Glyburide, a NLRP3 Inhibitor, Decreases Inflammatory Response and Is a Candidate to Reduce Pathology in Leishmania braziliensis Infection

TO THE EDITOR

Cutaneous leishmaniasis (CL) is caused by Leishmania braziliensis and is characterized by an exaggerated inflammatory response that leads to parasite control, but it is also the cause of tissue damage and ulcer formation (Carvalho et al., 2012). The NLRP3 inflammasome is an intracellular protein complex that is activated by pathogen-associated molecular patterns or danger-associated molecular patterns, promoting activation of caspase 1 and release of active IL-1β (Latz et al., 2013). In C57BL/6 mice infected with a non-healing L. major strain, NLRP3 inflammasome and IL-1β were associated with severe pathology (Charmoy et al., 2016), and IL-1β caused severe disease in BALB/C mice infected with L. major (Voronov et al., 2010). Recently, we showed that CD8⁺ T cells induce immunopathology by NLRP3 inflammasome activation and IL-1β production in mice infected with L. braziliensis, and inhibitors of NLRP3 reduces pathology (Novais et al., 2017).

Glyburide is an FDA-approved ATP-sensitive K⁺ channel inhibitor used for the treatment of type 2 diabetes. Recently, it was shown that glyburide has anti-inflammatory effects mainly by inhibition of NLRP3 inflammasome and decreasing IL-1β release. (Lamkanfi et al., 2009). Furthermore, anti-Leishmania activity by glyburide was previously documented (Ponte-Sucre et al., 1997; Serrano-Martín et al., 2006). Additionally, glyburide may act in synergy with pentavalent antimony (Sb^v) promoting L. major killing (Padrón-nieves et al., 2009).

Here, we evaluated the ability of glyburide to reduce the systemic and local inflammatory response of patients with CL and it is leishmanicidal effect against intracellular amastigotes of L. braziliensis.

This study was conducted in an area of L. braziliensis transmission and participants included 11 patients with CL. This study was approved by the Ethical Committee of the Federal University of Bahia Medical School and written informed consent was obtained from all participants. A detailed description of the methods is presented in the Supplementary Materials. Peripheral blood mononuclear cells were cultured with soluble Leishmania antigens in the presence or absence of glyburide. Unstimulated cells produced low or undetectable levels of cytokines. The levels of Leishmania-specific IL-1β significantly decreased after addition of glyburide in a dose dependent manner (Figure 1a). Overall, the median of IL-1β suppression was 72%, 86%, and 97% at the concentrations of 50, 100, and 200 μM, respectively. IL-17 and tumor necrosis factor (TNF) levels also decreased in the presence of the drug (Figure 1b and c). The percentage of suppression of IL-17 and TNF with 200 μM of glyburide was 95% and 69%, respectively. No changes in IFN-γ, IL-6, and IL-10 levels were observed (Figure 1d–f).

Figure 1. Glyburide downmodulates SLA-induced cytokine production in PBMC from patients with CL.

PBMC from 11 patients with CL were cultured in presence of SLA (5 μg) plus glyburide in different concentrations or plus drug diluent (DMSO) for 72 hours. Cytokine levels in culture supernatants were measured by ELISA. Levels of (a) IL-1β, (b) IL-17, (c) TNF, (d) IFN-γ, (e) IL-6, and (f) IL-10 are shown. Central lines represent median values. *P < 0.05, **P < 0.01, ****P < 0.0001. CL, cutaneous leishmaniasis; PBMC, peripheral blood mononuclear cells; SLA, soluble Leishmania antigen; TNF, tumor necrosis factor.

Next, we evaluated the ability of glyburide to downmodulate the inflammatory response in L. braziliensis lesions. Addition of glyburide decreased the levels of IL-1β (Figure 2a), IL-17 (Figure 2b), and TNF (Figure 2c) by 95%, 65%, and 83%, respectively. There was no change in the levels of IFN-γ, IL-6, and IL-10. These results indicated that glyburide was able to reduce both systemic and tissue inflammatory response in patients with CL, without toxic effects to host cells (Supplementary Figure S1).

Figure 2. Glyburide downmodulates pro-inflammatory cytokine release from lesion biopsies of patients with CL.

L. braziliensis lesions skin biopsies from 11 patients with CL were cut in half, and one half was cultured in media plus DMSO and the other half with media plus glyburide (200 μM) for 48 hours. Cytokine levels in biopsy supernatants were measured by ELISA. Levels of (a) IL-1β, (b) IL-17, (c) TNF, (d) IFN-γ, (e) IL-6, and (f) IL-10 are shown. **P < 0.01. CL, cutaneous leishmaniasis; TNF, tumor necrosis factor.

As an exaggerated inflammatory response is associated with pathology in L. braziliensis infection, we performed a correlation between cytokine levels in biopsy cultures and lesion size and illness duration. Despite the small sample size, we found a positive correlation between IL-1β levels and lesion size (r = 0.6, P = 0.04) and illness duration (Supplementary Figure S2a and b).

Next, we evaluated the anti-amastigote activity of glyburide in L. braziliensis infected macrophages. Treatment with glyburide did not modify the infection rates, nor impaired the ability of amphotericin B to kill L. braziliensis in infected macrophages (Supplementary Figure S3a and b).

Macrophage activation by IFN-γ and TNF is the main defense mechanism against Leishmania parasites. However, an exaggerated inflammatory response may lead to tissue damage and consequently the development of ulcerated lesions. NLRP3 inflammasome is involved in activation of caspase 1, which is important for maturation and secretion of IL-1β. Here, we found a modulatory activity of glyburide in Leishmania-specific IL-1β levels in a dose dependent manner. In peripheral blood mononuclear cells, IL-1β only occurred in soluble Leishmania antigens-stimulated cultures, but glyburide act mainly in monocytes as IFN-γ production was not affected. IL-1β production in CL seems dependent on NLRP3 activation. This result is in concordance with our previous finding in mice infected with L. braziliensis treated with glyburide or in NLRP3-deficient mice (Novais et al., 2017; Santos et al., 2018). In addition, NLRP3 inflammasome and IL-1β have been associated with disease severity in leishmaniasis in both mice and humans (Novais et al., 2017; Fernández-Figueroa et al., 2012; Santos et al., 2018). Our documentation of a positive correlation between IL-1β levels and lesion size, although with a small number of patients evaluated, argues in favor of IL-1β overproduction promoting pathology in human CL caused by L. braziliensis. Moreover, we found that high dose of glyburide induced a significant reduction of IL-17 and TNF levels, inflammatory cytokines that are also associated with pathology in Leishmania infection (Gonzalez-Lombana et al., 2013; Carvalho et al., 2013).

Here, we did not find any change in infection rates of L. braziliensis in macrophages after treatment with glyburide. This is in agreement with our previous observation that treatment with glyburide did not affect parasite load in mice infected with L. braziliensis (Novais et al., 2017). In contrast, deficiency in NLRP3 inflammasome was associated with a decrease in NO production and impaired parasite control in mice infected with Leishmania amazonensis (Lima-Junior et al., 2013). As NO seems to be less important to L. braziliensis killing in humans (Carneiro et al., 2016), it is likely that NLRP3 inflammasome does not play an important role in parasite control in patients with CL because of L. braziliensis.

The standard drug for CL treatment is Sb^V, a highly toxic drug with growing therapeutic failure rates. Since the inflammatory response is a major cause of pathology in CL, a combination therapy composed by an anti-Leishmania drug plus an anti-inflammatory agent should be considered. Here, we showed that in vitro addition of glyburide decreases the inflammatory response without decreasing IFN-γ production, suggesting that this drug may reduce pathology without affecting the main mechanism of parasite control. Therefore, we encourage clinical trials using glyburide as an adjuvant therapy to treat CL.

Abbreviations:

CL

cutaneous leishmaniasis

TNF

tumor necrosis factor