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. Author manuscript; available in PMC: 2015 Jun 19.
Published in final edited form as: Immunity. 2014 May 29;40(6):896–909. doi: 10.1016/j.immuni.2014.05.002

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,#
PMCID: PMC4104614  NIHMSID: NIHMS598830  PMID: 24882217

Abstract

Animal host defense against infection requires the expression of defense genes at the right place and the right time. To understand such tight control of host defense requires the elucidation of the transcription factors involved. 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.

INTRODUCTION

Innate mechanisms represent the first line of defense against microbial infection, not only for highly evolved vertebrates but also for the simplest metazoans (Hoffmann et al., 1999). How hosts are able to detect the presence of pathogens, and in response trigger the expression of innate defense genes, is a major question in biology. Without such gene expression, the host is unable to deploy both innate and adaptive immune responses (Ayres and Schneider, 2012; Hoffmann et al., 1999; Medzhitov, 2007). In recent years, pathways of signal transduction to transcription factors that drive defense gene expression have become better understood (Medzhitov and Horng, 2009). For instance, Toll-like receptor (TLR) and nucleotide-binding domain, leucine-rich repeat containing (NLR) signaling pathways that activate NF-κB transcription factors have emerged as major paradigms of control of defense gene expression (Ishii et al., 2008). However, the complete set of transcriptional regulators that control innate host defense remains poorly defined (Amit et al., 2009; 2011).

Evidence of the existence of undiscovered host defense transcription factors has partly emerged from the study of nematodes, the most abundant animals on the planet. These invertebrates lack NF-κB and other transcription factors known to participate in innate immunity in higher organisms (Irazoqui et al., 2010b). Furthermore, nematodes lack NLR and TLR pathways (Ishii et al., 2008). Nonetheless, bacterivorous nematodes, such as the model organism Caenorhabditis elegans, are capable of detecting infection and of discriminating infectious agents. As a result, they induce the expression of pathogen-specific transcriptional host responses that aid host survival (Engelmann et al., 2011; O’rourke et al., 2006; Sinha et al., 2012; Troemel et al., 2006). Because the transcription factors that control the induction of such responses are only partially identified, these findings strongly suggest that important host defense transcription factors remain unknown.

The initial discovery of NF-κB transcription factors and subsequent genetic studies performed in the invertebrate Drosophila melanogaster led to the elucidation of TLR signaling in mammalian innate immunity (Medzhitov and Horng, 2009). Inspired by this approach, we set out to identify C. elegans transcription factors required for the induction of the host response to Staphylococcus aureus. Infection of C. elegans with S. aureus by the oral route entails colonization of the intestinal lumen, intestinal epithelial cell destruction, and nematode death within 48 h (Irazoqui et al., 2010a; Sifri et al., 2003). Nematode killing requires S. aureus virulence factors that are also involved in human disease, indicating that S. aureus uses overlapping virulence mechanisms in worms and in humans (Bae et al., 2004; Begun et al., 2005).

As with other infection paradigms in C. elegans, S. aureus elicits a pathogen-specific transcriptional host response that is important for defense (Irazoqui et al., 2010a). However, the transcription factor(s) required for such response were not known. In the present study, we report that the evolutionarily conserved transcription factor HLH-30 is critical for the induction of the host response to S. aureus in C. elegans.

In mammalian cells, the HLH-30 ortholog TFEB is known to control the transcription of autophagy and lysosomal biogenesis genes in response to nutritional stress (Settembre and Ballabio, 2011). Upstream negative regulation by the kinases mTORC1 and ERK2 maintains TFEB inactive until intracellular amino acids become depleted (Settembre et al., 2011; 2012) or lysosomal function is disrupted (Martina et al., 2012; Roczniak-Ferguson et al., 2012; Settembre et al., 2012). Furthermore, we and others have shown that TFEB controls lipid store mobilization under conditions of nutritional deprivation, a function that is conserved between mammals and nematodes (Cuervo, 2013; O’Rourke and Ruvkun, 2013; Settembre et al., 2013). Due to its role in stress responses, enhancement of TFEB activity has emerged as a potential therapeutic approach for multiple lysosomal and protein aggregation disorders (Decressac et al., 2013; Pastore et al., 2013; Spampanato et al., 2013). Likewise, C. elegans HLH-30 was implicated in autophagy-mediated longevity extension in long-lived gonad-deficient animals (Lapierre et al., 2013b). Thus, TFEB has recently emerged as a nutritionally controlled stress-response factor.

Here we report that, in addition to its known role in nutritional stress, TFEB is also important for host defense against infection. HLH-30 was activated early during infection, and mutants lacking HLH-30 exhibited a profound host defense defect. Mechanistically, we observed that HLH-30 drove the vast majority of the transcriptional host response, and that both HLH-30-regulated antibacterial and autophagy genes were required for host tolerance of infection. In murine macrophages, we observe that TFEB was similarly activated following S. aureus infection and was required for induction of a repertoire of cytokine and chemokine genes, suggesting that TFEB may perform evolutionarily conserved defense functions in cells of the mammalian innate immune system. Taken together, our observations identify C. elegans HLH-30 and its mammalian ortholog TFEB as previously unknown transcription factors in the host response to infection.

RESULTS

Infection induces rapid nuclear accumulation of HLH-30, the sole C. elegans MiT transcription factor

We previously showed that infection with S. aureus induces a strong transcriptional host response that enhances C. elegans survival. Because this response occurs in the absence of NF-κB, this observation strongly suggested that an alternative transcription factor(s) is important for host response induction. To identify such factor(s), we examined which transcription factor binding sites were over-represented in the promoters of S. aureus-induced C. elegans genes. Using the software MAGMA (Ihuegbu et al., 2012), we analyzed the transcription start site (TSS)-proximal upstream 2 kb, and detected over-representation of the E-box DNA motif (CACGTG, P<0.001) (Fig. 1A). We found that E-boxes were most frequently located within the first 500 bp upstream of potential target TSS (Fig. 1A). The E-box is recognized in many organisms by basic helix-loop-helix (bHLH) transcription factors (Massari and Murre, 2000). Furthermore, we detected over-representation of the related M-box motif (P<0.001), which is specifically recognized by the MiT subfamily of bHLH transcription factors [comprised in humans by microphthalmia-associated transcription factor (MITF), and transcription factors E3 (TFE3), EB (TFEB), and EC (TFEC)] (Hemesath et al., 1994). These results suggested that an MiT-class transcription factor may be involved in the induction of the C. elegans host response to S. aureus.

Figure 1. HLH-30 acutely responds to S. aureus infection.

Figure 1

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(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).

Phylogenetic analysis of genes encoding bHLH proteins revealed that the gene hlh-30 encodes the sole MiT-class homolog in the C. elegans genome (Fig. 1B). Furthermore, HLH-30 protein had previously been shown to bind the E-box motif in vitro (Grove et al., 2009). Because the bulk of S. aureus-triggered transcriptional changes occur in the intestinal epithelial cells (Irazoqui et al., 2010a), we examined whether HLH-30 protein was also expressed in the intestine. We generated a C. elegans strain that carries a multicopy transgene composed of the hlh-30 promoter followed by GFP-tagged HLH-30a cDNA (hlh-30p::hlh-30::gfp). In uninfected animals, HLH-30::GFP protein was expressed throughout development (Fig. 1C). In L4 larvae and young adults, the stages used in our infection model, expression was highest in the intestine, rectal epithelial cells, vulval epithelial cells, spermatheca and pharynx, and absent from the gonads (Fig. 1C). Thus, HLH-30 protein appeared to be expressed in most discernable tissues, including the intestine.

The GFP signal was equally distributed between nucleus and cytoplasm of expressing cells, suggesting that HLH-30 may reside in both compartments in uninfected animals. In contrast, in infected animals HLH-30::GFP dramatically concentrated in the nucleus in all discernable tissues after just 30 min of infection (Fig. 1F-H). Importantly, control animals transferred to plates without food (to control for possible short-term starvation effects due to the transition from E. coli to S. aureus lawns) exhibited diffuse HLH-30::GFP localization (Fig. 1D-E and 1H), similar to uninfected animals fed nonpathogenic E. coli (Fig. 1C). These observations suggested that HLH-30 quickly reacts to infectious stimuli in adult animals by translocating and accumulating in the nucleus.

HLH-30 controls expression of host defense genes

To directly test the hypothesis that HLH-30 is important for defense gene induction, we performed transcriptional profiling by RNA-seq of S. aureus-infected wild type and hlh-30(tm1978) mutant animals, compared with controls fed nonpathogenic E. coli (Table S1). hlh-30(tm1978) mutant animals harbor a deletion that eliminates the HLH DNA binding domain and therefore is considered a null allele (Grove et al., 2009). After 8 h of infection, 825 genes were upregulated in wild type animals, defining the normal transcriptional host response to S. aureus (Fig. 2A and Table S2). 637 (77 %) of these genes were hlh-30-dependent, as they were not upregulated in hlh-30 mutants (Fig. 2A and Table S3 and Table S4). Thus, we concluded that HLH-30 was required for the vast majority of gene expression changes in infected animals, indicating that HLH-30 performed a key role in host defense.

Figure 2. HLH-30 is required for the host response to S. aureus infection.

Figure 2

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(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

An additional 188 genes were upregulated in both hlh-30 and wild type animals (Fig. 2A and Table S5), indicating that these genes were hlh-30-independent and suggesting that additional pathways may be involved in the host response. De novo discovery using MAGMA showed that the E-box motif was significantly over-represented only among hlh-30-dependent genes, suggesting that such gene set may be specifically enriched for direct HLH-30 target genes (Fig. 2A and Table S6).

To examine the biological consequence of such a striking transcriptional defect, we monitored infection survival of adult animals lacking hlh-30 function. hlh-30 mutant animals exhibited compromised survival of S. aureus infection, consistent with a role for HLH-30 in host defense (Fig. 2B and Table S7). Performing colony forming unit (C.F.U.) assays, we verified that similar amount of bacteria accumulated in the intestine of wild type and mutant animals (Fig. 2D). Thus, compared to wild-type animals, HLH-30 appeared less tolerant to infection, understood as the ability of the host to endure infection at a given pathogen load (Ayres and Schneider, 2012). hlh-30 animals also exhibited defective survival of Enterococcus faecalis, Salmonella enterica, and Pseudomonas aeruginosa infections (Fig. S1A-C and Table S7), indicating that HLH-30 is involved in defense against a range of Gram-positive and -negative pathogens. However, hlh-30 mutants also exhibited shortened survival on nonpathogenic food (heretofore referred to as ‘longevity’, Fig. 2C, S1D,E and Table S7). In addition, we previously showed that HLH-30 is required for survival of starvation (Settembre et al., 2013). In contrast, hlh-30 mutants did not exhibit a defect in resistance to oxidative stress, ruling out a generalized stress response defect in these animals (Fig. S1F). Together, these data suggested that HLH-30 may perform important functions for host defense and for longevity determination.

We previously showed that certain S. aureus-induced genes are individually required for survival of infection but not for longevity (Irazoqui et al., 2010a). Interestingly, we found that expression of 3 such genes (F43C11.7, math-38, and cyp-37B1) was HLH-30-dependent (Table S3), raising the possibility that their decreased expression in hlh-30 mutants might be causally linked to the observed defect in host defense. Using qRT-PCR, we confirmed that induction of these three genes required HLH-30 (Fig. S1G). In addition, we verified that RNAi of F43C11.7, math-38, and cyp-37B1 reduced the infection survival of wild-type animals, but not their longevity (Fig. S1H-J and Table S7).

In contrast, knockdown of math-38, cyp-37B1 or F43C11.7 did not affect survival of hlh-30 mutants (Fig. S1H-J and Table S7), consistent with the model that HLH-30 is required for their expression. These results suggested that HLH-30 may control host defense by driving the expression of genes that are important for survival of infection.

Overexpression of HLH-30::GFP in hlh-30 mutants rescued the longevity defect of hlh-30 mutants (Fig. 2C and Table S7), confirming the functionality of the HLH-30::GFP construct. Interestingly, the same construct further enhanced hlh-30 host survival of infection beyond wild type (Fig. 2B and Table S7). Taken together, these observations suggest that HLH-30 enhances host tolerance of infection by induction of downstream host defense genes.

HLH-30 controls expression of signaling pathways

Within the hlh-30-dependent gene set, we identified hlh-30 itself to be induced in an HLH-30-dependent manner (Table S3). Using qRT-PCR, we verified that hlh-30 transcript was induced 2-fold after 12 h of infection (Fig. 3A). Both hlh-30 basal and induced expression were diminished in animals carrying the hlh-30(tm1978) allele (Fig. 3B and S2). Together, these data suggested that HLH-30 participates in a positive feedback loop during infection.

Figure 3. HLH-30 is required for expression of host response components.

Figure 3

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(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

To elucidate downstream mechanisms by which HLH-30 may mediate host defense, we sought to define the cellular and physiological processes regulated by hlh-30. To this end, we performed gene-set enrichment analysis (GSEA) of HLH-30-dependent genes. GSEA showed that HLH-30 orchestrated a complex host response composed of cellular homeostasis genes, metabolic genes, and antimicrobial genes (Fig. 3C and Table S8). To facilitate analysis and discussion, we grouped such genes according to overall functional commonalities in four groups, labeled “Signaling”, “Cytoprotective”, “Antimicrobial”, and “Other” (Fig. 3D).

We observed that HLH-30 regulated the expression of known and putative signaling components, such as cell surface receptors, predicted protein kinases, transcription factors, and genes that encode components of signaling pathways implicated in host defense in many organisms (e.g. JNK, p38 MAPK, and TGF-β pathways, Fig. 3D). Of these, we verified hlh-30-dependent induction of kgb-1 (human JNK homolog), nsy-1 (homologous to human ASK1) and mdl-1 (MAD-like bHLH transcription factor) (Fig. 3E and S2). We also confirmed hlh-30-dependent induction of components of the insulin signaling pathway: ins-11 (insulin), sgk-1 (SGK), dct-1 (target of DAF-16/FOXO) (Fig. 3E and S2). These insulin signaling genes, together with mdl-1, are known longevity regulators (Hertweck et al., 2004; Kawano et al., 2006; Murphy et al., 2003; Pinkston-Gosse and Kenyon, 2007), and may be involved in longevity control downstream of HLH-30. Thus, HLH-30 controls the expression of signaling pathways relevant to immunity and to longevity.

HLH-30 controls expression of antimicrobial genes

In addition to signaling pathways, HLH-30 regulated an ‘Antimicrobial’ component of the host response (Fig. 3C-D) including genes that encode proteins with proposed or demonstrated antimicrobial activity, such as lysozymes, C-type lectins, antimicrobial peptides, and ferritin (Boehnisch et al., 2011; Hoeckendorf et al., 2012; Schulenburg et al., 2008; Simonsen et al., 2011; Tarr, 2012). We verified hlh-30-dependent induction of 14 genes in this group (Fig. 4A-D and Fig. S3A), demonstrating that HLH-30 is necessary for induction of the antimicrobial response. Furthermore, expression was restored by complementation with HLH-30::GFP for a majority of genes tested (Fig. 4E and S3B). The overexpression of HLH-30 in these animals was sufficient to drive overexpression of a few target genes, providing a plausible explanation for their enhanced infection survival (Fig. 2B).

Figure 4. HLH-30-regulated antimicrobial genes are required for defense.

Figure 4

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(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

HLH-30-controlled antimicrobial genes are necessary for host defense

We next addressed the significance of HLH-30-dependent antimicrobial genes. We focused on lysozymes, whose antibacterial activity by enzymatic degradation of bacterial peptidoglycan is well understood. To circumvent functional redundancy among them, we simultaneously knocked down expression of lys-5 and ilys-2, the two lysozymes whose expression was most affected by loss of hlh-30 (Fig. 4A). This treatment caused a drastic reduction in survival of infection in wild type, but not in hlh-30 animals (Fig. 4F, 4I and Table S7). We verified that RNAi treatment did not interfere with pathogen accumulation in the intestine (Fig. 4I). These results are consistent with the notion that lysozymes function downstream of HLH-30 and appear to participate in host tolerance of infection. In contrast, similar treatment did not result in major reduction of longevity in uninfected wild type animals (Fig. 4G-H and Table S7), showing that the observed reduction of survival is specific to infection. As a result, we concluded that antibacterial genes controlled by HLH-30 are required for host defense.

HLH-30 controls expression of cytoprotective mechanisms

In addition to signaling and antimicrobial genes, HLH-30 was necessary for the induction of ‘Cytoprotective’ genes associated with cellular homeostasis and repair (Fig. 3C-D), such as genes important for protein folding and for xenobiotic detoxification. A major fraction of this response belonged to the autophagy-lysosomal pathway of cell catabolism and clearance (Levine et al., 2011).

To examine the importance of cytoprotection in C. elegans defense against S. aureus, we first used qRT-PCR to verify the induction of autophagy genes lgg-1 and lgg-2 (homologous to human MAP1LC3), unc-51 (homologous to human ULK1), and atg-2, - 13, and −16.2 (homologous to human ATG2, ATG13, and ATG16L1, respectively)(Fig. 5A and S4A), which participate in several steps throughout the autophagic cycle (Meléndez and Levine, 2009) (Fig. S4B). Additionally, we verified induction of lysosomal genes with clinically important human homologs, such as cpr-1 and cpr-2 (homologous to human lysosomal Cathepsin B), asm-1 and asm-3 (homologous to human sphingomyeline phosphodiesterase SMPD1), nuc-1 (homologous to human DNAse), and tre-5 (homologous to human trehalase TREH)(Fig. 5B and S4A). These observations demonstrated that infection results in the induction of genes that participate in autophagy, and suggested that autophagy may play a significant role in C. elegans defense against S. aureus.

Figure 5. HLH-30-regulated autophagy genes are required for defense.

Figure 5

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(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

In all cases, infection-induced expression was abrogated by deletion of hlh-30 (Fig. 5A-B and S4A). Consistently, we observed markedly reduced levels of GFP::LGG-1 protein expression in hlh-30 mutants compared with wild type (Fig. S4C-G). Moreover, expression of a subset of genes was increased by over-expression of HLH-30::GFP, indicating that HLH-30 is both necessary and sufficient for cytoprotective gene induction (Fig. 5C and S4H).

HLH-30-controlled autophagy genes are required for host defense

To examine whether functional autophagy was differentially regulated during S. aureus infection, we used animals expressing GFP::LGG-1. GFP::LGG-1 localizes to autophagosomes and is used as a marker to quantify autophagosome abundance, in the form of GFP-positive intracellular foci (Fig. S4B) (Meléndez et al., 2003). Compared with uninfected controls (Fig. 5D-E,H), infection by S. aureus significantly increased the abundance of GFP::LGG-1 foci in intestinal cells (Fig. 5F-H), consistent with infection-induced autophagosome formation.

Next, we tested whether autophagy was required for defense against S. aureus. RNAi-mediated depletion of lgg-1, unc-51, or vps-34 (homologous to human phosphoinositide 3-kinase VPS34), which participate in early steps of the autophagic cycle (Meléndez and Levine, 2009) (Fig. S4B), impaired survival of infection (Fig. 5I-K and Table S7). Strikingly, none of such RNAi treatments caused detectable longevity changes (Fig. 5L-N and Table S7). In contrast, RNAi of autophagy genes did not affect the susceptibility of hlh-30 mutants (Fig. 5I-K and Table S7), nor their longevity (Fig. 5L-N and Table S7). Furthermore, vps-34 RNAi did not affect intestinal pathogen load (Fig. 5O), suggesting that autophagy may function as a mechanism of tolerance of infection. In conclusion, these data support the model that autophagy functions downstream of HLH-30 specifically for host defense.

TFEB is activated by infection in murine macrophages

Until this point, our evidence suggested that HLH-30 was critical for host defense in nematodes. Most importantly, we had found that HLH-30 became activated early during infection. Because HLH-30 is orthologous to mammalian TFEB, we hypothesized that mammalian TFEB may also become activated in innate immune cells during stimulation with bacterial pathogens.

Several independent studies reported that mammalian TFEB is regulated by phosphorylation. Phospho-TFEB is retained in an inactive form in the cytosol, whereas dephosphorylated TFEB is imported into the nucleus and drives transcription (Peña-Llopis et al., 2011; Roczniak-Ferguson et al., 2012; Settembre et al., 2011; 2012). To test whether TFEB is activated during infection in macrophages, we examined TFEB phosphorylation by anti-TFEB immunoblot of extracts from murine macrophage RAW264.7 cells infected with live or heat-killed S. aureus for 1 h. Regardless of the type of S. aureus used, we observed a quantitative shift of the TFEB band to a lower molecular weight (Fig. 6A), suggesting that a reduction in TFEB phosphorylation had taken place. A similar molecular weight shift was observed in cells stably expressing FLAG-tagged TFEB (TFEB-FLAG) after 1 h of infection (Fig. 6B). To verify that the molecular weight shift was due to differential phosphorylation, we treated the samples with λ phosphatase. As a result, both uninfected and infected samples exhibited an even faster migrating TFEB band (Fig. 6C). Therefore, we concluded that TFEB-FLAG was phosphorylated in both infected and uninfected cells, with a smaller extent of phosphorylation in infected cells. Collectively, these observations support the notion that TFEB becomes activated in macrophages during stimulation with S. aureus. Furthermore, this activation did not require the pathogen to be alive, and thus may not result from damage caused by the infection but rather from detection of pathogen-associated molecules.

Figure 6. S. aureus infection of murine macrophages activates TFEB.

Figure 6

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(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).

To further test TFEB activation, we examined the subcellular localization of TFEB-FLAG. Subcellular fractionation showed nuclear exclusion of TFEB in uninfected cells, and its redistribution to the nucleus in infected cells after just 1 h (Fig. 6D). Furthermore, immunofluorescence in uninfected controls revealed TFEB-FLAG mainly in the cytosol (Fig. 6E-F), whereas in contrast infected cells exhibited quantitative relocalization of TFEB-FLAG to the nucleus (Fig. 6E-F). Collectively, these observations strongly suggested that TFEB is activated early during infection in macrophages.

TFEB is required for the transcription of cytokines and chemokines in murine macrophages

To determine the physiological relevance of TFEB in stimulated macrophages, we knocked down endogenous TFEB expression using siRNA in RAW264.7 cells (Fig. 7A-B). After 48 h of treatment with a single siRNA (siTFEB #1) or a pool of siRNAs (siTFEB #2) against TFEB, we observed an approx. 50% reduction of TFEB transcript, and 65% and 84% reduction of total TFEB protein compared to control siRNA (siCtrl)(Fig. 7A-B).

Figure 7. TFEB is required for the macrophage proinflammatory response.

Figure 7

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(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.

To identify genes that required TFEB for their induction, we examined cytokine and chemokine transcript levels in infected siRNA-treated cells using commercial qRT-PCR assays. Using infected siCtrl macrophages as reference, we found that knockdown of TFEB using siTFEB #2 resulted in decreased transcript levels of several cytokines and chemokines such as the proinflammatory interleukins (IL) IL-1β and IL-6, or the chemokine CCL5 and CCL17 (Fig. 7C). In independently designed assays, we found that IL-1β and IL-6, as well as tumor necrosis factor (TNF) α, exhibited reduced transcript levels in infected cells with decreased TFEB expression (Fig. 7D). We obtained similar results for CCL5 (Fig. 7D), CCL17, IL-27 and IL-1rn (Fig. S5A).

Additionally, we found significant overlap between sets of genes induced by TFEB overexpression in HeLa cells and induced by S. aureus in human macrophages (Koziel et al., 2009; Kupershmidt et al., 2010; Sardiello et al., 2009) (Fig. S5B), with a strong positive correlation between their sets of upregulated genes (Fig. S5C). The set of overlap genes was significantly enriched for functional categories relevant to host defense (Fig. S5D). We verified that a subset of these genes was also induced in mouse macrophages (Fig. S5E). While TNFα-induced protein 3 (TNFAIP3) and TNF superfamily member 9 (TNFSF9) did not seem to require TFEB for their induction (Fig. S5F), TFEB siRNA significantly reduced induction of the autophagy gene optineurin (OPTN) (Fig. 7E). Genes encoding suppressor of cytokine signaling 3 (SOCS3) (Fig. 7E), 2′-5′-oligoadenylate synthetase 2 (OAS2), and intracellular adhesion molecule 1 (ICAM1) (Fig. S5G) also trended towards reduction. Conversely, TFEB overexpression in TFEB-FLAG RAW264.7 cells was sufficient for expression of target genes: OPTN and CCL5 were both more highly expressed in these cells compared with vector-transfected cells or parental RAW264.7 cells (Fig. 7F). Additionally, their induction by S. aureus was proportionally higher (Fig. 7F). Phagocytosis controls ruled out an effect of TFEB knockdown on bacterial uptake, since cells pretreated with siTFEB#1 and siTFEB#2 were indistinguishable from siCtrl-treated cells in terms of bacterial uptake and killing after 1 and 4 h (Fig. 7G). Taken together, these data show that TFEB is necessary and sufficient for the expression of proinflammatory signaling molecules in a murine macrophage cell line.

DISCUSSION

The evidence in this report supports an important role for TFEB in innate host defense. We report a C. elegans transcription factor that is acutely activated during infection and that controls the vast majority of the induced host response. Furthermore, to our knowledge this is the first report of transcriptional induction of autophagy and lysosomal biogenesis during infection in any organism, and provides a strong rationale for examining the transcriptional control of those processes during infection in mammals. Starting from unbiased de novo motif discovery, we identified HLH-30 as a critical transcription factor for host defense against infection. We showed that HLH-30 nuclear accumulation is immediately induced by infection. This is evidence that HLH-30 is acutely activated by infection stimuli by analogy to the human MiT transcription factors, including TFEB, which translocate into the nucleus upon activation (Roczniak-Ferguson et al., 2012; Sardiello et al., 2009). Activation of TFEB early during infection is evolutionarily conserved, based on our observations that murine TFEB is activated and required for a proper host response in macrophages infected by S. aureus. Therefore, it is likely that TFEB is an important component of host defense signaling in cells of the mammalian innate immune system.

Because both mammalian TFEB (Settembre et al., 2011) and its C. elegans homolog HLH-30 are major regulators of autophagy and lysosomal gene expression, we submit that HLH-30 is the C. elegans TFEB functional homolog. This conclusion is independently supported by observations that HLH-30 controls autophagy and lysosomal gene expression in long-lived gonadless worms (Lapierre et al., 2013a) and in starved animals (O’Rourke and Ruvkun, 2013). Furthermore, we previously showed that HLH-30 controls the expression of lipid metabolism genes during starvation, a function that it also shares with human TFEB (Cuervo, 2013; O’Rourke and Ruvkun, 2013; Settembre et al., 2013). Therefore, data showing that HLH-30 exhibits similar subcellular localization and functional significance as human TFEB in diverse physiological scenarios strongly support the identification of HLH-30 as the C. elegans TFEB functional homolog.

Our findings resemble the initial findings concerning the fundamental role of Drosophila DIF or 7NF-κB in host response induction (Lemaitre and Hoffmann, 2007). In the case of DIF, its identification as an important innate immunity transcription factor led to the discovery of Toll signaling as a key signaling pathway for host defense in flies and in mammals (Lemaitre, 2004). As in C. elegans, we found that murine TFEB is activated during phagocytosis of bacteria in macrophages, where it is important for the expression of pro-inflammatory mediators. Therefore, we hypothesize that the last common ancestor of invertebrates and vertebrates may have used at least two signaling axes to induce the complex host response: one axis composed of the well-studied pathways that lead to NF-κB activation, and another that controlled TFEB activity. While TFEB mainly controls cytoprotective genes, NF-κB may have been specifically focused on other types of defense genes. In flies, for example, NF-κB mainly controls antimicrobial peptide production (Ganesan et al., 2011). Over evolution, nematode HLH-30 may have concentrated both antimicrobial and cytoprotective functions, thus becoming fully redundant with NF-κB and allowing its loss from the genome. Because TFEB appears to have remained involved in the host response of mammals, it is likely that further study of the signaling pathways that control HLH-30 during infection may increase understanding of innate immunity signaling in higher organisms.

Although in the present study we focused on host defense, our results also indicate that HLH-30 performs important longevity functions, such as the transcription of longevity regulators and its effect on longevity assurance. This raises the possibility that HLH-30 could control host defense and longevity through overlapping downstream processes. In this scenario, inhibition of a downstream pathway would be expected to produce similar effects during host survival of infection and aging. Alternatively, HLH-30 could control downstream pathways that specifically function either in host defense or longevity. In this scenario, inhibition of defense-specific downstream pathways would be expected to affect host survival of infection and not longevity. Our observations better support the latter scenario: inhibition of autophagy caused a defect in host defense against S. aureus, but did not alter longevity. Similar results were obtained when inhibiting the antimicrobial response. This is evidence that the pathways that operate downstream of HLH-30 for host defense are separable from those that function in longevity.

Autophagy has previously been implicated in intestinal epithelial host defense in C. elegans (Jia et al., 2009) and in mammals (Benjamin et al., 2013; Patel and Stappenbeck, 2013). The proposed mechanism is the clearance of intracellular pathogens, such as Salmonella enterica (Jia et al., 2009; Madeo et al., 2010). However, we previously showed that S. aureus does not invade the intestinal epithelial cells of C. elegans (Irazoqui et al., 2010a). Alternatively, autophagy is believed to mediate cytoprotection under conditions of stress (Kroemer et al., 2010). One attractive hypothesis stemming from our observations is that autophagy mediates cell repair to enhance the host’s ability to survive large burdens of pathogens, by limiting pathogen- and self-inflicted cellular damage and thus providing the time window necessary for antimicrobial responses to achieve maximal efficacy.

Mechanisms of host defense that limit the damage caused by infection, but that do not directly affect pathogen burden, have been termed mechanisms of “tolerance of infection” (not to be confused with immunological tolerance) (Ayres and Schneider, 2012). Mechanisms of tolerance of infection are poorly understood, especially in animals (Medzhitov, 2009). Because defects in HLH-30 and its downstream pathways did not appear to affect pathogen burden over the course of infection, we favor the notion that HLH-30/TFEB controls the expression of genes involved in infection tolerance. Further study is required to elucidate precisely how TFEB-regulated pathways effect host tolerance of infection in nematodes and in mammals.

EXPERIMENTAL PROCEDURES

C. elegans strains and growth

All strains used in this study are detailed in supplemental information. C. elegans was grown on nematode-growth media (NGM) plates seeded with E. coli OP50 according to standard procedures (Brenner, 1974).

Bacterial strains

The bacterial strains used is study are: Escherichia coli OP50 (Ura.StrR); Staphylococcus aureus NCTC8325 (wild type strain; rsbU mutant); Salmonella enterica serovar typhimurium SL1344 (VanB GmR); Enterococcus faecalis V583 (VanB GmR); Pseudomonas aeruginosa PA14 (Pathogenic clinical isolate); Escherichia coli HT115 [F-, mcrA, mcrB, IN(rrnD-rrnE)1, lambda-, rnc14::Tn10(DE3 lysogen:lacUV5 promoter-T7 polymerase)].

Identification of M-box and E-box motifs in the promoters of S. aureus induced genes

A Grubb’s test was performed on the normalized expression values of the previously described microarray dataset in the GSE21819 record to remove potential outlier gene probes (16; p ≤ 10-9). Differentially expressed probes were then detected using a Z-score test on the log-fold changes. 249 genes were identified whose probe intensity were higher than expected by chance (15, p < 0.01). In parallel, we discovered a catalog of 2,309 potentially functional DNA motifs with MAGMA using its default parameters (Ihuegbu et al., 2012). MAGMA uses C. elegans as the reference genome and compares segments of the genome that are conserved across five other nematode species, to identify motifs within the reference genome that occur in intergenic, intronic, and 5′ and 3′ untranslated regions of annotated genes (Ihuegbu et al., 2012). To narrow the search space, conserved sites for each motif (the “exemplar sites”) were associated with nearby genes using PeakAnalyzer (Salmon-Divon et al., 2010), defining 664 motifs that were associated with at least 20 genes and were present within 2 kb of their translation start sites. Next, significantly over-represented M-box and E-box motifs were identified in promoters from the 249 significantly up-regulated S. aureus-induced genes by comparison to the catalog of exemplar sites from the 664 putative cis-regulatory motifs.

Survival Assays

All bacterial pathogenesis (killing) assays were performed at 25 °C as described in (Powell and Ausubel, 2008). For consistency with bacterial pathogenesis assays, all longevity assays were also performed at 25 °C. Bacterial strains and detailed procedure are described in supplemental information.

Cell culture and siRNA transfection

RAW264.7 cells (ATCC) were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 μg/ml streptomycin, and 100 μg/ml penicillin. RAW264.7 cells stably expressing TFEB-3xFLAG (Ferron et al., 2013) were grown in 250 μg/ml Geneticin. 50 nM ON-TARGET Plus siRNA Control, TFEB #1 (single siRNA) and TFEB #2 (smart pool) (Thermo Scientific Dharmacon) were transfected using Lipofectamine LTX (Life technologies) incubated 48 h prior to analysis, according to (Carralot et al., 2009).

S. aureus infection in macrophages

S. aureus NCTC8325 was grown at 37 °C in Columbia medium (Difco, BD) supplemented with 10 μg/ml Nalidixic acid overnight, cultured the next day to the mid-exponential phase (OD600 = 0.8 - 1), washed twice in cold PBS and resuspended in DMEM 10% FBS without antibiotic. RAW264.7 cells were infected at MOI 10. After 30 min, infected cells were washed twice in PBS and incubated 30 min (1 h time point), 210 min (4 h time point) or 450 min (8 h time point) in DMEM 10% FBS supplemented with 100 μg/ml gentamicin prior to analysis.

qRT-PCR

C. elegans were washed twice in water and lysed in TRI Reagent (Molecular Research Center). RAW264.7 cells were washed twice in PBS and directly lysed in TRI Reagent. cDNA was obtained using SuperScript III (Invitrogen) and analyzed as in (Irazoqui et al., 2008). For the cytokine and chemokine screen, cDNA was obtained using RT2 first strand kit (Qiagen) and analyzed using Mouse Cytokine and Chemokine RT2 profiler plates (Qiagen). Data analysis was performed using the Pfaffl method (Pfaffl, 2001).

Supplementary Material

01
02

HIGHLIGHTS.

  • ■

    HLH-30 is a key regulator of the C. elegans transcriptional response to infection.

  • ■

    HLH-30 induces autophagy and antimicrobial genes required for tolerance of infection.

  • ■

    TFEB, a murine homolog of HLH-30, is activated during infection in macrophages.

  • ■

    TFEB is required for proper induction of several cytokines and chemokines.

ACKNOWLEDGMENTS

We thank A. Lacy-Hulbert for helpful discussions, S. Margolis for critical reading of the manuscript, A. Sokolovska and A. Tanne for assistance with macrophage experiments, L. R. Lapierre and M. Hansen for sharing unpublished data, M. Ferron and G. Karsenty for their generous gift of TFEB-3xFLAG RAW264.7 cells, and V. Ambros for the hlh-30(tm1978) strain. We thank the Genome Access Technology Center at Washington University Medical School for their sequencing assistance, especially N. Rockweiler for helpful discussions. Some C. elegans strains were provided by the Caenorhabditis Genetics Center, which is funded by the NIH Office of Research Infrastructure Programs (P40-OD010440). This study was funded in part by the National Institute of General Medical Sciences of the National Institutes of Health, under awards numbers P30-GM092431, R01-GM101056, and R01-GM101056-S1 (to J. E. I.) and R01-HG000249 from the NHGRI (to G. D. S.). O.V. was funded by a Fund for Medical Discovery postdoctoral fellowship from the Massachusetts General Hospital. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Footnotes

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AUTHOR CONTRIBUTIONS

OV performed, analyzed, and reported most experiments. NI performed bioinformatic analysis of microarray and RNAseq data, identifying DNA motifs and differentially expressed genes. SAL, LGL, AFA, ACW, and JEI contributed to specific experiments. LMS provided key intellectual input. GDS and JEI provided overall guidance. All authors were involved in writing the manuscript.

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Supplementary Materials

01
NIHMS598830-supplement-01.pdf (4.4MB, pdf)
02
NIHMS598830-supplement-02.xlsx (299.8KB, xlsx)

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