Monocytes in disseminated leishmaniasis

<b>Figure 1</b>: <b>Frequency of infected monocytes and parasite load after infection with different isolates of <i>L. braziliensis</i>: </b>PBMC-derived monocytes from patients with DL (n=12) or CL (n=12) were infected with different isolates of <i>L. braziliensis</i>at a ratio of 5:1 for 2 h or 48 h. The number of infected cells (A) and the number of intracellular parasites (B) were evaluated by optical microscopy in 100 monocytes following Romanowsky staining. <b>Figure 2</b>: <b>Viability of extracellular promastigotes in the supernatants of cultured monocytes from patients with DL, CL or healthy subjects following infection with different <i>L. braziliensis</i>isolates: </b>PBMC-derived monocytes from patients with DL (n=12), CL (n=12) or HS (n=12) were infected with different isolates of<i>L. braziliensis </i>(DL or CL) for 48 hours. After this period RPMI medium was replaced by Schneider culture medium for another 48 hours. The number of viable promastigotes was evaluated by optical microscopy. A) Monocytes from patients with DL; B) Monocytes from patients with CL; C) Monocytes from HS. D) Viability of promastigotes in CL and DL monocytes following infection with different isolates. <b>Figure 3: Induction of oxidative burst by monocytes from patients with DL, CL or healthy subjects following infection with different <i>L. braziliensis</i>isolates: </b>PBMC-derived<b></b>monocytes from patients with DL (n=12), CL (n=12) or HS (n=8) were treated with DHR (10ng/mL; 10 min) and infected with <i>L. braziliensis</i>isolates from DL or CL patients for 25 minutes at a ratio of 5:1 cells. PMA (1ug/ml) was used as positive control. Cells were stained with anti-CD14 for flow cytometric evaluation. (A) Representative gating strategy detailing CD14+ and DHR expression in monocytes from a CL patient. (B) Data representative of median mean fluorescence intensity (MFI) (MIF) of oxidative burst induction in DL and CL monocytes infected with different isolates. (C) Representative oxidative burst expression in monocytes from different clinical forms compared to PMA (100% induction). <b>Figure 4</b>:<b>TLR2 and TLR4 expression in monocytes from DL and CL patients</b>. PBMC-derived<b></b>monocytes from DL (n=12) and CL (n=12) patients were infected with different isolates of <i>L. braziliensis </i>at a ratio of 5:1 for 2 h. Following stimulation, monocytes were marked with anti-CD14 antibodies, and with anti-TLR2 or anti-TLR4 for flow cytometry analysis. (A) Figure representative of flow cytometry gating stragegy<b>;</b>(B) Expression of TLR2 (C) Expression of TLR4. <b>Figure 5: Production of TNF, CXCL9 and CXCL10 by monocytes from patients with DL and CL following infection with different <i>L. braziliensis</i>isolates: </b>PBMC-derived<b></b>monocytes from patients with DL (n=9) and CL (n=6) were infected with different isolates of <i>L. braziliensis </i>(5:1) for 2 h. Cells were treated for 6 h with Stop Golgi, followed by surface staining with an anti-CD14 antibody for monocyte characterization. After permeative treatment, cells were marked with anti-TNF, anti-CXCL9 and anti-CXCL10 antibodies for flow cytometric analysis. (A) Representative graph illustrating intracellular production by flow cytometry. (B) TNF expression (C) CXCL9 expression. (D) CXCL10 expression. <b></b><b> </b><b>Sup. Fig. 1: </b><b>Frequency of infected monocytes and parasite load after infection with different isolates of <i>L. braziliensis: </i></b>PBMC-derived monocytes from healthy subjects were infected with different isolates of <i>L. braziliensis</i>at a ratio of 5:1 for 2 h or 48hrs. The number of infected cells <b>(A)</b>and the number of intracellular parasites <b>(B)</b>were evaluated by optical microscopy in 100 monocytes following Romanowsky staining. <b> </b><b>Sup. Fig. 2. Expression of oxidative burst by monocytes from healthy subjects infected with different <i>Leishmania braziliensis</i>isolates: </b>Peripheral blood mononuclear cells (PBMC) from healthy subjects (n = 8) were stimulated with dihidrohodamine 123 (DHR) for 10 minutes. Monocytes were infected with different isolates of <i>Leishmania braziliensis</i>at a 5:1 ration for 20 minutes and stained with antibody anti-CD14. The data represent the median of mean intensity of fluorescence (MIF) of oxidative burst production by monocytes. <b> </b><b>Sup. Fig. 3: Expression of TLR2 and TLR4 in monocytes from healthy subjects infected with different <i>Leishmania braziliensis</i>isolates: </b>Peripheral blood mononuclear cells (PBMC) from healthy subjects (N=7) were infected with the different isolates of <i>Leishmania braziliensis</i>(5: 1) for 2 hours and stained with anti-CD14, anti-TLR2 and anti-TLR4 antibodies. <b>(A) </b>The data represent the median of mean intensity of fluorescence (MIF) of TLR2 expression. <b>(B)</b>The data represent the median of mean intensity of fluorescence (MIF) of TLR4 expression<br><br>