The
During the process of infection, after the engagement of innate immunity into the defence system, adaptive immunity is activated. This type of immunity consists of dual branches of cellular and humoral immunity. The principal effectors of cellular immunity are T lymphocytes, while the principle effectors of humoral immunity are B lymphocytes [
Abnormal cell-mediated immunity, including changes in main immunoglobulins as well as their subclasses that play a specific role in the immune response cascades, could further reduce immune functions [
The immune system has been studied in patients with diabetes mellitus but with conflicting results [
Therefore, we could hypothesised that the impairment of immune system function could be present also in patients with diabetic foot ulcers (DFUs), in whom infection related complications occur quite frequently and could lead to lower limb amputation [
The aim of our current study was to analyse in detail the presence of possible immunological abnormalities in humoral as well as in cell-mediated immunity in patients with more severe, chronically infected DFUs and the potential relationship between immunological alterations and glycaemic control. In addition, we analysed differences in selected immune parameters between the patients with ulcers infected by sensitive pathogens and those infected by resistant bacterial strains recruited presumably from repeated chronic infections and antibiotic therapy [
68 patients with type 1 and type 2 diabetes mellitus aged between 30 and 70 years with infected DFUs were consecutively included into our cross-sectional study. Control group was formed by 34 patients matched by age, gender, and type of diabetes but without a history of diabetic foot disease.
We included those patients with DFUs who had been treated according to current guidance by the IWGDF [
Before inclusion, patients with DFUs had to have at least two positive wound cultures from swabs taken from the deep tissue after local debridement [
All patients with infected chronic DFUs included into our study were further divided into two subgroups: patients from whom sensitive microbial strains were isolated (subgroup S,
The local ethics committee approved our study. Prior to enrolment into the study, each patient signed an informed consent form.
The following were measured in all participants: blood glucose level (by spectrophotometry; Abbott Architect, USA), creatinine (detected enzymatically; Abbott Architect, USA), and glycosylated haemoglobin (HbA1c: normal values 20–42 mmol/mol; by HPLC Method; Tosoh G8, Japan).
From laboratory markers of infection were assessed CRP (determined turbidimetrically; Abbott Architect, USA), procalcitonin (by electrochemiluminescence; ECLIA, Cobac 6000, Roche, Switzerland), and blood cell counts (by spectrophotometry; SYSMEX, Japan).
Complements represented by C3, C4 (by immunonephelometry; Abbott Architect, USA), the absolute amounts and percentages of NK cells (CD16/56+ cells), and CD14+HLA-DR cells (monocytes, which serve as an important prognostic factor for the progression of an infection, especially in septic stage) were measured. NK cells and CD14+HLA-DR cells were determined together with subpopulations of lymphocytes (see below) by flow cytometry.
Phagocytosis was defined by the percentage of phagocytic cells and FAGSI (phagocyte stimulation index). Phagocytosis was assessed by the FagoFlowEx® Kit (Exbio Prague, Czech Republic). Phagocytic activity of granulocytes was tested by measuring the respiratory (oxidative) burst after their stimulation with
CD3+, CD4+, CD8+, naive inactive (CD4+CD45RA+CD62L+), memory inactive (CD4+CD45RA−CD62L+), naive effector (CD4+CD45RA+CD62L−) and memory effector CD4+ T lymphocytes (CD4+CD45RA−CD62L−) and naive inactive (CD8+CD45RA+CD62L+), memory inactive (CD8+CD45RA−CD62L+), naive effector (CD8+CD45RA+CD62L−), and memory effector CD8+ T lymphocytes (CD8+CD45RA−CD62L−) were assessed by flow cytometry. Also we assessed their indexes counted as naive/memory cells.
During flow cytometry venous blood samples were collected into sterile tubes containing EDTA. Lymphocytes from peripheral blood (100
Serum levels of immunoglobulins (IgM, IgA, and IgG, diagnosed by immunoturbidimetry; Abbott Architect, USA) and IgG subclasses (determined by immunonephelometry; Immage/Immage 800, Beckman Coulter, USA) were also measured. In addition to this, we also compared the number of patients with deficits, particularly in IgG subclasses. Deficiencies were defined as serum levels of certain immunoglo-bulin or its subclass below physiological ranges that were determined by several studies [
The characteristics of patients, laboratory markers of infection, and selected measures of innate and adaptive immunity, including lymphocyte subpopulations and IgG subclasses, were compared between the study groups and subgroups. Data analyses were performed using BMDP software (PC 90). Descriptive data were presented as means ± SD; differences between all study groups were determined using
Patients with infected DFUs did not differ significantly in basic characteristics from the diabetic controls apart from higher serum creatinine levels (Table
A comparison of basic characteristics and certain inflammatory markers between the study groups.
Evaluated parameters | Patients with DFUs ( | Diabetic controls ( | |
---|---|---|---|
Age (years) | 60.3 ± 7.7 | 58.5 ± 6.9 | NS |
Type of diabetes (type 1/type 2/other types; %) | 16.2/80.9/2.9 | 15.6/84.4/0 | NS |
Serum glucose level (mmol/L) | 10.8 ± 5.4 | 9.6 ± 3.7 | NS |
HbA1c according to IFCC (mmol/mol) | 67 ± 19 | 62 ± 21 | NS |
Serum level of creatinine ( | 107.7 ± 58.8 | 84.5 ± 14.3 | |
Serum level of CRP (mg/L) | 10.7 ± 14.7 | 2.7 ± 2.2 | |
Serum level of procalcitonin ( | 0.07 ± 0.08 | 0.1 ± 0 | NS |
Data are presented as means ± SD; types of diabetes mellitus are in percentages; patients with DFUs (diabetic foot ulcers;
Total numbers of leukocytes (7.7 ± 1.9 versus 8.5 ± 2.4 × 109/L; NS), neutrophils (5.1 ± 1.5 versus 5.3 ± 1.8 × 109/L; NS), and other laboratory markers of infection instead of CRP (Table
The differences in selected parameters of innate immunity between the study groups.
Evaluated parameters | Patients with DFUs ( | Diabetic controls ( | |
---|---|---|---|
C3 (g/L) | 1.32 ± 0.27 | 1.35 ± 0.19 | NS |
C4 (g/L) | 0.3 ± 0.08 | 0.28 ± 0.07 | NS |
Percentage of phagocyting PMN cells (%) | 98.2 ± 1.7 | 97.9 ± 1.4 | NS |
FAGSI | 67.6 ± 27.7 | 65.8 ± 30.1 | NS |
Percentage of NK cells (%) | 13.45 ± 6.46 | 13.15 ± 5.83 | NS |
Absolute numbers of NK cells (cells/ | 226 ± 113 | 327 ± 190 | |
CD14+HLA-DR (%) | 80.9 ± 16.5 | 86.2 ± 14.7 | NS |
Data are presented as means ± SD; patients with DFUs (diabetic foot ulcers;
Differences were observed predominantly in measures as of cellular as of humoral branch arm of adaptive immunity. Reductions of percentages and absolute values of total lymphocytes and decreased absolute numbers of almost all types of lymphocytes subpopulations including B lymphocytes, CD4+, CD8+ T lymphocytes, and their effector and memory cells (Table
The differences in selected parameters of cell-mediated immunity between the study groups.
Evaluated parameters | Patients with DFUs ( | Diabetic controls ( | |
---|---|---|---|
% of total lymphocytes (calculated from blood counts) | 23.08 ± 6.2 | 27.5 ± 6.9 | |
Absolute number of total lymphocytes (×109/L) | 1.78 ± 0.61 | 2.4 ± 0.83 | |
% of CD3+ lymphocytes | 75.96 ± 7.64 | 75.77 ± 6.73 | NS |
Absolute number of CD3+ lymphocytes (cells/ | 1341 ± 484 | 1847 ± 669 | |
% of CD4+ lymphocytes | 49.33 ± 8.74 | 47.24 ± 9.78 | NS |
Absolute number of CD4+ lymphocytes (cells/ | 870 ± 331 | 1122 ± 382 | |
% of CD8+ lymphocytes | 25.4 ± 9.66 | 27.83 ± 10.84 | NS |
Absolute number of CD8+ lymphocytes (cells/ | 451 ± 252 | 712 ± 454 | |
% of CD19+ lymphocytes | 10.1 ± 4.84 | 10.64 ± 4.11 | NS |
Absolute numbers of CD19+ lymphocytes (cells/ | 188 ± 124 | 251 ± 111 | |
% of CD4+ naive effector cells | 1.43 ± 2.79 | 3.03 ± 5.45 | NS |
Absolute number of CD4+ naive effector cells (cells/ | 12.88 ± 27.42 | 34.72 ± 59.76 | |
% of CD4+ memory effector cells | 21.74 ± 9.08 | 19.44 ± 8.64 | NS |
Absolute numbers of CD4+ memory effector cells (cells/ | 180 ± 85 | 220 ± 140 | NS |
Index of CD4+ naive/memory effector cells | 0.09 ± 0.26 | 0.18 ± 0.38 | NS |
% of CD4+ naive inactive cells | 22.8 ± 12.9 | 27 ± 13.4 | NS |
Absolute numbers of CD4+ naive inactive cells (cells/ | 207 ± 154 | 305 ± 194 | |
% of CD4+ memory inactive cells | 54.3 ± 9.8 | 50.6 ± 10.5 | NS |
Absolute numbers of CD4+ memory inactive cells (cells/ | 472 ± 194 | 562 ± 214 | |
Index of CD4+ naive/memory inactive cells | 0.47 ± 0.35 | 0.59 ± 0.37 | NS |
% of CD8+ naive effector cells | 26.4 ± 17.5 | 24.1 ± 13.3 | NS |
Absolute number of CD8+ naive effector cells (cells/ | 128 ± 130 | 189 ± 180 | |
% of CD8+ memory effector cells | 31.5 ± 11.2 | 32.4 ± 12.8 | NS |
Absolute number of CD8+ memory effector cells (cells/ | 145 ± 106 | 243 ± 260 | |
Index of CD8+ naive/memory effector cells | 1.14 ± 1.28 | 1.02 ± 1.08 | NS |
% of CD8+ naive inactive cells | 17.9 ± 10.1 | 21.9 ± 12.1 | NS |
Absolute number of CD8+ naive inactive cells (cells/ | 73.2 ± 47.4 | 144.2 ± 131.8 | |
% of CD8+ memory inactive cells | 24.2 ± 10.4 | 21.7 ± 10.7 | NS |
Absolute number of CD8+ memory inactive cells (cells/ | 105 ± 72 | 136 ± 79 | |
Index of CD8+ naive/memory inactive cells | 0.98 ± 1.02 | 1.31 ± 1.2 | NS |
Data are presented as means ± SD; patients with DFUs (diabetic foot ulcers;
The differences in selected parameters of humoral immunity between the study groups.
Evaluated parameters | Patients with DFUs ( | Diabetic controls ( | |
---|---|---|---|
IgM (g/L) | 1.1 ± 0.82 | 0.94 ± 0.42 | NS |
IgA (g/L) | 3.56 ± 1.32 | 2.62 ± 1 | |
IgG (g/L) | 12.24 ± 3.19 | 10.83 ± 1.69 | |
IgG1 (g/L) | 6.85 ± 2.19 | 5.3 ± 1.28 | |
IgG2 (g/L) | 4.12 ± 1.85 | 4.12 ± 1.4 | NS |
IgG3 (g/L) | 0.81 ± 0.51 | 0.62 ± 0.27 | |
IgG4 (g/L) | 0.47 ± 0.4 | 0.43 ± 0.38 | NS |
Deficit of IgG1 (<3,82 g/L) | 2/68 (3%) | 3/34 (9%) | NS |
Deficit of IgG2 (<2,42 g/L) | 15/68 (22%) | 5/34 (15%) | NS |
Deficit of IgG3 (<0,22 g/L) | 0 | 0 | NS |
Deficit of IgG4 (<0,04 g/L) | 3/68 (4%) | 2/34 (6%) | NS |
Data are presented as means ± SD; patients with DFUs (diabetic foot ulcers;
Patients infected by resistant pathogens differed significantly from those infected by sensitive microorganisms in the percentage of basophils (0.43 ± 0.24 versus 0.66 ± 0.38 × 109/L;
The comparison of serum levels of immunoglobulins including IgG subclasses among patients with chronic DFUs infected by sensitive and resistant pathogens and diabetic controls. Data are presented as means ± SD; patients with DFUs (diabetic foot ulcers) infected by sensitive (subgroup S;
There were no significant differences in deficits of each immunological subclass between the study groups (Table
In our study, we focused on the evaluation of systemic immunity changes to determine the occurrence of immune deficiencies at the level as of innate as of adaptive immunity. We aimed to clarify whether some patients with any kind of immunodeficiency exist among those treated for nonhealing chronically infected wounds. It could help in several cases potentially explain the prolonged DFUs healing and inadequate inflammatory response to local infection. We did not assess any immune parameters or mediators at the local level since their concentrations could be modified by a variety of factors including, for example, the mild forms of peripheral arterial disease or previous applications of biological active local dressings.
The main findings in patients with chronically infected DFUs were the significantly reduced percentages and absolute numbers of lymphocytes in contrast to diabetic controls without DFUs. We could explain the lower serum levels of lymphocytes by a previously described impairment of immune system at the level of lymphoid stem cells or mesenchymal cells [
The reduced number of lymphocytes seen in our study in patients with the DFUs was associated with a decline in the absolute numbers of almost all subpopulations of lymphocytes including T-helper, cytotoxic lymphocytes, B lymphocytes and naive, memory inactive, and effector cells as of CD4+ as ofCD8+ T lymphocytes. The reason for this finding is not still clear. Similar findings have been demonstrated in experimental animal models [
Naive cells are defined by the expression of CD45RA antigen, which is present on the surface of approximately 50% of T lymphocytes. T lymphocytes expressing on their surface the CD45RO antigen are antigen activated cells (memory cells). CD45RO is usually present on the surface of approximately 40% of T cells (CD4+). During the activation of T lymphocytes (i.e., due to infection), the expression of CD45RA decreases simultaneously with the increase of the expression of CD45RO isoforms. The rise of effector cells, particularly CD8+ effector memory cells [
Other significant changes that were found in our study were alterations in the humoral arm of the adaptive immune system, in immunoglobulins levels. In addition to elevated serum levels of total IgA and IgG, patients with DFUs had in comparison to diabetic controls significant differences in IgG subclasses, specifically increased levels of IgG1 and IgG3 suggesting activation of immune system by inflammation. Both molecules serve as complement activators via binding to the protein antigen of the pathogen. Therefore, the elevation of their values is not surprising in infection [
Since a response to bacterial infection could be altered in patients with DFUs, especially in the presence of immunoglobulin deficiencies particularly in IgG subclasses [
Looking at the possible alterations of the immune system in patients infected by resistant pathogens, we did not find the marked deficits of immune system that we initially supposed in such cohort in contrast to patients infected by sensitive microorganisms. Differences were seen predominantly in humoral immunity in the patients with resistant pathogens, in whom the serum levels of individual immunoglobulins and even IgG subclasses did not reach the levels detected in patients infected by sensitive microorganisms. In fact, IgG4 values were significantly lower, less than half of those found in the subgroup with sensitive pathogens and diabetic controls. We speculate that patients with DFUs infected by resistant pathogens have a predisposition to the long persistence of these bacteria possibly due to insufficiently activated humoral immunity, which in turn leads to reduced opsonisation, activation of cell-mediated immunity, and reduced elimination of pathogens by the immune system.
Our study did not show significant alterations of the immune system at the level of innate immune system including measures of phagocytosis. No changes were found in values of C3, which is usually reduced during infection due to its consumption [
Significantly increased laboratory markers of infection in patients with infected DFUs are closely related to physiologically stimulated immune response to inflammation. This confirms the results of other studies [
We conclude that in this group of patients with infected chronic DFUs mild activation of a systemic inflammatory response and significantly reduced numbers of lymphocytes, including nearly all of their subpopulations, were found. There was no evidence, however, of their activation, especially an increase of effector cells or reduction of appropriate subpopulations indexes (naive/memory cells) as may have been expected. In patients with infected DFUs we further demonstrated abnormalities of the humoral components of immunity particularly at the level of IgG subclasses combined with a relatively high incidence of their deficits, especially of IgG2. Changes in IgG subclasses were more prevalent in patients whose DFUs were infected by resistant pathogens.
We did not include into our study patients with noninfected diabetic foot ulcers since they represent only a minority group of our complicated patients with the diabetic foot in our foot clinic which provides a comprehensive care for patients from the whole republic, other centers, transplant and immunocompromised patients, and so forth [
We recommend performing more detailed immunological investigations in patients with DFUs with recurrent or chronic infectious complications particularly caused by resistant pathogens. These tests should focus on the determination of cell-mediated and humoral immunity, concretely on lymphocyte subpopulations and IgG subclasses.
All participated authors of this study have no conflict of interests.
Great acknowledgement belongs to Jindřiška Volková, Alena Stříbrská, Zdeněk Hunal, Eva Fáberová, and Adéla Sajdlová for their practical and theoretical support during the performance of our study. This study was supported by the project (Ministry of Health, Czech Republic) for development of research organization 00023001 (IKEM, Prague, Czech Republic), institutional support.