Non-canonical inflammasome drives intrinsic anti-microbial responses in human Natural Killer cells

Human NK cells engage cell-intrinsic non-canonical inflammasome function | 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 Human NK cells engage cell-intrinsic non-canonical inflammasome function View ORCID Profile Nathan G.F. Leborgne , View ORCID Profile Jadie Acklam , Alexander J. Hogg , Zarema Albakova , Karen G. Hogg , View ORCID Profile Kim Samirah Robinson , Grant Calder , Arnel Villamin Jr , View ORCID Profile Callum Robson , View ORCID Profile Freya Pealing , Matthew A. Care , Monika Gonka , James M. Fox , View ORCID Profile James Iremonger , View ORCID Profile Frances E. Pearson , View ORCID Profile Max E. Noble , View ORCID Profile Alison M. Layton , View ORCID Profile James P. Hewitson , View ORCID Profile David G. Kent , View ORCID Profile William Grey , View ORCID Profile Jillian L. Barlow , View ORCID Profile Dave Boucher doi: https://doi.org/10.1101/2025.10.20.682800 Nathan G.F. Leborgne 1 York Biomedical Research Institute, University of York, Biology Department , Wentworth Way, York, YO10 5DD 3 Center for Blood Research Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nathan G.F. Leborgne Jadie Acklam 1 York Biomedical Research Institute, University of York, Biology Department , Wentworth Way, York, YO10 5DD 3 Center for Blood Research Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jadie Acklam Alexander J. Hogg 1 York Biomedical Research Institute, University of York, Biology Department , Wentworth Way, York, YO10 5DD 3 Center for Blood Research Find this author on Google Scholar Find this author on PubMed Search for this author on this site Zarema Albakova 1 York Biomedical Research Institute, University of York, Biology Department , Wentworth Way, York, YO10 5DD 3 Center for Blood Research Find this author on Google Scholar Find this author on PubMed Search for this author on this site Karen G. Hogg 1 York Biomedical Research Institute, University of York, Biology Department , Wentworth Way, York, YO10 5DD Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kim Samirah Robinson 1 York Biomedical Research Institute, University of York, Biology Department , Wentworth Way, York, YO10 5DD 2 Hull York Medical School, University of York , York, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kim Samirah Robinson Grant Calder 1 York Biomedical Research Institute, University of York, Biology Department , Wentworth Way, York, YO10 5DD Find this author on Google Scholar Find this author on PubMed Search for this author on this site Arnel Villamin Jr 1 York Biomedical Research Institute, University of York, Biology Department , Wentworth Way, York, YO10 5DD 3 Center for Blood Research Find this author on Google Scholar Find this author on PubMed Search for this author on this site Callum Robson 1 York Biomedical Research Institute, University of York, Biology Department , Wentworth Way, York, YO10 5DD 3 Center for Blood Research Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Callum Robson Freya Pealing 1 York Biomedical Research Institute, University of York, Biology Department , Wentworth Way, York, YO10 5DD 3 Center for Blood Research Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Freya Pealing Matthew A. Care 1 York Biomedical Research Institute, University of York, Biology Department , Wentworth Way, York, YO10 5DD 3 Center for Blood Research Find this author on Google Scholar Find this author on PubMed Search for this author on this site Monika Gonka 1 York Biomedical Research Institute, University of York, Biology Department , Wentworth Way, York, YO10 5DD 3 Center for Blood Research Find this author on Google Scholar Find this author on PubMed Search for this author on this site James M. Fox 1 York Biomedical Research Institute, University of York, Biology Department , Wentworth Way, York, YO10 5DD Find this author on Google Scholar Find this author on PubMed Search for this author on this site James Iremonger 4 VISFO, The Old Flour Mill , Mill Royd Street, Brighouse, West Yorkshire, HD6 1EY Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for James Iremonger Frances E. Pearson 4 VISFO, The Old Flour Mill , Mill Royd Street, Brighouse, West Yorkshire, HD6 1EY Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Frances E. Pearson Max E. Noble 4 VISFO, The Old Flour Mill , Mill Royd Street, Brighouse, West Yorkshire, HD6 1EY Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Max E. Noble Alison M. Layton 2 Hull York Medical School, University of York , York, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Alison M. Layton James P. Hewitson 1 York Biomedical Research Institute, University of York, Biology Department , Wentworth Way, York, YO10 5DD 3 Center for Blood Research Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for James P. Hewitson David G. Kent 1 York Biomedical Research Institute, University of York, Biology Department , Wentworth Way, York, YO10 5DD 3 Center for Blood Research Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for David G. Kent William Grey 1 York Biomedical Research Institute, University of York, Biology Department , Wentworth Way, York, YO10 5DD 3 Center for Blood Research Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for William Grey Jillian L. Barlow 1 York Biomedical Research Institute, University of York, Biology Department , Wentworth Way, York, YO10 5DD 3 Center for Blood Research Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jillian L. Barlow Dave Boucher 1 York Biomedical Research Institute, University of York, Biology Department , Wentworth Way, York, YO10 5DD 3 Center for Blood Research Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Dave Boucher For correspondence: dave.boucher{at}york.ac.uk Abstract Full Text Info/History Metrics Preview PDF Abstract Inflammasome pathways are critical for detecting pathogens and initiating inflammatory responses, yet how individual cell types configure these signalling platforms remains poorly understood. Here, we demonstrate that human Natural Killer cells possess distinct inflammasome machinery, lacking canonical components and innate sensors (NLRP3, NLRC4, TLR4) but harbour a functional non-canonical inflammasome (NCI). Using Salmonella enterica Typhimurium infection of primary human NK cells, we show that cytosolic bacterial access triggers caspase-4-dependent pyroptosis and IL-18 secretion, without canonical inflammasome activation or IL-1β production. Strikingly, murine NK cells express inflammasome transcripts but lack functional caspase-11 protein and do not undergo pyroptosis upon infection, revealing species-specific post-transcriptional regulation. We further identify IL-12, but not IFNγ, as a priming signal for NCI components in human NK cells, a functional distinction from macrophages and epithelial cells. Together, these findings reveal that human NK cells have evolved a specialised inflammasome with unique regulatory inputs, establishing these cells as autonomous sensors of intracellular Gram-negative infection. Introduction Innate immune responses are controlled by cellular pathways that coordinate immunity to rapidly eliminate pathogens and restore homeostasis ( 1 ). Inflammasomes are critical signalling platforms controlling responses to sterile and infectious agents in immune and epithelial cells ( 2 , 3 ). The inflammasome pathway senses triggers using pattern-recognition receptors (PRRs), which include members of the Nod-like Receptor family (Nod-like receptor protein-1, NLRP1, and −3, NLRP3) and the NLR family CARD domain-containing protein 4 (NLRC4) ( 2 ). Unlike the canonical inflammasome, which activates caspase-1 upon detection of bacterial ligands ( e.g., flagellin), and self-derived danger signals ( e.g., ATP, nucleic acids), the non-canonical inflammasome (NCI) activates caspase-4 and caspase-5 (humans) and caspase-11 (mice) upon recognition of cytosolic bacterial lipopolysaccharides (LPS) ( 4 – 6 ) or from intracellular delivery of outer membrane vesicles ( 7 ). In non-phagocytic cells, such as epithelial cells, LPS is presented to caspase-4 through interferon-inducible proteins called guanylate-binding proteins (GBPs) ( 8 – 12 ). GBP1 is a direct LPS sensor and is involved in NCI activation in human cells upon Salmonella infection ( 9 , 12 ). NCI signalling leads to the maturation and secretion of pro-inflammatory cytokines ( 13 ) and to pyroptosis, a form of pro-inflammatory cell death ( 14 ). Pyroptosis is driven by the caspase-mediated cleavage of the pore-forming protein Gasdermin D (GSDMD) ( 14 – 16 ), which activates a Ninjurin-1 (NINJ1)-dependent plasma membrane rupture ( 17 ), and is critical to control infections, limiting their spread and mobilising further immune cells for pathogen clearance ( 18 ). Pyroptosis mediates the release of antigens, which support adaptive responses ( 19 ). Additionally, GSDMD pores mediate potassium influx, which activates the NLRP3 canonical inflammasome downstream of caspase-4/11 ( 20 – 22 ). Inflammasome activation in macrophages and epithelial cells induces the maturation and secretion of interleukin-18 (IL-18) and IL-1β, which are released through the GSDMD pores ( 23 , 24 ). These cytokines generate antimicrobial responses in myeloid, lymphoid, and epithelial cells ( 25 ). Discovered in the 1970s, NK cells are part of the now-expanded family of innate lymphoid cells (ILCs), including ILC1s, ILC2s, ILC3s, and lymphoid tissue-inducer cells. They play key roles in viral and tumour responses and are increasingly studied ex vivo as part of novel immunotherapy against cancer ( 26 , 27 ). Functionally, these cells are broadly divided into cytotoxic NK cells (release of perforin, granzyme and defined as CD56 dim CD16 + ) and immunoregulatory NK cells (IL-12- and IL-18-dependent release of proinflammatory cytokines, IFNγ, CCL3, GM-CSF and defined as CD56 high CD16 low ) ( 28 , 29 ). Progress has been made in recent years on the contribution of NK cells to early responses during microbial infection. Several studies have shown that NK cells indirectly respond to bacterial infection via IL-12 and IL-18 secreted by accessory cells such as macrophages and dendritic cells, leading to IFN-γ production. ( 30 – 32 ). Human NK cells can also directly recognise bacterial PAMPs, such as outer membrane protein A (OmpA), flagellin, and bacillus Calmette-Guérin (BCG), via TLR2, TLR5, or NKp44, respectively, triggering NK proliferation, IFN-γ production, and α-defensin release without requiring accessory cells ( 33 , 34 ). However, our knowledge of direct bacterial recognition by NK cells remains limited. Inflammasomes influence adaptive responses in NK, B, and T cells through cytokine-driven and pyroptosis-mediated antigen release from myeloid and epithelial cells ( 19 ). As such, inflammasomes are an important part of strong immune responses to vaccines and long-term defence against pathogens ( 35 , 36 ). Recently, cell-intrinsic inflammasome-dependent pyroptosis was demonstrated in naive T cells ( 37 , 38 ). Moreover, it is now established that inflammasome derived-IL-18 can activate NK cells and promote the expression of interferon-γ (IFNγ) and cytotoxic effectors ( 39 ). Thus, NK cells are currently viewed as a downstream effector in the inflammasome pathway. To date, inflammasome functions have mainly been studied in myeloid and epithelial cells where Gram-negative bacteria, such as Salmonella , are detected through exposure to a range of Pathogen-Associated Molecular Patterns (PAMPs), including type III secretion system components, flagellin and LPS ( 40 , 41 ). Whether NK cells possess effector and cell-intrinsic inflammasome activity has not been investigated. Here, we investigate how inflammasomes shape NK cell behaviours during infection with the Gram-negative bacteria Salmonella enterica Typhimurium. Using primary human NK cells isolated from blood, we identified a unique inflammasome signature that drives NCI activation but not canonical inflammasome activation. This signature was further enhanced upon IL-12 priming and was absent in mouse NK cells. Our findings highlight the unique regulation and roles of the inflammasome in human NK cells and describe direct intracellular sensing functions during gram-negative infections. Results Human NK cells possess a functional non-canonical inflammasome We took advantage of a substantial dataset of healthy human blood NK cells to profile the expression of inflammasome pathway components in these cells where gene and protein expression data in human blood NK cells from 13 healthy donors (36,270 high-quality filtered cells) were grouped functionally as, NK1 (CD56 dim CD16 high CD3 - CD127 - CD57 - CD2 - , further divided as subtype A, B, and C), NK2 (CD56 high CD16 - CD3 - CD127 + CD57 - CD2 + ), NK3 (CD56 dim CD16 high CD3 - CD127 - CD57 + CD2 - ), and NKint ( 42 ). As expected, GZMB, GZMA, and PRF1 showed higher mean expression in NK1 and NK3 cells ( Figure 1A ). Strikingly, canonical inflammasome components ( NLRP3 , NLRC4 ), the pro-inflammatory cytokine IL-1β, and the receptor detecting extracellular LPS ( TLR4 ), were not expressed ( 40 , 43 , 44 ). CASP1 and GSDMD were detected in NK1 and NK3, but not in NK2 subtype. However, we were able to observe increased mean expression of NCI components, including CASP4 , GBP1 , and GBP2 , in NK1 and NK3 cells, compared to NK2 cells. Other inflammasome-related genes were further restricted, with GBP4 expressed only in NK1B and NK2, and PYCARD in NK1B and NK1C ( Figure 1A ). To exclude the possibility that this was blood-specific, we examined gene expression in human foetal thymic (tissue-resident) NK cells and confirmed that NLRP3 , NLRC4 , and TLR4 were not expressed. We identified a CASP4 + GBP2 + NK cell population, in which CASP1, GSDMD , and PRF1 were upregulated in cycling cells, as compared to non-cycling cells ( Supp. Figure 1A ). This suggests the ability of specific NK cell populations to respond to intracellular Gram-negative infections in different tissues. Download figure Open in new tab Figure 1. Human blood NK cells express components of the non-canonical inflammasome. ( A ) scRNA-seq data of inflammasome components in subsets of human peripheral blood NK cells showing the relative abundance (standard-scaled) and percentage of cells expressing inflammasome components, (n=19). ( B ) Quantitative PCR analysis of non-canonical inflammasome components in unexpanded human peripheral blood NK cells, n = 4 donors ( C ) Quantitative PCR analysis of TLR4 and NLRP3 in unexpanded peripheral blood NK cells and neutrophils from four matched donors. ( D ) Western blot analysis of expanded human peripheral blood NK cells and macrophages from 2 matched donors compared to loading controls of αTubulin and H3. * 1 and * 2 indicate NK cell-specific protein isoforms of GBP1 and GBP5, respectively. Western blot is representative of 4 blood donors. To confirm the findings from these datasets, we next isolated human peripheral blood NK cells and neutrophils, paired from the same donor and performed quantitative PCR for inflammasome-related genes. Alongside GZMA expression, NK cells also showed low expression of CASP1 , CASP4 , and GSDMD , with greater expression of GBP2 ( Figure 1B ). This was compared to expression of these genes in neutrophils, but whilst neutrophils showed gene expression for TLR4 and NLRP3, NK cells did not ( Figure 1C and Supp. Figure 1B ). Analysis of human blood NK cells, using macrophages as a comparison, by western blot also indicated lower protein expression of caspase-1, caspase-4, and GBP1 in NK cells, and similar levels of GBP2 and GBP5. We identified NK-specific proteoforms of GBP1 and GBP5 ( Figure 1D ), also previously reported in HUVEC cells ( 45 ). Overall, these data reveal that human NK cells operate a minimalist inflammasome system, lacking the canonical machinery (NLRP3, NLRC4, TLR4) that defines macrophage responses to Salmonella . Instead, this suggests they rely exclusively on the NCI pathway for bacterial detection. To test the activation of the NCI in the context of infection, we next expanded negatively-selected human peripheral blood NK cells in vitro for 14 days, resulting in a highly enriched population containing all NK cell subpopulations, as compared to NK cells directly isolated from fresh blood ( Supp. Figure 2 ). We infected the NK cells with Salmonella enterica Typhimurium, strain SL1344 and LT2, which infect cell types such as macrophages and epithelial cells, activating canonical (NLRP3, NLRC4; caspase-1) and non-canonical (GBPs; caspase-4,-5) inflammasomes. Infection resulted in a statistically significant increase in lytic cell death, as measured by LDH release ( Figure 2A ), compared to uninfected cells. This cell death was decreased with the addition of the caspase-1/-4 inhibitor, VX-765 ( 46 ) ( Figure 2A ) and upon infection with SalΔorgA, a Salmonella mutant lacking the functional SPI1 injectisome required for the invasion of non-phagocytic cells ( 47 ) ( Figure 2B ). Indeed, Salmonella enter the cytosol ( 9 , 10 ) by using a subset of effectors ( 48 ), including the needle injectisome protein, OrgA, where its deletion strongly reduces Salmonella invasion of non-phagocytic cells ( 40 ). Thus, our data not only suggest a caspase-1/-4-dependent cell death mechanism, but also that NK cell lytic death requires intracellular Salmonella invasion. Download figure Open in new tab Figure 2. Salmonella Typhimurium infection induces cell death and IL-18 production in human blood NK cells. Expanded human peripheral blood NK cells were infected with S. Typhimurium or S. Typhimurium ΔorgA , and with or without VX-765. At 5 hrs post-infection, cells supernatants were assessed by ( A and B ) LDH assay for cell death, and ( D and E ) ELISA for IL-1beta and IL-18 production, n = 6 - 9 donors across 2 - 3 independent experiments. ( C ) Quantitative PCR analysis of IL1B following priming with LPS (for 3 hrs) and IFNgamma (for 16 hrs). (F) At 5 hrs post-infection, caspase-1 cleavage was characterised by western blot. * 1 indicate a p33 cleavage product of active caspase-1. In ( C ), ( D ) and ( F ) NK cells were compared to neutrophils and macrophages from matching donors. Data in (A, B and E) were analysed by one-way ANOVA with Dunnett’s multiple-comparison test. p values are displayed and p<0.05 was considered statistically significant. Central to the activation of the NCI is the maturation and secretion of pro-inflammatory cytokines, such as IL-1ꞵ and IL-18. Our data confirm that priming with LPS or IFNγ, or infection with S. Typhimurium induces IL-1ꞵ gene expression ( Figure 2C ) and its secretion ( Figure 2D ) in human neutrophils and macrophages. However, IL-1ꞵ was not detectable in NK cells under the same conditions in matched donors. Strikingly, S. Typhimurium infection of NK cells induced a statistically significant increase in IL-18 production, which was caspase-1/-4-dependent ( Figure 2E ). As Salmonella has been shown to activate multiple inflammasomes during infection, we investigated the pathways involved in such a response. Interestingly, we could not detect the cleaved caspase-1 fragments (p20/p10) upon Salmonella infection ( Figure 2F ). Additionally, Nigericin treatment induced an NLRP3-independent cell death in NK cells, as shown with the addition of its inhibitor, MCC950 ( 49 ) ( Supp. Figure 3A ). NLRP3-dependent caspase-1 cleavage was also not observed in NK cells ( Supp. Figure 3B ). Moreover, Val-boroPro (VbP, NLRP1 inflammasome activator, ( 50 )) did not lead to GSDMD cleavage in NK cells but was observed in a keratinocyte cell line (N/TERT) ( Supp. Figure 3C - D ) Our data suggest a distinct mechanism of Salmonella sensing in NK cells that does not involve canonical inflammasome signalling, as evidenced by the undetectable gene expression level of NLRC4 and NLRP3 and the absence of response to NLRP3 and NLRP1 triggers. Instead, NK cells utilise a pathway that leads to inflammatory caspase-dependent pyroptosis, and IL-18 secretion, but not IL-1ꞵ. In addition, the absence of TLR4 expression in NK cells, which has been shown to be important for priming the canonical inflammasome pathway, may explain NK cell non responsiveness to NLRP3 triggers and the absence of IL-1β. This differs from the classical activation of these pathways in macrophages where NLRC4 and NLRP3 are major contributors to the innate detection of Salmonella ( 43 , 51 , 52 ). The inflammatory caspase-dependent secretion of IL-18 is consistent with previous literature showing its cleavage by caspase-4 ( 9 , 10 , 53 – 55 ) and suggests that NK cells use an autocrine mechanism to potentiate their activation during bacterial infection. Salmonella Typhimurium accesses the cytosol in human NK cells Gram-negative bacteria can activate the NCI through a range of mechanisms, including direct invasion of the host cell cytoplasm or secretion of outer-membrane vesicles ( 7 ). Upon infection of epithelial and myeloid cells, Salmonella localises and replicates in specialised vacuoles, where it is protected from immune detection. However, as established in gut epithelial cells, Salmonella can escape these vacuoles, allowing cytosolic hyper-replication but also its detection by cytoplasmic sensors ( 56 ). Therefore, we next sought to identify whether Salmonella invade NK cells by infecting them with S. Typhimurium LT2-TdTomato. Using flow cytometry, we showed that ∼40% of NK cells were TdTomato + ( Figure 3A ), suggesting an efficient invasion of NK cells. Moreover, fluorescence microscopy on NK cells indicates the presence of Tdtomato + S. Typhimurium in both CD56 + vacuole-like structures and in the cytosol ( Figure 3B ). We observed a range of NK cell- Salmonella interactions as shown in Supp. Figure 4A . However, we could not fully differentiate between cytosolic and vacuole-associated Salmonella . To confirm that Salmonella could access the cytosol, we used Salmonella expressing a glucose-6-phosphate-dependent cytosolic eGFP-reporter ( 9 ). As glucose-6-phosphate is only accessible in the cytosol, only cytosolic Salmonella will express the fluorescent GFP reporter. At 5hrs post-infection, a significant percentage of NK cells (10-15%), were eGFP + ( Figure 3C ), similar to levels in epithelial cells ( 9 ), suggesting that Salmonella can invade the cytosol of a proportion of NK cells. This correlates with NCI activation, as shown by western blot for the active caspase-4 p32 cleavage product ( 13 ) and the reduction in full-length GSDMD in NK cells in response to S. Typhimurium ( Figure 3D ). In comparison, infection with a Gram-positive bacteria, Staphylococcus aureus, which is known to activate the canonical inflammasome ( 57 ), did not induce caspase-4 cleavage or reduce full-length GSDMD protein. Download figure Open in new tab Figure 3. Salmonella Typhimurium accesses the cytosol and persists in human blood NK cells. Expanded human peripheral blood NK cells were infected with S. Typhimurium-TdTomato. ( A ) At 5 hrs post-infection NK cells were subjected to analysis by flow cytometry or ( B ), stained on fibronectin coated slides with anti-CD56 and DAPI and visualised by fluorescence microscopy (white bar indicates 5 µm). ( C ) Alternatively, these NK cells were infected with S. Typhimurium, stably expressing a cytosolic-induced eGFP reporter and analysed by flow cytometry. ( D ) Representative western blot analysis of expanded human peripheral blood NK cells infected with either S. Typhimurium or Staphylococcus aureus and with or without VX-765 for 5 hrs compared to loading controls of H3. * 1 indicates a p32 cleavage product of active caspase-4 ( E ) Caspase-1 and −4 activation was measured by flow cytometry using the inflammatory caspase probe FLICA660. ( F ) Percentage of S. Typhimurium - TdTomato in NK cells was measured at days 1, 2, 5, 7, and 14 post-infection. Data from 2 independent experiments, n = 4 - 6 donors. Data in (A, C and E) were analysed by paired t tests. Data in (F) were analysed by one-way ANOVA with Dunnett’s multiple-comparison test. p values are displayed and p<0.05 was considered statistically significant. To confirm inflammatory caspase activation, we used the inflammatory caspase activity-based probe FLICA660 and showed that ∼5% of total NK cells were FLICA + upon Salmonella infection, compared to ∼1% in uninfected cells, validating caspase-4 activation ( Figure 3E ). In alignment with our previous microscopy and CellView TM analysis indicates a range of activation and infection states, with the existence of FLICA + Salmonella- TdTomato - ,FLICA - Salmonella- TdTomato + ,and FLICA + Salmonella- TdTomato + NK cell populations ( Supp. Figure 4C ). To test the potential of NK cells to act as an infectious reservoir for Salmonella, we tracked S. Typhimurium LT2-TdTomato infection over 14 days. After 5 hrs and 24 hrs, initial infection persists in a significant subset of NK cells, ∼25 - 32%. Between day 1 and day 14 post-infection, this proportion decreases from ∼30-20% to 10-5% ( Figure 3F ), indicating a small NK population in which Salmonella persists. Thus, our study suggests heterogeneity in infective states in NK cells, with persistence, potentially in the vacuole, and access to cytosolic niches. Further study will be required to determine whether these differences in persistence and access are associated with specific NK cell subsets. Overall, these mechanisms converge on Salmonella’s ability to create an intracellular niche in NK cells. Mouse NK cells do not activate the non-canonical inflammasome We next asked whether murine NK cells displayed similar features, using published RNA-seq datasets ( 58 ), where gene expression changes were measured in innate-like lymphoid populations, as compared to macrophages, following LPS priming. Our analyses showed that, in contrast to humans, mouse NK cells express Tlr4 . Other non-canonical inflammasome-associated genes, such as Gbp2 , Casp4 , and Gsdmd , were expressed, similarly to human NK cells and other mouse innate lymphoid populations ( Supp. Figure 5A ) ( 59 ). These results were supported by qPCR analysis of murine wild-type splenic NK cells ( Supp. Figure 5B ). In naive NK cells, we detected transcripts for Casp1 , Casp4 , Gbp2 , Gsdmd , and Ninj1 , which was comparable to expression seen in macrophages. Macrophages were additionally found to express transcripts for Il18 , Il1b , and Il1a (also upregulated with LPS priming), but these transcripts were absent in NK cells ( Supp. Figure 5C ). In contrast to human NK cells, Salmonella infection did not induce cell death in murine NK cells ( Supp. Figure 5D ). Furthermore, we did not detect protein expression of full-length caspase-11 (the murine orthologue of human caspase-4) in NK cells compared with matched neutrophils ( Supp. Figure 5F ). Unlike human NK cells, which express inflammasome components at the gene level, murine NK cells do not express a functional caspase-11 and do not activate the NCI in response to Salmonella infection. In contrast, human NK cells do not express TLR4 but instead use the non-canonical inflammasome to detect intracellular bacterial infection. These data highlight a critical difference between human and murine inflammasomes at the cellular level. The non-canonical inflammasome in human NK cells is primed by IL-12 IFNγ priming in non-phagocytic and phagocytic cells has previously been identified to impact NCI activation ( 9 – 11 ). To investigate the effect of IFNγ on NK cells, we primed resting NK cells in vitro for 16 hrs with recombinant human IFNγ or with the NK-activating cytokine IL-12. This cytokine is also important for the human response to Salmonella, as individuals with a deficiency in IL-12 signalling are especially sensitive to Salmonella infection ( 60 – 63 ). Surprisingly, IFNγ did not induce expression of any NCI-related genes ( CASP4 , CASP1 , GBP1 , GBP2 , GBP5 , GSDMD , and NINJ1 ), as compared to unstimulated cells ( Figure 4A - C ). In contrast, IL-12 stimulation resulted in statistically significant increases in GBP1 and GSDMD . Although this did not increase NK cell death, as measured by LDH assay at 5 hrs post-infection ( Figure 4D ), immunoblotting showed increased cleavage of caspase-4 and GSDMD ( Figure 4E ). Ninj1 levels remained unchanged. IL-12 priming significantly increased IFNγ and CCL3 secretion by NK cells, but did not further enhance this response upon S. Typhimurium infection ( Figure 4F and G ). Importantly, the Salmonella -dependent IL-18 secretion previously observed ( Figure 2E ) was increased upon IL-12, but not IFNγ priming, indicating a potentiation of NCI activity in human NK cells ( Figure 4H ). Download figure Open in new tab Figure 4. IL-12 but not IFN γ -mediated activation of human blood NK cells leads to upregulation of non-canonical inflammasome components. Expanded human peripheral blood NK cells were treated with and without rhIL-12 or rhIFN γ for 16 hrs. Quantitative PCR was used to assess expression of ( A ) CASP4 and CASP1 , ( B ) GBP1 and GBP2 , and ( C ) GBP5 , GSDMD , and NINJ1 . Post-treatment, NK cells were additionally infected with S. Typhimurium alone, or in combination with VX-765. Supernatant was taken at 5 hrs post-infection in order to assess ( D ) cell death by LDH release and ( F ) IFN γ , ( G ) CCL3, and ( H ) IL-18 production by ELISA. ( E ) Representative western blot analysis of expanded and primed human NK cells infected with either S. Typhimurium alone or in combination with VX-765 for 5 hrs compared to loading controls of H3. * 1 indicates a p32 cleavage product of caspase-4. * 2 indicates a p30 cleavage product of GSDMD. Data from 2 - 3 independent experiments, n = 3 - 8 donors. Data in (A, B C and H) were analysed by one-way ANOVA with Dunnett’s multiple-comparison test. p values are displayed and p<0.05 was considered statistically significant. Discussion Inflammasome pathways have been extensively characterised in macrophages and epithelial cells, yet whether lymphocytes engage these signalling platforms remains unclear. Here, we demonstrate that human NK cells possess a functional non-canonical inflammasome (NCI) that enables cell-intrinsic detection of cytosolic Gram-negative bacteria. This finding repositions NK cells from passive recipients of inflammasome-derived cytokines to autonomous and active participants in anti-bacterial immunity. The NK cell NCI displays a species-specific protein profile and is primed by IL-12 rather than IFNγ. Human NK cells use a limited inflammasome system. They lack the canonical sensors NLRP3 and NLRC4, do not express TLR4, and fail to produce IL-1β upon bacterial infection. Yet they retain functional caspase-4, guanylate-binding proteins (GBP1, GBP2, GBP5), and GSDMD, enabling detection of cytosolic LPS and execution of pyroptosis. The NK NCI produces a specific output profile with IL-18 secretion and inflammatory cell death without IL-1β production, highlighting fundamental differences from macrophage responses to the same pathogen. However, the prevalence of such mechanisms across lymphocyte populations remains to be fully investigated. Previous work suggested that T cell subsets activate the canonical CARD8 inflammasome upon treatment with chemotherapeutics, triggering cell death without cytokine production ( 38 ). Strikingly, murine NK cells, despite expressing inflammasome component transcripts at levels comparable to macrophages, lack functional caspase-11 protein and do not undergo pyroptosis upon Salmonella infection. This transcript-protein discordance suggests post-transcriptional or translational regulation that differs between species. This species difference has practical implications: mouse models have been used extensively to study NK cell responses to Salmonella , including in cancer immunotherapy contexts ( 64 ). Our findings urge caution in extrapolating from these studies to human NK cell biology and suggest that human-specific experimental systems are essential for understanding the contributions of inflammasomes to human NK cell function. The selective responsiveness of human NK cells to IL-12 rather than IFNγ priming represents an unexpected regulatory distinction from other cell types. IFNγ, signalling through JAK-STAT1 ( 65 ), is the canonical priming agent for non-canonical inflammasome components in macrophages and epithelial cells ( 9 – 11 ). In NK cells, however, IFNγ had no effect on caspase-4, GBPs, or GSDMD expression. Recent transcriptomic analyses support this interpretation, demonstrating that NK cells show minimal response to IFNγ compared with T and B cells ( 66 ). IL-12, via STAT4 signalling ( 61 ), significantly upregulated these components and enhanced infection-induced IL-18 secretion. This suggests that the promoters of key NCI genes may harbour STAT4-responsive elements that are preferentially active in NK cells. The IL-12 dependency also aligns with clinical observations that individuals with defects in IL-12 signalling are particularly susceptible to Salmonella infection, raising the possibility that impaired NK cell inflammasome priming contributes to this phenotype ( 60 , 63 ). Our observation that Salmonella infection induces IL-18 secretion from NK cells indicates a potential for autocrine amplification of NK cell responses. IL-18 receptor expression varies across NK cell subsets, with recent single-cell analyses indicating the highest expression in NK2 (CD56high, CD16low) populations. If NCI-competent NK cells (predominantly NK1 and NK3, based on our expression data) secrete IL-18 upon infection, this could preferentially activate nearby NK2 cells, thereby amplifying the local immune response across tissues. Such subset-specific interactions deserve investigation, as they may shape NK cell responses to bacterial infection. More broadly, the heterogeneity of inflammasome component expression across NK cell subsets with PYCARD/ASC expression being restricted to NK1B and NK1C, highlighting that different NK populations may have distinct capacities for inflammasome activation, a complexity that functional studies have yet to address. Our finding that a subset of NK cells remains infected with Salmonella over 14 days raises questions about bacterial persistence and potential immune evasion. Salmonella persistence in macrophage vacuoles is well documented and enables antibiotic evasion and cellular reprogramming ( 67 ). Whether similar mechanisms operate in NK cells and whether persistent infection affects NK cell function or memory-like phenotypes remain to be determined. Our data indicate that, at least during the initial infection, Salmonella can invade NK cell cytosol and activate the NCI. The absence of NLRP3 and NLRC4 in these cells suggests that cytosolic LPS is likely the primary PAMP sensed by NK cells upon Salmonella infection. However, we cannot exclude other inflammatory conditions that may prime caspase-1 activation in these cells (e.g., other pathogens). In conclusion, we have identified a specialised non-canonical inflammasome pathway in human NK cells that operates independently of canonical inflammasome components and is regulated by IL-12 rather than IFNγ. This pathway enables NK cells to directly sense intracellular Gram-negative bacteria, adding to the network of innate immune cells capable of autonomous pathogen detection. The species-specific nature of this response highlights the importance of human experimental systems for understanding NK cell inflammasome biology. Future work should address how this pathway shapes tissue responses to infection and whether it contributes to diseases such as sepsis, in which reduced NK cell numbers are observed and are associated with poor patient outcomes ( 68 ). Material and Methods Study approval Work with human primary cells was performed under approval by the University of York Biology Ethic Committee (DB202111). Mice were bred and maintained in specific pathogen-free conditions in the University of York, Biological Services Facility. Surplus C57 BL/6 mouse tissue was used with the approval of the local AWERB committee, and under license with the UK Home Office. Human immune cell isolation and expansion Human NK cells, PBMCs and neutrophils were isolated from fresh blood in EDTA-coated tubes (BD Bioscience) or from blood cones (apheresis product, NHS) collected from anonymous healthy donors. Neutrophils (only from fresh blood) and NK cells were isolated from whole blood by negative selection using the EasySep Direct Neutrophil isolation kit (StemCell Technology, 19666) and EasySep Direct human NK isolation kit (StemCell Technology, 19665). To ensure a maximal NK purity, isolated NK cells were expanded for 14 days with Immunocult NK Cell Expansion Kit (StemCell Technology, 100-0711), according to manufacturer instructions. After 14 days, dead cells from NK culture were removed using EasySep Dead Cell Removal (Annexin V) Kit. PBMCs were isolated from whole blood using Lymphocyte Separation Medium (Lonza, BESP1109E). Red blood cells were lysed using ACK lysis buffer (Gibco, A1049201) for 5 min at room temperature. PBMCs were differentiated into macrophages with 50 ng/ml rhM-CSF (Proteintech, HZ-1192) in RPMI, 10% FBS, 1% penicillin/streptomycin, and 1% glutamine for 7 days at 37°C, 5% CO 2 . Murine immune cell isolation and priming C57BL/6J mouse spleens were collected in PBS with 2% FBS. Single cell suspensions were obtained by passing tissue through a 70µm cell strainer (Corning, 352350). Red blood cells were lysed using ammonium chloride solution for 5 minutes at room temperature. Neutrophils were isolated using an EasySep Direct Neutrophil isolation kit (StemCell Technology, 19855). NK cells (LiveCD45 + CD3CD4CD8CD19CD11bCD11cFcεR1Gr1 (lineage) - NK1.1 + ) and macrophages (LiveCD45 + CD3 - CD11b high ) were stained with flow cytometry antibodies (see Supplementary Table 1 ) and isolated by FACS using a MoFlo Astrios (Beckman Coulter, Summit software). Mouse NK cells and macrophages were primed with 100 ng/mL of LPS EK (Invivogen) for 3hrs or used directly for RNA extraction after isolation. Bacterial infection, cell priming, and cell death analysis NK cells, neutrophils, and differentiated macrophages were plated in 12-well plates in Opti-MEM (GIBCO) at 1×10 6 cells/ml, 2.5×10 6 cells/m and 0.5×10 6 cells/ml, respectively. Cells were infected with either Salmonella enterica Serovar Typhimurium SL1344 or LT2, or S. entericaΔorgA , LT2-TdTomato, LT2-pPuhpT-GFP, or Staphylococcus aureus grown in log phase. Briefly, bacterial strains were cultured in lysogeny broth (LB) medium overnight at 37°C on an orbital shaker. These were subcultured 1:40 and grown for 3h at 37°C to generate log phase cultures. Before infection, bacteria were collected by centrifugation, and washed twice with pre-warmed PBS. Cells were infected at MOI 50 for 1h at 37°C. After 1 hr, gentamicin was added at a final concentration of 100 µg/ml to kill extracellular bacteria and cells were incubated for 4 hrs at 37°C, 5% CO 2 . As indicated, VX-765 (25 µM in DMSO, Sigma-Aldrich, 5.31372) was added 30 mins before inflammasome activation. In some experiments, NK, rested for 6 hours in NK medium without NK cell Expansion Supplement (StemCell Technology, 100-0711) and neutrophils were primed 3 hours with 100 ng/ml ultrapure LPS-EK (Invivogen, tlrl-peklps) or with 25 ng/ml rhIFNγ (Proteintech, HZ-1301) or 100 ng/ml rhIL-12 (Proteintech, HZ-1256) for 16 hours. NLRP3 inflammasome was activated using Nigericin (20 µM in EtOH, Sigma-Aldrich, N7143) for 3 hrs at 37°C. As indicated MCC950 (10 µM in H 2 O, Sigma-Aldrich, PZ0280) were added to the cell culture media 30 min before inflammasome activation. NLRP1 was activated using 3 µM Val-boroPro for 6 hrs at 37°C (Vbp, 5.31465). For cell death measurements lactate dehydrogenase release in the supernatants was measured using a CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, G1780). Absorbance was measured on a plate reader (Infinite M Plex, TECAN) at 490nm with a 650nm background subtraction following manufacturer’s instructions. Quantitative PCR analysis mRNA from primary cells (mouse and human) were isolated using the Arcturus PicoPure Isolation kit (ThermoFisher, KIT0204) and cDNA was generated using the SuperScript IV First Strand kit (ThermoFischer, 18091050), using manufacturer’s instructions. qPCR was performed using TaqMan probes (Life Technologies, Supplementary Table 1 ) on an AriaMx Real-time PCR system (Agilent). Immunoblotting Cell extracts were lysed with cell sample buffer (2% SDS, 66mM Tris pH7.6 reconstituted in NuPage 1X LDS buffer (Novex, P5878547) and run on 10% or 4 - 20% acrylamide gels (BioRad) with methanol-chloroform precipitated cell supernatant mixes ( 69 ). Gels were transferred on 45 µM nitrocellulose membrane using the TurboBlot transfer system (BioRad), and blocked in 5% skimmed milk TBST. Samples were then incubated overnight at room temperature in the primary antibody at 4°C and for 2 hrs the following day in the secondary antibody ( Supplementary Table 1 ). Membrane was imaged using the Immobilon Forte ECL Substrate (VWR, WBLUF) using a ThermoFisher iBright 1500. Flow cytometry Single cell suspensions were incubated with anti-CD16/CD32 antibodies (Biolegend, Clone 93) followed by fluorochrome-conjugated antibodies as detailed in Supplementary Table 1 . All samples were co-stained with a cell viability dye (Zombie YG581 or Aqua, Biolegend, 423123 and 423102). In some experiments with human blood NK cells after Salmonella infection, and following the addition of gentamicin, YVAD-FMK FLICA660 or FLICA-FAM was added following manufacturer instructions (1:1000, FLICA Caspase-1 kit, BIO-RAD, 9120 and ICT098) and cells were incubated for 1 hr at 37°C. Cells were washed twice with the 1x kit wash buffer. Untreated NK cells stained for FLICA were used as control. Sample acquisition used an LSR Fortessa, CytoFLEX LX (Beckman Coulter), or FACSDiscover™ S8 (BD Biosciences, CellView™ Image) and then visualised using FlowJo (version 10.10) software. For cell sorting, an Astrios (70 µm nozzle, Beckman Coulter, Summit software) was used. Immunofluorescence microscopy 8-well chamber slides (Ibidi, 80821) were coated with 12.5 µg/ml fibronectin for 1 hr, washed with PBS and transferred to 4°C until use. NK cells were seeded directly into chamber slides at 4×10 5 cells/well and immediately infected with log phase Salmonella LT2-TdTomato at MOI 50 for 1h at 37°C, 5% CO 2 . Gentamicin was added directly to the culture medium, and cells were cultured for 4 hrs at 37°C, 5% CO 2 . Cells were subsequently fixed with 4% PFA (in PBS) for 10 min at room temperature and washed with PBS. Cells were blocked with 5% goat serum for 2 hrs, stained with rabbit anti-human NCAM (CD56, Cell Signalling, 99746) overnight, washed with PBS and stained with goat anti-rabbit IgG (H+L), AF488 conjugate (Cell Signalling, 4412) for 1 hr. Cells were washed with PBS and stained with DAPI (2µg/ml) (Biotum, 40043), ready for imaging on Zeiss Elyra 7. Cytokine assays Cytokine secretion in cell culture supernatants was assessed using standard sandwich ELISA, IL-1β (Invitrogen, 88-7261), CCL3 (Invitrogen, 88-7035), IL-18 (R&D systems, DY318-05), GM-CSF (Invitrogen, 88-8337), IL-22 (Invitrogen, 88-7522), according to the manufacturer’s instructions. IFNγ secretion was measured using the Lumit IFNγ immunoassay (Promega, W6040) following the manufacturer’s instructions in a 384 well plate. Luminescence was measured on a plate reader (Infinite M Plex, TECAN). Bioinformatical data analysis and statistics Mouse RNA sequencing data was obtained from the GEO database (GSE143943, ( 49 ). The data was filtered for genes relevant to NCI and the mean expression values log transformed. Heat maps were generated in R Studio using the pheatmap package. Clustering was automatically applied to the gene list. Human scRNAseq datasets were obtained from either the foetal immune cell atlas ( https://developmental.cellatlas.io/# ) or peripheral blood ( https://storage.googleapis.com/haniffalab/meta-nk/PBMC_Noreg.h5ad ) and mined to analyse tissue resident or blood NK cells, respectively. The published data was loaded with scanpy and the inflammasome component genes visualised in a violin or dotplot . The dotplot was grouped by the 6 NK clusters discovered in the original study, and the genes were standard-scaled across these clusters to visualize relative abundance of each gene across cell contexts. Statistical analysis for most experiments was performed using Prism10 (GraphPad, San Diego, CA). Authors contribution N.G.F.L, J.A, A.H, Z.A, K.S.R, M.G, C.R, A.V, J.L.B, M.A.C, F.P. and D.B performed experiments. N.G.F.L, Z.A, J.H, J.L.B and D.B designed experiments, D.B secured funding, supervised, designed and oversaw the project. N.G.F.L, J.A, A.H, K.S.R, K.H, G.C, J.L.B, D.B analysed data. D.B, N.G.F.L, and J.L.B wrote the paper with inputs from all authors. Supplementary Figure Legends Download figure Open in new tab Supplementary Figure 1. Human thymic NK cells express components of the non-canonical inflammasome. ( A ) scRNA-seq data of inflammasome components in CASP4 + GBP2 + and CASP4 - GBP2 - human foetal thymic non-cycling and cycling NK cells showing the median expression and number of cells expressing inflammasome components. ( B ) Quantitative PCR analysis of non-canonical inflammasome components in neutrophils, n = 4 donors, matched with NK cell phenotypes in Fig. 1B . Download figure Open in new tab Supplementary Figure 2. Characterisation of NK cell subpopulations in human blood. Representative density plots of ( A ) PBMCs, ( B ) negatively-selected NK cells and ( C ) 14 days expanded NK cells from human peripheral blood showing typical percentages of CD56 int CD16 + and CD56 high CD16 - . Human peripheral blood from ( C ) before and ( D ) after NK cell negative selection, analysed by flow cytometry to show typical percentages of CD56 int CD16 + and CD56 high CD16 - , and within this NK1, NK2, and NK3 subsets, as defined by Rebuffet et al (2024). Download figure Open in new tab Supplementary Figure 3. NLRP1, NLRP3, and FLICA activation in human blood NK cells. Expanded human peripheral blood NK cells were treated with nigericin (20 µM), with or without MCC950 (10 µM) for 3 hrs. Cells were then assessed for ( A ) the release of LDH for cell death and ( B ) the cleavage of caspase-1 by western blot. ( C ) NK cells or ( D ) N/TERT keratinocytes were treated with Val-boroPro (3µM, VbP) for 6hrs, with and without VX-765, and assessed by western blot. n = 4 - 6 donors. * 1 and * 2 indicate possible NK cell-specific protein isoforms of CASPASE-1. * 3 indicates the p30 cleavage fragment of GSDMD. Data in (A) were analysed by one-way ANOVA with Dunnett’s multiple-comparison test. p values are displayed and p<0.05 was considered statistically significant. Download figure Open in new tab Supplementary Figure 4. Salmonella Typhimurium interacts with and enters NK cells, activating inflammatory caspases. Expanded human peripheral blood NK cells were infected with S. Typhimurium-TdTomato. ( A ) At 5 hrs post-infection NK cells were stained on fibronectin coated slides with anti-CD56 and DAPI and visualised by fluorescence microscopy (white bar indicates 5 µm). Panel shows range of NK cell- Salmonella interactions. Control represents non-infected NK cells stained with anti-CD56 and DAPI. ( B ) Representative images of CD56 + cells were reconstructed, using CellView TM beam-splitting technology, to show human NK cells that were FLICA-FAM + Salmonella- TdTomato - (NK cell 1), FLICA-FAM - Salmonella- TdTomato + (NK cell 2), and FLICA-FAM + Salmonella- TdTomato + (NK cell 3), respectively. Download figure Open in new tab Supplementary Figure 5. Canonical and non-canonical inflammasome components are expressed in mouse NK cells at the gene, but not the protein level. ( A ) RNASeq dataset extracted from flow cytometry sorted immune cells showing a heatmap expression analysis of canonical and non-canonical inflammasome-associated genes ( 58 ). Murine spleen NK cells and CD11b high cells were sorted and cDNA was extracted from either ( B ) naive populations or ( C ) from LPS (100 ng/mL, 3 hrs)-primed cells. Expression of canonical and non-canonical inflammasome-associated genes was assessed by quantitative PCR using Gapdh as a house-keeping gene, n = 4-8, collected across 2 independent experiments. ( D ) Representative western blot analysis of caspase-11 expression in LPS-primed murine spleen NK cells. View this table: View inline View popup Download powerpoint Supplementary Table 1. (A) Quantitative PCR Taqman primer sets for human and mouse targets. (B) Antibodies used in flow cytometry (FC), immunofluorescence (IF), and western blot (WB). Acknowledgement D.B. is supported by an MRC New Investigator Award (MR/Z504221/1), a Springboard Award (Academy of Medical Sciences and Wellcome Trust) and a Royal Society Grant. J.A. was supported by a GenerationResearch MSc Studentship and VISFO. J.L.B., D.G.K, and M.C. are supported by the NIHR Leeds Biomedical Research Centre (BRC) (NIHR203331), and J.L.B and D.G.K. by an MRC National Mouse Genetics Network grant (MC_PC_21043). D.G.K is additionally supported by a CRUK Programme Foundation Award (DCRPGF\100008), an MRC-AMED joint award (MR/V005502/1), and Blood Cancer UK (24014). M.G. was in receipt of a BBSRC White Rose DTP PhD Studentship (BB/T007222/1). W.G. is supported by an MRC Career Development Fellowship (MR/X007146/1), Leukaemia U.K. (2021/JGF/002), an EHA bilateral collaborative award and the Children’s Cancer and Leukaemia Group (CCLGA 2022 13 Grey). K.S.R is supported by the Academy of Medical Sciences Springboard (SBF009\1153), Royal Society (RG\R1\241058) and Hull York Medical School Start-up. The authors thank the Centre for Blood Research (CBR), SUKIT and the InflammaZoom UK networks for helpful discussions and constructive criticism on this research. The authors thank the phlebotomist team at the University of York for their support with collecting fresh blood for experimental purposes and the donors who kindly provided the blood. N/TERT was a kind donation from Prof. James Rheinwald ( 70 ). We would like to thank Prof. Petr Broz (University of Lausanne) and Prof. Marjan van der Woude (University of York) for providing the bacterial strains used in this study. Funder Information Declared Medical Research Council, https://ror.org/03x94j517 , MR/Z504221/1 Footnotes ↵ 6 Senior Author ↵ 7 Lead contact Additional results have been added regarding the murine NK cells to further support our conclusions. We have added additional microscopy images as well of infected NK cells. References 1. ↵ Janeway CA . Approaching the asymptote? Evolution and revolution in immunology . Cold Spring Harb Symp Quant Biol . 1989 ; 54 Pt 1 : 1 – 13 . OpenUrl Abstract / FREE Full Text 2. ↵ Fu J , Schroder K , Wu H . Mechanistic insights from inflammasome structures . Nat Rev Immunol . 2024 July ; 24 ( 7 ): 518 – 35 . OpenUrl CrossRef PubMed 3. ↵ Schroder K , Tschopp J . The inflammasomes . Cell . 2010 Mar Mar ; 140 ( 6 ): 821 – 32 . OpenUrl CrossRef PubMed Web of Science 4. ↵ Ross C , Chan AH , von Pein JB , Maddugoda MP , Boucher D , Schroder K . Inflammatory Caspases: Toward a Unified Model for Caspase Activation by Inflammasomes . Annu Rev Immunol . 2022 Apr Apr ; 40 : 249 – 69 . OpenUrl CrossRef PubMed 5. Rathinam VAK , Zhao Y , Shao F . Innate immunity to intracellular LPS . Nat Immunol . 2019 May ; 20 ( 5 ): 527 – 33 . OpenUrl CrossRef PubMed 6. ↵ Grant S , Boucher D . 10 things you wish you knew about inflammatory caspases: a holistic review of their biochemistry, activation, and signalling . J Biol Chem . 2025 Aug Aug ; 110557 . 7. ↵ Vanaja SK , Russo AJ , Behl B , Banerjee I , Yankova M , Deshmukh SD , et al. Bacterial Outer Membrane Vesicles Mediate Cytosolic Localization of LPS and Caspase-11 Activation . Cell . 2016 May ; 165 ( 5 ): 1106 – 19 . OpenUrl CrossRef PubMed 8. ↵ Tretina K , Park ES , Maminska A , MacMicking JD . Interferon-induced guanylate-binding proteins: Guardians of host defense in health and disease . J Exp Med . 2019 Mar Mar ; 216 ( 3 ): 482 – 500 . OpenUrl Abstract / FREE Full Text 9. ↵ Santos JC , Boucher D , Schneider LK , Demarco B , Dilucca M , Shkarina K , et al. Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria . Nat Commun . 2020 June June ; 11 ( 1 ): 3276 . OpenUrl CrossRef PubMed 10. ↵ Wandel MP , Kim BH , Park ES , Boyle KB , Nayak K , Lagrange B , et al. Guanylate-binding proteins convert cytosolic bacteria into caspase-4 signaling platforms . Nat Immunol . 2020 Aug ; 21 ( 8 ): 880 – 91 . OpenUrl CrossRef PubMed 11. ↵ Fisch D , Bando H , Clough B , Hornung V , Yamamoto M , Shenoy AR , et al. Human GBP1 is a microbe-specific gatekeeper of macrophage apoptosis and pyroptosis . EMBO J . 2019 July July ; 38 ( 13 ): e100926 . OpenUrl CrossRef PubMed 12. ↵ Kutsch M , Sistemich L , Lesser CF , Goldberg MB , Herrmann C , Coers J . Direct binding of polymeric GBP1 to LPS disrupts bacterial cell envelope functions . EMBO J . 2020 July July ; 39 ( 13 ): e104926 . OpenUrl CrossRef PubMed 13. ↵ Chan AH , Burgener SS , Vezyrgiannis K , Wang X , Acklam J , Von Pein JB , et al. Caspase-4 dimerisation and D289 auto-processing elicit an interleukin-1β-converting enzyme . Life Sci Alliance . 2023 Oct ; 6 ( 10 ): e202301908 . OpenUrl Abstract / FREE Full Text 14. ↵ Broz P , Pelegrín P , Shao F . The gasdermins, a protein family executing cell death and inflammation . Nat Rev Immunol . 2020 Mar ; 20 ( 3 ): 143 – 57 . OpenUrl CrossRef PubMed 15. Shi J , Zhao Y , Wang K , Shi X , Wang Y , Huang H , et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death . Nature . 2015 Oct Oct ; 526 ( 7575 ): 660 – 5 . OpenUrl CrossRef PubMed 16. ↵ Kayagaki N , Stowe IB , Lee BL , O’Rourke K , Anderson K , Warming S , et al. Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling . Nature . 2015 Oct Oct ; 526 ( 7575 ): 666 – 71 . OpenUrl CrossRef PubMed 17. ↵ Kayagaki N , Kornfeld OS , Lee BL , Stowe IB , O’Rourke K , Li Q , et al. NINJ1 mediates plasma membrane rupture during lytic cell death . Nature . 2021 Mar ; 591 ( 7848 ): 131 – 6 . OpenUrl CrossRef PubMed 18. ↵ Chen KW , Broz P . Gasdermins as evolutionarily conserved executors of inflammation and cell death . Nat Cell Biol . 2024 Sept ; 26 ( 9 ): 1394 – 406 . OpenUrl CrossRef PubMed 19. ↵ Deets KA , Vance RE . Inflammasomes and adaptive immune responses . Nat Immunol . 2021 Apr ; 22 ( 4 ): 412 – 22 . OpenUrl CrossRef PubMed 20. ↵ Rühl S , Broz P . Caspase-11 activates a canonical NLRP3 inflammasome by promoting K(+) efflux . Eur J Immunol . 2015 Oct ; 45 ( 10 ): 2927 – 36 . OpenUrl CrossRef PubMed 21. Baker PJ , Boucher D , Bierschenk D , Tebartz C , Whitney PG , D’Silva DB , et al. NLRP3 inflammasome activation downstream of cytoplasmic LPS recognition by both caspase-4 and caspase-5 . Eur J Immunol . 2015 Oct ; 45 ( 10 ): 2918 – 26 . OpenUrl CrossRef PubMed 22. ↵ Schmid-Burgk JL , Gaidt MM , Schmidt T , Ebert TS , Bartok E , Hornung V . Caspase-4 mediates non-canonical activation of the NLRP3 inflammasome in human myeloid cells . Eur J Immunol . 2015 Oct ; 45 ( 10 ): 2911 – 7 . OpenUrl CrossRef PubMed 23. ↵ Monteleone M , Stanley AC , Chen KW , Brown DL , Bezbradica JS , von Pein JB , et al. Interleukin-1β Maturation Triggers Its Relocation to the Plasma Membrane for Gasdermin-D-Dependent and -Independent Secretion . Cell Rep . 2018 Aug Aug ; 24 ( 6 ): 1425 – 33 . OpenUrl CrossRef PubMed 24. ↵ Evavold CL , Ruan J , Tan Y , Xia S , Wu H , Kagan JC . The Pore-Forming Protein Gasdermin D Regulates Interleukin-1 Secretion from Living Macrophages . Immunity . 2018 Jan Jan ; 48 ( 1 ): 35 – 44.e6 . OpenUrl CrossRef PubMed 25. ↵ Chan AH , Schroder K . Inflammasome signaling and regulation of interleukin-1 family cytokines . J Exp Med . 2020 Jan Jan ; 217 ( 1 ): e20190314 . OpenUrl CrossRef PubMed 26. ↵ Vivier E , Artis D , Colonna M , Diefenbach A , Di Santo JP , Eberl G , et al. Innate Lymphoid Cells: 10 Years On . Cell . 2018 Aug Aug ; 174 ( 5 ): 1054 – 66 . OpenUrl CrossRef PubMed 27. ↵ Tang F , Li J , Qi L , Liu D , Bo Y , Qin S , et al. A pan-cancer single-cell panorama of human natural killer cells . Cell . 2023 Sept ; 186 ( 19 ): 4235 – 4251.e20 . OpenUrl CrossRef PubMed 28. ↵ Lanier LL , Le AM , Civin CI , Loken MR , Phillips JH . The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes . J Immunol Baltim Md 1950 . 1986 June June ; 136 ( 12 ): 4480 – 6 . OpenUrl 29. ↵ Nagler A , Lanier LL , Cwirla S , Phillips JH . Comparative studies of human FcRIII-positive and negative natural killer cells . J Immunol Baltim Md 1950 . 1989 Nov Nov ; 143 ( 10 ): 3183 – 91 . OpenUrl 30. ↵ Lusty E , Poznanski SM , Kwofie K , Mandur TS , Lee DA , Richards CD , et al. IL-18/IL-15/IL-12 synergy induces elevated and prolonged IFN-γ production by ex vivo expanded NK cells which is not due to enhanced STAT4 activation . Mol Immunol . 2017 Aug ; 88 : 138 – 47 . OpenUrl PubMed 31. Hook CE , Matyszak MK , Gaston JSH . Infection of epithelial and dendritic cells by Chlamydia trachomatis results in IL-18 and IL-12 production, leading to interferon-Î 3 production by human natural killer cells . FEMS Immunol Med Microbiol . 2005 Aug ; 45 ( 2 ): 113 – 20 . OpenUrl CrossRef PubMed 32. ↵ Humann J , Lenz LL . Activation of Naive NK Cells in Response to Listeria monocytogenes Requires IL-18 and Contact with Infected Dendritic Cells . J Immunol . 2010 May May ; 184 ( 9 ): 5172 – 8 . OpenUrl Abstract / FREE Full Text 33. ↵ Esin S , Batoni G , Counoupas C , Stringaro A , Brancatisano FL , Colone M , et al. Direct Binding of Human NK Cell Natural Cytotoxicity Receptor NKp44 to the Surfaces of Mycobacteria and Other Bacteria . Infect Immun . 2008 Apr ; 76 ( 4 ): 1719 – 27 . OpenUrl Abstract / FREE Full Text 34. ↵ Chalifour A , Jeannin P , Gauchat JF , Blaecke A , Malissard M , N’Guyen T , et al. Direct bacterial protein PAMP recognition by human NK cells involves TLRs and triggers α-defensin production . Blood . 2004 Sept Sept ; 104 ( 6 ): 1778 – 83 . OpenUrl Abstract / FREE Full Text 35. ↵ Reinke S , Pantazi E , Chappell GR , Sanchez-Martinez A , Guyon R , Fergusson JR , et al. Emulsion and liposome-based adjuvanted R21 vaccine formulations mediate protection against malaria through distinct immune mechanisms . Cell Rep Med . 2023 Nov Nov ; 4 ( 11 ): 101245 . OpenUrl PubMed 36. ↵ Muñoz-Wolf N , Ward RW , Hearnden CH , Sharp FA , Geoghegan J , O’Grady K , et al. Non-canonical inflammasome activation mediates the adjuvanticity of nanoparticles . Cell Rep Med . 2023 Jan ; 4 ( 1 ): 100899 . OpenUrl PubMed 37. ↵ Linder A , Hornung V . Inflammasomes in T cells . J Mol Biol . 2022 Feb Feb ; 434 ( 4 ): 167275 . OpenUrl CrossRef PubMed 38. ↵ Linder A , Bauernfried S , Cheng Y , Albanese M , Jung C , Keppler OT , et al. CARD8 inflammasome activation triggers pyroptosis in human T cells . EMBO J . 2020 Oct Oct ; 39 ( 19 ): e105071 . OpenUrl CrossRef PubMed 39. ↵ Kroes MM , Mariman R , Hijdra D , Hamstra HJ , Van Boxtel KJWM , Van Putten JPM , et al. Activation of Human NK Cells by Bordetella pertussis Requires Inflammasome Activation in Macrophages . Front Immunol . 2019 Aug Aug ; 10 : 2030 . OpenUrl PubMed 40. ↵ Bierschenk D , Boucher D , Schroder K . Salmonella-induced inflammasome activation in humans . Mol Immunol . 2017 June ; 86 : 38 – 43 . OpenUrl CrossRef PubMed 41. ↵ Clare B . Inflammasome activation by Salmonella . Curr Opin Microbiol . 2021 Dec ; 64 : 27 – 32 . OpenUrl CrossRef PubMed 42. ↵ Rebuffet L , Melsen JE , Escalière B , Basurto-Lozada D , Bhandoola A , Björkström NK , et al. High-dimensional single-cell analysis of human natural killer cell heterogeneity . Nat Immunol . 2024 Aug ; 25 ( 8 ): 1474 – 88 . OpenUrl CrossRef PubMed 43. ↵ Gram AM , Wright JA , Pickering RJ , Lam NL , Booty LM , Webster SJ , et al. Salmonella Flagellin Activates NAIP/NLRC4 and Canonical NLRP3 Inflammasomes in Human Macrophages . J Immunol Baltim Md 1950 . 2021 Feb Feb ; 206 ( 3 ): 631 – 40 . OpenUrl 44. ↵ Naseer N , Egan MS , Reyes Ruiz VM , Scott WP , Hunter EN , Demissie T , et al. Human NAIP/NLRC4 and NLRP3 inflammasomes detect Salmonella type III secretion system activities to restrict intracellular bacterial replication . PLoS Pathog . 2022 Jan ; 18 ( 1 ): e1009718 . OpenUrl CrossRef PubMed 45. ↵ Naschberger E , Geißdörfer W , Bogdan C , Tripal P , Kremmer E , Stürzl M , et al. Processing and secretion of guanylate binding protein-1 depend on inflammatory caspase activity . J Cell Mol Med . 2017 Sept ; 21 ( 9 ): 1954 – 66 . OpenUrl PubMed 46. ↵ Wannamaker W , Davies R , Namchuk M , Pollard J , Ford P , Ku G , et al. (S)-1-((S)-2-{[1-(4-Amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2 R,3 S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide (VX-765), an Orally Available Selective Interleukin (IL)-Converting Enzyme/Caspase-1 Inhibitor, Exhibits Potent Anti-Inflammatory Activities by Inhibiting the Release of IL-1β and IL-18 . J Pharmacol Exp Ther . 2007 May ; 321 ( 2 ): 509 – 16 . OpenUrl Abstract / FREE Full Text 47. ↵ Srikanth CV , Mercado-Lubo R , Hallstrom K , McCormick BA . Salmonella effector proteins and host-cell responses . Cell Mol Life Sci CMLS . 2011 Nov ; 68 ( 22 ): 3687 – 97 . OpenUrl PubMed 48. ↵ Agbor TA , McCormick BA . Salmonella effectors: important players modulating host cell function during infection . Cell Microbiol . 2011 Dec ; 13 ( 12 ): 1858 – 69 . OpenUrl CrossRef PubMed 49. ↵ Coll RC , Hill JR , Day CJ , Zamoshnikova A , Boucher D , Massey NL , et al. MCC950 directly targets the NLRP3 ATP-hydrolysis motif for inflammasome inhibition . Nat Chem Biol . 2019 June ; 15 ( 6 ): 556 – 9 . OpenUrl CrossRef PubMed 50. ↵ Okondo MC , Johnson DC , Sridharan R , Go EB , Chui AJ , Wang MS , et al. DPP8 and DPP9 inhibition induces pro-caspase-1-dependent monocyte and macrophage pyroptosis . Nat Chem Biol . 2017 Jan ; 13 ( 1 ): 46 – 53 . OpenUrl CrossRef PubMed 51. ↵ Man SM , Hopkins LJ , Nugent E , Cox S , Glück IM , Tourlomousis P , et al. Inflammasome activation causes dual recruitment of NLRC4 and NLRP3 to the same macromolecular complex . Proc Natl Acad Sci U S A . 2014 May May ; 111 ( 20 ): 7403 – 8 . OpenUrl Abstract / FREE Full Text 52. ↵ Miao EA , Alpuche-Aranda CM , Dors M , Clark AE , Bader MW , Miller SI , et al. Cytoplasmic flagellin activates caspase-1 and secretion of interleukin 1beta via Ipaf . Nat Immunol . 2006 June ; 7 ( 6 ): 569 – 75 . OpenUrl CrossRef PubMed Web of Science 53. ↵ Wang K , Sun Q , Zhong X , Zeng M , Zeng H , Shi X , et al. Structural Mechanism for GSDMD Targeting by Autoprocessed Caspases in Pyroptosis . Cell . 2020 Mar Mar ; 180 ( 5 ): 941 – 955.e20 . OpenUrl CrossRef PubMed 54. Devant P , Dong Y , Mintseris J , Ma W , Gygi SP , Wu H , et al. Structural insights into cytokine cleavage by inflammatory caspase-4 . Nature . 2023 Dec ; 624 ( 7991 ): 451 – 9 . OpenUrl CrossRef PubMed 55. ↵ Exconde PM , Hernandez-Chavez C , Bourne CM , Richards RM , Bray MB , Lopez JL , et al. The tetrapeptide sequence of IL-18 and IL-1β regulates their recruitment and activation by inflammatory caspases . Cell Rep . 2023 Dec Dec ; 42 ( 12 ): 113581 . OpenUrl CrossRef PubMed 56. ↵ Chong A , Cooper KG , Kari L , Nilsson OR , Hillman C , Fleming BA , et al. Cytosolic replication in epithelial cells fuels intestinal expansion and chronic fecal shedding of Salmonella Typhimurium . Cell Host Microbe . 2021 July ; 29 ( 7 ): 1177 – 1185.e6 . OpenUrl PubMed 57. ↵ Craven RR , Gao X , Allen IC , Gris D , Wardenburg JB , McElvania-TeKippe E , et al. Staphylococcus aureus α-Hemolysin Activates the NLRP3-Inflammasome in Human and Mouse Monocytic Cells . Ratner AJ , editor. PLoS ONE . 2009 Oct 14 ; 4 ( 10 ): e7446 . OpenUrl CrossRef PubMed 58. ↵ Kim EY , Ner-Gaon H , Varon J , Cullen AM , Guo J , Choi J , et al. Post-sepsis immunosuppression depends on NKT cell regulation of mTOR/IFN-γ in NK cells . J Clin Invest . 2020 June June ; 130 ( 6 ): 3238 – 52 . OpenUrl PubMed 59. ↵ Yang S , Sheffer M , Kaplan IE , Wang Z , Tarannum M , Dinh K , et al. Non-pathogenic E. coli displaying decoy-resistant IL18 mutein boosts anti-tumor and CAR NK cell responses . Nat Biotechnol . 2024 Oct Oct ; 60. ↵ Jong RD , Altare F , Haagen IA , Elferink DG , Boer TD , Van Breda Vriesman PJC , et al. Severe Mycobacterial and Salmonella Infections in Interleukin-12 Receptor-Deficient Patients . Science . 1998 May May ; 280 ( 5368 ): 1435 – 8 . OpenUrl Abstract / FREE Full Text 61. ↵ Tait Wojno ED , Hunter CA , Stumhofer JS . The Immunobiology of the Interleukin-12 Family: Room for Discovery . Immunity . 2019 Apr ; 50 ( 4 ): 851 – 70 . OpenUrl CrossRef PubMed 62. Ottenhoff THM , Verreck FAW , Lichtenauer-Kaligis EGR , Hoeve MA , Sanal O , van Dissel JT . Genetics, cytokines and human infectious disease: lessons from weakly pathogenic mycobacteria and salmonellae . Nat Genet . 2002 Sept ; 32 ( 1 ): 97 – 105 . OpenUrl CrossRef PubMed Web of Science 63. ↵ Staretz-Haham O , Melamed R , Lifshitz M , Porat N , Fieschi C , Casanova JL , et al. Interleukin-12 Receptor β1 Deficiency Presenting as Recurrent Salmonella Infection . Clin Infect Dis . 2003 July July ; 37 ( 1 ): 137 – 40 . OpenUrl CrossRef PubMed Web of Science 64. ↵ Lin Q , Rong L , Jia X , Li R , Yu B , Hu J , et al. IFN-γ-dependent NK cell activation is essential to metastasis suppression by engineered Salmonella . Nat Commun . 2021 May May ; 12 ( 1 ): 2537 . OpenUrl CrossRef PubMed 65. ↵ Schroder K , Hertzog PJ , Ravasi T , Hume DA . Interferon-gamma: an overview of signals, mechanisms and functions . J Leukoc Biol . 2004 Feb ; 75 ( 2 ): 163 – 89 . OpenUrl CrossRef PubMed Web of Science 66. ↵ Moss N , Sakai C , Kaul SN , Graybuck LT , Rachid Zaim S , Angus-Hill ML , et al. Dissecting type I and II interferon impacts on human immune cells in disease by a cell type-specific interferon response atlas [Internet] . Immunology ; 2025 [cited 2026 Jan 20 ]. Available from: http://biorxiv.org/lookup/doi/10.64898/2025.12.02.691676 67. ↵ Stapels DAC , Hill PWS , Westermann AJ , Fisher RA , Thurston TL , Saliba AE , et al. Salmonella persisters undermine host immune defenses during antibiotic treatment . Science . 2018 Dec Dec ; 362 ( 6419 ): 1156 – 60 . OpenUrl Abstract / FREE Full Text 68. ↵ Andaluz-Ojeda D , Iglesias V , Bobillo F , Almansa R , Rico L , Gandía F , et al. Early natural killer cell counts in blood predict mortality in severe sepsis . Crit Care . 2011 Oct Oct ; 15 ( 5 ): R243 . OpenUrl CrossRef PubMed 69. ↵ Groß O . Measuring the Inflammasome . In: Ashman RB , editor. Leucocytes [Internet] . Totowa, NJ : Humana Press ; 2012 [cited 2025 July 6 ]. p. 199 – 222 . (Methods in Molecular Biology; vol. 844 ). Available from: https://link.springer.com/10.1007/978-1-61779-527-5_15 OpenUrl 70. ↵ Dickson MA , Hahn WC , Ino Y , Ronfard V , Wu JY , Weinberg RA , et al. Human Keratinocytes That Express hTERT and Also Bypass a p16 INK4a -Enforced Mechanism That Limits Life Span Become Immortal yet Retain Normal Growth and Differentiation Characteristics . Mol Cell Biol . 2000 Feb Feb ; 20 ( 4 ): 1436 – 47 . OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted January 22, 2026. Download PDF 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. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Human NK cells engage cell-intrinsic non-canonical inflammasome function Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Human NK cells engage cell-intrinsic non-canonical inflammasome function Nathan G.F. Leborgne , Jadie Acklam , Alexander J. Hogg , Zarema Albakova , Karen G. Hogg , Kim Samirah Robinson , Grant Calder , Arnel Villamin Jr , Callum Robson , Freya Pealing , Matthew A. Care , Monika Gonka , James M. Fox , James Iremonger , Frances E. Pearson , Max E. Noble , Alison M. Layton , James P. Hewitson , David G. Kent , William Grey , Jillian L. Barlow , Dave Boucher bioRxiv 2025.10.20.682800; doi: https://doi.org/10.1101/2025.10.20.682800 Share This Article: Copy Citation Tools Human NK cells engage cell-intrinsic non-canonical inflammasome function Nathan G.F. Leborgne , Jadie Acklam , Alexander J. Hogg , Zarema Albakova , Karen G. Hogg , Kim Samirah Robinson , Grant Calder , Arnel Villamin Jr , Callum Robson , Freya Pealing , Matthew A. Care , Monika Gonka , James M. Fox , James Iremonger , Frances E. Pearson , Max E. Noble , Alison M. Layton , James P. Hewitson , David G. Kent , William Grey , Jillian L. Barlow , Dave Boucher bioRxiv 2025.10.20.682800; doi: https://doi.org/10.1101/2025.10.20.682800 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 (7624) Biochemistry (17651) Bioengineering (13871) Bioinformatics (41882) Biophysics (21424) Cancer Biology (18566) Cell Biology (25461) Clinical Trials (138) Developmental Biology (13365) Ecology (19867) Epidemiology (2067) Evolutionary Biology (24290) Genetics (15590) Genomics (22476) Immunology (17714) Microbiology (40331) Molecular Biology (17148) Neuroscience (88483) Paleontology (666) Pathology (2828) Pharmacology and Toxicology (4817) Physiology (7635) Plant Biology (15114) Scientific Communication and Education (2044) Synthetic Biology (4286) Systems Biology (9815) Zoology (2268)