The Transcription Factors TFEB and TFE3 Link the FLCN-AMPK Signaling Axis to Innate Immune Response and Pathogen Resistance

Abstract

TFEB and TFE3 are transcriptional regulators of the innate immune response, but the mechanisms regulating their activation upon pathogen infection are poorly elucidated. Using C. elegans and mammalian models, we report that the master metabolic modulator 5'-AMP-activated protein kinase (AMPK) and its negative regulator Folliculin (FLCN) act upstream of TFEB/TFE3 in the innate immune response, independently of the mTORC1 signaling pathway. In nematodes, loss of FLCN or overexpression of AMPK confers pathogen resistance via activation of TFEB/TFE3-dependent antimicrobial genes, whereas ablation of total AMPK activity abolishes this phenotype. Similarly, in mammalian cells, loss of FLCN or pharmacological activation of AMPK induces TFEB/TFE3-dependent pro-inflammatory cytokine expression. Importantly, a rapid reduction in cellular ATP levels in murine macrophages is observed upon lipopolysaccharide (LPS) treatment accompanied by an acute AMPK activation and TFEB nuclear localization. These results uncover an ancient, highly conserved, and pharmacologically actionable mechanism coupling energy status with innate immunity.

Keywords: AMPK; FLCN; TFE3; TFEB; autophagy; innate immune response; lysosomal biogenesis; pathogen resistance; phagocytosis.

Figure 1:. Loss of flcn-1 increases the expression of antimicrobial genes and confers resistance to bacterial pathogens

(A) Pie chart of functional gene ontology analysis of the genes upregulated in flcn-1(ok975) mutant animals at basal level. (B) Relative mRNA expression of stress response and antimicrobial peptide genes in wild-type and flcn-1 mutant animals. Data represents the average of three independent experiments, each done in triplicates ± SEM. Significance was determined using student’s t-test (*p<0.05, **p<0.01, ***p<0.001). (C-E) Percent survival of indicated strains upon infection with S. aureus and P. aeruginosa. Refer to Table S1 for details on number of animals utilized and number of repeats. The Statistical analysis was obtained using Mantel Cox test on the pooled curve.

Figure 2:. Loss of flcn-1 increases pathogen resistance via HLH-30 activation

(A) Representative micrographs of HLH-30::GFP at basal level or after infection with S. aureus for 30 min. The signal is found in the nuclei of enterocytes, a cell-type in which lipids are stored in nematodes. Scale bars in i, iii and in ii, iv represent 100 μm and 50 μm respectively. (B) Percent animals showing HLH-30 nuclear translocation in hlh-30p::hlh-30::GFP and flcn-1; hlh-30p::hlh-30::GFP worm strains upon S. aureus infection for indicated time points to determine HLH-30 nuclear localization upon flcn-1 loss at basal level (time 0) and upon S. aureus infection. Data represents the mean ± SEM from three independent repeats, n ≥ 30 animals/condition for every repeat. Significance was determined using student’s t-test (*p<0.05, **p<0.01, ***p<0.001). (C, D) Percent survival of indicated worm strains upon infection with S. aureus and P. aeruginosa. Refer to Table S1 for details on number of animals utilized and number of repeats. Statistics obtained using Mantel-Cox analysis on the pooled curve. (E-K) Relative mRNA expression of indicated target genes in wild-type, flcn-1(ok975), flcn-1(ok975); hlh-30 (tm1978), and hlh-30 (tm1978) animals at basal level and after treatment with S. aureus for 4 h. Data represents the average of three independent experiments done in triplicates ± SEM. Significance was determined using one-way ANOVA with the application of Bonferroni correction (*p<0.05, **p<0.01, ***p<0.001).

Figure 3:. The regulation of TFEB/TFE3 by FLCN is evolutionarily conserved through mTOR independent mechanisms

(A) Immunoblot of isolated cytosolic-soluble fractions and nuclear fractions of wild-type and FLCN KO mouse embryonic fibroblasts (MEFs). (B) Relative mRNA levels measured by qRT-PCR of indicated genes in wild-type and FLCN KO MEFs. Data represent the average of three independent experiments done in triplicates ± SEM. Significance was determined using student’s t-test (**p<0.01, ***p<0.001). (C) Quantification of the percentage of cells showing TFEB nuclear staining treated with mTORC1 inhibitor; Torin1 (1 μM) for 2 h. Data represents the average of three independent experiments, each done in triplicates ± SEM. Significance was determined using one-way ANOVA with the application of Bonferroni correction (*p<0.05). (D) Immunoblot analysis of whole cell extracts with or without Torin1 (1 μM) for 2 h. (E) Percent animals showing HLH-30 nuclear translocation in indicated hlh-30p::hlh-30::GFP worm strains treated with or without let-363 RNAi at basal level or upon S. aureus infection. Data represent the mean ± SEM with 3 independent repeats, n ≥ 30 animals/condition for every repeat. Significance was determined using one-way ANOVA with the application of the Bonferroni correction (**p<0.01, ***p<0.001). (F) Relative IL-6 mRNA levels measured by qRT-PCR in empty vector (EV) or shTFEB/TFE3-treated wild-type or FLCN KO MEFs, stimulated with or without 10 ng/ml TNF-α for 2 h. Data represents the average of three independent experiments, each done in triplicates ± SEM. Significance was determined using one-way ANOVA with the application of Bonferroni correction (*p<0.05, **p<0.01). (G) Immunoblot analysis of wild-type and FLCN KO MEFs transfected with EV or shTFEB/TFE3. (H) Immunoblot analysis of RAW 264.7 cells transfected with EV or shFLCN. (I) Relative IL-6 mRNA levels measured by qRT-PCR in EV or shFLCN-treated RAW264.7 cells, stimulated with or without 1 μg/ml LPS for 3 h. (J) Quantification of IL-6 levels of conditions described in (I) using Mouse Protein Cytokine Array. Data represent the average of three independent experiments ± SEM. Significance was determined using one-way ANOVA with the application of Bonferroni correction (*p<0.05, **p<0.01). (K) Hierarchical clustering of cytokine and chemokine secretion in the supernatant using Mouse Protein Cytokine Array in EV or shFLCN-treated RAW264.7 cells stimulated with 1 μg/ml LPS for 3 and 24 h. Each square in a column represents the average of triplicate experiments, and each column represents an independent replicate. Fold increase was normalized against EV and color-coded (dark red indicates 2 or more-fold increase, dark blue indicates no change). (L) Fold increase in cytokine and chemokine secretion levels as described in (J). Data represent the average of three independent experiments, each done in triplicates ± SEM. Significance was determined using student’s t-test in comparison to the EV stimulated with LPS for 3h and 24, respectively (*p<0.05, **p<0.01, ***p<0.001).

Figure 4:. FLCN depletion in macrophages enhances their energy metabolism and phagocytic potential

(A) Glucose production and (B) lactate consumption levels measured using NOVA Bioanalysis flux analyzer in empty vector (EV) or shFLCN RAW264.7 at basal level. (C, D) Extracellular acidification rate (ECAR) and (E, F) oxygen consumption rate (OCR) of EV or shFLCN RAW264.7 at basal level as measured by Seahorse Bioscience XF96 extracellular flux analyzer. After establishing a baseline, oligomycin (10 μM), FCCP (15 μM), and rotenone/antimycin A (1 μM, and 10 μM, respectively) were added. (G) Fold change in ATP levels in EV or shFLCN RAW264.7 after 24 h of seeding as measured by CellTiter-Glo Luminescent Cell Viability Assay. Data represent the average of three independent experiments, each done in triplicates ± SEM. Significance was determined using student’s t-test (**p<0.01, ***p<0.001). (H) Immunoblot analysis of EV and TFEB/TFE3 DKO RAW264.7 cells transfected with EV or shFLCN. (I) Phagocytic activities of EV, TFEB/TFE3 DKO, and TFEB/TFE3 DKO shFLCN RAW264.7 cells measured using Red pHrodo S.aureus BioParticles by flow cytometry. Data represents the average of three independent experiments, each done in triplicates ± SEM. Significance was determined using one-way ANOVA with the application of Bonferroni correction (**p<0.01, ****p<0.0001).

Figure 5:. AMPK regulates HLH-30 activation and antimicrobial response upon infection with bacterial pathogens

(A-C) Percent survival of indicated worm strains upon infection with S. aureus or P. aeruginosa. Refer to Table S1 for details on number of animals utilized and number of repeats. Statistics obtained by Mantel-Cox analysis on the pooled curve. (D) Percentage of animals showing HLH-30 nuclear translocation in indicated hlh-30p::hlh-30::GFP worm strains upon infection with S. aureus for the indicated amount of time. Data represent the mean ± SEM with 3 independent repeats, n ≥ 30 animals/condition for every repeat. Significance was determined using student’s t-test (*p<0.05, **p<0.01, ***p<0.001). (E) Venn diagram of the overlapping set of genes between S. aureus-induced genes in wild-type animals and genes downregulated in aak-1(tm1944); aak-2(ok524) mutant animals upon infection. (F) Venn diagram and (G) pie chart of functional gene ontology analysis of AMPK-dependent genes obtained by the overlap analysis between genes downregulated in aak-1(tm1944); aak-2(ok524) mutant animals in comparison to wild-type animals upon S. aureus infection and the hlh-30-dependent list of genes published in (40). Comparisons were performed using the “compare two lists” online software and the significance was obtained using “nemates” software. (H-R) Relative mRNA levels measured by qRT-PCR of AMPK-dependent genes in wild-type and aak-1(tm1944); aak-2(ok524) mutant animals infected with or without S. aureus for 4 h. Results are normalized to non-treated wild-type animals. Validation of RNA-seq using three biological replicates per condition and three technical replicates per biological repeat. Significance was determined using one-way ANOVA with the application of the Bonferroni correction (*p<0.05, **p<0.01, ***p<0.001, ****p<0.001).

Figure 6:. AMPK regulates TFEB/TFE3-mediated innate immune response

(A) Immunoblot of wild-type or AMPKα1/α2 double knock out (DKO) MEFs stimulated with the AMPK activator; GSK-621 (30 μM) for 1 h. (B) Representative images of TFEB and TFE3 staining in wild-type and AMPK DKO MEFs before and after treatment with GSK-621 (30 μM) for 1 h. Scale bars represent 20 μm. (C) Quantification of the percentage of cells showing TFEB and TFE3 nuclear staining of the conditions described in (B). (D) Relative IL-6 mRNA levels measured by qRT-PCR in wild-type MEFs transfected with EV or shTFEB/TFE3, stimulated with GSK-621 for 2 h. Data represents the average of three independent experiments, each done in triplicates ± SEM. Significance was determined using one-way ANOVA with the application of Bonferroni correction (*p<0.05, ***p<0.001). (E) Immunoblot analysis of RAW264.7 macrophages treated with GSK-621 (30 μM) for 2 h. (F) Quantification of the percentage of RAW264.7 cells showing TFEB nuclear staining of the conditions described in (D). (G) Quantification of the significant fold increases in cytokine and chemokine protein levels in RAW264.7 macrophages, treated with GSK-621 (30 μM) for 2 h as compared to control. (H) Fold change in ATP levels in RAW264.7 treated with LPS (1 μg/ml) for up to 1 h as measured by CellTiter-Glo Luminescent Cell Viability Assay. (I) Immunoblot analysis of RAW264.7 macrophages treated with LPS (1 μg/ml) for up to 1 h. (J) Quantification of the percentage of RAW264.7 cells with TFEB nuclear localization of the conditions described in (H). Data represent the average of three independent experiments, each done in triplicates ± SEM. Significance was determined using student’s t-test (*p<0.05, **p<0.01, ***p<0.001).