Reis AS et al. (2020), Inflammasome activation and IL-1 signaling duri...

ECB-ART-48517

Sci Adv 2020 Mar 04;610:eaax6346. doi: 10.1126/sciadv.aax6346.

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Inflammasome activation and IL-1 signaling during placental malaria induce poor pregnancy outcomes.

Reis AS , Barboza R , Murillo O , Barateiro A , Peixoto EPM , Lima FA , Gomes VM , Dombrowski JG , Leal VNC , Araujo F , Bandeira CL , Araujo RBD , Neres R , Souza RM , Costa FTM , Pontillo A , Bevilacqua E , Wrenger C , Wunderlich G , Palmisano G , Labriola L , Bortoluci KR , Penha-Gonçalves C , Gonçalves LA , Epiphanio S , Marinho CRF .

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Placental malaria (PM) is associated with severe inflammation leading to abortion, preterm delivery, and intrauterine growth restriction. Innate immunity responses play critical roles, but the mechanisms underlying placental immunopathology are still unclear. Here, we investigated the role of inflammasome activation in PM by scrutinizing human placenta samples from an endemic area and ablating inflammasome components in a PM mouse model. The reduction in birth weight in babies from infected mothers is paralleled by increased placental expression of AIM2 and NLRP3 inflammasomes. Using genetic dissection, we reveal that inflammasome activation pathways are involved in the production and detrimental action of interleukin-1β (IL-1β) in the infected placenta. The IL-1R pharmacological antagonist Anakinra improved pregnancy outcomes by restoring fetal growth and reducing resorption in an experimental model. These findings unveil that IL-1β-mediated signaling is a determinant of PM pathogenesis, suggesting that IL-1R antagonists can improve clinical outcomes of malaria infection in pregnancy.

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	Fig. 1. Pf infection during pregnancy induces placental pathology, poor outcomes, and inflammasome activation.(A to F) Representative histology images of placentas collected from NI (A) and Pf-I (B to F) women. Black arrows indicate the presence of iRBCs in the IVS (B), hemozoin deposition (C), necrotic villi (D), syncytial nuclear aggregates (E), and fibrotic necrosis areas (F). AU, arbitrary units; DAB, 3,3′-diaminobenzidine. Tissue imaged with ×40 (A and D to F), ×63 (B), and ×20 (C) magnification. Quantification of the histopathological parameters (G) and newborn (H) and placental (I) weight. (J) Quantification of monocytes within the IVS in 10 different microscopic fields. (K) Placental plasma levels of IL-1β, TNF-α, IL-6, and IL-10 cytokines measured by CBA. (L and M) Relative expression of placental IL-1R protein measured by Western blot (WB) (L) and immunohistochemistry (M) normalized by a fraction of the area. (N and O) Placental caspase-1 activity (N) and protein expression of AIM2, NLRP3, and NLRC4 receptors, and ASC adaptor (O) identified by WB. WB data were quantified by densitometry and normalized by tubulin (L and O) or β-actin (N). Data are expressed as scatter plot and group mean (dash). Number of individuals: (G to K) NI = 59 and Pf-I = 19; (L) NI = 5 and Pf-I = 5; (M) NI = 49 and Pf-I = 16; (N) NI = 6 and Pf-I = 8; and (O) NI = 5 and Pf-I = 5. The differences between NI and infected pregnant women were determined by Student’s t test (H and I) or Mann-Whitney rank sum test (G and J to O). P < 0.05 and *P < 0.01. (A to F) Photo credit: Rodrigo M. Souza, University of São Paulo.
	Fig. 2. IL-1β production in human monocytes and trophoblasts exposed to Pf-iRBCsA.(A and C) Flow cytometry histograms and column bar graph of the caspase-1 median fluorescence intensity (MFI) of THP-1 monocytic (A) and BeWo trophoblast (C) cell lines, stimulated with an extract of Pf-I (iRBCs, green) or NI (RBCs, red) RBCs for a 12-hour period in a 20:1 RBCs:cell ratio. THP-1 cells were prestimulated with 100 nM lipopolysaccharide. Medium was added to the nonstimulated control group (gray histogram). (B and D to F) Secreted IL-1β was evaluated in the supernatant of THP-1 (B) and BeWo (D) cells, as well as in primary cultures of human monocytes (E) and trophoblasts (F) stimulated with an extract of RBCs (ratio, 40:1) and iRBCs at different ratios (10:1, 20:1, and 40:1) for 24 (E) or 48 hours (B, D, and F). Data are represented as means ± SD of triplicate cultures that were performed in two (A, C, E, and F) and three (B and D) independent experiments. The differences between each group were determined by one-way ANOVA with Bonferroni’s post hoc correction. P < 0.05, P < 0.01, and **P < 0.001.
	Fig. 3. Pb infection during murine pregnancy induces placental pathology and caspase-1 activation.(A to C) Images illustrated the NI (Ct, left) and infected (Inf, right) placental vascular spaces (A), fetus (B), and uterus (C) of mice. (A) Relative quantification of placental vascular space. (B) Fetal weight measured at G19. (C) Relative quantification of the resorption number (arrows) in relation to the total fetuses’ number (viable and resorptions). (D and E) Protein levels of IL-1β [enzyme-linked immunosorbent assay (ELISA)], TNF-α, IFN-γ, IL-6, and IL-10 (CBA) in placentas (D) and plasma (E) collected from NI (Ct) or infected (Inf) mice. (F) Placental caspase-1 (p20) activity by WB in NI (Ct) or infected (Inf) mice, normalized by β-actin. Data are represented as means ± SD of 29 to 49 placentas or fetuses (n = 8 to 12 pregnant mice) (A and B) and 5 to 12 pregnant mice (C to F) per group, which were performed in two or more independent experiments. The differences between the control group (Ct) and infected (Inf) pregnant mice were determined by Student’s t test. P < 0.05, P < 0.01, and **P < 0.001. (A to C) Photo credit: Aramys S. Reis, University of São Paulo.
	Fig. 4. AIM2 and NLRP3 inflammasomes are associated with poor pregnancy outcomes in Pb-infected mice.Evaluation of pregnancy outcomes in mice deficient for Casp1/11, Asc, Nlrc4, Nlrp3, and Aim2 genes of the inflammasome complexes. (A) Percentage of weight reduction of fetuses born from infected pregnant mice in relation to their NI controls at G19. (B) Relative resorption rates of infected in relation to their NI controls. (C) Measure of IL-1β levels in placentas collected from infected (Inf) and NI (Ct) mice by ELISA. Pooled placenta samples, each containing four mouse placentas per sample, were used for protein extraction. WT refers to C57BL/6 mice. Data are represented as means ± SD of 37 to 39 fetuses (n = 5 to 14 pregnant mice) (A) and 5 to 14 pregnant mice (B and C) per group, which were performed in two or more independent experiments. The differences between each group were determined by one-way ANOVA with Bonferroni’s post hoc correction (A and B) and Student’s t test (C). P < 0.05 and **P < 0.001.
	Fig. 5. Impaired pregnancy outcomes are reverted by IL-1 axis genetic ablation and pharmacological blocking.Assessment of IL-1β signaling impact in fetal by genetic ablation of Il1b and Il1r1 or by blocking the receptor with Anakinra, a recombinant of the IL-1Ra. (A and B) Weight reduction of fetuses born from infected pregnant mice expressed in percentage of their NI controls at G19. (C and D) Relative resorption rates of infected in relation to their NI controls. (B and D) Anakinra treatment was performed in WT (C57BL/6) mice, and vehicle represents an infected group treated with Anakinra denaturated in phosphate-buffered saline. Data are represented as means ± SD of 24 to 46 fetuses (n = 3 to 12 pregnant mice) (A and C) or 3 to 12 pregnant mice (B and D) per group and are cumulative of two or more independent experiments. The differences between each group were determined by one-way ANOVA with Bonferroni’s post hoc correction (A and C) or Student’s t test (B and D). P < 0.05 and **P < 0.001.
	Fig. 6. Proposed mechanism of inflammasome activation during placental malaria.Plasmodium antigens and cellular danger signals like GPI, hemozoin, dsDNA, and heme are recognized by TLR4 and TLR9 expressed in placental macrophages and trophoblasts. This phenomenon triggers these pathways through MyD88, activating the nuclear factor κB (NF-κB) transcription factor, which induces transcription of proinflammatory cytokines such as TNF-α, IFN-γ, and pro–IL-1β. In addition, hemozoin and dsDNA activate NLRP3 and AIM2 inflammasomes, respectively, which induce pro–IL-1β cleavage by caspase-1, which ultimately will be secreted in its active form. Active IL-1β will bind, with high affinity, to its receptor (IL-1R), triggering the downstream events that sustain the production of more proinflammatory cytokines and, possibly, impairing the expression and function of nutrient transporters, such as SNAT1, SNAT2, and GLUT1. Dysregulation of nutrient transportation will promote IUGR, consequently leading to poor pregnancy outcomes such as LBW. Furthermore, increasing levels of IL-1β, TNF-α, and IFN-γ in the placenta also contribute to local inflammation, causing severe damage in placental tissue and poor pregnancy outcomes. The archetype of placental malaria, reduced fetal development, can be reverted in our experimental model with Anakinra treatment, a recombinant of the IL-1Ra. PAMPs, pathogen-associated molecular patterns; DAMPs, damage-associated molecular patterns.

References [+] :

Abrahams, Toll-like receptors and their role in the trophoblast. 2005, Pubmed

	Fig. 1. Pf infection during pregnancy induces placental pathology, poor outcomes, and inflammasome activation.(A to F) Representative histology images of placentas collected from NI (A) and Pf-I (B to F) women. Black arrows indicate the presence of iRBCs in the IVS (B), hemozoin deposition (C), necrotic villi (D), syncytial nuclear aggregates (E), and fibrotic necrosis areas (F). AU, arbitrary units; DAB, 3,3′-diaminobenzidine. Tissue imaged with ×40 (A and D to F), ×63 (B), and ×20 (C) magnification. Quantification of the histopathological parameters (G) and newborn (H) and placental (I) weight. (J) Quantification of monocytes within the IVS in 10 different microscopic fields. (K) Placental plasma levels of IL-1β, TNF-α, IL-6, and IL-10 cytokines measured by CBA. (L and M) Relative expression of placental IL-1R protein measured by Western blot (WB) (L) and immunohistochemistry (M) normalized by a fraction of the area. (N and O) Placental caspase-1 activity (N) and protein expression of AIM2, NLRP3, and NLRC4 receptors, and ASC adaptor (O) identified by WB. WB data were quantified by densitometry and normalized by tubulin (L and O) or β-actin (N). Data are expressed as scatter plot and group mean (dash). Number of individuals: (G to K) NI = 59 and Pf-I = 19; (L) NI = 5 and Pf-I = 5; (M) NI = 49 and Pf-I = 16; (N) NI = 6 and Pf-I = 8; and (O) NI = 5 and Pf-I = 5. The differences between NI and infected pregnant women were determined by Student’s t test (H and I) or Mann-Whitney rank sum test (G and J to O). P < 0.05 and *P < 0.01. (A to F) Photo credit: Rodrigo M. Souza, University of São Paulo.
	Fig. 2. IL-1β production in human monocytes and trophoblasts exposed to Pf-iRBCsA.(A and C) Flow cytometry histograms and column bar graph of the caspase-1 median fluorescence intensity (MFI) of THP-1 monocytic (A) and BeWo trophoblast (C) cell lines, stimulated with an extract of Pf-I (iRBCs, green) or NI (RBCs, red) RBCs for a 12-hour period in a 20:1 RBCs:cell ratio. THP-1 cells were prestimulated with 100 nM lipopolysaccharide. Medium was added to the nonstimulated control group (gray histogram). (B and D to F) Secreted IL-1β was evaluated in the supernatant of THP-1 (B) and BeWo (D) cells, as well as in primary cultures of human monocytes (E) and trophoblasts (F) stimulated with an extract of RBCs (ratio, 40:1) and iRBCs at different ratios (10:1, 20:1, and 40:1) for 24 (E) or 48 hours (B, D, and F). Data are represented as means ± SD of triplicate cultures that were performed in two (A, C, E, and F) and three (B and D) independent experiments. The differences between each group were determined by one-way ANOVA with Bonferroni’s post hoc correction. P < 0.05, P < 0.01, and **P < 0.001.
	Fig. 3. Pb infection during murine pregnancy induces placental pathology and caspase-1 activation.(A to C) Images illustrated the NI (Ct, left) and infected (Inf, right) placental vascular spaces (A), fetus (B), and uterus (C) of mice. (A) Relative quantification of placental vascular space. (B) Fetal weight measured at G19. (C) Relative quantification of the resorption number (arrows) in relation to the total fetuses’ number (viable and resorptions). (D and E) Protein levels of IL-1β [enzyme-linked immunosorbent assay (ELISA)], TNF-α, IFN-γ, IL-6, and IL-10 (CBA) in placentas (D) and plasma (E) collected from NI (Ct) or infected (Inf) mice. (F) Placental caspase-1 (p20) activity by WB in NI (Ct) or infected (Inf) mice, normalized by β-actin. Data are represented as means ± SD of 29 to 49 placentas or fetuses (n = 8 to 12 pregnant mice) (A and B) and 5 to 12 pregnant mice (C to F) per group, which were performed in two or more independent experiments. The differences between the control group (Ct) and infected (Inf) pregnant mice were determined by Student’s t test. P < 0.05, P < 0.01, and **P < 0.001. (A to C) Photo credit: Aramys S. Reis, University of São Paulo.
	Fig. 4. AIM2 and NLRP3 inflammasomes are associated with poor pregnancy outcomes in Pb-infected mice.Evaluation of pregnancy outcomes in mice deficient for Casp1/11, Asc, Nlrc4, Nlrp3, and Aim2 genes of the inflammasome complexes. (A) Percentage of weight reduction of fetuses born from infected pregnant mice in relation to their NI controls at G19. (B) Relative resorption rates of infected in relation to their NI controls. (C) Measure of IL-1β levels in placentas collected from infected (Inf) and NI (Ct) mice by ELISA. Pooled placenta samples, each containing four mouse placentas per sample, were used for protein extraction. WT refers to C57BL/6 mice. Data are represented as means ± SD of 37 to 39 fetuses (n = 5 to 14 pregnant mice) (A) and 5 to 14 pregnant mice (B and C) per group, which were performed in two or more independent experiments. The differences between each group were determined by one-way ANOVA with Bonferroni’s post hoc correction (A and B) and Student’s t test (C). P < 0.05 and **P < 0.001.
	Fig. 5. Impaired pregnancy outcomes are reverted by IL-1 axis genetic ablation and pharmacological blocking.Assessment of IL-1β signaling impact in fetal by genetic ablation of Il1b and Il1r1 or by blocking the receptor with Anakinra, a recombinant of the IL-1Ra. (A and B) Weight reduction of fetuses born from infected pregnant mice expressed in percentage of their NI controls at G19. (C and D) Relative resorption rates of infected in relation to their NI controls. (B and D) Anakinra treatment was performed in WT (C57BL/6) mice, and vehicle represents an infected group treated with Anakinra denaturated in phosphate-buffered saline. Data are represented as means ± SD of 24 to 46 fetuses (n = 3 to 12 pregnant mice) (A and C) or 3 to 12 pregnant mice (B and D) per group and are cumulative of two or more independent experiments. The differences between each group were determined by one-way ANOVA with Bonferroni’s post hoc correction (A and C) or Student’s t test (B and D). P < 0.05 and **P < 0.001.
	Fig. 6. Proposed mechanism of inflammasome activation during placental malaria.Plasmodium antigens and cellular danger signals like GPI, hemozoin, dsDNA, and heme are recognized by TLR4 and TLR9 expressed in placental macrophages and trophoblasts. This phenomenon triggers these pathways through MyD88, activating the nuclear factor κB (NF-κB) transcription factor, which induces transcription of proinflammatory cytokines such as TNF-α, IFN-γ, and pro–IL-1β. In addition, hemozoin and dsDNA activate NLRP3 and AIM2 inflammasomes, respectively, which induce pro–IL-1β cleavage by caspase-1, which ultimately will be secreted in its active form. Active IL-1β will bind, with high affinity, to its receptor (IL-1R), triggering the downstream events that sustain the production of more proinflammatory cytokines and, possibly, impairing the expression and function of nutrient transporters, such as SNAT1, SNAT2, and GLUT1. Dysregulation of nutrient transportation will promote IUGR, consequently leading to poor pregnancy outcomes such as LBW. Furthermore, increasing levels of IL-1β, TNF-α, and IFN-γ in the placenta also contribute to local inflammation, causing severe damage in placental tissue and poor pregnancy outcomes. The archetype of placental malaria, reduced fetal development, can be reverted in our experimental model with Anakinra treatment, a recombinant of the IL-1Ra. PAMPs, pathogen-associated molecular patterns; DAMPs, damage-associated molecular patterns.