Aloe vera downregulates LPS-induced inflammatory cytokine production and expression of NLRP3 inflammasome in human macrophages

Highlights

• Aloe vera reduces inflammatory cytokine production in LPS-treated human macrophages.

• Aloe vera inhibits the expression of IL-1beta and Nlrp3 inflammasome components.

• P2X7 receptor expression is downregulated in Aloe-treated activated macrophages.

• LPS-induced activation of signaling pathways like NFkB, p38 and JNK is inhibited.

Abstract

Aloe vera has been used in traditional herbal medicine as an immunomodulatory agent inducing anti-inflammatory effects. However, its role on the IL-1β inflammatory cytokine production has not been studied. IL-1β production is strictly regulated both at transcriptional and posttranslational levels through the activity of Nlrp3 inflammasome. In this study we aimed to determine the effect of Aloe vera on the molecular mechanisms of Nlrp3 inflammasome-mediated IL-1β production in LPS-activated human THP-1 cells and monocyte-derived macrophages. Our results show that Aloe vera significantly reduced IL-8, TNFα, IL-6 and IL-1β cytokine production in a dose dependent manner. The inhibitory effect was substantially more pronounced in the primary cells. We found that Aloe vera inhibited the expression of pro-IL-1β, Nlrp3, caspase-1 as well as that of the P2X7 receptor in the LPS-induced primary macrophages. Furthermore, LPS-induced activation of signaling pathways like NF-κB, p38, JNK and ERK were inhibited by Aloe vera in these cells.

Altogether, we show for the first time that Aloe vera-mediated strong reduction of IL-1β appears to be the consequence of the reduced expression of both pro-IL-1β as well as Nlrp3 inflammasome components via suppressing specific signal transduction pathways. Furthermore, we show that the expression of the ATP sensor P2X7 receptor is also downregulated by Aloe vera that could also contribute to the attenuated IL-1β cytokine secretion. These results may provide a new therapeutic approach to regulate inflammasome-mediated responses.

Introduction

Inflammatory responses are characterized by a sequence of complex, interrelated events that ultimately lead to the recruitment of phagocytes, the elimination of harmful particles and the initiation of tissue repair. These events rely on an orchestrated network of cellular components including soluble factors like that of the inflammatory cytokines. Among pro-inflammatory cytokines, IL-1β functions as a “master” cytokine that has an indispensable role in orchestrating effective innate and adaptive immune responses (Dinarello, 2009). It induces the activation of various cell types like phagocytes, epithelial and endothelial cells, helps the activation and polarization of T-lymphocytes, and enhances the expression of further pro-inflammatory cytokines like that of the IL-6, TNFα (Ciraci et al., 2012, Dinarello, 1997, Dinarello, 2011a). However, prolonged production of IL-1β can lead to severe tissue and organ damages as it is reported in several chronic inflammatory and autoimmune diseases (Dinarello, 2011b, Gabay et al., 2010).

Production of IL-1β strongly depends on the function of multiprotein complexes called inflammasomes. Among inflammasomes, one of the most intensively studied ones is the Nlrp3 inflammasome that contains an Nlrp3 sensor, an ASC adaptor and a caspase-1 enzyme (Cassel et al., 2009, De Nardo and Latz, 2011, Menu and Vince, 2011). Due to its critical function in inflammatory responses, and as part of the safety feature of the immune system, IL-1β production is regulated at various levels and requires distinct signals. Some of these signals induce the expression of the inactive pro-IL-1β and Nlrp3 via the activation of NF-κB and stress-activated protein kinases such as JNK and p38 MAPK, while other signals like ATP trigger the processing of pro-IL-1β to mature IL-1β by caspase-1 (Franchi et al., 2012, Gross et al., 2011, Lopez-Castejon and Brough, 2011, Rathinam et al., 2012, Schroder et al., 2012). ATP induces Nlrp3 inflammasome activation through stimulation of purinergic receptor P2X ligand-gated ion channel 7 (P2X7) which induces K⁺ efflux.

Aloe vera is a medical plant used traditionally in diverse therapeutic applications. The beneficial properties of the plant have been ascribed to the inner, colorless leaf gel that can function both orally and topically as an active immunomodulator. The gel of Aloe vera has been reported to stimulate wound-healing and skin hydration, induce hematopoiesis, and possess anti-diabetic, anti-carcinogenic, antimicrobial, anti-oxidant as well as anti-inflammatory activities (Dat et al., 2012, Hamman, 2008a, Im et al., 2010, Surjushe et al., 2008, Vazquez et al., 1996).

Over 75 active components have already been identified in Aloe vera leaf gels (Hamman, 2008b), and some of them have been implicated as immunomodulatory compounds based on animal studies. Complex carbohydrates were shown to increase macrophage phagocytosis by increasing NO production (Karaca et al., 1995). Aloe-emodin, an anthraquinon compound has been shown to promote natural killer cell activity and macrophage phagocytosis (Yu et al., 2006). Aloins and low molecular fractions of Aloe also containing them together with other anthraquinons might influence the immune response by inhibiting H₂O₂ formation (’t Hart et al., 1990) and/or by inhibiting proteases (Barrantes and Guinea, 2003).

The anti-inflammatory activity of Aloe vera has been evaluated in a number of inflammation models. It has been shown that in experimental animals Aloe vera reduces inflammation detected as reduced neutrophil migration and edema formation (Davis et al., 1989). In Helicobacter pylori-infected rats Aloe vera significantly reduced leukocyte adhesion and TNFα levels (Prabjone et al., 2006). Other studies showed that mRNA expression of colonic mucosa pro-inflammatory cytokines such as IL-6 and TNFα was significantly reduced in colitic animals fed by Aloe vera gel (Park et al., 2011). Similar effect was found in a mouse sepsis model and in a human colorectal mucosa model, in particular that treatment with Aloe vera significantly inhibited the elevation of TNFα, IL-6 and IL-1β levels (Eamlamnam et al., 2006, Langmead et al., 2004, Yun et al., 2009).

Regarding human models, the effect of Aloe vera is usually studied in skin and epidermis related disorders (Reuter et al., 2008, Vogler and Ernst, 1999). Aloe vera has recently been shown to inhibit inflammatory responses by downregulating MMP-9 in peripheral blood leukocytes (PBLs), and to reduce cytokine (TNFα and IL-1β) production in undifferentiated THP-1 cells and PBLs (Vijayalakshmi et al., 2012, Habeeb et al., 2007). The anti-inflammatory effect of the supernatant of a commercial stabilized gel (from Forever Living Product) was found to have very similar effects to those found with a reconstituted lyophilized powder. However, to our knowledge no studies have been performed so far on the effect of Aloe vera on primary human monocyte-derived macrophages, and on the involved molecular mechanisms on its capacity to inhibit cytokine production.

Macrophages are indispensable participants of inflammatory responses and they are the main sources of pro-inflammatory cytokines including IL-1β⋅ In this study, we aimed to determine the anti-inflammatory effect of Aloe vera on LPS-activated THP-1 cells as well as human monocyte-derived macrophages. We show that Aloe vera significantly downregulated the expression of pro-inflammatory cytokines, including IL-1β of these cells. Also, we show that the activation of NF-κB, p38, JNK and ERK signaling pathways is significantly inhibited by Aloe vera treatment in the activated primary macrophages. Furthermore, we show for the first time that Aloe vera could significantly inhibit the LPS-induced expression of Nlrp3 inflammasome components and that of the P2X7 receptor, a phenomenon that likely contributes to the reduced IL-1β production by human macrophages.