IL-1β Production by Intermediate Monocytes Is Associated with Immunopathology in Cutaneous Leishmaniasis

Abstract

Cutaneous leishmaniasis due to Leishmania braziliensis infection is an inflammatory disease in which skin ulcer development is associated with mononuclear cell infiltrate and high levels of inflammatory cytokine production. Recently, NLRP3 inflammasome activation and IL-1β production have been associated with increased pathology in murine cutaneous leishmaniasis. We hypothesized that cutaneous leishmaniasis patients have increased expression of NLRP3, leading to high levels of IL-1β production. In this article we show high production of IL-1β in biopsy samples and Leishmania antigen-stimulated peripheral blood mononuclear cells from patients infected with L. braziliensis and reduced IL-1β levels after cure. IL-1β production positively correlated with the area of necrosis in lesions and duration of the lesions. The main source of IL-1β was intermediate monocytes (CD14⁺⁺CD16⁺). Furthermore, our murine experiments show that IL-1β production in response to L. braziliensis was dependent on NLRP3, caspase-1, and caspase-recruiting domain (ASC). Additionally, we observed an increased expression of the NLRP3 gene in macrophages and the NLRP3 protein in intermediate monocytes from cutaneous leishmaniasis patients. These results identify an important role for human intermediate monocytes in the production of IL-1β, which contributes to the immunopathology observed in cutaneous leishmaniasis patients.

Figure 1. IL-1β is produced by L. braziliensis-infected individuals during active disease

IL-1β concentrations were determined by ELISA on supernatants of PBMC (a and b) and biopsies (c). (a) PBMC from HS (N=10), ECL (N=20), CL (N=19) and CCL (N=19) were cultured in the presence or absence of soluble Leishmania antigen (SLA). (b) PBMC from CL on days 0 and 60 post treatment were cultured with or not SLA. (c) Biopsies from healthy skin from HS (N=15) and lesions from CL patients (N=15) were cultured for 72 hours. (d) Correlation of IL-1β⁺ cells and areas of necrosis (%) (N=26). (e) Immunohistochemistry for IL-1β was performed on lesion of CL patients (20X). Scale bar = 0.1 mm. Statistical analysis was performed using Manny Whitney and Pearson correlation tests. * p<0.05, ** p<0.001; ***p<0.0001.

Figure 2. IL-1β levels correlates with disease progression

Correlation of IL-1β concentration on supernatants of PBMC with lesion size (a), time since lesion appearance (b) and time to heal (c). Statistical analysis was performed using Pearson correlation test.

Figure 3. NLRP3, ASC and Caspase-1 are necessary for IL-1β production in mouse macrophages

BMDMs from wild-type C57BL/6 mices and deficient for NLRP3, ASC, Caspase-1/11, AIM2 and IL-1R were prepared, pulsed with LPS (500 ng/ml) and infected with L. braziliensis (MOI 10:1) or stimulated with monosodium urate (MSU) (250 µg/ml). After 48 hours of culture ELISA for IL-1β was performed on supernatants. * p<0.001, two-way ANOVA with Bonferroni’s posttest, compared with wild-type C57BL/6.

Figure 4. Intermediate monocytes express NLRP3

(a) NLRP3 and AIM2 gene expression, represented as 2^−ΔΔCT, following RT-PCR of RNA from macrophages-derived monocytes of HS (N=5) and CL (N=5) stimulated or not with SLA for 2 hours. (b) Gating strategy to assess monocyte subsets based on size and complexicity, followed by CD14 and CD16 expression and histograms representative of NLRP3 expression in infected monocytes. The gate strategy was done based on all minus one staining. (c) Frequency of NLRP3 ex vivo expression was determined by intracellular staining in monocyte subsets. (d) Monocytes from CL patients were infected or not with L. braziliensis (ratio 5:1) and labeled for CD14, CD16 and NLRP3. Statistical analyses were performed using the Mann-Whitney test and the Wilcoxon rank test. * p<0.05, ** p<0.01.

Figure 5. Intermediate monocytes are the main source of IL-1β

PBMC were obtained from HS (N =5), and cultured in presence or absence of SLA for 8 hours in the presence of Golgi Stop. Staining for CD14, CD16 and the frequency of monocyte subsets producing IL-1β was determined by intracellular staining. (a) Representative plots showing frequencies of monocyte subsets producing IL-1β. (b) Frequency of IL-1β producing cells from each monocyte subset. Statistical analysis was performed using the Wilcoxon rank test and results were considered significant with a **p< 0.005.

Figure 6. Phagocytosis and killing of Leishmania does not depend on presence of IL-1β

Monocytes-derived macrophages from HS (N=5) were infected with L. braziliensis in the stationary phase (ratio 5:1), stimulated with recombinant (rhIL-1β) (20 ng/ml) or anti-IL-1β (5µg/mL) and cultured for 2, 48 and 96 hours. (a) Frequency of infected cells at different time points. (b) Number of Leishmania amastigotes/100 macrophages. Data represent the mean ± SD.