Beta-Hydroxybutyrate Attenuates Bronchial Smooth Muscle Pro-Inflammatory Cytokine Production

Beta-Hydroxybutyrate Attenuates Bronchial Smooth Muscle Pro-Inflammatory Cytokine Production | 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 Beta-Hydroxybutyrate Attenuates Bronchial Smooth Muscle Pro-Inflammatory Cytokine Production View ORCID Profile V. Amanda Fastiggi , View ORCID Profile Madeleine M. Mank , View ORCID Profile Matthew E. Poynter doi: https://doi.org/10.1101/2025.02.19.639048 V. Amanda Fastiggi 1 Department of Medicine, Division of Pulmonary Disease and Critical Care, University of Vermont, and The Vermont Lung Center , Burlington, VT, 05405, USA 2 Cellular, Molecular, and Biomedical Sciences Doctoral Program, University of Vermont , Burlington, VT, 05405, USA BS Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for V. Amanda Fastiggi Madeleine M. Mank 1 Department of Medicine, Division of Pulmonary Disease and Critical Care, University of Vermont, and The Vermont Lung Center , Burlington, VT, 05405, USA MPH Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Madeleine M. Mank Matthew E. Poynter 1 Department of Medicine, Division of Pulmonary Disease and Critical Care, University of Vermont, and The Vermont Lung Center , Burlington, VT, 05405, USA PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Matthew E. Poynter For correspondence: matthew.poynter{at}uvm.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Asthma is a common airway condition causing breathing difficulties due to reversible airflow obstruction. It often affects obese individuals, with symptoms triggered by environmental factors that induce immune responses, leading to inflammation and bronchoconstriction. Bronchial smooth muscle (BSM) plays a central role in airway narrowing, driven by type 2 immune responses involving cytokines like IL-4, IL-5, and IL-13, along with leukocytes including eosinophils and type 2 T-helper cells. These responses cause structural changes such as fibrosis and airway thickening, while BSM cells worsen asthma by releasing pro-inflammatory cytokines in response to allergens, microbial signals, or inflammatory cytokines from other cells. While current treatments manage asthma in most patients, alternative therapies are needed for difficult-to-treat cases, particularly prevalent in obese, allergic individuals. Emerging research suggests that therapeutic ketosis, induced by dietary changes or ketone supplementation, may reduce airway hyperresponsiveness and inflammation. The primary ketone body, β-hydroxybutyrate (BHB), produced during carbohydrate scarcity, acts via cell-surface receptors and transporters, potentially mitigating asthma symptoms. Weight loss and caloric restriction increase ketone levels, correlating with reduced inflammation and improved asthma outcomes. We hypothesized that β-hydroxybutyrate (BHB) reduces bronchoconstriction and inflammation in asthma by targeting bronchial smooth muscle. Using human bronchial smooth muscle cells (HBSMC) in vitro, we demonstrate herein that BHB suppresses IL-1β-induced pro-inflammatory cytokine production through Free Fatty Acid Receptor 3 (FFAR3) activation. These findings suggest that bronchial smooth muscle is a key target of therapeutic ketosis, supporting BHB’s potential benefits in preclinical asthma models. Introduction Asthma, a condition affecting the airways, is typically provoked by environmental triggers that initiate immune reactions in the lungs, leading to excessive inflammation and bronchoconstriction, and affects approximately 8.9% of adults and 6.7% of children in the United States 1 . Asthma-associated airway hyperresponsiveness can be evaluated using the methacholine challenge test, a diagnostic procedure designed to assess the responsiveness of airway smooth muscle. This test involves the inhalation of methacholine, a substance that causes airway constriction in individuals with asthma or other airway hyperreactivity conditions. By monitoring the degree of airway narrowing in response to methacholine, the test helps healthcare providers evaluate the functionality of the airway smooth muscle and plays a crucial role in confirming the diagnosis of asthma 2 , 3 . Bronchial smooth muscle is central to the pathophysiology of asthma, as its contraction causes airway narrowing, resulting in obstruction and breathing difficulties 4 . Bronchial smooth muscle has been implicated in the pathophysiology of bronchial inflammation both as a target as well as a mediator of inflammatory reactions 5 , 6 . Type 2 inflammation is most common in asthmatics and is associated with the cytokines interleukin(IL)-4, IL-5, and IL-13, inflammatory cells including eosinophils, mast cells, basophils, type 2 innate lymphoid (ILC2) and T helper (Th2) lymphocytes, and immunoglobulin E (IgE)-producing plasma cells 7 , 8 . These asthma-relevant cell types can release pro-inflammatory mediators such as cytokines and chemokines that can affect bronchial smooth muscle cells 6 , 9 , 10 , the products of which further exacerbate airway hyperresponsiveness and remodeling 4 , 5 , 10 – 12 . Over time, this chronic inflammation can lead to structural changes in the airways, including thickening of the airway walls, fibrosis, and increased mucus production, which in aggregate contribute to the persistent symptoms of asthma 11 , 13 . We and others have previously reported that bronchial smooth muscle cells secrete pro-inflammatory cytokines in response to immune stimulants, including lipopolysaccharide (LPS) and house dust mite (HDM) extract, as well as cytokines such as IL-1β 10 , 14 produced from the other asthma-relevant cell types. IL-1β is a potent pro-inflammatory cytokine produced from both immune (leukocytes) and non-immune cells 12 , 15 . Importantly, IL-1β is elevated in the airways 16 and serum of allergic and non-allergic asthmatic subjects compared to controls 17 . Current treatments for asthma include broncho-relaxing β-agonists, anti-inflammatory corticosteroids, and biological immunotherapies targeting the type 2 immune response 18 . While these therapies provide effective disease control for most patients, individuals with ’difficult-to-treat’ asthma often require alternative or additional approaches, especially as severe asthmatics with type 2 inflammation exhibit poor response to corticosteroids 19 . Recently, we have demonstrated the beneficial effects of increasing circulating ketone bodies, known as therapeutic ketosis, in multiple mouse models of obesity-associated 20 and allergic asthma 21 . Ketone bodies, β-hydroxybutyrate (BHB) and acetoacetate (AcAc), are synthesized in the liver from fatty acids 22 , 23 , either through dietary modifications 23 , 24 or the mobilization of adipose tissue during periods of increased energy demand 23 . Once produced, these ketone bodies are transported via the bloodstream to cells throughout the body. Ketone bodies have been implicated in modulating key pathological processes in asthma. Initially recognized as an energy substrate for ATP production in the Krebs cycle during carbohydrate scarcity, ketone bodies have since been shown to influence cellular processes through various mechanisms. They can stimulate cell-surface receptors such as the G-protein coupled receptors Hydroxycarboxylic Acid Receptor 2 (HCAR2 or GPR109a) and Free Fatty Acid Receptor 3 (FFAR3 or GPR41) 23 , 25 – 29 , or by uptake through transporters such as Monocarboxylate Transporter 1 (MCT1) 27 , 30 . Beyond these roles, ketone bodies also function as antioxidants 31 , 32 and exert effects through transcriptional and epigenetic regulation 22 , 27 , 33 , including suppression of nuclear factor-κB (NF-κB) activation 31 , 25 . Furthermore, they possess anti-inflammatory properties, notably through inhibition of the NLRP3 inflammasome, which decreases IL-1β production 34 – 36 . In vivo , dietary interventions such as weight loss and alternate-day caloric restriction raise BHB levels, which correlate with reduced asthmatic symptoms, including lower oxidative stress and inflammation in obese asthmatic patients 14 , 37 , 38 . Early ketone body elevations are linked to improved asthma symptoms in obese patients following bariatric surgery 39 , 40 , alternate-day caloric restriction 37 , and during treatment with GLP-1R agonists 41 . Independent of weight loss, therapeutic ketosis can be achieved through providing exogenous ketones or ketogenic precursors ( e.g., ketone esters). Therapeutic ketosis can be induced by providing exogenous ketones or ketogenic precursors (e.g., ketone esters). Notably, increasing ketone levels is generally well-tolerated in humans 42 , 43 , highlighting its potential as a viable therapeutic approach. Therapeutic ketosis achieved through feeding a high fat/low carbohydrate diet or by supplementing the normal diet with ketone esters, significantly and substantially decreases methacholine hyperresponsiveness in mouse models of asthma 20 , 21 and also decreases methacholine responsiveness in non-asthmatic mice 20 . As methacholine functions through the activation of bronchial smooth muscle 44 – 47 , we sought to study these cells more directly to explore the impact of ketone bodies on the ability of bronchial smooth muscle to contribute to the inflammatory environment in asthma. We have established a well-functioning in vitro model using human bronchial smooth muscle cells (HBSMC), allowing us to directly observe the effects of BHB on this cell type 21 . Herein, we used HBSMC as an in vitro model of bronchial smooth muscle to address gaps and provide a valuable platform for investigating the therapeutic potential of ketone bodies and their underlying mechanisms of action. We hypothesized that BHB can mitigate asthma-related pathologies, in part, by inhibiting pro-inflammatory cytokine production from bronchial smooth muscle cells. Our objectives were to assess the effectiveness of BHB in reducing IL-1β-induced pro-inflammatory cytokine secretion in vitro and to identify the mechanisms by which BHB may influence these effects. Gaining a deeper understanding of the efficacy and mechanisms by which BHB reduces pro-inflammatory cytokine production by human bronchial smooth muscle in vitro could offer valuable insights into potential novel targets for treating asthma and its associated symptoms. Materials and Methods Study Approval Studies involving potentially hazardous material were reviewed and approved by the University of Vermont’s Institutional Biosafety Committee (REG201900052). Human Bronchial Smooth Muscle Cell Culture Primary human bronchial smooth muscle cells (HBSMC) isolated from a 45-yr-old female patient with asthma (Lonza, Morristown, NJ, Lot No. 00194850, Batch No. 0000195154) were cultured in smooth muscle cell growth medium-02 BulletKit (Lonza, CC-3182) according to the manufacturer’s instructions at 37°C in 95% humidified air containing 5% CO 2 . The cells were used within the first seven passages to ensure proper smooth muscle phenotype. Cell authentication was performed by Lonza (negative Factor VIII-related antigen, positive α-Actin expression) and cells tested negative for mycoplasma (MycoDect Mycoplasma Detection Kit (Alstem, Richmond, CA)) before being utilized for experiments. For agonist-induced cytokine production experiments, human bronchial smooth muscle cells (HBSMC) were stimulated with 50 ug/mL House Dust Mite extract (HDM) (Stagrallery/Greer, Cat No.B70, Lot No.390992), 100 ng/mL ultrapure lipopolysaccharide (LPS) from Escherichia coli 0111:B4 (Invivogen, Cat No.tlrl-3pelps), or 10 ng/mL recombinant human IL-1β (Stem Cell Technologies, Cat No.78034.1). For simultaneous exposure and stimulation experiments, HBSMC were plated at 5×10 4 cells/cm 2 in 1mL of media in a 12-well plate and allowed to grow for 48 hours at 37°C and 5% CO 2 . HBSMC were exposed for 24 hours with vehicle or 1.25-10 mM beta-hydroxybutyric acid (BHBA) (Sigma-Aldrich, St. Louis, MO, Cat No.166898), sodium beta-hydroxybutyrate (NaBHB)(Sigma-Aldrich, Cat No.54965), (R)-beta-hydroxybutyric acid ((R)-BHBA) (Sigma-Aldrich, Cat No.54920), (S)-beta-hydroxybutyric acid ((S)-BHBA) (Sigma-Aldrich, Cat No.54925), FFAR3 agonist, AR420626 (AR) (Caymen, Cat No.17531), or MCT1 inhibitor, AZD3965 (Med Chem Express, Cat No.HY-12750), butyric acid (BA), sodium butyrate (SB), nicotinic acid (NA), and sodium nicotinic acid (NaNA) while being stimulated with 10 ng/mL recombinant human IL-1β (Stem Cell Technologies, Cat No.78034.1). In pre-exposure experiments with subsequent stimulation, HBSMC were plated at 5×10 4 cells/cm 2 in 1mL of media in a 12-well plate and allowed to grow for 24 hours at 37°C and 5% CO 2 . After the initial 24 hours, cells were exposed to vehicle or 1.25-10 mM BHBA, NaBHB, (R)- BHBA, (S)-BHBA, or AR for another 24 hours before being washed and stimulated with 10 ng/mL recombinant human IL-1β. Cytokine Immunoassays Conditioned medium from the cell culture studies was collected at the indicated time points, centrifuged for 10 minutes at 3300 x g to eliminate debris, transferred into new tubes or multi-well plates, and frozen at -20°C until analysis. ELISAs to quantitate human IL-8/CXCL8 and IL-6 levels (DuoSets from R&D Systems) were used according to the manufacturer recommendations, with samples diluted to coincide with the range of the standards. A custom human magnetic Luminex assay (R&D Systems, Minneapolis, MN) was performed per manufacturers’ instructions to measure cytokines present in the HBSMC supernatants, including CCL2, CCL4, CCL20, CXCL1, CXCL2, and G-CSF. Luminex assays were performed using the Bio-Rad Bio-Plex suspension array system, Bio-Rad Bio-Plex Pro II wash station, and Bio-Plex Manager 6.0 software (Bio-Rad, Hercules, CA). Concentrations were calculated by five-place logistic regression from standards within 70%-130% of the expected values using Bio-Rad Manager 6.0. Studies were conducted with all BHB compounds used in the experiments to validate that they did not interfere substantially with the ability of the ELISAs to accurately quantify the recombinant standards. Statistical Analyses All experiments included multiple biological replicates for each condition. Outliers were identified and removed using the ROUT method (Q=1%) and the cleaned data were analyzed using unpaired one-way ANOVA with post-hoc multiple comparisons: Tukey’s test (for comparing all means) or Dunnett’s test (for comparing each mean to a control), were performed using GraphPad Prism 10.2.3 (GraphPad Software, Inc., La Jolla, CA). Data are presented as means ± SEM from representative experiments. P values below 0.05 are considered statistically significant, and significance levels are indicated in the figure legends. Results Human Bronchial Smooth Muscle Cells (HBSMC) Inducibly Secrete Pro-Inflammatory Cytokines Although known primarily as a contractile cell type, bronchial smooth muscle can produce many chemokines and cytokines in response to various agonists 10 , 14 , 48 . A custom human magnetic Luminex assay was performed to measure cytokines present in the HBSMC supernatants following stimulation for 24 hours with House Dust Mite extract (HDM), lipopolysaccharide (LPS), and human recombinant IL-1β to determine which pro-inflammatory cytokines were produced. This analysis revealed that CCL2 ( Figure 1A ) , CCL4 ( Figure 1B ) , CCL20 ( Figure 1C ) , CXCL1 ( Figure 1D ) , CXCL2 ( Figure 1E ) , G-CSF ( Figure 1F ) , IL-6 ( Figure 1G ) , and CXCL8/IL-8 ( Figure 1H ) were produced significantly when HBSMC were stimulated with 10 ng/mL of recombinant human IL-1β. Download figure Open in new tab Figure 1. IL-1β induces pro-inflammatory cytokine secretion from HBSMC. HBSMCs were unstimulated (Unstim) or stimulated in vitro with 50 mg/mL House Dust Mite extract (HDM), 100 ng/mL lipopolysaccharide (LPS), or 10 ng/mL recombinant human IL-1β for 24 hours and (A) CCL2, (B) CCL20, (C) G-CSF, (D) CCL4, (E) CXCL2, (F) CXCL1 (G) IL-6 and (H) CXCL8/IL-8 levels were measured. ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001, ∗∗∗∗ P ≤ 0.0001 compared to the vehicle. BHB Attenuates IL-1β-Induced Pro-Inflammatory Cytokine Production From HBSMC Ketone bodies, acetoacetate (AcAc) and especially β-hydroxybutyrate (BHB), have been reported to exert anti-inflammatory effects, especially via inhibition of NLRP3 inflammasome activation 34 – 36 . In our recent studies, we demonstrated the therapeutic benefits of elevating circulating ketone bodies—a state known as therapeutic ketosis—in multiple mouse models of both obese 20 and allergic asthma 21 , which effectively mitigated methacholine-induced airway hyperresponsiveness and significantly improved lung function in vivo 20 , 21 . We also demonstrated in vitro that BHB attenuated pro-inflammatory cytokine secretion from several asthma-relevant cell types, including macrophages, CD4 T lymphocytes, and airway epithelium 21 . Building on these findings, we investigated the effects of ketone bodies on IL-1β-induced pro-inflammatory cytokine production from human bronchial smooth muscle cells (HBSMC). We determined that simultaneous exposure for 24 hours to beta-hydroxybutyric acid (BHBA) or sodium beta-hydroxybutyrate (NaBHB) in the presence of 10ng/mL human recombinant IL-1β caused a dose-dependent decrease in the concentrations of the pro-inflammatory cytokines IL-6 ( Figure 2A ) and CXCL8/IL-8 ( Figure 2B ) compared to cells stimulated with IL-1β in the absence of BHB or in the presence of appropriate pH controls. BHBA significantly decreased both IL-6 and IL-8 concentrations in a dose-dependent manner, but when neutralized to a pH of 7 the robust effect in attenuating IL-6 secretion at all concentrations and in attenuating IL-8 secretion at a BHBA concentration of 10mM was lost. NaBHB did not exert a robust inhibitory effect on pro-inflammatory cytokine production until the pH of the 10mM working solution was matched to the pH of the working concentration of 10mM BHBA. The acidified NaBHB exerted a more robust inhibitory dose response than the non-acidified NaBHB or the neutralized BHBA. An equivalent concentration of HCl matching the acidity (pKa) of the acidic BHB molecules (10mM) modestly attenuated IL-1β-induced secretion of IL-6 and IL-8, but not to the extent of the BHBA or the acidified NaBHB. Download figure Open in new tab Figure 2. BHB dose-dependently attenuates IL-1β-stimulated pro-inflammatory cytokine production from HBMSC. HBSMCs were unstimulated (Unstim) or stimulated in vitro with 10 ng/mL recombinant human IL-1β in the presence of increasing concentrations of BHBA, NaBHB, or the appropriate pH controls of each for 24 hours and (A) IL-6 or (B) CXCL8/IL-8 were measured. n=4 per group; values are inclusive of studies performed three times. ∗∗∗∗ P ≤ 0.0001 compared to the vehicle, # P ≤ 0.05, ## P ≤ 0.0, ### P ≤ 0.001, #### P ≤ 0.0001 compared to IL-1β. Inhibition of Pro-Inflammatory Cytokine Production from HBSMC by BHB Is Not Enantioselective To determine if the inhibitory effect elicited by BHB on IL-1β-induced IL-6 and IL-8 production was through a mechanism dependent on the metabolism of BHB as an energetic substrate, we utilized (R)-beta-hydroxybutyric acid ((R)-BHBA) and (S)-beta-hydroxybutyric acid ((S)-BHBA), the individual enantiomers of the racemic BHBA, as well as included exposure to the (R) enantiomer of NaBHB, and a cocktail representative of circulating ketone bodies in vivo . The BHB cocktail consists of 78% BHB, 20% AcAc, and 2% acetone, which are biologically consistent with the concentrations of ketone bodies in circulation during fasting 49 . In this study, with simultaneous exposure to the BHB compounds and 10 ng/mL IL-1β for 24 hours, we determined that both the (R) and (S) enantiomers, the (R) enantiomer of NaBHB, and the BHB cocktail significantly inhibited IL-1β-induced secretion of IL-6 ( Figure 3A ) and CXCL8/IL-8 ( Figure 3B ) from HBSMC. In this study, all conditions except the (R) enantiomer of NaBHB demonstrated dose-dependent inhibition of pro-inflammatory cytokine production. Download figure Open in new tab Figure 3. BHB enantiomers dose dependently attenuate IL-1β-stimulated pro-inflammatory cytokine production from HBSMC. HBSMCs were unstimulated (Unstim) or stimulated in vitro with 10 ng/mL recombinant human IL-1β in the presence of increasing concentrations of (R)-BHBA, (S)-BHBA, Na-(R)-BHBA, or a BHB cocktail composed of 78% (R)-BHBA, 20% AcAc, and 2% acetone, for 24 hours and (A) IL-6 or (B) CXCL8/IL-8 were measured. n=4 per group; values are inclusive of studies performed three times. ∗∗∗∗ P ≤ 0.0001 compared to the vehicle, # P ≤ 0.05, ## P ≤ 0.01, ### P ≤ 0.001, #### P ≤ 0.0001 compared to IL-1β. Short-Chain Carboxylic Acids Inhibit IL-1β-Induced Pro-Inflammatory Cytokine Production From HBSMC As BHB has been proposed to elicit effects through activation of cell surface receptors such as hydroxycarboxylic acid receptor 2 (HCAR2) 22 23 25 26 27 , we sought to determine if a pharmacological HCAR2 agonist would elicit similar beneficial inhibitory effects as BHB. Furthermore, as we have previously shown that small molecules structurally similar to BHB have anti-inflammatory effects 34 , and that short-chain carboxylic acids exert anti-inflammatory functions 50 , 51 , we wanted to determine if butyric acid and sodium butyrate could elicit the same inhibitory effects as BHB. When HBSMC were simultaneously exposed to butyric acid, sodium butyrate, nicotinic acid, and sodium nicotinic acid, as well as the HCAR2 agonist, and stimulated with 10ng/mL IL-1β, production of IL-6 ( Figure 4A ) and CXCL8/IL-8 ( Figure 4B ) was dose-dependently attenuated. Whereas the nicotinic acid and the sodium nicotinic acid both significantly inhibited secretion of the pro-inflammatory cytokines, the butyric acid and sodium butyrate had a much more substantial effect, with the lowest doses of butyric acid and sodium butyrate having a greater impact than the highest dose of exposure, which was 10mM of the nicotinic acid or sodium nicotinic acid. Download figure Open in new tab Figure 4. Short-chain carboxylic acids inhibit IL-1β-induced pro-inflammatory cytokine secretion. HBSMCs were unstimulated (Unstim) or stimulated in vitro with 10 ng/mL recombinant human IL-1β in the presence of increasing concentrations of butyric acid (BA), sodium butyrate (SB), nicotinic acid (NA), or sodium nicotinic acid (NaNA) for 24 hours and (A) IL-6 or (B) CXCL8/IL-8 were measured. n=4 per group; values are inclusive of studies performed three times. ∗∗∗∗ P ≤ 0.0001 compared to the vehicle, # P ≤ 0.05, ## P ≤ 0.01, ### P ≤ 0.001, #### P ≤ 0.0001 compared to IL-1β. Pre-Exposure of HBSMC to BHBA Attenuates IL-1β-Induced Pro-Inflammatory Cytokine Production As our previously reported in vivo studies have modeled endogenous ketone augmentation through dietary interventions that provide elevated systemic concentrations of these molecules over a protracted period, we sought to model this extended exposure in an in vitro system. HBSMC were pre-exposed to biologically relevant concentrations of beta-hydroxybutyric acid (BHBA) for 24 hours, followed by washing and subsequent stimulation with 10 ng/mL human recombinant IL-1β. Pre-exposure to BHBA significantly attenuated IL1β-induced IL-6 ( Figure 5A ) and CXCL8/IL-8 ( Figure 5B ) production in a dose-dependent manner, consistent with the inhibitory effects observed during simultaneous exposure of BHBA and IL-1β. While not as robust as simultaneous stimulation, pretreatment with the higher concentrations of 5mM and 10mM BHBA significantly attenuated IL-6 and IL-8 production from these cells. Although not statistically significant, a trend towards a dose-responsive effect was observed. Download figure Open in new tab Figure 5. Pre-exposure to BHBA dose-dependently decreases IL-1β-induced pro-inflammatory cytokine secretion. HBSMCs remained unexposed or were exposed in vitro to increasing concentrations of beta hydroxybutyric acid (BHBA) for 24 hours, washed and then stimulated with 10 ng/mL recombinant human IL-1β for another 24 hours. (A) IL-6 or (B) CXCL8/IL-8 were then measured. n=4 per group; values are inclusive of studies performed three times. ∗∗∗ P ≤ 0.001 compared to the vehicle, # P ≤ 0.05, ## P ≤ 0.01, ### P ≤ 0.001, #### P ≤ 0.0001 compared to IL-1β. Simultaneous Exposure of BHBA with FFAR3 Agonist Dose-Dependently Attenuates IL-1β-Induced Pro-Inflammatory Cytokine Production Ketone bodies, particularly BHB, have been proposed to mediate their beneficial effects via uptake through Monocarboxylic Transporter 1 (MCT1) 26 , 43 or through a Free Fatty Acid 3 (FFAR3)-dependent pathway 26 – 28 , 52 . To evaluate the involvement of these receptors, we tested the MCT1 inhibitor AZD3965 (AZD) in the presence of the BHB compounds. HBSMCs were exposed to biologically relevant concentrations of BHB to model endogenous ketone augmentation, a combination of both the increasing concentrations of BHB and 50µM of the FFAR3 agonist, or a combination of increasing concentrations of BHB and 40nM MCT1 inhibitor (AZD) while stimulated with 10 ng/mL recombinant human IL-1β for 24 hours. The FFAR3 agonist in combination with BHB dose-dependently attenuated IL-6 ( Figure 6A ) and CXCL8/IL-8 ( Figure 6B ) production, and to a greater extent than BHBA alone. In contrast, the presence of AZD did not block the inhibitory effects of BHB on IL-1β-induced pro-inflammatory cytokine production, indicating that these effects are independent of MCT1-mediated uptake of BHB. Download figure Open in new tab Figure 6. Simultaneous exposure to BHBA with a FFAR3 agonist or MCT1 inhibitor dose-dependently attenuates IL-1β induced pro-inflammatory cytokine secretion. HBSMCs were unstimulated (Unstim) or stimulated in vitro with 10 ng/mL recombinant human IL-1β in the presence of increasing concentrations of beta hydroxybutyric acid (BHBA), a combination of FFAR3 agonist (AR) and BHBA, or a combination of MCT1 inhibitor (AZD) and BHBA for 24 hours and (A) IL-6 or (B) CXCL8/IL-8 were measured. n=4 per group; values are inclusive of studies performed three times. ∗∗∗∗ P ≤ 0.0001 compared to the vehicle, # P ≤ 0.05, ## P ≤ 0.01, #### P ≤ 0.0001 compared to IL-1β. FFAR3 Activation Is Sufficient to Inhibit IL-1β-Induced HBSMC Pro-Inflammatory Cytokine Production Given that beta-hydroxybutyrate (BHB) has been suggested to act as a ligand for FFAR3 and exert its beneficial effects through a FFAR3-mediated pathway, we investigated whether activating FFAR3 could replicate the inhibitory effects of BHB on IL-1β-induced pro-inflammatory cytokine production from HBSMC. Whereas in our previous study we evaluated the combination of BHBA and FFAR3 agonist, we sought to determine whether FFAR3 activation alone could elicit the same effects. HBSMC were exposed to biologically relevant concentrations of BHBA, 50µM FFAR3 agonist (AR) alone, or to a combination of the concentrations of BHBA and 50µM FFAR3 agonist. HBSMCs exposed to BHBA and AR individually, as well as those exposed to the combination of BHBA and the FFAR3 agonist, significantly attenuated IL-1β-induced HBSMC production of IL-6 ( Figure 7A ) and CXCL8/IL-8 ( Figure 7B ) , suggesting that the observed inhibitory effects of BHB may indeed be mediated, at least in part, through the activation of FFAR3. Notably, whereas the FFAR3 agonist (AR) had a robust effect alone, the combination of the FFAR3 agonist and BHBA exerted the greatest inhibition, signifying a synergistic effect. Download figure Open in new tab Figure 7. FFAR3 activation is sufficient to inhibit IL-1β induced pro-inflammatory cytokine secretion. HBSMCs were unstimulated (Unstim) or stimulated in vitro with 10 ng/mL recombinant human IL-1β in the presence of increasing concentrations of beta hydroxybutyric acid (BHBA), a combination of FFAR3 agonist (AR) and BHBA, or a combination of MCT1 inhibitor (AZD) and BHBA for 24 hours and (A) IL-6 or (B) CXCL8/IL-8 were measured. n=4 per group; values are inclusive of studies performed three times. ∗∗∗∗ P ≤ 0.0001 compared to the vehicle, # P ≤ 0.05, ## P ≤ 0.01, #### P ≤ 0.0001 compared to IL-1β. Discussion The growing asthma epidemic underscores the urgency for new treatment approaches aimed at managing severe and challenging endotypes, ultimately enhancing the well-being of individuals affected by this complex and chronic disorder. One promising strategy that has attracted increasing interest is therapeutic ketosis, recognized for its wide-ranging beneficial effects across various pathological conditions and diseases 53 – 56 . In our previous studies, we reported that therapeutic ketosis, achieved through dietary interventions such as a ketogenic diet, ketogenic precursor supplementation, or ketone ester intake, augments circulating concentrations of BHB and reduces methacholine hyperresponsiveness, a pathophysiological feature in models of preclinical allergic-associated and obesity-associated asthma 20 , 21 , decreases airway inflammation, and inhibits a multitude of pathological activities of asthma relevant cell types 20 , 21 . Several other studies have shown the in vivo anti-inflammatory activities of therapeutic ketosis 36 , 57 , 58 , with some affecting lung inflammation. Although we previously demonstrated that exogenous ketones reduce pro-inflammatory cytokine production and exert anti-inflammatory effects in vitro 20 , 21 , their impact on bronchial smooth muscle cells remains unexplored. Given that bronchial smooth muscle cells are key players in bronchial inflammation, acting as both targets and mediators of inflammatory responses 5 , 6 , investigating their cytokine production represents a critical research area in airway inflammation and asthma management. In preclinical asthma models, distal airway and peripheral lung dysfunction are commonly observed 59 . Our prior work showed that elevated BHB concentrations confer protective effects on these regions 20 , 21 , including reductions in methacholine-induced hyperresponsiveness, airway resistance, tissue damping, and tissue elastance—physiological markers that increase due to heterogeneous ventilation caused by uneven airway narrowing 59 , 60 that are closely linked to peripheral airway contraction. These findings suggest that ketones may directly modulate bronchial smooth muscle to reduce airway hyperresponsiveness. As reported herein, our studies demonstrate that BHB inhibits bronchial smooth muscle pro-inflammatory cytokine production induced by the asthma-relevant agonist, IL-1β 10 , 12 , 14 – 17 . The mechanisms by which BHB can elicit these effects may include activation of cell surface receptors such as free fatty acid receptor 3 (FFAR3) 22 , 23 , 27 , 28 or hydroxycarboxylic acid 2 (HCAR2) 25 , 26 , 29 . Given that BHB has been proposed to function across a wide range of applications and through many G-coupled protein receptors (GCPRs) 22 , 25 – 29 , including perhaps inducing tachyphylaxis, it likely operates through a fundamental mechanism that is universally relevant to these processes, such as ion exchange 27 , 36 , 61 , calcium signaling 27 , 28 , or altering membrane potential 61 . Once determining that IL-1β significantly caused pro-inflammatory cytokines to be produced from human bronchial smooth muscle cells (HBSMC), our in vitro studies demonstrated that BHB attenuated the production of IL-6 and IL-8 (CXCL8) in a dose-dependent manner. These studies included conditions using beta-hydroxybutyric acid (BHBA) as well as sodium beta hydroxybutyrate (NaBHB). The inclusion of both of these compounds in our studies allowed us to explore the influence of pH by comparing BHBA with a 1M stock pH of 2.09 and the NaBHB, with a 1M stock pH of 9.73. When diluted to biologically relevant concentrations in buffered media, which resulted in near-neutral pH in the cell culture media ( i.e. , pH 7.2), only BHBA had a significant inhibitory effect on IL-1β-induced production of pro-inflammatory cytokines in vitro . These results suggest that the acidic nature of the BHBA influences its ability to inhibit pro-inflammatory cytokine production. The inclusion of pH controls supports this notion in that when the 1M stock pH of the NaBHB was changed to be reminiscent of the 1M stock pH of the BHBA, the inhibitory effects of BHB were then present. Conversely, when the 1M stock pH of the BHBA was adjusted to the 1M stock pH of the NaBHB, the inhibitory effects were lost. The inclusion of a hydrochloric acid (HCl) pH-equivalent control, which did not inhibit pro-inflammatory cytokine production from IL-1β stimulated HBSMCs, implies that although the acid is sufficient to elicit modest inhibitory effects, the inclusion of BHB is necessary to enhance the effect. BHBA is a racemic mixture containing both enantiomers (R)-BHB and (S)-BHB. Since only (R)-BHB can be efficiently metabolized by β-hydroxybutyrate dehydrogenase to form acetyl-CoA that is subsequently converted into ATP and used as an energy substrate 62 , we confirmed that the attenuation of pro-inflammatory cytokine production occurs independently of BHB’s metabolic function. Additionally, the MCT1 inhibitor, AZD3965 (AZD) failed to block BHB’s effects, indicating that MCT1-mediated uptake is not involved in the inhibitory capacity of BHB. The comparison between the (R)-BHB and the (R) enantiomer of the NaBHB (Na-R-BHB) reiterates that the acid allows for a robust pro-inflammatory cytokine production to be inhibited. The HCAR2 agonists, nicotinic acid and sodium nicotinic acid, elicited similar inhibitory effects, but not to the extent of BHBA or the structurally similar compounds butyric acid and sodium butyrate, suggesting that some inhibitory effects may be mediated though HCAR2. Most likely, BHB does not elicit its attenuating effects on IL-1β-induced pro-inflammatory cytokine production through activation of HCAR2. Furthermore, the FFAR3 agonist AR420626 elicited inhibitory effects similar to and in an additive manner with BHB, suggesting that FFAR3 activation contributes substantially to the inhibitory action of BHB. Confirmation of this action would require an FFAR3 antagonist, a compound that is not currently available. Despite this limitation, these findings support future investigations, including studies using FFAR3 knockdown cells and FFAR3 knockout mice, to clarify the receptor’s involvement. Simultaneous exposure to BHBA during IL-1β stimulation was more effective at reducing IL-1β-induced pro-inflammatory cytokine production in HBSMCs compared to BHBA pre-exposure and subsequent stimulation with IL-1β. This effect may be partly due to greater cytokine induction during simultaneous exposure, likely caused by increased cell confluency from the additional 24 hours in culture. Nonetheless, in a clinical setting, ‘therapeutic ketosis’ would maintain consistently elevated BHB concentrations, which could be modeled in vitro using the pre-exposure approach without removing BHBA before assessing IL-1β-induced pro-inflammatory cytokine production from HBSMCs. There are several limitations to our findings. Namely, we conducted in vitro studies using primary human bronchial smooth muscle cells instead of ex vivo studies or human subjects. While informative, human cell studies fail to capture the prolonged and complex nature of human asthma. They offer a reductionist approach by focusing on a single cell type and a specific agonist that readily initiates an inflammatory cascade, allowing us to observe only the inhibitory effects of BHB at that initial step without assessing its role in maintaining suppression or facilitating recovery. Another notable limitation of this study is the lack of assessment of the fibrotic remodeling response of bronchial smooth muscle to IL-1β stimulation. In our previous work, we used an in vivo mouse model of severe asthma characterized by fibrotic remodeling and found that therapeutic ketosis still produced beneficial effects 21 . Overall, these findings support the potential of therapeutic ketosis as a promising treatment strategy for asthma, demonstrating its efficacy in both fibrotic and non-fibrotic contexts and highlighting its broad anti-inflammatory and protective effects. To gain a more comprehensive understanding of BHB’s impact on the long-term outcomes of an asthma exacerbation, diverse models, including in vivo , ex vivo , more advanced in vitro systems, and in clinic studies could be used to evaluate not only the initiation but also the maintenance and resolution of inflammation. Therapeutic ketosis is being explored in the clinical treatment of respiratory diseases, including asthma 63 , 64 and Cystic fibrosis 65 . In asthma trials, medium-chain triglyceride supplementation is used as a substrate to promote ketone body formation in vivo , whereas Cystic fibrosis trial uses a ketone ester precursor, which more rapidly and efficiently increases circulating ketone body concentrations and is the same compound we have employed in mouse models of obese asthma and allergic asthma 20 , 21 . The inclusion of therapeutic ketosis in clinical trials highlights the increasing scientific interest in its potential benefits. The positive results of the studies reported herein would strengthen the case for investigating therapeutic ketosis into asthma treatment regimens and stimulate further mechanistic and clinical studies into its capacity to serve as a complementary or alternative asthma therapy. This ongoing exploration emphasizes the importance of understanding ketone-based interventions and calls for continued examination of their underlying mechanisms and therapeutic potential. Grant Support This work was funded by National Institute of Health grants R01 HL142081 and T32 HL076122, the Vermont Space Grant Consortium (VTSGC) Graduate Fellowship Program, and the CMB-Graduate Assistance in Areas of National Need (CMB-GAANN) Training Grant. Disclosures All authors were supported by NIH grants and have declared that no relevant conflicts of interest exist. Graphical Abstract In accordance with journal requirements, a graphical abstract will be submitted if we are invited to revise this manuscript. Abbreviations AcAc acetoacetate ANOVA analysis of variance BHB beta-hydroxybutyrate FFAR free fatty acid receptor GPR G protein-coupled receptor HBSMC human bronchial smooth muscle cells HCAR hydroxycarboxylic acid receptor HDM house dust mite IL- interleukin- PBS phosphate-buffered saline MCT1 monocarboxylate transporter 1 SEM standard error of the mean Footnotes This work was funded by National Institute of Health grants R01 HL142081 and T32 HL076122, the Vermont Space Grant Consortium (VTSGC) Graduate Fellowship Program, and the CMB-Graduate Assistance in Areas of National Need (CMB-GAANN) Training Grant. VAF and MEP designed research studies, conducted experiments, acquired data, analyzed data, prepared figures, and wrote the manuscript. MMM conducted experiments, acquired data, and analyzed data. All authors edited the manuscript and approved the final version. The authors all declare no conflicts of interest. References 1. ↵ Statistics, N. C. f. H . 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