The FOXO Transcription Factor DAF-16 Bypasses ire-1 Requirement to Promote Endoplasmic Reticulum Homeostasis

SummaryThe unfolded protein response (UPR) allows cells to adjust the capacity of the endoplasmic reticulum (ER) to the load of ER-associated tasks. We show that activation of the Caenorhabditis elegans transcription factor DAF-16 and its human homolog FOXO3 restore secretory protein metabolism when the UPR is dysfunctional. We show that DAF-16 establishes alternative ER-associated degradation systems that degrade misfolded proteins independently of the ER stress sensor ire-1 and the ER-associated E3 ubiquitin ligase complex sel-11/sel-1. This is achieved by enabling autophagy-mediated degradation and by increasing the levels of skr-5, a component of an ER-associated ubiquitin ligase complex. These degradation systems can act together with the conserved UPR to improve ER homeostasis and ER stress resistance, beyond wild-type levels. Because there is no sensor in the ER that activates DAF-16 in response to intrinsic ER stress, natural or artificial interventions that activate DAF-16 may be useful therapeutic approaches to maintain ER homeostasis

Altered somatic hypermutation patterns in COVID-19 patients classifies disease severity

IntroductionThe success of the human body in fighting SARS-CoV2 infection relies on lymphocytes and their antigen receptors. Identifying and characterizing clinically relevant receptors is of utmost importance.MethodsWe report here the application of a machine learning approach, utilizing B cell receptor repertoire sequencing data from severely and mildly infected individuals with SARS-CoV2 compared with uninfected controls.ResultsIn contrast to previous studies, our approach successfully stratifies non-infected from infected individuals, as well as disease level of severity. The features that drive this classification are based on somatic hypermutation patterns, and point to alterations in the somatic hypermutation process in COVID-19 patients.DiscussionThese features may be used to build and adapt therapeutic strategies to COVID-19, in particular to quantitatively assess potential diagnostic and therapeutic antibodies. These results constitute a proof of concept for future epidemiological challenges

Innate immunity mediated longevity and longevity induced by germ cell removal converge on the C-type lectin domain protein IRG-7.

In C. elegans, removal of the germline triggers molecular events in the neighboring intestine, which sends an anti-aging signal to the rest of the animal. In this study, we identified an innate immunity related gene, named irg-7, as a novel mediator of longevity in germlineless animals. We consider irg-7 to be an integral downstream component of the germline longevity pathway because its expression increases upon germ cell removal and its depletion interferes with the activation of the longevity-promoting transcription factors DAF-16 and DAF-12 in germlineless animals. Furthermore, irg-7 activation by itself sensitizes the animals' innate immune response and extends the lifespan of animals exposed to live bacteria. This lifespan-extending pathogen resistance relies on the somatic gonad as well as on many genes previously associated with the reproductive longevity pathway. This suggests that these genes are also relevant in animals with an intact gonad, and can affect their resistance to pathogens. Altogether, this study demonstrates the tight association between germline homeostasis and the immune response of animals, and raises the possibility that the reproductive system can act as a signaling center to divert resources towards defending against putative pathogen attacks

Innate immunity mediated longevity and longevity induced by germ cell removal converge on the C-type lectin domain protein IRG-7

<div><p>In <i>C</i>. <i>elegans</i>, removal of the germline triggers molecular events in the neighboring intestine, which sends an anti-aging signal to the rest of the animal. In this study, we identified an innate immunity related gene, named <i>irg-7</i>, as a novel mediator of longevity in germlineless animals. We consider <i>irg-7</i> to be an integral downstream component of the germline longevity pathway because its expression increases upon germ cell removal and its depletion interferes with the activation of the longevity-promoting transcription factors DAF-16 and DAF-12 in germlineless animals. Furthermore, <i>irg-7</i> activation by itself sensitizes the animals' innate immune response and extends the lifespan of animals exposed to live bacteria. This lifespan-extending pathogen resistance relies on the somatic gonad as well as on many genes previously associated with the reproductive longevity pathway. This suggests that these genes are also relevant in animals with an intact gonad, and can affect their resistance to pathogens. Altogether, this study demonstrates the tight association between germline homeostasis and the immune response of animals, and raises the possibility that the reproductive system can act as a signaling center to divert resources towards defending against putative pathogen attacks.</p></div

The transcription factor DAF-12 is activated in <i>irg-7(zc6)</i> mutants.

<p>(A-B) The <i>irg-7(zc6)</i> mutation is sufficient to increase the expression of the <i>Pcdr-6</i>::<i>gfp</i> reporter (Student's t-test, p<0.0001) in a <i>daf-12(+)</i> background but not in a <i>daf-12(-)</i> background (Student's t-test, p = 0.075). Asterisks mark Student's t-test values of P<0.001 compared to the fluorescence in wild-type animals. 30–35 animals analyzed per genotype. Error bars represent SE of 3 independent biological replicates. (C) The <i>irg-7(zc6)</i> mutation is not sufficient to increase the expression of the DAF-16 reporter <i>Psod-3</i>::<i>gfp</i> (Student's t-test, P = 0.067). (D) The <i>irg-7(zc6)</i> mutation is not sufficient for the accumulation of DAF-16::GFP translational reporter in the intestine.</p

<i>irg-7(zc6)</i> is a gain of function mutation in F40F4.6.

<p>(A) <i>F40F4</i>.<i>6</i> RNAi significantly shortened the lifespan of <i>irg-7(zc6)</i> mutants (Mantel-Cox, P<0.0001) but did not affect the lifespan of otherwise wild-type animals (Mantel-Cox,P = 0.9). Mean lifespans are indicated within each graph. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s004" target="_blank">S2 Table</a></b> for more lifespan data. (B) Representative fluorescence micrographs (100-fold magnification) of day-1 adults harboring an integrated <i>Phsp-4</i>::<i>gfp</i> transgene. <i>F40F4</i>.<i>6</i> RNAi significantly reduced the expression of the <i>Phsp-4</i>::<i>gfp</i> in <i>zc6</i> mutants. Asterisks mark Student's T-test values of P<0.001 compared to fluorescence on control RNAi. 40 animals were analyzed per genotype. Error bars represent SE of 3 independent biological replicates. (C) Expression of fosmid WRM0619aB08 (which includes F40F4.6) significantly extended the lifespan of wild-type animals (Mantel-Cox, P<0.001), whereas expression of the partially overlapping fosmid WRM0635bF05 (which does not include F40F4.6) did not extend the lifespan of wild-type animals (Mantel-Cox, P = 0.4). Mean lifespans are indicated within each graph. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s004" target="_blank">S2 Table</a></b> for more lifespan data. (D) Expression of an NLS-RFP reporter fused to the putative promoter upstream of the F40F4.6 gene drives expression in the posterior cells of the intestine. Fluorescence of this reporter increased upon exposure of L4 animals to Photorhabdus luminescens subsp Hb bacteria (HB) or Enterococcus faecalis bacteria (EF). Asterisks mark Student's T-test values of P<0.001 compared to wild-type fluorescence on OP50 bacteria. (E) Schematic representation of the major domains in the F40F4.6 protein. The region deleted by the <i>zc6</i> mutation is indicated by a dashed line. This region includes an EGF domain. Note that the deletion preserves the ORF of the original gene. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s002" target="_blank">S2 Fig</a> for sequence data.</p

The longevitiy induced by germ cell removal and by the <i>irg-7(zc6)</i> mutation extend lifespan by a shared mechanism.

<p>(A) Bar graph presenting the percentage of lifespan extension conferred by the <i>irg-7(zc6)</i> mutation in the indicated genetic backgrounds. Each bar is the average of at least three independent lifespans. Error bars represent SE of at least 3 independent biological replicates. Asterisks mark bars in which all the lifespan experiments were increased significantly by the <i>irg-7(zc6)</i> mutation (P<0.05). Colored boxes indicate longevity pathways associated with each longevity gene: the reproductive longevity pathway induced by germ cell removal (Red), the insulin/IGF1 signaling pathway (Blue), the dietary restriction pathway (Orange) and the mitochondrial respiration pathway (Green). Note that the dietary restriction pathway is only partly dependent of <i>daf-</i>16 (marked with an Orange triangle), depending on the dietary regimen used. Note that the longevity of <i>irg-7(zc6) gof</i> mutants requires many genes implicated in the longevity of germline-less animals with the exception of <i>nhr-80</i>, which is not required for <i>irg-7(zc6)</i> longevity although it is required for the longevity of germlineless animals. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s008" target="_blank">S6 Table</a></b> for more lifespan data. (B-C) The <i>irg-7(zc6) gof</i> mutation extended the lifespan of wild-type animals with an intact reproductive system but shortened the lifespan of animals that lack a somatic gonad (B). It did not further extend the lifespan of germline-less animals (C). Mean lifespan and Mantel-Cox P-values are indicated within each graph. Mantel Cox P-value for the wild type vs. <i>irg-7(zc6)</i> single mutant comparison is in green; Mantel Cox P-value for the mutant vs. the mutant; <i>irg-7(zc6)</i> double mutant comparison is in black. <b>See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s008" target="_blank">S6 Table</a></b> for additional lifespan data.</p

<i>irg-7(zc6)</i> mutation extends lifespan by increasing pathogen resistance.

<p>(A) Feeding animals with dead OP50 bacteria extended the lifespan of wild-type animals (Mantel-Cox, P<0.001), but did not extend the lifespan of <i>irg-7(zc6)</i> mutants (Mantel-Cox, P = 0.53). (B) The <i>irg-7(zc6) gof</i> mutation improved the survival of animals fed with pathogenic HB bacteria (Mantel-Cox, P<0.0001). (C-D) <i>irg-7</i> inactivation by pre-treatment with F40F4.6 RNAi from eggs to early adulthood hindered survival of animals fed henceforth with pathogenic HB bacteria (C) (Mantel-Cox, P<0.0001), but did not affect the survival of animals fed henceforth with OP50 bacteria (D) (Mantel-Cox, P = 0.7). (E) <i>irg-7</i> inactivation by pre-treatment with F40F4.6 RNAi from eggs to early adulthood did not affect the survival of <i>daf-16(mu86)</i> mutants fed henceforth with HB bacteria (Mantel-Cox, P = 0.43). Mean lifespan are indicated within each graph. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s005" target="_blank">S3 Table</a></b> for additional lifespan data.</p

The reproductive innate immunity pathway model.

<p>Pathogens are one of the factors that limit lifespan in <i>C</i>. <i>elegans</i> and in higher organisms. Our data implies that exposure to pathogens and/or their toxins can directly or indirectly modulate germline proliferation and survival resulting in a reproductive system with less germ cells. Germline depletion induces the transcription of <i>irg-7</i>, an innate immunity-related secreted protein whose induction promotes the activation of a somatic defense response. On one hand, <i>irg-7</i> enables the activation of bona-fida longevity-associated transcription factors, which support the induction of a longevity-promoting transcriptome. At the same time, <i>irg-7</i> enables the activation of <i>atf-7</i>, a dedicated innate immunity-related transcription factor. In this way, exposure to pathogens can be sensed by perturbations in germline homeostasis. Consequently, the reproductive system can serve as a signaling center to divert key metabolic resources towards defending against the putative pathogen attack by activating the innate immune response.</p