*Article* **Monitoring 10-m LST from the Combination MODIS**/**Sentinel-2, Validation in a High Contrast Semi-Arid Agroecosystem**

**Juan M. Sánchez 1,\* , Joan M. Galve 1 , José González-Piqueras 1 , Ramón López-Urrea 2 , Raquel Niclòs 3 and Alfonso Calera 1**

Received: 25 March 2020; Accepted: 1 May 2020; Published: 4 May 2020

**Abstract:** Downscaling techniques offer a solution to the lack of high-resolution satellite Thermal InfraRed (TIR) data and can bridge the gap until operational TIR missions accomplishing spatio-temporal requirements are available. These techniques are generally based on the Visible Near InfraRed (VNIR)-TIR variable relations at a coarse spatial resolution, and the assumption that the relationship between spectral bands is independent of the spatial resolution. In this work, we adopted a previous downscaling method and introduced some adjustments to the original formulation to improve the model performance. Maps of Land Surface Temperature (LST) with 10-m spatial resolution were obtained as output from the combination of MODIS/Sentinel-2 images. An experiment was conducted in an agricultural area located in the Barrax test site, Spain (39◦03′35" N, 2 ◦06′ W), for the summer of 2018. Ground measurements of LST transects collocated with the MODIS overpasses were used for a robust local validation of the downscaling approach. Data from 6 different dates were available, covering a variety of croplands and surface conditions, with LST values ranging 300–325 K. Differences within ±4.0 K were observed between measured and modeled temperatures, with an average estimation error of ±2.2 K and a systematic deviation of 0.2 K for the full ground dataset. A further cross-validation of the disaggregated 10-m LST products was conducted using an additional set of Landsat-7/ETM+ images. A similar uncertainty of ±2.0 K was obtained as an average. These results are encouraging for the adaptation of this methodology to the tandem Sentinel-3/Sentinel-2, and are promising since the 10-m pixel size, together with the 3–5 days revisit frequency of Sentinel-2 satellites can fulfill the LST input requirements of the surface energy balance methods for a variety of hydrological, climatological or agricultural applications. However, certain limitations to capture the variability of extreme LST, or in recently sprinkler irrigated fields, claim the necessity to explore the implementation of soil moisture or vegetation indices sensitive to soil water content as inputs in the downscaling approach. The ground LST dataset introduced in this paper will be of great value for further refinements and assessments.

**Keywords:** Downscaling; thermal infrared; land surface temperature; disaggregation; Copernicus

#### **1. Introduction**

Time series of fine spatial and temporal resolution Thermal Infrared Images (TIR) are essential in a variety of agricultural applications, water resources management or irrigation scheduling, based on surface energy balance modeling [1–4]. However, spatio-temporal resolution of the operational TIR satellite sensors results are insufficient for some applications and services, including agriculture. The importance of high-resolution TIR images is being claimed [5–9]. The limitation in the TIR domain remains, since the revisit time for high spatial resolution TIR sensors is typically poor, while the spatial resolution for those with a high revisit frequency is too coarse. In practice, the spatial resolution requirements of satellite-derived surface temperature for agricultural applications are <50 m to face certain physical limitations related to the sensor's point spread function in TIR observations [2,10,11]. As for the temporal resolution, daily TIR observations are desired, although this requirement could be relaxed to 3 days as a minimum threshold [7,11].

The Copernicus conceptual mission LSTM [12] could complement other planned high-resolution TIR missions (e.g., the JPL-NASA Landsat 9-10 or the Indian-French TRISHNA mission [13]) and fulfill the spatio-temporal requirements stated above. In the meantime, downscaling methods are contributing to filling this gap by downscaling the TIR coarse resolution to finer resolutions [3,14–17]. Several techniques have been proposed in the literature to enhance the spatial resolution of the TIR domain over vegetated areas by linking TIR and reflectance information in the Visible Near Infrared (VNIR) [18–21]. These techniques are generally based on the assumption that there exists a relation between the vegetation cover and the LST. According to these approaches, a relation between the TIR and VNIR bands is first obtained at coarse spatial resolution, and then applied at the finer resolution of the VNIR bands, assuming that this relation is scale invariant.

The Normalized Difference Vegetation Index (NDVI) or the Fractional Vegetation Cover (FVC) are the most commonly used inputs in sharpening techniques, although some studies have recently explored the possibility of implementing other combinations of reflectance values that can better characterize the surface response [17,22,23]. There are also some efforts attempting to integrate soil moisture delineated vegetation indices [24], and even radar-derived soil moisture [25] in the formulations of the LST downscaling.

For years, the Moderate Resolution Imaging Spectroradiometer (MODIS) or the Advanced Along-Track Scanning Radiometer (AATSR) were combined with Landsat or Advanced Spaceborne Thermal Emission and Reflection Radiometer (ASTER) imagery to downscale LST from 1000 m × 1000 m to ~1 ha (10,000 m2 ) scale. Higher resolution VNIR sensors, such as Formosat or the Satellite pour l´Observation de la Terre (SPOT), have been also used to improve the disaggregated LST pixel size [10,26].

The synergistic use of Copernicus Sentinel-2 (S2) and Sentinel-3 (S3) imagery could offer the desired solution of high spatial and temporal resolution [8,26,27]. Although no TIR information is provided, the Sentinel-2A and -2B tandem offers a 3–5-day repeat cycle, and a 10–20 m spatial resolution in the VNIR bands. Revisit time for S3 reduces to 1–2 days, with a spatial resolution of 1000 m for their thermal channels. The relationship between TIR and VNIR bands could be extracted from S3 and then applied to S2 VNIR data. Sobrino et al. [27] explored the conceptual combination of the MultiSpectral Instrument (MSI), on board the Sentinel-2, and the Ocean and Land Color Instrument/Sea and Land Surface Temperature Radiometer (OLCI/SLSTR), on board the Sentinel-3, to show an improvement in LST products derived from AATSR at that time, before the Sentinel-3 data were available. High spatial resolution data from S2 was used to improve the characterization of the sub-pixel heterogeneity through a better parameterization of surface emissivity, although no downscaling was applied by these authors. A machine learning algorithm was proposed by Guzinski and Nieto [8] to sharpen low-resolution TIR observations from S3 using high-resolution VNIR S2 imagery. Huryna et al. [28] applied the methodology introduced by Agam et al. [19] to the combination of S3–S2 imagery. However, the methodology was tested using Terra/MODIS or Landsat observations in both works, due to the lack of high-resolution TIR data to use for cross-validation.

Despite the extraordinary growth of downscaling studies in the past decade, the assessment of the thermal sharpening techniques has been traditionally conducted by cross-validation with derived LST products at original Landsat or ASTER TIR spatial resolutions, 60–100 m and 90 m, respectively [1,8,17,21,28]. Comprehensive ground validations of disaggregated LST are quite scarce, due to the lack of robust datasets covering high contrast heterogeneous areas.

This paper continues the work initiated by Bisquert et al. [1]. These authors tested the application of different downscaling techniques in an experimental site in Barrax (Albacete, Spain) from the combination MODIS-Landsat to provide LST at fine spatial and temporal resolutions, to fulfill the requirements in the estimation of surface energy fluxes and evapotranspiration in the agricultural areas of semi-arid regions, where small land holdings dominate. Bisquert et al. [1] analyzed both classical methods based on the VNIR-LST regression, as well as more advanced approaches based on Neural Networks (NN) or Data Mining (DM). Linear, quadratic and exponential relationships, proposed in the literature, were tested and results were compared to those obtained by applying NN and regression trees in a DM approach using reflectance values from all the spectral bands available. These authors observed that NN and DM, as well as the nonlinear regression tested, have the risk of overfitting, being very sensitive to noise in the samples. They concluded that the simpler NDVI-LST linear regression led to the better results in this case. Bisquert et al. [1] explored the technique results for the different land covers in the Barrax area, and found the largest uncertainties for irrigated croplands, especially in summer when cover heterogeneity and irrigation effects are stressed. As a follow-up, Bisquert et al. [26] extracted disaggregated LST maps at a 10-m spatial resolution for the first time, using high-resolution SPOT-5 images in the framework of the Spot-5 Take 5 project. Results shown in [26] were encouraging for the further application of the model to operational S2 images.

In this context, the objective of this paper is to revise and adapt the downscaling technique to the combination MODIS-S2 to derive operational LST maps with a spatial resolution of 10 m. Some adjustments to the original formulation of the approach were introduced to reduce the model uncertainty by adding an additional image-based parametrization of the residual as a function of the VNIR response. Ground LST data from an experimental campaign carried out in the summer of 2018 were used for the model evaluation. The variety of croplands and the contrast in the surface conditions during the experiment in the selected area allowed a comprehensive analysis of the performance of the downscaling technique, not achieved before. Strengths and limitations of the models were discussed, and also some guidelines for the optimal use of this technique with Sentinel-3 and Sentinel-2 imagery are given.

This paper is structured as follows. Section 2 describes the study site, the field measurements and the satellite imagery used, as well as the downscaling methodology. Results of the ground validation and distributed assessment are shown in Section 3. Interpretation of the results and comparison with previous studies comprise Section 4. Finally, Section 5 summarizes the main conclusions of this work.

#### **2. Materials and Methods**

### *2.1. Study Site and Measurements*

This work was conducted in the semi-arid area of Barrax, southeast Spain (39◦03′ N, 2◦06′ W). This is a very flat area with an average altitude of 700 m a.s.l, close to Albacete (Figure 1), traditionally used by ESA (European Space Agency) as a test site in different international campaigns [29–31]. Irrigated and rainfed crops combine in this agroecosystem, with field size ranging from small terrains below 1 ha to large pivots over 50 ha (Figure 1). This large variety makes Barrax a perfect target to assess the performance of a downscaling technique and explore its strengths and weaknesses.

Ground measurements of LST (LSTg) were registered in "Las Tiesas" experimental farm during the summer of 2018, concurrent with EOS-Terra/MODIS overpasses, and covering a total of 10 different crops in 13 independent fields (Figure 1). The temperatures were measured using four hand-held infrared radiometers (IRTs) Apogee MI-210. These radiometers have a broad thermal band (8–14 µm), with a 22◦ field of view and an accuracy of ±0.2 K, according to the manufacturer (Apogee Instruments, Inc.). In fact, the similar Apogee SI-121 radiometers (same radiometer, but with a field of view of 18◦ and without datalogger) were calibrated against a National Institute of Standards and Technology (NIST) blackbody, during a comparison of TIR radiometers carried out in Miami by the Committee on Earth Observation Satellites (CEOS), and the accuracy was established at 0.2 K [32]. Special care was taken with the ground measurements in the sparse crops (vineyard and almond orchards), by averaging soil and canopy component temperatures to obtain representative values of the target LST. The radiometers were manually carried back and forth along transects on the fields pointing at nadir view, at a height of 1.5–2 m above the ground surface. Temperatures were registered at a rate of 5–10 measurements/min, in transect distances of 30–50 m/min, and then covering several hectares with each IRT. The 10-min averages centered at the satellite overpass time were considered. Radiometric temperatures were corrected from atmospheric and emissivity effects [33]. Downwelling sky radiance was measured with each radiometer and emissivity data were obtained through the Temperature-Emissivity Separation (TES) procedure [34,35] from in situ thermal radiance measurements using a multispectral radiometer CIMEL Electronique CE 312-2 [36].

**Figure 1.** Overview of "Las Tiesas" experimental farm. Measurement sites are located over a S2 false color composition corresponding to date 25 July 2018. Labels for the different study fields are explained in the adjacent Table, together with indication of crop type and field size.


*2.2.2 Semectite minerals*

**Smectites** are TOT phyllosilicates type. The association between smectite layers leads to a constant van der Waals break between the layers, known as "interlayer" [35].

Two smectite subgroups are existing:

i.Dioctahedral smectites, as montmorillonite, non-tronit, beidellite, etc.

*New Trends in Clay-Based Nanohybrid Applications: Essential Oil Encapsulation Strategies... DOI: http://dx.doi.org/10.5772/intechopen.106855*

ii.Trioctahedral smectites, as hectorite, saponite, sauconite, etc. [22].

The smectite clays structure is able to expand and depreciate without losing its crystallinity.

Smectite layers are typified by plentiful isomorphic substitutions in tetrahedral (Si4+ replaced by Al3+) and octahedral (Al3+ replaced by Mg2+ or Fe2+ and Mg2+ substituted by Li+ ) positions generating a layer charge that is neutralized by hydrated cations in the interlayer space, named interlayer cations (Na+ , Ca2+, K+ , etc.) [36].

**Montmorillonite** (MMT) is the most important smectite clay owing to its commercial value. It emanates from the replacement of the Si4+ in the silica tetrahedral sheets by Al3+ and Al3+ in the alumina octahedral sheets by Mg2+ leading to a negative charge that is neutralized by the Na+ and Ca2+ interlayer cations.

These cations can be facilely supplied by other organic or inorganic cations, due to the incomparable hydrophilicity, swelling, adsorption, and fluidity properties of montmorillonite [37].

#### *2.2.3 Palygorskite and sepiolite minerals*

**Sepiolite** (Sep) [Mg8Si12O30(OH)4(OH2)4.n(R2 + (H2O)8] and **palygorskite** (Pal) [MgAl3Si8O20(OH)3(H2O)4.n(R2 + (H2O)4] are 2:1 layer silicates being distinct from other clay minerals owing to the existence of continued two-dimensional tetrahedral sheet and broken octahedral sheets. Their form can be defined as ribbons of 2:1 phyllsilicate structure. Each ribbon is bonded to the next by inversion of SiO4 tetrahedra along a set of Si-O-Si bonds. Channels between the ribbons are generated due to these structural properties, which are larger in Sep (4 Å x 9.5 Å) in comparison with Pal (4 Å x 6 Å). Pal is dioctahedral and Sep is trioctahedral. Both minerals have elongated habits often forming bundles of lath-like or fibrous crystals. They contain two types of water, structural water coordinated to the octahedral cations and zeolitic water, which is loosely bound in the channels. Due to the existence of channels, Pal and Sep have great microporous volumes. They are also of a big interest and importance due to their large specific surface, exceptional swelling, and good absorbability [25, 38].

#### **2.3 Clay minerals modification**

Modification of clay minerals' native physicochemical and structural characteristics (porosity, CEC, acidity, surface area, etc.) is usually required to adapt and extend their use in different fields of application [20].

Several physical and chemical methods have been investigated to modify clay minerals features, such as acid activation, intercalation or adsorption of organic components, clay pillarization, synthesis of porous clays heterostructures, ultrasonication, and thermal treatment [39–42].

### **3. Essential oil: chemical composition and properties**

Essential oils (EOs) are volatile liquids extracted from numerous plants. They are hydrophobic and contain volatile aromatic compounds or vegetable essences. These aromatic compounds are volatile organic compounds (VOCs), which result from the plant secondary metabolism [43]. According to the European Pharmacopoeia, an essential oil is defined as an "odorous product, commonly with a complex

composition, obtained from a plant raw material by steam distillation, dry distillation, Hydrodistillation or other suitable methods without heating" [44].

Essential oils could be obtained from flowers (Pelargonium Rosat, Lavanduladentata), leaves (Eucalyptus globulus, Thymus vulgaris, Origanum vulgare, melaleuca alternifolia), seeds (Coriandrumsativum, Carumcarvi, Foeniculum vulgare, Pimpinellaanisum), peel (Citrus sinensis, CitrusLimon), rhizomes (Zingiberofficinale), and woods (Cinnamomum Cassia, Santalum album) [45].

Generally, EOs present less than 5% of the vegetal dry matter. Their composition may vary depending on the plant organ utilized, the soil and climatic conditions, and the harvesting season. Essential oils are insoluble in inorganic solvents, while soluble in organic ones (alcohol, ether, and oils). They are colorless, liquid, and volatile at room temperature, having a characteristic odor with a density less than water density [8].

EOs are very interesting natural products with several biological properties, such as antioxidative, anti-inflammatory, cytoprotective, antitumor, antimicrobial, antihypertensive, analgesic, larvicidal, insecticidal, antiparasitic, and other biological activities [5]. Thus, they have been largely used in agriculture, pharmaceutical field, medicine, and cosmetics. Indeed, numerous studies have shown insect-repellant and biocide activities, which are usable in agronomy and food industry [46, 47]. Due to their incredible properties, interest in EOs has massively increased in recent years. They are subsequently useful as complementary medicinal treatments, due to their availability and to synergistic therapeutic effects with conventional medicines, such as antibiotics [48]. Aspects related to the significant antimicrobial effect against multiresistant pathogens of the EOs, and their synergetic effect when one or more oils are mixed used in combination with known drugs [49, 50], are also presented in a recent review [51].

#### **3.1 Chemical composition of essential oils**

The number of known compounds present in essential oils has recently increased with the improvement in instrumental analytical chemistry. Nowadays, there are more than 300 components present in pure EOs [52, 53] that can be classified into two categories:

As mentioned before, the main component of the chemical composition of EOs is a complex mixture of hydrocarbon terpenes and terpenoids. Obviously, chemical compounds contain carbon and hydrogen as their building blocks that are isoprene (C5H8), the basic hydrocarbon unit found in EOs [46]. Moreover, terpenes are a class of natural products represented by general structural formula (C5H8)n. Containing more than 30.000 compounds, these unsaturated hydrocarbons are developed by plants, particularly conifers. The classification of terpenes is based on the number of isoprene units: Two, three, or four isoprene units are joined head to tail form monoterpenes, sesquiterpene, and diterpenes, respectively. Monoterpenes are present *New Trends in Clay-Based Nanohybrid Applications: Essential Oil Encapsulation Strategies... DOI: http://dx.doi.org/10.5772/intechopen.106855*

with more than 80% of EOs composition with 10 carbons, while sesquiterpenes and diterpenes are composed of 15 and 20 carbons, respectively. However, hemiterpenes (C5), diterpenes (C20), triterpenes (C30), and tetraterpenes (C40) are also observed in nature [55–57].

The second class of terpenes containing oxygen is called terpenoids. Those oxygenated derivatives of hydrocarbon terpenes can be aldehydes, alcohols, esters, ketones, acids, and phenols. Terpenoids also present many biological properties such as anti-inflammatory, antifungal, antiseptic, bactericidal, antiviral, sedative, and antitumor [58–60].

Furthermore, non-terpene components, defined as phenylpropanoids, are also found in EOs providing odor and a particular flavor (Eugenol and Cinnamaldehyde) [55].

#### **3.2 biological properties**

EOs have, for a long time, been recognized to possess several biological actions on humans, animals, and plants, namely [61–65]:


*Article* **Gasdermin D Deficiency Limits the Transition of Atherosclerotic Plaques to an Inflammatory Phenotype in** *ApoE* **Knock-Out Mice**

**Pauline Puylaert, Melissa Van Praet, Frederik Vaes, Cédric H. G. Neutel, Lynn Roth, Pieter-Jan Guns, Guido R. Y. De Meyer and Wim Martinet \***

> Laboratory of Physiopharmacology, University of Antwerp, 2610 Antwerp, Belgium<EMAIL_ADDRESS>(P.P<EMAIL_ADDRESS>(M.V.P<EMAIL_ADDRESS>(F.V<EMAIL_ADDRESS>(C.H.G.N<EMAIL_ADDRESS>(L.R<EMAIL_ADDRESS>(P.-J.G<EMAIL_ADDRESS>(G.R.Y.D.M.) **\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** Gasdermin D (GSDMD) is the key executor of pyroptotic cell death. Recent studies suggest that GSDMD-mediated pyroptosis is involved in atherosclerotic plaque destabilization. We report that cleaved GSDMD is expressed in macrophage- and smooth muscle cell-rich areas of human plaques. To determine the effects of GSDMD deficiency on atherogenesis, *ApoE*−*/*− *Gsdmd*−/− (*n* = 16) and *ApoE*−*/*− *Gsdmd+/+* (*n* = 18) mice were fed a western-type diet for 16 weeks. Plaque initiation and formation of stable proximal aortic plaques were not altered. However, plaques in the brachiocephalic artery (representing more advanced lesions compared to aortic plaques) of *ApoE*−*/*− *Gsdmd*−*/*− mice were significantly smaller (115 ± 18 vs. 186 ± 16 × 103 μm2, *p* = 0.006) and showed features of increased stability, such as decreased necrotic core area (19 ± 4 vs. 37 ± 7 × 103 μm2, *p* = 0.03) and increased αSMA/MAC3 ratio (1.6 ± 0.3 vs. 0.7 ± 0.1, *p* = 0.01), which was also observed in proximal aortic plaques. Interestingly, a significant increase in TUNEL positive cells was observed in brachiocephalic artery plaques from *ApoE*−*/*− *Gsdmd*−*/*− mice (141 ± 25 vs. 62 ± 8 cells/mm2, *p* = 0.005), indicating a switch to apoptosis. This switch from pyroptosis to apoptosis was also observed in vitro in *Gsdmd*−*/*− macrophages. In conclusion, targeting GSDMD appears to be a promising approach for limiting the transition to an inflammatory, vulnerable plaque phenotype.

**Keywords:** gasdermin D; pyroptosis; inflammation; atherosclerosis

#### **1. Introduction**

Vulnerable atherosclerotic plaques are characterized by a large necrotic core formed by excessive necrotic cell death and inflammation [1]. Plaque cells can undergo different types of regulated necrosis although their significance in atherosclerosis is not always clear-cut. One of the best-defined forms of regulated necrosis is pyroptosis, a pro-inflammatory form of regulated cell death that is characterized by the formation of plasma membrane pores via members of the gasdermin (GSDM) protein family [2,3]. Six members of this family have been identified, including gasdermin D (GSDMD). GSDMD is N-terminally (NT) cleaved and is activated by caspase 1 and caspase 4/5 (homologous to caspase 11 in mouse) [3–5]. Subsequently, NT-GSDMDs oligomerize, translocate to the cell membrane and induce pore formation, which allows the release of cellular content and pro-inflammatory cytokines such as IL-1β and IL-18, and finally results in membrane disruption and cell lysis [6,7].

In canonical pyroptosis induction, cleavage and activation of caspase 1 is mediated by inflammasomes, which are large supramolecular complexes that sense intracellular danger signals through nucleotide-binding oligomerization (NOD)-like receptor sensor molecules [8]. NLRP3 (NOD-like, leucine-rich repeat (LRR) and pyrin domain containing

**Citation:** Puylaert, P.; Van Praet, M.; Vaes, F.; Neutel, C.H.G.; Roth, L.; Guns, P.-J.; De Meyer, G.R.Y.; Martinet, W. Gasdermin D Deficiency Limits the Transition of Atherosclerotic Plaques to an Inflammatory Phenotype in *ApoE* Knock-Out Mice. *Biomedicines* **2022**, *10*, 1171. https://doi.org/10.3390/ biomedicines10051171

Academic Editors: Tânia Martins-Marques, Gonçalo F. Coutinho and Attila Kiss

Received: 17 April 2022 Accepted: 17 May 2022 Published: 19 May 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

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receptor 3) inflammasome-mediated pyroptosis is currently the best characterized pathway described in atherosclerotic plaques. Activation of the NLRP3 inflammasome can be initiated by oxidized LDL, cholesterol and calcium phosphate crystals inducing lysosomal lysis, cathepsin release, and potassium efflux [9–12]. In human atherosclerotic plaques, mRNA and protein levels of caspase 1, NLRP3, IL-1β, and IL-18 are increased compared to normal arteries [13–16]. Immunohistochemical analyses of human carotid plaques have revealed that NLRP3 immunoreactivity colocalizes with CD68-positive macrophages and occasionally with smooth muscle cells [15,16], and that plaque rupture is associated with strong immunoreactivity for caspase 1 [17]. In mice, several studies have demonstrated beneficial effects on atherogenesis and plaque stability when components of the NLRP3 inflammasome, caspase 1 activity, IL-1 or IL-18 are lacking [18–26]. Together, these studies suggest that modulation of NLRP3- and caspase 1-mediated pyroptosis offers significant health benefits for patients with advanced atherosclerosis [27]. This assumption is supported by the CANTOS trial showing beneficial effects of IL-1β antibody canakinumab in statin-treated patients at risk [28]. In contrast to canonical pyroptosis, caspase 4/5 and caspase 11 can be directly activated without the need for an inflammasome, resulting in non-canonical pyroptosis induction [29,30]. Recently, caspase 11 deficiency was reported to reduce plaque burden and macrophage infiltration in *ApoE*−*/*− mice [30], demonstrating that also non-canonical pyroptosis plays a role in atherogenesis.

GSDMD is the common executor of both canonical pyroptosis (mediated by NLRP3 and other inflammasomes) and non-canonical pyroptosis. Moreover, GSDMD is required for IL-1β release, not only during pyroptosis but also in viable macrophages [7,31]. Interestingly, *Gsdmd* mRNA is upregulated in peripheral blood monocytes from patients with coronary artery disease and expression of GSDMD and NT-GSDMD is increased in *ApoE*−*/*− mice fed a high fat diet as compared to chow-fed controls [30]. These experiments indicate that GSDMD is actively involved in pyroptosis during atherogenesis in both humans and mice, and thus represents a promising target in plaques for inhibiting pyroptosis and inflammation. Genetic deletion of *Gsdmd* or pharmacological inhibition with necrosulfonamide reduces infarct size and heart failure in a mouse model of acute myocardial infarction [32], underlining the involvement of GSDMD in cardiovascular disease and the possibility for using it as a pharmacological target in atherosclerosis. Therefore, we aimed to evaluate the impact of *Gsdmd* gene deletion in atherogenesis. First, the effects of *Gsdmd* gene deletion were evaluated in macrophages and smooth muscle cells in vitro. We also analyzed the expression of cleaved GSDMD in human carotid lesions. Next, the effect of *Gsdmd* deletion on advanced atherogenesis was evaluated in *ApoE*−*/*− mice.

#### **2. Materials and Methods**

#### *2.1. Human Atherosclerotic Plaques*

Human carotid endarterectomy specimens were obtained from patients (71 ± 3 years, 70% men) with a carotid stenosis of >70% [33]. Specimens were fixed in 4% formaldehyde (pH 7.4) within 2 min after surgical removal and paraffin embedded. To identify specific cell types expressing cleaved GSDMD, double immunohistochemical staining was performed using anti-cleaved GSDMD (Cell signaling, 36425, Danvers, MA, USA) combined with anti-CD68 (clone PG-M1, ab9498, Abcam, Cambridge, UK), anti-α-smooth muscle actin (αSMA; clone 1A4, A2547, Sigma-Aldrich, St. Louis, MO, USA), or anti-CD31 (clone JC/70A, ab783, Abcam, Cambridge, UK). Images were acquired with an Olympus BX43 microscope and quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).

#### *2.2. Mice*

Standard *ApoE*−*/*− mice (Jackson Laboratory, 002052, Bar Harbor, ME, USA) were crossbred with *Gsdmd*−*/*− mice (Genentech, South San Francisco, CA, USA), carrying a 1 bp insertion in the *Gsdmd* coding sequence (GAGTGATGTTGTCAGGCATGGGA becomes GAGTGATGTT**t**GTCAGGCATGGGA) created with CRISPR/Cas9. Litters were screened for the *Gsdmd*−*/*− genotype by PCR analysis using Gsdmd-specific primers (forward primer: GTTTCTTGTCGATGGGAACATTCAG, reverse primer: TGAGTCACACGCAGTATA) followed by Sanger sequencing using the reverse primer. Genotyping of the *ApoE* alleles was performed by PCR according to the instructions from the Jackson Laboratory.  The animals were housed in a temperature-controlled room with a 12 h light/dark cycle and had free access to water and food. After 16 weeks WD, an overdose of sodium pentobarbital (250 mg/kg, i.p.) was administered and blood samples were collected via the retro-orbital plexus. Plasma levels of total cholesterol were measured using a commercially available kit (Randox laboratories, Crumlin, UK). Blood leukocyte subsets were analyzed on a BD accuri C6 flow cytometer as previously described [34]. All experiments were approved by the Ethical Committee of the University of Antwerp (Code 2019-24) and carried out in accordance with European Directive 2010/63/EEC.

#### *2.3. Histological Analyses*

The thoracic aorta was stained en face with Oil Red O to determine lipid burden. The heart, brachiocephalic artery, and proximal ascending aorta of *ApoE*−*/*− *Gsdmd*−*/*− and *ApoE*−*/*− *Gsdmd+/+* mice were fixed in 4% formaldehyde (pH 7.4) for 24 h, dehydrated overnight in 60% isopropanol, and subsequently embedded in paraffin. The proximal ascending aorta was marked on the distal arch end and the brachiocephalic artery on the distal carotid end to ensure that they were always cut on the proximal side. Serial crosssections (5 μm) of the proximal parts of the brachiocephalic artery, proximal aorta and aortic root were prepared in random for histological analyses. Atherosclerotic plaque size and necrotic core area (defined as acellular areas with a threshold of 3000 μm2) were analyzed on hematoxylin-eosin (H&E) stained sections. Total collagen content was measured on Sirius red stained sections. Apoptosis was analyzed using an ApopTag Plus Peroxidase In Situ Apoptosis kit (Millipore, S7101, Burlington, VT, USA). For immunohistochemistry, the following antibodies were used: anti-MAC3 (BD Pharmingen, 550292, San Diego, CA, USA) and anti-α-smooth muscle actin (αSMA, 12547, Sigma-Aldrich, St. Louis, MO, USA). Images were acquired with an Olympus BX43 microscope, which was calibrated for each magnification. Plaque size was measured based on pixels per μm, which was determined during the calibration of the microscope. Per mouse, one section was analyzed. Plaques were manually delineated in Image J software (National Institutes of Health) to establish the region of interest (ROI). Further analyses within the ROIs were performed using color thresholding or manual counting (apoptotic cells).

#### *2.4. Cell Culture*

Bone marrow-derived macrophages (BMDMs) were harvested by flushing bone marrow of the femur with a 25 Gauge needle and heparinized (10 IU/mL) RPMI 1640 medium (Gibco Life Technology, Merelbeke, Belgium). After washing and filtration, cells were cultured in RPMI 1640 medium supplemented with Glutamax (Gibco Life Technology, Merelbeke, Belgium) and 15% L929-cell conditioned medium (LCCM) containing monocyte colony stimulating factor (M-CSF) for 7 days in 95% air/5% CO2 until 80–90% confluency was reached. To induce pyroptosis, BMDMs were primed with 100 ng/mL lipopolysaccharide (LPS, Sigma-Aldrich, St. Louis, MO, USA) for 4 h followed by treatment with 2.5–20 μM nigericin (Enzo Life Sciences, BML-CA421-0005, Brussels, Belgium) or 5 mM ATP (Calbiochem, 1191, Sigma-Aldrich, St. Louis, MO, USA). Vascular smooth muscle cells (VSMCs) were isolated as previously described [35,36]. Briefly, aortas were incubated in an enzyme solution containing 1 mg/mL collagenase type II (Worthington, Lakewood, NJ, USA), 1 mg/mL soybean trypsin inhibitor (Worthington, Lakewood, NJ, USA), and 0.744 units/mL elastase (Worthington) for 15 min at 37 ◦C to remove the adventitia. Subsequently, aortas were placed in a fresh enzyme solution for 75 min at 37 ◦C. Isolated cells were collected, washed, and resuspended in DMEM/F12 medium (Gibco Life Technology)

supplemented with 20% heat-inactivated FBS (Gibco Life Technology). Cells were used from passage 4 till 10 and cultured in DMEM/F12 medium supplemented with 10% heatinactivated FBS. To induce pyroptosis, VSMCs were primed with 50 ng/mL TNFα (Abcam, ab9740, Cambridge, UK) for 2 h followed by treatment with 5 mM ATP (Calbiochem, 1191) for 2 h. Alternatively, non-primed cells were treated with 300 μg/mL oxidized LDL (oxLDL, L34357, Life Technologies, Carlsbad, CA, USA) for 48 h. Necrosis was evaluated by labeling BMDMs or VSMCs with 1 μg/mL propidium iodide (PI, Molecular Probes, Eugene, OR, USA) and 10 μg/mL Hoechst (Life Technologies, Carlsbad, CA, USA), followed by visualization of PI/Hoechst-labeled cells using a Celena S digital Imaging System (Logos Biosystems, Dongan-gu, Anyang-si, Korea). To measure the release of IL-1β and IL-18, primed BMDMs were treated with 10 μM nigericin. After 2 h, cell supernatant was collected and cellular debris was removed by centrifugation. IL-1β and IL-18 secretions were quantified in the supernatant using a mouse IL-1β Quantikine ELISA kit (R&D Systems, MLB00C, Minneapolis, MN, USA) and IL-18 mouse ELISA kit (Invitrogen, BMS618-3, Waltham, MA, USA), respectively.

#### *2.5. Flow Cytometry*

Apoptosis was quantified with TdT-mediated dUTP-X nick end labeling (TUNEL). Briefly, BMDMs were detached with 0.25% trypsin-EDTA (Thermo Fischer Scientific, 25200072, Waltham, MA, USA). Detached BMDMs were fixed in 4% paraformaldehyde for 1 h at room temperature. After washing with PBS, BMDMs were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate solution for 2 min on ice. BMDMs were washed again and incubated with TUNEL reaction mixture using an in situ cell death detection kit (fluorescein,<PHONE_NUMBER>0, Roche, Switzerland) for 1 h at 37 ◦C. The samples were washed twice with FACS buffer (PBS with 0.1% bovine serum albumin and 0.05% sodium azide) and measured in the FL-1 channel on a BD Accuri C6 flow cytometer. At least 10,000 cells were measured. Debris was always gated out based on FSC/SSC scatter. Positive controls consisted of BMDMs treated with TNFα combined with cycloheximide. Negative controls consisted of untreated BMDMs. Unstained controls were included to exclude background signal.

#### *2.6. Western Blotting*

Tissues were homogenized in RIPA buffer containing protease and phosphatase inhibitors. Protein concentrations were determined using the BCA method. Samples were then 1:1 diluted in Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) containing 5% β-mercaptoethanol (Sigma-Aldrich) and heat-denatured for 5 min at 100 ◦C. Samples were loaded on Bolt 4–12% Bis-Tris gels (Invitrogen) and after electrophoresis transferred to Immobilon-FL PVDF membranes (Millipore) according to standard procedures. Subsequently, membranes were blocked for one hour in Odyssey Li-COR blocking buffer. After blocking, membranes were probed with primary antibodies diluted in Odyssey Li-COR blocking buffer followed by 1 h incubation with IRDye-labeled secondary antibodies at room temperature. Membranes were visualized with an Odyssey SA infrared imaging system (Li-COR Biosciences, Lincoln, NE, USA).

The following primary antibodies were used: rabbit anti-GSDMD (Abcam, ab219800, Cambridge, UK), rabbit anti-cleaved GSDMD (Cell signaling, 10137, Danvers, MA, USA), rabbit anti-NLRP3 (Abcam, ab263899), rabbit anti-caspase 1 (Abcam, ab179515), and mouse anti-β-actin (Abcam, ab8226).

#### *2.7. Statistical Analyses*

Statistical analyses were performed using Graphpad Prism 9. All data were expressed as mean ± SEM, except in boxplots where medians are shown. Dots represent the number of samples from independent experiments or individual mice. Statistical tests are specified in the text and figure legends. Differences were considered significant when *p* < 0.05.

#### **3. Results**

#### *3.1. Pyroptosis Is Inhibited in Gsdmd*−*/*− *BMDMs but a Switch to Apoptosis Is Induced*

Because pyroptosis is mainly characterized in myeloid cells [37,38], BMDMs were isolated from *Gsdmd*−*/*− and *Gsdmd+/+* mice. First, GSDMD deficiency was confirmed in *Gsdmd*−*/*− BMDMs via Western blotting (Figure 1A). To induce pyroptosis, BMDMs were primed with LPS followed by treatment with nigericin, a canonical pyroptosis inducer. Significant induction of cell death was observed in *Gsdmd+/+* cells while *Gsdmd*−*/*− cells were clearly more resistant to pyroptosis induction (Figure 1B). The release of IL-1β and IL-18 was quantified in BMDM supernatant after LPS priming and treatment with nigericin. Both IL-1β and IL-18 were released by *Gsdmd+/+* BMDMs and this was significantly decreased in *Gsdmd*−*/*− BMDMs (Figure 1C). IL-1β and IL-18 were not detected in supernatant of untreated BMDMs (data not shown). LPS priming induced the expression of NLRP3 and procaspase 1, both in *Gsdmd+/+* and *Gsdmd*−*/*− BMDMs (Figure 1D). Accordingly, Western blot analysis further showed that LPS-primed *Gsdmd+/+* BMDMs treated with 2.5 to 20 μM nigericin expressed GSDMD and cleaved GSDMD, while *Gsdmd*−*/*− BMDMs did not (Figure 1E). Moreover, both *Gsdmd+/+* and *Gsdmd*−*/*− BMDMs expressed procaspase 1 and, upon treatment with nigericin, the active caspase 1 p10 subunit was expressed (Figure 1E). Altogether, these results confirm that GSDMD-pore formation and pyroptosis induction are inhibited in *Gsdmd*−*/*− BMDMs while upstream NLRP3 assembly and procaspase 1 recruitment (and cleavage) are not affected.

Interestingly, the expression of caspase 1 p10 was significantly higher in *Gsdmd*−*/*− BMDMs as compared to *Gsdmd+/+* controls (Figure 1E). Because active caspase 1 can also act in a pro-apoptotic fashion [39,40], a TUNEL assay was performed on LPS-primed BMDMs after treatment with nigericin for 1 h (Figure 1F). A significant increase in TUNEL positivity was observed after nigericin treatment in *Gsdmd*−*/*− BMDMs, while PI positivity was not increased, indicating that the plasma membranes of *Gsdmd*−*/*− cells were intact but that DNA fragmentation typical of apoptosis occurred. In contrast, TUNEL positivity did not change in *Gsdmd+/+* BMDMs after nigericin treatment while PI positivity did increase significantly (Figure 1F). Similar findings were observed after treatment with ATP, another classical caspase 1-dependent pyroptosis inducer (Figure 1G).

#### *3.2. Gsdmd*−*/*− *VSMCs Are Less Sensitive to Pyroptosis Inducers*

There is increasing evidence that pyroptosis is not limited to inflammatory cells [22,41–43]. Therefore, VSMCs were isolated from *Gsdmd+/*+ and *Gsdmd*−*/*− mice. Absence of GSDMD in *Gsdmd*−*/*− VSMCs was confirmed via Western blotting (Figure 2A). To induce pyroptosis, VSMCs were primed with TNFα followed by ATP treatment. Cell death increased significantly in *Gsdmd+/+* VSMCs but not in *Gsdmd*−*/*− VSMCs (Figure 2B). Similar to TNFα/ATP treatment, oxLDL, which is also reported to induce pyroptosis [12,44], increased cell death significantly in *Gsdmd+/+* VSMCs but not in *Gsdmd*−*/*− cells (Figure 2C).

#### *3.3. Cleaved GSDMD Is Expressed in Human Carotid Lesions*

To confirm that GSDMD is not only active in mice, cell-specific expression of cleaved GSDMD was evaluated in human carotid lesions (Figure 3). In general, cleaved GSDMD was expressed in the plaque, especially in the shoulder regions, and media (Figure 3B–D). Double immunohistochemical staining of cleaved GSDMD and CD68 showed that GSDMD is cleaved in plaque areas rich in macrophages, albeit not all CD68-positive cells contained cleaved GSDMD (Figure 3B). On sections stained separately for cleaved GSDMD and αSMA (Figure 3C), the positivity pattern appeared very similar. Indeed, double immunohistochemical staining of cleaved GSDMD and αSMA showed a clear overlapping of red (cl-GSDMD) and purple (αSMA) signal, confirming colocalization of cleaved GSDMD and smooth muscle cells. The CD31-positive intima delineated the vessel lumen (Figure 3D). However, no cleaved GSDMD positivity was observed in CD31-positive endothelial cells.

**Figure 1.** Pyroptosis is inhibited in *Gsdmd*−*/*− BMDMs and a switch to apoptosis occurs. Bone marrowderived macrophages (BMDMs) were isolated from *Gsdmd+/+* and *Gsdmd*−*/*− mice. (**A**) Deficiency of GSDMD in *Gsdmd*−*/*− BMDMs was confirmed via Western blotting. (**B**–**D**) *Gsdmd*−*/*− and *Gsdmd+/+* BMDMs were primed with 100 ng/mL LPS for 4 h followed by treatment with 10 μM nigericin for 2 h. (**B**) Cell death was measured using propidium iodide (PI) labelling (two-way ANOVA followed by Sidak's multiple comparison, *n* = 5 independent experiments) and (**C**) the release of IL-1β and IL-18 was quantified in the cell supernatant (Mann–Whitney test, *n* = 5 independent experiments). (**D**) Western blot analyses of NLRP3 and procaspase (procasp) 1 on lysates of LPS-primed BMDMs (two-way ANOVA followed by Sidak's multiple comparison, *n* = 4–7 independent experiments, data are expressed as fold change of target/β-actin ratio). (**E**) *Gsdmd*−*/*− and *Gsdmd+/+* BMDMs were primed with 100 ng/mL LPS for 4 h followed by treatment with 2.5–20 μM nigericin for 2 h. Western blot analyses of GSDMD, cleaved (cl)-GSDMD, procaspase 1, and caspase (casp) 1 p10 (two-way ANOVA followed by Dunnett's multiple comparison between concentrations per genotype, *\* p* < 0.05, *\*\*\* p* < 0.001; two-way ANOVA followed by Sidak's multiple comparison between genotypes per concentration, *# p* < 0.05; *n* = 3 independent experiments, data are expressed as fold change of target/βactin ratio). (**F**,**G**) LPS-primed BMDMs were treated for 1 h with (**F**) 20 μM nigericin or (**G**) 5 mM ATP. TUNEL labeling was performed and analyzed via flow cytometry and cell death was measured using PI labelling (two-way ANOVA followed by Sidak's multiple comparison, *n* = 3–5 independent experiments). \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

**Figure 2.** *Gsdmd*−*/*− VSMCs are less sensitive to pyroptosis inducers compared to controls. Vascular smooth muscle cells (VSMCs) were isolated from *Gsdmd+/+* and *Gsdmd*−*/*− mice. (**A**) Deficiency of GSDMD in *Gsdmd*−*/*− VSMCs was confirmed via Western blotting. (**B**) VSMCs were primed with 50 ng/mL TNFα for 2 h followed by treatment with 5mM ATP for 1 h. (**C**) VSMCs were treated with 300 μg/mL oxLDL for 48 h. Cell death was measured using PI labelling. \* *p* < 0.05 (two-way ANOVA followed by Sidak's multiple comparison, *n* = 4 independent experiments).

#### *3.4. Atherogenesis Is Delayed in ApoE*−*/*− *Gsdmd*−*/*− *Mice but a Switch to Apoptosis Occurs in Plaques of the Brachiocephalic Artery*

To evaluate the effect of *Gsdmd* gene deletion on atherogenesis, *ApoE*−*/*− *Gsdmd*−*/*− and *ApoE*−*/*− *Gsdmd+/+* mice were fed a western-type diet (WD) for 16 weeks. First, deletion of *Gsdmd* was confirmed via Western blotting in the aortic arch, descending thoracic aorta (desc TA), lung, liver, heart, spleen, and kidney of *ApoE*−*/*− *Gsdmd*−*/*− mice (Supplementary Figure S1). After 16 weeks WD, body weight (27.2 ± 0.7 vs. 29.3 ± 1.0 g, 1.5 ± 0.03 vs. 1.5 ± 0.05 fold change compared to starting weight; independent samples *t*-test, *p* > 0.05), plasma cholesterol (685.8 ± 18.2 vs. 652.1 ± 20.0 mg/dL; independent samples *t*-test, *p* > 0.05), and circulating leukocyte subsets (Supplementary Figure S2) were not different between *ApoE*−*/*− *Gsdmd+/+* and *ApoE*−*/*− *Gsdmd*−*/*− mice. En face Oil Red O staining of the thoracic aorta showed no differences in lipid burden between *ApoE*−*/*− *Gsdmd+/+* and *ApoE*−*/*− *Gsdmd*−*/*− mice in the whole thoracic aorta, as well as in the aortic arch and descending aorta separately (Figure 4).

**Figure 3.** Cleaved GSDMD is expressed in human carotid plaques. (**A**) Overview image of a section from a human carotid artery lesion stained with hematoxylin/eosin. The boxed area corresponds with the region shown in (**B**–**D**). (**B**–**D**) Immunohistochemical staining of cleaved (cl)-GSDMD combined with (**B**) CD68, (**C**) α-smooth muscle actin (αSMA) or (**D**) CD31. From top to bottom: 1. Image of section stained for (B) CD68 (red), (**C**) αSMA (brown), or (**D**) CD31 (red). 2. Image of section stained for cl-GSDMD (red). 3. Image of double stained section for cl-GSDMD (red) combined with (**B**) CD68, (**C**) αSMA, or (**D**) CD31 (purple). 4–7. Magnifications of dotted frames in images 3–5. Scale bar = 500 μm (**A**,**B**–**D**: 1–3), 100 μm (**B**–**D**: 4–7). Representative images are shown. M = media, L = lumen, I = intima, P = plaque.

**Figure 4.** Lipid burden in the thoracic aorta is not different between *ApoE*−*/*− *Gsdmd*−*/*− and *ApoE*−*/*− *Gsdmd+/+* mice. *ApoE*−*/*− *Gsdmd*−*/*− and *ApoE*−*/*− *Gsdmd+/+* mice were fed a WD for 16 weeks. The aortic arch and descending thoracic aorta (TA) were stained en face with Oil Red O to evaluate the plaque burden (Mann–Whitney test, *n* = 6 mice per group). The dotted line seperates the aortic arch (top) from the descending TA (bottom). Scale bar = 5 mm.

In contrast, plaque size was significantly decreased in the brachiocephalic artery of *ApoE*−*/*− *Gsdmd*−*/*− mice as compared to *ApoE*−*/*− *Gsdmd+/+* controls (115 ± 18 vs. 186 ± 16 × 103 μm2; independent samples *t*-test, *p* = 0.006, Figure 5A). This suggests that plaque progression is delayed in *ApoE*−*/*− *Gsdmd*−*/*− mice. Moreover, the necrotic core area was significantly decreased in *ApoE*−*/*− *Gsdmd*−*/*− plaques of the brachiocephalic artery as compared to *ApoE*−*/*− *Gsdmd+/+* controls (19 ± 4 vs. 37 ± 7 × 103 μm2; independent samples *t*-test, *p* = 0.03), although the relative necrotic core area was not altered (Figure 5A). Similarly, the number of cells infiltrated in plaques of the brachiocephalic artery of *ApoE*−*/*− *Gsdmd*−*/*− mice was significantly lower as compared to *ApoE*−*/*− *Gsdmd+/+* mice (Figure 5A). The total collagen content did not differ between *ApoE*−*/*− *Gsdmd*−*/*− and *ApoE*−*/*− *Gsdmd+/+* plaques in the brachiocephalic artery (Figure 5B). Moreover, αSMA and MAC3 immunoreactivity did not change significantly, however, the ratio of αSMA to MAC3 immunoreactivity increased significantly in *ApoE*−*/*− *Gsdmd*−*/*− plaques of the brachiocephalic artery as compared to controls (Figure 5C), indicating a shift in plaque composition and inflammatory state. Because a switch in cell death modality was observed in vitro in *Gsdmd*−*/*− BMDMs, TUNEL staining was performed on plaques *of ApoE*−*/*− *Gsdmd+/+* and *ApoE*−*/*− *Gsdmd*−*/*− mice. Similarly to what was observed in vitro, TUNEL positivity in vivo was significantly increased in plaques of the brachiocephalic artery from *ApoE*−*/*− *Gsdmd*−*/*− mice compared to *ApoE*−*/*− *Gsdmd+/+* controls (Figure 5D).

In plaques of the proximal aorta (Figure 6) and aortic root (Supplementary Figure S3), no differences in plaque size, necrotic core area, cell infiltration, and total collagen content were observed between *ApoE*−*/*− *Gsdmd*−*/*− and *ApoE*−*/*− *Gsdmd+/+* mice. Nevertheless, MAC3 immunoreactivity was significantly decreased while the αSMA to MAC3 immunoreactivity ratio was significantly increased in plaques of the proximal aorta from *ApoE*−*/*− *Gsdmd*−*/*− mice, again indicating a shift in plaque composition and inflammatory state (Figure 6C). In contrast to what was observed in vitro and in the brachiocephalic artery from *ApoE*−*/*− *Gsdmd*−*/*− mice, TUNEL positivity was not increased in plaques in the proximal aorta (Figure 6D).

**Figure 5.** Plaques in the brachiocephalic artery from *ApoE*−*/*− *Gsdmd*−*/*− mice are smaller but show increased apoptosis. *ApoE*−*/*− *Gsdmd*−*/*− and *ApoE*−*/*− *Gsdmd+/+* mice were fed a WD for 16 weeks. Sections of the brachiocephalic artery were stained with (**A**) hematoxylin/eosin to quantify plaque size, necrotic cores (# hash signs), and cell infiltration; (**B**) Sirius red to measure total collagen content; (**C**) anti-MAC3 and anti-α-smooth muscle actin (αSMA) to determine macrophage and smooth muscle cell content, respectively, and to calculate the ratio of αSMA/MAC3 immunoreactivity; (**D**) TUNEL to count apoptotic cells (dotted boxes are magnified, scale bar = 20 μm). \* *p* < 0.05, \*\* *p* < 0.01 (independent samples *t*-test, boxplot: Mann–Whitney test, *n* = 10–18 mice per group). Scale bar = 100 μm. Representative images are shown.

**Figure 6.** Plaques in the proximal aorta from *ApoE*−*/*− *Gsdmd*−*/*− mice show decreased macrophage infiltration as compared to *ApoE*−*/*− *Gsdmd+/+* controls. *ApoE*−*/*− *Gsdmd*−*/*− and *ApoE*−*/*− *Gsdmd+/+* mice were fed a WD for 16 weeks. Sections of the proximal aorta were stained with (**A**) hematoxylin/eosin to quantify plaque size, necrotic cores (# hash signs), and cell infiltration; (**B**) Sirius red to measure total collagen content; (**C**) anti-MAC3 and anti-α-smooth muscle actin (αSMA) to determine macrophage and smooth muscle cell content, respectively, and to calculate the ratio of αSMA/MAC3 immunoreactivity; (**D**) TUNEL to count apoptotic cells (dotted boxes are magnified, scale bar = 20 μm). \* *p* < 0.05, \*\* *p* < 0.01 (independent samples *t*-test, boxplot: Mann–Whitney test, *n* = 13–18 mice per group). Scale bar = 100 μm. Representative images are shown.

#### **4. Discussion**

GSDMD is the common executor of canonical and non-canonical pyroptosis. Increasing evidence suggests that pyroptosis occurs in atherosclerotic plaques in both humans and mice [13–26]. In the present study, we isolated BMDMs and VSMCs from *Gsdmd+/+* and *Gsdmd*−*/*− mice and confirmed that canonical pyroptosis is GSDMD-dependent in BMDMs and VSMCs. Indeed, treatment of LPS-primed BMDMs with classical caspase 1 activators such as nigericin and ATP induced significant pyroptosis in *Gsdmd+/+* controls, but not in *Gsdmd*−*/*− cells. Concurrently, *Gsdmd*−*/*− BMDMs showed increased DNA fragmentation, measured with TUNEL staining, which was absent in *Gsdmd+/+* BMDMs. Thus, *Gsdmd*−*/*− BMDMs are resistant to canonical pyroptosis induction, albeit they switch to another type of cell death that is characterized by DNA fragmentation, strongly suggestive of apoptosis. A switch from pyroptosis to apoptosis is further supported by the observation that caspase 1 p10 is upregulated in LPS/nigericin-treated *Gsdmd*−*/*− BMDMs, and not in *Gsdmd+/+* controls, and may act in a pro-apoptotic manner in the absence of GSDMD [39,40,45].

Previous studies have demonstrated that GSDMD, NT-GSDMD, and *Gsdmd* mRNA are upregulated in aortas of hyperlipidemic mice and in LPS/oxLDL-treated mouse peritoneal macrophages [30]. Moreover, the expression of NT-GSDMD is increased in plaques of LDLr−/− mice [45]. *Gsdmd* mRNA is also upregulated in peripheral blood monocytes of atherosclerotic patients [30]. However, to the best of our knowledge, the expression of cleaved NT-GSDMD has not yet been evaluated in human atherosclerotic plaques. Therefore, we performed immunostaining on human carotid lesions. We demonstrated that NT-GSDMD was expressed in human plaques, both in macrophage- and VSMC-rich regions. However, it should be noted that this finding was based on analysis of a limited number of plaques and comparison with plaque-free arteries was not made. Other groups have shown that components of the canonical pyroptosis pathway colocalize with plaque macrophages and contribute to macrophage pyroptosis and atherogenesis [12,15,24,27]. Interestingly, in the present study, colocalization of NT-GSDMD with αSMA-positive smooth muscle cells was even more pronounced than with CD68-positive macrophages. This is in line with a recent study reporting that caspase 1-dependent pyroptosis occurs in VSMCs and contributes to the progression of atherosclerosis [22]. Importantly, pyroptosis has also been described in plaque endothelial cells [46–48]. However, we did not observe any NT-GSDMD immunoreactivity in the luminal endothelial layer of human carotid lesions. Similarly, Rajamäki and colleagues also did not observe any NLRP3 immunoreactivity in the endothelium of human coronary plaques [15].

Disulfiram (used to treat alcohol use disorder) has recently been identified as a potent GSDMD inhibitor [49]. Furthermore, dimethyl fumarate (used as an immunomodulator in multiple sclerosis) also covalently binds GSDMD thereby inhibiting pyroptosis in vitro and in vivo in a mouse model of LPS-induced shock [50]. The availability of safe, EMAand FDA-approved drugs that inhibit GSDMD makes it an interesting therapeutic target to address pyroptosis in atherosclerosis. Therefore, we crossbred atherosclerotic *ApoE*−*/*− mice with *Gsdmd*−*/*− mice to evaluate the effect of GSDMD deficiency on atherogenesis. After 16 weeks WD, Oil red O staining was performed on the thoracic aorta but no change in overall lipid burden was observed. In line with this finding, plaque size was not altered in the proximal aorta. However, the plaque size in the brachiocephalic artery of *ApoE*−*/*− *Gsdmd*−*/*− mice was significantly decreased as compared to *ApoE*−*/*− *Gsdmd+/+* mice. Although both the ascending proximal aorta and the brachiocephalic artery of *ApoE*−*/*− mice are atherogenesis-prone sites [51], plaques in the brachiocephalic artery enter an advanced, human-like stage more rapidly [52,53]. Indeed, plaques in the brachiocephalic artery of *ApoE*−*/*− mice easily reveal a vulnerable plaque phenotype and thinning of the fibrous cap may even lead to plaque rupture, similarly to human plaques [53–55]. In contrast, aortic plaques of *ApoE*−*/*− mice resemble stable lesions in humans, based on fibrous cap stress analysis [53]. Altogether, these observations suggest that GSDMD deficiency does not affect plaque initiation (as lipid burden in the thoracic aorta and proximal aortic plaque size were not decreased in *ApoE*−*/*− *Gsdmd*−*/*− mice) but rather the transition to and growth

of a vulnerable plaque. This is supported by the increased αSMA/MAC3-immunoreactivity ratio observed in plaques of both the brachiocephalic artery and the proximal aorta of *ApoE*−*/*− *Gsdmd*−*/*− mice as compared to controls. This finding suggests a lower degree of plaque inflammation and vulnerability, and was also observed in plaques of the proximal aorta despite the unchanged plaque size, demonstrating a general decrease in plaque inflammation. Moreover, macrophage content was significantly decreased in plaques in the proximal aorta of *ApoE*−*/*− *Gsdmd*−*/*− mice as compared to controls. This is in line with a recent study in which GSDMD expression was suppressed in *ApoE*−*/*− mice using an adeno-associated virus-5 (AAV-5) delivery system [30]. The authors reported a decrease in F4/80-positive macrophage content in the aorta and aortic valve of AAV-5-GSDMD-treated *ApoE*−*/*− mice as compared to AAV-5-control-treated *ApoE*−*/*− mice. Of note, the authors also reported a decrease in lipid burden and plaque size in the aorta and aortic valve when GSDMD expression was suppressed, however, only a limited number of mice was included and preliminary data were reported [30]. Another study reported a decreased lesion area in the aortic root of LDLr antisense oligonucleotide-treated *Gsdmd*−*/*− mice compared to *Gsdmd+/+* controls, which was attributed to decreased IL-1β release resulting in decreased foam cell formation and ATP release in plasma [45]. Importantly, experimental differences such as a longer period of feeding WD in the present study, evaluation of different vascular sites, and the use of different genetic models make comparison with these studies difficult.

In plaques of the brachiocephalic artery, the number of TUNEL positive cells per mm2 was significantly increased in *ApoE*−*/*− *Gsdmd*−*/*− mice compared to controls. Increased TUNEL positivity is not specific and DNA fragmentation is reported to occur during apoptotic death as well as during different forms of necrotic death [56,57]. However, as necrosis is decreased in plaques of *ApoE*−*/*− *Gsdmd*−*/*− mice, it is plausible to conclude that a switch to apoptosis occurs when pyroptosis is defective. Accordingly, in vitro TUNEL positivity and levels of pro-apoptotic caspase 1 p10 were increased in *Gsdmd*−*/*− BMDMs while PI positivity did not increase. Similarly, Opoku and colleagues recently reported increased apoptosis, characterized by phosphatidyl serine exposure on the cell surface, when pyroptosis was defective in *Gsdmd*−*/*− macrophages [45]. Importantly, a switch to apoptosis, which is non-lytic and non-inflammatory (in contrast to pyroptosis), will limit plaque progression, inflammation, and destabilization and thus, is regarded to be atheroprotective at this stage of atherosclerosis in *ApoE*−*/*− mice. However, as previously reported by our lab, accumulation of apoptotic bodies can induce secondary necrosis, eventually resulting in expansion of the necrotic core and plaque size during later stages of atherosclerosis [58]. Accordingly, large plaques with an inflammatory phenotype were observed in the aortic root from both *ApoE*−*/*− *Gsdmd*−*/*− and *ApoE*−*/*− *Gsdmd+/+* mice, possibly because after 16 weeks WD plaques in the aortic root are in a more advanced stage compared to plaques in the brachiocephalic artery. Thus, the effects in the longer term of targeting GSDMD and the concomitant switch in cell death modality remain to be elucidated.

#### **5. Conclusions**

We report that cleaved GSDMD is present in human atherosclerotic plaques and is required for inflammatory pyroptosis in murine macrophages and smooth muscle cells in vitro. GSDMD deficiency in *ApoE*−*/*− mice does not inhibit plaque initiation and the formation of stable aortic plaques, but plays a role in the formation of inflammatory plaques in the brachiocephalic artery. Indeed, a shift toward a less inflammatory plaque composition and delayed plaque progression were observed in plaques of the brachiocephalic artery from *ApoE*−*/*− *Gsdmd*−*/*− mice as compared to plaques with a more vulnerable phenotype observed in *ApoE*−*/*− *Gsdmd+/+* controls. This is accompanied by less plaque necrosis and a switch to apoptosis when GSDMD is deficient, which is also observed in vitro in BMDMs. Therefore, targeting GSDMD appears to be a promising approach for limiting the transition to an inflammatory and vulnerable plaque phenotype, and subsequently plaque destabilization. However, the pharmacological translation and therapeutic value in atherosclerosis patients should be further investigated.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biomedicines10051171/s1, Figure S1: Validation of GSDMD deficiency in tissues from *ApoE*−*/*− *Gsdmd*−*/*− mice; Figure S2: Analysis of leukocyte subsets in plasma from *ApoE*−*/*− *Gsdmd*−*/*− and *ApoE*−*/*− *Gsdmd+/+* mice. Figure S3: Plaque analysis in the aortic root from *ApoE*−*/*− *Gsdmd*−*/*− and *ApoE*−*/*− *Gsdmd+/+* mice.

**Author Contributions:** Conceptualization, P.P., W.M. and G.R.Y.D.M.; methodology, P.P., M.V.P., C.H.G.N., W.M. and G.R.Y.D.M.; validation, P.P., M.V.P. and F.V.; formal analysis, P.P.; investigation, P.P., M.V.P., F.V.; resources, W.M., G.R.Y.D.M., L.R. and P.-J.G.; data curation, P.P.; writing—original draft preparation, P.P.; writing—review and editing, P.P., W.M., G.R.Y.D.M., M.V.P., C.H.G.N., L.R. and P.-J.G.; visualization, P.P.; supervision, W.M. and G.R.Y.D.M.; project administration, P.P. and W.M.; funding acquisition, W.M., L.R., P.-J.G. and G.R.Y.D.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Fund for Scientific Research (FWO)-Flanders (EOS 30826052) and the Hercules Foundation (grant number AUHA/13/03). Pauline Puylaert and Cédric H.G. Neutel are fellows of the FWO-Flanders.

**Institutional Review Board Statement:** The animal study protocol was approved by the Ethics Committee of the University of Antwerp (Code: 2019-24, date of approval: 11 December 2019).

**Informed Consent Statement:** Patient samples were collected anonymously and untraceably approximately 25 years ago.

**Data Availability Statement:** The data that support the findings of this study are available from the corresponding author upon reasonable request.

**Acknowledgments:** The authors are grateful to Mandy Vermont and Hermine Fret for excellent technical support. Bronwen Martin is acknowledged for critical reading of the manuscript. Gsdmd−/− mice were a kind gift from Genentech and delivered thanks to the help of Andy Wullaert (University of Ghent).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

*Systematic Review*


*4.13. Experimental Validation*

In addition to numerical verification, it is strongly suggested that, if at all possible, experimental validation is carried out, due to the complex geometries and complex physics encountered after fluid-based topology optimisation. One thing is that a simulation tool shows that the optimised geometry performs better than a reference design but should preferably be validated experimentally.

Only 12 papers, or 7%, contain some form of experimental investigation of the topology-optimised designs. These cover fixed-geometry fluid diodes [43,68], small scale rotary pumps [62], forced convection heat sinks [116,128,141,146], passive coolers for light-emitting diode lamps [154,155], porous bioreactors [186], particle separators [102], and conformal cooling channels [142].

#### **5. Conclusions**

This review paper provides an overview of the development of topology optimisation for fluid flow and fluid-based problems. Since the seminal paper by Borrvall and Petersson [7] in 2003, 186 papers have been published treating a large variety of phenomena and component design, ranging from creeping flow in pipes and microfluidic mixers to turbulent flow and heat transfer, from steady to time-dependent flows, and from simple academic problems to real-life industrial examples. A wide range of topology optimisation methods have been applied to the field, with most being classified as density-based methods and significantly less using level set methods. This is surprising since the potential strength of level set methods, in combination with surface-capturing discretisation schemes, is to provide a better definition of the interface, which can be necessary for more complicated fluid problems. On the contrary, the limited ability to create a new topology favours the density-based methods.

Recommendations for future research directions are outlined based on the extensive literature review, the quantitative analysis, as well as the authors' personal experience and opinions. The community is encouraged to focus on moving the field to more complicated applications, such as transient, turbulent and compressible flows. Generally, previously published examples should serve only the purpose of benchmarks for verifying a new method or implementation. However, if the new method does not show clear improvements in accuracy and efficiency over the previously published

works, it should not be published. It is suggested that published works in the future only present improvements and extensions to the previous .

**Author Contributions:** The two authors have contributed equally to this work. All authors have read and agreed to the published version of the manuscript.

**Funding:** The second author was partly funded by the InnoTop project through the Villum Foundation.

**Conflicts of Interest:** The authors declare no conflicts of interest.

## **References**

c 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).


**2. Principles of tissue regeneration**

Tissue regeneration requires 3 key elements: 1) cells that are harvested and dissociated from the donor tissue, 2) scaffold substrates as biomaterials in which cells are attached and cultured resulting in the implantation at the desired site of the functioning tissue, and 3) cell signals that promote or prevent cell adhesion, proliferation, migration, and differentiation by upre‐ gulating or downregulating the synthesis of proteins, growth factors, and receptors (Figure 1). Combined with these 3 key elements at different rates, timings, and so on, the clinical strategy of tissue regeneration mainly comprises cell transplantation, implantation of bioarti‐ ficial tissue constructs, and chemical induction of regeneration from tissue at the site of injury. According to the nature of the target tissue and the extent of the damage to be repaired, the treatment strategy needs to be modified. For bone tissue regeneration, mesenchymal cell

© 2013 Hibi and Ueda; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Hibi and Ueda; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

transplantation and osteogenic chemical induction are preferred for small tissue deficiencies, while bioartificial mineral construct implantation is considered suitable for large deficiencies.

**Figure 1.** Elements required for tissue regeneration


Preface

The human microbiome is a collection of all microbiota that live in the human body: in the oral cavity and gastrointestinal tract, on the upper respiratory tract, in the urinary and genital tracts, in biological media (saliva, sputum, bile, feces, urine, seminal fluid, etc.). Humans are colonized by many microorganisms because the term «human microbiome» is sometimes used to refer to the collective genomes of all resident microorganisms including bacteria, archaea, fungi, protists, and viruses. The term «human microbiota» is used more often in scientific research when referring to microorganisms that do not cause diseases under normal conditions (non-pathogenic). Some micro-organisms that colonize humans are commensal, meaning they co-exist without harming humans; others have a mutualistic relationship with their human hosts.

Interest in the role of the human microbiome is extremely high, and for a long time, it was limited by the difficulties of cultivating numerous types of microorganisms that are part of the community. Modern laboratory technologies have made it possible to fully assess not only the taxonomic composition of microbial communities but also to gain new knowledge about the functional characteristics of the relationship between the microbiota and the human body.

This book presents data from recent years confirming the huge role that the microbiome plays in the human body. The results of scientific research using state-of-the-art technologies allowed us to establish that not only normal taxonomic diversity is important for human health, but also adequate functional activity of the microbiota. The book provides data on the features of changes in the microbiota in a number of problematic diseases and discusses hypotheses about cause-and-effect relationships in the microbiome-metabolome system.

In different chapters of the book, the results of fundamental and clinical research concerning the features of the microbiome in inflammatory, critical state, other pathological conditions, and diseases (stroke, cancer, autism, allergy, psoriasis, colitis, liver diseases, etc.) are presented; much attention is paid to the interaction of the microbiota and its metabolites with drugs and natural products. The conceptual data synthesis can be a basis for the development of new approaches to treatment when one of the important therapeutic targets will be the microbiota and its key metabolites.

The problem of the human microbiome is huge and immeasurable, it goes far beyond microbiology and includes almost all of medicine. Experience shows that young scientists and doctors actively participate in scientific research, feeling the prospects of this direction for solving problems of difficult-to-treat diseases. We would like to sincerely thank the authors for the pleasure of working on this book and express the hope that it will be interesting and useful to our dear readers.

**1**

Section 1

Microbiome Research

Technologies

Section 1


**2. Gaussian processes**

A stochastic process *W t*ð Þ, *t*∈ *T* is a collection of random variables. That is, for each *t*∈*T*, *W t*ð Þ is a random variable [24]; if the stochastic process is indexed by a coordinate space *s*∈ *A* ⊂ *d*, then the stochastic process is called a random field [25]. A realization of the random field, *W s*ð Þ, *s*∈ *A*, is given by ð Þ *W s*ð Þ¼ 1 *y s*ð Þ1 , … ,*W s*ð Þ¼ *n y s*ð Þ*n* . Generally from the sample *y s*ð Þ1 , … , *y s*ð Þ*n* one tries to know the characteristics of the process *W* in *si*, *i* ¼ 1, … , *n*; and with this information to make inference of the process *W s*ð Þ on all *A* ⊂ *d* , a convex set where *s* varies continuously. To the geo-referenced data *y s*ð Þ1 , … , *y s*ð Þ*n* is often referred to as geocoded, geostatistical data or point-referenced data. The study of this type of data is known as geostatistics, which is a part of spatial statistics that studies phenomena with continuous variation in space, a convex region denoted *A* [26].

A process *W* is second order stationary if it has finite variance, constant mean and its covariance function depends only on distance. Having second-order stationarity in a stochastic process implies having intrinsic stationarity, i.e., second-order stationarity is stronger than intrinsic stationarity. On the other hand, weak stationarity and second-order stationarity are equivalent in the space [27]. The following defines what is known as a Gaussian process (field).

**Definition 1.** A stochastic process *W s*ð Þ : *s*∈ *A* ⊂ 2 , where *s* varies continuously on a fixed subset *A* content in 2 , is a Gaussian process if for any collection of locations *s*1, … , *sn* with *si* ∈ *A*, the joint distribution of ð Þ *W s*ð Þ1 , … ,*W s*ð Þ*n* is multivariate Gaussian [1].

What is known as a stationary Gaussian process is defined below.

**Definition 2.** A Gaussian process *W s*ð Þ : *s*∈ *A* ⊂ 2 , is stationary if ∀*s*∈ *A*:

$$E(\mathcal{W}(\mathfrak{s})) = \mathbf{0},\tag{1}$$

$$\operatorname{Var}(\mathcal{W}(\mathfrak{s})) = \sigma^2,\tag{2}$$

and its correlation function depends only on the distance, i.e.

$$\operatorname{Corr}(\mathcal{W}(\mathfrak{s}), \mathcal{W}(\mathfrak{s'})) = \rho(h), \tag{3}$$

where *h* ¼ *s* � *s* 0 k k is the Euclidean distance that exists between *s* and *s* 0 .

That is, the mean and variance of *W s*ð Þ are constant and its correlation function only depends on the distance, so that

$$\mathcal{W} \sim \mathcal{N}\left(\mathbf{0}, \sigma^2 \rho(h)\right) \tag{4}$$

Given **W** ¼ ð Þ *W*1, … ,*Wn* , where *Wi* ¼ *W s*ð Þ*i* , the distribution of **W** is normal multivariate ð Þ NM , i.e.

**Table 1.**

*Models for the spatial correlation structure of a spatial process.*

$$\mathbf{W} \sim \mathcal{N} \mathcal{M}(\mathbf{0}, \sigma^2 \mathbf{R}),\tag{5}$$

where the ð Þ *i*, *j* element of **R** is given by ð Þ **R** *ij* ¼ *Corr W s*ð Þ*i* ,*W sj* � � � � ¼ *ρ hij* � �, *hij* ¼ *si* � *sj* � � � � is the Euclidean distance between *si* and *sj*. Note that the covariance of the Gaussian process is given by *Cov*ð Þ¼ **W** *σ*2**R**.

In this way, the correlation structure of a stationary Gaussian process can be studied through the *ρ*ð Þ *h* function. Several parametric expressions for this function are shown in the **Table 1**. In these correlation functions, *ϕ*>0 is a range parameter controlling the spatial decay over distance; *h* ¼ *s* � *s* 0 k k is the Euclidean distance between *s* and *s* 0 and *h*≥0; Γð Þ� denotes the gamma function. *κ* >0, in theory of spatial extremes *Jκ*ð Þ� and *Kκ*ð Þ� are the Bessel and modified Bessel function of the third kind of order *κ* [28], while in the spatial survival analysis and generalized linear models *Kκ*ð Þ� is the modified Bessel function of the second kind of order *κ* [29]; *κ* is a shape parameter that determines the analytic smoothness of the underlying process *W* [1]. In the powered exponential correlation function 0 <*κ* ≤2 and in the Bessel correlation function *κ* ≥0.


*2.5. Manuscript Selections*

Two independent reviewers of two different universities (Messina and Naples) singularly analyzed the obtained papers in order to select inclusion and exclusion criteria as follows. Reviewers correlated their evaluations and analyzed differences through comparing the manuscripts and consulting a third experienced senor independent reviewer (Loma Linda University) when consensus could not be reached. For the stage of the full-text articles revision, a complete independent dual analysis was performed.