More than just exosomes: distinct Leishmania infantum extracellular products potentiate the establishment of infection

Abstract

The use of secretion pathways for effector molecule delivery by microorganisms is a trademark of pathogenesis. Leishmania extracellular vesicles (EVs) were shown to have significant immunomodulatory potential. Still, they will act in conjunction with other released parasite-derived products that might modify the EVs effects. Notwithstanding, the immunomodulatory properties of these non-vesicular components and their influence in the infectious process remains unknown. To address this, we explored both in vitro and in vivo the immunomodulatory potential of promastigotes extracellular material (EXO), obtained as a whole or separated in two different fractions: EVs or vesicle depleted EXO (VDE). Using an air pouch model, we observed that EVs and VDE induced a dose-dependent cell recruitment profile different from the one obtained with parasites, attracting significantly fewer neutrophils and more dendritic cells (DCs). Additionally, when we co-inoculated parasites with extracellular products a drop in cell recruitment was observed. Moreover, in vitro, while VDE (but not EVs) downregulated the expression of DCs and macrophages activation markers, both products were able to diminish the responsiveness of these cells to LPS. Finally, the presence of Leishmania infantum extracellular products in the inoculum promoted a dose-dependent infection potentiation in vivo, highlighting their relevance for the infectious process. In conclusion, our data demonstrate that EVs are not the only relevant players among the parasite exogenous products. This, together with the dose-dependency observed, opens new avenues to the comprehension of Leishmania infectious process. The approach presented here should be exploited to revisit existing data and considered for future studies in other infection models.

Keywords: Leishmania; air pouch; dendritic cells; exosomes; extracellular vesicles; immunomodulation; infection; macrophages.

Figure 1.

EVs and VDE represent distinct aspects of the total EXO. Representative TEM images of equivalent amounts of EXO (I) and the respective fractions VDE (II) and EVs (III) stained with acetate uracil (a). Average diameter of EVs preparations determined by TEM, DLS and NTA (b). Polydispersity index (PDI) and average particle number/ml of the preparations are also represented (b). Average dispersion of particle size (diameter) in the EVs population determined by direct measurement from TEM preparations (c). All the data are presented as average ± SD from a minimum of 5 independent preparations.

Figure 2.

Leishmania infantum extracellular products promote a dose-dependent differential cell recruitment. Relative number of total cells (a), eosinophils, neutrophils, macrophages/monocytes and dendritic cells (normalized against the PBS recruitment) (b) and relative cellular abundance (c) in mice pre-formed air pouches 6 h after the injection of PBS (crossed dots), LPS (dark grey dots), 10⁶, 10⁷ and 10⁸ L. infantum promastigotes (black dots) or the EXO (medium grey dots), VDE (light grey dots) and EVs (white dots) produced by the same number of parasites (e⁶, e⁷ and e⁸). Cells were quantified using a Neubauer chamber and the phenotype analysed by flow cytometry. Each dot represents an animal. Bars represent the average (± SEM) obtained from the animals studied in three different experiments. One-sample t-test was used to assess statistical significances comparing to the PBS control group. Significant differences are highlighted in the figure: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 3.

Leishmania infantum extracellular products alter the cell recruitment profile induced by the parasite. Relative number of total cells (a), frequencies of eosinophils, neutrophils, macrophages and dendritic cells (b) and relative cellular abundance (c) in mice pre-formed air pouches, 6 h after the injection of 10⁶ L. infantum promastigotes (black), alone or together with EXO (dark grey), VDE (light grey) and EVs (white) produced by 10⁷ parasites (e⁷). Each dot represents an animal; bars (a) and horizontal lines (b) indicate group means (± SEM) of each experimental group. One-sample t-test was used to assess statistical significances compared to parasites only. Significant differences are highlighted in the figure: ** p ≤ 0.01.

Figure 4.

In vitro dose-dependent immunomodulatory effect of L. infantum extracellular material on BMDC and BMMØ. Normalized expression levels of CD86, CD40 and MHC-II in BMDC and BMMØ cultured for 48 h in the presence of increasing amounts of EXO (dark grey boxes), VDE (light grey boxes) and EVs (white boxes), without (a) or with (b) LPS re-stimulation 24 h after the addition of the primary stimuli. In re-stimulation assays, black boxes represent the results obtained for the positive control (LPS stimulation during the last 24 h of culture). Results are relative to the values obtained for non-stimulated cells (dotted line) and boxes illustrate the average, minimum and maximum values of three different experiments. One-sample t-test was used to assess statistical significances to the hypothetical negative (a) and positive (b) values. Significant differences are highlighted in the figure: * p ≤ 0.05; ** p ≤ 0.01.

Figure 5.

Leishmania infantum extracellular material favours infection. (a) BMDC and BMMØ were infected during 4 h with L. infantum promastigotes in the presence or not of Leishmania extracellular products. Parasites were then washed away and cells recovered or left in culture. Four, twenty-four or forty-eight  hour after infection cells were cytospined, stained by the Giemsa method and the infection analysed under the microscope. The percentage of infected cells at the different time-points is indicated. Symbols and bars represent the mean ± SEM of three independent experiments. (b) Splenic parasite burdens of mice infected intraperitoneally with 10⁷ L. infantum promastigotes together with EXO (dark grey dots), VDE (light grey dots) or EVs (white dots) produced by10⁷ (e⁷), 10⁸ (e⁸) or 10⁹ (e⁹) parasites. Every symbol represents a mouse. Lines represent the mean of each experimental group. Statistical significance was attained comparing each group with the control group (mice infected only with 10⁷ L. infantum promastigotes, dashed line, n = 6), using the One-sample t-test. Significant differences are highlighted in the figure: * p ≤ 0.05, *** p ≤ 0.001.