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. 2014 Jun 19;40(6):896-909.
doi: 10.1016/j.immuni.2014.05.002. Epub 2014 May 29.

Innate host defense requires TFEB-mediated transcription of cytoprotective and antimicrobial genes

Orane Visvikis #  1 , Nnamdi Ihuegbu #  2 , Sid A Labed  1 , Lyly G Luhachack  1 , Anna-Maria F Alves  1 , Amanda C Wollenberg  1 , Lynda M Stuart  3 , Gary D Stormo  2 , Javier E Irazoqui  1
Affiliations

Innate host defense requires TFEB-mediated transcription of cytoprotective and antimicrobial genes

Orane Visvikis et al. Immunity. .

Abstract

Animal host defense against infection requires the expression of defense genes at the right place and the right time. Understanding such tight control of host defense requires the elucidation of the transcription factors involved. By using an unbiased approach in the model Caenorhabditis elegans, we discovered that HLH-30 (known as TFEB in mammals) is a key transcription factor for host defense. HLH-30 was activated shortly after Staphylococcus aureus infection, and drove the expression of close to 80% of the host response, including antimicrobial and autophagy genes that were essential for host tolerance of infection. TFEB was also rapidly activated in murine macrophages upon S. aureus infection and was required for proper transcriptional induction of several proinflammatory cytokines and chemokines. Thus, our data suggest that TFEB is a previously unappreciated, evolutionarily ancient transcription factor in the host response to infection.

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Figures

Figure 1
Figure 1. HLH-30 acutely responds to S. aureus infection
(A) Distribution of the identified E-box upstream of C. elegans genes. Insert, logo representation of identified E-box. (B) Phylogenetic relationships among human MiT proteins and C. elegans HLH protein isoforms. (C) Representative micrographs of HLH-30::GFP expression in embryos, larval stages, and adults. Bottom row: higher magnification of adult head, midbody, and tail showing expression in pharynx (ph), intestine (in), spermatheca (sp), rectal epithelial cells (rec), and vulval epithelial cells (vec). (D-G) Representative micrographs of HLH-30::GFP animals infected 30 min with S. aureus (F, G) and uninfected controls (D, E). (E) and (G): higher magnification of areas indicated in (D) and (F). (H) HLH-30::GFP nuclear accumulation. Data are mean ± S.E.M. (two biological replicates, N ≥ 50/condition). ***: p < 0.001 (two-sample t test).
Figure 2
Figure 2. HLH-30 is required for the host response to S. aureus infection
(A) Proportions of HLH-30-dependent and –independent S. aureus-induced genes. (B,C). Survival of N2 [rol-6] (wild-type), hlh-30;[rol-6] (hlh-30), and hlh-30;[hlh-30p::hlh-30::gfp,rol-6] (hlh-30;[hlh-30p::hlh-30::gfp]) animals infected with S. aureus (B) or fed E. coli OP50 (C). ***: p < 0.0001 (Log-Rank test). Statistical analysis can be found in Table S7. Experiments are representative of at least two independent trials. (D) Intestinal accumulation of S. aureus, expressed in colony-forming units (C.F.U.) per animal. Data are mean ± S.E.M. (N = 2 biological replicates). See also Figure S1
Figure 3
Figure 3. HLH-30 is required for expression of host response components
(A) hlh-30 expression (qRT-PCR), in wild type animals infected with S. aureus for 4, 8, or 12 h, normalized to uninfected animals. (B) hlh-30 expression (3′UTR qRT-PCR) in wild type and hlh-30 mutant animals exposed to S. aureus or control E. coli for 8 h. (C) Over-represented functional categories of hlh-30-dependent S. aureus-induced genes (also see Table S8). (D) Four functional gene sets of HLH-30-dependent S. aureus-induced genes. (E) hlh-30-dependent gene expression, measured as in B. Data are mean ± S.E.M. (N = 3 biological replicates). *: p < 0.05, **: p< 0.01, ***: p<0.001 (two-sample t test). See also Figure S2
Figure 4
Figure 4. HLH-30-regulated antimicrobial genes are required for defense
(A-D) HLH-30-dependent antimicrobial gene expression (qRT-PCR) in animals exposed for 8 h to S. aureus or nonpathogenic E. coli. (E) Unsupervised hierarchical clustering of expression levels (qRT-PCR) of S. aureus-induced HLH-30-dependent genes, in infected hlh-30 mutants and in hlh-30;[hlh-30p::hlh-30::gfp] animals (hlh-30 overexpression), normalized to infected wild type animals (8h of infection). Each column represents an independent replicate. Primary data can be found in Fig. S3. (F-G) Survival of wild type and hlh-30 animals, treated with E. coli HT115 carrying vector L4440 (empty vector) or expressing dsRNA targeting (RNAi) ilys-2 and lys-5, and subsequently infected with S. aureus (F) or maintained on RNAi bacteria (G). **: p < 0.001; ***: p < 0.0001 (Log-Rank test). Statistical analyses can be found in Table S7. Experiments are representative of at least three independent trials. (H) Time of 50% death (TD50) of wild type animals treated with ilys-2 and lys-5 RNAi, normalized to empty vector controls. Data show mean ± S.E.M. (N = 3 independent trials); *: p < 0.05 (two-sample t test). (I) S. aureus accumulation in RNAi-treated animals after 27 h of infection, expressed as C.F.U./animal. Representative experiment (2 independent trials). Data are mean ± S.E.M. (N = 3 replicates). Differences between groups were not significant (two-sample t test). See also Figure S3
Figure 5
Figure 5. HLH-30-regulated autophagy genes are required for defense
(A-B) qRT-PCR of HLH-30-dependent autophagy and lysosomal genes, of animals exposed to S. aureus or control E. coli (8 h). (C) Unsupervised hierarchical clustering of HLH-30-dependent gene expression (qRT-PCR), as in Fig. 4E. Primary data can be found in Fig. S4. (D-G) Confocal micrographs of anterior intestinal cells containing GFP::LGG-1 puncta in animals fed nonpathogenic E. coli (D, E) or infected 8 h with S. aureus (F, G). Insets are Nomarski micrographs of the same field. Red puncta indicate auto-fluorescent gut granules. (E) and (G): higher magnification of areas indicated in (D) and (F), respectively. Bar = 6 μm. (H) Quantification of GFP::LGG-1 puncta. Data are mean ± S.E.M., N = 13 animals each. **: p<0.01 (two-sample t test). (I-N). Survival of wild type and hlh-30 animals, empty vector or lgg-1 (I,L), unc-51 (J, M) and vps-34 RNAi-treated (K, N), and subsequently infected with S. aureus (I-K) or maintained on RNAi bacteria (L-M). Experiments are representative of at least two independent trials. **: p < 0.001; ***: p < 0.0001 (Log-Rank test). Statistical analyses can be found in Table S7. (O) S. aureus accumulation in RNAi-treated animals after 27 h of infection, expressed as C.F.U./animal. Representative experiment of two independent trials. Data are mean ± S.E.M. (N = 3 replicates). Differences between groups were not significant (two-sample t test). See also Figure S4
Figure 6
Figure 6. S. aureus infection of murine macrophages activates TFEB
(A) anti-TFEB immunoblot (IB) of whole-cell extracts (WCE) from RAW264.7 cells (1 h treatment with live or heat-killed (HK) S. aureus, or vehicle control). Anti-actin IB is loading control. (B-E) TFEB-3xFLAG RAW264.7 cells were vehicle-treated or infected 1 h with S. aureus. (B) Anti-FLAG IB of WCE. Anti-actin IB is loading control. (C) WCE treated with λ phosphatase for 30 min before IB as in B. (D) Subcellular fractionation of WCE followed by anti-FLAG IB. Nuclear fraction is marked with LSD1 and cytosolic fraction is marked with GAPDH. (E) Anti-FLAG immunofluorescence (Green). Hoechst for DNA (Red). (F) Quantification of TFEB localization from (E). Data are mean percentage ± S.E.M. (N = 3 independent trials, n > 200 cells per condition per trial). **: p < 0.01; ***: p< 0.001 (two-sample t test).
Figure 7
Figure 7. TFEB is required for the macrophage proinflammatory response
(A) IB of WCE for TFEB from RAW264.7 cells transfected with control siRNA (siCtrl) or two different TFEB siRNA (siTFEB #1 and #2). Anti-actin IB is loading control. (B) TFEB expression (qRT-PCR) in RAW264.7 cells treated as in A. (C) Hierarchical clustering of cytokine and chemokine expression (qRT-PCR) in infected RAW264.7 cells. Cells were transfected with siCtrl or siTFEB #2, and subsequently infected with S. aureus for 4 h. Each column represents an independent replicate. ΔCt values are row-normalized and color-coded (Red indicates maximal value, blue indicates minimal value for each gene). (D-E) Gene expression (qRT-PCR) in siRNA-treated cells, subsequently vehicle-treated (Uninf.) or infected 4 h with S. aureus. Data are mean ± S.E.M. (N = 5 biological replicates). (F) Gene expression (qRT-PCR) in RAW264.7 cells, TFEB-3xFLAG RAW264.7 cells or vector control RAW264.7 cells, either vehicle-treated (Uninf.) or infected 4 h with S. aureus. Data are mean ± S.E.M. (N = 3 biological replicates). (G) Quantification of cell-associated bacteria, expressed as C.F.U. per well (~5×105 cells). Representative experiment of three independent trials. Data are mean ± S.E.M. (N = 3 replicates). Differences between groups were not significant (two-sample t test). *: p < 0.05, **: p< 0.01, ***: p<0.001 (two-sample t test). See also Figure S5.
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