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Frequencies of CD4+ T Regulatory Cells and their CD25high and FoxP3high Subsets Augment in Peripheral Blood of Patients with Acute and Chronic Brucellosis

Objectives Brucellosis remains one of the most common zoonotic diseases worldwide. In humans, brucellosis can be a serious, debilitating, and sometimes chronic disease. Different mechanisms can be postulated as to the basis for the induction of the chronic status of infectious diseases that T regulatory cells are one of the most important related mechanisms. The current study was designed to determine whether percentage of CD4+Treg cells and their CD25high and FoxP3high subpopulations in peripheral blood are changed in human brucellosis samples in comparison to a control group. Methods In total, 68 brucellosis patients (acute form: n = 43, chronic form: n = 25) and 36 healthy volunteers entered our study. After isolating of peripheral blood mononuclear cells, heparinized venous blood samples were obtained from both patients and healthy donors, CD4, CD25, and FoxP3 molecules were evaluated by two- and three-color flow cytometric methods. Results The results revealed a new finding in relation to Treg cells and human brucellosis. The numbers of CD4+Treg cells and their CD25high and FoxP3high subsets increase significantly in the peripheral blood of acute and chronic forms of brucellosis samples compared with healthy groups, with this increase being greater in the chronic group. Conclusion There seems to be a correlation between increase of CD4+Treg cells and their subsets and the disease progress from healthy state to acute and chronic brucellosis.


Introduction
Brucellosis is an intracellular bacterial infection with worldwide distribution with > 500,000 human cases reported annually [1,2]. The disease is caused by organisms of the genus Brucella, especially by Brucella melitensis, and is known as the most common zoonotic disease [3]. In humans, the disease culminates in high clinical morbidity and diverse clinical manifestations, as any organ can be affected [4]. Despite early diagnosis and treatment, approximately 10e30% of patients develop chronic disease [5,6].
Host protection against intracellular pathogens such as Brucella spp. depends on cell-mediated immunity (CMI) [7,8]. Brucella can survive within macrophages of the host and invades the normal mechanisms of bacterial killing, causing chronic disease [6,7]. Th1 related cytokines [interleukin (IL)-12 and interferon-g], which are fundamental for the clearance of brucellosis agent, and percentages of ex vivo and phytohemagglutinin (PHA)-cultured CD4þ T lymphocytes are found to be diminished in chronic patients [9e11].
In recent years, regulatory T cells (Tregs) have become a popular subject for immunological research. Numerous reports shed light on the major aspects of Treg biology in humans, with the characterization of different T-cell subpopulations, including naturally occurring CD4þ CD25þTregs, induced Tregs [IL-10 producing CD4þ type I regulatory T cells (Tr1) and T helper type 3 (Th3) cells], and CD4þ CD25þ T cells that develop in the periphery by conversion of CD4þ CD25e T cells. All these different T-cell populations with regulatory function coexist and contribute to immune suppression [12e14].
There is now considerable evidence that CD4þ CD25þ Tregs represent a stable population of peripheral lymphocytes. These CD4þ CD25þ Tregs represent about 5e10% of human CD4þ T cells, and are commonly identified by the constitutive expression of IL-2 receptor a (IL-2Ra; CD25), as well as the transcription factor scurfin, encoded by the forkhead family transcription factor 3 (Foxp3) gene [15], and are also characterized by their very low levels of proliferation on T-cell receptor (TCR) stimulation in vitro. The CD4þ CD25 high subset (which has high levels of CD25 expression) in normal individuals comprises about 1e3% of human circulating CD4þ T cells. Unlike the total population of CD4þ CD25þ T cells, these CD4þ CD25 high cells constitutively expressing FoxP3 molecules at high levels can significantly inhibit the proliferation and cytokine secretion induced by TCR cross-linking of CD4þ CD25À responder T cells, CD8þ T cells, dendritic cells, natural killer cells, and B cells [16e20]. However, FoxP3 is emerged transiently in CD25 low CD4þT lymphocytes, which may be without suppressive activities; but they contain effector functions [21].
The role of CD4þCD25 hi T and CD4þFoxP3 hi T cells in immunopathology of Brucella infection has not yet been elucidated [22]. So, in our study these cells were evaluated to determine two substantial questions: is the frequency of CD4þCD25 high T, CD4þFoxP3 high T, CD4þCD25 high FoxP3þ, and CD4þCD25þFoxP3 high cells increased in the peripheral blood (PB) of patients with acute or chronic brucellosis as a direct result and, if so, can these Treg cells impinge on antibacterial immune responses as an inductive result, like other infectious diseases.

Participants
There were 104 unrelated participants enrolled in the study: 68 brucellosis patients; and 36 healthy age-and gender-matched volunteers who were used as controls. According to disease history, clinical picture, and laboratory findings, patients were divided into acute (AB) and chronic (CB) brucellosis groups ( Table 1).
The AB group included 43 consecutive patients. All AB patients had a disease duration 8 weeks (mean AE standard deviation, 3.5 AE 2.3 weeks). The diagnosis was based on compatible clinical picture ( Table  2) in combination to high serum titers of anti-brucellar antibodies or fourfold increase of the initial titers in two paired samples drawn 2 weeks apart. In addition, in 19 out of 43 AB patients, Brucella melitensis was isolated in blood culture. In 33 patients brucellar DNA was detected by PCR analysis in the blood and the serum.
The CB group was composed of 25 patients. All CB patients had disease duration !6 months [23]. Thirteen of 22 CB patients had also positive PCR analysis in the blood.

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A. Bahador, et al High specific titers were defined by anti-brucellar antibodies, which were considered for the Wright agglutination test !1:320, for the Coombs' agglutination test !1:320, and for the complement fixation test !32. Healthy volunteers were tested serologically for brucellosis and found to be negative.

Flow cytometry
Surface CD4 and CD25, and intracellular FoxP3 expression were assessed by flow cytometry using the eBioscience human regulatory T cell staining kit Number 3 (eBioscience, San Diego, CA, USA). In brief, PB mononuclear cells (PBMCs) were obtained by FicolleHypaque density gradient centrifugation. Cells were incubated with FITC anti-human CD4 (RPA-T4) and PE anti-human CD25 (BC96) for 30 minutes at 4 C.Then, after twice washing and fixing with fixation/ permeabilization solutions, the cells were stained with PE-Cy5 anti-human FoxP3 (PH101), and incubated similarly. Finally, two-and three-color fluorescence flow cytometric analysis by a BD FACS Calibur system were used to demonstrate the frequency of CD4þCD25þ, CD4þ FoxP3þ and CD4þCD25þFoxP3þ cells in PB samples.

Statistical analysis
Parametric statistical tests were applied as the variables were distributed normally (KolmogoroveSmirnov test). Data were analyzed by one-way analysis of variance using SPSS version 21 (SPSS Inc., Chicago, IL, USA) and were represented as mean AE SD. A p value <0$05 was considered to be statistically significant.

Ethics statement
Our study was confirmed by the ethical committee of Tehran University of Medical Sciences, Tehran, Iran. The participants were informed about the study and they chose whether or not to participate in this study.
The proportions of CD4þCD25þ cells included 16.6% in AB and 27.6% in CB patients. Also, the CD25 high subset of these cells contained 3.9% in AB and 6.8% in CB patients (Table 3, Figures 1 and 2).
CD4þFoxP3þ cells accounted for 5.7% of CD4þTcells in AB and 10.6% of CD4þT cells in CB patients. In total CD4þlymphocytes, FoxP3 high subpopulation consisted of 1.6% and 2.9% in AB and CB, respectively.
In patients with AB, CD4þCD25þFoxP3þcells comprised 5.06% of total CD4þ lymphocytes; in CB patients, percentage of these cells was 9.5% of total CD4þ lymphocytes (Table 3).
CD4þCD25 high FoxP3þcells subtended 1.28% of CD4þ lymphocytes in AB and 2.45% in CB patients. Moreover, frequencies of CD4þCD25þFoxP3 high cells encompassed 1.41% and 2.03% of CD4þ lymphocytes in attribution of AB and CB patients, respectively (Table 3, Figure 3).

Correlation between the level of CD25 expression in CD4D lymphocytes and CD4DFoxP3Dcells
Correlation coefficients of CD4þCD25 high cells and CD4þFoxP3þ cells in the three groups consisted of R Z 0.792, p < 0.001 (healthy control), R Z 0.817,   Figure 4).

Discussion
Regulatory T cells (Treg cells) were originally identified through their function in controlling autoimmunity, where they mediate immunological tolerance to self-antigens [24]. However, it later became apparent

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A. Bahador, et al that Treg cells also control immune responses to foreign antigens on pathogens [25]. The function of Treg cells during infection appears to be primarily to prevent collateral damage from unrestrained immune responses to the pathogen, with a number of studies demonstrating enhanced immunopathology with defective or depleted Treg cells. It also seems that the induction of Treg cells during infection is an immune subversion strategy engaged by certain pathogens in order to last their survival in the host [26].
In agreement with other studies [11,27], we found that the percentage expression of IL-2 receptor a on peripheral CD4þlymphocytes in the healthy group was <10% (data not shown). Also, in the current study, > 90% of FoxP3þ cells in all three groups were CD4þFoxP3þ (data not shown), which implies that the majority of FoxP3þTreg cells in healthy individuals, AB, and CB patients manifest surface CD4 molecules.
In our study, the percentage of peripheral CD4þCD25þ(low or high) T-lymphocytes was increased in AB patients, as compared to controls, suggesting that helper T-cells are activated in AB. This finding adds further support to data presented by other researchers [11,28,29] and it approves that CD25 is upregulated in AB patients. CB patients showed significantly increased percentage of CD25 expression on peripheral CD4þCD25 high lymphocytes in comparison to AB patients. This seems to be in contrast with a previous study by Skendros et al [11], which showed diminished percentage in CD25 expression on peripheral CD4þT cells in compared with AB. But, they use PHA in the culture of obtained PBMC for activating and proliferating of lymphocytes and for this the acute brucellosis group has the higher level of CD4þCD25 low cells as CD4þT-helper cells, were activated and proliferated more than other groups. By contrast, the increase of CD4þCD25 high cells in PB of CB patients and induced suppression of T cells by this Treg cells, appears to be a substantial reason for the significant reduction in the percentage of PHA-proliferating TCD4þCD25þ cells and anergy induction in CB [30].
In addition, in our research, there were significant increases in the proportions of CD4þFoxP3þcells and their FoxP3 high subpopulation, as well as CD4þCD25þFoxP3þ cells and their CD25 high or FoxP3 high subsets in CB in comparison to AB patients   Each row was assigned to the one group, in which the left and the right diagrams depict the correlation among frequencies of CD4þFoxP3þ cells with CD4þCD25 low or CD4þCD25 high cells, respectively.

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A. Bahador, et al and healthy controls. Also, CD4þCD25 high Treg cells in the AB group showed a significant increase in frequencies compared with the control group. According to Curiel et al [31], FoxP3 expression in low levels can cause differentiation of Treg cells to Th2 or Th17 cells. So, the intensity of FoxP3 expression is crucial in the state of Treg suppressive activity. In fact, the presence of FoxP3 molecule maybe not only function as an on-and-off switch to offer suppressive activity of Treg cells but also control the regulatory capacity of these cells owing to the decreased function of Treg cells due to attenuated FoxP3 expression [32]. Consequently, our report about an increase of FoxP3þCD4þTreg subsets in AB and CB patients versus control volunteers indicates a significant rising trend in suppressive activities of the immune response in parallel with an ongoing infection such as brucellosis.
By contrast, we found the relationship between CD4þCD25 high and CD4þFoxP3þ cells displayed a stronger power of correlation coefficient than CD4þCD25 low and CD4þFoxP3þ cells in all three groups. This positive correlation verifies the direct relationship between the high-level intensity of CD25 (CD25 high ) and FoxP3 expression. Also, the AB group (R Z 0.817) showed a better correlation of CD4þCD25 high and CD4þFoxP3þ cells compared with the two other groups [R (healthy group) Z 0.792; R (CB) Z 0.661], which may be due to the larger sample size of the AB group ( Figure 4).
In conclusion, our results demonstrate that there is a marked increase in circulating CD4þ CD25þ, CD4þFoxP3þ, and CD4þCD25þFoxP3þ Tregs and their CD25 high or FoxP3 high subpopulations in AB and CB patients. Tregs play a negative role not only in modulating the effectors of immune responses by inhibiting interferon-g secretion and cellular proliferation upon Brucella antigen stimulation, but also in influencing the disease persistence in CB. These findings suggest that modulation of CD4þ CD25þ Tregs might be one potential therapeutic strategy for the treatment of the chronic form of this infection. However, this study was limited by analysis of the PB compartment only, and further detailed investigation of the level and function of CD4þ CD25þ and CD4þFoxP3þTregs in brucellosis needs to be carried out.

Conflicts of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.