Serum levels of CC chemokine ligands in cutaneous leishmaniasis patients

Abstract

The crucial functions of chemokine/receptors in numerous parasitic infections, including leishmaniasis, are well documented. This study aimed to assess the serum levels of CC ligand (CCL) 2, CCL5, and CCL11 in cutaneous leishmaniasis (CL) patients. 64 patients, suffering from CL and 100 healthy people were selected, and their blood serum concentrations of CCL2, CCL5, and CCL11 were measured using enzyme-linked immunosorbent assay. The results demonstrated that while the mean serum levels of CCL5 and CCL11 increased significantly in CL patients, the mean serum levels of CCL2 decreased, compared to the control group. Despite the sufficient production of CCL5 and CCL11 in CL patients, they suffered from CCL2 deficiency, as the defense mechanism against parasitic infection. These findings suggest a mechanism that might partially explain the patients’ susceptibility to persistent infection and their inability to clear the parasites.

Introduction

Leishmaniasis is one of the most important vector-borne diseases and occurs as a result of infection with several Leishmania species (Boutsini et al. 2018; Sofizadeh et al. 2018). This disease has three main types, including cutaneous, mucocutaneous, and visceral (Nieto-Meneses et al. 2018). In the mucocutaneous form, parasites damage mucosal membranes (often nasal mucosal), resulting in disfiguring lesions often in the nose, while in the visceral form, which is a life-threatening disease, internal organs, such as the liver, spleen, and bone marrow, involve (Kedzierski and Evans 2018; Nieto-Meneses et al. 2018). Leishmania species are obligate intracellular parasites (Rabhi et al. 2016). To develop the successful infection of leishmaniasis, the parasite must evade the innate and adaptive immune responses (Kedzierski and Evans 2018). The most well-defined host cell receptors for the recognition of Leishmania species are FcRs, scavenger receptor, CR3, MR, and some TLRs (Polando et al. 2013). Initial interactions between the host’s innate immune cells and the Leishmania parasites affect the subsequent adaptive immune response and the extent of pathogenesis (Banerjee et al. 2016).

Chemokines are a group of small proteins and immune system members recruit immunoreactive cells. Four distinct categories of chemokines involve in leishmaniasis development. Chemokines are subdivided according to the position of cysteine motifs in the N-terminus of their biochemical structure as CXC, CX3C, CC, and C classes (Aminzadeh et al. 2012; Hassanshahi et al. 2007b). All chemokines have an “L” suffix to indicate that they are ligands, followed by a number. CCL1, CCL2, and CXCL12 can be exemplified as chemokine ligands (Vinader and Afarinkia 2012). These chemotactic cytokines are essential for the recruitment of white blood cells (lymphocytes) and tissues and are involved in regulating cellular adhesion and transendothelial migration (Aminzadeh et al. 2012; Hassanshahi et al. 2007b). The trafficking of leukocytes and immune cells throughout the secondary lymphoid tissues is finely tuned by these chemotactic mediators (Hughes and Nibbs 2018). Although the role of cyto/chemokine network in the pathogenesis of CL and DCL has been yet to be fully understood, previous studies reported more negative results of skin test against leishmanial antigens with lower IFN-γ and TNF-α in DCL, compared to those patients affected by CL (Guerin et al. 2002). Unconventional T cells (γδ T cells) are able to recognize a broad range of infected or transformed cells. When an infected or a transformed cell is recognized, γδ T cells generate various cytokines, such as IFN-γ and TNF-α, and various chemokines, such as CCL5, CXCL10, and lymphotactin, and also induce the cytolysis of infected or transformed target cells by the use of various means, such as perforin, granzymes, and TNF-related apoptosis-inducing ligand (TRAIL) (Keselowsky et al. 2020). As both TNF-α and IFN-γ are the upstream activators of chemokine production (Ahmadi et al. 2013), the reduction of T cell responses is proposed as a risk factor for the abnormal chemokine production and occurrence of parasite dissemination. It has been well known that chemokines link innate immunity to adaptive immunity and recruit tissues inflammatory cells; however, this role is poorly understood in the case of CL. For instance, CCL2 and CCL3 have been found as anti-leishmanial agents (Teixeira et al. 2006), and it has been reported that the tissue expression of CCL2 is decreased in DCL (Ritter and Körner 2002). Also, it has been demonstrated that CCL chemokines contribute to the development of skin ulcers and nodules. Skin ulcer healing is composed of four distinct but overlapping stages including hemostasis, inflammation, proliferation, and remodeling. Chemokines have a critical role in regulating the sequence of these stages and recall of inflammatory cells, which produce various cytokines and growth factors to boost ulcer healing (Ridiandries et al. 2018).

Although many studies have been conducted to address the role of chemokines in leishmaniasis, few studies examined the serum levels of CCL chemokines in CL. Due to the crucial roles of CC chemokines subfamily, especially chemokines involved in the recruitment of eosinophils, which are key cells against parasitic infections, the present study aimed to examine the serum levels of CCL2, CCL5, and CCL11, as the recruiters of eosinophils.

Materials and methods

Materials

Sera were obtained from 64 patients (32 males and 32 females), suffering from CL and 100 healthy subjects as the control. Human CCL2, CCL5, and CCL11 ELISA kits were obtained from R&D Systems, Minneapolis, MN (USA). All other chemicals were of analytical grade. Distilled water was used throughout the study.

Study subjects

In the present cross-sectional study, serum CC chemokines (CCL2, CCL5, and CCL11) were measured in 64 patients, including 32 males and 32 females and 100 healthy subjects as the control group, with CL. Forty two out of 64 patients suffered from dry CL (18 males, 24 females), while others suffered from wet CL (14 males, 8 females). Also, 56 (27 males, 29 females) were Iranian, while others (5 males, 3 females) had Afghani nationality. The disease was diagnosed with an expert parasitologist. The laboratory examinations and findings of amastigotes in the samples confirmed the active form of CL. Two major diagnostic criteria were a) the isolation of Leishmania parasites (Leishmania body) and b) the positivity of the Leishmania skin test. The patients were infected with Leishmania major and Leishmania tropica. Almost all the patients were from a highly endemic area for leishmaniasis in Iran. Also, all of them had typical ulcerative lesions with raised edges. They suffered from active CL for 45–80 days without any other dermatological diseases and were HIV negative. All the control participants were healthy and non-affected. The participants filled a consent form out before taking part in the study. Also, a legally authorized representative of participants was provided as an illiterate population is also involved in the study. The methods and protocols of this study were in accordance with the relevant institutional regulations and were approved by the local Ethical and Research Committees of Yazd and Rafsanjan Universities of Medical Sciences.

Measurement of serum chemokines

Peripheral blood samples were collected from each participant via venous puncture. Sera were isolated and kept at − 20 °C for further use. Hemolyzed samples were removed from the study. The serum concentration of CCL2, CCL5, and CCL11 was measured using the ELISA technique according to the manufacturer’s instructions. The sensitivity of ELISA kits was 2 pg/mL, and inter and intra-assay assessments for their reliability were conducted.

Statistical analysis

The data were analyzed using student’s t test and SPSS software version 18. Differences were considered significant if P < 0.05.

Results

The mean serum levels of CCL2 in CL patients and controls were 298.84 ± 5.97 pg/mL and 320.45 ± 7.93 pg/mL, respectively (Fig. 1a). These values for CCL5 were 1201.10 ± 50.19 pg/mL and 170.04 ± 8.78 pg/mL in patients and control groups, respectively, and the differences were statistically significant (Fig. 1b). Also, the mean serum levels of CCL11 were 189.98 ± 3.53 pg/mL in the patients and 143.25 ± 3.59 pg/mL in the control group (Fig. 1c). The mean serum levels of CCL2, CCL5, and CCL11 in the dry type of leishmaniasis were 300.32 ± 42.8, 1221.8 ± 403.2, and 187.75 ± 28.62 pg/mL, respectively, while these values in the wet type of leishmaniasis were 327.49 ± 17.27, 1112.29 ± 58.35, and 189.27 ± 9.28 pg/mL, respectively, indicating that there was no significant difference between wet and dry types of leishmaniasis. In addition, there was no difference in the chemokine expression profile between Iranian and Afghani patients (Table 1).

Fig. 1.

Serum levels of a CCL2, b CCL5, and c CCL11 chemokines in CL patients and healthy controls. As the figure shows, the concentration of CCL5 and CCL11 increased in CL patients, while the CCL2 concentration decreased in CL patients

Table 1.

Demographic and clinical findings of the patients and controls

Variable	Control	Patient
Age (year)	40 ± 12	45 ± 9.5
Gender
Female	25	32
Male	25	32
Nationality
Iranian	25	55
Afghani	25	9
Education
Literated	30	52
Illiterated	20	12
Location of the lesion
Foot	Nil	10
Face	Nil	20
Hand	Nil	17
Hands and foot	Nil	5
Hands and face	Nil	7
Face and foot	Nil	2
Type of lesion
Cutaneous wet leishmania	Nil	22
Cutaneous dry leishmania	Nil	42
Number of lesions
3–5	Nil	30
2–4	Nil	34

Discussion

Leishmaniasis is a complex set of disorders with important clinical and epidemiological diversity (Desjeux 2004). The current study aimed to analyze the serum concentrations of different CC chemokines in patients with CL. The findings revealed that CCL5 and CCL11 serum levels increased significantly with leishmaniasis, while there was no statistical relationship between serum CCL2 levels and leishmaniasis. The results also showed that the serum chemokine concentrations were not affected by the gender and nationality of patients. According to the results, it can be concluded that the chemokines might play important roles in the pathology of leishmaniasis.

CCL2 is an angiogenic chemokine inherently, and its balanced levels might contribute to the process of wound and scar healing. Most of the efficacious functions, such as an increase in TNFα expression in response to extracellular stimulators are regulated by MAPK (Arababadi et al. 2011; Gschwandtner et al. 2019; Hassanshahi et al. 2007a). The parasite-triggered reciprocal MAPK signaling via ERK1/2 and p38 MAPK governs the counteracting effect of the host immune responses (Mathur et al. 2004). In agreement with this report, other researchers demonstrated that CL, in both humans and mice, is associated with rapid changes in chemokine expression (Navas et al. 2014; Teixeira et al. 2006). This might propose the possible involvement of these chemokines in the disease etiology and pathogenesis.

The strongly enhanced levels of CCL11 observed in leishmania infected patients in the present study might confirm its potential involvement role in leishmaniasis prevention and consider as a therapy candidate for immunotherapy, due to its role in the recruitment and activation of Th1 responses (Salomon et al. 2002; Wildbaum et al. 2002). Another mechanism, which might explain the elevated serum concentration of CCL11, is its role in response to medical therapy and allergic reactions as eosinophils are important cells in response to food and drug allergies as well as anti-parasitic immune responses (Radman et al. 2013).

The results of the present study showed that the leishmaniasis patients were unable to produce sufficient serum concentration of CCL2 in leishmaniasis. This might be a pathogenesis mechanism, explaining that the patients were not able to eradicate the parasites. Cellular immunity is the main constituent of the immune system against leishmaniasis. The patients with chronic CL are also unable to eradicate the parasite completely. It seems that some patients in the chronic phase of the disease have incompatible levels of cellular immunity against the parasite (Dassoni et al. 2017). However, there are only few reports evaluating these specific chemokines in leishmaniasis patients. The results of the current study were in agreement with the results of Ritter and Körner (2002) study, in which the serum levels of CXC chemokine members, such as CXCL9 and CXCL10, were highly expressed in the patients with localized CL, while CCL3 expression was marginally increased in these patients. CCL2 is a known recruiter of monocytes; therefore, it might conclude that local immune cells, such as monocytes, are unable to respond to the trace level of CCL2 and further destroy the parasites. In agreement with the present study, Ritter et al. (Ritter and Körner 2002) reported that the serum level of CCL2 increased in localized CL, while its serum level decreased in progressive DCL, indicating that CCL2 might contribute to the healing process. To the best of our knowledge, the present study is the first to address the role of CCL5 and CCL11 in leishmaniasis.

Also, researchers showed that the serum concentrations of CXCL9 and CXCL10, as IFN-γ inducible chemokines, were locally increased in leishmaniasis lesions (Gupta et al. 2019). Furthermore, it could be assumed that chemokines play important roles in the CL pathogenesis. It has been demonstrated that the systemic concentrations of IFN and TNF were significantly decreased in DL comparative to CL patients. However, it was found that the majority of these patients produce IFN, and some of them produced IFN as much as CL patients. Chemokines play fundamental roles in the migration of activated T-cells while they are induced by T-cell-derived cytokines (David and Kubes 2019).

CXCL11 can induce the production of IL-12 by dendritic cells, while CCL2 and CCL3 enable to attract monocytic cells and induce anti-leishmanial activity (Oghumu et al. 2010; Schaerli et al. 2000). Transient impairment of primary T-cell responses in Leishmania infection might contribute to parasite invasion. However, as the infection is in progress, it can be controlled by the local immune response, and only a few amastigotes remain in the lesion site. Later on, as a result of the local amatory response, the lesion becomes progressed to and extended (Desjeux 2004). This study pointed out that other possibilities should be evaluated to explain the inflammation observed in this clinical form of leishmaniasis. Future studies are needed to focus on the involvement of other members of the chemokine family and the therapeutic properties of these mediators.

Conclusion

Chemokines have critical roles in the recruitment of various immune cells, such as activated T-cells and monocytic cells, and the induction of various cytokines, such as IL-12. They participate in the cytolysis of infected or transformed target cells and in the induction of antileishmanial activity. In the present study, both CCL5 and CCL11 but not CCL2 were found to be significantly increased in CL. This suggests that chemokines might have important roles in the pathogenesis of the disease. Therefore, it could probably be applicable to use these chemokines as disease markers or even serve as promising targets for therapeutic studies.

Abbreviations

CCL

Chemokine (C–C motif) ligand

CL

Cutaneous leishmaniasis

CR3

Complement receptor 3

CXCL

Chemokine (C-X-C motif) ligand

DCL

Diffuse cutaneous leishmaniasis

DL

Disseminated leishmaniasis

ELISA

Enzyme-linked immune-sorbent assay

ERK

Extracellular signal-regulated kinase

FcRs

Fc receptors

MR

Mannose receptor

IFN

Interferon

IL-12

Interleukin-12

MAPK

Mitogen-activated protein kinase

PBMC

Peripheral blood mononuclear cell

SPSS

Statistical package for the social sciences

Th1

T helper 1

TNF

Tumor necrosis factor

TLRs

Toll-like receptors

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest.

Ethical standards

This article is prepared and presented by the author in accordance with the ethical standards.