Impact of human monocyte and macrophage polarization on NLR expression and NLRP3 inflammasome activation

Abstract

Inflammasomes are multiprotein complexes nucleating around an NLR (Nucleotide-binding domain and Leucine-rich Repeat containing protein), which regulate the secretion of the pro-inflammatory interleukin (IL)-1β and IL-18 cytokines. Monocytes and macrophages, the main cells expressing the inflammasome genes, adapt to their surrounding microenvironment by a phenotypic polarization towards a pro-inflammatory M1 phenotype that promotes inflammation or an anti-inflammatory M2 phenotype important for resolution of inflammation. Despite the importance of inflammasomes in health and disease, little is known about inflammasome gene expression in relevant human cells and the impact of monocyte and macrophage polarization in inflammasome gene expression. We examined the expression of several members of the NLR, caspase and cytokine family, and we studied the activation of the well-described NLRP3 inflammasome in an experimental model of polarized human primary monocytes and monocyte-derived macrophages (M1/M2 phenotypes) before and after activation with LPS, a well-characterized microbial pattern used in inflammasome activation studies. Our results show that the differentiation of monocytes to macrophages alters NLR expression. Polarization using IFN-γ (M1 phenotype), induces among the NLRs studied, only the expression of NOD2. One of the key results of our study is that the induction of NLRP3 expression by LPS is inhibited in the presence of IL-4+IL-13 (M2 phenotype) at both mRNA and protein level in monocytes and macrophages. Unlike caspase-3, the expression of inflammasome-related CASP1 (encodes caspase-1) and CASP4 (encodes caspase-4) is up-regulated in M1 but not in M2 cells. Interestingly, the presence of LPS marginally influenced IL18 mRNA expression and secretion, unlike its impact on IL1B. Our data provide the basis for a better understanding of the role of different inflammasomes within a given environment (M1 and M2) in human cells and their impact in the pathophysiology of several important inflammatory disorders.

Fig 1. NLR expression in human PBMCs, monocytes and macrophages.

(A) mRNA gene expression was measured by RT-qPCR and presented as relative fold change of PBMCs. Data represent the mean ± standard error of the mean (SEM) of ≥ 4 experiments performed in duplicate in cells isolated from ≥ 4 independent donors. Mono: monocytes; Macro: macrophages. Asterisks indicate significant differences as compared to PBMCs (Mann Whitney test: * p < 0.05; ** p < 0.01). (B) Western blot analysis in total lysates of human PBMCs, monocytes and macrophages using anti-NLRP3 antibody. GAPDH was used as loading control. The blot shown is representative of at least 5 experiments from independent donors. Mono: monocytes; Macro: macrophages.

Fig 2. CASP and cytokine expression in human PBMCs, monocytes and macrophages.

(A) mRNA gene expression was measured by RT-qPCR and expressed as relative fold change of PBMCs. Data represent the mean ± SEM of ≥ 4 experiments performed in duplicate in cells isolated from ≥ 4 independent donors. Asterisks indicate significant differences as compared to PBMCs (Mann Whitney test: * p < 0.05; ** p < 0.01; *** p < 0.001); (#) indicates significant differences between monocytes and macrophages (Mann Whitney test: # p < 0.05). (B&C) Western blot analysis in total lysates from human PBMCs, monocytes and macrophages using an anti-ASC, an anti-caspase-1 or an anti-IL-1β antibody. GAPDH was used as loading control. Western blots are representative of at least 5 independent experiments. Mono: monocytes; Macro: macrophages; casp-1: caspase-1.

Fig 3. NLR, CASP and cytokine gene expression in human polarized monocytes.

Monocytes were polarized towards M1 or M2 and stimulated with 100 ng/ml LPS for 3h as described in the methods. mRNA was isolated and gene expression was measured by RT-qPCR and expressed as relative fold change of M0. Data represent the mean ± SEM of ≥ 4 experiments performed in duplicates in cells isolated from ≥ 4 independent donors. M0: cells treated with complete medium (control); M1: cells treated with 100 ng/ml IFN-γ (polarized towards M1); M2: cells treated with 10 ng/ml IL-4+IL-13 (polarized towards M2). Asterisks indicate significant differences as compared to M0 (Mann Whitney test: * p < 0.05; ** p < 0.01); (#) points out significant differences between the indicated groups (Mann Whitney test: # p < 0.05; ## p < 0.01).

Fig 4. NLRP3 inflammasome protein expression in polarized monocytes.

(A) Western blot analysis from total cell lysates of M0, M1 or M2 monocytes in the presence or absence of LPS using an anti-NLRP3, an anti-ASC, an anti-caspase-1 or an anti-IL-1β antibody. GAPDH was used as loading control. Blots are representative of ≥ 3 independent experiments. (B-D) IL-1β, IL-1α and IL-18 secretion as assessed by ELISA in cell culture supernatants of M0, M1, or M2 monocytes after activation of NLRP3 inflammasome with 100 ng/ml LPS for the indicated time in the presence or absence of 5mM ATP for the last 30 minutes. M0: cells treated with complete medium (control); M1: cells treated with 100 ng/ml IFN-γ (polarized towards M1); M2: cells treated with 10 ng/ml IL-4+IL-13 (polarized towards M2). Data represent the mean ± SEM of ≥ 4 independent experiments done in monocytes isolated from buffy coats of ≥ 4 independent donors.

Fig 5. NLR, CASP and cytokine gene expression in human polarized macrophages.

Monocyte derived macrophages were polarized towards M1, M2, or M0 and stimulated with 100 ng/ml LPS for 3h as described in the methods. mRNA was isolated and gene expression was measured by RT-qPCR and expressed as relative fold change of M0. Data represent the mean ± SEM of ≥ 4 experiments performed in duplicates in cells isolated from independent donors. M0: cells treated with complete medium (control); M1: cells treated with 100 ng/ml IFN-γ (polarized towards M1); M2: cells treated with 10 ng/ml IL-4+IL-13 (polarized towards M2). Asterisks indicate significant differences as compared to M0 (Mann Whitney test: * p < 0.05, ** p < 0.01); (#) points out significant differences between the indicated groups (Mann Whitney test: # p < 0.05; ## p < 0.01).

Fig 6. NLRP3 inflammasome protein expression in polarized macrophages.

(A) Western blot analysis from total cell lysates of M0, M1 or M2 macrophages in the presence or absence of LPS using an anti-NLRP3, an anti-ASC, an anti-caspase-1 or an anti-IL-1β antibody. GAPDH was used as loading control. Blots are representative of ≥ 3 independent experiments. (B-D) IL-1β, IL-1α and IL-18 secretion as assessed by ELISA in cell culture supernatants of M0, M1, or M2 monocytes after activation of NLRP3 inflammasome with 100 ng/ml LPS for the indicated period of times in the presence or absence of 5mM ATP for the last 30 minutes. M0: cells treated with complete medium (control); M1: cells treated with 100 ng/ml IFN-γ (polarized towards M1); M2: cells treated with 10 ng/ml IL-4+IL-13 (polarized towards M2). Data represent the mean ± SEM of ≥ 4 independent experiments done in macrophages derived from monocytes of ≥ 4 independent donors.