Deubiquitinase USP-14 controls intestinal distension-induced immune activation in Caenorhabditis elegans via Wnt/β-catenin signaling

Deubiquitinase USP-14 controls intestinal distension-induced immune activation in Caenorhabditis elegans via Wnt/β-catenin signaling | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Deubiquitinase USP-14 controls intestinal distension-induced immune activation in Caenorhabditis elegans via Wnt/β-catenin signaling View ORCID Profile Annesha Ghosh , View ORCID Profile Jogender Singh doi: https://doi.org/10.1101/2025.11.25.690479 Annesha Ghosh 1 Department of Biological Sciences, Indian Institute of Science Education and Research , Mohali, Punjab, 140306, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Annesha Ghosh Jogender Singh 1 Department of Biological Sciences, Indian Institute of Science Education and Research , Mohali, Punjab, 140306, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jogender Singh For correspondence: jogender{at}iisermohali.ac.in Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Pathogen infections disrupt multiple host cellular processes, and hosts have consequently evolved mechanisms to detect these perturbations and initiate appropriate immune responses. In Caenorhabditis elegans , gut distension caused by bacterial colonization is known to activate innate immunity, yet the molecular mechanisms linking intestinal distension to immune activation remain poorly understood. Here, we perform a forward genetic screen to identify suppressors of intestinal distension-induced immune activation in C. elegans . This screen identifies a loss-of-function mutation in the gene encoding the deubiquitinase (DUB) USP-14 as a potent suppressor of immune activation triggered by gut distension. We show that usp-14 knockout mutants exhibit increased sensitivity to the bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus . Global transcriptomic profiling further demonstrates that USP-14 modulates innate immune responses induced by intestinal distension. Epistasis analyses establish that USP-14 functions through the Wnt/β-catenin signaling pathway to regulate these immune responses. Together, our findings reveal a previously unrecognized role for the conserved DUB USP-14 in host immunity, providing a foundation for future studies investigating how USP-14 modulates immune signaling. Introduction Living in microbe-rich environments, animals have evolved sophisticated strategies to discriminate between harmful and beneficial microorganisms ( Blander & Sander, 2012 ). A central component of this defense is the detection of microbial-associated molecular patterns by conserved pattern-recognition receptors ( Akira et al , 2006 ). In parallel, organisms have also evolved mechanisms to sense disruptions in their own cellular physiology, a process termed surveillance immunity ( Willmann & Moita, 2025 ; Pukkila-Worley, 2016 ). This mode of immune activation is widely conserved across plants, invertebrates, and vertebrates ( Cook et al , 2015 ; Pukkila-Worley, 2016 ; Willmann & Moita, 2025 ). In Caenorhabditis elegans , perturbations of core cellular processes, including translation, endoplasmic reticulum function, mitochondrial function, and the ubiquitin-proteasome system, trigger robust innate immune responses ( Melo & Ruvkun, 2012 ; Pukkila-Worley, 2016 ; Dunbar et al , 2012 ; McEwan et al , 2012 ; Ghosh & Singh, 2024 ; Rao et al , 2025 ; Grover et al , 2024 ). Because such perturbations can arise from diverse pathogens, surveillance of cellular integrity likely provides a broad evolutionary advantage ( Willmann & Moita, 2025 ). In addition to monitoring cell-intrinsic damage, organisms can detect disruptions at the tissue level ( Pugin, 2012 ). For example, epidermal injury in C. elegans activates multiple immune pathways, including the p38 MAPK cascade ( Taffoni & Pujol, 2015 ). More recently, intestinal distension caused by pathogenic colonization has been shown to induce innate immune responses in C. elegans ( Singh & Aballay, 2019a , 2019c ; Kumar et al , 2019 ). Remarkably, intestinal distension alone, independent of pathogen exposure, is sufficient to activate immune defenses, indicating that distension is detected as a danger signal ( Singh & Aballay, 2019a ; Kumar et al , 2019 ). Further studies revealed that this response is mediated by the neuronal acetylcholine receptor ACC-4, which signals to the intestine through the Wnt pathway ( Ren et al , 2022 ). Nonetheless, beyond this work, the molecular components that link intestinal distension to immune activation remain poorly defined. In this study, we conducted a forward genetic screen to identify regulators of immune responses triggered by intestinal distension. Our screen uncovered a loss-of-function mutation in the gene encoding the deubiquitinase (DUB) USP-14, which suppresses distension-induced immune activation and increases susceptibility to Staphylococcus aureus and Pseudomonas aeruginosa . We showed that intestinal expression of usp-14 is sufficient to confer immunity to both pathogens. Transcriptomic analyses further revealed that intestinal distension elicits both USP-14-dependent and USP-14-independent innate immune programs. Genetic epistasis experiments demonstrated that USP-14 acts through the canonical Wnt/β-catenin signaling pathway, and notably, USP-14 influences the transcription of multiple Wnt pathway components. Together, our findings identify a critical role for the conserved DUB USP-14 in orchestrating intestinal distension-mediated immune responses through activation of Wnt/β-catenin-dependent innate immunity. Results The DUB USP-14 is required for immune activation during C. elegans intestinal distension Previous studies have shown that intestinal distension, even in the absence of pathogenic infection, can trigger the activation of innate immune responses ( Singh & Aballay, 2019a , 2019c ; Kumar et al , 2019 ). Furthermore, it has been reported that a member of the acetylcholine receptor family in the nervous system is required for the activation of immune responses following intestinal distension ( Ren et al , 2022 ). To identify additional pathways that mediate the activation of innate immunity upon intestinal distension, we conducted a forward genetic screen using the aex-5(sa23);clec-60p::gfp strain. Inhibition of aex-5 , which encodes a furin-like prohormone convertase, disrupts the defecation motor program, leading to intestinal distension ( Branicky & Hekimi, 2006 ). This distension, in turn, induces the expression of several immune genes, including the C-type lectin gene clec-60 ( Singh & Aballay, 2019a ). The forward genetic screen was designed to isolate mutants exhibiting reduced green fluorescent protein (GFP) expression in aex-5(sa23);clec-60p::gfp worms ( Figure 1A ). We identified four independent mutants with reduced GFP fluorescence ( Figure 1B, C ). Two of these mutants also showed a marked decrease in the myo-2p::mCherry co-injection marker ( Figure 1D ), suggesting that they might carry mutations causing transgene silencing. The remaining two mutants (#1.5 and #3.2) were backcrossed six times with the parental strain, followed by whole-genome sequencing. Single-nucleotide polymorphism analysis combined with RNA interference (RNAi)-based validation identified usp-14 and cec-10 as the causal genes for mutants #1.5 and #3.2, respectively ( Figure S1A, B ). The cec-10(jsn20) allele in mutant #3.2 carried a single G→T substitution mutation, leading to a premature stop codon for glutamic acid (E) codon at 259 position (E259*). Knockdown of cec-10 led to only a marginal decrease in survival during P. aeruginosa infection ( Figure S1C ). Because cec-10 lacks a human homolog and has minimal impact on C. elegans survival during infection, we did not pursue it further in the context of intestinal distension-induced immune activation. Download figure Open in new tab Figure 1: The deubiquitinase USP-14 is required for immune activation during C. elegans intestinal distension (A) Scheme for a forward genetic screen for mutants that have a suppressed clec-60p::gfp in aex-5(sa23);clec-60p::gfp worms. (B) Representative fluorescence images of the control parental strain aex-5(sa23);clec-60p::gfp and the mutants isolated from the forward genetic screen with reduced GFP levels. Scale bar = 200 μm. (C) Quantification of GFP levels of the control parental strain aex-5(sa23);clec-60p::gfp and the mutants isolated from the forward genetic screen. *** p < 0.001 via ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test ( n = 30-31 worms each). (D) Quantification of mCherry levels of myo-2p::mCherry in the control parental strain aex-5(sa23);clec-60p::gfp and the mutants isolated from the forward genetic screen. *** p < 0.001 and ns, non-significant via ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test ( n = 30-31 worms each). (E) Mapping of the usp-14(jsn19) allele identified in the forward genetic screen. (F) Summary of the usp-14(jsn19) allele. (G) Quantitative reverse transcription-PCR (qRT-PCR) analysis of the immune gene clec-60 in wild-type N2, usp-14(jsn19) , and usp-14(tm1481) worms after treatment with the empty vector (EV) control and aex-5 RNAi. ** p < 0.01 via t-test. Data represents the mean and standard deviation from three independent experiments. (H) Representative survival plots of clec-60p::gfp , usp-14(jsn19);clec-60p::gfp , and usp-14(tm1481);clec-60p::gfp worms on P. aeruginosa PA14 at 25°C. *** p < 0.001 for the mutants as compared to the control worms ( n = 105 per condition). (I) Representative survival plots of clec-60p::gfp , usp-14(jsn19);clec-60p::gfp , and usp-14(tm1481);clec-60p::gfp worms on S. aureus NCTC832 5 at 25°C. *** p < 0.001 for the mutants as compared to the control worms ( n = 70 for clec-60p::gfp and 105 for each usp-14(jsn19) and usp-14(tm1481) ). (J) Representative fluorescence images of clec-60p::gfp , usp-14(jsn19);clec-60p::gfp , and usp-14p::usp-14;usp-14(jsn19);clec-60p::gfp worms exposed to the EV control and aex-5 RNAi. Scale bar = 200 μm. (K) Quantification of GFP levels of clec-60p::gfp , usp-14(jsn19);clec-60p::gfp , and usp-14p::usp-14;usp-14(jsn19);clec-60p::gfp worms exposed to the EV control and aex-5 RNAi. *** p < 0.001 via t-test ( n = 30-31 worms each). (L) Representative survival plots of clec-60p::gfp , usp-14(jsn19);clec-60p::gfp , and usp-14p::usp-14;usp-14(jsn19);clec-60p::gfp worms on P. aeruginosa PA14 at 25°C. *** p < 0.001 between clec-60p::gfp and usp-14(jsn19);clec-60p::gfp , and non-significant between clec-60p::gfp and usp-14p::usp-14;usp-14(jsn19);clec-60p::gfp ( n = 105 per condition). (M) Representative survival plots of clec-60p::gfp , usp-14(jsn19);clec-60p::gfp , and usp-14p::usp-14;usp-14(jsn19);clec-60p::gfp worms on S. aureus NCTC832 5 at 25°C. *** p < 0.001 between clec-60p::gfp and usp-14(jsn19);clec-60p::gfp , and ** p < 0.01 between clec-60p::gfp and usp-14p::usp-14;usp-14(jsn19);clec-60p::gfp ( n = 105 for clec-60p::gfp and usp-14(jsn19) , and 60 for usp-14p::usp-14;usp-14(jsn19);clec-60p::gfp ). The usp-14(jsn19) allele in mutant #1.5 carried a single C→T substitution, resulting in a glutamic acid to lysine (K) change at residue 122 ( Figure 1E, F ). USP-14 is a cysteine-type DUB that regulates proteasomal protein degradation and is the C. elegans homolog of human USP14 (ubiquitin-specific peptidase 14) ( Wang et al , 2021 ). To determine whether USP-14 regulates clec-60 expression at the transcriptional level, we performed quantitative reverse transcription PCR (qRT-PCR) on usp-14(jsn19) worms. As expected, RNAi-mediated knockdown of aex-5 led to robust upregulation of clec-60 in wild-type N2 worms ( Figure 1G ). However, usp-14(jsn19) worms exhibited significantly attenuated clec-60 induction, indicating that USP-14 positively regulates clec-60 transcription. To validate that this effect resulted from usp-14 loss of function, we examined clec-60 levels in an independent allele, usp-14(tm1481) , which carries a 696 bp deletion encompassing most of the third exon and is likely a null allele. Similar to usp-14(jsn19) worms, usp-14(tm1481) mutants displayed a significantly reduced induction of clec-60 upon aex-5 RNAi compared with wild-type worms ( Figure 1G ). We next tested whether USP-14 contributes to host defense against bacterial infection. Both usp-14(jsn19) and usp-14(tm1481) mutants exhibited reduced survival when exposed to P. aeruginosa compared with wild-type worms ( Figure 1H ). Because clec-60 expression is also induced by the Gram-positive pathogen S. aureus ( Irazoqui et al , 2010 ), we examined whether usp-14 mutants were similarly sensitive to S. aureus infection. Consistent with this hypothesis, both usp-14(jsn19) and usp-14(tm1481) worms showed significantly reduced survival upon S. aureus infection ( Figure 1I ). To confirm that these phenotypes were specifically due to the loss of USP-14 function, we expressed usp-14 under its endogenous promoter in usp-14(jsn19) mutants. Transgenic expression of usp-14 restored clec-60p::gfp induction upon aex-5 RNAi ( Figure 1J, K ) and rescued the survival defects of usp-14(jsn19) worms during both P. aeruginosa and S. aureus infections ( Figure 1L, M ). Together, these results demonstrate that USP-14 is essential for the activation of innate immune responses triggered by intestinal distension in C. elegans and plays a critical role in protecting the host from bacterial infection. Intestinal expression of USP-14 is sufficient to activate immunity during intestinal distension We next examined the spatial expression pattern of usp-14 in C. elegans . To this end, we generated transgenic worms expressing GFP under the control of the usp-14 promoter ( usp-14p::gfp ). GFP fluorescence was primarily detected in a subset of neuronal cells ( Figure 2A ). Based on this expression pattern, we hypothesized that neuronal usp-14 might be required for regulating innate immune responses. To test this, we expressed usp-14 under the pan-neuronal promoter rgef-1p in usp-14(jsn19) mutants and assessed both clec-60 expression and survival upon bacterial infection. Surprisingly, neuronal expression of usp-14 failed to restore clec-60 expression ( Figure 2B, C ) or improve the survival of usp-14(jsn19) worms upon bacterial infection ( Figure 2D, E ). Download figure Open in new tab Figure 2: Intestinal expression of USP-14 is sufficient to activate immunity during intestinal distension (A) Representative fluorescence images of usp-14p::gfp worm showing GFP in some head neurons (highlighted by the dotted line) along with a control non-transgenic worm. The fluorescence in the pharynx in the transgenic worm is from myo-2p::mCherry . Scale bar = 10 μm. (B) Representative fluorescence images of clec-60p::gfp , usp-14(jsn19);clec-60p::gfp , and rgef-1p::usp-14;usp-14(jsn19);clec-60p::gfp worms exposed to the empty vector (EV) control and aex-5 RNAi. Scale bar = 200 μm. (C) Quantification of GFP levels of clec-60p::gfp , usp-14(jsn19);clec-60p::gfp , and rgef-1p::usp-14;usp-14(jsn19);clec-60p::gfp worms exposed to the EV control and aex-5 RNAi. *** p < 0.001 and * p < 0.05 via unpaired t-test ( n = 30-31 worms each). The controls clec-60p::gfp and usp-14(jsn19);clec-60p::gfp for this experiment are the same as shown in Figures 1K and 2G . (D) Representative survival plots of clec-60p::gfp , usp-14(jsn19);clec-60p::gfp , and rgef-1p::usp-14;usp-14(jsn19);clec-60p::gfp worms on P. aeruginosa PA14 at 25°C. *** p < 0.001 between clec-60p::gfp and usp-14(jsn19);clec-60p::gfp , and non-significant between usp-14(jsn19);clec-60p::gfp and rgef-1p::usp-14;usp-14(jsn19);clec-60p::gfp ( n = 105 per condition). (E) Representative survival plots of clec-60p::gfp , usp-14(jsn19);clec-60p::gfp , and rgef-1p::usp-14;usp-14(jsn19);clec-60p::gfp worms on S. aureus NCTC832 5 at 25°C. *** p < 0.001 between clec-60p::gfp and usp-14(jsn19);clec-60p::gfp , and non-significant between usp-14(jsn19);clec-60p::gfp and rgef-1p::usp-14;usp-14(jsn19);clec-60p::gfp ( n = 105 per condition). The curves shown for clec-60p::gfp and usp-14(jsn19);clec-60p::gfp on S. aureus NCTC8325 are the same as shown in Figure 1M . (F) Representative fluorescence images of clec-60p::gfp , usp-14(jsn19);clec-60p::gfp , and vha- 6p::usp-14;usp-14(jsn19);clec-60p::gfp worms exposed to the EV control and aex-5 RNAi. Scale bar = 200 μm. (G) Quantification of GFP levels of clec-60p::gfp , usp-14(jsn19);clec-60p::gfp , and vha-6p::usp-14;usp-14(jsn19);clec-60p::gfp worms exposed to the EV control and aex-5 RNAi. *** p < 0.001 via t-test ( n = 30-31 worms each). The controls clec-60p::gfp and usp-14(jsn19);clec-60p::gfp for this experiment are the same as shown in Figures 1K and 2C . (H) Representative survival plots of clec-60p::gfp , usp-14(jsn19);clec-60p::gfp , and vha-6p::usp-14;usp-14(jsn19);clec-60p::gfp worms on P. aeruginosa PA14 at 25°C. *** p < 0.001 between clec-60p::gfp and usp-14(jsn19);clec-60p::gfp , and non-significant between clec-60p::gfp and vha-6p::usp-14;usp-14(jsn19);clec-60p::gfp ( n = 105 per condition). The curves shown for clec-60p::gfp and usp-14(jsn19);clec-60p::gfp on P. aeruginosa PA14 are the same as shown in Figure 2D . (I) Representative survival plots of clec-60p::gfp , usp-14(jsn19);clec-60p::gfp , and vha-6p::usp-14;usp-14(jsn19);clec-60p::gfp worms on S. aureus NCTC832 5 at 25°C. *** p < 0.001 between clec-60p::gfp and usp-14(jsn19);clec-60p::gfp , and non-significant between clec-60p::gfp and vha-6p::usp-14;usp-14(jsn19);clec-60p::gfp ( n = 70 for clec-60p::gfp , 105 for usp-14(jsn19);clec-60p::gfp , and 90 for vha-6p::usp-14;usp-14(jsn19);clec-60p::gfp). The curves shown for clec-60p::gfp and usp-14(jsn19);clec-60p::gfp on S. aureus NCTC8325 are the same as shown in Figure 1I . Given that clec-60 expression is primarily localized to intestinal cells, the main immune barrier against bacterial pathogens in C. elegans , we next asked whether USP-14 functions in the intestine to mediate immune activation. To address this, we expressed usp-14 under the intestine-specific promoter vha-6p in usp-14(jsn19) mutants and evaluated both clec-60 induction and pathogen resistance. Remarkably, intestinal expression of usp-14 completely rescued clec-60 induction upon aex-5 RNAi ( Figure 2F, G ). Consistently, intestinal expression also restored the survival of usp-14(jsn19) worms during both P. aeruginosa and S. aureus infections ( Figure 2H, I ). Together, these findings demonstrate that intestinal, but not neuronal, expression of USP-14 is sufficient to activate innate immune responses during intestinal distension in C. elegans . Intestinal distension induces USP-14-dependent and -independent innate immune response genes To elucidate the role of USP-14 in intestinal distension-mediated immune activation, we performed transcriptomic profiling by RNA sequencing. We compared the global gene expression patterns of wild-type N2 and usp-14(tm1481) worms grown to adulthood on the empty vector (EV) control or aex-5 RNAi (Table S1 and S2). We first examined whether USP-14 influences the transcriptome under basal conditions, in the absence of intestinal distension. Comparison of usp-14(tm1481) mutants and wild-type worms grown on EV revealed 127 differentially expressed genes, of which 86 were upregulated and 41 were downregulated ( Figure 3A , Table S3). Gene Ontology (GO) enrichment analysis of both upregulated ( Figure 3B ) and downregulated ( Figure 3C ) genes indicated a strong overrepresentation of innate immune response genes. These results suggested that loss of usp-14 perturbs immune homeostasis, potentially leading to constitutive activation of MAP kinase signaling pathways ( Figure S2A, B ). Download figure Open in new tab Figure 3: Intestinal distension induces USP-14-dependent and -independent innate immune response genes (A) Volcano plot of upregulated and downregulated genes in usp-14(tm1481) worms compared to wild-type N2 worms grown on empty vector (EV) control RNAi. Green and red dots represent significantly upregulated and downregulated genes, respectively, while the gray dots represent the genes that are not differentially regulated. (B)-(C) Gene Ontology (GO) enrichment analysis for upregulated (B) and downregulated (C) genes in usp-14(tm1481) worms compared to wild-type N2 worms grown on the EV control RNAi. (D) Venn diagram showing the overlap between genes upregulated upon aex-5 RNAi in N2 and usp-14(tm1481) worms. The GO analysis for the biological processes of unique and common genes is shown. (E) qRT-PCR analysis of innate immune response genes in wild-type N2 and usp-14(tm1481) worms after treatment with the EV control and aex-5 RNAi. *** p < 0.001, ** p < 0.01, * p < 0.05, ns, non-significant via t-test. Data represent the mean and standard deviation from three independent experiments. Intestinal distension is known to activate innate immune responses ( Singh & Aballay, 2019a ; Kumar et al , 2019 ). Therefore, we analyzed whether aex-5 RNAi in N2 worms also led to the induction of innate immune response genes. The knockdown of aex-5 resulted in the upregulation of 1241 genes and downregulation of 209 genes (Table S1). As expected, the upregulated genes were strongly enriched in innate immune responses ( Figure S2C ). We next investigated which of these aex-5 RNAi-induced genes required USP-14 for their activation. A comparative analysis between aex-5 RNAi-treated N2 and usp-14(tm1481) worms identified 633 genes whose induction was USP-14-dependent ( Figure 3D , Table S4). GO enrichment analysis revealed that these USP-14-dependent genes were highly enriched for innate immune response functions ( Figure 3D ). Because USP-14 also regulated a subset of immune genes under basal conditions ( Figure 3C ), we examined whether the USP-14-dependent genes induced during intestinal distension overlap with those regulated in the absence of distension. Notably, no overlap was observed between the genes downregulated in usp-14(tm1481) mutants under basal conditions and those dependent on USP-14 during intestinal distension ( Figure S2D ). This finding indicated that USP-14 specifically regulates a distinct set of immunity genes upon intestinal distension, beyond its basal role in immune homeostasis. To validate the RNA sequencing results, we performed qRT-PCR on a subset of immunity genes that were upregulated in N2 worms but not in usp-14(tm1481) mutants following aex-5 RNAi treatment ( Figure 3E ). Most of these genes showed significantly reduced expression in usp-14(tm1481) worms compared to N2 worms under aex-5 RNAi, confirming the transcriptomic results. We then analyzed the GO enrichment for the 595 genes that were upregulated in both N2 and usp-14(tm1481) worms upon aex-5 RNAi. These genes were also strongly enriched in innate immune response terms ( Figure 3D ), whereas genes uniquely upregulated in usp-14(tm1481) mutants showed no such enrichment. Collectively, these findings demonstrate that intestinal distension triggers both USP-14-dependent and USP-14-independent innate immune response pathways in C. elegans , highlighting a dual regulatory mechanism underlying distension-induced immune activation. USP-14 modulates immunity via the Wnt/β-catenin signaling pathway We next investigated whether USP-14 regulates intestinal distension-mediated immune activation through known immune signaling pathways. To this end, we generated several double mutants in the clec-60p::gfp background, including hlh-30(tm1978);usp-14(tm1481) , nhr-49(nr2041);usp-14(tm1481) , and pmk-1(km25);usp-14(tm1481) . The TFEB ortholog HLH-30, the nuclear hormone receptor NHR-49, and the NSY-1/SEK-1/PMK-1 MAP kinase cascade are all known to mediate host defense against P. aeruginosa and S. aureus infections ( Kim et al , 2002 ; Naim et al , 2021 ; Rao & Singh, 2025 ; Sifri et al , 2003 ; Wani et al , 2021 ), similar to USP-14 as shown in this study. RNAi-mediated knockdown of aex-5 resulted in robust induction of clec-60p::gfp in hlh-30(tm1978) , nhr-49(nr2041) , and pmk-1(km25) mutants ( Figure S3A, B ). Likewise, exposure of these mutants to S. aureus infection also induced clec-60p::gfp expression ( Figure S4A, B ). However, this induction was strongly suppressed when these mutations were combined with usp-14(tm1481) , both under aex-5 RNAi and during S. aureus infection (Figures S3, S4). These findings indicated that USP-14 regulates intestinal distension-mediated activation of clec-60 independently of HLH-30, NHR-49, and PMK-1. Furthermore, double mutants of usp-14(tm1481) with hlh-30(tm1978) , nhr-49(nr2041) , or pmk-1(km25) exhibited enhanced susceptibility to P. aeruginosa and S. aureus infections compared to either single mutant ( Figure S5A-F ), further supporting that USP-14 acts independently of these canonical immune pathways. We next investigated whether USP-14-mediated immune regulation is associated with the Wnt/β-catenin signaling pathway. The β-catenin homolog BAR-1 is known to regulate innate immunity in C. elegans intestinal cells ( Irazoqui et al , 2008 ; Labed et al , 2018 ), and intestinal distension has been shown to activate immune responses through components of the Wnt pathway ( Ren et al , 2022 ). To test whether USP-14 acts through this pathway, we first confirmed that knockdown of bar-1 suppressed intestinal distension-induced clec-60 expression ( Figure 4A, B ). Knockdown of bar-1 did not further reduce expression of clec-60 in the usp-14(jsn19) background. Notably, while bar-1 knockdown increased the susceptibility of N2 worms to P. aeruginosa and S. aureus , it did not further enhance the sensitivity of usp-14(tm1481) worms ( Figure 4C-F ), suggesting that USP-14 and BAR-1 function in the same pathway. Download figure Open in new tab Figure 4: USP-14 modulates immunity via the Wnt/β-catenin signaling pathway (A) Representative fluorescence images of clec-60p::gfp , aex-5(sa23);clec-60p::gfp , and usp-14(jsn19);aex-5(sa23);clec-60p::gfp worms exposed to the empty vector (EV) control and bar-1 RNAi. Scale bar = 200 μm. (B) Quantification of GFP levels of clec-60p::gfp , aex-5(sa23);clec-60p::gfp , and usp-14(jsn19);aex-5(sa23);clec-60p::gfp worms exposed to the EV control and bar-1 RNAi. *** p < 0.001 and ** p < 0.01 via unpaired t-test ( n = 28-31 worms each). (C) Representative survival plots of control N2 worms exposed to the EV and bar-1 RNAi on P. aeruginosa PA14 at 25°C. *** p < 0.001 ( n = 60 on EV and 90 on bar-1 RNAi). (D) Representative survival plots of usp-14(tm1481) worms exposed to the EV and bar-1 RNAi on P. aeruginosa PA14 at 25°C. ns, non-significant ( n = 105 per condition). (E) Representative survival plots of control N2 worms exposed to the EV and bar-1 RNAi on S. aureus NCTC8325 at 25°C. *** p < 0.001 ( n = 90 per condition). (F) Representative survival plots of control usp-14(tm1481) worms exposed to the EV and bar-1 RNAi on S. aureus NCTC8325 at 25°C. ns, non-significant ( n = 90 per condition). (G) qRT-PCR analysis of the Wnt/β-catenin signaling pathway genes in wild-type N2 and usp-14(tm1481) worms after 12 hours of infection with S. aureus NCTC8325, compared with E. coli OP50 controls at 25 °C. *** p < 0.001, ** p < 0.01, * p < 0.05, and ns, non-significant via t-test. Data represent the mean and standard deviation from three independent experiments. Previous studies have shown that S. aureus infection induces the expression of bar-1 (encoding the transcriptional effector of Wnt signaling), pop-1 (encoding the TCF transcription factor), and other Wnt pathway components such as mig-1 (encoding a Wnt receptor) and cwn-2 (encoding a Wnt ligand) ( Labed et al , 2018 ; Ren et al , 2022 ). To determine whether USP-14 regulates Wnt signaling at the transcriptional level, we quantified the mRNA levels of these genes upon S. aureus infection. The results revealed that S. aureus -induced expression of Wnt signaling pathway genes was significantly reduced in usp-14(tm1481) worms compared to wild type ( Figure 4G ). Together, these findings demonstrate that USP-14 modulates innate immune responses by regulating the transcriptional activation of multiple components of the Wnt/β-catenin signaling pathway, positioning USP-14 as an upstream regulator of Wnt-mediated immunity during intestinal distension in C. elegans . Discussions Recent work across multiple organisms has demonstrated that intestinal distension profoundly influences food-seeking behavior, immunity, and lifespan ( Das et al , 2024 ; Min et al , 2021 ; Duvall et al , 2019 ; Kumar et al , 2019 ; Singh & Aballay, 2019b ; Wang et al , 2025 ). In C. elegans , gut distension modulates pathogen avoidance by activating defined neuroendocrine pathways ( Filipowicz et al , 2021 ; Singh & Aballay, 2019a , 2019c ). These distension-induced avoidance behaviors can also be inherited by progeny through histone H4 Lys8 acetylation in the germline ( Hong et al , 2021 ). Beyond behavioral outputs, intestinal distension also initiates robust innate immune activation ( Singh & Aballay, 2019a ; Kumar et al , 2019 ). This response is mediated by the neuronal acetylcholine receptor ACC-4, which signals to the intestine via the Wnt/β-catenin pathway ( Ren et al , 2022 ). In this study, we identify the DUB USP-14 as an intestinal factor required for this distension-mediated immune activation. Both ACC-4 and USP-14 converge on the Wnt/β-catenin axis; however, it remains unclear whether they function within a single pathway or act in parallel to control Wnt-dependent immune gene expression. Moreover, because intestinal distension also activates USP-14-independent immune responses, additional signaling mechanisms must operate downstream of gut distension. USP14 is a highly conserved proteasome-associated DUB with dual activities in regulating protein stability. It can inhibit proteasomal degradation by trimming ubiquitin chains from substrates bound to the proteasome ( Lee et al , 2016 , 2010 ), yet it can also promote protein degradation by facilitating 20S proteasome gate opening ( Peth et al , 2009 ). In mammals, USP14 has been implicated in cancer progression by stabilizing oncogenic factors such as hypoxia-inducible factor and Jun kinase ( Du et al , 2023 ; Lv et al , 2021 ), and it has become a therapeutic target in several malignancies ( Tian et al , 2014 ; Morgan et al , 2023 ). USP14 can also suppress T-cell function, thereby contributing to immune evasion in tumors ( Shi et al , 2022 ; Yuan et al , 2025 ). Beyond cancer, USP14 also negatively regulates RIG-I-dependent antiviral immunity ( Li et al , 2019 ). Conversely, USP14 activity in lung epithelial cells promotes cytokine release by enhancing I-κB degradation ( Mialki et al , 2013 ). Our findings add to this complex landscape by demonstrating that USP-14 is required for the activation of innate immune responses triggered by intestinal distension in C. elegans . Thus, the immunomodulatory functions of USP14 appear to be strongly context dependent. USP14 has also been linked to the Wnt/β-catenin pathway at multiple mechanistic levels. In mammalian cells, USP14 enhances Wnt signaling by deubiquitinating Dishevelled ( Jung et al , 2013 ). Ubiquitin immunoprecipitation studies in USP14 inactive mutants have further identified β-catenin as a USP14 substrate ( Chadchankar et al , 2019 ), and subsequent work demonstrated that USP14-mediated stabilization of β-catenin promotes diffuse large B-cell lymphoma proliferation ( Wang et al , 2024 ). Consistently, curcumin-mediated inhibition of cancer progression has been attributed, in part, to modulation of β-catenin through USP-14 ( Shen et al , 2025 ). These findings collectively highlight the evolutionarily conserved connection between USP14 and Wnt/β-catenin signaling. Our data now extend this relationship to innate immunity in C. elegans , showing that USP-14 regulates the transcriptional activation of Wnt/β-catenin pathway components during intestinal distension. Interestingly, the mode of USP-14 action in C. elegans appears distinct from previously described post-translational regulation of Wnt components in mammalian systems. The ability of USP-14 to influence Wnt signaling at the transcriptional level suggests the existence of additional substrates or interacting proteins that modulate transcriptional outputs downstream of intestinal distension. In future work, it will be important to identify the direct USP-14 substrates that enable transcriptional activation of Wnt/β-catenin signaling and to determine how these molecular mechanisms interface with neuronal ACC-4 signaling and other distension-responsive pathways. Materials and methods Bacterial strains The following bacterial strains were used in this study: Escherichia coli OP50, E. coli HT115(DE3), Pseudomonas aeruginosa PA14, and Staphylococcus aureus NCTC8325. Cultures of E. coli OP50, E. coli HT115(DE3), and P. aeruginosa PA14 were grown in Luria-Bertani (LB) broth at 37°C, while S. aureus NCTC8325 was cultured in tryptic soy (TS) broth at 37°C. C. elegans strains and growth conditions C. elegans hermaphrodites were maintained at 20°C on nematode growth medium (NGM) plates seeded with E. coli OP50 as the food source, unless stated otherwise. The following strains were used in the study: JIN810 clec-60p::gfp , JT23 aex-5(sa23) , usp-14(tm1481) , KU25 pmk-1(km25) , JIN1375 hlh-30(tm1978), STE68 nhr-49(nr2041). The aex-5(sa23);clec-60p::gfp , usp-14(jsn19);clec-60p::gfp, usp-14(tm1481);clec-60p::gfp , pmk-1(km25);clec-60p::gfp, pmk-1(km25);usp-14(tm1481);clec-60p::gfp, hlh-30(tm1978);clec-60p::gfp, hlh-30(tm1978);usp-14(tm1481);clec-60p::gfp, nhr-49(nr2041);clec-60p::gfp , and nhr-49(nr2041);usp-14(tm1481);clec-60p::gfp strains were obtained by standard genetic crosses. The following strains were generated in this study: JSJ19 usp-14(jsn19), JSJ20 cec-10(jsn20) , usp-14(jsn19);clec-60p::gfp;jsnEx6[usp-14p::usp-14 + myo-3p::mCherry], usp-14(jsn19);clec-60p::gfp;jsnEx7[rgef-1p::usp-14 + myo-3p::mCherry], usp-14(jsn19);clec-60p::gfp;jsnEx8[vha-6p::usp-14 + myo-3p::mCherry], and jsnEx9[usp-14p::gfp + myo-2p::mCherry]. Some of the strains were obtained from the Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, MN). The usp-14(tm1481) allele was obtained from the National BioResource Project (NBRP), Japan. RNA interference (RNAi) RNAi was used to induce gene-specific loss-of-function phenotypes by feeding C. elegans with E. coli HT115(DE3) strains expressing double-stranded RNA corresponding to the target gene. RNAi experiments were performed as described previously ( Ghosh & Singh, 2024 ). Briefly, E. coli HT115(DE3) carrying the appropriate RNAi plasmids were grown overnight in LB broth containing ampicillin (100 µg/mL) at 37°C overnight on a shaker, concentrated 10 times, and plated onto RNAi NGM plates containing 100 µg/mL ampicillin and 3 mM isopropyl β-D-thiogalactoside. The plated bacteria were allowed to grow overnight at 37°C. Gravid adults were transferred to RNAi-expressing bacterial lawns and allowed to lay eggs for 2 hours. Adults were then removed, and the eggs were incubated at 20°C for 72 hours before being used for downstream assays. All RNAi clones were obtained from the Ahringer RNAi library and verified by sequencing. For bar-1 RNAi experiments, worms were grown on empty vector-containing E. coli until the L4 stage and subsequently transferred to bar-1 RNAi plates. Forward genetic screen for mutants with suppressed clec-60p::gfp expression Ethyl methanesulfonate (EMS) mutagenesis screens were performed using the aex-5(sa23);clec-60p::gfp strain as described previously ( Singh, 2021 ). Approximately 2,500 synchronized late L4 larvae were treated with 50 mM EMS for 4 hours, followed by three washes with M9 buffer. The EMS-treated animals (P0 generation) were transferred to 9-cm NGM plates seeded with E. coli OP50 and allowed to lay eggs (F1 progeny) overnight. Subsequently, the P0 animals were washed away with M9 medium, while the F1 eggs remained attached to the bacterial lawn. The F1 progeny were allowed to develop into adults, which were then bleached to obtain F2 eggs. F2 eggs were transferred to E. coli OP50-seeded NGM plates and incubated at 20°C for 96 hours. Plates were screened for worms exhibiting suppressed clec-60p::gfp expression levels. A total of ∼50,000 haploid genomes were screened, resulting in the isolation of four mutants with reduced GFP expression. Each mutant was backcrossed six times with the parental aex-5(sa23);clec-60p::gfp strain before further analysis. Whole-genome sequencing (WGS) and data analysis Genomic DNA was isolated following previously described methods ( Gokul & Singh, 2022 ; Ravi et al , 2023 ). Mutant worms were cultivated at 20°C on NGM plates seeded with E. coli OP50 until starvation. For each strain, animals from four 9-cm plates were collected using M9 buffer, washed three times, and incubated in M9 with gentle rotation for 2 hours to clear intestinal contents. The worms were washed three additional times with distilled water and stored at -80°C until DNA extraction. Genomic DNA was extracted using the Gentra Puregene Kit (Qiagen, Netherlands). DNA libraries were prepared according to the standard Illumina protocol (San Diego, CA). Whole-genome sequencing was performed on an Illumina NovaSeq 6000 platform utilizing 150 paired-end nucleotide reads. Library preparation and sequencing were carried out at the National Genomics Core, National Institute of Biomedical Genomics, Kalyani, India. Whole-genome sequence data were analyzed using the Galaxy web platform, as described earlier ( Gokul & Singh, 2022 ). Briefly, the forward and reverse FASTQ reads, C. elegans reference genome Fasta file (ce11M.fa), and the gene annotation file (SnpEff4.3 WBcel235.86) were input into the Galaxy workflow. Low-quality bases were trimmed using the Sickle tool, and the filtered reads were aligned to the reference genome using BWA-MEM. Duplicate reads mapping to multiple genomic locations were removed using MarkDuplicates. Variant calling was performed using FreeBayes, which identifies small polymorphisms, including single-nucleotide polymorphisms (SNPs), insertions and deletions (indels), multi-nucleotide polymorphisms (MNPs), and complex variants smaller than the typical short-read alignment length. Shared variants among different mutants were filtered out to identify unique mutations. Genetic variants were annotated using the SnpEff 4.3 WBcel235.86 database to predict their effects, including amino acid substitutions. Finally, linkage maps for each mutant were generated based on the identified variant profiles. C. elegans killing assays on P. aeruginosa PA14 Full lawn killing assays of C. elegans on P. aeruginosa PA14 were performed as previously described ( Ghosh & Singh, 2024 ; Das et al , 2024 ). Briefly, single colonies of P. aeruginosa PA14 were inoculated into 2 mL of LB broth and incubated at 37°C with shaking for 10-12 hours. Bacterial lawns were prepared by spreading 20 µL of the culture onto modified NGM agar plates (3.5 cm diameter) containing 0.35% peptone. The seeded plates were incubated at 37°C for 10-12 hours and then cooled to room temperature for at least 30 minutes before use. After that, synchronized 1-day-old adult worms were transferred to the P. aeruginosa -seeded plates and maintained at 25°C. Live animals were transferred daily to freshly prepared P. aeruginosa plates to avoid confounding effects of progeny. Worms were examined at the indicated time points and scored as dead if they failed to respond to gentle prodding. For killing assays involving bar-1 RNAi, worms were grown on empty vector-containing E. coli until the L4 stage and subsequently transferred to bar-1 RNAi plates. The 1-day-old adult worms were then transferred to P. aeruginosa -seeded plates and maintained at 25°C. Each experimental condition was assayed in at least three independent biological replicates. Detailed statistical analyses for all survival assays are provided in Table S5. C. elegans killing assays on S. aureus Individual S. aureus colonies were inoculated into 2 mL of TS broth and cultured overnight at 37°C with shaking. The overnight culture was diluted 1:10 in fresh TS broth, and 20 µL of the diluted suspension was spread onto 3.5 cm NGM plates. The plates were incubated at 37°C for 6 hours to allow lawn formation, then cooled to room temperature for at least 30 minutes before use. After that, synchronized 1-day-old adult worms were transferred to the S. aureus -seeded plates and incubated at 25°C. Animals were transferred daily to freshly seeded plates, and viability was assessed at the indicated time points. Worms were considered dead if they failed to respond to touch with a platinum wire. For killing assays involving bar-1 RNAi, worms were grown on empty vector-containing E. coli until the L4 stage and subsequently transferred to bar-1 RNAi plates. The 1-day-old adult worms were then transferred to S. aureus -seeded plates and maintained at 25°C. All assays were performed in triplicate with independent biological replicates. Detailed statistical analyses for all survival assays are provided in Table S5. C. elegans RNA isolation and quantitative reverse transcription-PCR Wild-type N2 and usp-14(tm1481) worms were synchronized by timed egg laying. Approximately 50 gravid adults were transferred to 9-cm NGM plates seeded with either aex-5 RNAi or control empty vector RNAi bacteria and allowed to lay eggs for 4 hours. The adults were then removed, and the plates were incubated at 20°C for 96 hours. The resulting adult worms were collected, washed with M9 buffer, and preserved in TRIzol reagent (Life Technologies, Carlsbad, CA, USA) for RNA extraction. Total RNA was isolated using the RNeasy Plus Universal Kit (Qiagen, Netherlands). For cDNA synthesis, 1 µg of total RNA was reverse transcribed using random primers and the PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa), according to the manufacturer’s protocol. Quantitative RT-PCR was performed using TB Green fluorescence (TaKaRa) on a MasterCycler EP Realplex 4 thermal cycler (Eppendorf) in a 96-well plate format. Reactions were prepared in 15 µL volumes and analyzed following the manufacturer’s instructions. Relative transcript levels were calculated using the comparative CT (2 −ΔΔ CT ) method, normalized to pan-actin ( act-1 , -3 , -4 ) expression as previously described ( Singh & Aballay, 2017 ). All qRT-PCR reactions were performed in triplicate (technical replicates) and repeated across at least three independent biological replicates. Primer sequences are provided in Table S6 . RNA sequencing and data analysis For RNA sequencing, worm synchronization and RNA extraction for N2 and usp-14(tm1481) strains grown on aex-5 RNAi were performed as described above for qRT-PCR. Three independent biological replicates were used for each condition. Library preparation and sequencing were performed at Neuberg Supratech Reference Laboratories, India, with cDNA libraries sequenced on the NovaSeq 6000 platform using 50-bp paired-end reads. RNA sequencing data were analyzed via the Galaxy web platform ( https://usegalaxy.org/ ) following established protocols ( Ghosh & Singh, 2024 ). Briefly, paired-end reads underwent quality trimming using Trimmomatic before being aligned to the C. elegans reference genome (WS220) using STAR. Read counts per gene were obtained using htseq-count , and differential gene expression analysis was carried out with DESeq2. Genes showing a ≥2-fold change in expression and a P -value < 0.01 were considered differentially expressed. Gene Ontology analysis utilized the DAVID Bioinformatics Database ( https://david.ncifcrf.gov/tools.jsp ), and Venn diagrams were generated using BioVenn ( https://www.biovenn.nl/ ) ( Hulsen et al , 2008 ). Plasmid constructs and generation of transgenic C. elegans To generate the usp-14p::gfp transcriptional reporter, the 714 bp promoter region upstream of the usp-14 start codon was amplified from N2 genomic DNA and cloned into the pPD95.77 plasmid upstream of GFP using SalI and KpnI restriction sites. The resulting construct was microinjected into wild-type N2 animals along with pCFJ90 ( myo-2p::mCherry ) as a co-injection marker to generate the usp-14p::gfp reporter strain. For rescuing the usp-14(jsn19) mutant phenotype, the usp-14 promoter and its complete coding sequence, including the stop codon, were amplified from N2 genomic DNA and cloned into pPD95.77 upstream of GFP using SphI and SmaI restriction sites. The construct was microinjected into usp-14(jsn19);clec-60p::gfp worms together with pCFJ104 ( myo-3p::mCherry ) as a co-injection marker to generate the strain usp-14(jsn19);clec-60p::gfp;jsnEx6[usp-14p::usp-14 + myo-3p::mCherry] . For neuronal rescue, the 3,392 bp promoter region upstream of the rgef-1 start codon was first cloned into the pPD95.77 plasmid using BamHI and SmaI restriction sites. The usp-14 coding sequence was then inserted downstream of the rgef-1 promoter using the In-Fusion HD Cloning Kit (Takara Bio, Tokyo, Japan). The resulting rgef-1p::usp-14 construct was microinjected into usp-14(jsn19);clec-60p::gfp worms along with pCFJ104 as a co-injection marker to generate usp-14(jsn19);clec-60p::gfp;jsnEx7[rgef-1p::usp-14 + myo-3p::mCherry] . To generate intestine-specific rescue strains, the usp-14 coding sequence was cloned into the pDB24 plasmid (kindly provided by the laboratory of Alejandro Aballay), which carries the vha-6 promoter, using BamHI and AgeI restriction sites. The construct was microinjected into usp-14(jsn19);clec-60p::gfp worms with pCFJ104 as a co-injection marker to generate usp-14(jsn19);clec-60p::gfp;jsnEx8[vha-6p::usp-14 + myo-3p::mCherry] . For all injections, usp-14p::gfp , usp-14p::usp-14 , vha-6p::usp-14 , and rgef-1p::usp-14 plasmids were used at a concentration of 50 ng/µL. The co-injection markers myo-2p::mCherry and myo-3p::mCherry were used at final concentrations of 5 ng/µL and 25 ng/µL, respectively. All transgenes were maintained as extrachromosomal arrays, and at least two independent transgenic lines were established and analyzed for each construct. Primer sequences are provided in Table S6 . Fluorescence imaging of C. elegans Fluorescence imaging was performed as described previously (Das et al , 2025; Rao et al , 2025 ). Briefly, animals were picked under a non-fluorescence stereomicroscope to avoid potential bias. Worms were anesthetized in M9 buffer containing 50 mM sodium azide and mounted on 2% agarose pads for imaging. Fluorescence visualization was conducted using either a Nikon SMZ-1000 or SMZ18 stereomicroscope. Fluorescence intensity was quantified using ImageJ software. High-resolution images of the usp-14p::gfp reporter strain were captured using a ZEISS Imager.Z2 microscope. Quantification and statistical analysis All statistical analyses were performed with Prism 8 (GraphPad). Data are presented as mean ± standard deviation. Statistical significance between two groups was assessed using an unpaired, two-tailed t -test. For comparisons involving more than two groups, an ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test was applied. Statistical significance was defined as p < 0.05. In all figures, asterisks indicate significance levels as follows: * p < 0.05; ** p < 0.01; and *** p < 0.001, relative to the corresponding controls. Survival data were analyzed using the Kaplan-Meier method, and differences between survival curves were evaluated using the log-rank test. All experiments were performed independently at least three times unless otherwise stated. Funding This work was supported by the following grants: Har-Gobind Khorana-Innovative Young Biotechnologist Fellowship (File No. HRD-17011/2/2023-HRD-DBT) and Ramalingaswami Re-entry Fellowship (Ref. No. BT/RLF/Re-entry/50/2020) awarded by the Department of Biotechnology, India; STARS grant (File No. MoE-STARS/STARS-2/2023-0116) awarded by the Ministry of Education, India; Anusandhan National Research Foundation (ANRF) Core Research Grant (Ref. No. CRG/2023/001136) awarded by DST, India; Research Grant (Ref. No. 37/1741/23/EMR-II) awarded by the Council of Scientific & Industrial Research (CSIR), India; and IISER Mohali intramural funds. Author Contributions A.G. and J.S. conceived and designed the experiments. A.G. performed the experiments. A.G. and J.S. analyzed the data and wrote the paper. Declaration of interests The authors declare no competing interests. Data availability The whole-genome sequence data for JSJ19 and JSJ20 have been submitted to the public repository, the Sequence Read Archive, with BioProject ID PRJNA1367285. The RNA sequencing data for N2 and usp-14(tm1481) worms grown on the empty vector control and aex-5 RNAi have been submitted to the public repository, the Sequence Read Archive, with BioProject ID PRJNA1364750. All data generated or analyzed during this study are included in the manuscript and supporting files. Supplementary figures Download figure Open in new tab Figure S1: Knockdown of usp-14 and cec-10 suppresses intestinal distension-mediated clec-60 induction (A) Representative fluorescence images of aex-5(sa23);clec-60p::gfp worms grown on the empty vector (EV) control, usp-14 , and cec-10 RNAi. Scale bar = 200 μm. (B) Quantification of GFP levels of aex-5(sa23);clec-60p::gfp worms grown on the EV control, usp-14 , and cec-10 RNAi. *** p < 0.001 via ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test ( n = 20 worms each). (C) Representative survival plots of N2 worms exposed to the EV and cec-10 RNAi on P. aeruginosa PA14 at 25°C. *** p < 0.001 ( n = 60 on EV and 90 on cec-10 RNAi). Download figure Open in new tab Figure S2: Intestinal distension induces USP-14-dependent and -independent innate immune response genes (A)-(B) Gene Ontology (GO) enrichment analysis for molecular functions for upregulated (A) and downregulated (B) genes in usp-14(tm1481) worms compared to wild-type N2 worms grown on the empty vector (EV) control RNAi. (C) GO enrichment analysis for biological process for upregulated genes in N2 worms grown on aex-5 RNAi compared to N2 worms grown on the EV control RNAi. (D) Venn diagram showing the overlap between intestinal distension-induced USP-14-dependent genes and genes downregulated in usp-14(tm1481) mutants independent of intestinal distension. Download figure Open in new tab Figure S3: USP-14 mediates intestinal distension-induced immunity independent of known canonical innate immune pathways (A) Representative fluorescence images of clec-60p::gfp , usp-14(tm1481);clec-60p::gfp, hlh-30(tm1978);clec-60p::gfp, hlh-30(tm1978);usp-14(tm1481);clec-60p::gfp , nhr-49(nr2041);clec-60p::gfp , nhr-49(nr2041);usp-14(tm1481);clec-60p::gfp , pmk-1(km25);clec-60p::gfp , and pmk-1(km25);usp-14(tm1481);clec-60p::gfp worms exposed to empty vector (EV) control and aex-5 RNAi. Scale bar = 200 μm (B) Quantification of GFP levels of clec-60p::gfp , usp-14(tm1481);clec-60p::gfp, hlh-30(tm1978);clec-60p::gfp, hlh-30(tm1978);usp-14(tm1481);clec-60p::gfp , nhr-49(nr2041);clec-60p::gfp , nhr-49(nr2041);usp-14(tm1481);clec-60p::gfp , pmk-1(km25);clec-60p::gfp , and pmk-1(km25);usp-14(tm1481);clec-60p::gfp worms exposed to EV control and aex-5 RNAi. *** p < 0.001 via unpaired t-test ( n = 30 worms each). Download figure Open in new tab Figure S4: USP-14 mediates S. aureus -induced immunity independent of known canonical innate immune pathways (A) Representative fluorescence images of clec-60p::gfp , usp-14(tm1481);clec-60p::gfp, hlh-30(tm1978);clec-60p::gfp, hlh-30(tm1978);usp-14(tm1481);clec-60p::gfp , nhr-49(nr2041);clec-60p::gfp , nhr-49(nr2041);usp-14(tm1481);clec-60p::gfp , pmk-1(km25);clec-60p::gfp , and pmk-1(km25);usp-14(tm1481);clec-60p::gfp worms after 12 hours of infection with S. aureus NCTC8325, compared with E. coli OP50 controls at 25 °C. Scale bar = 200 μm (B) Quantification of GFP levels of clec-60p::gfp , usp-14(tm1481);clec-60p::gfp, hlh-30(tm1978);clec-60p::gfp, hlh-30(tm1978);usp-14(tm1481);clec-60p::gfp , nhr-49(nr2041);clec-60p::gfp , nhr-49(nr2041);usp-14(tm1481);clec-60p::gfp , pmk-1(km25);clec-60p::gfp , and pmk-1(km25);usp-14(tm1481);clec-60p::gfp worms after 12 hours of infection with S. aureus NCTC8325, compared with E. coli OP50 controls at 25 °C. *** p < 0.001 and * p < 0.05 via unpaired t-test ( n = 27-32 worms each). Download figure Open in new tab Figure S5: USP-14 modulates immunity independent of known canonical innate immune pathways (A) Representative survival plots of hlh-30(tm1978);clec-60p::gfp and hlh-30(tm1978);usp-14(jsn19);clec-60p::gfp worms on P. aeruginosa PA14 at 25°C. *** p < 0.001 ( n = 90 per condition). (B) Representative survival plots of nhr-49(nr2041);clec-60p::gfp and nhr-49(nr2041);usp-14(tm1481);clec-60p::gfp worms on P. aeruginosa PA14 at 25°C. *** p < 0.001 ( n = 90 for nhr-49(nr2041);clec-60p::gfp and 91 for nhr-49(nr2041);usp-14(tm1481);clec-60p::gfp ). (C) Representative survival plots of control pmk-1(km25);clec-60p::gfp and pmk-1(km25);usp-14(tm1481);clec-60p::gfp worms on P. aeruginosa PA14 at 25°C. *** p < 0.001 ( n = 90 per condition). (D) Representative survival plots of control hlh-30(tm1978);clec-60p::gfp and hlh-30(tm1978);usp-14(jsn19);clec-60p::gfp worms on S. aureus NCTC8325 at 25°C. *** p < 0.001 ( n = 90 per condition). (E) Representative survival plots of control nhr-49(nr2041);clec-60p::gfp and nhr-49(nr2041);usp-14(tm1481);clec-60p::gfp worms on S. aureus NCTC8325 at 25°C. *** p < 0.001 ( n = 90 per condition). (F) Representative survival plots of control pmk-1(km25);clec-60p::gfp and pmk-1(km25);usp-14(tm1481);clec-60p::gfp worms on S. aureus NCTC8325 at 25°C. *** p < 0.001 ( n = 90 per condition). Supplementary tables Table S1: Upregulated and downregulated genes in aex-5 RNAi versus empty vector control RNAi in N2 worms. Genes exhibiting at least two-fold change and P -value <0.01 were considered differentially expressed. A separate Excel file is provided. Table S2: Upregulated and downregulated genes in aex-5 RNAi versus empty vector control RNAi in usp-14(tm1481) worms. Genes exhibiting at least two-fold change and P -value <0.01 were considered differentially expressed. A separate Excel file is provided. Table S3: Upregulated and downregulated genes in usp-14(tm1481) worms versus N2 worms grown on empty vector control RNAi. Genes exhibiting at least two-fold change and P -value <0.01 were considered differentially expressed. A separate Excel file is provided. Table S4: Comparison of genes upregulated upon aex-5 RNAi in N2 and usp-14(tm1481) worms. A separate Excel file is provided. Table S5: Statistical analysis of survival curves from three independent experiments for each condition. A separate Excel file is provided. View this table: View inline View popup Table S6: Primers used in the study Acknowledgments We thank the Caenorhabditis Genetics Center [funded by the NIH Office of Research Infrastructure Programs (P40 OD010440)] for providing the strains used in this study. Funder Information Declared Department of Biotechnology, https://ror.org/03tjsyq23 , HRD-17011/2/2023-HRD-DBT , BT/RLF/Re-entry/50/2020 Ministry of Education, India , MoE-STARS/STARS-2/2023-0116 Science and Engineering Research Board, https://ror.org/03ffdsr55 , CRG/2023/001136 Council of Scientific and Industrial Research , 37/1741/23/EMR-II References ↵ Akira S , Uematsu S & Takeuchi O ( 2006 ) Pathogen Recognition and Innate Immunity . Cell 124 : 783 – 801 OpenUrl CrossRef PubMed Web of Science ↵ Blander JM & Sander LE ( 2012 ) Beyond pattern recognition: five immune checkpoints for scaling the microbial threat . Nat Rev Immunol 12 : 215 – 225 OpenUrl CrossRef PubMed ↵ Branicky R & Hekimi S ( 2006 ) What keeps C. elegans regular: the genetics of defecation . Trends Genet 22 : 571 – 579 OpenUrl CrossRef PubMed Web of Science ↵ Chadchankar J , Korboukh V , Conway LC , Wobst HJ , Walker CA , Doig P , Jacobsen SJ , Brandon NJ , Moss SJ & Wang Q ( 2019 ) Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells . PLOS ONE 14 : e0225145 OpenUrl CrossRef PubMed ↵ Cook DE , Mesarich CH & Thomma BPHJ ( 2015 ) Understanding Plant Immunity as a Surveillance System to Detect Invasion . Annu Rev Phytopathol 53 : 541 – 563 OpenUrl CrossRef PubMed ↵ Das P , Aballay A & Singh J ( 2024 ) Calcineurin inhibition enhances Caenorhabditis elegans lifespan by defecation defects-mediated calorie restriction and nuclear hormone signaling . eLife 12 : RP89572 Das P , Ravi & Singh J ( 2025 ) Iron-deplete diet enhances Caenorhabditis elegans lifespan via oxidative stress response pathways . EMBO J : doi: 10.1038/s44318-025-00634–7 OpenUrl CrossRef ↵ Du X-H , Ke S-B , Liang X-Y , Gao J , Xie X-X , Qi L-Z , Liu X-Y , Xu G-Y , Zhang X-D , Du R-L , et al. ( 2023 ) USP14 promotes colorectal cancer progression by targeting JNK for stabilization . Cell Death Dis 14 : 56 OpenUrl PubMed ↵ Dunbar TL , Yan Z , Balla KM , Smelkinson MG & Troemel ER ( 2012 ) C. elegans detects pathogen-induced translational inhibition to activate immune signaling . Cell Host Microbe 11 : 375 – 386 OpenUrl CrossRef PubMed Web of Science ↵ Duvall LB , Ramos-Espiritu L , Barsoum KE , Glickman JF & Vosshall LB ( 2019 ) Small-Molecule Agonists of Ae. aegypti Neuropeptide Y Receptor Block Mosquito Biting . Cell 176 : 687 – 701 .e5 OpenUrl CrossRef PubMed ↵ Filipowicz A , Lalsiamthara J & Aballay A ( 2021 ) TRPM channels mediate learned pathogen avoidance following intestinal distention . eLife 10 : e65935 OpenUrl CrossRef PubMed ↵ Ghosh A & Singh J ( 2024 ) Translation initiation or elongation inhibition triggers contrasting effects on Caenorhabditis elegans survival during pathogen infection . mBio 15 : e0248524 OpenUrl CrossRef PubMed ↵ Gokul G & Singh J ( 2022 ) Dithiothreitol causes toxicity in C. elegans by modulating the methionine-homocysteine cycle . eLife 11 : e76021 OpenUrl CrossRef PubMed ↵ Grover M , Gang SS , Troemel ER & Barkoulas M ( 2024 ) Proteasome inhibition triggers tissue-specific immune responses against different pathogens in C. elegans . PLOS Biol 22 : e3002543 OpenUrl CrossRef PubMed ↵ Hong C , Lalsiamthara J , Ren J , Sang Y & Aballay A ( 2021 ) Microbial colonization induces histone acetylation critical for inherited gut-germline-neural signaling . PLOS Biol 19 : e3001169 OpenUrl CrossRef PubMed ↵ Hulsen T , de Vlieg J & Alkema W ( 2008 ) BioVenn - a web application for the comparison and visualization of biological lists using area-proportional Venn diagrams . BMC Genomics 9 : 488 OpenUrl CrossRef PubMed ↵ Irazoqui JE , Ng A , Xavier RJ & Ausubel FM ( 2008 ) Role for β-catenin and HOX transcription factors in Caenorhabditis elegans and mammalian host epithelial-pathogen interactions . Proc Natl Acad Sci 105 : 17469 – 17474 OpenUrl Abstract / FREE Full Text ↵ Irazoqui JE , Troemel ER , Feinbaum RL , Luhachack LG , Cezairliyan BO & Ausubel FM ( 2010 ) Distinct Pathogenesis and Host Responses during Infection of C. elegans by P. aeruginosa and S. aureus . PLOS Pathog 6 : e1000982 OpenUrl CrossRef PubMed ↵ Jung H , Kim B-G , Han WH , Lee JH , Cho J-Y , Park WS , Maurice MM , Han J-K , Lee MJ , Finley D , et al. ( 2013 ) Deubiquitination of Dishevelled by Usp14 is required for Wnt signaling . Oncogenesis 2 : e64 OpenUrl CrossRef PubMed ↵ Kim DH , Feinbaum R , Alloing G , Emerson FE , Garsin DA , Inoue H , Tanaka-Hino M , Hisamoto N , Matsumoto K , Tan M-W , et al. ( 2002 ) A conserved p38 MAP kinase pathway in Caenorhabditis elegans innate immunity . Science 297 : 623 – 626 OpenUrl Abstract / FREE Full Text ↵ Kumar S , Egan BM , Kocsisova Z , Schneider DL , Murphy JT , Diwan A & Kornfeld K ( 2019 ) Lifespan Extension in C. elegans Caused by Bacterial Colonization of the Intestine and Subsequent Activation of an Innate Immune Response . Dev Cell 49 : 100 – 117 .e6 OpenUrl CrossRef PubMed ↵ Labed SA , Wani KA , Jagadeesan S , Hakkim A , Najibi M & Irazoqui JE ( 2018 ) Intestinal Epithelial Wnt Signaling Mediates Acetylcholine-Triggered Host Defense against Infection . Immunity 48 : 963 – 978 .e4 OpenUrl CrossRef PubMed ↵ Lee B-H , Lee MJ , Park S , Oh D-C , Elsasser S , Chen P-C , Gartner C , Dimova N , Hanna J , Gygi SP , et al. ( 2010 ) Enhancement of proteasome activity by a small-molecule inhibitor of USP14 . Nature 467 : 179 – 184 OpenUrl CrossRef PubMed Web of Science ↵ Lee B-H , Lu Y , Prado MA , Shi Y , Tian G , Sun S , Elsasser S , Gygi SP , King RW & Finley D ( 2016 ) USP14 deubiquitinates proteasome-bound substrates that are ubiquitinated at multiple sites . Nature 532 : 398 – 401 OpenUrl CrossRef PubMed ↵ Li H , Zhao Z , Ling J , Pan L , Zhao X , Zhu H , Yu J , Xie B , Shen J & Chen W ( 2019 ) USP14 promotes K63-linked RIG-I deubiquitination and suppresses antiviral immune responses . Eur J Immunol 49 : 42 – 53 OpenUrl CrossRef PubMed ↵ Lv C , Wang S , Lin L , Wang C , Zeng K , Meng Y , Sun G , Wei S , Liu Y & Zhao Y ( 2021 ) USP14 maintains HIF1-α stabilization via its deubiquitination activity in hepatocellular carcinoma . Cell Death Dis 12 : 803 OpenUrl PubMed ↵ McEwan DL , Kirienko NV & Ausubel FM ( 2012 ) Host Translational Inhibition by Pseudomonas aeruginosa Exotoxin A Triggers an Immune Response in Caenorhabditis elegans . Cell Host Microbe 11 : 364 – 374 OpenUrl CrossRef PubMed Web of Science ↵ Melo JA & Ruvkun G ( 2012 ) Inactivation of Conserved C . elegans Genes Engages Pathogen- and Xenobiotic-Associated Defenses . Cell 149 : 452 – 466 OpenUrl CrossRef PubMed Web of Science ↵ Mialki RK , Zhao J , Wei J , Mallampalli DF & Zhao Y ( 2013 ) Overexpression of USP14 Protease Reduces I-κB Protein Levels and Increases Cytokine Release in Lung Epithelial Cells* . J Biol Chem 288 : 15437 – 15441 OpenUrl Abstract / FREE Full Text ↵ Min S , Oh Y , Verma P , Whitehead SC , Yapici N , Van Vactor D , Suh GS & Liberles S ( 2021 ) Control of feeding by Piezo-mediated gut mechanosensation in Drosophila . eLife 10 : e63049 OpenUrl CrossRef PubMed ↵ Morgan EL , Toni T , Viswanathan R , Robbins Y , Yang X , Cheng H , Gunti S , Huynh A , Sowers AL , Mitchell JB , et al. ( 2023 ) Inhibition of USP14 promotes TNFα-induced cell death in head and neck squamous cell carcinoma (HNSCC) . Cell Death Differ 30 : 1382 – 1396 OpenUrl PubMed ↵ Naim N , Amrit FRG , Ratnappan R , DelBuono N , Loose JA & Ghazi A ( 2021 ) Cell nonautonomous roles of NHR-49 in promoting longevity and innate immunity . Aging Cell 20 : e13413 OpenUrl CrossRef PubMed ↵ Peth A , Besche HC & Goldberg AL ( 2009 ) Ubiquitinated Proteins Activate the Proteasome by Binding to Usp14/Ubp6, which Causes 20S Gate Opening . Mol Cell 36 : 794 – 804 OpenUrl CrossRef PubMed Web of Science ↵ Pugin J ( 2012 ) How tissue injury alarms the immune system and causes a systemic inflammatory response syndrome . Ann Intensive Care 2 : 27 OpenUrl CrossRef PubMed ↵ Pukkila-Worley R ( 2016 ) Surveillance Immunity: An Emerging Paradigm of Innate Defense Activation in Caenorhabditis elegans . PLOS Pathog 12 : e1005795 OpenUrl CrossRef PubMed ↵ Rao R , Aballay A & Singh J ( 2025 ) Inhibition of the UFD-1-NPL-4 complex triggers an inflammation-like response in Caenorhabditis elegans . eLife 13 : RP94310 ↵ Rao R & Singh J ( 2025 ) Innate immune and endoplasmic reticulum unfolded protein response pathways protect Caenorhabditis elegans against chloroquine toxicity . J Biosci 50 : 15 OpenUrl CrossRef PubMed ↵ Ravi , Kumar A , Bhattacharyya S & Singh J ( 2023 ) Thiol reductive stress activates the hypoxia response pathway . EMBO J 42 : e114093 OpenUrl CrossRef PubMed ↵ Ren J , Sang Y & Aballay A ( 2022 ) Cholinergic receptor-Wnt pathway controls immune activation by sensing intestinal dysfunction . Cell Rep 41 : 111575 OpenUrl PubMed ↵ Shen M , Chen L , Jiang J , Wang Z , Gong Q , Zhang X , Ke X & Qu Y ( 2025 ) Curcumin modulates β-catenin stabilization via targeting proteasomal deubiquitinating enzyme USP14⋆ . Pharmacol Res 216 : 107745 OpenUrl PubMed ↵ Shi D , Wu X , Jian Y , Wang J , Huang C , Mo S , Li Y , Li F , Zhang C , Zhang D , et al. ( 2022 ) USP14 promotes tryptophan metabolism and immune suppression by stabilizing IDO1 in colorectal cancer . Nat Commun 13 : 5644 OpenUrl PubMed ↵ Sifri CD , Begun J , Ausubel FM & Calderwood SB ( 2003 ) Caenorhabditis elegans as a model host for Staphylococcus aureus pathogenesis . Infect Immun 71 : 2208 – 2217 OpenUrl Abstract / FREE Full Text ↵ Singh J ( 2021 ) Harnessing the power of genetics: fast forward genetics in Caenorhabditis elegans . Mol Genet Genomics 296 : 1 – 20 OpenUrl CrossRef PubMed ↵ Singh J & Aballay A ( 2017 ) Endoplasmic Reticulum Stress Caused by Lipoprotein Accumulation Suppresses Immunity against Bacterial Pathogens and Contributes to Immunosenescence . mBio 8 : e00778 – 17 OpenUrl CrossRef PubMed ↵ Singh J & Aballay A ( 2019a ) Microbial Colonization Activates an Immune Fight-and-Flight Response via Neuroendocrine Signaling . Dev Cell 49 : 89 – 99 .e4 OpenUrl CrossRef PubMed ↵ Singh J & Aballay A ( 2019b ) Similar Neural Pathways Control Foraging in Mosquitoes and Worms . mBio 10 : doi: 10.1128/mbio.00656-19 OpenUrl CrossRef ↵ Singh J & Aballay A ( 2019c ) Intestinal infection regulates behavior and learning via neuroendocrine signaling . eLife 8 : e50033 OpenUrl CrossRef PubMed ↵ Taffoni C & Pujol N ( 2015 ) Mechanisms of innate immunity in C. elegans epidermis . Tissue Barriers 3 : e1078432 OpenUrl ↵ Tian Z , D’Arcy P , Wang X , Ray A , Tai Y-T , Hu Y , Carrasco RD , Richardson P , Linder S , Chauhan D , et al. ( 2014 ) A novel small molecule inhibitor of deubiquitylating enzyme USP14 and UCHL5 induces apoptosis in multiple myeloma and overcomes bortezomib resistance . Blood 123 : 706 – 716 OpenUrl Abstract / FREE Full Text ↵ Wang C , Fu W , Zhang Y , Hu X , Xu Q & Tong X ( 2024 ) C-MYC-activated lncRNA SNHG20 accelerates the proliferation of diffuse large B cell lymphoma via USP14-mediated deubiquitination of β-catenin . Biol Direct 19 : 47 OpenUrl PubMed ↵ Wang F , Ning S , Yu B & Wang Y ( 2021 ) USP14: Structure, Function, and Target Inhibition . Front Pharmacol 12 : 801328 OpenUrl CrossRef PubMed ↵ Wang H , Lou R , Wang Y , Hao L , Wang Q , Li R , Su J , Liu S , Zhou X , Gao X , et al. ( 2025 ) Parallel gut-to-brain pathways orchestrate feeding behaviors . Nat Neurosci 28 : 320 – 335 OpenUrl CrossRef PubMed ↵ Wani KA , Goswamy D , Taubert S , Ratnappan R , Ghazi A & Irazoqui JE ( 2021 ) NHR-49/PPAR-α and HLH-30/TFEB cooperate for C. elegans host defense via a flavin-containing monooxygenase . eLife 10 : e62775 OpenUrl PubMed ↵ Willmann K & Moita LF ( 2025 ) Biological and clinical implications of a model of surveillance immunity . J Clin Invest 135 ↵ Yuan Y , Niu Y , Huang Z , Shi Y , Lin Z , Zuo D , Zhong C , Li K , Guan X-Y , Yuan Y , et al. ( 2025 ) USP14-mediated metabolic competition impairs CD8+ T cell immunosurveillance in hepatocellular carcinoma . Proc Natl Acad Sci 122 : e2510576122 OpenUrl PubMed View the discussion thread. Back to top Previous Next Posted November 28, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. 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Share Deubiquitinase USP-14 controls intestinal distension-induced immune activation in Caenorhabditis elegans via Wnt/β-catenin signaling Annesha Ghosh , Jogender Singh bioRxiv 2025.11.25.690479; doi: https://doi.org/10.1101/2025.11.25.690479 Share This Article: Copy Citation Tools Deubiquitinase USP-14 controls intestinal distension-induced immune activation in Caenorhabditis elegans via Wnt/β-catenin signaling Annesha Ghosh , Jogender Singh bioRxiv 2025.11.25.690479; doi: https://doi.org/10.1101/2025.11.25.690479 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Immunology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17691) Bioengineering (13892) Bioinformatics (41937) Biophysics (21452) Cancer Biology (18588) Cell Biology (25504) Clinical Trials (138) Developmental Biology (13378) Ecology (19899) Epidemiology (2067) Evolutionary Biology (24320) Genetics (15609) Genomics (22506) Immunology (17736) Microbiology (40394) Molecular Biology (17181) Neuroscience (88605) Paleontology (666) Pathology (2832) Pharmacology and Toxicology (4824) Physiology (7641) Plant Biology (15156) Scientific Communication and Education (2045) Synthetic Biology (4294) Systems Biology (9825) Zoology (2271)