Human visceral leishmaniasis is not associated with expansion or accumulation of Foxp3+ CD4 cells in blood or spleen

Abstract

Natural regulatory T cells (CD4⁺CD25⁺ Foxp3⁺), nTreg, play an important role in the regulation of inflammatory immune responses. However, the immunosuppressive properties of nTreg may unfavorably affect the host's ability to clear certain infections. In human visceral leishmanaisis (VL), reports on the frequency and function of nTreg are not conclusive. A limitation of our own previous studies that did not indicate a major role for Foxp3⁺ nTreg in VL pathogenesis was that Foxp3 was measured by mRNA expression alone, as other tools were not available at the time. We have in this study assessed CD4⁺CD25⁺Foxp3⁺ cells in splenic aspirates and peripheral blood mononuclear cells (PBMC) from an extensive series of VL patients and endemic controls (EC) by flow cytometry (FACS). The results do not show increased frequencies of Foxp3⁺ cells in VL patient pre and post treatment, neither were they elevated as compared to PBMC of endemic controls. We conclude that active VL is not associated with increased frequencies of peripheral Foxp3 Treg or accumulation at the site of infection.

Introduction

Visceral leishmaniasis (VL) is a life threatening disease characterized by uncontrolled parasitization of organs, such as spleen, liver, and bone marrow. A hallmark of VL patients is the inability of PBMC to mount curative, antigen specific immune responses, as reflected by their failure to proliferate or produce IFN-γ in response to stimulation with leishmanial antigens (1, 2). After cure, however, individuals are resistant to re-infection and generally become leishmanin skin test (LST) positive, and mount antigen specific IFN-γ responses in vitro. As VL patients mount proper responses after cure and respond normally to other antigens like PPD during active disease (2), there would not appear to be an intrinsic Leishmania driven Th1 response defect, nor a generalized failure in mounting antigen specific responses in patients with active VL. Thus other immunosuppressive mechanisms may contribute to the pathogenesis of VL.

CD4⁺CD25⁺, T cells constitute 5-10% of peripheral CD4+ T cells in humans, approximately 50-60% of these express the transcription factor Foxp3 (3) and are here referred to as natural regulatory T cells (nTreg). nTreg suppress a number of potentially tissue damaging responses in vivo, most notably T cell responses directed against self-antigens. nTreg may also suppress potentially beneficial immune responses, such as those directed against tumors and microbial pathogens.

In experimental models of cutaneous leishmaniasis (CL) nTreg cells have been proposed to promote survival of Leishmania parasites and reactivation of disease (4). Experimental infection with L. infantum in mice, indicate that nTreg accumulate in spleen and draining lymph nodes during infection (5) and intralesional nTreg have been associated with pathology of human CL (6, 7). In human VL, there is limited data on nTreg. In our previous studies we found little evidence to support a role for these cells in VL pathogenesis; their numbers were not increased in either peripheral blood nor spleen in pre-treatment as compared to post-treatment tissue, and their depletion did not reconstitute antigen-specific responses in PBMCs (8). However, a study by Saha et al. (9) indicated an increased frequency of CD4⁺CD25⁺ cells in PBMC of active VL cases, their decline with clinical cure and their re-expansion in post kala-azar dermal leishmaniasis (PKDL). CD25, which was used as a marker of nTreg in these studies, is not, however, a reliable marker of nTreg during active disease, since it can be upregulated upon activation of conventional T cells. Furthermore, Foxp3 expression, which provides a more reliable molecular signature for regulatory T cells, can be expressed by both CD25⁺ and CD25^- CD4 T cells (10). The combined expression of both CD25 and Foxp3, however, remains a valid approach for identification of most nTreg (11). In our previous study, Foxp3 expression was only assessed at the level of mRNA. In an attempt to clarify and extend our original findings, we have assessed nTreg by FACS staining of Foxp3 protein in splenic aspirates and PBMC from a large series of samples obtained from VL patients pre- and post-treatment, and in comparison to PBMC from endemic controls.

Material and Methods

Study groups

Patients presenting with symptoms at the Kala-azar Research Center, Muzaffarpur, Bihar, India, and confirmed to be VL positive by detection of amastigotes in splenic aspirates, were included in the study. Patients positive for human immunodeficiency virus (HIV) were excluded. Splenic needle aspirates were collected for diagnostic purpose before treatment (n=53) and 3-4 weeks after initiation of Amphotericin B treatment (n=30) to evaluate parasitologic cure. Aspirates were first used for preparation of smears. The remaining volume in the syringe was rinsed out with in 1ml sterile heparinized RPMI-1640 medium supplemented with 2 mM L-glutamine, 100U/ml penicillin, 100μg/ml streptomycin and 10% fetal calf serum (FCS) and collected for staining purposes.

Heparinized venous blood was obtained from VL patients prior to and post treatment and from endemic control (EC) volunteers; (VL before treatment (BT) n= 48, VL post treatment (PT) n=29, and EC n=35). EC were healthy adult household members of an active case. Clinical data for the patient and normal donors are listed in table 1. Due to difficulty with logistics, longitudinal studies were not carried out and cells from different patients were assessed before and after treatment. Blood samples were not obtained from all patients.

Table 1.

Aggregate clinical data for VL patients and ECs.

a: Post treatment samples are not from the same donor as samples collected before treatment. Clinical data is routinely collected, and is, thus, available from all patients that have been treated at the KMRC hospital

b: Mean values ± SD of aggregated data are shown, and median values are given within parenthesis.

c: Scoring of parasite load is on a logarithmic scale from 1 to 6, were 0 is no parasites per 1,000 microscopic fields (1,000×), 1 is 1–10 parasites per 1,000 fields, and 6 is >100 parasites per field

N/D, not done; N/A, not applicable.

	Before treatment	Post treatment a	Endemic Control
n	53	30	35
Age	25.1 ± 17.7 (20)b	26.5 ± 14.9 (25)	40.0 ± 11.2 (40)
Sex % (M/F)	59:41	60:40	40:60
Duration of illness (in days)	40.1 ± 38.8 (25)	25.3 ± 24.7 (20)	N/A
Infection score at Day 0c	1.5 ± 1.2 (1)	1.7 ± 1 (1)	N/A
Spleen size (cm) at Day 0	4.3 ± 3.2 (3)	4.4 ± 2.7 (4)	N/A
At Day 15	0.7 ± 1.5 (0)	0.8 ± 1.5 (0)	N/A
WBC (×103/mm3) at Day 0	4.7 ± 7.5 (3.3)	3.3 ± 1.6 (3.1)	N/D
At Day 15	10.2 ± 13.5 (7.9)	6.3 ± 2.7 (5.8)	N/D
Platelets (×105/mm3) at Day 0	1.0 ± 0.6 (1)	1.1 ± 0.4 (1.0)	N/D
At Day 15	2.4 ± 0.7 (2.4)	1.9 ± 0.7 (1.8)	N/D

The use of human subjects followed recommendations outlined in the Helsinki Declaration. Ethical approval for the study was obtained from the ethical review committee of Banaras Hindu University, Varanasi, India.

Assessment of Foxp3⁺ cells

PBMC were isolated by ficoll-hypaque (Amersham) gradient centrifugation. 1-2 ×10⁵ PBMC or splenic leukocytes were stained for expression of CD4, Foxp3, and CD25 using the “Human regulatory T cells staining kit” (eBioscience, USA) according to manufactures instructions. Antibodies were conjugated with FITC, PE and APC respectively. Isotype matched control antibodies (IgG2FITC, IgG1PE and IgG2 APC) were used to gate the negative populations. Cells were acquired on a FACSCalibur (BD Biosciences, USA) and analyzed by BD CellQuest software (BD). Analysis was performed on at least 2.5×10⁴ gated lymphocytes (figure 1a).

Figure 1.

Expression of Treg associated markers CD25 and Foxp3 in spleen and blood of VL patients. a) Schematic overview of FACS plots illustrating assement of surface marker expression. Lymphocytes were gated based on forward side scatter profile, followed by assessment of CD4+ and CD4+CD25+ on total lymphocytes and expression of FoxP3 and CD25 on gated CD4+ cells. b) Frequencies of splenic lymphocytes expressing CD4 and CD25 (left panel) and CD4⁺ lymphocytes expressing Foxp3⁺ (center panel) and CD4 lymphocytes expressing both CD25 and Foxp3 (right panel), before (circles) and after (squares) treatment. c) Frequencies of PBMC expressing CD4 and CD25 (left panel) and peripheral blood lymphocytes expressing Foxp3⁺ (center panel) and CD4 lymphocytes expressing both CD25 and Foxp3 (right panel), before (circles) and after (squares) treatment or in endemic controls (triangles).

While the clinical samples were collected on several different occasions, samples from the different groups were represented each time samples were assessed. Statistical analysis was done using PRISM 5 (GraphPad Software Inc.). For differences between multiple groups one-way ANOVA followed by Turkey's multiple comparison was performed or, in case normality test failed, Kruskal-Wallis test followed by Dunn's multiple comparison test. For comparison between two groups students t-test was used or, in case normality test failed, Mann-Whitney test.

Results & Discussion

Foxp3 expression was mainly found in the CD4 subset, and was expressed on both CD4⁺CD25⁺ and CD4⁺CD25^- cells, albeit at lower levels in the later population. Due to limitation in the number of cells available for analysis, we were not able to make the distinction between CD25^high and CD25^intermediate expression. Comparison of CD4⁺ cells pre- and post-treatment did not reveal any differences in the frequency of CD25⁺ and/or Foxp3⁺ cells in PBMC or splenic aspirate cells (figure 1b, c). If anything, the expression of Treg associated markers in PBMC tended to be lower in patients with on-going VL as compared to endemic controls (figure 1c).

Frequencies of cells (percentage of gated lymphocytes) ± standard deviation (SD) were as follows;

• Splenic CD4⁺, VL BT: 18.8±5.6, VL PT: 19±7.5; PBMC CD4⁺, VL BT: 31±10.5, VL PT; 32.6±12, EC: 33±7.6.

• Splenic CD4⁺CD25⁺, VL BT: 1.3±0.8, VL PT: 1.2±0.9; PBMC CD4⁺CD25⁺, VL BT: 1.6±0.8, VL PT: 2.0±1.0, EC: 1.4±0.7.

Percentages of gated CD4⁺ lymphocytes:

• Splenic CD25⁺, VL BT: 7.1± 4.1, VL PT 6.4±2.8; PBMC CD25⁺, VL BT: 5.4±2.8, VL PT: 6.4±2.7, EC 4.3±1.9

• Splenic Foxp3⁺, VL BT: 3.9±2,4, VL PT: 4.4±1.4; PBMC Foxp3⁺, VL BT: 2.4±1.2, VL PT: 3.2±2.0, EC: 3.7±1.7.

• Splenic CD25⁺Foxp3⁺, VL BT: 1.4±0.8, VL PT: 2.1±1.4; PBMC CD25⁺Foxp3⁺, VL BT: 0.9±0.5, VL PT: 1.0 ±0.7, EC: 1.4±1.0.

The frequencies of nTreg in spleen and PBMC, as defined by CD4⁺ cells bearing both CD25 and Foxp3 markers, is substantially lower than the frequencies of CD4⁺CD25⁺ cells in PBMC from VL patients that we previously reported (8). This may be explained by the fact that CD25 is not always co-expressed on Foxp3⁺ cells, and that the intranuclear staining of Foxp3, which involves fixation and permeabilization, may compromise CD25 staining. Furthermore, different antibodies and fluorochromes were used for detection of CD25. The results, nonetheless, are in general agreement that the frequencies of nTreg do not differ considerably between VL patients pre- and post-treatment, or between VL patients and healthy, endemic controls.

These findings, therefore, do not support the expansion or accumulation of nTreg (Foxp3⁺CD4⁺CD25⁺) cells in VL patients, either in peripheral blood or in lesional tissue. We cannot rule out that the nTreg are more activated, i.e. suppressive, and that therapeutic intervention of nTreg function could be effective in VL. Targeting other immune regulatory mechanisms, like IL-10 and/or TGFβ, is better supported by studies of human VL and by experimental data, indicating that CD25^-Foxp3^- cells may be more important in parasite persistence and pathology of leishmanial disease (8, 12, 13).