EMBO/soda-nel huggingface dataset:
Mild mitochondrial impairment enhances innate immunity and longevity through ATFS-1 and p38 signaling (2021)
Figure 1

Figure 1. Innate immunity genes are upregulated in long-lived mitochondrial mutants.

A. Genes involved in innate immunity are significantly upregulated in nuo-6 and isp-1 worms. Gene expression changes in the mitochondrial mutants were determined by RNA sequencing and compared to wild-type N2 worms. Results represent counts per million (CPM) expressed as a percentage of wild-type of six biological replicates per strain.

B. Upregulation of innate immunity genes is dependent on the p38-mediated innate immune signaling pathway including NSY-1, SEK-1, PMK-1 and ATF-7. Gene expression changes were examined by quantitative RT-PCR of three biological replicates per strain.

C. Using a fluorescent reporter strain for the innate immunity gene T24B8.5 confirms that innate immunity genes are upregulated in the long-lived mitochondrial mutants and that components of the p38-mediated innate immune signaling pathway are required for their upregulation. Scale bar indicates 250 µM. Three biological replicates per strain were quantified.

D. Differentially expressed genes in long-lived mitochondrial mutants show significant overlap with genetic targets of the p38-mediated innate immune signaling pathway. Significantly modulated genes in long-lived mitochondrial mutants nuo-6 and isp-1 (six biological replicates per strain) were compared to genes that are modulated in response to exposure to the bacterial pathogen P. aeruginosa strain PA14 in a PMK-1- and ATF-7-dependent manner as identified by Fletcher et al., 2019. There is a highly significant degree of overlap between genes upregulated by activation of the p38-mediated innate immune pathway and genes upregulated in nuo-6 and isp-1 mutants. Similarly, there is a highly significantly degree of overlap between genes downregulated by activation of the p38-mediated innate immune pathway and genes downregulated in nuo-6 and isp-1 mutants. The p-values indicate the significance of the difference between the observed number of overlapping genes between the two gene sets and the expected number of overlapping genes if the genes were picked at random.

Data information: Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001. Statistical significance was determined using a one-way ANOVA with Dunnett's multiple comparison test in panels A and B. Statistical significance was determined using a two-way ANOVA with Bonferroni post-test in panel C.
Mild mitochondrial impairment enhances innate immunity and longevity through ATFS-1 and p38 signaling (2021)
Figure 2

Figure 2. Long-lived mitochondrial mutants exhibit increased resistance to bacterial pathogens that is dependent on presence of p38-mediated innate immune signaling pathway. A-D. Resistance to bacterial pathogens was tested by exposing worms to Pseudomonas aeruginosa strain PA14 in a slow kill assay. Both nuo-6 and isp-1 long-lived mitochondrial mutants show increased resistance compared to wild-type worms (A). Disruption of components of the p38-mediated innate immune signaling pathway (nsy-1, sek-1, pmk-1, atf-7) markedly decreases resistance to PA14 in wild-type (B), nuo-6 (C) and isp-1 (D) worms. All strains were tested in a single parallel experiment. Two biological replicates per strain were performed with a total of at least 175 animals per strain. A bar graph of all of these results can be found in Appendix Fig S7.

Data information: ***p<0.001. Statistical significance for survival plots was determined with the log-rank test. Significance is indicated between the strain listed on top and all other strains. Data from panel A is repeated in panels B, C, and D for direct comparison.

Source data are available online for this figure.

Source Data for Figure 2.
Mild mitochondrial impairment enhances innate immunity and longevity through ATFS-1 and p38 signaling (2021)
Figure 3

Figure 3. Disruption of the p38-mediated innate immune signaling pathway markedly ablates extended longevity of long-lived mitochondrial mutants.

A-H. To examine the role of the p38-mediated innate immune signaling pathway in the long-lifespan of the nuo-6 and isp-1 mitochondrial mutants, we crossed the long-lived mitochondrial mutants to worms with mutations in nsy-1, sek-1, pmk-1 and atf-7(gof). In each case, mutation of genes involved in p38-mediated innate immunity signaling markedly reduces the lifespan of the long-lived mitochondrial mutant but have little or no impact on wild-type lifespan. In panel G, we utilized pmk-1 RNAi instead of the pmk-1 mutation because of the close proximity of isp-1 and pmk-1 on the same chromosome. EV indicates empty vector. The lifespan results for panels A, B, D, E, F, and H were performed in a single parallel experiment. Results are from a minimum of three biological replicates per strain.

Data information: Statistical significance for survival plots was determined with the log-rank test. p-values indicate significance between red and purple lines. Control strains are shown in multiple panels for direct comparison.

Source data are available online for this figure.

Source Data for Figure 3.
Mild mitochondrial impairment enhances innate immunity and longevity through ATFS-1 and p38 signaling (2021)
Figure 4

Figure 4. p38-mediated innate immune signaling pathway is not activated in long-lived mitochondrial mutants.

A,B. To determine the extent to which the p38-mediated innate immunity pathway is activated in nuo-6 and isp-1 mutants, we measured the ratio of phosphorylated PMK-1/p38 to total PMK-1/p38 by Western blotting. Representative blots are shown (A). There is no difference in the ratio of phosphorylated to total PMK-1/p38 in the long-lived mitochondrial mutants compared to wild-type (B). Eight biological replicates of wild-type worms and nine of mitochondrial mutants were quantified.

C. Both long-lived mitochondrial mutants, nuo-6 and isp-1, exhibit markedly decreased food consumption compared to wild-type worms. Six biological replicates per strain were measured, each including at least three technical replicates.

D. Deletion of daf-16 significantly increases food consumption in both wild-type and isp-1 mutants. Three biological replicates per strain were measured, each including at least three technical replicates.

E. While disruption of atfs-1 does not affect food intake in wild-type worms, it increases food consumption in nuo-6 worms back to wild-type. atfs-1 loss of function allele was gk3094. Three biological replicates per strain were measured, each including at least three technical replicates.

F. Constitutive activation of atfs-1 is sufficient to decrease food consumption to the same level as in long-lived mitochondrial mutants. Three biological replicates per strain were measured, each including at least three technical replicates. atfs-1(lof) allele is gk3094, atfs-1(gof) allele is et17.

Data information: Error bars indicate SEM. ***p<0.001. NS = not significant. Statistical significance was assessed using a one-way ANOVA with Dunnett's multiple comparison test in panels B, C and F. Statistical significance was assessed using a two-way ANOVA with Bonferroni posttest in panels D and E.

Source data are available online for this figure.

Source Data for Figure 4, panel B.
Mild mitochondrial impairment enhances innate immunity and longevity through ATFS-1 and p38 signaling (2021)
Figure 5

Figure 5. DAF-16/FOXO is required for increased resistance to bacterial pathogens in long-lived mitochondrial mutants.

A. A deletion of daf-16 completely abolishes the increased resistance to bacterial pathogens in isp-1 mutants. p-value indicates the significance of difference between red and purple lines. Three biological replicates per strain were performed.

B,C. While daf-16 RNAi effectively decreases the expression of DAF-16 target genes (B, sod-3, dod-3, mtl-1, ftn-1, icl-1, sodh-1), it does not 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 indicates that DAF-16 is not required for expression of innate immune signaling pathway target genes in isp-1 mutants. daf-16 expression was knocked down using RNAi beginning at the L4 stage of the parental generation. 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.

Data information: Statistical significance in panel A was assessed using log-rank test.

Source data are available online for this figure.

Source Data for Figure 5.
Mild mitochondrial impairment enhances innate immunity and longevity through ATFS-1 and p38 signaling (2021)
Figure 6

Figure. 6. ATFS-1 is required for increased bacterial pathogen resistance and upregulation of innate immunity genes in long-lived nuo-6 mitochondrial mutants.

A. To examine the role of the ATFS-1 transcription factor and the mitoUPR response in the enhanced bacterial pathogen resistance in nuo-6 mutants, we examined the effect of disrupting atfs-1 in nuo-6 mutants. Loss of atfs-1 completely abolishes the increased resistance to bacterial pathogens in nuo-6 worms. p-value indicates significance of difference between red and purple lines. nuo-6; atfs-1 contains the gk3094 deletion allele. Three biological replicates per strain were measured.

B. To examine the role of ATFS-1 in the upregulation of innate immunity genes in nuo-6 mutants, we compared gene expression between nuo-6 and nuo-6; atfs-1 mutants with wild-type worms and atfs-1 mutants as controls. Deletion of atfs-1 significantly decreases the expression of genes involved in innate immunity in nuo-6 worms but does not decrease the expression of these genes in wild-type worms. Gene expression changes were determined by RNA sequencing of six biological replicates of each genotype. Results represent counts per million (CPM) expressed as a percentage of wild-type. Worms in a wild-type background (control) are shown with white bars, while worms with atfs-1 deletion are shown with blue bars.

Data information: Error bars indicate SEM. **p<0.01, ***p<0.001. Statistical significance in panel A was assessed using log-rank test. Statistical significance in panel B was assessed using a two-way ANOVA with Bonferroni posttest.

Source data are available online for this figure.

Source Data for Figure 6, panel A.
Mild mitochondrial impairment enhances innate immunity and longevity through ATFS-1 and p38 signaling (2021)
Figure 7

Figure. 7. Constitutive activation of mitochondrial unfolded protein response results in upregulation of innate immunity genes.

A. Examination of mRNA levels of genes involved in innate immunity in two constitutively active atfs-1 mutants (et15 and et17) revealed that innate immunity genes are upregulated when ATFS-1 is activated. This indicates that activation of the mitoUPR can cause activation of genes that are regulated by the p38-mediated innate immune pathway. Gene expression changes were determined by RNA sequencing of six biological replicates of each genotype. Results represent counts per million (CPM) expressed as a percentage of wild-type.

B,C. Constitutive activation of ATFS-1 does not increase activation of PMK-1/p38 as measured by the ratio of phosphorylated-PMK-1/p38 to total PMK-1/p38 using Western blotting. Representative blots are shown in panel (B). There is also no significant difference in the levels of PMK-1/p38 or phosphorylated-PMK-1/p38 in atfs-1(gof) and atfs-1(lof) mutants compared to wild-type (C). atfs-1(gof) is the et17 allele. atfs-1(lof) is the gk3094 allele. Eight biological replicates per strain were quantified.

Data information: Error bars indicate SEM. *p<0.05 **p<0.01, ***p<0.001. NS = not significant. Statistical significance in panels A and C was assessed using a one-way ANOVA with Dunnett's multiple comparison test.

Source data are available online for this figure.

Source Data for Figure 7, panel C.
IL-10 producing type 2 innate lymphoid cells prolong islet allograft survival (2020)
Figure 1

Figure 1. IL-33 prolonged islet allograft survival.

(A) Streptozotocin-induced diabetic C57BL/6 (H2b) mice were treated with mouse recombinant IL-33 daily for 5 consecutive days before islet transplantation. On day 0, mice were transplanted with BALB/c (H2d) islets. Mice were sacrificed at day 80 post-islet transplantation or at the day when grafts were considered rejected after two consecutive BGLs >16 mmol/liter (mM) after a period of normoglycemia.

(B) Islet graft survival of mice receiving vehicle (PBS) or IL-33 was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. ***P<0.001 vs islet+vehicle.

(C) Blood glucose level of mice treated with IL-33 (the horizontal black line indicates a BGL of 16 mmol/liter, the threshold for rejection). Each line represents one mouse.

(D) Intraperitoneal glucose tolerance test (IPGTT) was assessed in normal mice, islet transplant mice receiving vehicle (on the day when grafts were considered rejected), and islet transplant mice treated with IL-33 (at day 30 and day 80 post-islet transplantation). Data shown are the mean ± SEM (n=6-9 per group).

(E) Area under the curve (AUC) for IPGTT was assessed. Data shown are the mean ± SEM (n=6-9 per group) and a one-way ANOVA was performed, ***P<0.001.

(F) Representative immunohistochemical staining for insulin in graft samples from mice receiving vehicle or IL-33. Scale bar = 100 µm.
IL-10 producing type 2 innate lymphoid cells prolong islet allograft survival (2020)
Figure 2

Figure 2. IL-33 induced Tregs and ILC2s in vivo

C57BL/6 mice were treated with mouse recombinant IL-33 or PBS daily for 5 consecutive days.

(A, B) Representative FACS analysis showing the proportion of Tregs (CD4+Foxp3+) in the CD4+ T cell compartment from the spleens (A) and kidneys (B) at day 3 after treatment in C57BL/6 mice receiving PBS (n=4) or IL-33 (n=6).

(C, D) Representative FACS analysis showing the proportion of ILC2s (Lin-GATA-3+) in the CD45+ leukocyte compartment from the spleens (C) and kidneys (D) at day 3 after treatment in C57BL/6 mice receiving PBS (n=4) or IL-33 (n=6).

(E, F) proportion of Tregs (E) and ILC2s (F) in the spleen and kidney in PBS-injected controls (n=4) and at weeks 1-12 after IL-33 treatment (n=4-6 per IL-33-treated group). Data information: Data shown are the mean ± SEM; Statistical analysis was performed with an unpaired t-test, **P<0.01, ***P<0.001.
IL-10 producing type 2 innate lymphoid cells prolong islet allograft survival (2020)
Figure 3

Figure 3. IL-33 induced Tregs and ILC2s in mice with islet transplantation

(A) Streptozotocin-induced diabetic C57BL/6 (H2b) mice with IL-33 treatment were transplanted with BALB/c (H2d) islets. Mice were sacrificed at day 7, 30 and 80 post-islet transplantation.

(B-D) Proportion or numbers of CD4+Foxp3+ Tregs in the spleens, kidneys and islet grafts of normal, islet transplant mice receiving vehicle and islet transplant mice with IL-33 treatment (at day 7, 30 and 80 post-islet transplantation). Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; ***P<0.001.

(E) Representative confocal microscopy images of immunostaining for CD4, Foxp3 and insulin in islet grafts. Scale bar = 50 µm.

(F-H) Proportion or numbers of ILC2s in the spleens, kidneys and islet graft of mice with or without IL-33 treatment. Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; **P<0.01, ***P<0.001.

(I) Representative confocal microscopy images of immunostaining for CD127, ST2, CD3 and insulin in islet grafts. Scale bar = 50 µm.

(J) The mRNA expression of IL-25, IL-33 and TSLP in islet grafts of mice with or without IL-33 treatment was examined by qPCR, and expressed relative to the control of each experiment. Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; **P<0.01, ***P<0.001.
IL-10 producing type 2 innate lymphoid cells prolong islet allograft survival (2020)
Figure 4

Figure 4. ILC2s and Tregs contributed to IL-33-mediated islet protection in vivo

(A) Streptozotocin-induced diabetic DEREG C57BL/6 mice were treated with mouse recombinant IL-33 daily for 5 consecutive days, as well as diphtheria toxin (DT), PC61 or DT+PC61 on days -4 and -1 prior to and on day 2 post islet transplantation. Mice were sacrificed at day 80 post-islet transplantation or at the day when grafts were considered rejected.

(B,C) Proportion of CD4+Foxp3+ Tregs (B) and Lin-GATA-3+ ILC2s (C) from the spleens and kidneys of islet transplant mice receiving vehicle, IL-33, IL-33/DT, IL-33/PC61, or IL-33/DT/PC61 at day 5 post-islet transplantation. Data shown are the mean ± SEM (n=4-5 per group) and a one-way ANOVA was performed; NS: non significant, ***P<0.001.

(D) Islet graft survival of five groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. *P<0.05, **P<0.01.

(E) Data are shown as blood glucose measurement in islet transplant mice treated with IL-33/DT, IL-33/PC61, or IL-33/DT/PC61. The horizontal black line indicates a BGL of 16 mmol/liter, the threshold for rejection. Each line represents one mouse.
IL-10 producing type 2 innate lymphoid cells prolong islet allograft survival (2020)
Figure 5

Figure 5. IL-33 and IL-2C induced ILC210

(A,B) ILC210 and non-IL-10 producing ILC2s were assessed in islet graft (A) and kidney (B) by intracellular IL-10 staining at day 5 post islet transplantation. Data shown are the mean ± SEM (n=5 per group) and an unpaired t-test was performed; ***P<0.001. ST2: suppression of tumorigenicity 2.

(C) IL-10 reporter C57BL/6 mice were treated with PBS, IL-33 alone or IL-33 and IL-2C daily for 5 consecutive days. Proportion of lin-CD127+ST2+IL-10-GFP ILC210 from the kidneys at day 3 after treatment in IL-10 reporter C57BL/6 mice. Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; ***P<0.001.

(D) Kidney ILC2s isolated from normal IL-10 reporter C57BL/6 mice were cultured with Medium only or Medium with IL-33 and IL-2C for six days. Proportion of ILC210 was assessed by flow cytometry. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed; ***P<0.001. ST2: suppression of tumorigenicity 2.

(E) The secreted cytokine IL-10 was analyzed via ELISA. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed; ***P<0.001.

(F) Kidney ILC2s isolated from C57BL/6 mice were cultured with Medium only or Medium with IL-33 and IL-2C for 30 minutes. The expression of phosphorylated STAT5 was examined in kidney ILC2s by flow cytometry. p-STAT5 (red lines) and isotype controls (gray-filled areas) are shown. Data represent the mean ± SEM of evaluations of MFI from each group (n=6 per group) and an unpaired t-test was performed; ***P<0.001. MFI, mean fluorescence intensity.

(G) The kidney ILC2s were cultured with Medium, IL-33/IL-2C, or IL-33/IL-2C and STAT5 inhibitor (STAT5-IN) for three days. The secreted cytokine IL-10 was analyzed via ELISA. Data shown are the mean ± SEM (n=6 per group) and a one-way ANOVA was performed; ***P<0.001.
IL-10 producing type 2 innate lymphoid cells prolong islet allograft survival (2020)
Figure 6

Figure 6. Transfused ILC210 suppressed islet rejection via IL-10.

(A) ILC210 and non-ILC210 were isolated from ex vivo-expanded kidney ILC2 by flow sorting. ILC210 were transfected with control (ILC210-C) or IL-10 CRISPR-Cas9 (ILC210-IL-10). IL-10 was measured in culture supernatant of ILC210-C and ILC210-IL-10 via ELISA. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed; ***P<0.001.

(B) Transfected ILC210-C and ILC210-IL-10, and non-ILC210 were adoptively transferred into diabetic C57BL/6 mice twice at one day prior to and two days post islet transplantation. Mice were sacrificed at the day when grafts were considered rejected.

(C) Transfused ILC2s (CFSE labeled, Green) were observed in islet graft at day 5 post islet transplantation. Scale bar = 50 µm. The numbers of CFSE-labeled ILC2s in the islet graft were counted. Data shown are the mean ± SEM and a one-way ANOVA was performed (n=4 per group). NS, not significant.

(D) The mRNA expression of IL-10 in islet grafts at day 5 post islet transplantation was examined by qPCR. Data shown are the mean ± SEM (n=4 per group) and a one-way ANOVA was performed; **P<0.01, ***P<0.001.

(E) Islet graft survival of four groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. ***P<0.001.

(F) CD4+ T cells isolated from C57BL/6 splenocytes were cultured with irradiated splenocytes derived from BALB/c in the presence of ILC210 at the indicated ratios for 4 days. CD4+ T cells proliferation was assessed using [3H]-thymidine incorporation assays. Data shown are the mean ± SEM (n=4-6 per group).

(G) Neutralizing anti-IL-10 antibodies or genetic ablation of IL-10 were used to block the effect of ILC210 on CD4+ T cells proliferation. Data shown are the mean ± SEM (n=6 per group) and an unpaired t-test was performed. ***P<0.001.
IL-10 producing type 2 innate lymphoid cells prolong islet allograft survival (2020)
Figure 7

Figure 7. Co-transplantation of IL-210 with islet prolonged allograft survival.

(A) ILC210 were isolated from ex vivo-expanded kidney ILC2s by flow sorting. ILC210 were co-transplanted with islet locally or adoptively transferred into diabetic C57BL/6 mice twice at one day prior to and two days post islet transplantation. Mice were sacrificed at day 80 post-islet transplantation or at the day when grafts were considered rejected.

(B) Islet graft survival of five groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. ***P<0.001.

(C) To track ILC210 in vivo, CD45.2+ ILC210 were co-transplanted with islets in diabetic CD45.1+ C57BL/6 mice. Representative FACS analysis showing the proportion of locally transplanted ILC210 (CD45.2+ST2+) in the total CD45+ cell compartment from islet graft at day 5 and 80 post-islet transplantation or at the day when grafts were considered rejected.

(D,E) Proportion and numbers of locally transplanted CD45.2+ST2+ ILC210 in islet grafts of islet transplant mice over time. Data shown are the mean ± SEM (n=4-5 per group) and a one-way ANOVA was performed, **P<0.01, ***P<0.001.

(F) The expression of IL-10-GFP was examined in locally transplanted CD45.2+ST2+ ILC210 by flow cytometry. IL-10-GFP (red lines) and controls (gray-filled areas) are shown. Data represent the mean ± SEM of evaluations of MFI from each group (n=4-5 per group) and a one-way ANOVA was performed; ***P<0.001. MFI, mean fluorescence intensity.