2, TGFβ 74G>C, and TLR4, and low percentage of TGFβ 29T>C and TLR2 heterozygotes and homozygotes. The practical implications of finding high frequency of homozygotes TT of TLR4 1363C>T are indications for testing this gene in patients with obstructive lymphedema of lower limbs and early antibiotic prophylaxis of recurrent attacks of DLA, and during elective surgery of lymphedema. The obtained data are also important as a contribution to mapping of variations in acquired lymphedema of lower limbs.

Footnotes

Acknowledgment

This work was supported by project number NN 404 0851 40 founded by National Science Centre, Poland.

Author Disclosure Statement

No competing financial interests exist.

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