LIPL-1 and LIPL-2 are TCER-1-regulated Lysosomal Lipases with Distinct Roles in Immunity and Fertility

Abstract

Reproduction and immunity are fundamental, energy intensive processes that often compete for resources, leading to trade-offs observed across diverse species. Lipid metabolism plays a crucial role in integrating these processes, particularly during stressful conditions such as pathogenic infections. Yet the molecular mechanisms governing this integration remain poorly understood. TCER-1, the C. elegans homolog of mammalian TCERG1, suppresses immunity and promotes fertility, especially upon maternal infection. Here, we show that TCER-1 regulates two conserved lysosomal lipases, lipl-1 and lipl-2, to balance reproduction, immunity and lifespan. Using transcriptomic, lipidomic, and molecular-genetic analyses, we demonstrate that while both lipl-1 and lipl-2 mediate infection-induced lipid remodeling, lipl-1 enhances immunity and catalyzes the accumulation of ceramide species linked to stress response and longevity, whereas, lipl-2 unexpectedly does not. Both lipases contribute towards fertility outcomes, but lipl-2 is especially critical for maintaining embryonic-eggshell integrity during maternal infection and aging. Strikingly, expression of human lysosomal acid lipase (LAL), the ortholog of lipl genes, rescues the immune defects triggered by lipl-l loss and enhances immune resilience. Together, these findings uncover functionally distinct roles for lipl-1 and lipl-2 in modulating lipid species that shape immune fitness, healthspan and reproductive health, and suggest a potentially conserved mechanism by which lipid metabolism links fertility and immunity.

Fig 1:. TCER-1 regulates lipid metabolism upon PA14 infection.

A) RNAseq experimental paradigm. Age-matched wild type (WT) and tcer-1 L4s raised on OP50 exposed to PA14 for 8 hours. B) Overlaps of groups of differentially expressed genes (DEGs) identified. C-F) Overlap of genes upregulated (C, D) and downregulated (E, F) upon PA14 exposure with previously-identified PA14-responsive genes by Troemel et al. 2006. RF: Representation Factor. Statistical significance of overlap between gene sets calculated using hypergeometric probability formula with normal approximation (see Methods). Comparisons with additional studies in Fig. S1 and Table S2. G) Gene ontology (GO) term analysis of PA14- and TCER-1- driven DEGs using WormCat. Metabolism (Category 1), particularly lipid metabolism, (Category 2) (blue boxes) identified one of the most differentially impacted processes amongst genes upregulated (UP, left) and downregulated (DOWN, right) in WT and tcer-1 mutants. Key shown in middle (black box). H, I) lipl-1 and lipl-2 are transcriptionally upregulated in tcer-1 mutants on PA14 infection. mRNA levels of lipl-1 (H) and lipl-2 (I) measured by qPCR in WT (black) and tcer-1 mutant (blue) adults maintained on OP50 (solid bars) or exposed as L4s to PA14 for 8 h (hashed bars). Data from at least 3 independent trials/biological replicates. Asterisks represent statistical significance of differences observed in unpaired, two-tailed t-tests with P values * ≤ 0.05, *** p≤0.001 and ****p≤0.0001.

Figure 2:. tcer-1 inactivation and pathogen exposure induce lipl-1 transcriptional upregulation.

A-F: lipl-1 transcriptional changes visualized in vivo using Plipl-1::mCherry. Animals raised on bacteria expressing control empty vector (Ctrl, A, B) or tcer-1 dsRNA (RNAi, C, D) until pre-adult, L4 larval stage and transferred to plates seeded with P. aeruginosa PA14 (PA14, C, D) or E. coli OP50 (OP50, A, B) and incubated for 8 hours at 25°C. Expression visible in intestine and head, and in a normal adult population, varied from low levels to high or very high fluorescence intensities in intestine and head, respectively (categorization and quantification detailed in Fig. S2). A-D show representative images pseudocolored with ImageJ LUT Fire. Fraction of population with high or very high expression quantified in intestine (E) or head (F). E: Control (Ctrl) RNAi OP50 (n=81, 3.7%), tcer-1 RNAi, OP50 (n=83, 21.69%), Ctrl RNAi, PA14 (n=69, 23.19%), tcer-1 RNAi, PA14 (n=68, 58.82%). F: Ctrl RNAi, OP50 (n=81, 10.52%), tcer-1 RNAi, OP50 (n=83, 7.895%), Control RNAi PA14 (n=69, 7.895%), tcer-1 RNAi, PA14 (n=67, 23.68%). Comparisons performed using two-tailed Fisher’s exact test on contingency tables of data from 3 pooled independent biological replicates. *p≤ 0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001. G-K: LIPL-1 protein levels observed using translational reporter Plipl-1::LIPL-1::RFP. Growth and exposure conditions similar to those in A-D. Representative images of Day 1 adults showing expression that was predominantly seen in coelomocytes (G-J) and rare animals with punctate expression in the head (K).

Figure 3:. TCER-1 transcriptionally suppresses lipl-2 and LIPL-2 protein levels are stringently controlled.

lipl-2 expression levels observed using the transcriptional reporter Plipl-2::mCherry (A-E) or translational reporter Plipl-2:LIPL-2::RFP (F-J). A-E: Animals expressing Plipl-2:mCherry were raised on bacteria expressing control empty vector (Ctrl) or tcer-1 dsRNA (RNAi) until young adulthood, transferred to plates seeded with pathogen (PA14) or E. coli (OP50) bacteria and incubated for 8 hours at 25°C. Expression visible predominantly in the intestine and varied from low to high fluorescence intensities (detailed in Fig. S3). A-D show representative images pseudocolored with ImageJ LUT Fire. E: Fraction of populations showing high intestinal expression. Comparisons performed using two-tailed Fisher’s exact test on contingency tables of data from 3 pooled biological replicates of Control (Ctrl) RNAi OP50 (n=86, 1.16%), tcer-1 RNAi, OP50 (n=83, 7.22%), Ctrl RNAi, PA14 (n=81, 1.23%), tcer-1 RNAi, PA14 (n=72, 12.5%). *p≤ 0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001. F-J: LIPL-1 protein levels observed using translational reporter Plipl-1::LIPL-1::RFP. Growth and exposure conditions similar to those in A-D. Representative images of Day 1 adults pseudocolored with ImageJ LUT RedHOT showing very low expression predominantly in coelomocytes (with high intestinal autoflourescence in F-I). J: Rare animals with punctate expression in the head.

Fig 4:. Enhanced immunity of tcer-1 mutants is dependent upon lipl-1 but not lipl-2.

A, B: Impact of lipl-1 deletion on survival upon pathogen infection and lifespan. Survival of wild type (WT, black), tcer-1 (blue), tcer-1;lipl-1 (dark green) and lipl-1 (light green) raised on OP50 till L4 stage and exposed to PA14 (A) or retained on OP50 (B). A: WT (m= 51.13 ± 1.32, n= 86/113), tcer-1 (m=70.61 ± 2.09, n=87/112), lipl-1 (m=52.17 ± 1.1, n=102/134), lipl-1;tcer-1 (dark green, m=56.04 ± 1.23, n=85/120). B: WT (m=14.96+−0.34, n=94/121), tcer-1 (m=14.23 ± 0.48, n=92/120), lipl-1 (m=15.79 ± 0.57, n=80/106), lipl-1;tcer-1 (m=15.36 ± 0.51, n=77/120). C, D: Impact of lipl-2 deletion on survival upon pathogen infection and lifespan. Survival of wild type (WT, black), tcer-1 (blue), tcer-1;lipl-2 (grape) and lipl-2 (magenta) raised on OP50 till L4 stage and exposed to PA14 (C) or retained on OP50 (D). C: WT (m= 51.13 ± 1.32, n= 86/113), tcer-1 (m=70.61 ± 2.09, n=87/112), lipl-2 (m=70.64 ± 1.5, n=61/123), tcer-1; lipl-2 (m=78.23 ± 2.66, n=66/124). D) Survival on OP50. WT (m=14.96 ± 0.34, n=94/121), tcer-1 (m=14.23 ± 0.48, n=92/120), lipl-2 (m= 15.93 ± 0.56, n= 89/113), tcer-1; lipl-2 (m= 16.72 ± 0.58, n=81/103). Data from additional PA14 survival and lifespan trials in Table S3 and Table S4, respectively. Statistical significance was determined using log-rank Mantel Cox method and shown in each panel next to a given strain/condition. Asterisks are color coded to indicate the strain/condition being used for the comparison. p≤ 0.05(*), p < 0.01 (**), <0.001 (***), <0.0001 (****).

Fig 5:. lipl-1 and lipl-2 lof result in decreased brood size, sterility, and embryonic inviability, particularly in combination with tcer-1 lof.

A-C: Comparisons of maternal reproductive success in wild type (WT, black), lipl-1 (light green), lipl-2 (pink), lipl-1 lipl-2 (light brown), tcer-1 (blue), tcer-1;lipl-1 (dark green), tcer-1;lipl-2 (grape) and tcer-1;lipl-1 lipl-2 (dark brown) strains. A: Brood size: Total number of live progeny counted per worm per strain. Each dot represents one worm from an aggregate of 3 independent trials. WT (n=28, m=330.9 ± 59.19), lipl-1 (n=29, m=275.8 ± 51.97), lipl-2 (n=23, m=288.2 ± 39.97), lipl-1;lipl-2 (n=24, m=316.3 ± 33.23) , tcer-1 (n=25, m=236.2 ± 97.14) tcer-1;lipl-1 (n=23, m=68.09 ± 68.12), tcer-1;lipl-2 (n=36, m=99.31 ± 116.1), tcer-1;lipl-1 lipl-2 (n=20, m=32.80 ± 44.87). B: Sterility: Percentage of animals which never produced a live progeny counted for each strain. WT (n=28, m=0), lipl-1 (n=29, m=0), lipl-2 (n=23, m=0), lipl-1;lipl-2 (n=24, m=0), tcer-1 (n=25, m=0), tcer-1;lipl-1 (n=23, m=27.48 ± 8.82), tcer-1;lipl-2 (n=36, m=48.87 ± 27.60), tcer-1;lipl-1 lipl-2 (n=20, m=30.67 ± 9.81). C: Hatch Rate: calculated from [#progeny/(#progeny + unhatched eggs)]. WT (m=99.01 ± 0.29), lipl-1 (m=97.09 ± 1.24), lipl-2 (m=98.81 ± 0.65), lipl-1 lipl-2 (m=99.47 ± 0.15), tcer-1 (m=97.75 ± 1.36), tcer-1;lipl-1 (m=59.95 ± 18.07), tcer-1;lipl-2 (m=81.75 ± 7.78), tcer-1;lipl-1 lipl-2 (m=74.79 ± 3.24). Data obtained from 3 independent trials in all cases. D-F: Embryonic eggshell defects induced upon maternal PA14 infection. Representative images of embryos expressing Pcpg-2::mCherry::CPG-2 and pie-1p::GFP::PH PLC1delta1, labeling eggshell chondroitin proteoglycan layer and embryonic plasma membrane, respectively, incubated with lipid-labeling dye, BODIPY. D: BODIPY is excluded in healthy embryo with intact lipid-permeability barrier (LBP) and CPG-2 is sequestered away from the embryo, in a characteristic ‘wavy’ pattern in the perivitelline space of the eggshell. E: Embryo with defective LPB exhibits widespread BODIPY staining, and mCherry::CPG-2 which freely diffuses between the outer eggshell and the embryo. F: Quantification of BODIPY-permeable embryos. Average fold-change of the fraction of BODIPY stained embryos laid by Day 4 mothers, normalized to tcer-1 mutants. tcer-1 (n=1964, m=0), WT (n=1930, m=0), lipl-1 (n=2811, m=0.7153 ± 0.7889), tcer-1;lipl-2 (n=1940, m=6.222 ± 3.030), tcer-1;lipl-1 (n=2257, m=1.563 ± 1.587), lipl-2 (n=2546, m=2.244 ± 1.980). Data obtained from 4 independent trials in all cases except lipl-2 for which 3 trials were conducted. In A-C, statistical significance was calculated using one-way ANOVA with Tukey’s correction. In F, student’s t-test was used. p≤ 0.05(*), p <0.01 (**), <0.001 (***), <0.0001 (****).

Fig 6:. lipl-1 and lipl-2 have both shared and distinct impacts on the lipidome of tcer-1 mutants.

A-C: Effects of lipl-1 (A) and lipl-2 (B) loss on broad neutral lipid (NL) and phospholipid (PL) populations in wild type (WT, black) and tcer-1 mutant (blue) animals. Bars indicate the fold-change of each data point normalized to the WT average. Statistical significance calculated using mixed-effects analysis with Tukey’s correction. C: Relative abundance of overall phosphatidylcholine (PC) levels. D: Categorization of NL (top, blue) and PL (bottom, green) species whose abundance is altered in tcer-1 mutants. For each class, number of species identified and those changed in tcer-1 mutants shown in the table. E: Relative abundance of PC 34:2 and PC 34:3 compared to all PCs in different strains. F-I: Distinct impacts of lipl-1 (F-H) and lipl-2 (I) on lipidome of tcer-1 mutants. F-H: Relative abundance of Cer17:1;O2/22:0 (F), Cer 17:1;O2 /24:0 (G) and Cer 17:1;O2 /26:0 (H) compared to all ceramides. I: Relative abundance of lysophospatidyl cholines (LPC) 18:2 compared to all LPCs. Data derived from HPLC-MS/MS analysis of Day 1 young adult hermaphrodites isolated in 4–10 independent biological trials. Statistical significance was calculated using two-way ANOVA with Tukey’s correction. Asterisks indicate the statistical significance and their color the strain being used for the comparison. p≤ 0.05(*), p<0.01 (**), <0.001 (***), <0.0001 (****), n.s: not significant.

Fig 7:. Human Lysosomal Acid Lipase (hLAL) rescues immune deficits induced by lipl-1 mutation and enhances survival upon PA14 infection.

A: hLAL rescues survival of tcer-1;lipl-1 mutants on PA14 exposure to tcer-1 level. Survival upon PA14 exposure from late-L4 stage onwards compared between wild type WT (black, m= 35.06 ± 1.91, n= 54/90), tcer-1 (blue, m=54.8 ± 3.25, n= 63/90), tcer-1;lipl-1 (green, m= 36.65 ± 1.79, n= 52/90), tcer-1;lipl-1;hLAL (green, m= 55.77 ± 2.53, n= 73/90). B: hLAL expression in C. elegans enhances survival upon PA14 infection. Survival upon PA14 exposure from late-L4 stage onwards compared between WT (m= 57.24 ± 2.01, n= 35/110) and two transgenic strains expressing hLAL broadly in somatic tissues. COP2983 (orange, m= 83.18 ± 2.22, n= 46/96) and COP2589 (red, m= 82.84 ± 2.22, n= 63/103). Statistical significance was calculated using the log-rank Mantel Cox method and is shown on each panel next to a given strain/condition with the color of the asterisk indicating strain being used for comparison. p < 0.01 (**), <0.001 (***), <0.0001 (****), n.s: not significant. Data from additional trials in Table S8.