Low-dose IL-2 therapy limits the reduction in absolute numbers of peripheral lymphocytes in systemic lupus erythematosus patients with infection

Abstract

Background

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disorder characterized by disturbed cellular and humoral immune responses. Dysregulations of immune system and immunosuppressive medications predispose SLE patients to infection. This study aims to investigate the alterations and absolute concentrations of lymphocyte subpopulations in SLE patients with different infection and their responses of low-dose IL-2 therapy.

Methods

A total of 333 patients with SLE without recent infection, 162 patients suffering infection, and age and sex-matched 132 healthy controls (HCs) were recruited. Of them, 54 SLE patients (including 41 non-infected group and 13 infected group) received a 5-day course of low-dose IL-2 administration at a dose of 0.5 million IU per day. Lymphocyte subpopulations were analyzed by flow cytometry.

Results

Patients with SLE had lower levels of lymphocyte subpopulations in peripheral blood such as T, B, NK, CD4 + T, CD8+ T, Th1, Th2, Th17, and Treg cells, and the reduction in these cells was more obvious in patients with infection (p <.05 to p <.01). Low-dose IL-2 effectively expanded T (p <.001), B (p <.001), CD4 + T (p <.01), CD8 + T (p <.001), Th1 (p <.01), Th17 (p <.1), and Treg cells (p <.01) of SLE patients, these cells were comparable to that of HCs after the IL-2 treatment.

Conclusions

Patients with SLE had insufficiency of circulating lymphocyte subsets. This phenomenon was more obverse in those accompanying infection, suggesting the low concentration of lymphocytes may be used as indicators of high infection risk in SLE patients. Low-dose IL-2 induced expansion of Treg cells and NK cells, which may contribute to the restoration of immune homeostasis in SLE patients.

Keywords:

• Systemic lupus erythematosus
• infection
• interleukin 2
• lymphocyte subpopulations

Introduction

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disorder characterized by a variety of autoantibodies production that affects multiple systems in the body with a vast array of clinical manifestations¹. Despite advanced awareness of this disease and multiple autoantibodies, infections are a most common cause of morbidity and mortality in this patient population^2–4 and at least 50% of patients with SLE suffered with infections during the course of their disease⁵ and around 30% of deaths are related to infections^6,7. The deregulation of intrinsic and adaptive immune systems plays a central role in the susceptibility of patients to infections. Recent studies revealed that patients with SLE had immunologic abnormalities, especially those treated by immunosuppressive agents^8–12.

Interleukin-2 (IL-2), a cytokine mediator with bacteriolytic activity of immune system, plays an important role in the proliferation and differentiation of lymphocyte cells^13,14. One study has shown that human IL-2 is able to exhibit bacteriolytic activity¹³. IL-2 is capable of lysing Escherichia coli and Lactobacillus plantarum cells, but the mechanism of the bacteriolytic action of IL-2 still remains unknown. Based on this activity, it has entered clinical development for treating infectious diseases¹⁵. Recent studies revealed that low-dose IL-2 alleviates disease activity in SLE patients by electively modulates CD4 + T cell subsets^16–19. However, most previous studies focused on the proportion of CD4 + T cell subsets, which can be affected by levels of their subsets (such as Th1, Th2, and Th17 cells), rather than the absolute counts, and did not include large amounts of clinical data²⁰. Furthermore, the status of lymphocyte subpopulations in patients with SLE and the association of these cells and infection remain unclear, and the effect of low dose IL-2 on these subpopulations in these patients with infection and without infection is also unknown.

In this retrospective study, we mainly measured the absolute counts of peripheral blood (PB) lymphocytes and CD4 + T cell subsets of SLE patients with infection to those without infection and/or those of HCs to elucidate the immune mechanism of SLE, and further explored whether low-dose IL-2 could effectively correct the immunologic abnormalities in SLE, especially infected patients.

Methods

Recruitment of participants

Between July 2014 and December 2016, 495 patients with SLE (333 patients without recent infection, 162 patients suffering infection) from the inpatient population of the Second Hospital of Shanxi Medical College were enrolled in the study. All patients fulfilled the 1997 SLE revised classification criteria of the American College of Rheumatology²¹. Age- and sex-matched healthy individuals (n = 132) were recruited from the physical examination center of the Second Hospital. The history, physical examination, and auxiliary examination were based to determine whether infection is bacterial or viral. The presence of an infection was confirmed by a positive pathogen test from various specimens (blood, sputum, pus, stool, and urine) or clear evidence of infection, such as an abscess on computed tomography. Patients who had fever (body temperature over 38.0 °C) for at least 3 days not caused by disease activity, and effectively reversed by anti-infective treatment, were also considered as infected. All of these patients who met the criteria for infection were enrolled in the study. All patients were on prednisone and/or other immunosuppressive medications to control disease activity (Table 1).

Table 1. Comparison of baseline characteristics between the non-infected group and infected group in patients with SLE.

Characteristics	Non-infected (n = 333)	Infected (n = 162)	p value
Age, years, mean (SD)	35.9 (14.0)	39.0 (15.8)	.025
Female, n (%)	302 (90.1)	144 (18)	.525
Duration of SLE, years, median (range)	3.0 (0.02–34.0)	4.0 (0.03–39.9)	.098
WBC, ×10^9/L, mean (SD)	5.49 (2.98)	5.34 (3.42)	.611
Platelet, ×10^12/L, mean (SD)	188 (82)	158 (94)	.001
Lymphocyte, ×10^9/L, mean (SD)	1.35 (0.69)	1.06 (0.66)	.601
Neutrophil, ×10^9/L, mean (SD)	3.80 (2.84)	3.95 (3.06)	<.001
Prednisone dose, mg/day, median (range)	30 (0, 60)	40 (0, 60)	.008
Use of concomitant agents (no. of patients)
Hydroxychloroquine	251	112	—
Methotrexate	38	11	—
Leflunomide	73	24	—
Cyclophsphamide	158	67	—
Mycophenolate mofetil	69	33	—

Low-dose IL-2 (recombinant human interleukin-2, Beijing, China) was administered in 54 patients (including 41 in the non-infected group and 13 in the infected group) at a dose of 0.5 million IU per day for 5 consecutive days subcutaneously. The IL-2 was approved by the State Food and Drug Administration of China and had comparable bioactivity to Proleukin (aldesleukin). The exclusion criteria included an IL-2 allergy or intolerance; any other autoimmune disease; a malignant tumor; any heart, kidney, or liver dysfunction. All subjects recruited for this study provided written the prior informed consent and agreed to be assessed for absolute number of lymphocyte subsets. This study was approved by the Ethics Committee of the Second Hospital of Shanxi Medical College (Taiyuan, China).

Flow cytometric analysis

Materials

Phorbol myristate acetate (PMA), Anti-human FoxP3-PE and IL-17-PE, Permeabilization Buffer 10× Fixation/Permeabilization Diluent, and Fixation/Permeabilization Concentrate were from Invitrogen, eBioscience, Affymetrix Inc. (by Thermo Fisher Scientific, San Diego, CA). GolgiStop was from BD Biosciences Pharmingen (San Diego, CA). Monoclonal antibodies including CD3FITC/CD8PE/CD45PercP/CD4APC, CD3FITC/CD16 + 56-PE/CD45 PercP/CD19APC, CD4-FITC, IL-4PE, IFN-γ-APC, CD25-APC, and Trucount tube (contain a set number of beads) are from Becton-Dickinson (BD; Franklin Lakes, NJ, USA).

Absolute lymphocyte count

Briefly, the absolute number of T cell subsets (cell/µl) were established from fresh blood samples using reference beads in a Trucount tube from BD as an internal standard for cell concentration. This BD Trucount Tube with beads is ready to use and avoid variation produced by adding standard beads one by one.

Analysis of lymphocyte surface and intracellular markers

The total absolute number of CD4 + T cells was assessed by flow cytometry (FACS Calibur, BD) according to our modified stain and then lyse and wash protocol in manufacturers’ directions of BD Trucount TM tubes. Briefly, 50 μl EDTA-anticoagulated venous blood was added into Trucount tubes A and B separately by reverse pipetting and stained by 20 μl antiCD3-FITC/CD8-PE/CD45-PercP/CD4-APC antibodies in tube A and 20 μl CD3-FITC/CD16 + 56-PE/CD45-PercP/CD19-APC antibodies in tube B (do not touch the blood). Then cells were mixed with 450 μl 1X FACS. Fifteen thousand cells were acquired and detected by MultiSET software within 24 h. The absolute number of T, B, CD8 + T, and NK cells could also be counted in the same way.

Analysis of Th1/Th2/Th17 cells

Cells in 80 μl heparin-anticoagulated venous blood were stimulated by 10 μl PMA, 10 μl Ionomycin (final concentration was 750 ng/ml), and 1 μl GolgiStop in 37 °C for 5 h and then divided into two tubes (tube A and tube B) followed by staining with human anti-CD4-FITC antibodies at room temperature away from light for 30 min. One milliliter fresh Fixation/Permeabilization was used to fix and permeabilize cells and then they were stained by IL-4-PE and IFN-γ-APC in tube A and human anti-IL-17-PE in tube B. Cells were washed with PBS and analyzed using flow cytometry.

Analysis of CD4Treg cells

Cells in 80 μl of heparin-anticoagulated vein blood were surface-labeled with anti-CD4-FITC and anti-CD25-APC and fixed and permeabilized by 1 ml fresh Fixation/Permeabilization followed by staining with human anti-FOXP3-PE.

Statistical analyses

All statistical analyses were performed with SPSS 22.0 software (IBM, Armonk, NY, USA). The categorical demographic characteristics of the patients were compared with the use of the χ² test. Continuous data that satisfy the homogeneity of normality and variance are presented as mean (±SD). An independent Student’s t-test was used for comparisons between two groups, and one-way analysis of variance (ANOVA) was used for comparisons among three or more groups. Paired-sample t-test was used for paired comparison of differences in immunological features between values at baseline and those after IL-2. Data that dose note satisfy the homogeneity of normality or variance are presented as median (range) and compared by Mann–Whitney U test. A p value of less than .05 was considered statistically significant.

Results

The baseline demographic and clinical characteristics of the patients

Of a total of 495 SLE patients, 302 (60.1%) were female and 162 (32.7%) were suffering from infection. As shown in Table 1, there was no significant difference in gender between the patients with and without infection (p >.05). The patients with infection were older than those without infection (p <.05). The average duration of disease in the infected patients was longer than that of the non-infected, but no significant difference was observed (p =.098). All the patients received conventional steroids and immunosuppressive therapies and a higher dosage of prednisone was used in the infection groups (p <.01). Compared with the non-infected SLE, infected patients had a lower level of platelets (p <.01), but a higher proportion of neutrophils (p <.001).

Regarding localization of infection, the respiratory tract was most frequently involved (its infection was found in 71.6% patients), followed by the gastrointestinal (18.5%) and urinary tract (13.0%). Concerning pathogens, bacterial infection was more common (59.9%); viral (10.5%) including Epsteim-Barr virus (6.8%), cytomegalovirus (3.7%), and respiratory syncytial virus (0.6%); 33 patients had unknown pathogens (20.4%) (Table 2).

Table 2. Localization and cause of infection (n = 162).

	N	%
Localization
Respiratory	116	71.6
Gastrointestinal	30	18.5
Urinary tract	21	13
Others	18	11.1
Pathogens
Bacteria	97	59.9
Fungus	20	12.3
Virus	17	10.5
Unknown	33	20.4

The absolute numbers of T and CD4 + T cells in the non-infected group were significantly lower than that of HCs (p <.001), but still dramatically higher than that in infected patients (p <.001). The levels of B, CD8 + T, and NK cells in the infected patients were significantly decreased when compared with that of both non-infected patients and healthy controls (p <.01), while there was no significant difference in these cells between healthy controls and the non-infected patients (p >.05) (Figure 1a).

Figure 1. Multiple subpopulations of lymphocytes decreased in peripheral blood of SLE patients with infection. Absolute numbers of peripheral lymphocytes subpopulations were analyzed by flow cytometry. Data were presented as mean ± SD and statistical analysis was determined by one-way ANOVA. *p <.05, **p <.01, ***p <.001. (a) SLE patients (n = 495) have lower levels of T, B, NK, CD4 + T, and CD8 + T cells in PB compared with healthy controls. The numbers of PB lymphocytes in the infected groups (n = 162) was also much lower than those in non-infected patients (n = 333). (b) Comparison of numbers of CD4 + T cell subsets among different groups. Patients with SLE had lower levels of Th1 cells, Th2 cells, Th17 cells, as well as Treg cells compared with healthy donors (n = 132), especially the infected groups.

As for CD4 + T cell subsets, the numbers of Th1 and Th2 cells in the infected group were lower than that of non-infected patients and healthy controls (p <. 001), though there was no significant difference in these cells between the non-infected patients and HCs (p >.05). The non-infected patients had a higher level of Th17 cells than that of HCs (p <.05), but the absolute numbers of Th17 cells in infected patients was the lowest among the three groups (p <.05). The number of Treg cells in SLE patients was significantly lower than that of HCs (p <.01), and the infected patients had the fewest Treg cells among all these group (p <.05) (Figure 1b).

Low-dose IL-2 corrected the decreased lymphocyte subpopulations in SLE patients

Fifty-four patients with SLE with or without infection received the treatment of IL-2 at 0.5 million IU per day for 5 days subcutaneously (Supplementary Table S1). Of note, low-dose IL-2 treatment also raised the absolute numbers of these lymphocyte subpopulations in the patients with infection as efficiently as those without infection (Table 3). After the treatment, compared with the baseline, there was a significant increase in the number of studied lymphocyte subpopulations except CD4 + T cells: T (p <.001), B (p <.001), CD8 + T (p <.01), NK (p <.001), Th1 (p <.01), Th17 (p <.1), and Treg cells (p <.01) (Figure 2 and Table 4). CD4 + T cells in SLE patients were still lower than that of healthy subjects (p <.001). Furthermore, low-dose IL-2 raised the cell numbers of these subpopulations in SLE patients to a comparable level of those in healthy controls and an even higher level of Treg cells was detected in SLE as compared to healthy donors (p <.001) (Figure 3 and Table 5).

Figure 2. Multiple subpopulations of peripheral lymphocytes of SLE patients were significantly upregulated after low-dose IL-2 treatment (n = 54). Absolute numbers of lymphocyte subpopulations in peripheral blood were analyzed by flow cytometry. (a–i) Changes in the numbers of T, B, NK, CD4 + T, CD8 + T, Th1, Th2, Th17, and Treg cells. Comparison between amounts of cells at baseline and after the treatment. Data were presented as mean ± SD and were calculated and compared by paired-sample t-test. *p <.05, **p <.01, ***p <.001.

Figure 3. Lymphocyte subpopulations in patients with SLE (n = 54) were upregulated by IL-2 treatment and got to levels near healthy controls (n = 132). Absolute numbers of lymphocyte subpopulations in peripheral blood were analyzed by flow cytometry. Data were presented as mean ± SD and statistical analysis was determined by one-way ANOVA. *p <.05, ***p <.001. (a) SLE patients had a comparable level of T, B, CD4 + T, and CD8 + T cells in PB to healthy controls. (b) Comparison of the number of CD4 + T cell subsets among different groups. Patients with SLE had a comparable number of Th1, Th2, and Th17, and even higher Treg cells than healthy donors, especially the infected groups.

Table 3. Comparison of the number of PB lymphocyte subpopulations in patients with SLE before and after IL-2 treatment (n = 54).

	Without infection (n = 41)			With infection (n = 13)
	Before	After	p value	Before	After	p value
T, cells/μl, mean (SD)	911.4 (508.1)	1,334.6 (760.4)	<.001	493.9 (349.2)	760.7 (545.1)	.042
B, cells/μl, mean (SD)	160.8 (134.5)	341.6 (324.5)	<.001	110.0 (120.9)	182.9 (267.1)	.207
CD4 + T, cells/μl, mean (SD)	454.8 (274.9)	657.8 (368)	.003	228.6 (219.2)	327.5 (224.6)	.097
CD8 + T, cells/μl, mean (SD)	419.3 (234.5)	618.7 (416.1)	<.001	242.3 (144.3)	406.4 (322.4)	.044
NK, cells/μl, mean (SD)	94.3 (91.9)	115.4 (126.1)	.199	50.3 (31.5)	67.7 (45.1)	.205
Th1, cells/μl, mean (SD)	51.7 (44)	86.9 (87.5)	.007	34.9 (30.0)	54.2 (43.4)	.120
Th2, cells/μl, mean (SD)	10.2 (8.7)	13.2 (11.7)	.144	5.4 (6.7)	8.1 (10.2)	.424
Th17, cells/μl, mean (SD)	8.5 (6.6)	15.3 (21.4)	.050	4.0 (3.8)	7.8 (5.9)	.041
Treg, cells/μl, mean (SD)	28.0 (33.0)	80.0 (50.0)	<.001	9.1 (7.8)	34.8 (32.1)	.005

The lymphocyte subpopulations of the patients were compared respectably. p value <.05 was considered statistically significant.

Abbreviation: PB, peripheral blood.

Table 4. Comparison of peripheral lymphocyte subsets in patients with SLE before and after IL-2 treatment (mean ± SD).

Characteristics, cells/μl	Before (n = 54)	After (n = 54)	t	p
T	810.907 ± 504.871	1,196.463 ± 751.697	4.539	<.001
B	148.565 ± 132.061	303.366 ± 316.717	3.992	<.001
CD4 + T	400.318 ± 278.241	578.319 ± 366.027	3.564	.001
CD8 + T	376.683 ± 228.111	567.607 ± 403.275	4.807	<.001
NK	83.694 ± 83.409	103.908 ± 113.545	1.603	.115
Th1	47.668 ± 41.421	79.066 ± 80.022	3.195	.002
Th2	9.042 ± 8.435	11.946 ± 11.484	1.720	.091
Th17	7.435 ± 6.321	13.527 ± 19.071	2.357	.022
Treg	13.832 ± 19.141	69.098 ± 50.018	8.940	<.001

Table 5. Comparison of peripheral lymphocyte subsets in patients with SLE after IL-2 treatment and HCs (mean ± SD).

Characteristics, cells/μl	HCs (n = 132)	After (n = 54)	t	p
T	1,335.345 ± 332.566	1,196.463 ± 751.697	1.306	.196
B	205.252 ± 81.324	303.366 ± 316.717	‒2.246	.029
CD4 + T	712.159 ± 203.548	578.319 ± 366.027	2.532	.014
CD8 + T	485.889 ± 199.919	567.607 ± 403.275	‒1.419	.161
NK	297.961 ± 134.334	103.908 ± 113.545	10.015	<.001
Th1	96.367 ± 69.859	79.066 ± 80.022	1.468	.144
Th2	10.588 ± 5.995	11.946 ± 11.484	‒.824	.413
Th17	7.645 ± 4.052	13.527 ± 19.071	‒2.246	.029
Treg	34.133 ± 12.663	69.098 ± 50.018	‒5.071	<.001

In addition, some of the patients received blood routine and inflammatory indicators both before and after the treatments. Paired-sample t-test suggested a decrease in erythrocyte sedimentation rate (p <.001) and an increase in blood routine measures such as platelets (p <.05), WBC, and lymphocyte neutrophils (p <.001) after the treatment (Table 6).

Table 6. Comparison of baseline characteristics in patients with SLE before and after IL-2 treatment.

Characteristics	Before	After	p value
Blood routine (n = 48)
WBC, ×10^9/L, mean (SD)	4.08 (2.70)	8.45 (4.47)	<.001
Platelet, ×10^12/L, mean (SD)	158 (85)	191 (90)	.017
Lymphocyte, ×10^9/L, mean (SD)	1.12 (.63)	2.09 (1.18)	<.001
Neutrophil, ×10^9/L, mean (SD)	3.49 (2.59)	5.87 (3.90)	<.001
Complement (n = 18)
C3, mean (SD)	.51 (.20)	.55 (.23)	.373
C4, mean (SD)	.10 (.07)	.11 (.13)	.558
ESR, mm/h, mean (SD) (n = 42)	45.69 (36.78)	24.24 (23.85)	<.001
CRP, mg/dL, mean (SD) (n = 20)	10.18 (16.56)	8.14 (10.35)	.584

Abbreviations: ESR, erythrocyte sedimentation rate; CRP, C-reaction protein.

Discussion

Infection remains a major cause of mortality and morbidity in SLE^2,6,22. Epidemiological studies found the hospitalization rate of SLE patients for infections was 11–23%^23–25, which was 12-times higher than that of patients without SLE²⁶. Our result showed that the total incidence of infection in SLE (32.7%) was not very different from previous reports (11–45%)^6,23,27–32. Our patients had a high dose of Prednisone (30 mg/Day median). Considering that, this study was only a short-term course of treatment for patients with newly-diagnosed SLE, and 5-days observation is not sufficient to adjust glucocorticoid dose. During the course of diagnosis and treatment, the dose of prednisone depends on the patient’s condition. At each visit, the investigators assessed patients to determine whether it was necessary to adjust the dose of glucocorticoids. Some viruses, bacteria, and protozoa were revealed to cause immune dysfunction in a variety of ways³³. Our data confirm respiratory infection and bacteria pathogens were the most common in SLE patients with infection, which were consistent with previous studies^6,23,34.

The possible cause of the infection accounts for the abnormality in immune system by disturbed lymphocytes and its signaling^1,12,35–37. Our results revealed that patients with SLE had a lower absolute number of T cells, B cells, NK cells, CD4 + T cells, CD8 + T cells, Th1 cells, Th2 cells, Th17, or Treg cells in PB, and these decreases were more obvious in patients suffering infection. The decrease in these cells suggested disturbances of both innate and adaptive immune systems and thereby more vulnerability to infection. Wu et al.³⁸ reported a lower level of CD4 + T cells but a greater percentage of CD8 + T cell levels in SLE patients with infection, however, the increased CD8 + T cell percentage did not mean a higher level of these cells; as a matter of fact, the absolute number of all these lymphocyte cells are lower in SLE patients with infection. Infected SLE patients had a lower level of platelets. Thrombocytopenia is the most common manifestation of blood system damage in SLE³⁹, which is associated with the presence of anti-platelet antibodies, anti-phospholipid antibodies in serum, and impaired bone marrow megakaryocyte maturation⁴⁰. The level of platelet <100 × 10⁹/L at least once is considered thrombocytopenia^41,42. Therefore, physicians should carefully consider the possible effects of low absolute numbers of lymphocyte subpopulations on infection. The low absolute number of these cells may be more susceptible to infection in SLE patients.

IL-2 is known as a T cell growth factor, playing a central role in maintaining proper function of the immune system of the host⁴³. Though concanavalin A-generated blast cells in SLE patients responded normally to exogenous IL-2, the ability of lupus lymphocyte cells to produce IL-2 significantly decreased⁴⁴. This inability of producing adequate amounts of IL-2 may account for the increased rates of infections. Recently, IL-2 treatment represents a novel strategy to control immune tolerance by influencing functions and survival of Treg cells and to control inflammation and autoimmune diseases^45,46. The IL-2 pathway is an attractive target for the treatment in acute and chronic phases of Chagas’ disease, dampening T cell activation through the expansion/maintenance of Treg cells and regulation of IL-17 production⁴⁷. IL-2 therapy has been used to stimulate an immune response to increase T cell numbers in vivo, particularly in treating cancers and human immunodeficiency virus-infected patients^48,49. In this study, we observed an increase in various lymphocyte subsets, most of them comparable with that of HCs, suggesting a potential restoration of abnormal immune function and responses in patients with SLE, which may not only benefit the alleviation of disease activity by the increase of Treg cells¹⁶, but also help in enhancing the potential of anti-infection.

Besides expansion of regulatory T cells, low-dose IL-2 may also sustain cellular immunity with enhanced natural killer cells¹⁸. In our study, though total CD4+ T cells remained unchanged after IL-2 injection, low-dose IL-2 therapy induced a significant expansion of Treg cells and CD8+ T cells. Some findings suggest that sequential IL-2 treatment can restore Hepatitis B virus-specific CD8+ T cell responses, showing efficacy in rescuing immune function in non-responder patients with refractory chronic hepatitis B⁵⁰.

IL-2 treatment may enhance virus-specific CD8+ T cell responses and promote the activity of NK cells against infections^51,52. Similar to our clinical observation, He etc.¹⁸ observed significantly increased expression of IFNγ and NKp46 by NK cells in response to low-dose IL-2 treatment, which implicated potential augmentation of anti-infectious cellular immunity. In fact, NK cells are important in protection against various infections^51,52. Infection is a major cause of relapse, hospitalization, and death in patients with SLE^23,53, and that low-dose IL-2 might increase anti-infectious immune response^54,55 in agreement with previous studies¹⁶. Whether low-dose IL-2 treatment could decrease viral and bacterial loads in infected patients should be carried out in the future.

IL-2 is a regulatory protein that plays a role in oncological and infectious diseases. It is reported that IL-2 had the properties of a bacteriolytic agent via the mechanism of bacteriolytic enzyme¹³. In current medical practice, IL-2 is used as a regulator of the immune system but not as a bacteriolytic factor, since its bacteriolytic properties had not been previously known. However, it is possible that antimicrobial properties also play an important role in some cases when the effectiveness of IL-2 is confirmed. IL-2 is used both in the case of sepsis, where the role of bacteria is obvious, and in the treatment of cancer, where the role of bacteria is less obvious but there may be a combination of bacterial tissue damage and the underlying disease^13,56,57. In a randomized, double-blind, placebo-controlled trial of Ld-IL2 therapy in SLE, no serious infections were observed in the IL-2 group compared with the control group^16,58. Zhou et al.⁵⁸ showed that Ld-IL2 therapy reduced the risk of infections in SLE patients and enhanced the control of viral infection. Saadoun et al.⁵⁴ reported an in vivo expansion of potent suppressive Tregs in response to low-dose IL-2 immunotherapy in patients with autoimmune-related diseases in the absence of concomitant use of glucocorticoids or immunosuppressive treatments. Besides, low-dose IL-2 was associated with clinical improvement in patients of mixed cryoglobulinemic vasculitis associated with HCV infection and had a modest effect on viral load. These results give a new understanding of the biological functions of IL-2. The mechanism of bacteriolytic action of IL-2 has not yet been established and the mechanism of action of effectors on IL-2 activity also requires further investigation.

This study provides supportive data to confirm the therapeutic effects of low-dose IL-2 in SLE treatment. However, some limitations of this study must be acknowledged. First, several descriptions such as measures of disease activity and damage were not assessed. Furthermore, detailed comparison of lymphocyte subsets in different etiologic agents and locations was not presented due to the unmatched samples. Finally, only 5-days observation is not enough to estimate the direct effect of IL-2 treatment on infection and whether IL-2 therapy could reduce infection rates will be studied further.

Safety

Low-dose IL-2 was well tolerated in SLE patients. None of them displayed severe adverse effects. Non-severe adverse events were characterized by skin rashes at the injection site that resolved spontaneously without special intervention.

Conclusions

Patients with SLE had a disturbance in immune system by decreased number of various lymphocyte subsets, especially those suffering infections. This preliminary finding suggests that the low absolute numbers of these cells may be used as indicators of possible infection in SLE patients and low-dose IL-2 may enhance the ability to resistant infection in SLE patients by restoring the decreased number of lymphocyte subpopulations. Further studies are needed.

Transparency

Declaration of funding

This project was supported by the National Natural Science Foundation of China (No. 82001740) and Graduate Students Outstanding Innovation Project Foundation of Shanxi Province (2C592020079). The funding body had a role in the design of the study and collection, analysis, and interpretation of data.

Declaration of financial/other relationships

The authors declare that there is no conflict of interest. Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Author contributions

Study design and manuscript writing: JZ and SZ. Data extraction, quality assessment, analysis, and interpretation of data: JW. All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Xiao-Feng Li had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Ethical approval

This study was approved by the Ethics Committee of the Second Hospital of Shanxi Medical University (2016 KY-007).

Acknowledgements

Some of this work was presented as a poster in the International Journal of Rheumatic Diseases and published as an abstract.

Data availability statement

All data generated or analyzed during this study are included in this published article.