Entire Mitral Valve Reconstruction Using Porcine Extracellular Matrix: Adding a Ring Annuloplasty.

PURPOSE: This study investigated the implications of inserting a flexible annuloplasty ring after reconstructing the entire mitral valve in a porcine model using a previously investigated tube graft design made of 2-ply small intestinal submucosa extracellular matrix (CorMatrix®). METHODS: An acute model with eight 80-kg pigs, each acting as its own control, was used. The entire mitral valve was reconstructed with a 2-ply small intestinal submucosa extracellular matrix tube graft (CorMatrix®). Subsequently, a Simulus® flexible ring was inserted. The characterization was based on mitral annular geometry and valvular dynamics with sonomicrometry and echocardiography. RESULTS: After adding the ring annuloplasty, the in-plane annular dynamics were more constant throughout the cardiac cycle compared to the reconstruction alone. However, the commissure-commissure distance was statistically significantly decreased [35.0 ± 3.4 mm vs. 27.4 ± 1.9 mm, P < 0.001, diff = - 7.6 mm, 95% CI, - 9.8 to (-5.4) mm] after ring insertion, changing the physiological annular D-shape into a circular shape which created folds at the coaptation zone resulting in a central regurgitant jet on color Doppler. CONCLUSION: We successfully reconstructed the entire mitral valve using 2-ply small intestinal submucosal extracellular matrix (CorMatrix®) combined with a flexible annuloplasty. The annuloplasty reduced the unphysiological systolic widening previously found with this reconstructive technique. However, the Simulus flex ring changed the physiological annular D-shape into a circular shape and hindered a correct unfolding of the leaflets. Thus, we do not recommend a flexible ring in conjunction with this reconstructive technique; further investigations are needed to discover a more suitable remodelling annuloplasty.

Induction of CD8+ immune memory and enhanced inflammation in a skin inflammation model through pre-immunization with inactivated pathogens.

Laboratory mice live in specific pathogen-free (SPF) conditions, resulting in an immature immune system comparable to that of newborns rather than adult humans or mice from pet shops. This condition may compromise their translational value. Reintroducing pathogens would lead to the uncontrolled spread of infections and associated diseases, so research facilities should seek safer alternatives. We immunized laboratory mice with a cocktail of pathogens, which were inactivated by ultraviolet irradiation and mixed with the adjuvant AddaVax. This immunization resulted in a higher percentage of CD8+ effector memory T cells compared to untreated mice, although the response was not as robust as in pet shop mice. In a model of skin inflammation, pre-immunization led to an increased skin inflammatory response compared to non-immunized mice. All immunized mice seroconverted to the pathogens in the mixture, while none of the non-immunized mice housed together seroconverted to the pathogens applied to the pre-immunized mice. In conclusion, pre-immunization of mice impacts the immune system, which includes increasing the levels of CD8+ effector memory T cells.

Clinical Staphylococcus aureus inhibits human T-cell activity through interaction with the PD-1 receptor.

IMPORTANCE: Therapies that target and aid the host immune defense to repel cancer cells or invading pathogens are rapidly emerging. Antibiotic resistance is among the largest threats to human health globally. Staphylococcus aureus (S. aureus) is the most common bacterial infection, and it poses a challenge to the healthcare system due to its significant ability to develop resistance toward current available therapies. In long-term infections, S. aureus further adapt to avoid clearance by the host immune defense. In this study, we discover a new interaction that allows S. aureus to avoid elimination by the immune system, which likely supports its persistence in the host. Moreover, we find that blocking the specific receptor (PD-1) using antibodies significantly relieves the S. aureus-imposed inhibition. Our findings suggest that therapeutically targeting PD-1 is a possible future strategy for treating certain antibiotic-resistant staphylococcal infections.

Effect of Systemic Administration of CD4+ T cells and Local Administration of T-cell Stimulants on T-cell Activity in Psoriatic Skin Xenografts on NOG Mice.

Immunodeficient mice engrafted with psoriatic human skin are widely used for the preclinical evaluation of new drug candidates. However, the T-cell activity, including the IL23/IL17 pathway, declines in the graft over time after engraftment, which likely affects the study data. Here, we investigated whether the T-cell activity could be sustained in xenografted psoriatic skin by local stimulation of T cells or systemic injection of autologous CD4 + T cells. We surgically transplanted human psoriatic skin from 5 untreated patients onto female NOG mice. Six days after surgery, mice received an intraperitoneal injection of autologous human CD4+ T cells, a subcutaneous injection under the grafts of a T-cell stimulation cocktail consisting of recombinant human IL2, human IL23, antihuman CD3, and antihuman CD28, or saline. Mice were euthanized 21 d after surgery and spleens and graft biopsies were collected for analysis. Human T cells were present in the grafts, and 60% of the grafts maintained the psoriatic phenotype. However, neither local T-cell stimulation nor systemic injection of autologous CD4+ T cells affected the protein levels of human IL17A, IL22, IFN γ, and TNF α in the grafts. In conclusion, NOG mice seem to accept psoriatic skin grafts, but the 2 approaches studied here did not affect human T-cell activity in the grafts. Therefore, NOG mice do not appear in this regard to be superior to other immunodeficient mice used for psoriasis xenografts.

Vibrational characterization of cavitation in left ventricular assist device.

BACKGROUND: The left ventricular assist device (LVAD) is a mechanical circulatory support device for patients with severe heart failure. Microbubbles caused by cavitation in the LVAD can potentially lead to physiological and pump-related complications. The aim of this study is to characterize the vibrational patterns in the LVAD during cavitation. METHODS: The LVAD was integrated into an in vitro circuit and mounted with a high-frequency accelerometer. Accelerometry signals were acquired with different relative pump inlet pressures ranging from baseline (+20 mmHg) to -600 mmHg in order to induce cavitation. Microbubbles were monitored with dedicated sensors at the pump inlet and outlet to quantify the degree of cavitation. Acceleration signals were analyzed in the frequency domain to identify changes in the frequency patterns when cavitation occurred. RESULTS: Significant cavitation occurred at the low inlet pressure (-600 mmHg) and was detected in the frequency range between 1800 and 9000 Hz. Minor degrees of cavitation at higher inlet pressures (-300 to -500 mmHg) were detected in the frequency range between 500-700, 1600-1700 Hz, and around 12 000 Hz. The signal power of the dominating frequency ranges was statistically significantly different from baseline signals. CONCLUSION: Vibrational measurements in the LVAD can be used to detect cavitation. A significant degree of cavitation could be detected in a wide frequency range, while minor cavitation activity could only be detected in more narrow frequency ranges. Continuous vibrational LVAD monitoring can potentially be used to detect cavitation and minimize the damaging effect associated with cavitation.

Imiquimod induces skin inflammation in humanized BRGSF mice with limited human immune cell activity.

Human immune system (HIS) mouse models can be valuable when cross-reactivity of drug candidates to mouse systems is missing. However, no HIS mouse models of psoriasis have been established. In this study, it was investigated if imiquimod (IMQ) induced psoriasis-like skin inflammation was driven by human immune cells in human FMS-related tyrosine kinase 3 ligand (hFlt3L) boosted (BRGSF-HIS mice). BRGSF-HIS mice were boosted with hFlt3L prior to two or three topical applications of IMQ. Despite clinical skin inflammation, increased epidermal thickness and influx of human immune cells, a human derived response was not pronounced in IMQ treated mice. However, the number of murine neutrophils and murine cytokines and chemokines were increased in the skin and systemically after IMQ application. In conclusion, IMQ did induce skin inflammation in hFlt3L boosted BRGSF-HIS mice, although, a limited human immune response suggest that the main driving cellular mechanisms were of murine origin.

Sustaining the T-cell activity in xenografted psoriasis skin.

Xenografting of psoriasis skin onto immune deficient mice has been widely used to obtain proof-of-principle of new drug candidates. However, the lack of human T-cell activity in the grafts limits the use of the model. Here, we show that xenografting of lesional skin from psoriasis patients onto human IL-2 NOG mice results in increased numbers of human CD3+ cells in the grafts, axillary lymph nodes and blood from human IL-2 NOG mice compared to C.B-17 scid and NOG mice. In addition, disease relevant human cytokine levels were higher in graft lysates and serum from human IL-2 NOG mice. However, the epidermis was lacking and no efficacy of ustekinumab, a human anti-P40 antibody targeting both IL-12 and IL-23, was shown. Thus, despite the sustained T-cell activity, the model needs further investigations and validation to capture more aspects of psoriasis.

CTLA4-Ig prevents development of neutralizing antibody formation after continuous treatment with human FVIII in HA rats.

INTRODUCTION: Immunogenicity causing development of anti-drug antibodies (ADAs) are major challenges in the treatment of haemophilia, as well as other diseases where proteins are used for treatment. Furthermore, it is a complication for preclinical testing of such therapies in animal models. AIM: To investigate if the immunosuppressive drug CTLA4 immunoglobulin (CTLA4-Ig) can induce tolerance in haemophilia A (HA) rats receiving recombinant human coagulation factor VIII (rhFVIII) treatment. METHODS: Two different prophylactic rhFVIII compounds were given intravenously to HA rats for 4 weeks. Both rhFVIII compounds were co-administered with commercially available CTLA4-Ig or human IgG subclass 4 (hIgG4) as control, and blood samples were collected. To functionally test if pharmacological efficacy was retained, rats were subjected to a bleeding experiment under anaesthesia at end of study. RESULTS: The mean inhibitory level after 4 weeks in rats receiving rhFVIII and hIgG4 was 85.7 BU for octocog alfa and 37.4 BU for rurioctocog alfa pegol. In contrast, co-administration with CTLA4-Ig during rhFVIII therapy prevented the formation of ADAs (both binding and inhibitory) in 14/14 rats receiving octocog alfa and in 7/7 rats receiving rurioctocog alfa pegol. Moreover, we were able to show that the pharmacological efficacy of rhFVIII was preserved. CONCLUSION: In a rat model with spontaneous bleeding, co-administration of CTLA4-Ig with rhFVIII prevented antibody formation. No FVIII antibodies were detected, demonstrating that CTLA4-Ig co-administration can be applicable as a method to prevent immunogenicity, when evaluating human proteins in preclinical systems permitting continuous pharmacokinetic and pharmacodynamic assessment.

Effect of gluten-free diet and antibiotics on murine gut microbiota and immune response to tetanus vaccination.

The purpose of this study was to compare the effect of a gluten-free diet and/or antibiotics on tetanus vaccine induced immunoglobulin G titers and immune cell levels in BALB/c mice. The gluten-free diet was associated with a reduced anti-tetanus IgG response, and it increased the relative abundance of the anti-inflammatory Bifidobacterium significantly in some of the mice. Antibiotics also led to gut microbiota changes and lower initial vaccine titer. After a second vaccination, neither gluten-free diet nor antibiotics reduced the titers. In the spleen, the gluten-free diet significantly increased regulatory T cell (Treg) fractions, CD4+ T cell activation, and tolerogenic dendritic cell fractions and activation, which extend the downregulating effect of the Treg. Therefore, the systemic effect of the gluten-free diet seems mainly tolerogenic. Antibiotics reduced the fractions of CD4+ T and B cells in the mesenteric lymph nodes. These results suggest that vaccine response in mice is under influence of their diet, the gut microbiota and the interplay between them. However, a gluten-free diet seems to work through mechanisms different from those induced by antibiotics. Therefore, diet should be considered when testing vaccines in mice and developing vaccines for humans.

A porcine model of subcutaneous Staphylococcus aureus infection: a pilot study.

In this descriptive pilot study, we aim to establish a porcine Staphylococcus aureus skin infection model by subcutaneous injection (s.c.) of the porcine S54F9 S. aureus strain in the groin area. Six pigs were used in the study: Five pigs were injected with S. aureus, inocula ranging from 7 × 103 to 5 × 107 colony-forming units per kg bodyweight; one pig was injected with saline exclusively. Lesions were recorded up to 6 days postinoculation using clinical evaluation, ultrasound evaluation, microbiology, flow cytometry, and pathology. Inoculation gave rise to lesions ranging from localized skin infection, that is, minute histological changes, intracellular infection, and macroscopic abscess formation with sequestration of soft tissue, to generalized infection and development of disseminated intravascular coagulation necessitating euthanasia only 10 h after inoculation. Ultrasound assessment of maximum width and characteristics was not able to disclose the progress of the local infection. Flow cytometry and immunohistochemistry revealed the participation of γδT cells in the immune response. In conclusion, we did see a graded inflammatory response associated with the dose of s.c. inoculated bacteria, which may be useful for studying, in particular, the interaction of bacteria and inflammatory mononuclear cell populations. It needs to be investigated if the model is discriminatory and robust.

Monocyte Infiltration and Differentiation in 3D Multicellular Spheroid Cancer Models.

Tumor-associated macrophages often correlate with tumor progression, and therapies targeting immune cells in tumors have emerged as promising treatments. To select effective therapies, we established an in vitro 3D multicellular spheroid model including cancer cells, fibroblasts, and monocytes. We analyzed monocyte infiltration and differentiation in spheroids generated from fibroblasts and either of the cancer cell lines MCF-7, HT-29, PANC-1, or MIA PaCa-2. Monocytes rapidly infiltrated spheroids and differentiated into mature macrophages with diverse phenotypes in a cancer cell line-dependent manner. MIA PaCa-2 spheroids polarized infiltrating monocytes to M2-like macrophages with high CD206 and CD14 expression, whereas monocytes polarized by MCF-7 spheroids displayed an M1-like phenotype. Monocytes in HT-29 and PANC-1 primarily obtained an M2-like phenotype but also showed upregulation of M1 markers. Analysis of the secretion of 43 soluble factors demonstrated that the cytokine profile between spheroid cultures differed considerably depending on the cancer cell line. Secretion of most of the cytokines increased upon the addition of monocytes resulting in a more inflammatory and pro-tumorigenic environment. These multicellular spheroids can be used to recapitulate the tumor microenvironment and the phenotype of tumor-associated macrophages in vitro and provide more realistic 3D cancer models allowing the in vitro screening of immunotherapeutic compounds.

Emissions of soot, PAHs, ultrafine particles, NOx, and other health relevant compounds from stressed burning of candles in indoor air.

Burning candles release a variety of pollutants to indoor air, some of which are of concern for human health. We studied emissions of particles and gases from the stressed burning of five types of pillar candles with different wax and wick compositions. The stressed burning was introduced by controlled fluctuating air velocities in a 21.6 m3 laboratory chamber. The aerosol physicochemical properties were measured both in well-mixed chamber air and directly above the candle flame with online and offline techniques. All candles showed different emission profiles over time with high repeatability among replicates. The particle mass emissions from stressed burning for all candle types were dominated by soot (black carbon; BC). The wax and wick composition strongly influenced emissions of BC, PM2.5 , and particle-phase polycyclic aromatic hydrocarbons (PAHs), and to lower degree ultrafine particles, inorganic and organic carbon fraction of PM, but did not influence NOx , formaldehyde, and gas-phase PAHs. Measurements directly above the flame showed empirical evidence of short-lived strong emission peaks of soot particles. The results show the importance of including the entire burn time of candles in exposure assessments, as their emissions can vary strongly over time. Preventing stressed burning of candles can reduce exposure to pollutants in indoor air.

Annular and subvalvular dynamics after extracellular matrix mitral tube graft implantation in pigs.

OBJECTIVES: Entire mitral valve reconstruction with an extracellular matrix tube graft is a potential candidate to overcome the current limitations of mechanical and bioprosthetic valves. However, clinical data have raised concern with respect to patch failure. The aim of our study was to evaluate the impact of extracellular matrix mitral tube graft implantation on mitral annular and subvalvular regional dynamics in pigs. METHODS: A modified tube graft design made of 2-ply extracellular matrix was used (CorMatrix®; Cardiovascular Inc., Alpharetta, GA, USA). The reconstructions were performed in an acute 80-kg porcine model (N = 8), where each pig acted as its own control. Haemodynamics were assessed with Mikro-Tip pressure catheters and mitral annular and subvalvular geometry and dynamics with sonomicrometry. RESULTS: Catheter-based peak left atrial pressure and pressure difference across the mitral and aortic valves in the reconstructions were comparable to the values seen in the native mitral valves. Also comparable were maximum mitral annular area (755 ± 100 mm2), maximum septal-lateral distance (29.7 ± 1.7 mm), maximum commissure-commissure distance (35.0 ± 3.4 mm), end-systolic annular height-to-commissural width ratio (10.2 ± 1.0%) and end-diastolic interpapillary muscle distance (27.7 ± 3.3 mm). Systolic expansion of the mitral annulus was, however, observed after reconstruction. CONCLUSIONS: The reconstructed mitral valves were fully functional without regurgitation, obstruction or stenosis. The reconstructed mitral annular and subvalvular geometry and subvalvular dynamics were found in the same range to those in the native mitral valve. A regional annular ballooning effect occurred that might predispose to patch failure. However, the greatest risk was found at the papillary muscle attachments.

Emissions of ultrafine particles from five types of candles during steady burn conditions.

Emissions from candles are of concern for indoor air quality. In this work, five different types of pillar candles were burned under steady burn conditions in a new laboratory scale system for repeatable and controlled comparison of candle emissions (temperature ~25°C, relative humidity ~13%, O2 >18%, air exchange rate 1.9 h-1 ). Burn rate, particle number concentrations, mass concentrations, and mode diameters varied between candle types. Based on the results, the burning period was divided in two phases: initial (0-1 h) and stable (1-6 h). Burn rates were in the range 4.4-7.3 and 4.7-7.1 g/h during initial and stable phase, respectively. Relative particle number emissions, mode diameters, and mass concentrations were higher during the initial phase compared to the stable phase for a majority of the candles. We hypothesize that this is due to elevated emissions of wick additives upon ignition of the candle together with a slightly higher burn rate in the initial phase. Experiments at higher relative humidity (~40%) gave similar results with a tendency toward larger particle sizes at the higher relative humidity. Chemical composition with respect to inorganic salts was similar in the emitted particles (dry conditions) compared to the candlewicks, but with variations between different candles.

Evaluation of Fluorescent Light Energy for the Treatment of Acute Second-degree Burns.

INTRODUCTION: The use of photobiomodulation has been proposed to improve wound healing for the last two decades. Recent development in photobiomodulation has led to the development of a novel biophotonic platform that utilizes fluorescent light energy (FLE) within the visible spectrum of light for healing of skin inflammation and wounds. MATERIALS AND METHODS: In this article, FLE was used in preliminary analysis on 18 case studies of acute second-degree burns and in a pilot study using an ex vivo human skin model. Efficacy of FLE on wound healing and tissue remodeling was evaluated by monitoring improvements in the treated tissues, assessing pain for the patients, and by performing human genome microarray analysis of FLE-treated human skin samples. RESULTS: Healing was reported for all 18 patients treated with FLE for acute second-degree burns without reported adverse effects or development of infections. Furthermore, preliminary ex vivo skin model data suggest that FLE impacts different cellular pathways including essential immune-modulatory mechanisms. CONCLUSIONS: The results presented in this article are encouraging and suggest that FLE balances different stages of wound healing, which opens the door to initiating randomized controlled clinical trials for establishing the efficacy of FLE treatment in different phases of wound healing of second-degree burns.

Mitral Annular Forces and Their Potential Impact on Annuloplasty Ring Selection.

Objectives: To provide an overview that describes the characteristics of a mitral annuloplasty device when treating patients with a specific type of mitral regurgitation according to Carpentier's classification of mitral regurgitation. Methods: Starting with the key search term "mitral valve annuloplasty," a literature search was performed utilising PubMed, Google Scholar, and Web of Science to identify relevant studies. A systematic approach was used to assess all publications. Results: Mitral annuloplasty rings are traditionally categorised by their mechanical compliance in rigid-, semi-rigid-, and flexible rings. There is a direct correlation between remodelling capabilities and rigidity. Thus, a rigid annuloplasty ring will have the highest remodelling capability, while a flexible ring will have the lowest. Rigid- and semi-rigid rings can furthermore be divided into flat and saddled-shaped rings. Saddle-shaped rings are generally preferred over flat rings since they decrease annular and leaflet stress accumulation and provide superior leaflet coaptation. Finally, mitral annuloplasty rings can either be complete or partial. Conclusions: A downsized rigid- or semi-rigid ring is advantageous when higher remodelling capabilities are required to correct dilation of the mitral annulus, as seen in type I, type IIIa, and type IIIb mitral regurgitation. In type II mitral regurgitation, a normosized flexible ring might be sufficient and allow for a more physiological repair since there is no annular dilatation, which diminishes the need for remodelling capabilities. However, mitral annuloplasty ring selection should always be based on the specific morphology in each patient.

Annular Dynamics and Leaflet Geometry in Patch Reconstruction of the Posterior Mitral Leaflet After Adding a Flexible Annuloplasty Ring.

PURPOSE: Patch reconstruction of the posterior mitral leaflet using small intestinal submucosa extracellular matrix has been successfully performed in a porcine study. The patch reconstruction, however, resulted in non-physiological systolic widening of the mitral annulus, suggesting the need for an annuloplasty ring. The objective was to characterize the impact on annular dynamics and leaflet geometry of adding a flexible annuloplasty ring to the posterior mitral leaflet patch reconstruction. METHODS: Measurements were performed in an acute 80-kg porcine model, with seven pigs acting as their own controls. The posterior mitral leaflet was reconstructed with a 2-ply small intestinal submucosa extracellular matrix patch (CorMatrix®). Additionally, a Simulus® Flexible Annuloplasty Ring (Medtronic Inc., Minneapolis, MN, USA) was inserted. Mitral annular dynamics were evaluated using sonomicrometry, and leaflet geometry was described using echocardiography. RESULTS: The annuloplasty ring reduced mitral annular dimensions and restricted cyclic changes in mitral annular area (126 ± 19 vs. 30 ± 13 mm2, p < 0.001), septal-lateral and commisure-commisure distances. Ring annuloplasty prevented systolic widening in the mitral annulus after posterior mitral leaflet reconstruction. The annular saddle shape and leaflet coaptation length (8.7 ± 2.3 vs. 9.7 ± 1.3 mm, p = 0.221) were comparable before and after ring insertion. CONCLUSIONS: The flexible annuloplasty ring resulted in a downsized annulus with restriction of cyclic annular changes in the reconstructed mitral valve. Ring insertion preserved the annular saddle shape and coaptation length. The ring annuloplasty counteracted the non-physiological annular dynamics, and this may improve durability of the posterior mitral leaflet patch reconstruction.

The importance of collagen composition and biomechanics for the porcine aortic root.

A thorough understanding of the aortic root structure and biomechanics is necessary when performing aortic valve-sparing procedures in patients with aortic root aneurysms. This study aimed to evaluate the amount of collagen and biomechanics at different levels and segments of the aortic root. Ten aortic roots from healthy pigs were excised including the aortic annulus, the sinuses of Valsalva, and the sinotubular junction (STJ). Specimens were further divided into three circumferential segments; left coronary (LC)-, right coronary (RC)-, and non-coronary (NC) sinus. Collagen was determined using hydroxyproline analysis and specimens were tested biomechanically for stress-strain relations. The annulus showed significantly larger average maximum stiffness (9.6 ± 4.5 N/mm) compared with the sinus (4.5 ± 2.0 N/mm) and STJ (4.8 ± 1.8 N/mm). The average collagen content was likewise higher in the annulus (4.0 ± 1.0 mg/ml) compared with the sinus (2.4 ± 0.6 mg/ml) and STJ (2.2 ± 0.5 mg/ml) for all three segments. The NC sinus segment exhibited a significantly larger maximum stiffness and stress under static conditions compared with the RC. These results suggest that the aortic root is heterogeneous in both structure and biomechanical properties and that it varies both in levels and segments of the aortic root. Future surgical approaches should consider enhanced strength parameters for specific areas of the aortic root to achieve the best results when performing aortic valve-sparing techniques. From this study, we conclude that the aortic annulus needs special attention to imitate normal physiologic properties during aortic valve-sparing surgery due to its higher maximum stiffness, stress, and load. Modified future surgical procedures could potentially prevent recurrent aneurysmal formation.

Metabolism of short-chain fatty acid propionate induces surface expression of NKG2D ligands on cancer cells.

SCFAs are primarily produced in the colon by bacterial fermentation of nondigestible carbohydrates. Besides providing energy, SCFAs can suppress development of colon cancer. The mechanism, however, remains elusive. Here, we demonstrate that the SCFA propionate upregulates surface expression of the immune stimulatory NKG2D ligands, MICA/B by imposing metabolic changes in dividing cells. Propionate-mediated MICA/B expression did not rely on GPR41/GPR43 receptors but depended on functional mitochondria. By siRNA-directed knockdown, we could further link phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme in gluconeogenesis to propionate regulation of MICA/B expression. Moreover, knockdown of Rictor and specific mTOR inhibitors implicated mTORC2 activity with metabolic changes that control MICA/B expression. SCFAs are precursors to short-chain acyl-CoAs that are used for histone acylation thereby linking the metabolic state to chromatin structure and gene expression. Propionate increased the overall acetylation and propionylation and inhibition of lysine acetyltransferases (KATs) that are responsible for adding acyl-CoAs to histones reduced propionate-mediated MICA/B expression, suggesting that propionate-induced acylation increases MICA/B expression. Notably, propionate upregulated MICA/B surface expression on colon cancer cells in an acylation-dependent manner; however, the impact of mitochondrial metabolism on MICA/B expression was different in colon cancer cells compared with Jurkat cells, suggesting that continuous exposure to propionate in the colon may provide an enhanced capacity to metabolize propionate. Together, our findings support that propionate causes metabolic changes resulting in NKG2D ligand surface expression, which holds potential as an immune activating anticancer therapy.

Cytoplasmic Citrate Flux Modulates the Immune Stimulatory NKG2D Ligand MICA in Cancer Cells.

Immune surveillance of cancer cells is facilitated by the Natural Killer Group 2D (NKG2D) receptor expressed by different lymphocyte subsets. It recognizes NKG2D ligands that are rarely expressed on healthy cells, but upregulated by tumorigenesis, presenting a target for immunological clearance. The molecular mechanisms responsible for NKG2D ligand regulation remain complex. Here we report that cancer cell metabolism supports constitutive surface expression of the NKG2D ligand MHC class I chain-related proteins A (MICA). Knockout of the N-glycosylation gene N-acetylglucosaminyltransferase V (MGAT5) in HEK293 cells induced altered metabolism and continuous high MICA surface expression. MGAT5 knockout cells were used to examine the association of cell metabolism and MICA expression through genetic, pharmacological and metabolic assays. Findings were verified in cancer cell lines. Cells with constitutive high MICA expression showed enhanced spare respiratory capacity and elevated mitochondrial efflux of citrate, determined by extracellular flux analysis and metabolomics. MICA expression was reduced by inhibitors of mitochondrial function, FCCP and etomoxir e.g., and depended on conversion of citrate to acetyl-CoA and oxaloacetate by ATP citrate lyase, which was also observed in several cancer cell types. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analysis revealed that upregulated MICA transcription was associated with an open chromatin structure at the MICA transcription start site. We identify mitochondria and cytoplasmic citrate as key regulators of constitutive MICA expression and we propose that metabolic reprogramming of certain cancer cells facilitates MICA expression and NKG2D-mediated immune recognition.