An essential role for XBP-1 in host protection against immune activation in C. elegans

Abstract

The detection and compensatory response to the accumulation of unfolded proteins in the endoplasmic reticulum (ER), termed the unfolded protein response (UPR), represents a conserved cellular homeostatic mechanism with important roles in normal development and in the pathogenesis of disease. The IRE1-XBP1/Hac1 pathway is a major branch of the UPR that has been conserved from yeast to human. X-box binding protein 1 (XBP1) is required for the differentiation of the highly secretory plasma cells of the mammalian adaptive immune system, but recent work also points to reciprocal interactions between the UPR and other aspects of immunity and inflammation. We have been studying innate immunity in the nematode Caenorhabditis elegans, having established a principal role for a conserved PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway in mediating resistance to microbial pathogens. Here we show that during C. elegans development, XBP-1 has an essential role in protecting the host during activation of innate immunity. Activation of the PMK-1-mediated response to infection with Pseudomonas aeruginosa induces the XBP-1-dependent UPR. Whereas a loss-of-function xbp-1 mutant develops normally in the presence of relatively non-pathogenic bacteria, infection of the xbp-1 mutant with P. aeruginosa leads to disruption of ER morphology and larval lethality. Unexpectedly, the larval lethality phenotype on pathogenic P. aeruginosa is suppressed by loss of PMK-1-mediated immunity. Furthermore, hyperactivation of PMK-1 causes larval lethality in the xbp-1 mutant even in the absence of pathogenic bacteria. Our data establish innate immunity as a physiologically relevant inducer of ER stress during C. elegans development and indicate that an ancient, conserved role for XBP-1 may be to protect the host organism from the detrimental effects of mounting an innate immune response to microbes.

Figure 1. PMK-1 p38 MAPK-dependent activation of the IRE-1-XBP-1-dependent UPR by P. aeruginosa infection

(a) Fluorescence and DIC microscopy of WT and mutant larvae carrying the Phsp-4∷GFP(zcIs4) transgene 24 h after egg lay on indicated treatments (Scale bar = 100μm). (b) qRT-PCR of endogenous hsp-4 mRNA levels. (c) qRT-PCR analysis of total and spliced xbp-1 mRNA levels. Values represent fold change relative to WT on OP50 ± s.e.m. (n = 4 independent experiments, *P < 0.05 and ***P < 0.001, two-way ANOVA with Bonferroni post test).

Figure 2. XBP-1 is required for C. elegans development and survival on P. aeruginosa

(a) Development of indicated mutants to the L4 larval stage or older after 3 d at 25° C on either P. aeruginosa PA14 or E. coli OP50, or (b) mixture of PA14 and E. coli HB101. Plotted is mean ± s.d. (n = 3-4 plates, ***P<0.001, two-way ANOVA with Bonferroni post test in (a), and n = 4 plates, *** p<0.001 two tailed t-test in (b)). Results in (a) and (b) are representative of 3 independent experiments. (c) Transmission electron microscopy of L3 larvae at 60,000x magnification (Scale bar = 500 nm). (d) Fluorescence and DIC microscopy overlay of larvae 40 h after egg-lay on P. aeruginosa PA14 expressing GFP. Strain Construction

Figure 3. Suppression of the P. aeruginosa-induced larval lethality phenotype of xbp-1(zc12) by pmk-1(km25)

(a) DIC microscopy of representative worms of each genotype 3 d after egg-lay on P. aeruginosa PA14 (Scale bar = 100 μm). (b) Development of indicated mutants to the L4 larval stage or older after 3 d at 25° C on either P. aeruginosa PA14 or E. coli OP50. Plotted is the mean ± s.d. (n = 3-4 plates, ***P < 0.001, two-way ANOVA with Bonferroni post test). Representative results of 3 independent experiments.

Figure 4. XBP-1 is required for development and survival during pathogen-independent constitutive activation of PMK-1

(a) Fluorescence and DIC microscopy of WT and xbp-1 larvae carrying the Phsp-4∷GFP(zcIs4) transgene, which have been treated with control (empty vector) or vhp-1 RNAi, 24 h after egg lay. (b) Development to the L4 larval stage or older after 3 d at 25° C of WT, xbp-1, and xbp-1;pmk-1 mutants that have been subjected to RNAi of vhp-1 and propagated on E. coli OP50. Plotted is mean ± s.e.m (n = 3 independent experiments, ***P < 0.001, two-way ANOVA with Bonferroni post test).