Mild impairment of mitochondrial function increases longevity and pathogen resistance through ATFS-1-driven activation of p38-regulated innate immunity

While mitochondrial function is essential for life in all multicellular organisms, a mild impairment of mitochondrial function can extend longevity. By understanding the molecular mechanisms involved, these pathways might be targeted to promote healthy aging. In studying two long-lived mitochondrial mutants in C. elegans, we found that disrupting subunits of the mitochondrial electron transport chain resulted in upregulation of genes involved in innate immunity, which we found to be dependent on not only the canonical p38-mediated innate immune signaling pathway but also on the mitochondrial unfolded protein response. Both of these pathways are absolutely required for the increased resistance to bacterial pathogens and extended longevity of the long-lived mitochondrial mutants, as is the FOXO transcription factor DAF-16. This work demonstrates that both the p38-mediated innate immune signaling pathway and the mitochondrial unfolded protein response can act on the same innate immunity genes to promote resistance to bacterial pathogens, and that input from the mitochondria can extend longevity by signaling through these two pathways. Combined, this indicates that multiple evolutionarily conserved genetic pathways controlling innate immunity also function to modulate lifespan. Significance Statement In this work, we explore the relationship between mitochondrial function, aging and innate immunity. We find that mild impairment of mitochondrial function results in upregulation of genes involved in innate immunity, increased resistance to bacterial pathogens and lifespan extension, all of which are dependent on two evolutionarily conserved signaling pathways. This work demonstrates how changes in functional status of the mitochondria can trigger activation of innate immunity, and that the underlying mechanisms are important for the longevity of the organism. This work advances our understanding of connections between metabolism and immunity. As the pathways studied here are conserved up to mammals, these insights may help us to understand the role of mitochondrial health, innate immunity and lifespan in humans.


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In this work, we explore the relationship between mitochondrial function, aging and innate 23 immunity. We find that mild impairment of mitochondrial function results in upregulation of 24 genes involved in innate immunity, increased resistance to bacterial pathogens and lifespan 25 extension, all of which are dependent on two evolutionarily conserved signaling pathways. This 26 work demonstrates how changes in functional status of the mitochondria can trigger activation 27 of innate immunity, and that the underlying mechanisms are important for the longevity of the 28 organism. This work advances our understanding of connections between metabolism and 29 immunity. As the pathways studied here are conserved up to mammals, these insights may help 30 us to understand the role of mitochondrial health, innate immunity and lifespan in humans.

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Introduction 32 33 While aging was long believed to be a stochastic process of damage accumulation, research 34 during the past three decades has demonstrated that lifespan can be strongly influenced by 35 genetics. Single-gene mutations have been shown to extend longevity in model organisms, 36 including yeast, worms, flies and mice. Importantly, genes and interventions that increase 37 lifespan tend to be conserved across species. For example, decreasing insulin-IGF1 signaling, 38 which was first shown to increase lifespan in the worm C. elegans(1-3), has subsequently been 39 shown to extend longevity in flies(4) and mice(5), and to be associated with longevity in 40 humans(6). This suggests that studying the aging process in model organisms can provide 41 insights that are relevant to human aging.

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The first single gene mutation that was shown to extend lifespan was identified in C. elegans(2, 44 3), and since then this organism has been extensively used to find additional genetic pathways 45 associated with lifespan extension and to elucidate the underlying mechanisms. Among the 46 earliest genes that were shown to influence longevity were genes involved in mitochondrial 47 function. Mutations in clk-1, nuo-6 and isp-1 affect different components of the mitochondrial 48 electron transport chain, and all lead to increased lifespan(7-10). In the case of nuo-6 and isp-1, While initially it was believed that the mechanism by which mild impairment of mitochondrial 57 function increased lifespan was through a decrease in the production of reactive oxygen species 58 (ROS) and the resulting oxidative damage(9), more recent studies show that mutations affecting 59 mitochondrial function actually increase the levels of ROS(16). The increase in ROS is required 60 6 34) (Fig. S1). Downstream of this pathway, the transcription factor ATF-7/ATF2/ATF7/CREB5 90 acts to modulate the expression of genes involved in innate immunity (35,36). While ATF-7 91 normally acts as a repressor of gene function, when it is phosphorylated by PMK-1, ATF-7 92 functions as an activator of p38/ATF-7-regulated immunity gene expression (Fig. S1).

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In this work, we show that the p38-mediated innate immune signaling pathway is essential for 95 the longevity and pathogen resistance of the long-lived mitochondrial mutants, nuo-6 and isp-1.

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We find that both strains exhibit an upregulation of genes involved in innate immunity that is 97 driven by the activation of the mitoUPR, but also dependent on the p38-mediated innate 98 immune signaling pathway, leading to an increased resistance to bacterial pathogens. The p38-99 mediated innate immune signaling pathway is absolutely required for the long lifespan of nuo-6 100 and isp-1 mutants. Finally, we demonstrate that activation of the mitoUPR is sufficient to 101 upregulate innate immunity genes, and is also required for their upregulation in nuo-6 mutants.

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Overall, this work demonstrates the importance of the mitoUPR in upregulating innate 103 immunity in response to signals from the mitochondria, and delineates a clear role of innate 104 immune signaling pathways in determining lifespan. 7

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Long-lived mitochondrial mutants exhibit broad upregulation of genes involved in innate 108 immunity that is dependent on p38-mediated innate immune signaling pathway 109 110 While mild impairment of mitochondrial function has been shown to extend longevity, the 111 underlying mechanisms are yet to be fully elucidated. When mitochondrial function is impaired, 112 mitochondria are able to communicate with the nucleus to alter nuclear gene expression. To 113 obtain a comprehensive, unbiased view of the transcriptional changes that result from 114 impairment of mitochondrial function, we used RNA sequencing (RNA-seq) to examine gene 115 expression in two long-lived mitochondrial mutants, nuo-6 and isp-1. After determining which 116 genes were differentially expressed compared to wild-type worms, we identified groups of 117 genes that showed enrichment. Among the genes that showed enrichment were genes involved 118 in innate immunity. These genes encode proteins that function to inhibit the growth and 119 survival of pathogenic bacteria, and to repair or remove damage to the worm(37, 38). 120 Accordingly, we decided to investigate the role of innate immunity in the long lifespan of these 121 long-lived mitochondrial mutants.

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To determine the extent to which genes involved in innate immunity are upregulated in the 124 long-lived mitochondrial mutants, nuo-6 and isp-1, we first examined eight genes, which others 125 have used to monitor innate immune activity(29, 35, 37, 39-41). These genes included T24B8.5,

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K08D8.5, F55G11.8, clec-65, clec-67, dod-22, Y9C9A.8 and C32H11.4. All of these genes are 127 upregulated in response to exposure to the bacterial pathogen Pseudomonas aeruginosa strain 128 PA14 and five of eight have been shown to be direct targets of the p38-mediated innate 129 immune signaling pathway in a ChIP-seq analysis of ATF-7(36). In examining the expression of 130 these genes in our RNA-seq data, we found that all eight genes were significantly upregulated 131 in both nuo-6 and isp-1 worms (Fig. 1A). To determine whether the upregulation of innate immunity genes in the long-lived 134 mitochondrial mutants requires the p38-mediated innate immune signaling pathway (NSY-1  135 SEK-1  PMK-1  ATF-7), we generated double mutants of nuo-6 and isp-1 with all of these 136 genes before measuring gene expression with quantitative RT-PCR (qPCR). We used loss of 137 function mutants for nsy-1, sek-1 and pmk-1. Since ATF-7 normally acts as a repressor, we used 138 the qd22 gain-of-function mutation for this gene. This mutation prevents phosphorylation of 139 ATF-7 by PMK-1 thereby making the mutant ATF-7 a constitutive repressor(35) (Fig. S1). As in 140 the RNA-seq data, the results from the qPCR experiments showed that innate immune genes 141 are upregulated in nuo-6 and isp-1 worms. Importantly, we found that in both nuo-6 and isp-1 142 worms that disruption of genes involved in the p38-mediated innate immune signaling pathway 143 (nsy-1, sek-1, pmk-1, atf-7(gof)) prevented the upregulation of innate immunity genes (

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To confirm these results using an alternative approach, we crossed a fluorescent reporter strain 147 for one of the innate immunity genes (T24B8.5)(35) to nuo-6 and isp-1 mutants and examined 148 the effect of knocking down sek-1 through RNA interference (RNAi). Again, using this approach, 149 we found that T24B8.5 is upregulated in nuo-6 and isp-1 worms, and that this upregulation is 150 dependent on SEK-1 (Fig. 1C). Combined, these results demonstrate that innate immunity 151 genes are upregulated in the long-lived mitochondrial mutants, nuo-6 and isp-1, and that this 152 upregulation is dependent on the p38-mediated innate immune signaling pathway.

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To further examine the expression of innate immunity genes in nuo-6 and isp-1 mutants, we 155 compared the differentially expressed genes in these mutants to a more comprehensive and 156 unbiased list of genes involved in innate immunity. A recent study defined the changes in gene 157 expression that result from exposure to the bacterial pathogen Pseudomonas aeruginosa strain 158 PA14 and determined which of these changes in gene expression are dependent on PMK-1 and 159 ATF-7(36). In total, they reported 300 genes that were upregulated by exposure to PA14 in a 160 PMK-1 and ATF-7-dependent manner, and 230 genes that were downregulated by exposure to 161 PA14 in a PMK-1 and ATF-7-dependent manner. We compared these lists of PA14-modulated, PMK-1-dependent, ATF-7-dependent genes to 164 genes that we found to be significantly upregulated or downregulated in nuo-6 and isp-1 165 mutants. We found that of the genes that are upregulated by PA14 exposure in a PMK-1 and 166 ATF-7-dependent manner, 38% (p=1.5 x 10 -35 ) and 30% (p=3.7 x 10 -27 ) are also upregulated in 167 nuo-6 and isp-1 mutants, respectively (Fig. 1D). There is a high degree of overlap between 168 genes upregulated in nuo-6 and isp-1 mutants (71%), and this includes the genes involved in 169 innate immunity (Table S1; 71 overlapping genes of 89/115 innate immunity genes upregulated 170 in nuo-6 or isp-1, respectively). In contrast, there was no significant overlap of the same genes 171 upregulated by PA14 exposure with genes downregulated in nuo-6 and isp-1 mutants. In 172 examining the list of genes that are downregulated by exposure to PA14 in a PMK-1 and ATF-7-173 dependent manner, we found that 20% (p=8.4 x 10 -7 ) and 20% (p=7.5 x 10 -5 ) were also 174 downregulated in nuo-6 and isp-1 mutants, respectively.

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To more comprehensively compare the PA14-modulated, PMK-1-dependent, ATF-7-dependent 177 gene expression changes to gene expression changes in nuo-6 and isp-1 mutants, we generated 178 heat maps comparing the expression of PA14-modulated, PMK-1-dependent, ATF-7-dependent 179 genes between wild-type worms and nuo-6 or isp-1 mutants. Among the genes that are 180 upregulated by PA14 exposure in a PMK-1 and ATF-7-dependent manner, many of these genes 181 are upregulated in nuo-6 and isp-1 mutants compared to wild-type worms, while a small subset 182 show decreased expression (Figs. S3,S4). Among the genes that are downregulated by PA14 183 exposure in a PMK-1 and ATF-7-dependent manner, many of these genes are downregulated in 184 nuo-6 and isp-1 mutants compared to wild-type worms, while a number of these genes also  Based on our observation that the long-lived mitochondrial mutants, nuo-6 and isp-1, have 198 increased expression of many genes involved in innate immunity, we next sought to determine 199 whether this increase in their expression resulted in enhanced resistance to bacterial 200 pathogens. To test pathogen resistance, we exposed worms to PA14 in a slow kill assay where 201 worms die from the ingestion and internal proliferation of the PA14 bacteria(42-44). We found 202 that both nuo-6 and isp-1 worms exhibited significantly increased survival on PA14 bacteria 203 compared to wild-type worms ( Fig. 2A). The increase in survival of nuo-6 and isp-1 worms did 204 not result from reduced exposure to the pathogenic bacteria as their tendency to avoid PA14 205 was equivalent to wild-type worms (Fig. S7).

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To determine the extent to which their enhanced resistance to bacterial pathogens is 208 dependent on the p38-mediated innate immune signaling pathway, we next examined PA14 209 resistance in nuo-6 and isp-1 worms in which genes in this pathway were disrupted. In wild-type 210 worms, mutations in nsy-1, sek-1, pmk-1 or atf-7(gof) significantly decrease the survival of 211 worms exposed to PA14 (Fig. 2B). Similarly, we found that disruptions of these innate immune 212 signaling genes in nuo-6 ( Fig. 2C) or isp-1 (Fig. 2D) worms also results in a significant decrease in 213 survival on PA14 bacteria. In nuo-6 worms, survival was decreased back to wild-type by 214 mutations in nsy-1, pmk-1 or atf-7(gof), while a larger decrease in survival was observed with 215 the sek-1 mutation. In isp-1 worms, mutations in nsy-1, sek-1 and atf-7(gof) all decreased 216 survival to a greater extent than in nuo-6 mutants, and in each case the survival of the double 217 mutant was less than in wild-type worms (Fig. S8). Combined, this shows that nuo-6 and isp-1 218 worms have increased resistance to bacterial pathogens, which is dependent on the p38-219 mediated innate immune signaling pathway. The p38-mediated innate immune signaling pathway is required for the lifespan extension 225 resulting from decreased insulin-IGF1 signaling (daf-2 mutants) and dietary restriction(38, 39).

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Interestingly, in both daf-2 mutants and dietary restricted worms expression of p38-regulated 227 innate immunity genes is largely decreased, and activation of this pathway to higher levels is 228 deleterious for lifespan extension, suggesting that a lower level of immune activation is optimal 229 under these conditions(39). We decided to examine the role of the p38-mediated innate 230 immune signaling pathway in the long lifespan of nuo-6 and isp-1 mutants, in which we found 231 these immunity genes were largely upregulated. To do this, we genetically disrupted 232 components of the p38-mediated innate immune signaling pathway in nuo-6 and isp-1 mutants 233 and quantified the resulting effect on lifespan. In nuo-6 mutants, we found that mutation of 234 nsy-1, sek-1, pmk-1 or atf-7(gof) reduced the long lifespan of nuo-6 worms to near wild-type 235 lifespan, almost completely preventing any increase in lifespan resulting from the nuo-6 236 mutation ( Fig. 3A-D). Similarly, we found that mutations in the p38-mediated innate immune 237 signaling pathway also markedly reduced the lifespan of isp-1 worms ( Fig. 3E-H). Mutations in 238 nsy-1, sek-1, or atf-7(gof) decreased isp-1 lifespan to near wild-type lifespan, while pmk-1 RNAi 239 reduced the lifespan extension in isp-1 worms by half (we were unable to generate isp-1 pmk-1 240 double mutants because of the proximity of the two genes on the same chromosome).

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Although OP50 bacteria, which is typically used for C. elegans maintenance and lifespan assays, 243 is considered to be non-pathogenic, it can have detrimental effects on lifespan through 244 proliferation in the pharynx and intestine (45, 46). Accordingly, we also measured the lifespan 245 of nuo-6 and isp-1 mutants on non-proliferating OP50 bacteria, as well as the dependency of 246 their long lifespan on the p38-mediated innate immunity pathway under these conditions.

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Similar to what we observed on live bacteria, we found that nuo-6 and isp-1 mutants are long-12 lived on non-proliferating bacteria, and that their long lifespan is still completely dependent on 249 the p38-mediated innate immune signaling pathway (Fig S9). isp-1 mutants(16), we hypothesized that the upregulation of innate immunity genes in these 264 mutants resulted from increased signaling through the p38-mediated innate immune signaling 265 pathway. To test this, we examined the extent to which this pathway might be activated in 266 these mutants by quantifying the ratio of phosphorylated (active) p38/PMK-1 to total p38/PMK-267 1 by Western blotting(47). To validate this approach, we showed that decreasing innate 268 immune signaling with sek-1 RNAi and increasing innate immune signaling with vhp-1 RNAi 269 (VHP-1 is a phosphatase that inhibits PMK-1/p38 signaling) alters the ratio of phospho-p38 to 270 p38 as expected (Fig. S10).

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Having shown that known modulators of PMK-1/p38 activation alter PMK-1/p38 273 phosphorylation in a predictable manner, we examined PMK-1/p38 activation in nuo-6 and isp-274 1 mutants. In both cases we found that neither the levels of PMK-1/p38 protein nor the 275 proportion of PMK-1/p38 protein that is phosphorylated are increased compared to wild-type 276 ( Fig. 4A,B). Thus, even though nuo-6 and isp-1 mutants have increased expression of ATF-7 277 13 target genes, there is no detectable increase in PMK-1/p38 activation. Since the p38-mediated 278 innate immune signaling pathway is required for the increased expression of ATF-7 target genes 279 even though p38 signaling is not increased, this suggests that this pathway is playing a 280 permissive role, and that other mechanisms are driving the innate immune activation in the 281 long-lived mitochondrial mutants. We next wanted to define the mechanism by which feeding is decreased in the long-lived 303 mitochondrial mutants. Since DAF-16 is activated in nuo-6 and isp-1 mutants (20) and 304 disruption of daf-16 has been shown to increase food intake in wild-type and daf-2 worms(39), 305 we sought to determine whether daf-16 is required for the decreased food intake observed in 306 14 the long-lived mitochondrial mutants. We found that deletion of daf-16 increased food 307 consumption in both wild-type and isp-1 worms (Fig. 4D), thereby indicating that DAF-16 is 308 required for the decreased food consumption in isp-1 mutants.

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Since activation of ATFS-1 is sufficient to upregulate DAF-16 target genes(19) and ATFS-1 is 311 activated in the long-lived mitochondrial mutants(19), we wondered whether ATFS-1 also 312 contributes to the decreased food intake in the long-lived mitochondrial mutants. While 313 disruption of atfs-1 (using the gk3094 deletion mutation) had no effect on feeding in a wild-314 type background, loss of atfs-1 reverted food consumption in nuo-6 worms to wild-type ( Having shown that activation of ATFS-1 contributes to the decreased food intake in nuo-6 319 mutants, we wondered whether activation of ATFS-1 alone is sufficient to decrease food 320 consumption. To test this idea, we measured food consumption in a constitutively activated 321 atfs-1 mutant (et17). In this mutant, the mitochondrial targeting sequence of ATFS-1 is 322 disrupted resulting in nuclear localization and constitutive activation of ATFS-1 target 323 genes(49). We found that the constitutively activated atfs-1 mutant et17 showed a marked 324 decrease in food consumption, equivalent to that observed in nuo-6 and isp-1 mutants (Fig. 4F).

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Combined, this indicates that activation of ATFS-1 is sufficient to decrease food consumption, 326 and is required for the diminished food intake in nuo-6 worms.

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The FOXO transcription factor DAF-16 is required for bacterial pathogen resistance in long-329 lived mitochondrial mutants but does not account for activation of innate immunity genes In our previous studies, we have shown that the mitoUPR is activated in nuo-6 and isp-1 worms, 356 and that this activation is required for their increased lifespan(19). As activation of the mitoUPR 357 by RNAi against spg-7 (the worm homolog of SPG7/paraplegin) has been shown to increase 358 resistance to PA14 and upregulate innate immunity genes(29), we hypothesized that the ATFS-1 359 activation that occurs in nuo-6 and isp-1 mutants(19) might contribute to their enhanced 360 resistance to PA14.

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To test this, we examined the effect of inhibiting the mitoUPR on the survival of nuo-6 worms 363 under bacterial pathogen stress. We found that disruption of atfs-1 completely ablates the 364 16 bacterial pathogen resistance of nuo-6 worms such that nuo-6;atfs-1 worms have decreased 365 survival compared to wild-type or atfs-1 single mutants (Fig. 6A). This indicates that activation 366 of the mitoUPR is required for the enhanced pathogen resistance in nuo-6 mutants.

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To determine if ATFS-1 is also responsible for the upregulation of innate immunity genes in nuo-369 6 mutants, we examined the effect of atfs-1 deletion. While deletion of atfs-1 did not decrease 370 the expression of any of the innate immunity genes examined in wild-type worms, loss of atfs-1 371 markedly decreased the expression of innate immunity genes in nuo-6 worms, in most cases 372 reverting expression levels to wild-type (Fig. 6B). Thus, ATFS-1 is required for the upregulation 373 of innate immunity genes in nuo-6 mutants, but is dispensable for the normal nutrient-driven 374 regulation of innate immune gene expression that is seen in wild-type worms(39).

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Activation of ATFS-1 is sufficient to upregulate innate immune signaling genes 377 378 Since loss of atfs-1 prevented the upregulation of innate immunity genes in nuo-6 mutants, we 379 next sought to determine if activation of ATFS-1 is sufficient to cause upregulation of innate 380 immunity genes. To do this, we examined gene expression in two constitutively active atfs-1 381 mutants, et15 and et17(49). We found constitutive activation of ATFS-1 resulted in a significant 382 upregulation of innate immunity genes (Fig. 7A). As a positive control, we found that the ATFS-1 383 target gene hsp-6 is significantly upregulated in both constitutively activated atfs-1 mutants 384 ( Fig. S12).

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To determine if activation of ATFS-1 causes upregulation of innate immunity genes through 387 activation of the p38-mediated innate immune signaling pathway, we examined PMK-1/p38 388 phosphorylation in a constitutively activated atfs-1 mutant (et17) and an atfs-1 loss-of-function 389 mutant (gk3094). Similar to what we observed in the long-lived mitochondrial mutants, we 390 found that the levels of PMK-1/p38 protein, phosphorylated PMK-1/p38 protein and the ratio 391 of phosphorylated p38/p38 are equivalent to wild-type in both atfs-1 mutants (Fig. 7B). This 392 suggests that activation of ATFS-1 can upregulate innate immunity genes without increased 393 activation of the p38-mediated innate immune signaling pathway.

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To determine if ATFS-1 might be able to directly bind to the same innate immunity genes as 396 ATF-7, we compared data from two previous CHiP-seq studies(36, 50). For this comparison, we 397 defined innate immunity genes as genes that are upregulated in response to a 4-hour exposure 398 to PA14(36). After exposure to PA14, ATF-7 was found to be bound to 345 of these innate 399 immunity genes(36). Following exposure to mitochondrial stress induced by spg-7 RNAi, ATFS-1 400 was found to be bound to 51 of these innate immunity genes(50). Of the 51 innate immunity 401 genes that can be bound by ATFS-1, 49% (25 genes) can also be bound by ATF-7 (Fig. S13; Table   402 S3). This binding likely occurs at different sites since the predicted binding site motifs of ATFS-1 403 and ATF-7 bear little similarity(36, 50). The fact that ATFS-1 and ATF-7 are able to bind to the 404 same genes involved in innate immunity indicates that the mitoUPR and p38-mediated innate 405 immune signaling pathway act in parallel to modulate the expression of innate immunity genes, 406 which are critical for lifespan extension. While both the long-lived mitochondrial mutants and constitutively active atfs-1 mutants, 437 exhibit increased expression of ATF-7 target genes overall, these mutants show no increase in 438 p38-mediated innate immune signaling (Fig. 7). Moreover, since ATFS-1 activation is sufficient 439 to decrease food intake, these mutants would be predicted to have decreased ATF-7 target 440 gene expression because decreasing food intake diminishes p38-mediated innate immune 441 signaling(39). Combined this suggests that ATFS-1 can activate innate immunity genes directly.

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This conclusion is supported by the fact that ATFS-1 can bind to the same innate immunity 443 genes as ATF-7 (Fig. S12).

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Previous work examining the relationship between the mitoUPR and p38-mediated innate 446 immune signaling pathway has generated conflicting results. In an earlier study, activation of 447 the mitoUPR by spg-7 RNAi was found to increase the survival of pmk-1 and sek-1 mutants on 448 PA14, leading the authors to conclude that the mitoUPR was increasing pathogen resistance 449 independently of the p38-mediated innate immune signaling pathway(29). Subsequently, it was 450 shown that overexpression of the mitoUPR target gene hsp-60 is sufficient to activate the p38-451 mediated innate immune signaling pathway leading to increased expression of genes involved 452 in innate immunity and increased PA14 resistance, all of which was dependent on p38-453 mediated innate immune signaling pathway (nsy-1, sek-1 and pmk-1)(40). Our current results 454 resolve this discrepancy by demonstrating that the mitoUPR can affect the expression of innate 455 immunity genes and pathogen resistance both directly through binding of ATFS-1 to innate 456 immunity genes, and indirectly through the p38-mediated innate immune signaling and ATF-7 457 (Fig. 7).

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The increased expression of innate immunity genes in nuo-6 and isp-1 worms results in 460 enhanced resistance to bacterial pathogens, which is dependent on both ATFS-1 and the p38-   Strains were grown on OP50 bacteria at 20°C. All of the strains were genotyped or sequenced 551 to confirm the presence of the mutation in the gene of interest. Double mutants were 552 generated by crossing WT males to nuo-6 or isp-1 mutants and crossing the resulting 553 heterozygous male progeny (nuo-6/+ or isp-1/+) to the mutant of interest (nsy-1, sek-1, pmk-1, 554 atf-7). After selfing, slow growing progeny were singled and genotyped or sequenced to identify 555 double mutants. We were unable to generate isp-1 pmk-1 and nuo-6 daf-16 double mutants 556 due to the close proximity of these two genes on the same chromosome. to these prepared plates, with 10 plates scored per strain. The assays were conducted at 20°C.

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Animals that did not respond to gentle prodding from a platinum wire were scored as dead.   Table S2.  Table S2.   Tables S1 to S3 Figure S1. Overview of p38-mediated innate immune signaling pathway. The p38-mediated innate immune signaling pathway is a MAPK signaling pathway. Exposure to bacterial pathogens or increased food intake results in the activation of this pathway through the phosphorylation of NSY-1/ASK1 (MAPK kinase kinase). NSY-1 then phosphorylates SEK-1/MKK3/MKK6 (MAPK kinase), which phosphorylates PMK-1/p38 (MAPK), which phosphorylates the transcription factor ATF-7/ATF2/ATF7/CREB5. Under normal conditions, ATF-7 acts as a repressor inhibiting the expression of innate immunity genes. When ATF-7 is phosphorylated by PMK-1, it becomes an activator promoting the expression of innate immunity genes. The qd22 mutation prevents phosphorylation of ATF-7 by PMK-1. As a result, ATF-7 with the qd22 mutation acts as a constitutive repressor even in the presence of bacterial pathogens.  Figure S2. Upregulation of innate immunity genes in long-lived mitochondrial mutants requires the p38mediated innate immune signaling pathway. Mutation of genes involved in the p38-mediated innate immune signaling pathway (nsy-1, sek-1, pmk-1, atfs-7(gof)) decrease the expression of genes involved in innate immunity in wild-type (A), nuo-6 (B), and isp-1 (C) worms. Gene expression was determined by quantitative real-time RT-PCR on three biological replicates of pre-fertile young adult worms. Error bars indicate SEM. All differences from control are significant p<0.05.  A B C Figure S3. Genes that are upregulated by activation of the p38-mediated innate immune pathway are primarily upregulated in the long-lived mitochondrial mutant nuo-6. This heatmap includes genes that are upregulated by exposure to the bacteria pathogen P. aeruginosa PA14 in a PMK-1-and ATF-7-dependent manner and for which the expression is significantly changed in nuo-6 mutants. Figure S4. Genes that are upregulated by activation of the p38-mediated innate immune pathway are primarily upregulated in the long-lived mitochondrial mutant isp-1. This heatmap includes genes that are upregulated by exposure to the bacteria pathogen P. aeruginosa PA14 in a PMK-1-and ATF-7dependent manner and for which the expression is significantly changed in isp-1 mutants. Figure S5. Expression of genes that are downregulated by activation of the p38-mediated innate immune pathway is altered in the long-lived mitochondrial mutant nuo-6. This heatmap includes genes that are downregulated by exposure to the bacteria pathogen P. aeruginosa PA14 in a PMK-1and ATF-7--dependent manner and for which the expression is significantly changed in nuo-6 mutants. Figure S6. Expression of genes that are downregulated by activation of the p38-mediated innate immune pathway is altered in the long-lived mitochondrial mutant isp-1. This heatmap includes genes that are downregulated by exposure to the bacteria pathogen P. aeruginosa PA14 in a PMK-1and ATF-7--dependent manner and for which the expression is significantly changed in isp-1 mutants. To explore the mechanisms underlying the increased resistance to bacterial pathogens in nuo-6 and isp-1 mutants, bacterial avoidance and food consumption were examined. There was no significant difference in bacterial avoidance between wild-type worms and nuo-6 or isp-1 mutants on OP50 bacteria (A) or P. aeruginosa (B). Three biological replicates per strain were quantified. Error bars indicate SEM.  Figure S8. Disruption of p38-mediated innate immune signaling pathway decreases resistance to bacterial pathogens. Resistance to bacterial pathogens was tested by exposing worms to Pseudomonas aeruginosa strain PA14 in a slow kill assay. Long-lived mitochondrial mutants, nuo-6 and isp-1, have increased survival on pathogenic PA14 bacteria. Mutations affecting genes involved in the p38-mediated innate immune signaling pathway including nsy-1, sek-1, pmk-1 and atf-7(gof) cause decreased resistance to bacterial pathogens in wild-type (white bars), nuo-6 (blue bars) and isp-1 (red bars) worms. Black dotted line indicates wild-type survival. Blue dotted line indicates nuo-6 survival. Red dotted line indicates isp-1 survival. Survival is measured as the number of days from exposure to PA14 (day 3 of adulthood) until death. Error bars indicate SEM of survival of individual animals. This bar graph is a summary of data shown in Figure 2 to facilitate comparison across all strains. Raw data and total N per strain are provided in Table S2. sek-1 atf-7(gof) nuo-6 isp-1 Figure S9. Disruption of genes involved in the p38-mediated innate immune signaling pathway abolishes the extended longevity of long-lived mitochondrial mutants independently of bacterial proliferation. Quantification of nuo-6 and isp-1 lifespan on non-proliferating bacteria revealed that their long lifespan is independent of bacterial proliferation. Similarly, lifespan extension in nuo-6 and isp-1 mutants is completely dependent on having a function p38-mediated innate immune signaling pathways as deletion of nsy-1, sek-1, or atf-7(gof) completely prevented the increase in lifespan in long-lived nuo-6 and isp-1 mutants. Lifespans were performed in liquid culture with worms fed ad libitum. Bacteria proliferation was prevented through treatment with cold and antibiotics. Statistical analysis on survival plots was performed with logrank test. p-value indicates significance of difference between blue and purple lines. All strains were tested in a single parallel experiment. Control strains are shown in multiple panels for direct comparison. Raw data and total N per strain can be found in Table S2. Figure S10. Proportion of activated PMK-1/p38 is not increased in nuo-6 and isp-1 worms. Activation of PMK-1/p38 was measured by levels of phosphorylated p38/PMK-1 compared to total levels of PMK-1 by western blotting. As a positive and negative controls, we examined the effect of vhp-1 RNAi and sek-1 RNAi. RNAi against sek-1 resulted in decreased phosphorylation of PMK-1/p38 without affecting total PMK-1 levels. RNAi against vhp-1 increased phospho-p38/PMK-1 levels without affecting total PMK-1 levels. RNAi treatment was begun at the L3 developmental stage and worms were collected 2 days later.   Figure S11. DAF-16 is not required for expression of innate immune signaling pathway target genes. daf-16 expression was knocked down using RNAi beginning at the L4 stage of the parental generation. While daf-16 RNAi effectively decreased the expression of DAF-16 target genes (b, sod-3, dod-3, mtl-1, ftn-1, icl-1, sodh-1) in both wild-type and nuo-6 mutants, it did not markedly affect the expression of any of the innate immunity genes (c, T24B8.5, K08D8.5, F55G11.8, clec-65, clec-67, dod-22, Y9C9A.8 and C32H11.4). This suggests that DAF-16 is not required for expression of innate immune signaling pathway target genes in wild-type worms and nuo-6 mutants. RNA was isolated from six biological replicates at the young adult stage of the experimental generation. RNA from the six biological replicates was pooled for RNA sequencing.
Innate immunity genes DAF-16 target genes  Figure S13. ATFS-1 and ATF-7 bind to the same genes involved in innate immunity. Innate immunity genes are defined as genes that are upregulated by a 4-hour exposure to PA14. This list was obtained from Fletcher et al., 2019. ATF-7-bound genes are genes bound by ATF-7 after exposure to PA14 from a CHIP-seq experiment conducted by Fletcher et al., 2019. ATFS-1-bound genes are genes bound by ATFS-1 after mitochondrial stress resulting from RNAi against spg-7 as determined by a CHIP-seq experiment conducted by Nargund et al., 2015. ATF-7 was found to be bound to 345 innate immunity genes after exposure to PA14. ATFS-1 was found to be bound to 51 innate immunity genes after exposure to mitochondrial stress. Of these 51 genes, 25 were found to be in common with ATF-7-bound innate immunity genes. This highly significant overlap (p=1.28X10 -29 ) clearly demonstrates that ATF-7 and ATFS-1 can bind to and regulate the same innate immunity genes. See supplemental Table 2  Phospho-p38 WT isp-1 nuo-6 atfs-1 gof(et17) lof nuo-6 isp-1 isp-1 WT