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. 2022 Jan 31;11:e74206. doi: 10.7554/eLife.74206

Figure 3. Cholesterol scarcity activates intestinal innate immune defenses.

Images of T24B8.5p::gfp (A) and irg-5p::GFP (B) transcriptional immune reporters in wild-type animals grown on standard nematode growth media ( + 5 μg/mL cholesterol) and in the absence of supplemented cholesterol ( + 0 μg/mL cholesterol). (C) qRT-PCR data of the indicated innate immune effector genes in wild-type C. elegans grown in the presence ( + 5 μg/mL) and absence ( + 0 μg/mL) of supplemented cholesterol. *equals p < 0.05 (unpaired t-test). (D and E) Data from mRNA-seq experiments comparing genes differentially regulated in uninfected nhr-8(hd117) mutants versus wild-type animals (D) or uninfected wild-type animals grown in the absence (0 μg/mL) versus presence (5 μg/mL) of supplemental cholesterol (E) (y-axis) are compared with genes differentially expressed in wild-type animals during P. aeruginosa infection (x-axis). All genes are shown in gray. Genes that are differentially expressed in both datasets are shown in black (Fold change >2, q < 0.01). Genes that are annotated as innate immune genes are shown in red. The location of the representative genes T24B8.5, irg-5, irg-4, and K08D8.4, whose expression is examined throughout this manuscript, are shown. (Of note, in the 0 μg/mL cholesterol mRNA-seq data set K08D8.4 did not meet our cut-off threshold, although was significantly upregulated, fold change = 1.79, q = 5.6 × 10–4). See also Supplementary files 1-3. (F, G, H) Images of T24B8.5p::gfp animals of the indicated genotypes grown under the indicated conditions are shown. (H) C. elegans were grown on media solidified with agarose rather than agar. (I, J, K, L) qRT-PCR data of the indicated genes in wild-type and nhr-8(hd117) mutant animals grown on standard nematode growth media ( + 5 μg/mL cholesterol) in the presence or absence of 0.1% Tergitol, as indicated. For the qRT-PCR studies in (C, I, J, K and L), data are the average of three to six independent biological replicates, each normalized to a control gene with error bars representing SEM and are presented as the value relative to the average expression from all replicates of the indicated gene in wild-type animals on standard nematode growth media ( + 5 μg/mL cholesterol). *equals p < 0.05 (two-way ANOVA with Tukey’s multiple comparison testing). (M, N) Survival curves for C. elegans pathogenesis assays with P. aeruginosa and C. elegans of the indicated genotypes at the L4 larval stage and exposed to the indicated conditions. Data are representative of three trials. The difference between the nhr-8(hd117) mutant and the other conditions in both M and N is significant (p < 0.05). The Kaplan-Meier method was used to estimate the survival curves for each group, and the log-rank test was used for all statistical comparisons. Sample sizes, mean lifespan and p-values for all trials are shown in Supplementary file 4. (O) P. aeruginosa, isolated from the intestines of animals with the indicated genotypes, were quantified after 24 hr of bacterial infection. Data are colony-forming units (CFU) of P. aeruginosa and are presented as the average of 10 separate biological replicates, with each replicate containing 10–11 animals. *equals p < 0.05 (unpaired t-test). Scale bars in all images equal 200 μm. See also Figure 3—figure supplement 1.

Figure 3—source data 1. Figure 3C qRT-PCR data of the indicated innate immune effector genes in wild-type C. elegans growing in the presence (+5 μg/mL) and absence (+0 μg/mL) of supplemented cholesterol.
Figure 3—source data 2. Figure 3I qRT-PCR data of T24B8.5 in wild-type and nhr-8(hd117) mutant animals grown on standard nematode growth media (+5 μg/mL cholesterol) in the presence or absence of 0.1% Tergitol, as indicated.
Figure 3—source data 3. Figure 3J qRT-PCR data of irg-4 in wild-type and nhr-8(hd117) mutant animals grown on standard nematode growth media (+5 μg/mL cholesterol) in the presence or absence of 0.1% Tergitol, as indicated.
Figure 3—source data 4. Figure 3K qRT-PCR data of irg-5 in wild-type and nhr-8(hd117) mutant animals grown on standard nematode growth media (+5 μg/mL cholesterol) in the presence or absence of 0.1% Tergitol, as indicated.
Figure 3—source data 5. Figure 3L qRT-PCR data of K08D8.4 in wild-type and nhr-8(hd117) mutant animals grown on standard nematode growth media (+5 μg/mL cholesterol) in the presence or absence of 0.1% Tergitol, as indicated.
Figure 3—source data 6. Figure 3O Colony-forming units (CFUs) of P. aeruginosa, isolated from the intestines of animals with the indicated genotypes, quantified after 24 hr of bacterial infection.

Figure 3.

Figure 3—figure supplement 1. Cholesterol scarcity activates intestinal innate immune defenses.

Figure 3—figure supplement 1.

(A) An mRNA-seq experiment as described in Figure 3D, except using a different nhr-8 mutant allele: nhr-8(ok186). (B) Data from mRNA-seq experiments as described in Figure 3D, except comparing genes differentially regulated in uninfected nhr-8(hd117) mutants versus wild-type animals (y-axis) with genes differentially expressed in uninfected wild-type animals grown in the absence (0 μg/mL) versus presence (5 μg/mL) of supplemental cholesterol (x-axis). (C) Images of T24B8.5p::gfp animals as described in Figure 3G. (D) Images of T24B8.5p::gfp on agarose media with 0.1% Tergitol in the presence of supplemented fatty acids, as indicated. See also Figure 3.