PDE10A Inhibition Reduces NLRP3 Activation and Pyroptosis in Sepsis and Nerve Injury

PDE10A Inhibition Reduces NLRP3 Activation and Pyroptosis in Sepsis and Nerve Injury | 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 PDE10A Inhibition Reduces NLRP3 Activation and Pyroptosis in Sepsis and Nerve Injury View ORCID Profile Bradford C. Berk , View ORCID Profile Camila Lage Chávez , View ORCID Profile Chia George Hsu doi: https://doi.org/10.1101/2025.03.17.643692 Bradford C. Berk 1 Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry , Rochester, NY, USA 2 Department of Physical Medicine and Rehabilitation, University of Rochester School of Medicine and Dentistry , Rochester, NY, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Bradford C. Berk For correspondence: Chia.Hsu{at}utsa.edu Bradford_Berk{at}urmc.rochester.edu Camila Lage Chávez 1 Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry , Rochester, NY, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Camila Lage Chávez Chia George Hsu 1 Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry , Rochester, NY, USA 3 Department of Kinesiology, The University of Texas at San Antonio , San Antonio, TX, USA 4 Department of Molecular Microbiology and Immunology, The University of Texas at San Antonio , San Antonio, TX, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Chia George Hsu For correspondence: Chia.Hsu{at}utsa.edu Bradford_Berk{at}urmc.rochester.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Cell death and inflammation are key innate immune responses, but excessive activation can cause tissue damage. The NLRP3 inflammasome is a promising target for reducing inflammation and promoting recovery. Immunometabolism regulates NLRP3 responses in neurological and inflammatory diseases through cyclic nucleotide signaling. Targeting phosphodiesterases (PDEs), which hydrolyze cAMP and cGMP, offers a novel approach to mitigate inflammation. While 14 PDE inhibitors are FDA-approved, PDE10A’s role in NLRP3 inflammasome activation remains unclear. This study investigates the effects of PDE10A inhibition on inflammasome-driven inflammation using two PDE10A inhibitors, MP-10 and TP-10, in macrophage and animal models of sepsis and traumatic nerve injury. Our results show that PDE10A inhibition reduces inflammasome activation by preventing ASC speck formation and by lowering levels of cleaved caspase-1, gasdermin D, and IL-1β, which are key mediators of pyroptosis. In the sepsis model, MP-10 significantly reduced inflammation, decreased plasma IL-1β, alleviated thrombocytopenia, and improved organ damage markers. In the nerve injury model, PDE10A inhibition enhanced motor function recovery and reduced muscle atrophy-related gene expression. These findings suggest that PDE10A inhibition could be a promising therapeutic approach for inflammatory and neuromuscular injuries. Given MP-10’s established safety in human trials, Phase 2 clinical studies for sepsis and nerve injury are highly promising. Introduction Inflammation is a critical response to injury and infection, but when it becomes unregulated, it can lead to tissue damage and contribute to the development of a wide range of diseases, including neuromuscular disorders, cardiovascular diseases, and immune dysfunction( 1 - 4 ). Central to the inflammatory process is the activation of the inflammasome, a multiprotein complex that triggers caspase-1 activation, leading to the production of pro-inflammatory cytokines such as IL-1β and IL-18( 5 , 6 ). This process not only amplifies the inflammatory response but also induces a form of programmed cell death known as pyroptosis, which further exacerbates tissue damage and promotes disease progression( 7 - 9 ). Consequently, targeting inflammasome activation has emerged as a promising therapeutic strategy to mitigate excessive inflammation and improve outcomes in inflammatory diseases. However, while general anti-inflammatory therapies aim to reduce inflammation systemically, they often lack specificity, resulting in limited efficacy and side effects( 6 ). Phosphodiesterases (PDEs) are enzymes that regulate intracellular levels of cyclic nucleotides, such as cyclic AMP (cAMP) and cyclic GMP (cGMP), by hydrolyzing them to their inactive forms( 10 ). These molecules play essential roles in various cellular processes, including immune cell activation and the regulation of inflammation. Given the diversity of PDE isoforms and their involvement in a wide range of physiological functions, PDEs represent promising pharmacological targets( 11 ). By 2024, a number of PDE inhibitors, including those targeting PDE4 and PDE5, have been FDA-approved for the treatment of various conditions( 12 ). For instance, sildenafil (Viagra), a PDE5 inhibitor, is used to treat erectile dysfunction by inducing smooth muscle relaxation and increasing blood flow( 13 ). Similarly, roflumilast, a PDE4 inhibitor, has been approved for managing exacerbations in chronic obstructive pulmonary disease (COPD)( 14 ). PDE10A, which has a unique expression profile in immune cells, regulates both cAMP and cGMP levels, and is emerging as a potential therapeutic target. In clinical trials, PDE10A inhibitors, such as TP-10 and MP-10 (PF-2545920), have demonstrated safety and efficacy for cognitive and motor control improvement, particularly in brain disorders( 15 , 16 ). Despite this, the role of PDE10A in regulating NLRP3 inflammasome activation and its involvement in inflammasome-related diseases remains poorly understood. In this study, we examined the effects of PDE10A inhibition on NLRP3 inflammasome activation and pyroptosis in vitro, using human THP-1 macrophages and mouse-derived macrophages, and in vivo, using sepsis( 17 , 18 ) and sciatic nerve injury mouse models( 19 , 20 ). We utilized two PDE10A inhibitors, MP-10 and TP-10, which have been systemically administered in clinical trials for neuropsychiatric conditions like schizophrenia( 21 ), as well as in preclinical models of heart failure( 22 , 23 ). Our results show that PDE10A inhibition effectively reduces inflammasome activation, protects against pyroptosis, and improves functional recovery in both sepsis and traumatic nerve injury models. These findings support the potential for targeting PDE10A in the treatment of inflammasome-driven diseases and suggest that PDE10A inhibitors may offer therapeutic benefits in managing inflammation and promoting recovery in various pathological conditions Materials and Methods Animal Female and male C57BL/6J mice (Jackson Laboratory, 000664) aged 10-14 weeks were used in the experiments. All mice were housed in a specific pathogen-free facility and kept in a temperature-controlled room set to a light and dark cycle of 12 hours each. The mice had ad libitum access to standard mouse chow and water. No sample or animal was excluded from the experiments. Experiments with animals were blinded to the researcher assessing markers. In vivo sepsis model, MP-10 treatments, and tissue harvesting Sepsis was induced in C57BL/6J mice by intraperitoneal (IP) injection of LPS (10 mg/kg) (Sigma-Aldrich, Escherichia coli O127:B8-L3129-10MG) for 3 hours, followed by an IP injection of 100 mM ATP (100 µL, pH 7.4) (Sigma-Aldrich, A7699), as previously described( 17 , 18 ). Mice were randomly assigned to experimental groups. MP-10 was dissolved in a vehicle containing 10% DMSO and 40% hydroxypropyl-β-cyclodextrin, based on previous studies( 22 ). Mice were given a subcutaneous (S.C.) injection of either vehicle or MP-10 (3 mg/kg) 30 minutes after LPS administration. Blood and peritoneal fluid samples were collected 30 minutes after ATP injection. Mice were administered an intraperitoneal (IP) injection of ketamine and xylazine for deep anesthesia. Whole blood was then collected in tubes containing 10 µL of 500 mM EDTA, followed by centrifugation at 1000 g for 10 minutes at 4°C. Plasma was collected and aliquoted for cytokine assays. Peritoneal fluid was harvested by washing the peritoneal cavity with 5 mL of ice-cold PBS supplemented with 1 mM EDTA. The cell suspension was centrifuged at 500 g for 5 minutes at 4°C, and the supernatant was collected and aliquoted for cytokine assays. Spleen tissues were harvested and fixed in 4% Paraformaldehyde (PFA) (VWR International, 100503-917) overnight at 4°C, followed by embedding in paraffin. Sciatic nerve crush and denervation injuries, MP-10 treatments, and tissue harvesting Sciatic nerve crush injury was performed on female C57BL/6J mice as previously described( 19 , 20 ). Briefly, Buprenorphine ER (0.5-1 mg/kg) was administered subcutaneously 1 hour prior to surgery for effective postoperative pain relief lasting 48-72 hours. After inducing anesthesia with intraperitoneal (IP) injections of ketamine (60 mg/kg) and xylazine (4 mg/kg), the mice were prepared aseptically. A lateral skin incision was made along the femur, and the sciatic nerve was bluntly exposed. The nerve was crushed proximal to the tibial and peroneal branches using smooth forceps with a metal calibration ring to standardize the pressure for 30 seconds. The wound was then closed, and the mice were monitored every 12 hours for the next 3 days. For MP-10 treatment, mice were randomized to receive daily subcutaneous injections of either MP-10 (10 mg/kg) or vehicle (10% DMSO in 40% β-cyclodextrin v/v) starting 1 hour after injury and continuing until day 7. On the day of sacrifice, the mice were given an intraperitoneal (IP) injection of ketamine and xylazine for deep anesthesia. Blood samples, sciatic nerves, and muscle tissue were subsequently collected. Gastrocnemius-soleus (GAS) muscles were harvested for gene expression analysis. Sciatic Functional Index (SFI) The effects of MP-10 treatment were evaluated using the Sciatic Functional Index (SFI), a noninvasive method for assessing in vivo functional recovery following sciatic nerve injury ( 19 , 20 ). The SFI is scored on a scale from 0 (normal function) to 100 (complete loss of function). Briefly, mice were trained to walk freely along a 77 cm by 7 cm corridor lined with white paper, and individual paw prints were obtained by painting each hind foot. Paw prints were measured by two blinded evaluators for toe spread (distance from the 1st to the 5th toe) and paw length (distance from the 3rd toe to the bottom of the print). Three prints from both the experimental (injured) and normal (uninjured) sides were measured, and the SFI was calculated for each animal by averaging these measurements and applying the following formula( 24 ): SFI = 118.9 ((ETS-NTS)/NTS)) - 5 1.2 ((EPL-NPL)/NPL)) -7.5. Where E refers to the experimental (injured: crush or denervated) paw, N refers to the normal (healthy: uninjured or control) paw, TS is toe spread, and PL is paw length. Bone marrow progenitor cell isolation and bone marrow-derived macrophage (BMDM) differentiation BMDMs preparation was performed as previously described( 17 , 18 ). L929 conditioned media which contains the macrophage growth factor M-CSF, was prepared by culturing L929 cells (ATCC) in complete DMEM (Thermo Fisher Scientific, MT10013CV) supplemented with 10% FBS, and 1% penicillin and streptomycin for 10 days at 37°C, 5% CO 2 . The L929 conditioned media, was collected, filtered (Vacuum Filter/Storage Bottle System, Corning, 431153), and stored at −80 °C until required. For isolation of BMDMs, tibias and femurs were removed from both C57BL/6J male and female mice. Bone marrow was collected at 500 x g for 2 min at 4 °C, resuspended with complete DMEM medium and filtered through a 70-μm cell strainer (VWR international, 10199-657). Bone marrow progenitor cells were cultured in 100 mm dishes for 6- 7 days in 70% complete DMEM medium and 30% L929-conditioned medium. Fresh medium (5 mL) was added on day 3. BMDMs were collected by scraping in cold PBS containing EDTA (1 mM). After centrifugation, BMDMs were seeded into 12-well plates at a density of 1.6 × 10 5 cells/well in DMEM and incubated overnight before use. THP-1 macrophage differentiation Human THP-1 monocytes were differentiated into macrophages by 24 hr incubation with 100 nM PMA (Sigma-Aldrich, P1585) in complete RPMI medium at 1.6 × 10 5 cells/well in 12-well plates. Cells were washed twice with 1x PBS and incubated with complete RPMI medium without PMA for 24 hr before experiment. ASC-GFP-overexpressed THP-1 macrophage A lentivirus expressing the ASC-GFP fusion protein was prepared by transfection of pLEX-MCS-ASC-GFP (Addgene 73957), psPAX2, and p MD.2G using Fugene 6 (Promega, PAE2693) in HEK293T cells. Culture medium was replaced after 24hr with fresh medium containing 5% FBS After a further 48 hrs virus containing medium was collected and stored in - 80°C. THP-1 monocytes at 1.6 × 10 5 cells/well on 12-well plates were infected with 400uL virus with polybrene (4 µg/ml) and centrifuged at 2500 rpm at 20°C for 90min. Fresh complete RPMI medium with 100 nM PMA was added and followed by 24 hr incubation. Cells were washed twice with 1x PBS and incubated with complete RPMI medium without PMA for 24 hr before experiment. Peritoneal macrophage isolation Peritoneal macrophage isolation was performed as previously described( 25 ). Bio-Gel preparation: Wash Bio-Gel P-100 gel three times with PBS. Pellet Bio-Gel by centrifugation for 5 min at 400 × g, then resuspend in PBS to yield 2% (w/v) Bio-Gel. Autoclave for 20 min before use. C57BL/6J mice between 10-12 weeks of age were used. Mice received intraperitoneal injection (IP) of 1mL 2% fine polyacrylamide beads. On day four after injection, the animals were euthanized by CO2 and macrophages were harvested by washing their peritoneal cavity with 5 mL ice-cold PBS supplemented with 1mM EDTA. The cell suspension was centrifuged at 500 g for 5 min at 4°C, and the supernatant was discarded. The cell pellet was washed one time in PBS and the supernatant was removed through centrifugation. The cell pellet was suspended in RPMI 1640 medium supplemented with 10% FBS, 1% streptomycin/penicillin, 0.1mM 2-Mercaptoethanol, and 1mM sodium pyruvate. Macrophages were cultured at 1.6 × 105 cells/well on 12-well plates. After incubated for 2 hours, cells were washed twice with PBS and media was replenished. Cells were used for experiments after 24 hours of culture. Inflammasome stimulation To activate the NLRP3 inflammasome, cells were primed for 3 h with LPS (100 ng/mL) followed by the addition of 2 µM (BMDMs, and peritoneal macrophages), 6µM (THP-1 differentiated macrophages) nigericin (Sigma-Aldrich, N7143-5MG), 2 mM ATP for 30 min. MP-10 (MedChemExpress, HY-50098) or TP-10 (3-5 µM) (MedChemExpress, HY-14550) was added together with LPS. Cell death by SYTOX TM green THP-1 macrophages or BMDMs were seeded in 96-well plates (2x10 4 cells/well) one day before the experiments. Cells were washed twice and incubated with LPS (100 ng/mL) in XF based medium (Agilent, 103334-100) supplemented with 4.5 g/L glucose, 2 mM glutamine, 1 mM sodium pyruvate, and 1 mM HEPES buffer at final pH of 7.4 for 3 hr. After 3 hr of LPS stimulation, SYTOX Green (final concentration 1µM) (Thermo Fisher Scientific, S7020) was added together with nigericin (2 µM for BMDMs; 6 µM for THP-1 macrophages) and fluorescence signals (Excitation wavelength: 485 nm, Emission wavelength: 550 nm) were analyzed using FLUOstar OPTIMA plate reader (BMG Labtech) at 36 °C for 120 min. The percentage of cell death was calculated by normalizing fluorescence signals from cells treated with Triton X-100 (0.1%). Cytokine assays Plasma, and peritoneal fluid levels of IL-1 β collected from mouse studies were determined by ELISA kits according to the manufacturer’s instructions. For in vitro studies, culture media was collected immediately after treatment. Samples were cleared by centrifugation at 16,000 ×g for 5 min and stored at −20 °C. Mouse IL-1 β (BioLegend, 432604) were measured in culture supernatants by ELISA. RNA extraction and Real-time PCR RNA was extracted from lung tissue or cultured cells using RNeasy kit (Qiagen, 74106) according to the manufacturer’s instructions. Complementary DNA was synthesized from 0.5 μg RNA by iScript™ cDNA Synthesis Kit (Bio-Rad, 1708891). Amplification reactions contained a target specific fraction of cDNA and 1 μM forward and reverse primers in iQ™ SYBR® Green Supermix (Bio-Rad, 1708882). Fluorescence was monitored and analyzed in a CFX connect real-time PCR system (Bio-Rad). Gene expression was normalized to β-actin using the delta delta cycle threshold method. Amplification of specific transcripts was confirmed by melting curve analysis at the end of each PCR experiment. The primers used are as follows: Mouse GLUT1 (Forward: GGTTGTGCCATACTCATGACC, Reverse: CAGATAGGACATCCAGGGTAGC); Mouse HK1(Forward: GTGGACGGGACGCTCTAC , Reverse: TTCACTGTTTGGTGCATGATT); Mouse β-actin (Forward: TTCAACACCCCAGCCATGT, Reverse: GTAGATGGGCACAGTGTGGGT); Mouse NLRP3 (Forward: TTCCCAGACACTCATGTTGC, Reverse: AGAAGAGACCACGGCAGAAG); Mouse Myogenin (Forward: CCTTGCTCAGCTCCCTCA, Reverse: TGGGAGTTGCATTCACTGG); Mouse Gadd45a (Forward: AGAGCAGAAGACCGAAGGA, Reverse: CGTAATGGTGCGCTGACTC); Mouse ASC (Forward: CTGCTCAGAGTACAGCCAGAAC, Reverse: CTGTCCTTCAGTCAGCACACTG); Mouse NLRP2 (Forward: TCTCATGTGCCTTCTACATCAGC, Reverse: CACAAAGGTCACGGATCAGAG); Mouse NAIP5 (Forward: TGCCAAACCTACAAGAGCTGA, Reverse: CAAGCGTTTAGACTGGGGATG); Mouse NAIP6 (Forward: TACAGGGAGTTTACAAGACCCC, Reverse: AGTGGCCTGGAGAGACTCAG). Western blot Proteins were separated by SDS-PAGE through 10% acrylamide gels and transferred to nitrocellulose membranes, blocked with 5% nonfat dry milk in Tween-TBS and reacted with the indicated antibody: Caspase-1(AdipoGen, AG-20B-0042-C100) 1:1000; GSDMD (Abcam, ab209845) 1:1000; IL-1β (GeneTex, GTX10750); 1:2000 β-actin (Cell Signaling Technology, 4970) overnight. Membranes were rinsed and incubated with horseradish peroxidase conjugated secondary antibody (Anti-mouse IgG, HRP-linked Antibody (Cell Signaling Technology, 7076), Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Technology, 7074), Anti-goat IgG, HRP-linked Antibody (Jackson Immuno Research, 805-035-180). Reactive proteins were detected by enhanced chemiluminescence, visualized by exposure to radiographic film and quantified by scanning densitometry normalized to β-actin expression measured in each sample on the same gel. All of the full and uncropped western blots were listed in supplemental material. Immunofluorescence Analysis Frozen and OCT-embedded sciatic nerves and Tibialis Anterior (TA) muscles from each group were sequentially cross-sectioned at 10 μm and stored at −80° C. Tissue sections were incubated with 0.2% Triton X-100 for 10 min and blocked in 10% normal goat serum (Jackson ImmunoResearch, 005-000-121) > for 30 min at room temperature. After overnight incubation with primary antibody (either 1:1000 PDE10A ( Santa Cruz, sc-515023), or 1:1000 cleaved IL-1β (Thermo Fisher Scientific, PA5-105048)) at 4°C, the fluorescent-labeled secondary antibody was incubated for 1 h at room temperature. Spleen tissues were drop fixed in 4% PFA, embedded in paraffin, cut into 5 μm sections, and mounted onto slides. Sections were deparaffinized before use. Sections were washed 3 times in PBS followed by antigen retrieval for 20 minutes with steam using 1X Citrate buffer (Millipore, 21545), pH=6.0. Sections were blocked in 10% normal goat serum (Vector Laboratories, S-1000) in PBS for 1 hour at room temperature followed by overnight incubation at 4°C with Fibrin (Millipore Sigmaat, MABS2155) in 2% normal goat serum in PBS overnight. After three washes with PBS, fluorescence-conjugated secondary antibodies (Molecular Probes, A-11001) 1:1000 were incubated for 1 hour at room temperature and followed by three washes with PBS. All slides were mounted with DAPI Fluoromount-G (SouthernBiotech, 0100-20) to detect nuclei. ASC speck formation ASC-GFP-expressing THP-1differentiated macrophages were seeded on glass coverslips in 12-well plates. Cells were incubated with 100 ng/ml LPS with or without TP-10 (5 µM) for 3 hr followed by incubation with 6 µM nigericin for 2 h. Cells were fixed in 4% PFA for 10 min. After three washes with 1x PBS, cells were mounted using fluoromount-G-DAPI. Immunofluorescence was analyzed by confocal microscope. The GFP 488 nm fluorescent signals were acquired by confocal microscopy. The data points represent biological replicates from 3 separate experiments. Statistics Unless otherwise specified, in vitro experiments were performed in triplicate, with results from duplicate or triplicate wells averaged before statistical analysis. Data are presented as mean ± SEM. Statistical analysis was conducted using GraphPad Prism 10.0. For cell culture experiments with PDE10A inhibitors, data were analyzed using one-way ANOVA. In vivo experiments with MP-10 treatment in sepsis and sciatic nerve injury models were analyzed using two-way ANOVA, followed by post hoc t-tests with Bonferroni correction for multiple comparisons. P values are denoted as follows: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. Conflict of interest The authors declare no conflict of interest. Results To investigate the regulation and function of phosphodiesterases (PDEs) in immune cells, we previously screened PDE genes in peritoneal and alveolar macrophages. Our results identified PDE4B and PDE10A as the most significantly induced by lipopolysaccharide (LPS)( 25 ). PDE4 inhibitor Roflumilast is FDA-approved for treating severe chronic obstructive pulmonary disease (COPD)( 26 ), but the specific role of PDE10A in NLRP3 inflammasome activation remains unclear. To explore this, we used two different PDE10A inhibitors, MP-10 and TP-10, in three types of macrophages: human THP-1 differentiated macrophages, mouse peritoneal macrophages, and mouse bone marrow-derived macrophages (BMDMs). NLRP3 inflammasome activation involves two key steps: priming (e.g., NF-κB-mediated expression of NLRP3) and subsequent assembly and activation (e.g., by nigericin or ATP to activate caspase cleavage)( 6 ). The inflammasome then activates caspase-1, which processes pro-inflammatory cytokines like IL-1β and cleaves gasdermin D (GSDMD). This cleavage results in the formation of pores in the cell membrane, causing cell swelling and rupture, a process called pyroptosis( 27 , 28 ). To assess the role of PDE10A in inflammasome-mediated pyroptosis, we treated human THP-1 differentiated macrophages with LPS, with or without MP-10, followed by nigericin. Cell death was quantified using real-time nucleic acid staining (SYTOX™)( 18 ). Treatment with the PDE10A inhibitor MP-10 significantly reduced LPS-nigericin-induced cell death in a dose-dependent manner ( Fig. 1A ), suggesting that PDE10A inhibition protects macrophages from pyroptosis. Download figure Open in new tab Fig. 1. PDE10A inhibition reduces NLRP3 inflammasome activation in macrophages. (A) THP-1 differentiated macrophages were stimulated with LPS (100 ng/mL) and co-incubated with MP-10 (3 or 10 μM) or vehicle (DMSO) for 3 hours, followed by 2 hours of nigericin (Nig, 6 μM) treatment. Real-time percentage cell death was determined by normalizing to maximal nuclear acid fluorescent intensity (0.1% Triton-X100 treated cells). (B) BMDMs were stimulated with LPS (100 ng/mL) for 3 hours with MP-10 (5μM) or TP-10 (5μM) or vehicle (DMSO) , followed by ATP (2 mM) stimulation for 1 hour. IL-1β in the medium was measured by ELISA. (C) THP-1 ASC-GFP overexpressing macrophages were stimulated with LPS (100 ng/mL) and nigericin (Nig, 6 μM) with or without TP-10 (5μM). ASC speck formation was measured by confocal microscopy. Arrowheads indicate ASC specks (green). Scale bar = 50 µm. (D) Peritoneal macrophages were stimulated with LPS (100 ng/mL) for 3 hours with or without TP-10 (5μM), followed by nigericin (2 μM) treatment. Pro- and cleaved caspase-1, IL-1β, and GSDMD were measured by western blotting. Data were expressed as mean ± SEM. Statistics in A-B were performed using one-way ANOVA. **** p: <0.001 Pyroptosis is a form of programmed cell death that amplifies inflammation, driven by inflammasome activation. This triggers caspase-1, which processes pro-inflammatory cytokines like IL-1β, intensifying both the inflammatory response and pyroptotic cell death. BMDMs were stimulated with LPS (100 ng/mL) for 3 hours in the presence of MP-10 (5 μM), TP-10 (5 μM), or vehicle (DMSO), followed by ATP (2 mM) stimulation for 1 hour. IL-1β levels in the medium were measured by ELISA. Both PDE10A inhibitors, MP-10 and TP-10, reduced IL-1β release ( Fig. 1B ). During NLRP3 inflammasome activation, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) is recruited by NLRP3 and forms large multimeric complexes known as ASC specks, which are a necessary step for caspase recruitment and cleavage of inflammatory cytokines ( 18 , 29 ). To further investigate the role of PDE10A in inflammasome activation, we overexpressed an ASC-GFP fusion protein in THP-1-differentiated macrophages and stimulated them with LPS followed by nigericin. Inhibition of PDE10A with TP-10 blocked ASC speck formation, as evidenced by reduced ASC speck immunofluorescence ( Fig. 1C ). To evaluate the effect of PDE10A inhibition on inflammasome activation, we measured the levels of cleaved caspase-1, cleaved gasdermin D (GSDMD), and mature IL-1β in macrophages treated with TP-10. GSDMD, a pore-forming protein, serves as the final effector in the activation of the inflammasome pathway, downstream of caspases-1, -4, and -11( 30 ). NLRP3 inflammasome activation triggers caspase-1 cleavage, resulting in the activation of caspases that cleave both GSDMD and IL-1β. This cleavage generates an N-terminal fragment of GSDMD (GSDMD-NT) that forms transmembrane pores, facilitating the release of mature IL-1β and driving pyroptosis( 31 ). Treatment with TP-10 significantly reduced the levels of cleaved caspase-1, cleaved GSDMD, and mature IL-1β ( Fig. 1D ), supporting the role of PDE10A in regulating inflammasome activation and pyroptosis. Activated macrophages exhibit a significant increase in glycolytic activity, suggesting that PDE10A inhibition could potentially influence the expression of genes involved in glucose metabolism. Specifically, glucose transporter 1 (GLUT1) and hexokinase 1 (HK1) are key regulators of glucose metabolism in LPS-treated macrophages. After 3 hours of LPS treatment, IL-6 mRNA expression was elevated by 20,000-fold compared to control. However, no significant changes were observed in the gene expression of GLUT1 or HK1. Treatment with the PDE10A inhibitor MP-10 significantly reduced LPS-induced IL-6 expression ( Fig. 2A ) but had no effect on the expression levels of GLUT1 or HK1 ( Fig. 2B-C ). These findings suggest that PDE10A does not play a role in modulating LPS-induced glucose metabolism at the transcriptional level. Download figure Open in new tab Fig. 2. PDE10A does not regulate LPS-induced glucose metabolism at the transcriptional level. BMDMs were stimulated with LPS (100 ng/mL) for 3 hours in the presence of MP-10 (5 μM) or vehicle (DMSO). mRNA expression in cell lysates was measured for (A) IL-6, (B) GLUT1, and (C) HK1. Data are expressed as mean ± SEM. Statistics in A-C were performed using one-way ANOVA. **** p: <0.001 The NLRP3 inflammasome can be activated by both sterile and non-sterile stimuli due to its capacity to detect a wide range of danger signals( 32 ). Non-sterile stimuli, such as pathogens or pathogen-associated molecular patterns (PAMPs)( 33 ), and sterile stimuli, including tissue damage or metabolic stress, activate the inflammasome through shared downstream signaling pathways( 34 , 35 ). To investigate the role of PDE10A in inflammasome activation in vivo, we employed a clinically relevant acute sepsis model, using LPS followed by ATP as a second signal. Mice were treated with MP-10 after LPS injection, and three hours later, ATP was administered to induce NLRP3 inflammasome activation. MP-10 treatment significantly reduced the severity of sepsis, as measured by the murine sepsis scoring (MSS) system ( Fig. 3A ), a reliable predictor of disease progression and mortality( 36 , 37 ). Plasma and peritoneal fluid were harvested 6 hours after ATP injection, revealing significantly lower IL-1β levels in both fluids in MP-10-treated mice compared to DMSO-treated controls ( Fig. 3B-C ). Thrombocytopenia, a common complication in sepsis associated with poor outcomes, was also mitigated by MP-10, which was shown to significantly increase total platelet counts. Additionally, MP-10 treatment reduced fibrin formation in the spleen, a hallmark of the inflammatory response in sepsis ( Fig. 3D ). These findings collectively demonstrate that PDE10A regulates inflammasome activation and the inflammatory response in a mouse model of non-sterile sepsis. Download figure Open in new tab Fig 3. PDE10A inhibition reduces sepsis severity. C57BL/6J mice underwent acute LPS (10 mg/kg body weight, i.p.) treatment with or without MP-10 (3 mg/kg, s.c. injection) for 3 hours, followed by ATP (100 mM in 100 µl, pH 7.4) i.p. (A) Murine sepsis score was measured at 3 and 6 hours after ATP injection. (B) IL-1β in plasma, (C) IL-1β in peritoneal fluid, (D) Total platelet count in whole blood, (E) Fibrin (green) and DAPI (Blue) staining in the spleen. Scale bar = 60 µm. N=4 for each group. Statistics in (A) were performed using one-way ANOVA. Statistics in (B-D) were performed using two-way ANOVA and Bonferroni’s post hoc test. *p < 0.05, **p < 0.01. Traumatic nerve injury causes mechanical damage followed by an inflammatory response, leading to axonal degeneration and demyelination(19, 20, 38). Numerous studies have shown that the activation of the NLRP3 inflammasome is a major inflammatory response following spinal cord injury( 39 , 40 ) and traumatic brain injury( 41 , 42 ). To investigate the role of PDE10A in this process, we employed a sterile sciatic nerve Injury (SNI) model to induce NLRP3 inflammasome activation. We hypothesize that inhibiting the NLRP3 inflammasome with a PDE10A inhibitor will reduce inflammation and improve motor function following SNI. Immunohistochemistry (IHC) performed on the sciatic nerve three days post-injury revealed a significant upregulation of PDE10A expression in response to nerve injury ( Fig. 4A ). To assess the impact of nerve injury on inflammasome-related gene transcription, we collected sciatic nerve tissue three days after injury and quantified mRNA levels of inflammasome-associated genes, including NLRP2, ASC, NIAP5, NIAP6, and NLRP3. Among these, ASC and NLRP3 showed the most substantial increases in expression in injured nerves when compared to uninjured controls ( Fig. 4B ). These findings suggest that both PDE10A and inflammasome signaling are involved in the pathophysiology of traumatic nerve injury. Download figure Open in new tab Fig. 4. Sciatic nerve injury-induced PDE10A protein expression and inflammasome gene Expression. (A) C57BL/6N mice underwent sham surgery and peripheral nerve injury (PNI). Sciatic nerves were harvested on day 3 for PDE10A staining (PDE10A, red; DAPI, blue). Representative immunofluorescence images of 2 animals from each group are shown. (B) Gene expression of inflammasomes. N=4 from each group. Data were expressed as mean ± SEM. Statistics in (A) were performed using one-way ANOVA. **p < 0.01; ***p < 0.001. To examine the effect of PDE10A inhibition on motor function recovery following nerve injury, we administered MP-10 or vehicle control subcutaneously, starting three hours after injury and continuing once daily for seven days. Motor recovery was assessed using the CatWalk gait analysis, and the sciatic functional index (SFI) was calculated based on walking footprint morphology on day 7 post-injury (normal SFI range: 0-10; impaired range: 80-100). The vehicle-treated group showed severe functional impairment, while the MP-10-treated group exhibited a significantly improved SFI ( Fig. 5A ). Download figure Open in new tab Fig. 5. PDE10A inhibition reduces gene expression of muscle atrophy and inflammasome activation Induced by sciatic nerve injury. (A) C57BL/6N mice underwent sciatic nerve crush injury and were treated with vehicle or MP-10 once daily (s.c. injection, 10 mg/kg). Sciatic functional index was measured by gait analysis on day 7 after injury (n=8 for each group). (B) Myogenin mRNA expression, (n=4 for each group), (C) Gadd45a mRNA expression in gastrocnemius muscle (n=4 for each group), (D) Cleaved IL-1β staining (green) in sciatic nerve. Representative immunofluorescence images of 4 animals from each group are shown. Scale bar = 200 µm. Data were expressed as mean ± SEM. Statistics in (A) were performed using one-way ANOVA. Statistics in (B-C) were performed using two-way ANOVA and Bonferroni’s post hoc test. *p < 0.05, **p < 0.01. Nerve injury also triggers the transcriptional regulation of muscle atrophy-related genes, such as Myogenin( 43 ) and Gadd45a( 44 , 45 ), which are critical regulators of muscle-related diseases and neuromuscular impairments. We observed that MP-10 treatment significantly reduced the expression of Myogenin and Gadd45a in the gastrocnemius muscle three days after crush injury, when compared to the vehicle-treated group ( Fig. 5B-C ). To investigate the effects of PDE10A inhibition on inflammasome activation, we analyzed cleaved IL-1β levels in the sciatic nerve by IHC three days after crush injury. Nerve injury induced a marked increase in IL-1β cleavage in the sciatic nerve ( Fig. 5D ). Importantly, MP-10 treatment significantly reduced IL-1β cleavage compared to the vehicle, indicating that PDE10A inhibition attenuates inflammasome activation ( Fig. 5D ). These results collectively suggest that PDE10A inhibition modulates inflammasome activation and improves functional recovery following traumatic nerve injury. Discussion This study provides compelling evidence that PDE10A plays a pivotal role in regulating inflammasome activation and pyroptosis in macrophages. Using both in vitro and in vivo models, we demonstrated that inhibition of PDE10A reduces inflammasome activation, mitigates inflammation, and improves functional recovery in conditions such as traumatic nerve injury and sepsis. Our findings suggest that PDE10A inhibitors, such as MP-10, may have therapeutic potential for treating diseases driven by excessive inflammation and inflammasome activation. One key observation was that PDE10A inhibition significantly reduced cell death in macrophages activated by the NLRP3 inflammasome. In a dose-dependent manner, the PDE10A inhibitor MP-10 protected human THP-1 macrophages from pyroptosis induced by LPS and nigericin. This suggests that PDE10A contributes to regulating inflammasome-induced cell death, and its inhibition may offer a protective effect in inflammatory conditions. The inflammasome pathway involves key events, including NLRP3 priming, ASC speck formation, and the activation of caspase-1 and gasdermin D (GSDMD). Activation of the NLRP3 inflammasome cleaves pro-caspase-1, leading to IL-1β processing and GSDMD cleavage. The resulting GSDMD fragment (GSDMD-NT) forms membrane pores, triggering cell rupture and the release of inflammatory mediators. Several studies have indicated that cAMP regulates NLRP3 inflammasome activation by inhibiting its assembly through multiple mechanisms, including cAMP-PKA inhibition of NF-ĸB activity( 46 ), direct cAMP binding to NLRP3( 47 ), and inhibition of caspase-1 activation( 48 ). Our results showed that MP-10 or TP-10 treatment significantly reduced cleaved caspase-1, cleaved GSDMD, and mature IL-1β levels, indicating that PDE10A is involved in regulating inflammasome activation. Furthermore, MP-10 inhibited ASC speck formation, a hallmark of inflammasome activation, suggesting that PDE10A modulates the assembly and activation of the NLRP3 inflammasome, but not downstream caspase-1 or GSDMD activity. These data underscore PDE10A’s role in regulating key steps of inflammasome activation and its potential to influence the severity of inflammasome-mediated inflammation. Excessive inflammasome activation and pyroptosis can exacerbate inflammation, contributing to the progression of inflammatory diseases( 31 ). In a clinically relevant acute sepsis model, PDE10A inhibition reduced inflammation and improved sepsis outcomes. Treatment with MP-10 led to a lower murine sepsis score (MSS), reduced plasma IL-1β levels, alleviated thrombocytopenia, and decreased fibrin deposition in the spleen. These findings indicated that PDE10A inhibition modulates inflammasome activation and improves inflammatory markers and organ damage in sepsis. Given that inflammasome overactivation is central to sepsis pathophysiology( 49 ), PDE10A represents a promising therapeutic target for diseases driven by inflammation, like sepsis. Additionally, PDE10A inhibition improved motor function recovery following traumatic nerve injury. In a sciatic nerve crush injury model, MP-10 treatment significantly enhanced motor recovery, as measured by the sciatic functional index (SFI), compared to vehicle-treated mice. This improvement was further supported by reduced expression of muscle atrophy-related genes, including Myogenin and Gadd45a, in the gastrocnemius muscle. By inhibiting PDE10A, MP-10 not only attenuated inflammasome activation but also mitigated some of the downstream effects of nerve injury. PDE10A expression has been associated with various injuries, including traumatic brain injury (TBI)( 50 , 51 ), ( 52 , 53 ), and spinal cord injury( 54 ). Consistent with our findings, PDE10A was highly induced after sciatic nerve crush injury. Emerging evidence also suggests that PDE10A inhibition can provide neuroprotection in TBI by reducing neuroinflammation and apoptosis via the cAMP/PKA pathway and downregulating NLRP3 inflammasome expression( 51 ). In our study, the reduced IL-1β cleavage in the sciatic nerve following MP-10 treatment further supported the idea that PDE10A inhibition modulates inflammasome activation in nerve injury, contributing to improved functional recovery and reduced muscle atrophy. These findings highlighted PDE10A as a critical regulator of inflammasome activation and neuroinflammation, with its inhibition offering therapeutic benefits in both immune-related disorders and recovery from traumatic nerve injury. While our study provides strong evidence that PDE10A inhibition modulates inflammasome activation and inflammation, the precise molecular mechanisms remain unclear. Future research should explore the downstream signaling pathways affected by PDE10A and its role in other immune cell types. Additionally, while our in vivo models suggest therapeutic potential for treating conditions like sepsis and traumatic nerve injury, further studies are needed to evaluate the clinical efficacy of PDE10A inhibitors. In conclusion, our study identified PDE10A as a key regulator of inflammasome activation and neuroinflammation. Inhibition of PDE10A reduced inflammasome-induced cytokine release, attenuated pyroptosis, and improved recovery following nerve injury and sepsis. These findings suggest that PDE10A represents a promising therapeutic target for treating inflammatory diseases and neuromuscular injuries driven by inflammasome activation. Acknowledgments This research was financially supported by the Department of Defense (DM190884, awarded to B.C.B.), the New York State Department of Health (C39071GG, awarded to B.C.B. and C.G.H.), the Trauma Research and Combat Casualty Care Collaborative (175153, awarded to C.G.H.), as well as The University of Texas at San Antonio Startup Funding and The College for Health, Community, and Policy (HCAP) Seed Grant (both awarded to C.G.H.). Footnotes Author Contributions C.G.H. designed research; C.G.H., and C.L.C. performed research; C.G.H., and B.C.B. contributed reagents/ analytic tools; C.G.H., and C.L.C. analyzed data; C.G.H., and B.C.B. wrote the paper. 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Berk , Camila Lage Chávez , Chia George Hsu bioRxiv 2025.03.17.643692; doi: https://doi.org/10.1101/2025.03.17.643692 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)