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Leishmania donovani Utilize Sialic Acids for Binding and Phagocytosis in the Macrophages through Selective Utilization of Siglecs and Impair the Innate Immune Arm

PLoS Neglected Tropical Diseases (2016) 10(8)
DOI: 10.1371/journal.pntd.0004904
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Abstract

Background: Leishmania donovani, belonging to a unicellular protozoan parasite, display the differential level of linkage-specific sialic acids on their surface. Sialic acids binding immunoglobulin-like lectins (siglecs) are a class of membrane-bound receptors present in the haematopoetic cell lineages interact with the linkage-specific sialic acids. Here we aimed to explore the utilization of sialic acids by Leishmania donovani for siglec-mediated binding, phagocytosis, modulation of innate immune response and signaling pathways for establishment of successful infection in the host. Methodology/Principle Findings: We have found enhanced binding of high sialic acids containing virulent strains (AG83+Sias) with siglec-1 and siglec-5 present on macrophages compared to sialidase treated AG83+Sias (AG83-Sias) and low sialic acids-containing avirulent strain (UR6) by flow cytometry. This specific receptor-ligand interaction between sialic acids and siglecs were further confirmed by confocal microscopy. Sialic acids-siglec-1-mediated interaction of AG83+Sias with macrophages induced enhanced phagocytosis. Additionally, sialic acids-siglec-5 interaction demonstrated reduced ROS, NO generation and Th2 dominant cytokine response upon infection with AG83+Sias in contrast to AG83-Sias and UR6. Sialic acids-siglecs binding also facilitated multiplication of intracellular amastigotes. Moreover, AG83+Sias induced sialic acids-siglec-5-mediated upregulation of host phosphatase SHP-1. Such sialic acids-siglec interaction was responsible for further downregulation of MAPKs (p38, ERK and JNK) and PI3K/Akt pathways followed by the reduced translocation of p65 subunit of NF-κβ to the nucleus from cytosol in the downstream signaling pathways. This sequence of events was reversed in AG83-Sias and UR6-infected macrophages. Besides, siglec-knockdown macrophages also showed the reversal of AG83+Sias infection-induced effector functions and downstream signaling events. Conclusions/Significances: Taken together, this study demonstrated that virulent parasite (AG83+Sias) establish a unique sialic acids-mediated binding and subsequent phagocytosis in the host cell through the selective exploitation of siglec-1. Additionally, sialic acids-siglec-5 interaction altered the downstream signaling pathways which contributed impairment of immune effector functions of macrophages. To the best of our knowledge, this is a comprehensive report describing sialic acids-siglec interactions and their role in facilitating uptake of the virulent parasite within the host.

Figures

  • Table 1. Primer sequences.
  • Fig 1. Identification of siglecs involved in binding of Leishmania promastigotes with the host cells. (A) Interaction between AG83+Sias promastigotes and different soluble siglec-Fc chimeras (Siglec-1, 3, 5, 7, 9 and 10) was quantified in overlay dot blot. Promastigotes (1×107), in PBS (50 μl) were soaked on nitrocellulose membrane. The air dried membrane was incubated with different soluble siglec-Fc chimeras followed by HRP-Protein-A and developed by DAB as described in materials and methods. (B) Sialic acids-siglec mediated binding of L. donovani
  • Fig 2. Comparison of Sias-siglec mediated binding between a virulent strain (AG83+Sias) and avirulent strain (UR6) with macrophages/monocytes. Sias-siglec mediated binding of AG83+Sias and UR6 with J774A.1 (A) and THP-1 (B) cells were measured by flow cytometry. AG83+Sias and UR6 were left untreated or treated with sialidase (AG83-Sias) and labeled with FITC. J774A.1/THP-1 cells were treated with cocktail of antisiglec antibodies. Cocktail of antibodies were used as inhibitors due to the presence of many siglecs in variable
  • Fig 3. Siglec-1 mediated phagocytosis of promastigotes in macrophages. (A) Sias-siglec dependent phagocytosis was quantitated by flow cytometry. J774A.1 (1× 106/well) cells were grown in a six well plate, either left untreated or blocked with anti-siglec-1 antibody (10 μg/ml) for 1 hr. Treated and untreated cells were then infected with FITC-AG83+Sias and AG83-Sias at 1:10 ratio for 2 hr at 37°C. Unbound parasite removed and extracellular fluorescence was quenched by trypan blue (0.02%) treatment. Cells were processed and visible fluorescence intensity was analyzed by flow cytometry which was due to phagocytosed parasites. (B) J774A.1 cells (2×104) were adhered on glass coverslip for 48 hr. The cells were left untreated or blocked with anti-siglec-1 antibody for 1 hr at 4°C. They were infected with FITC-AG83+Sias and FITC-AG83-Sias promastigotes for phagocytosis as described in Fig. 3A. Infected macrophages were stained with PE-anti-CD14 antibody, fixed in paraformaldehyde and mounted in mounting media containing DAPI. The image was visualized in Andor Spinning Disc Confocal microscopy.
  • Fig 4. Sias-siglec interaction mediated suppression of innate immune response. (A) J774A.1 cells (1×106/well) were left uninfected or infected with AG83+Sias or sialidase-treated (AG83-Sias) or UR6 promastigotes in a six well plate at 1:10 ratio for 1 hr. Unbound parasites were removed and intracellular ROS production was measured by H2DCFDA staining by FACS. Data was analyzed by CellQuestPro software. (B) J774A.1 cells (1×10 6/ well) were kept uninfected or infected with AG83+Sias or sialidase-treated (AG83-Sias) or UR6 similarly for 4 hr. Unbound parasites were washed and infection was allowed for additional 20 hr. The NO secretion was measured in the culture supernatant by Griess reaction. (C) J774A.1 cells (1×106/well) infected with AG83+Sias or AG83-Sias or UR6 for 4 hr as stated in Fig. 4B. Infection was allowed for additional 20 hr in the presence of LPS (2.5 μg/ml) or PMA (25 ng/ml)-ionomycin (1.0 μg/ml) as stimulator for cytokines secretion. The total RNA was isolated by Quigen RNeasy Mini Kit. cDNA was prepared from RNA by reverse transcriptase Kit. Then the specific expression of the Th1 (IL-2, IFNγ) and Th2 (IL-4, IL-10, TGFβ) cytokine genes along with iNOS expression was measured in PCR by using specific primers as listed in Table 1. GAPDH was used as loading control throughout. (D) Total RNA was isolated from infected and uninfected J774A.1 cells and cDNA was prepared similarly as stated in Fig. 4C. The level of mRNA expression of Th1 and Th2 cytokines along with iNOSwere quantified by real time PCR analysis. The mRNA level was normalized by 18s rRNA and the fold change was measured using uninfected cells as the control. Comparison was also made of AG83+Sias infection with AG83-Sias or UR6 infection. (E) Secreted cytokines were also measured in BMDM cells. BMDMs were isolated from the femur of healthy mice and macrophages were infected and stimulated by LPS or PMAionomycin as stated in Fig. 4C. The secreted cytokines (IL-4, IL-10 and IFNγ) in the culture supernatants were measured by respective ELISA kit as described in material and methods. (F) J774A.1 cells (1×104) were adhered overnight on glass coverslip. Macrophages were left untreated or blocked with anti-siglec antibody cocktail (anti-siglec-1,-E,-F) for 1hr. Untreated and treated cells were infected with AG83+Sias or AG83-Sias or UR6 for 4 hr at 1:10
  • Fig 5. Sias-mediated modulation in the SHP-1 and downstream signaling pathways. (A) J774A.1 cells (1×106/well) were uninfected or infected with AG83+Sias or AG83-Sias or UR6 at 1:10 ratio for 4 hr. Unbound parasites were removed. The cell lysate was prepared; proteins were quantified, separated in SDS-PAGE. They were transferred to nitrocellulose membrane and incubated overnight with anti-siglec-E and SHP-1 antibodies. α-tubulin was used as a control. The blots were incubated with respective secondary antibody and developed by ECL kit. Densitometric score mentioned below the each band was
  • Fig 6. Sias-mediated interaction by L. donovanimodulate a functional subunit of NF-κβ translocation in the nucleus. J774A.1 cells were uninfected or infected with AG83+Sias or AG83-Sias or UR6 at 1:10 for 12 hr, unbound parasites were removed. Cytosol (A) and Nuclear (B) fractions were separated as described in materials and methods and subjected to western blotting with indicated antibodies. Both α-tubulin and HDAC3 were used as loading controls for cytosol and nucleus respectively.
  • Fig 7. Siglec-E knockdown frommacrophages modulated macrophage effector functions and downstream signalingmolecules/pathways during L. donovani infection. (A) J774A.1 cells were either untransfected or transfected with siSiglec-E and infected with AG83+Sias for 1 hr. ROS generation was measured by H2DCFDA staining similarly as stated in Fig 4A. (B) J774A.1 cells were transfected with siSiglec-E and infected with AG83+Sias overnight. The culture supernatant was collected and nitric oxide was

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Roy, S., & Mandal, C. (2016). Leishmania donovani Utilize Sialic Acids for Binding and Phagocytosis in the Macrophages through Selective Utilization of Siglecs and Impair the Innate Immune Arm. PLoS Neglected Tropical Diseases, 10(8). https://doi.org/10.1371/journal.pntd.0004904

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