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DC-SIGN receptor is expressed by cells from cutaneous leishmaniasis lesions and differentially binds to Leishmania (Viannia) braziliensis and L. (Leishmania) amazonensis promastigotes

Abstract

BACKGROUND

Dendritic cells (DCs) specific intercellular adhesion molecule (ICAM)-3-grabbing non integrin receptor (DC-SIGN) binds to subgenera Leishmania promastigotes mediating its interaction with DC and neutrophils, potentially influencing the infection outcome.

OBJECTIVES

In this work, we investigated whether DC-SIGN receptor is expressed in cells from cutaneous leishmaniasis (CL) lesions as well as the in vitro binding pattern of Leishmania (Viannia) braziliensis (Lb) and L. (L.) amazonensis (La) promastigotes.

METHODS

DC-SIGN receptor was labeled by immunohistochemistry in cryopreserved CL tissue fragments. In vitro binding assay with CFSE-labeled Lb or La promastigotes and RAJI-transfecting cells expressing DC-SIGN (DC-SIGNPOS) or mock-transfected (DC-SIGNNEG) were monitored by flow cytometry at 2 h, 24 h and 48 h in co-culture.

RESULTS

In CL lesion infiltrate, DC-SIGNPOS cells were present in the dermis and near the epidermis. Both Lb and La bind to DC-SIGNPOS cells, while binding to DC-SIGNNEG was low. La showed precocious and higher affinity to DC-SIGNhi population than to DC-SIGNlow, while Lb binding was similar in these populations.

CONCLUSION

Our results demonstrate that DC-SIGN receptor is present in L. braziliensis CL lesions and interact with Lb promastigotes. Moreover, the differences in the binding pattern to Lb and La suggest DC-SIGN can influence in a difference way the intake of the parasites at the first hours after Leishmania infection. These results raise the hypothesis that DC-SIGN receptor could participate in the immunopathogenesis of American tegumentary leishmaniasis accounting for the differences in the outcome of the Leishmania spp. infection.

Key words:
DC-SIGN; cutaneous leishmaniasis; inflammatory infiltrate; Leishmania (Viannia) braziliensis; Leishmania (Leishmania) amazonensis


Cutaneous leishmaniasis (CL) due to Leishmania (Viannia) braziliensis is the most common clinical form of leishmaniasis in Brazil.11. Oliveira-Neto MP, Mattos MS, Perez MA, Da-Cruz AM, Fernandes O, Moreira J, et al. American tegumentary leishmaniasis (ATL) in Rio de Janeiro State, Brazil: main clinical and epidemiologic characteristics. Int J Dermatol. 2000; 39(7): 506-14. Skin CL lesions are characterized by a chronic granulomatous inflammatory infiltrate, with lymphocytes, plasmocytes, and macrophages.22. Magalhães AV, Moraes MA, Raick AN, Llanos-Cuentas A, Costa JM, Cuba CC, et al. [Histopathology of tegumentary leishmaniasis caused by Leishmania braziliensis braziliensis. 3. Cellular reactions in tissues]. Rev Inst Med Trop São Paulo. 1986; 28(5): 300-11. The presence of dendritic cells (DC) in CL lesions has been known since the 1980’s.33. Modlin RL, Tapia FJ, Bloom BR, Gallinoto ME, Castes M, Rondon AJ, et al. In situ characterization of the cellular immune response in American cutaneous leishmaniasis. Clin Exp Immunol. 1985; 60(2): 241-8. DCs are located in the epidermis, being capable to migrate to the dermis, and to uptake Leishmania amastigotes through the Fcγ receptor.44. Moll H, Fuchs H, Blank C, Rollinghoff M. Langerhans cells transport Leishmania major from the infected skin to the draining lymph node for presentation to antigen-specific T cells. Eur J Immunol. 1993; 23(7): 1595-601.,55. 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? receptors and facilitates acquisition of protective immunity. J Exp Med. 2006; 203(1): 177-88. In CL lesions, DCs can be found harboring Leishmania amastigotes.66. Isaza DM, Restrepo M, Restrepo R, Caceres-Dittmar G, Tapia FJ. Immunocytochemical and histopathologic characterization of lesions from patients with localized cutaneous leishmaniasis caused by Leishmania panamensis. Am J Trop Med Hyg. 1996; 55(4): 365-9.,77. Ridley MJ, Wells CW, Bartholomew S, Smithfield W. Lesions of cutaneous leishmaniasis. Am J Parasitol. 1985; 123(1): 79-85. However, infected DCs transport the parasite to draining lymph nodes and initiate the adaptative immune response.44. Moll H, Fuchs H, Blank C, Rollinghoff M. Langerhans cells transport Leishmania major from the infected skin to the draining lymph node for presentation to antigen-specific T cells. Eur J Immunol. 1993; 23(7): 1595-601. In the course of the infection, DCs producing IL-12 can be detected at the beginning of the inflammatory process.88. von Stebut E, Belkaid Y, Jakob T, Sacks DL, Udey MC. Uptake of Leishmania major amastigotes results in activation and interleukin 12 release from murine skin-derived dendritic cells: implications for the initiation of anti-Leishmania immunity. J Exp Med. 1998; 188(8): 1547-52. Moreover, DCs, professional antigen presenting cells, prime Leishmania specific CD4+ and CD8+ T cells and maintain the T cell memory activation.99. Zaph C, Uzonna J, Beverley SM, Scott P. Central memory T cells mediate long-term immunity to Leishmania major in the absence of persistent parasites. Nat Med. 2004; 10(10): 1104-10. However, depending on Leishmania species, different DC subpopulations are mobilized, which in turn influences the course of infection.1010. Falcão SAC, Jaramillo TMG, Ferreira LG, Bernardes DM, Santana JM, Favali CBF. Leishmania infantum and Leishmania braziliensis: differences and similarities to evade the innate immune system. Front Immunol. 2016; 7: 287.,1111. Carvalho AK, Silveira FT, Passero LF, Gomes CM, Corbett CE, Laurenti MD. Leishmania (V.) braziliensis and L. (L.) amazonensis promote differential expression of dendritic cells and cellular immune response in murine model. Parasite Immunol. 2012; 34(8-9): 395-403. It was shown that receptor signaling also contributes to the differentiation of protective inflammatory DC in L. (V.) braziliensis infection.1212. Costa DL, Lima-Júnior DS, Nascimento MS, Sacramento LA, Almeida RP, Carregaro V, et al. CCR2 signalling contributes to the differentiation of protective inflammatory dendritic cells in Leishmania braziliensis infection. J Leukoc Biol. 2016; 100(2): 423-32.

DC specific intercellular adhesion molecule (ICAM)-3-grabbing non-integrin receptor (DC-SIGN) is a member of the C-type lectin receptors family, also known as CD209.1313. Garg R, Trudel N, Tremblay MJ. Consequences of the natural propensity of Leishmania and HIV-1 to target dendritic cells. Trends Parasitol. 2007; 23(7): 317-24. DC-SIGN is expressed by immature DCs and macrophages in lymphoide organs and peripheral tissues.1414. Soilleux EJ, Morris LS, Leslie G, Chehimi J, Luo Q, Levroney E, et al. Constitutive and induced expression of DC-SIGN on dendritic cell and macrophage subpopulations in situ and in vitro. J Leukoc Biol. 2002; 71(3): 445-57. In normal skin, dermal DC, with the appearance of macrophages cells, express CD209 receptor.1515. Ochoa MT, Loncaric A, Krutzik SR, Becker TC, Modlin RL. "Dermal dendritic cells" comprise two distinct populations: CD1+ dendritic cells and CD209+ macrophages. J Invest Dermatol. 2008; 128(9): 2225-31. Many functions are associated with DC-SIGN receptor. The presence of DC-SIGN in DCs facilitates rolling and trans-endothelial migration by binding to ICAM-2 ligand.1616. van Kooyk Y, Geijtenbeek TB. A novel adhesion pathway that regulates dendritic cell trafficking and T cell interactions. Immunol Rev. 2002; 186: 47-56. DC-SIGN receptor in DCs surface interacts with ICAM-3 present in T cell membrane, suggesting that these molecules participate and stabilize the contact between DCs and T cells.1313. Garg R, Trudel N, Tremblay MJ. Consequences of the natural propensity of Leishmania and HIV-1 to target dendritic cells. Trends Parasitol. 2007; 23(7): 317-24. DC-SIGN receptor can act as a pathogen-recognized receptor, which recognized specific carbohydrates present in the surfaces of pathogens. Many pathogens utilize DC-SIGN receptor to invade DCs and escape the immune system.1717. Geijtenbeek TB, van Kooyk Y. DC-SIGN: a novel HIV receptor on DCs that mediates HIV-1 transmission. Curr Top Microbiol Immunol. 2003; 276: 31-54. In HIV infection, DC-SIGN acts as a receptor for HIV, capturing the virus and transmitting it to a targeted T cell.1313. Garg R, Trudel N, Tremblay MJ. Consequences of the natural propensity of Leishmania and HIV-1 to target dendritic cells. Trends Parasitol. 2007; 23(7): 317-24.Mycobacterium tuberculosis binds with high affinity to DC-SIGN receptor and can be uptaken DCs via this receptor. In vivo, M. tuberculosis antigens were detected in DC-SIGN positive cells.1818. Tallieux L, Schwartz O, Herrmann JL, Pivert E, Jackson M, Amara A, et al. DC-SIGN is the major Mycobacterium tuberculosis receptor on human dendritic cells. J Exp Med. 2003; 197(1): 121-7. In lepromatous leprosy lesions, DC-SIGN positive cells harbor M. leprae bacillus.1919. Soilleux EJ, Sarno EN, Hernandez MO, Moseley E, Horsley J, Lopes UG, et al. DC-SIGN association with the Th2 environment of lepromatous lesions: cause or effect? J Pathol. 2006; 209(2): 182-9. In non-infectious cutaneous inflammatory disease, like psoriasis, DC-SIGN positive cells can be detected in skin lesions. The expression levels of DC-SIGN receptor in inflammatory infiltrate were significantly higher in lesions than in normal skin, suggesting an involvement of this receptor in psoriasis pathogenesis.2020. Higaki M, Higaki Y, Kawashima M. Increased expression of CD208 (DC-LAMP) in epidermal keratinocytes of psoriatic lesions. J Dermatol. 2009; 36(3): 144-9.,2121. Wei-yuan M, Wen-ting L, Chen Z, Qing S. Significance of DC-LAMP and DC-SIGN expression in psoriasis vulgaris lesions. Exp Mol Pathol. 2011; 91(1): 461-5. Both in tegumentary or visceral leishmaniasis, the related species can bind to DC-SIGN receptor.2222. Colmenares M, Constant SL, Kima PE, McMahon-Pratt D. Leishmania pifanoi pathogenesis: selective lack of a local cutaneous response in the absence of circulating antibody. Infect Immun. 2002; 70(12): 6597-605.,2323. Colmenares M, Corbi AL, Turco SJ, Rivas L. The dendritic cell receptor DC-SIGN discriminates among species and life cycle forms of Leishmania. J Immunol. 2004; 172(2): 1186-90. Recently, it was shown that interaction between polymorphonuclear cells and L. (L.) amazonensis intermediated by DC-SIGN receptor is required for the release of inflammatory mediators.2424. Tiburcio R, Melo LD, Nunes S, Barbosa ALA, de Oliveira EC, Suarez M, et al. DC-SIGN mediates the interaction between neutrophils and Leishmania amazonensis-infected dendritic cells to promote DC maturation and parasite elimination. Front Immunol. 2021; 12: 1-13. In this report, we investigated whether DC-SIGN receptor is expressed in CL lesions caused by L. (V.) braziliensis, and explored the involvement of DC-SIGN on the initial infection process of two species with epidemiological importance in the Americas, L. (V.) braziliensis (Lb) and L. (L.) amazonensis (La) promastigotes.

SUBJECTS AND METHODS

Patients - We evaluated six CL patients who had acquired the disease in Rio de Janeiro, Brazil, which is an endemic area for L. (V.) braziliensis infection. All of them were men, mean age 39.2 ± 10.6 years old. The mean period of illness was 35.7 ± 19.8 days and the lesions were ulcerated. The Montenegro skin test was positive in five out six cases (mean 18.4 ± 5.8 mm). The polymerase chain reaction (PCR) assay to detect Leishmania kDNA was positive in all of them. Patients were successfully treated with antimonial pentavalent as recommended by the Brazilian Ministry of Health. Informed consent was obtained from each subject, and a skin biopsy was performed for diagnostic purposes. The skin fragment was cryopreserved in optimal cutting temperature (OCT) resin blocks at -196ºC (Tissue Tek; Sakura Finetek, Torrance, CA, USA) until the time of use. All procedures were approved by the Ethical Committee of the Fundação Oswaldo Cruz (CEP FIOCRUZ no. 291/05; CEP IPEC no.390/07), Ministério da Saúde, Rio de Janeiro, Brazil.

RAJI cells and Leishmania culture - RAJI-transfected cells expressing DC-SIGN (DC-SIGNPOS) and RAJI-mock transfected (DC-SIGNNEG) cells (Sigma Chemical Company, Saint Louis, USA) were maintained in culture in RPMI-1640 medium supplemented, with 2 mM L-glutamine (Gibco BRL, Gaithersburg, Germany), 100 U/mL penicillin (Sigma, USA), 100 µg/mL streptomycin (Sigma, USA) and 10% fetal bovine serum (FBS; Gibco BRL, Germany) (complete medium), at 37ºC in a humidified CO2 incubator.

Promastigotes of L. (V.) braziliensis (MCAN/BR/1998/619) and L. (L.) amazonensis (MHOM/BR/75/Josefa) were grown at 26ºC in Scheneider’s insect medium (Sigma, USA), supplemented with 2 mM L-glutamine (Gibco BRL, Germany), antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin (Sigma, USA) and 10% FBS (Gibco BRL, DE). Parasites were grown for four days in culture and used to perform the binding assay at stationary phase.

Leishmania - RAJI cell binding assay - RAJI (DC-SIGNNEG and DC-SIGNPOS) cells and carboxyfluorescein succinimidyl ester (CFSE) (CelltraceTM CFSE cell proliferation kit; Life Technologies, USA)-labeled promastigotes were adjusted to 1 cell:5 promastigotes in 24 well-plates containing RPMI-1640 complete medium and incubated at 37ºC in a humidified CO2 incubator. The binding assay was monitored for 2, 24 and 48 h. Later on, the co-cultured cells were collected and processed for flow cytometry.

Flow cytometry analysis for binding assay - RAJI cells (DC-SIGNNEG and DC-SIGNPOS) bound to Leishmania were collected from culture and washed with cold phosphate-buffered saline (PBS) containing 0·01% sodium azide (NaN3; Sigma, USA) and 5% FBS (PBS-Az/FBS). After that, 3 x 106/mL RAJI (DC-SIGNNEG and DC-SIGNPOS) cells were incubated for 30 min at 4ºC in presence of 10 µL of mouse anti-human DC-SIGN monoclonal antibody (R&D Systems, Minneapolis, USA). After incubation, the cells were washed in PBS-Az/FBS and incubated with goat anti-mouse PC7 secondary antibody (Santa Cruz Biotechnology, Texas, USA) for 30 min at 4ºC. The cells were fixed in a fixing solution containing 1% paraformaldehyde in PBS (PBS-PF 1%) for 20 min at 4ºC. The cells were then washed with PBS-PF 1% and resuspended in PBS-Az prior to analysis. The Leishmania promastigotes were labelled with carboxyfluorescein succinimidyl ester (CFSE, Invitrogen), which does not interfere in the parasite viability and infectivity, to be detected by flow cytometry assay. For flow cytometry analysis, 30.000 events in total lymphocyte gate (R1) per sample were acquired in a fluorescence activated cell sorter (CyAn ADP analyzes, Beckman Coulter). DC-SIGN surface receptor and CFSE-labelled Leishmania promastigote were analyzed using Summit 4.3 software. The total lymphocyte gate (R1) was settled based on size (forward scatter: FSC) and granularity (side-scatter: SSC). RAJI (DC-SIGNNEG and DC-SIGNPOS) cells were observed in lymphocyte gate. For binding assay analysis, a dot-plot graphic (DC-SIGN/PE-Cy7 x CFSE) gated in R1 was created, and the percentage results are observed in double positive quadrant. The results were expressed as a percentage mean of positive cells. Five independent experiments were performed.

Immunohistochemistry - To detect surface DC-SIGN receptors, the slides containing cells from CL lesions were fixed in acetone PA (Merck, Darmstadt, Germany/DE) and hydrated in phosphate-buffered saline (PBS) pH 7.4. The specimens were incubated with anti-human DC-SIGN (1:40; R&D Systems, USA). Dako Envision system (DakoCytomation, Carpinteria, CA, USA) was used to link anti-mouse primary antibodies to substrate. The staining was completed using 3-amino-9-ethylcarbazole (AEC; Sigma, USA) as the substrate-chromogen system. The slides were counterstained with Mayer’s hematoxylin (Merck, DE). For control, in the first step, antibody was omitted. The slides were examined under a light microscope (Nikon, Eclipse E600, Japan) with Cool Snap-Pro Color camera and acquired by ImagePro® Program (Media Cybernetics, Inc., USA). Only cells with visible nucleus and red-brown immune stain were counted as positive cells. All fields were counted in each section at a magnification of 1000x. The size of the section was measured using a millimeter paper.

Statistical analysis - Statistical analysis was performed by One-way or Two-way analysis of variance (Anova) tests using the GraphPad Prism software version 5·00 for Windows (GraphPad Software, San Diego, CA, USA). The results were expressed as the mean ± standard error or mean ± standard deviation. A p value ≤ 0.05 was considered significant.

RESULTS

Cells expressing DC-SIGN receptor were present in cutaneous leishmaniasis lesions - We evaluated the expression of DC-SIGN in the inflammatory cell infiltrate of American CL lesions due to L. (V.) braziliensis. The patients evaluated herein presented a clinical prolife similar to that observed in Rio de Janeiro State.2525. Geijtenbeek TB, Kwon DS, Torensma R, van Vliet SJ, van Duijnhoven GC, Middel J, et al. DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell. 2000; 100(5): 587-97. We observed DC-SIGNPOS cells (stained in red-brown color) were present in CL lesions (Fig. 1, panel A and C). The DC-SIGNPOS cells were located in the dermis and near the epidermis, isolated or in groups, and had similar morphology to macrophages. This indicates that this receptor can be used to uptake parasite forms. In the next step, we evaluated whether Lb and La promastigotes interact with DC-SIGN.

Fig. 1:
DC-SIGN positive cells in inflammatory infiltrate (arrows) of cutaneous leishmaniasis lesions caused by Leishmania (Viannia) braziliensis. Slide were stained with anti-human CD209 (A and C) followed by Dako Envision System. The slides were then developed with 3,3-diaminobenzidine (red-brown) and counterstained with Mayer’s hematoxylin. For control propose, anti-human CD209 antibody was omitted (B and D). Original magnification: 1000x (A and C), 400x (D) and 200x (B).

DC-SIGN expression in RAJI cells membranes - To determine the best day to perform the binding assay, we monitored the RAJI cells proliferation and DC-SIGN receptor expression during three days of culture (Fig. 2). During these three days in culture, the mean percentage of the DC-SIGN receptor expression in the surface of the RAJIPOS was not significantly different (0h: 61.5% ± 2.1%; 2h: 64.9% ± 6.0%; 24 h: 73.0% ± 2.7%; 48 h: 65.8% ± 1.8%; ANOVA p = 0.23) (Table).

Fig. 2:
representative flow cytometry histogram of DC-SIGN receptor analysis in RAJI cells in different time points of cell culture. Electronic gates R4 represents total RAJI transfected with DC-SIGN cells expressing DC-SIGN, R5 represents RAJI DC-SIGNPOS cells expressing low amount of DC-SIGN (DC-SIGNLow), and R6 represents RAJI DC-SIGNPOS cells expressing high amount of DC-SIGN (DC-SIGNHi).

TABLE
DC-SIGN expression in RAJI cells transfected or not with DC-SIGN during the 48 h in culture

The flow cytometry analysis showed that RAJI DC-SIGNPOS cell population can be divided into two minor populations, based on the intensity of DC-SIGN receptor expression, DC-SIGNLow or DC-SIGNHi (Fig. 2). Electronics gates were constructed to select RAJIPOS (R4), DC-SIGNLow(R5) or DC-SIGNHi (R6) cell populations (Fig. 2). After 24 h in cell culture, DC-SIGNPOS cell population was significantly enriched of DC-SIGNHi cell population (43.2% ± 5.7%; p = 0.05), when compared with other time points analyzed (0 h: 18.8% ± 3.6%; 2 h: 26.2% ± 4.5%; 48 h: 26.2% ± 5.5%) (Fig. 2 and Table).

American Leishmania species bind in RAJI DC-SIGN positive cells - To determine whether DC-SIGN receptor binds to Leishmania dermothropic species, we evaluated two species from subgenera Viannia and Leishmania that have epidemiological importance in America. For that, CFSE labelled L. (V.) braziliensis or L. (L.) amazonensis promastigotes were co-cultured with RAJI DC-SIGN positive and negative cells. The binding receptor percentage was evaluated by flow cytometry over different times from 0 h to 48 h. RAJI cells did not internalize Leishmania promastigotes.

Both Lb and La promastigotes presented high binding interaction with the DC-SIGNPOS cells, while the interaction with the DC-SIGNNEG cell was low (Fig. 3). After 2 hours in co-culture, the percentage of interaction between La and the DC-SIGNPOS cells was significantly higher (12.4% ± 2.11%; p < 0.001) when compared to DC-SIGNNEG (0.81% ± 0.30%) (Fig. 3A-B). The binding between La and DC-SIGNPOS cells was maintained even at 48 h of interaction (24 h - DC-SIGNPOS: 14.7% ± 2.07%; DC-SIGNNEG: 1.23% ± 0.15% p < 0.001; 48 h - DC-SIGNPOS: 11.9% ± 2.03%; DC-SIGNNEG: 1.17% ± 0.41%; p < 0.001) (Fig. 3B). For Lb, no binding differences was observed at 2 h of co-culture in the presence or absence of the DC-SIGN receptor (DC-SIGNPOS: 4.7% ± 0.94%; DC-SIGNNEG: 1.17% ± 0.27%; p > 0.05). The interaction between Lb and DC-SIGN receptor was observed only at 24 h in co-culture, maintaining the same binding levels at 48 h (24 h - DC-SIGNPOS: 18.2% ± 3.7%; DC-SIGNNEG: 1% ± 0.05%; p < 0.001; 48 h - DC-SIGNPOS: 19.3% ± 1.58%; DC-SIGNNEG: 1.9% ± 0.66%; p < 0.001) (Fig. 3C).

Fig. 3:
binding analysis of Leishmania (L.) amazonensis (La) or L. (Viannia) braziliensis (Lb) promastigotes to RAJI DC-SIGNNEG or RAJI DC-SIGNPOS cells after 2-48 h of co-culture. (A) Dotplot to double-staining analysis of Leishmania-CFSE (x axis) and DC-SIGN (CD209)-PE-Cy7 (y axis) from DC-SIGNNEG (left panels) or DC-SIGNPOS (right panels) cells after 24 h of co-culture. The percentage of Leishmania-RAJI cells binding in different time points were analised for L. (L.) amazonensis (B) or L. (V.) braziliensis (C). * p < 0.05; ***p < 0.001.

Leishmania (L.) amazonensis promastigotes bind preferentially to RAJI DC-SIGNHi , while L. braziliensis promastigotes bind similarly to DC-SIGN Hi or Low RAJI cells - To verify whether the Leishmania parasites bind specifically to cells with DC-SIGN receptor, we analyzed the binding of Leishmania to two RAJI DC-SIGNPOS populations: DC-SIGNHi or DC-SIGNLow (Fig. 4A). The percentage of binding between La and the DC-SIGNHi population was significantly higher and constant (2 h: 37.7% ± 7.6%; 24 h: 31.7% ± 5.7%; 48 h: 33.2% ± 5.7%) when compared to the DC-SIGNLow population, independent of the time point evaluated (2 h: 11% ± 1.9%; p < 0.001; 24 h:12.5% ± 1.12%; p < 0.001; 48 h: 13.2% ± 3.6%; p < 0.001) (Fig. 4B). Nevertheless, the binding between Lb and DC-SIGNHi or DC-SIGNLow did not present relevant differences (DC-SIGNLow, 2 h: 8.5% ± 1.16%; 24 h: 23.4% ± 4.28; 48 h: 35.3% ± 3.3%, and DC-SIGNHi, 2 h: 12.8% ± 2%; p > 0.05; 24 h: 26.7% ± 5.5%; p > 0.05; 48 h: 37% ± 3%; p > 0.05) (Fig. 4C). However, in Lb, significant differences were observed when we analyzed the percentage of binding during the different times of interaction (DC-SIGNLow: 2 h vs 24 h, p < 0.05; 24 h vs 48 h, p < 0.05; 2 h vs 48 h, p < 0.001; DC-SIGNHi: 2 h vs 24 h, p < 0.05; 24 h vs 48 h, p < 0.001; 2 h vs 48 h, p > 0.05) (Fig. 4C).

Fig. 4:
binding analysis of Leishmania (Leishmania) amazonensis (La) or L. (Viannia) braziliensis (Lb) in RAJI DC-SIGNLow or RAJI DC-SIGNHi populations after 2-48 h of co-culture. (A) Representatives histograms analysis of Leishmania-CFSE in DC-SIGNLow (left panels) or DC-SIGNHi (right panels) after 24 h of co-culture. The percentage of Leishmania-CFSE binding to DC-SIGNLow or DC-SIGNHi gated populations in different time points were analyzed for L. (L.) amazonensis (B) or L. (V.) braziliensis (C). * p < 0.05; ** p < 0.01; *** p < 0.001.

DISCUSSION

In this study, we observed that American dermotropic species of Leishmania bind to DC-SIGN receptor with different patterns. La promastigotes bind to RAJI DC-SIGNPOS cells promptly, and the percentual of interaction increases according to the receptor expression levels. Lb promastigotes delay to bind to this receptor, binding with the same affinity to the DC-SIGNLow and DC-SIGNHi population. Moreover, we found DC-SIGN positive cells in the inflammatory infiltrate of human CL lesions caused by L. braziliensis. Thus, it is possible that Leishmania uses DC-SIGN receptor to invade DCs or even to drive their functions.

Here we showed the presence of DC-SIGNPOS cells in CL lesions. It is the first description of presence of DC-SIGNPOS cells in CL lesions. Due to the cytoplasm characteristics, the histological aspects resemble macrophages. However, lymphocytes were also stained. In normal skin, DC-SIGN receptor is expressed in dermal DCs but not in Langerhans cells2525. Geijtenbeek TB, Kwon DS, Torensma R, van Vliet SJ, van Duijnhoven GC, Middel J, et al. DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell. 2000; 100(5): 587-97. or in DCs expressing an immature phenotype.1414. Soilleux EJ, Morris LS, Leslie G, Chehimi J, Luo Q, Levroney E, et al. Constitutive and induced expression of DC-SIGN on dendritic cell and macrophage subpopulations in situ and in vitro. J Leukoc Biol. 2002; 71(3): 445-57. DC-SIGN receptor was discovered to bind DCs by the ICAM-3 and, for similar structure, to ICAM-2, but not to ICAM-1. The interaction DC-SIGN-ICAM mediates trans endothelial migration of DCs, from blood to tissues.1616. van Kooyk Y, Geijtenbeek TB. A novel adhesion pathway that regulates dendritic cell trafficking and T cell interactions. Immunol Rev. 2002; 186: 47-56. DC-SIGNPOS cells presenting in CL lesions could be interacting with inflammed endothelial cells, especially those expressing ICAM-2 and ICAM-3, enabling them to exit skin and to home to secondary lymphoid organs. In other skin disorders, like leprosy and psoriasis, DC-SIGNPOS cells were already demonstrated in the inflammatory lesions.1919. Soilleux EJ, Sarno EN, Hernandez MO, Moseley E, Horsley J, Lopes UG, et al. DC-SIGN association with the Th2 environment of lepromatous lesions: cause or effect? J Pathol. 2006; 209(2): 182-9.,2020. Higaki M, Higaki Y, Kawashima M. Increased expression of CD208 (DC-LAMP) in epidermal keratinocytes of psoriatic lesions. J Dermatol. 2009; 36(3): 144-9.,2121. Wei-yuan M, Wen-ting L, Chen Z, Qing S. Significance of DC-LAMP and DC-SIGN expression in psoriasis vulgaris lesions. Exp Mol Pathol. 2011; 91(1): 461-5. In leprosy, the presence of DC-SIGNPOS cells was associated to Th2 environment, being the major bacilli reservoir in lepromatous lesions.1919. Soilleux EJ, Sarno EN, Hernandez MO, Moseley E, Horsley J, Lopes UG, et al. DC-SIGN association with the Th2 environment of lepromatous lesions: cause or effect? J Pathol. 2006; 209(2): 182-9. The function of DC-SIGNPOS cells in CL lesions is still unknown. It is possible that DC-SIGNPOS cells can be infected by Leishmania parasites. DCs with immature phenotype express DC-SIGN receptor and can harbor Leishmania parasites.1414. Soilleux EJ, Morris LS, Leslie G, Chehimi J, Luo Q, Levroney E, et al. Constitutive and induced expression of DC-SIGN on dendritic cell and macrophage subpopulations in situ and in vitro. J Leukoc Biol. 2002; 71(3): 445-57.,2626. Carvalho LP, Pearce EJ, Scott P. Functional dichotomy of dendritic cells following interaction with Leishmania braziliensis: infected cells produce high levels of TNF-a, whereas bystander dendritic cells are activated to promote t cell responses. J Immunol. 2008; 181(9): 6473-80. Interestingly, when co-cultivated with polimorphonuclear cells, L. (L.) amazonensis-infected DCs exhibited lower rates of infection and parasite load, and this phenomenon seemed to be mediated by DC-SIGNPOS in a direct contact-dependent manner.2424. Tiburcio R, Melo LD, Nunes S, Barbosa ALA, de Oliveira EC, Suarez M, et al. DC-SIGN mediates the interaction between neutrophils and Leishmania amazonensis-infected dendritic cells to promote DC maturation and parasite elimination. Front Immunol. 2021; 12: 1-13. This suggest that DC-SIGNPOS cells can uptake Leishmania amastigotes,2222. Colmenares M, Constant SL, Kima PE, McMahon-Pratt D. Leishmania pifanoi pathogenesis: selective lack of a local cutaneous response in the absence of circulating antibody. Infect Immun. 2002; 70(12): 6597-605. corroborating with the hypothesis that DC-SIGNPOS cells in lesions can harbor Leishmania parasites. After infected, DC-SIGNNEG cells monocytes-derivated reduce DC-SIGN expression,2424. Tiburcio R, Melo LD, Nunes S, Barbosa ALA, de Oliveira EC, Suarez M, et al. DC-SIGN mediates the interaction between neutrophils and Leishmania amazonensis-infected dendritic cells to promote DC maturation and parasite elimination. Front Immunol. 2021; 12: 1-13. and are able to migrate to the lymph node, and then present antigens to naïve T cells.

Our results showed that promastigotes from both Lb and La species bind to the DC-SIGN receptor. After two hours of interaction, La is already bound to DC-SIGN receptor, while Lb takes longer to interact with DC-SIGN receptor, i.e., at least 24 h of co-culture. Those differences can influence the clinical and immunological outcome. Both Lb and La cause cutaneous form of leishmaniasis. L. braziliensis can cause a more severe and disfiguring form, mucosal leishmaniasis (ML). The cellular immune response to Lb infection is present and can be hyperactivated in ML.2727. Coutinho SG, Pirmez C, Mendonça SCF, Conceição-Silva F, Dorea RCC. Pathogenesis and immunopathology of leishmaniasis. Mem Inst Oswaldo Cruz. 1987; 82(Suppl.): 214-28. Indeed, there are rare and severe forms of La infection as diffuse cutaneous leishmaniasis, presenting nodular lesions whiting many infected macrophages, absence of cellular immune response and high antibodies titles.2828. Castes M, Agnelli A, Rondon AJ. Mechanisms associated with immunoregulation in human American cutaneous leishmaniasis. Clin Exp Immunol. 1984; 57(2): 279-86.,2929. Xin L, Li K, Soong L. Down-regulation of dendritic cell signalling pathways by Leishmania amazonensis amastigotes. Mol Immunol. 2008; 45(12): 3371-82. Dermotropic Leishmania species induce different patterns of DCs infiltration in lesions, as observed by others.1111. Carvalho AK, Silveira FT, Passero LF, Gomes CM, Corbett CE, Laurenti MD. Leishmania (V.) braziliensis and L. (L.) amazonensis promote differential expression of dendritic cells and cellular immune response in murine model. Parasite Immunol. 2012; 34(8-9): 395-403. In murine cutaneous lesions, La induces a rapid infiltration of DCs with a development of large lesions within many infected macrophages.1111. Carvalho AK, Silveira FT, Passero LF, Gomes CM, Corbett CE, Laurenti MD. Leishmania (V.) braziliensis and L. (L.) amazonensis promote differential expression of dendritic cells and cellular immune response in murine model. Parasite Immunol. 2012; 34(8-9): 395-403.In vitro, La is unable to activate DCs and induce cytokines production.2929. Xin L, Li K, Soong L. Down-regulation of dendritic cell signalling pathways by Leishmania amazonensis amastigotes. Mol Immunol. 2008; 45(12): 3371-82. Moreover, this parasite is capable of altering a DC differentiation associated marker.3030. Favali C, Tavares N, Clarencio J, Barral A, Barral-Netto M, Brodskyn C. Leishmania amazonensis infection impairs differentiation and function of human dendritic cells. J Leukoc Biol. 2007; 82(6): 1401-6. In the other hand, in Lb murine infection, DCs infiltrate coincides with lesion regression and increased number of infiltrating T cells.1111. Carvalho AK, Silveira FT, Passero LF, Gomes CM, Corbett CE, Laurenti MD. Leishmania (V.) braziliensis and L. (L.) amazonensis promote differential expression of dendritic cells and cellular immune response in murine model. Parasite Immunol. 2012; 34(8-9): 395-403. In DCs cultures with Lb, both DC non-infected and activated cells and DC Leishmania-infected and deactivated cells were found. Both DCs were influenced by L. braziliensis infection and contributed to control and immunopathology of the CL.2626. Carvalho LP, Pearce EJ, Scott P. Functional dichotomy of dendritic cells following interaction with Leishmania braziliensis: infected cells produce high levels of TNF-a, whereas bystander dendritic cells are activated to promote t cell responses. J Immunol. 2008; 181(9): 6473-80. Besides, it was shown that DC-SIGN mediates contact between human polimophonuclear cells and DCs, resulting in increased release of proinflammatory markers and reduced rates of La infection.2424. Tiburcio R, Melo LD, Nunes S, Barbosa ALA, de Oliveira EC, Suarez M, et al. DC-SIGN mediates the interaction between neutrophils and Leishmania amazonensis-infected dendritic cells to promote DC maturation and parasite elimination. Front Immunol. 2021; 12: 1-13.

Our results addressing American Leishmania species are in consonance with previous data, in which lower L. major-DC-SIGN interaction was observed in comparison to L. pifanoi-DC-SIGN.2323. Colmenares M, Corbi AL, Turco SJ, Rivas L. The dendritic cell receptor DC-SIGN discriminates among species and life cycle forms of Leishmania. J Immunol. 2004; 172(2): 1186-90. Here, we showed that the Lb binding to DC-SIGN receptor is delayed while La promptly binds to the same receptor. Moreover, La binds to the DC-SIGNHi population 3.5-fold more than to DC-SIGNLow. On the order hand, Lb promastigotes do not discriminate between the DC-SIGNLow or DC-SIGNHi populations. Together, these results suggest that antigenic differences among the Leishmania species could be influencing the DC-SIGN receptor binding.

The present results extend the studies on the role of DC-SIGN receptor to species with epidemiological importance in the Americas. We showed, for the first time, that DC-SIGN positive cells are present in the inflammatory infiltrate of CL lesions caused by Lb. Curiously, although both Lb and La interact with DC-SIGN receptor, they differ in binding intensity and in the time for initiating the interaction. The exact function of DC-SIGNPOS cells in leishmaniasis has to be clarified, but our results indicate that Leishmania parasites can encounter this receptor in cells from infected skin and maybe they utilize it to infect DCs.

ACKNOWLEDGEMENTS

To Dr Andrea Henriques Pons and Alessandro Marins / Plataforma de Citometria de Fluxo, IOC, FIOCRUZ, RJ, Brazil for flow cytometry acquisition. We thank Dr Hebert L Guedes for his helpful suggestions and manuscript revision.

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    Geijtenbeek TB, Kwon DS, Torensma R, van Vliet SJ, van Duijnhoven GC, Middel J, et al. DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell. 2000; 100(5): 587-97.
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    Carvalho LP, Pearce EJ, Scott P. Functional dichotomy of dendritic cells following interaction with Leishmania braziliensis: infected cells produce high levels of TNF-a, whereas bystander dendritic cells are activated to promote t cell responses. J Immunol. 2008; 181(9): 6473-80.
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    Coutinho SG, Pirmez C, Mendonça SCF, Conceição-Silva F, Dorea RCC. Pathogenesis and immunopathology of leishmaniasis. Mem Inst Oswaldo Cruz. 1987; 82(Suppl.): 214-28.
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    Castes M, Agnelli A, Rondon AJ. Mechanisms associated with immunoregulation in human American cutaneous leishmaniasis. Clin Exp Immunol. 1984; 57(2): 279-86.
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    Xin L, Li K, Soong L. Down-regulation of dendritic cell signalling pathways by Leishmania amazonensis amastigotes. Mol Immunol. 2008; 45(12): 3371-82.
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    Favali C, Tavares N, Clarencio J, Barral A, Barral-Netto M, Brodskyn C. Leishmania amazonensis infection impairs differentiation and function of human dendritic cells. J Leukoc Biol. 2007; 82(6): 1401-6.
  • Financial support: This work was supported by IOC/FIOCRUZ internal funds and FAPERJ (E-26/102.457/2010). COM-A and pos-doctor are sponsored by FAPERJ/CAPES, Brazil; MYKO is a doctorate student sponsored by IOC/FIOCRUZ; AMD-C was sponsored by CNPq and FAPERJ research fellows.

Publication Dates

  • Publication in this collection
    27 Mar 2023
  • Date of issue
    2023

History

  • Received
    11 Apr 2022
  • Accepted
    06 Feb 2023
Instituto Oswaldo Cruz, Ministério da Saúde Av. Brasil, 4365 - Pavilhão Mourisco, Manguinhos, 21040-900 Rio de Janeiro RJ Brazil, Tel.: (55 21) 2562-1222, Fax: (55 21) 2562 1220 - Rio de Janeiro - RJ - Brazil
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