Analysis of human B cell response to recombinant Leishmania LPG3

Mostafa Haji Fatahaliha, Maryam Hosseini, Sanaz Rasolzadeh, Dariush Shane Bandi,Behzad Baradaran, Farhad Jadidi-Niaragh, Mehdi Yousefi*

1Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

2Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

3Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran

4Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

1. Introduction

Leishmaniasis is one of the important infectious diseases caused by an obligate intracellular parasite. It has been demonstrated that over 20 genus of Leishmania can induce this disease and their life cycle alternates between two separate developmental stages including promastigotes and amastigotes. The promastigote stage is related to flagellated extracellular stage in which parasite replicate into midgut of the insect vector, whereas amastigotes is referred to intracellular stage in which aflagellate parasite multiply within mammalian host macrophages[1,2]. The epidemiologic studies have been shown that Leishmaniasis is endemic in 88 countries, affecting 12 million people and an estimated 1.5 million to 2 million new cases occur worldwide annually. Leishmania infection is prevalent in tropical and subtropical areas of Asia, Africa, southern Europe,the Mediterranean (old world)and central and south America(new world). Leishmaniasis appears in various forms including cutaneous (the most common), visceral which also known as kalaazar (the most severe form of the disease)and mucocutaneous[1-3].The clinical manifestation of disease may various from selfhealing chronic cutaneous lesions to progressive and fatal systemic infection, if left untreated. It has been shown that while the cutaneous Leishmaniasis is caused by Leishmania aethiopica,Leishmania tropica and Leishmania major in the old world, it could be induced by Leishmania braziliensis, Leishmania mexicana,Leishmania guyanensis, and Leishmania amazonensis in the new world. CL is the most common in Pakistan, Saudi Arabia, Algeria,Syria, Tunisia, Afghanistan and Iran[4,5].

Paromomycin, pentavalent antimonial, amphotericin B and miltefosine are different chemical drugs used in the control and treatment of Leishmania[6,7]. However, application of these drugs was associated with several side effects and drug resistance which led to their limited prescription.

The several vaccination approaches such as whole killed parasites(as a first generation), poly-proteins, recombinant proteins or dendritic cells pulsed with Leishmania-derived peptides (as second generation)and DNA vaccines (as third generation)have recently been developed, however, none of them was sufficient to protect humans against infection[8,9].

Lipophosphoglycan (LPG)is a highly expressed surface molecule on Leishmania promastigotes. Regarding the protective role of LPG for parasite against phagocytosis, it plays an important role in parasite’s survival[10,11]. Leishmania lipophosphoglycan3 (LPG3)is essential for the synthesis of glycoconjugate molecules, particularly LPG. Thus, it has been thought that LPG3 may has a critical role in parasites infectivity[10,11].

In addition to antibody synthesis, B lymphocytes regulate immune responses and participate in inflammation via secretion of cytokines,growth factors, chemokines and also regulate T cell responses.Several studies showed that B cells can enhance Th1 and Th2 responses[12,13]. Human B lymphocytes are able to secrete IL-6 and TNF-α as pro inflammatory cytokines[14,15]. Some studies demonstrated that antibodies secreted by B cells result in killing of infected macrophages and intracellular parasite[16-18].

In the present study, we evaluated the capability of recombinant Leishmania LPG3 and its fragments in the activation of B cells. We assessed activation of B cells through the production of IL-6, TNF-α (as pro-inflammatory cytokines), and IL-10 (as an immuneregulatory cytokine)cytokines and expression of activation CD69 molecule.

2. Material and methods

2.1. B cells isolation by magnetic-activated cell sorting(MACS)

The heparinized peripheral blood samples were collected from 10 adult healthy volunteers with no history of Leishmania infection after obtaining their informed consent. Peripheral blood mononuclear cells (PBMCs)were isolated by density gradient centrifugationon Ficoll-Paque (Sigma, St. Louis, MO)from whole blood, as described previously[19]. Negative selection (depletion of unwanted cells)were used to isolate B cells from PBMC (B cell isolation kit Ⅱ, Miltenyi Biotech, Bergisch-Gladbach, Germany), according to the manufacturers instruction. For magnetic labeling, PBMCs were washed twice with MACS buffer reagent (300×g for 10 min)and supernatant was completely removed. PBMCs were incubated with 10 μL of B cell biotin-antibody cocktail per 107total cells for 5 min in the refrigerator (2 ℃-8 ℃). Subsequently, the cells were resuspended in 40 μL of MACS buffer per 107total cells. After incubation, 30 μL of MACS buffer and 20 μL of B cell microbead cocktail were added per 107total cells. Cells suspension incubated for 10 min in the refrigerator (2 ℃-8 ℃). Subsequently, MACS buffer (500 μL)was added to the cell suspension and B cells were isolated using magnetic separation.

2.2. B cell purity assessment

Purity of B cells was evaluated by flow cytometry using FITC-conjugated mouse anti-human CD19 (eBiosciences, San Diego, CA)

2.3. Treatment of purified B cells with different concentration of recombinant LPG3, NT-LPG3 and CT-LPG3 fragments

Purified B cells were co-incubated with recombinant LPG3, NTLPG3 and CT-LPG3 fragments that were produced at Pasteur institute, Iran[20]and was kindly gifted from professor Rafati.Briefly, 1×106B cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco, USA), penicillin(100 IU)and streptomycin (100 μg/mL)(Biosera, UK)The cells were treated with different concentrations of recombinant LPG3, NT and CT fragments (2, 10 and 20 μg/mL)for 24 and 48 h at 37 ℃ in a humidified 5% CO2incubator.

2.4. Cytokine assay

To evaluate the effects of LPG3 and its fragments on the production of IL-6, TNF-α and IL-10 by B cells, cell free supernatants of the cultured cells were harvested and the concentrations of IL-6, TNF-α and IL-10 were measured by standard sandwich ELISA kits (R&D,Minneapolis, MN)according to manufacturer’s instructions. Briefly,96-well microtiter plates were coated with anti-IL-6, anti-TNF-α and anti-IL-10 coating Abs in 100 mM Na2HPO4, pH 9.0, incubated for 12 h at 4 ℃, and blocked with blocking buffer containing 0.05%Tween20 and 10% FBS. Collected supernatants and recombinant IL-6, TNF-α and IL-10 standards were added (R&D Systems)and incubated at room temperature for 2 h. Following washing,biotinylated anti IL-6, TNF-α and IL-10 antibodies were added and incubated for 60 min at room temperature. Following washing,microtiter plates were incubated with streptavidine-polyHRP conjugate with p-nitrophenyl phosphate (4 mg/mL)as substrate. The absorbance at 405 nm was measured using a microtiter plate reader and the concentrations of IL-6, TNF-α and IL-10 were calculated from a standard curve of recombinant human IL-6, TNF-α and IL-10 standards, respectively. The concentrations of IL-6, TNF-α and IL-10 for each sample was calculated by regression analysis using the mean absorbance (average of triplicate readings of the samples added).

2.5. Assessment of B cell activation markers by flow cytometry

Expressions of CD69 were evaluated by flow cytometry to demonstrate activation state of B cells treated with rLPG3, CT and NT fragments. Briefly, 106cells were washed with PBS and stained with anti-CD69 PE and anti-CD19 FITC monoclonal antibodies(eBiosciences, San Diego, CA). Following 45 min incubation at 4 ℃in dark, the cells were washed and analyzed by flow cytometer and by FlowJo software.

2.6. Statistical analysis

For analysis of cytokine production, an intergroup comparison was performed by Kruskal-Wallis non-parametric ANOVA test. P-values below 0.05 were regarded as statistically significant.

3. Results

3.1. Recombinant LPG3 and its fragments stimulate the expression of CD69 on B cells

The representative dot plots illustrating the analysis method used for assessment of CD69 expressing B cells are shown in Figure 2.As shown in Figure 3, the stimulation of B cells with LPG-3 and NT fragment (but not CT fragment)could significantly increase the expression of CD69 on B cells (P＜0.05).

3.2. Recombinant LPG3 induced IL-6 production in B cells

The treatment of B cells with 10 and 20 μg/ml concentrations of recombinant LPG3 led to significant increase in secretion of IL-6 by B cells (P＜0.05)(Figure 4). Moreover, the 20 μg/mL (but not 2 and 10 μg/mL)concentration of recombinant NT and CT fragments of LPG3 could significantly increase the secretion of IL-6 by B cells.

3.3. Recombinant LPG3 induced TNF-α production in B cells

Our results showed that the high concentrations (10 and 20 μg/mL)of recombinant LPG3 could significantly increase the production of TNF-α in B cells. On the other hand, only the highest concentration(20 μg/mL)of NT and CT fragments had significant stimulatory effects on IL-6 secretion by B cells (Figure 5).

3.4. Effect of recombinant LPG3 and its fragments on IL-10 production in B cells

Our results showed that recombinant LPG3 and its fragments had no effects on the production of IL-10 by B cells (Figure 6).

4. Discussion

Active immunotherapy is the novel promising approach for eliminating and controlling of Leishmania infection. The presence of potent adjuvant is essential component of effective active immunotherapy.

In the present study, we were evaluated the B cell stimulating potential of different concentrations of LPG3, NT-LPG3 and CTLPG3 fragments. CD69 is an important activation marker that expresses on the surface of mature lymphocytes[21]. We found that rLPG3 and its NT fragment have a significant effect on upregulation of CD69 on the surface of purified B cells.

It has been demonstrated that B cell-derived antibodies facilitate the phagocytosis of Leishmania major. Moreover, it observed that the higher parasite burden was associated with a reduction of T cell response and IFN-γ production in mice lacking antibodies following infection[22]. Furthermore, reduced chemotaxy of monocytes and lymphocytes at the site of cutaneous lesion has been observed in B lymphocytes deficient mice compared to wild type[23]. B cells also produce several pro-inflammatory and anti-inflammatory cytokines which regulate the immune response during Leishmania infection[24].Our results indicate that recombinant LPG3 could activate B lymphocytes to produce and secrete IL-6 and TNF-α but not IL-10.Moreover, NT and CT fragments are able to induce IL-6 and TNF-α.

IL-6 as a multifunctional cytokine promotes inflammatory responses against infection. Moreover, IL-6 participates in the development of Th1 and Th2 immune responses in mammals[25,26].However, the function of IL-6 as a pro-inflammatory cytokines in cell-mediated immunity against leishmaniasis has not been clearly understood[27]. Akuffo et al[28]demonstrated that Leishmania aethiopica antigen enhances the production of IL-6 from PBMCs of localized cutaneous leishmaniasis patients more than in adults healthy volunteers.

IL-10 is an immune-modulatory cytokine and mediates a variety of both immunosuppressive and immunostimulatory activities in mouse and human immune cells[29]. IL-10 is the one of important anti-inflammatory cytokines[30], countered as a suppressor of macrophage functions[31,32]. It has been demonstrated that IL-10 can proceed parasite persistence[23]. Moreover, the immune suppressor activity of IL-10 has been indicated both in human[33]and mice visceral leishmaniasis[34]. A recent study demonstrated that IL-10-deficient BALB/c mice are highly resistant to Leishmania donovani infection[34]. However, our results showed that none of the LPG-3 and its fragments had effects on the production of IL-10 by B cells.It has been demonstrated that TNF-α has a protective effect in experimental mouse cutaneous leishmaniasis[35]. Consistently, it is reported that treatment of mice with TNF-α could decrease parasitic burden and lesion size[36,37].

Our data were consistent with previous report[20]regarding the immunogenic nature of recombinant LPG3 in BALB/c mice.Moreover, Larreta et al[38]reported that 84% of the dogs visceral leishmaniasis sera reacted with Leishmania infantum LPG3.

In conclusion, recombinant LPG3 stimulates human B cells to secrete pro-inflammatory IL-6 and TNF-α cytokines and inhibits production of regulatory IL-10 cytokine that may be considered as a promising candidate for Leishmania vaccine design in near future.

Conflict of interest statement

We declare that we have no conflict of interest.

[1]Descoteaux A, Avila HA, Zhang K, Turco SJ, Beverley SM. Leishmania LPG3 encodes a GRP94 homolog required for phosphoglycan synthesis implicated in parasite virulence but not viability. EMBO J 2002; 21(17):4458-4469.

[2]Franco LH, Beverley SM, Zamboni DS. Innate immune activation and subversion of mammalian functions by Leishmania lipophosphoglycan. J Parasitol Res 2012; 2012:165126

[3]Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J, et al.Leishmaniasis worldwide and global estimates of its incidence. PloS One 2012; 7(5): e35671.

[4]Joshi S, Rawat K, Yadav NK, Kumar V, Siddiqi MI, Dube A. Visceral leishmaniasis: advancements in vaccine development via classical and molecular approaches. Front Immunol 2014; 5: 380.

[5]Seyed N, Taheri T, Vauchy C, Dosset M, Godet Y, Eslamifar A, et al.Immunogenicity evaluation of a rationally designed polytope construct encoding HLA-A* 0201 restricted epitopes derived from Leishmania major related proteins in HLA-A2/DR1 transgenic mice: Steps toward polytope vaccine. PloS one 2014; 9(10): e108848.

[6]Croft SL, Barrett MP, Urbina JA. Chemotherapy of trypanosomiases and leishmaniasis. TRENDS Parasitol 2005; 21(11): 508-512.

[7]Natera S, Machuca C, Padrón-Nieves M, Romero A, Díaz E, Ponte-Sucre A. Leishmania spp.: proficiency of drug-resistant parasites. Int J Antimicrob Agents 2007; 29(6): 637-642.

[8]Khamesipour A, Rafati S, Davoudi N, Maboudi F, Modabber F.Leishmaniasis vaccine candidates for development: a global overview.Indian J Med Res 2006; 123(3): 423.

[9]Modabber F, Coler R, Reed S. Vaccines against Leishmania. Levine MM,Dougan M, Nabel G, Good MF, Nataro J, Liu MA, et al. New generation vaccines. 4th ed. New York, London: Informa Healthcare; 2010, p. 790-807.

[10]Beverley SM, Turco SJ. Lipophosphoglycan (LPG)and the identification of virulence genes in the protozoan parasite Leishmania. Trends Microbiol 1998; 6(1): 35-40.

[11]Ilg T. Lipophosphoglycan is not required for infection of macrophages or mice by Leishmania mexicana. EMBO J 2000; 19(9): 1953-1962.

[12]Ronet C, Hauyon-La Torre Y, Revaz-Breton M, Mastelic B, Tacchini-Cottier F, Louis J, Launois P. Regulatory B cells shape the development of Th2 immune responses in BALB/c mice infected with Leishmania major through IL-10 production. J Immunol 2010; 184(2): 886-894.

[13]Whitmire JK, Asano MS, Kaech SM, Sarkar S, Hannum LG, Shlomchik MJ, et al. Requirement of B cells for generating CD4+T cell memory. J Immunol 2009; 182(4): 1868-1876.

[14]Agrawal S, Gupta S. TLR1/2, TLR7, and TLR9 signals directly activate human peripheral blood naive and memory B cell subsets to produce cytokines, chemokines, and hematopoietic growth factors. J Clin Immunol 2011; 31(1): 89-98.

[15]Pistoia V. Production of cytokines by human B cells in health and disease. Immunol Today 1997; 18(7): 343-350.

[16]Gibson-Corley KN, Bockenstedt MM, Li H, Boggiatto PM, Phanse Y,Petersen CA, et al. An in vitro model of antibody-enhanced killing of the intracellular parasite Leishmania amazonensis. PloS One 2014; 9(9):e106426.

[17]Vouldoukis I, Mazier D, Moynet D, Thiolat D, Malvy D, Mossalayi MD.. IgE mediates killing of intracellular Toxoplasma gondii by human macrophages through CD23-dependent, interleukin-10 sensitive pathway.PLoS One 2011; 6(4): e18289.

[18]Vouldoukis I, Riveros-Moreno V, Dugas B, Ouaaz F, Bécherel P, Debré P,et al. The killing of Leishmania major by human macrophages is mediated by nitric oxide induced after ligation of the Fc epsilon RII/CD23 surface antigen. Proc Natl Acad Sci U S A 1995; 92(17): 7804-7808.

[19]Akuffo H, Maasho K, Howe R. Natural and acquired resistance to Leishmania: cellular activation by Leishmania aethiopica of mononuclear cells from unexposed individuals is through the stimulation of natural killer (NK)cells. Clin Exp Immunol 1993. 94(3): 516-521.

[20]Abdian N, Gholami E, Zahedifard F, Safaee N, Rafati S. Evaluation of DNA/DNA and prime-boost vaccination using LPG3 against Leishmania major infection in susceptible BALB/c mice and its antigenic properties in human leishmaniasis. Exp Parasitol 2011; 127(3): 627-636.

[21]Bankovich AJ, Shiow LR, Cyster JG. CD69 suppresses sphingosine 1-phosophate receptor-1 (S1P1)function through interaction with membrane helix 4. J Biol Chem 2010; 285(29): 22328-22337.

[22]Woelbing F, Kostka SL, Moelle K, Belkaid Y, Sunderkoetter C, Verbeek S, et al. Uptake of Leishmania major by dendritic cells is mediated by Fc γ receptors and facilitates acquisition of protective immunity. J Exp Med 2006; 203(1): 177-188.

[23]McMahon-Pratt D, Alexander J. Does the Leishmania major paradigm of pathogenesis and protection hold for New World cutaneous leishmaniases or the visceral disease? Immunol Rev 2004; 201(1): 206-224.

[24]Pistoia V, Corcione A. Relationships between B cell cytokine production in secondary lymphoid follicles and apoptosis of germinal center B lymphocytes. Stem Cells 1995. 13(5): 487-500.

[25]Dalrymple SA, Lucian LA, Slattery R, McNeil T, Aud DM, Fuchino S, et al. Interleukin-6-deficient mice are highly susceptible to Listeria monocytogenes infection: correlation with inefficient neutrophilia. Infect Immun 1995; 63(6): 2262-2268.

[26]Le J, Vilcek J. Interleukin 6: a multifunctional cytokine regulating immune reactions and the acute phase protein response. Lab Invest 1989.61(6): 588-602.

[27]Moskowitz NH, Brown DR, Reiner SL. Efficient immunity against Leishmania major in the absence of interleukin-6. Infect Immun 1997;65(6): 2448-2450.

[28]Akuffo H, Britton S. Contribution of non-Leishmania-specific immunity to resistance to Leishmania infection in humans. Clin Exp Immunol 1992;87(1): 58-64.

[29]Groux H, Cottrez F, Rouleau M, Mauze S, Antonenko S, Hurst S, et al. A transgenic model to analyze the immunoregulatory role of IL-10 secreted by antigen-presenting cells. J Immunol 1999; 162(3): 1723-1729.

[30]Mizoguchi A, Bhan AK. A case for regulatory B cells. J Immunol 2006;176(2): 705-710.

[31]Fillatreau S, Sweenie CH, McGeachy MJ, Gray D, Anderton SM. B cells regulate autoimmunity by provision of IL-10. Nat Immunol 2002; 3(10):944-950.

[32]Palanivel V, Posey C, Horauf AM, Solbach W, Piessens WF, Harn DA.B-cell outgrowth and ligand-specific production of IL-10 correlate with Th2 dominance in certain parasitic diseases. Exp Parasitol 1996; 84(2):168-177.

[33]Karp CL, el-Safi SH, Wynn TA, Satti MM, Kordofani AM, Hashim FA,et al. In vivo cytokine profiles in patients with kala-azar. Marked elevation of both interleukin-10 and interferon-gamma. J Clin Invest 1993; 91(4):1644.

[34]Murphy ML, Wille U, Villegas EN, Hunter CA, Farrell JP. IL-10 mediates susceptibility to Leishmania donovani infection. Eur J Immunol 2001; 31(10): 2848-2856.

[35]Theodos C, Povinelli L, Molina R, Sherry B, Titus RG. Role of tumor necrosis factor in macrophage leishmanicidal activity in vitro and resistance to cutaneous leishmaniasis in vivo. Infect Immun 1991; 59(8):2839-2842.

[36]Liew F, Li Y, Millott S. Tumor necrosis factor-alpha synergizes with IFN-gamma in mediating killing of Leishmania major through the induction of nitric oxide. J Immunol 1990; 145(12): 4306-4310.

[37]Liew FY, Parkinson C, Millott S, Severn A, Carrier M. Tumour necrosis factor (TNF alpha)in leishmaniasis. I. TNF alpha mediates host protection against cutaneous leishmaniasis. Immunology 1990; 69(4):570.

[38]Larreta R, Soto M, Alonso C, Requena JM. Leishmania infantum: gene cloning of the GRP94 homologue, its expression as recombinant protein,and analysis of antigenicity. Exp Parasitol 2000; 96(2): 108-115.