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Article The Chemically-Modified Tetracycline COL-3 and Its Parent Compound Prevent Microglial Inflammatory Responses by Reducing Glucose-Mediated

Nilson Carlos Ferreira Junior 1,2,3, Maurício dos Santos Pereira 1,2,3 , Nour Francis 1, Paola Ramirez 1, Paula Martorell 1, Florencia González-Lizarraga 4, Bruno Figadère 5, Rosana Chehin 4, Elaine Del Bel 2,3, Rita Raisman-Vozari 1,* and Patrick Pierre Michel 1,*

1 Sorbonne Université, Paris Brain Institute-ICM, Inserm, CNRS, APHP, Hôpital de la Pitié Salpêtrière, 75013 Paris, France; [email protected] (N.C.F.J.); [email protected] (M.d.S.P.); [email protected] (N.F.); [email protected] (P.R.); [email protected] (P.M.) 2 Department of Basic and Oral Biology, FORP, Campus USP, University of São Paulo, Av. Café, s/no, Ribeirão Preto 14040-904, Brazil; [email protected] 3 USP, Center for Interdisciplinary Research on Applied Neurosciences (NAPNA), São Paulo 05508-220, Brazil  4  Instituto de Investigación en Medicina Molecular y Celular Aplicada (IMMCA) (CONICET-UNT-SIPROSA), CP 4000 Tucumán, Argentina; mfl[email protected] (F.G.-L.); [email protected] (R.C.) Citation: Ferreira Junior, N.C.; dos 5 BioCIS, CNRS, Université Paris-Saclay, 92290 Châtenay-Malabry, France; [email protected] Santos Pereira, M.; Francis, N.; * Correspondence: [email protected] (R.R.-V.); [email protected] (P.P.M.); Ramirez, P.; Martorell, P.; Tel.: +33-(0)157274550 (R.R.-V.); +33-(0)157274534 (P.P.M.) González-Lizarraga, F.; Figadère, B.; Chehin, R.; Del Bel, E.; Abstract: We used mouse microglial cells in culture activated by lipopolysaccharide (LPS) or α- Raisman-Vozari, R.; et al. The synuclein amyloid aggregates (αSa) to study the anti-inflammatory effects of COL-3, a tetracycline Chemically-Modified Tetracycline derivative without antimicrobial activity. Under LPS or αSa stimulation, COL-3 (10, 20 µM) efficiently COL-3 and Its Parent Compound repressed the induction of the microglial activation marker Iba-1 and the stimulated-release Doxycycline Prevent Microglial of the pro-inflammatory cytokine TNF-α. COL-30s inhibitory effects on TNF-α were reproduced Inflammatory Responses by by the tetracycline doxycycline (DOX; 50 µM), the , and Reducing Glucose-Mediated (APO), an inhibitor of the -producing NADPH . This last Oxidative Stress. Cells 2021, 10, 2163. observation suggested that COL-3 and DOX might also operate themselves by restraining oxidative https://doi.org/10.3390/ stress-mediated signaling events. Quantitative measurement of intracellular reactive species cells10082163 (ROS) levels revealed that COL-3 and DOX were indeed as effective as APO in reducing oxidative

Academic Editor: Dominique Debanne stress and TNF-α release in activated microglia. ROS inhibition with COL-3 or DOX occurred together with a reduction of microglial glucose accumulation and NADPH synthesis. This suggested that Received: 29 June 2021 COL-3 and DOX might reduce microglial oxidative burst activity by limiting the glucose-dependent Accepted: 13 August 2021 synthesis of NADPH, the requisite substrate for NADPH oxidase. Coherent with this possibility, Published: 22 August 2021 the glycolysis inhibitor 2-deoxy-D-glucose reproduced the immunosuppressive action of COL-3 and DOX in activated microglia. Overall, we propose that COL-3 and its parent compound DOX Publisher’s Note: MDPI stays neutral exert anti-inflammatory effects in microglial cells by inhibiting glucose-dependent ROS production. with regard to jurisdictional claims in These effects might be strengthened by the intrinsic antioxidant properties of DOX and COL-3 in published maps and institutional affil- a self-reinforcing manner. iations.

Keywords: COL-3; glucose metabolism; microglia; NADPH oxidase; neuroinflammation; oxidative stress; tetracyclines

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article 1. Introduction distributed under the terms and Tetracyclines are a family of that inhibit bacterial protein synthesis by conditions of the Creative Commons Attribution (CC BY) license (https:// attaching to the ribosomal subunit and consequently blocking the binding of aminoacyl- creativecommons.org/licenses/by/ tRNAs to the A site [1]. Although the effects of tetracyclines as antibiotics have 4.0/). long been known, there has been a renewed interest in this family of molecules. Indeed, in

Cells 2021, 10, 2163. https://doi.org/10.3390/cells10082163 https://www.mdpi.com/journal/cells Cells 2021, 10, x FOR PEER REVIEW 2 of 16

1. Introduction Tetracyclines are a family of antibiotics that inhibit bacterial protein synthesis by at- taching to the ribosomal subunit and consequently blocking the binding of aminoacyl- Cells 2021, 10, 2163 tRNAs to the ribosome A site [1]. Although the effects of tetracyclines as antibiotics have2 of 16 long been known, there has been a renewed interest in this family of molecules. Indeed, in addition to their antibacterial activity, they can exert pharmacological effects that may have clinical applications in various disease states, including cancer [2], inflammation- relatedaddition disorders to their antibacterial[3], and CNS activity, neurodegenerative they can exert pathologies pharmacological [4,5]. effects that may have clinicalAs an applications illustration in of various that, doxycycline disease states, (DOX), including an old cancertetracycline [2], inflammation-related antibiotic currently disorders [3], and CNS neurodegenerative pathologies [4,5]. used for the treatment of problems [6], combines anti-tumoral [7], anti-inflammatory As an illustration of that, doxycycline (DOX), an old tetracycline antibiotic currently [8], and neuroprotective [9,10] activities. In particular, a number of studies reported neu- used for the treatment of skin problems [6], combines anti-tumoral [7], anti-inflammatory [8], roprotective effects of DOX in toxin-induced animal models of Parkinson’s disease (PD) and neuroprotective [9,10] activities. In particular, a number of studies reported neuropro- [9,10]. DOX was also found to decrease the expression of several inflammation markers in tective effects of DOX in toxin-induced animal models of Parkinson’s disease (PD) [9,10]. microglial cultures activated with the bacterial inflammogen lipopolysaccharide (LPS) [8], DOX was also found to decrease the expression of several inflammation markers in mi- which indicates that DOX’s neuroprotective effects may be due in part to its ability to limit croglial cultures activated with the bacterial inflammogen lipopolysaccharide (LPS) [8], inflammation-related events [9,10]. The anti-inflammatory properties of DOX may also which indicates that DOX’s neuroprotective effects may be due in part to its ability to explain why this tetracycline provided relief against L-DOPA-induced dyskinesia in a PD limit inflammation-related events [9,10]. The anti-inflammatory properties of DOX may rat model [11]. Interestingly, DOX was also found capable of preventing amyloid aggre- also explain why this tetracycline provided relief against L-DOPA-induced dyskinesia in gation of α Synuclein (αS) and tau, two seeding-prone involved in PD [12,13] and a PD rat model [11]. Interestingly, DOX was also found capable of preventing amyloid Alzheimer’saggregation ofdiseaseα Synuclein [14] pathologies, (αS) and tau, respectively, two seeding-prone suggesting proteins that involvedthis tetracycline in PD [12 has,13] theand potential Alzheimer’s of a multimod disease [14al] pathologies,neuroprotective respectively, drug. suggesting that this tetracycline has the potentialCOL-3, also of a multimodalnamed chemically neuroprotective modified drug. tetracycline 3 (CMT-3), 4-dedimethyla- minosancyclineCOL-3, also, or named incyclinide chemically, belongs modified to a group tetracycline of modified 3 (CMT-3), tetracyclines 4-dedimethylamin- that have beenosancycline, structurally or incyclinide, rearranged to belongs suppress to atheir group antibacterial of modified properti tetracyclineses. Removal that haveof the been an- timicrobialstructurally activity rearranged obtained to suppress by eliminating their antibacterial the dimethylamino properties. group Removal from of carbon the antimi- 4 in thecrobial A ring activity of the obtainedtetracycline by eliminatingcore structure the (Figure dimethylamino 1) was carried group out from with carbon the principle 4 in the ofA preserving ring of the other tetracycline activities core of structureinterest [15,16]. (Figure COL1) was-3 has carried been outtested with in theclinical principle trials ofin cancerpreserving patients other [17,18], activities and its of interestcapacity [of15 ,inhibiting16]. COL-3 matrix has been metalloproteinases tested in clinical offers trials the in possibilitycancer patients to reduce [17,18 the], and excessive its capacity breakdown of inhibiting of connective matrix metalloproteinasestissue in many pathological offers the conditionspossibility [19 to– reduce21]. COL the-3 excessive is highly breakdownlipophilic, so of it connective can cross the tissue blood in many–brain pathologicalbarrier and exertconditions CNS effects [19–21 [3,22].]. COL-3 In isthat highly respect, lipophilic, systemic so administration it can cross the of blood–brain COL-3 was barrier reported and toexert inhibit CNS brain effects microglial [3,22]. In activation that respect, induced systemic by the administration bacterial inflammogen of COL-3 LPS was [22]. reported How- to ever,inhibit the brain mechanism microglial through activation which induced COL-3 by reduces the bacterial neuroinflammation inflammogen LPS is still [22 ].unclear. However, the mechanism through which COL-3 reduces neuroinflammation is still unclear.

Figure 1. Chemical structures of the non-antibiotic tetracycline COL-3 and of its parent com- Figure 1. Chemical structures of the non-antibiotic tetracycline COL-3 and of its parent compound pound DOX. COL-3 differs from DOX by the absence of the DCBA naphthacene core structure DOX. COL-3 differs from DOX by the absence of the DCBA naphthacene core structure of dimethyl- aminoof dimethylamino-,-, hydroxyl-, and hydroxyl-, methyl- groups and methyl- in C-4, groupsC-5, and in C C-4,-6, respectively C-5, and C-6,. The respectively. loss of antimicrobial The loss activityof antimicrobial specifically activity results specifically from the removal results of from the thedimethylamino removal of thegroup dimethylamino at the C4 position. group at the C4 position. In the present study, we wished to further explore the mechanisms underlying the anti-inflammatory activity of COL-3 towards brain microglial cells, using its parent com- pound DOX as a reference tetracycline molecule. For this research project, we used brain microglial cells challenged with LPS and amyloid aggregates of the synaptic pro- tein αSa in order to model brain inflammatory-type reactions as they may occur in PD neurodegeneration [23–25]. We found that COL-3 was intrinsically more potent than DOX, Cells 2021, 10, 2163 3 of 16

as it was optimally effective at lower concentrations than its parent compound. Both COL-3 and DOX seemed to operate by limiting the capacity of microglia of generating glucose-dependent NADPH and, as a consequence, (ROS) via NADPH oxidase activation.

2. Materials and Methods 2.1. Drugs and Cell Culture Reagents Lipopolysaccharide (LPS; strain O26:B6; #L8274), 2-deoxy-D-glucose (2-DG; #D8375), Trolox (TROL; #238813), dexamethasone (DEX; #D4902), and DOX hyclate (#D9891) were obtained from Sigma-Aldrich (L’Isle d’Abeau Chesnes, France). COL-3, also named CMT-3, 4-dedimethyl aminosancycline, or incyclinide (#HY-13648), was purchased from MedChemExpress (Monmouth Junction, NJ, USA) and apocynin (APO; #4663) from R&D Systems Europe (Lille, France). Dulbecco’s modified Eagle’s medium (DMEM) and trypsin (0.05%)-EDTA solution, used to generate microglial subcultures, were obtained from ThermoFisher Scientific (Saint Aubin, France). Fetal calf serum (FCS) was provided by Biowest LLC (Eurobio, Les Ulis, France), and 2-[1,2-3H (N)]-deoxy-D-glucose (NET549A) was purchased from Perkin Elmer (Courtaboeuf, France).

2.2. Microglial Cell Cultures Ethics Statement To generate microglial cell cultures, we used newborn C57BL/6J mice obtained from Janvier LABS (Le Genest St. Isle, France). Mice were kept and cared for, following Euro- pean Union Council Directives (2010/63/EU). The Committee on the Ethics of Animal Experiments Charles Darwin no. 5 approved experimental protocols.

2.3. Polyethyleneimine Coating Polyethyleneimine (PEI; Mw: 750,000; Mn: 60,000; #P3143, Sigma-Aldrich, L’Isle d’Abeau Chesnes, France) was applied to culture flasks, as previously described [26]. PEI was diluted in a 40 mM borate buffer solution (1 mg/mL; pH 8.3) prepared with borax decahydrate (#B3545) [26,27]. After 2 h of incubation (37 ◦C), PEI was discarded, and culture flasks washed four times with Dulbecco’s phosphate-buffered saline (PBS) were kept aside until further use.

2.4. Isolation of Microglial Cells and Production of Microglial Subcultures The brains of mouse pups (postnatal day 1) were mechanically dissociated, as pre- viously described [26]. Microglial cells in suspension were plated in PEI-coated T-75 culture flasks (Corning) containing 12 mL of DMEM supplemented with FCS (10%) and a penicillin/ cocktail. Under these conditions, microglial cell isolation occurs spontaneously after 14–18 days of culture. When required, purified microglial cells were maintained for up to 1 more week in culture flasks by adding 2–3 mL of DMEM supple- mented with penicillin/streptomycin and only 1% of FCS [28]. Subcultures were produced by trypsinizing purified microglial cell cultures with 0.05% trypsin-EDTA for 5 min. After trypsin inactivation with 10% FCS, cells were triturated and plated onto uncoated Nunc 48-multiwell plates (105 cells per well), using DMEM supplemented with antibiotics and 1% FCS as maintenance medium.

2.5. Purification and Aggregation of Recombinant αS The expression and purification of recombinant human αS were performed as pre- viously described [12,29]. The purity of recombinant αS was evaluated by SDS-PAGE, and contaminating endotoxins were removed from protein samples using a Pierce Spin column (#88275; Thermo Fisher Scientific, Courtaboeuf, France). The Limulus amebocyte lysate assay revealed that monomeric αS stock solutions contain less than 0.1 endotoxin units (EU)/mL at this stage. The protein solutions were filtered and centrifuged, and the absorbance of the supernatant was measured at 280 nm using a Nanodrop 8000 spectropho- Cells 2021, 10, 2163 4 of 16

tometer (Thermo Fisher Scientific, Courtaboeuf, France). Finally, protein solutions were adjusted to obtain appropriate working concentrations. To generate amyloid aggregates of αS, we used a protocol reported by us before [12,30]. Briefly, αS samples incubated at 37 ◦C were submitted to continuous orbital agitation for 96 h (Thermomixer Comfort; Eppendorf, Montesson, France). Recovered samples sonicated for 2 min with a Branson B3510-DTH ultrasonic bath (VWR International, Fontenay sous Bois, France) were then kept at −20 ◦C until further use [13].

2.6. Transmission Electron Microscopy (TEM) We used TEM characterization to confirm that the process of αS fibrillization occurred in agitated samples [30]. Briefly, samples were adsorbed onto glow-discharged 200-mesh- carbon-film-coated copper grids. Then, samples were stained with 2% uranyl acetate. The liquid in excess was discarded, and the grids were left to dry at room temperature. TEM images were captured using a Hitachi HT7700 120 kV system.

2.7. Stimulation Protocols with Inflammogens and Drug Treatments Microglial cells were treated with either LPS or αSa for 24 h to model brain inflammatory-type reactions. Treatments with test compounds were initiated 2 h be- fore adding the inflammogens to the cultures and prolonged until the end of experimental protocols. Note that none of the test treatments exerted toxic effects on microglial cells under these conditions.

2.8. Immunofluorescence Detection of Microglial Cell Markers A MAC-1/CD11b rat monoclonal antibody (clone M1/70.15) from BioRad (Oxford, UK) and a rabbit antibody raised against ionized calcium-binding adaptor molecule-1 (Iba-1, #019-19741) from Wako Chemicals (Neuss, Germany) were used to monitor mi- croglial cell responses, at the cellular level. Briefly, we fixed microglial cells in 4% formalde- hyde in PBS (20 min at room temperature) and then incubated them sequentially with antibodies against MAC-1/CD11b (1:100 in PBS for 72 h) and Iba-1 (1:500 in PBS 0.2% Triton X-100 overnight). The immunodetection of MAC-1/CD11b and Iba-1 was performed with anti-rat Alexa-Fluor 488- and anti-rabbit Alexa-Fluor 555-conjugated antibodies, respec- tively (Invitrogen, Waltham, MA, USA). Nuclear counterstaining with the cell-permeable Hoechst-33342 dye (1 µg/mL for 5 min) was performed to enable automated focus for quantitative image analysis protocols (Arrayscan XTi workstation; Thermo Fisher Scien- tific, Courtaboeuf, France). Fluorescent images were acquired with a 20× objective, and variations in immunofluorescence signal intensities were quantified using the HCStudio software (Thermo Fisher Scientific, Courtaboeuf, France). Approximately 2500 microglial cells were analyzed for each treatment condition.

2.9. TNF-α Assay TNF-α was quantified using an ELISA kit from Thermo Fisher Scientific (Courtaboeuf, France) (#BMS607-3) according to the manufacturer’s protocol. Absorbance was measured at a wavelength of 450 nm using a SpectraMax i3X microplate reader (Molecular Devices, Sunnyvale, CA, USA).

2.10. Measurement of Intracellular NADPH A high-sensitivity NADPH quantitation fluorometric assay kit (#MAK216; Sigma- Aldrich, L’Isle d’Abeau Chesnes, France) was used to assess intracellular NADPH lev- els, as previously described [31]. The fluorescent reaction product was evaluated with a SpectraMax i3X microplate reader (Molecular Devices, Sunnyvale, CA, USA) using an excitation wavelength of 535 nm and an emission wavelength of 587 nm. Cells 2021, 10, 2163 5 of 16

2.11. Quantification of Intracellular ROS Levels Intracellular ROS levels were measured using the CellROX Deep Red reagent (Invitro- gen Life Technologies, Waltham, MA, USA), as previously described [31]. Signal acquisition was performed on an Arrayscan XTI automated workstation fitted with a 20× objective (Thermo Fisher Scientific, Courtaboeuf, France). Cellular fluorescence intensity was esti- mated using HCStudio software.

2.12. Assessment of [3H]-2-DG Uptake [3H]-2-DG uptake was assessed as previously described [31]. Briefly, after termination of test treatments, microglial cell cultures were incubated at 37 ◦C for 30 min in PBS containing 1 µCi [3H]-2-DG. 2-DG accumulation was interrupted by adding ice-cold PBS to the cultures. Microglial cells in culture were then scrapped in a 1% Triton-X–distilled water solution, and the radioactivity accumulated intracellularly was measured using a Tri-Carb 4910TR liquid scintillation counter (Villebon Sur Yvette, France). Blank values were measured in control wells containing 50 mM glucose.

2.13. Statistical Analysis Data values were collected from a minimum of two independent experiments, each performed in duplicate cultures. Experimental data are presented as the mean ± SEM. Statistical analyses were performed using one-way ANOVA followed by a Bonferroni post- hoc test using the GraphPad Prism version 8 for Windows (GraphPad Software, La Jolla, CA, USA).

3. Results 3.1. COL-3 Reduces the Inflammatory Response of Microglial Cells Exposed to LPS or αSa To assess the anti-inflammatory potential of the non-antibiotic tetracycline COL-3, we used primary microglial cell cultures treated with LPS (10 ng/mL), a prototypical agonist of Toll-like receptor (TLR)4 [32,33]. After 24 h of LPS exposure, the production/release of the pro-inflammatory cytokine TNF-α was strongly increased. In cultures treated with 10 or 20 µM COL-3, TNF-α production was reduced by more than 40% and 70%, respectively (Figure2A). No significant inhibitory effect was observed, however, at 1 µM, indicating that the anti-inflammatory action of COL-3 was concentration-dependent and only significant at 10 µM or above. At 20 µM, COL-3 appeared to be as effective as the reference tetracycline DOX at 50 µM. The reference anti-inflammatory drug DEX at 2.5 µM was more effective than COL-3 at 20 µM or DOX at 50 µM in reducing TNF-α release. We also performed immunosignal intensity measurements for the phenotypic activa- tion marker Iba-1 in the same experimental conditions. As expected, Iba-1 expression was significantly up-regulated in LPS-treated cultures. COL-3 suppressed Iba-1 induction at 10 and 20 µM but was without significant effect, at 1 µM (Figure2B). DOX (50 µM) also efficiently reduced the Iba-1 immunofluorescence signal in LPS-treated cultures. Note that 2.5 µM DEX reduced Iba-1 expression with an efficacy similar to that of 20 µM COL-3 or 50 µM DOX. Photomicrographs in Figure2C illustrate the impact of COL-3 (20 µM) or DOX (50 µM) treatments on Iba-1 immunosignal intensities in LPS-treated microglial cells immunostained with CD11b. To model inflammatory reactions as they may occur in PD [23,24], we used αSa to trigger an inflammogenic response in microglial cell cultures. The illustration from the insert of Figure2D confirms the presence of amyloid fibrils in αS samples shaken for 96 h (lower image), whereas non-shaken samples are free of such species (upper image). Figure2D shows that 24 h of exposure to 70 µg/mL αSa results in a robust increase in TNF-α release, whereas the monomeric form of the protein (αSm) induced only a limited but significant response in microglial cells. At 10 or 20 µM, COL-3 significantly reduced αSa-induced TNF-α release. DOX at 50 µM exerted a comparable repressive effect, while 2.5 µM DEX was as effective as 20 µM COL-3 and 50 µM DOX. Cells 2021, 10, x FOR PEER REVIEW 6 of 16

(lower image), whereas non-shaken samples are free of such species (upper image). Figure 2D shows that 24 h of exposure to 70 µg/mL αSa results in a robust increase in TNF-α release, whereas the monomeric form of the protein (αSm) induced only a limited but significant response in microglial cells. At 10 or 20 µM, COL-3 significantly reduced αSa- induced TNF-α release. DOX at 50 µM exerted a comparable repressive effect, while 2.5 µM DEX was as effective as 20 µM COL-3 and 50 µM DOX. Consistent with previous observations [24], we found that αSa induced Iba-1 overex- pression. COL-3 (10 or 20 µM), DOX (50 µM), and DEX (2.5 µM) were all effective in re- ducing the Iba-1 immunofluorescent signal in αSa-treated microglial cultures (Figure 2E). Note that the intensity of the Iba-1 immunosignal was not modified by αSm despite the fact that TNF-α release was slightly increased by such treatment. This may signify that quantifying TNF-α with an ELISA assay kit may be more sensitive than measuring changes in cellular Iba-1 expression levels when microglial inflammatory responses are of small intensity. Photomicrographs in Figure 2F represent microglial cell cultures treated Cells 2021, 10, 2163 with αSa in the presence or absence of COL-3 (20 µM) or DOX (50 µM), and then6 ofim- 16 munostained for CD11b and Iba-1.

α α Figure 2. COL-3 reduces inflammatory responses in microglial cells exposed to either LPS or Sa. (A) TNF- levels in microglial cell cultures exposed to LPS (10 ng/mL, 24 h) in the presence or absence of COL-3 (1, 10, or 20 µM), DOX (50 µM), Figure 2. COL-3 reduces inflammatory responses in microglial cells exposed to either LPS or αSa. (A) TNF-α levels in or the reference anti-inflammatory drug DEX (2.5 µM). Data are the mean ± SEM (n = 4). * p < 0.05 vs. control; # p < 0.05 vs. microglial cell cultures exposed to LPS (10 ng/mL, 24 h) in the presence or absence of COL-3 (1, 10, or 20 μM), DOX (50 µM),LPS. One-wayor the reference ANOVA anti followed-inflammatory by Bonferroni drug DEX post-hoc (2.5 μM). test. Data (B)Quantification are the mean ± of SEM Iba-1 (n immunofluorescence = 4). * p < 0.05 vs. control; signals # p in< 0.05microglial vs. LPS. cell One cultures-way treatedANOVA as infollowed (A). (C) by Merged Bonferroni images post of CD11b-hoc test. (green) (B) andQuantification Iba-1 (red) immunofluorescenceof Iba-1 immunofluorescence signals in signalsLPS (10 in ng/mL)-treated microglial cellmicroglial cultures treated cell cultures as in ( receivingA). (C) Merged COL-3 images (20 µM) of or CD11b DOX (50 (green)µM). Theand reddish/yellowIba-1 (red) immunofl fluorescenceuores- cencedenotes signals overexpression in LPS (10 ng/mL) of Iba-1.-treated Cell nuclei microglial are counterstained cell cultures receiving with Hoechst-33342 COL-3 (20 µM) (blue). or DOX (D) TNF- (50 µM).α levels The inreddish/yel- microglial lowcell culturesfluorescence treated denotes with α overexpressionSa (70 µg/mL, 24of h)Iba in-1. the Cell presence nuclei orare absence counterstained of COL-3 with (10 andHoechst 20 µM),-33342 DOX (blue). (50 µ M),(D) orTNF DEX-α levels(2.5 µ M).in microglial Some cultures cell cultures were also treated exposed with to αSaαSm (70 (70 μg/mL,µg/mL). 24 h) Data in the are presence the mean or± absenceSEM (n of= 4).COL * p-3< (10 0.05 and vs. 20 control; μM), DOX# p < 0.05(50 µM),vs. α orSa. DEX One-way (2.5 μM). ANOVA Some followed cultures bywere Bonferroni also exposed post-hoc to αSm test. (70 TEM μg/mL). images Data from are the the insert mean show ± SEM the presence(n = 4). * p < 0.05 vs. control; # p < 0.05 vs. αSa. One-way ANOVA followed by Bonferroni post-hoc test. TEM images from the insert of αS amyloid fibrils in αS samples shaken for 96 h (lower image) and the absence of aggregated species in non-shaken show the presence of αS amyloid fibrils in αS samples shaken for 96 h (lower image) and the absence of aggregated species samples (upper image). Scale bar = 1 µm. (E) Quantification of Iba-1 immunofluorescence signals in microglial cultures in non-shaken samples (upper image). Scale bar = 1 μm. (E) Quantification of Iba-1 immunofluorescence signals in micro- # α glialexposed cultures to the exposed same treatments to the same as intreatments (D). Data as are in the (D mean). Data± areSEM the (n mean= 4). *±p SEM< 0.05 (n vs. = 4). control; * p < 0.05p

3.2. COLConsistent-3 Reduces with Glucose previous Accumulation observations in Microglial [24], we C foundells Exposed that α toSa LPS induced or αSa Iba-1 over- expression. COL-3 (10 or 20 µM), DOX (50 µM), and DEX (2.5 µM) were all effective in Recent studies suggest that microglial inflammatory responses are profoundly de- reducing the Iba-1 immunofluorescent signal in αSa-treated microglial cultures (Figure2E ). pendent on glucose availability [31,34,35]. Here, we found that 24 h of treatment with 10 Note that the intensity of the Iba-1 immunosignal was not modified by αSm despite the fact that TNF-α release was slightly increased by such treatment. This may signify that quantifying TNF-α with an ELISA assay kit may be more sensitive than measuring changes

in cellular Iba-1 expression levels when microglial inflammatory responses are of small intensity. Photomicrographs in Figure2F represent microglial cell cultures treated with αSa in the presence or absence of COL-3 (20 µM) or DOX (50 µM), and then immunostained for CD11b and Iba-1.

3.2. COL-3 Reduces Glucose Accumulation in Microglial Cells Exposed to LPS or αSa Recent studies suggest that microglial inflammatory responses are profoundly de- pendent on glucose availability [31,34,35]. Here, we found that 24 h of treatment with 10 ng/mL of LPS led to an almost 2-fold increase in [3H]-2-DG uptake in microglial cell cultures (Figure3A). [ 3H]-2-DG uptake was significantly reduced when LPS-treated cell cultures were exposed to either 10 or 20 µM COL-3, whereas a lower COL-3 concentration (1 µM) had no significant effect on this parameter (Figure3A). DOX at 50 µM was also effective in reducing microglial glucose accumulation. Noticeably, DEX at 2.5 µM had no Cells 2021, 10, x FOR PEER REVIEW 7 of 16

ng/mL of LPS led to an almost 2-fold increase in [3H]-2-DG uptake in microglial cell cul- tures (Figure 3A). [3H]-2-DG uptake was significantly reduced when LPS-treated cell cul- tures were exposed to either 10 or 20 µM COL-3, whereas a lower COL-3 concentration (1 µM) had no significant effect on this parameter (Figure 3A). DOX at 50 µM was also ef- fective in reducing microglial glucose accumulation. Noticeably, DEX at 2.5 µM had no impact on [3H]-2-DG uptake, indicating that this compound may exert immunosuppres- sive effects in microglial cells without affecting glucose metabolism. In addition, 300 µM of the NADPH oxidase inhibitor APO or 500 µM of unlabeled 2-DG [a concentration not affecting microglial cell viability], efficiently reduced [3H]-2-DG uptake in agreement with previous observations [31]. As expected, 50 mM glucose added acutely to microglial cell cultures to compete with [3H]-2-DG during uptake assessment also efficiently prevented 3 Cells 2021, 10, 2163 [ H]-2-DG uptake (Figure 3B). 7 of 16 Interestingly, 70 µg/mL αSa also significantly increased [3H]-2-DG uptake in micro- glial cell cultures (Figure 3C), suggesting that the inflammatory response of microglial cells to αS fibril exposure is also fueled by glucose. By contrast, αSm had only a marginal 3 impactimpact on [glucoseH]-2-DG consumption. uptake, indicating Noticeably, that this COL compound-3 (10 or may20 µM) exert significantly immunosuppressive reduced glucoseeffects in accumulation microglial cells in cultur withoutes affectingexposed to glucose αSa; DOX metabolism. at 50 µM In mimicked addition, this 300 µeffect.M of theAs describedNADPH oxidase previously inhibitor for LPS, APO DEX or 500 (2.5µM)µM of failed unlabeled to inhibit 2-DG TNF [a concentrationα release stimulation not affecting by 3 αmicroglialSa. The treatment cell viability], of αSa efficiently-treated cultures reduced with [ H]-2-DG 300 µM uptake APO or in agreement500 µM 2-DG with also previous led to aobservations significant reduction [31]. As expected, in [3H]-2 50-DG mM uptake glucose (Figure added 3D). acutely Adding to microglial glucose in cell excess cultures when to 3 3 performingcompete with uptake [ H]-2-DG measurements during uptake also prevented assessment [3H] also-2-DG efficiently uptake prevented(Figure 3D). [ H]-2-DG uptake (Figure3B).

Figure 3. COL-3 reduces glucose accumulation in microglial cells exposed to either LPS or αSa. Figure(A) Quantification 3. COL-3 reducesof glucose glucose uptake accumulation in microglial in microglial cells exposed cells orexposed not to to LPS either (10 ng/mL,LPS or αSa. 24 h) (A in) Quantificationthe presence or of absence glucose of uptake COL-3 in (10 microglial or 20 µM), cells DOX exposed (50 µM), or ornot DEX to LPS (2.5 (10µM). ng/mL, (B) Quantification 24 h) in the presence or absence of COL-3 (10 or 20 μM), DOX (50 μM), or DEX (2.5 μM). (B) Quantification of of glucose uptake in microglial cells exposed or not to LPS treatment in the presence or absence of glucose uptake in microglial cells exposed or not to LPS treatment in the presence or absence of APO (300 µM) or 2-DG (500 µM). The specificity of the assay was confirmed by acutely exposing LPS- APO (300 µM) or 2-DG (500 µM). The specificity of the assay was confirmed by acutely exposing C LPStreated-treated cell cultures cell cultures to an excessto an excess of glucose of glucose (50 mM). (50 ( mM).) Quantification (C) Quantification of glucose of uptake glucose in microglialuptake in microglialcells exposed cells or exposed not to α orSa not (70 toµ g/mL,αSa (70 24μg/mL, h) in the24 h) presence in the presence or absence or absence of COL-3 of (COL10 and-3 (10 20 µ andM), 20DOX μM), (50 DOXµM), (50 or DEXμM), (2.5or DEXµM). (2.5 (D )μM). Quantification (D) Quantification of glucose of uptakeglucose in uptake microglial in microglial cells exposed cells exposedor not to orα Sanot in to the αSa presence in the presence or absence or absence of APO of (300 APOµM) (300 or µM) 2-DG or(500 2-DGµ M).(500 The µM). specificity The specificity of the ofassay the assay was confirmed was confirmed by acutely by acutely exposing exposingαSa-treated αSa-treated cell cultures cell cultures to an excessto an excess of glucose of glucose (50 mM). (50 mM).Data areData the are mean the ±meanSEM ± (SEMn = 6–8). (n = *6p–8).< 0.05* p < vs. 0.05 control; vs. control;# p < 0.05 # p < vs. 0.05 corresponding vs. corresponding inflammogen. inflam- mogen.One-way One ANOVA-way ANOVA followed followed by Bonferroni by Bonferroni post-hoc post test.-hoc test.

Interestingly, 70 µg/mL αSa also significantly increased [3H]-2-DG uptake in mi- croglial cell cultures (Figure3C), suggesting that the inflammatory response of microglial cells to αS fibril exposure is also fueled by glucose. By contrast, αSm had only a marginal impact on glucose consumption. Noticeably, COL-3 (10 or 20 µM) significantly reduced glucose accumulation in cultures exposed to αSa; DOX at 50 µM mimicked this effect. As described previously for LPS, DEX (2.5 µM) failed to inhibit TNFα release stimulation by αSa. The treatment of αSa-treated cultures with 300 µM APO or 500 µM 2-DG also led to a significant reduction in [3H]-2-DG uptake (Figure3D). Adding glucose in excess when performing uptake measurements also prevented [3H]-2-DG uptake (Figure3D).

3.3. COL-3 Prevents the Rise of NADPH in Microglial Cells Exposed to LPS or αSa Glucose may have a key impact on microglial cell inflammatory processes by activation of the pentose phosphate pathway and, consequently, by stimulating NADPH synthesis, a required substrate for the superoxide-producing enzyme NADPH oxidase [31,34]. Con- Cells 2021, 10, x FOR PEER REVIEW 8 of 16

3.3. COL-3 Prevents the Rise of NADPH in Microglial Cells Exposed to LPS or αSa Glucose may have a key impact on microglial cell inflammatory processes by activa- tion of the pentose phosphate pathway and, consequently, by stimulating NADPH syn- thesis, a required substrate for the superoxide-producing enzyme NADPH oxidase [31,34]. Consistent with this view, NADPH levels rose significantly in microglial cells treated with either 10 ng/mL of LPS or 70 µg/mL αSa (Figure 4A,B). No effect was ob- served, however, with αSm. Interestingly, COL-3 (20 µM) significantly reduced NADPH Cells 2021, 10, 2163 8 of 16 levels in both inflammation paradigms (Figure 4A,B). As expected, the inhibitory effects of COL-3 in each activation paradigm were mimicked with DOX at 50 µM. Both 2-DG (500 µM) and the NADPH oxidase inhibitor APO (300 µM) mimicked the inhibitorysistent with effect this of view, COL NADPH-3 on NADPH levels production rose significantly in microglial in microglial cells exposed cells treated to LPS with or αeitherSa (Figure 10 ng/mL 4A,B), of indicating LPS or 70 thatµg/mL the nonαSa-anti (Figurebiotic4A,B). tetracycline No effect and was its observed, parent compound however, DOXwith mayαSm. interfere Interestingly, with a glucose COL-3-dependent (20 µM) significantly mechanism reduced that promotes NADPH NADPH levels synthe- in both sisinflammation and, consequently, paradigms ROS (Figureproduction4A,B). via As NADPH expected, oxidase the inhibitory activation. effects of COL-3 in each activation paradigm were mimicked with DOX at 50 µM.

Figure 4. COL-3 prevents NADPH synthesis in microglial cells exposed to either LPS or αSa. Figure(A) Quantification 4. COL-3 preventsof NADPH NADPH in microglial synthesis cells in microglial exposed or cells not ex toposed LPS (10 to ng/mL, either LPS 24 h) or in αSa. the pres-(A) Quantificationence or absence of of NADPH COL-3 (20in microglialµM), DOX cells (50 µ exposedM), APO or (300 notµ toM), LPS or 2-DG(10 ng/mL, (500 µ M).24 h) (B in) Quantificationthe presence orof absence NADPH of in COL microglial-3 (20 μM), cells DOX exposed (50 μM), or not APO to α Sa(300 (70 μM),µg/mL, or 2- 24DG h) (500 in the µM). presence (B) Quantification or absence of of NADPH in microglial cells exposed or not to αSa (70 μg/mL, 24 h) in the presence or absence of COL-3 (20 µM), DOX (50 µM), APO (300 µM), or 2-DG (500 µM). Data are the mean ± SEM (n = 6–8). COL-3 (20 μM), DOX (50 μM), APO (300 μM), or 2-DG (500 µM). Data are the mean ± SEM (n = 6– * p < 0.05 vs. control; # p < 0.05 vs. corresponding inflammogen. One-way ANOVA followed by 8). * p < 0.05 vs. control; # p < 0.05 vs. corresponding inflammogen. One-way ANOVA followed by BonBonferroniferroni post post-hoc-hoc test. test. Both 2-DG (500 µM) and the NADPH oxidase inhibitor APO (300 µM) mimicked the 3.4. COL-3 Prevents Microglial Oxidative Stress Elicited by Inflammatory Signals inhibitory effect of COL-3 on NADPH production in microglial cells exposed to LPS or αSa (FigureA quantitative4A,B), indicating cell-based that the flu non-antibioticorescence assay tetracycline was performed and its to parent estimate compound ROS levels DOX inmay microglial interfere cells with treated a glucose-dependent with LPS or α mechanismSa and other that test promotes treatments NADPH (Figure synthesis 5). As and,ex- pected,consequently, 24 h of ROStreatment production with LPS via NADPH(10 ng/mL) oxidase or αSa activation. (70 µg/mL) significantly increased intracellular ROS levels in microglial cells. By contrast, αSm had no effect. Interestingly, ROS3.4. COL-3 production Prevents was Microglial maintained Oxidative near control Stress Elicited values by when Inflammatory microglial Signals cell cultures ex- posedA to quantitative either LPS (Figure cell-based 5A,B) fluorescence or αSa (Figure assay 5C,D) was performed were treated to estimate with 20 ROSµM levelsCOL-3, in confirmingmicroglial cellsthe view treated that with the LPSnon- orantibioticαSa and tetracycline other test treatments may exert (Figureanti-inflammatory5). As expected, ef- fects24 h byof treatmentcounteracting with oxidati LPS (10ve- ng/mL)stress-dependent or αSa (70 mechanisms.µg/mL) significantly As expected, increased the inhibi- intra- torycellular effect ROS of COL levels-3 inon microglialROS generation cells. Bywas contrast, mimickedαSm by had DOX no at effect. 50 µM. Interestingly, In each activa- ROS tionproduction paradigm, was the maintained NADPH nearoxidase control inhibitor values APO when (300 microglial µM) and cell the cultures antioxidant exposed TROL to ei- ther LPS (Figure5A,B) or αSa (Figure5C,D) were treated with 20 µM COL-3, confirming the view that the non-antibiotic tetracycline may exert anti-inflammatory effects by counteract- ing oxidative-stress-dependent mechanisms. As expected, the inhibitory effect of COL-3 on ROS generation was mimicked by DOX at 50 µM. In each activation paradigm, the NADPH oxidase inhibitor APO (300 µM) and the antioxidant TROL (10 µM) reduced microglial ROS production with an efficacy similar to that demonstrated by COL-3 and DOX. Cells 2021, 10, x FOR PEER REVIEW 9 of 16

Cells 2021, 10, 2163 (10 µM) reduced microglial ROS production with an efficacy similar to that demonstrated9 of 16 by COL-3 and DOX.

Figure 5. COL-3 prevents intracellular microglial oxidative stress generated by LPS or αSa exposure. Figure 5. COL-3 prevents intracellular microglial oxidative stress generated by LPS or αSa exposure. (A(A) )Quantification Quantification of of ROS ROS levels levels in in microglial microglial cells cells exposed exposed or or not not to to LPS LPS (10 (10 ng/m ng/mL,L, 24 24 h) h) in in the the presencepresence or or absence absence of of COL COL-3-3 (20 (20 μM),µM), DOX (50 µμM),M), APOAPO (300(300µ μM),M), or or TROL TROL (10 (10µ M).μM). Data Data are are the themean mean± SEM± SEM (n =(n6–8). = 6–8). * p *< p 0.05 < 0.05 vs. control;vs. control;# p < # 0.05p < 0.05 vs. LPS.vs. LPS. One-way One- ANOVAway ANOVA followed followed by Bonfer- by Bonferronironi post-hoc post test.-hoc (test.B) Visualization (B) Visualization of intracellular of intracellular ROS levelsROS levels in LPS in (10 LPS ng/mL)-treated (10 ng/mL)-treated microglial mi- croglialcell cultures cell cultures receiving receiving or not COL-3or not COL (20 µ-M)3 (20 or µM) DOX or (50 DOXµM). (50 Scale µM). bar Scale = 50 barµm. = 50(C µm) Quantification. (C) Quan- tificationof ROS levelsof ROS in levels microglial in microglial cells exposed cells exposed or not to orα Sanot (70 to αSaµg/mL, (70 μg/mL, 24 h) in 24 the h) presence in the presence or absence or absenceof COL-3 of COL (20 µ-3M), (20 DOX μM), (50DOXµM), (50 APOμM), (300APOµ (300M), orμM), TROL or TROL (10 µ M).(10 μM). Data areData the are mean the mean± SEM ± SEM (n = 6–8). * p < 0.05 vs. control; # p < 0.05 vs. αSa. One-way ANOVA followed by Bonferroni (n = 6–8).* p < 0.05 vs. control; # p < 0.05 vs. αSa. One-way ANOVA followed by Bonferroni post-hoc post-hoc test. (D) Visualization of intracellular ROS levels in αSa (70 μg/mL)-treated microglial cell test. (D) Visualization of intracellular ROS levels in αSa (70 µg/mL)-treated microglial cell cultures cultures exposed or not to COL-3 (20 µM) or DOX (50 µM) treatment. Scale bar = 50 µm. exposed or not to COL-3 (20 µM) or DOX (50 µM) treatment. Scale bar = 50 µm.

3.5.3.5. The The S Suppressiveuppressive E Effectffect of of COL COL-3-3 on on TNF TNF--αα RReleaseelease Is Is R Reproducedeproduced by I Interferingnterfering with GlucoseGlu- coseMetabolism Metabolism or NADPHor NADPH Oxidase Oxidase Activation Activation GivenGiven the the repressive repressive action action of of COL COL-3-3 on on microglial microglial glucose glucose consumption consumption and and ROS ROS production,production, we we wished wished to compare to compare its effect its effect to that to of that 2-DG of and 2-DG APO, and using APO, TNF using-α release TNF-α asrelease reference as referenceinflammation inflammation marker. Similar marker. to SimilarCOL-3, toAPO COL-3, (300 APOµM) and (300 2µ-DGM) (500 and 2-DGµM) were(500 highlyµM) were effective highly in effectivereducing in LPS reducing- (Figure LPS- 6A) (Figureor αSa-6inducedA) or α Sa-induced(Figure 6B) TNF (Figure-α re-6B) lease,TNF- furtherα release, supporting further supporting the view that the COL view-3 that exerts COL-3 its anti exerts-inflammatory its anti-inflammatory effect by inter- effect feringby interfering with a glucose with- adependent glucose-dependent mechanism mechanism that promotes that promotesoxidative s oxidativetress via NADPH stress via oxidaseNADPH activation oxidase activation(Figure 6B). (Figure 6B).

Cells 2021, 10, 2163 10 of 16 Cells 2021, 10, x FOR PEER REVIEW 10 of 16

Figure 6. Inhibitory effects of COL-3 on TNF-α release are reproduced by inhibition of either glucose Figuremetabolism 6. Inhibitory or NADPH effects oxidase of COL activity.-3 on TNF (A-)α Quantification release are reproduced of TNF-α bylevels inhibition in microglial of either cell glucose cultures metabolismexposed or or not NADPH to LPS (10oxidase ng/mL, activity. 24 h) in(A the) Quantification presence or absence of TNF of-α COL-3 levels (20in microglialµM), APO cell (300 cul-µM), turesor 2-DG exposed (500 orµM). not Data to LPS are (10 the ng/mL, mean ± 24SEM h) in ( nthe= 6).presence * p < 0.05 or vs.absence control; of COL# p <- 0.053 (20 vs. μM), LPS. APO One-way (300 µM), or 2-DG (500 µM). Data are the mean ± SEM (n = 6). * p < 0.05 vs. control; # p < 0.05 vs. LPS. One- ANOVA followed by Bonferroni post-hoc test. (B) Quantification of TNF-α levels in microglial cell way ANOVA followed by Bonferroni post-hoc test. (B) Quantification of TNF-α levels in microglial cultures exposed or not to αSa (70 µg/mL, 24 h) in the presence or absence of COL-3 (20 µM), APO cell cultures exposed or not to αSa (70 µg/mL, 24 h) in the presence or absence of COL-3 (20 μM), # APO(300 (300µM), µM), or 2-DG or 2-DG (500 (500µM). µM). Data Data are theare meanthe mean± SEM ± SEM (n =(n 6). = 6). * p *< p 0.05 < 0.05 vs. vs. control; control;p #

Cells 2021, 10, 2163 11 of 16

inflammatory effects. Incidentally, this confirms that the anti-inflammatory potential of tetracyclines is totally dissociated from their antimicrobial properties. Present findings are also consistent with studies reporting on the effects of COL-3 in experimental models of inflammation. For instance, COL-3 was found to inhibit - induced thermal hyperalgesia [36], a condition with a strong inflammatory component [37]. Cazalis et al. [38] showed that COL-3 was also capable of reducing cytokine secretion in an ex vivo human whole-blood model used to study periodontitis inflammation. In addition, COL-3 was reported to be effective in limiting microglial cell activation and TNF-α (but not IL-1β) production in the brain of mice receiving LPS intraperitoneal injections [22].

4.2. Anti-Inflammatory Properties of COL-3 Derive from Its Ability to Inhibit Glucose Uptake Together with others, we reported that inflammatory conditions cause dramatic changes in microglial glucose metabolism [31,34,39]. Precisely, we found that glucose (i.e., 2-DG) uptake was strongly enhanced in microglial cells activated by the bacterial inflammogen LPS [31]. We confirmed here this initial observation and showed that mi- croglial cells responded similarly to αSa, suggesting that changes in glucose metabolism are not restricted to TLR4-dependent mechanisms but also pertain to activation paradigms relying on TLR2/ P2X7 purinergic receptors [24,30]. Remarkably, COL-3 (10–20 µM) ro- bustly inhibited glucose uptake in both LPS- and αSa-treated microglial cells, suggesting that COL-3 may repress both inflammatory responses by reducing glucose utilization. At a concentration of 50 µM, the reference antibiotic tetracycline DOX mimicked the inhibitory effects of COL-3 on glucose uptake, which indicates that a reduction in glucose availability may also contribute to the anti-inflammatory effects of DOX. Incidentally, the fact that glucose uptake was measured after removal of COL-3 and DOX from the cultures indicates that these compounds did not repress glucose accumulation through a simple competitive inhibition mechanism. Note that DEX failed to repress microglial glucose uptake in activated microglia, regardless of the inflammation trigger. This is unexpected since it has been reported that DEX can inhibit glucose uptake in contracting myotubes [40]. Yet, classically inhibit the transcription of several that encode pro-inflammatory cy- tokines and chemokines [41], suggesting that alternative mechanisms are involved in DEX-immunosuppressive effects for microglial cells.

4.3. COL-3 Prevents Glucose-Dependent NADPH Production Glucose is known to fuel glycolysis and oxidative phosphorylation through glycolytic pyruvate [42]. In microglial cells, our group and others [31,34] have shown that a substantial fraction of glucose entering glycolysis is diverted at the level of Glucose-6-P to the pentose monophosphate shunt for regenerating NADPH, a dinucleotide operating as a requisite substrate for NADPH oxidase [42,43]. Interestingly, we show here that the increase in glucose consumption induced by LPS and αSa treatments resulted in a rise in NADPH synthesis that is preventable by COL-3. This indicates that the immunosuppressive action of COL-3 observed against the two inflammatory signals may result from its ability to limit the glucose-dependent synthesis of NADPH and, as a consequence, ROS production by NADPH oxidase. As expected, DOX also suppressed NADPH synthesis in microglial cells challenged with LPS and αSa, confirming the view that the two tetracyclines operate in a similar manner to mitigate microglial inflammatory reactions. Even if our results suggest that COL-3 and DOX act principally by reducing NADPH, generated via the pentose phosphate pathway, additional effects of the two tetracyclines on glycolytic cannot be totally excluded [39].

4.4. COL-3 Operates by Preventing Glucose-Dependent ROS Production The use of the fluorogenic probe CellROX Deep reagent revealed that ROS production was robustly increased in LPS and αSa-activated microglial cells in good agreement with previous reports [24,30,44]. Interestingly, ROS induction was efficiently inhibited by COL-3 Cells 2021, 10, x FOR PEER REVIEW 12 of 16 Cells 2021, 10, 2163 12 of 16 COL-3 and DOX. Both the antioxidant vitamin E derivative TROL and the inhibitor of the ROS producing enzyme NADPH oxidase APO mimicked the suppressive action of COL- 3and and DOX. DOX Bothon ROS the production. antioxidant This vitamin signifies E derivative that COL TROL-3 and and DOX the may inhibitor operate of theby pre- ROS ventingproducing a sequence enzyme of NADPH signaling oxidase events APO in which mimicked glucose the suppressivepromotes NADPH action ofoxidase COL-3-me- and diatedDOX onROS ROS production production. and This subsequent signifies thatinflammatory COL-3 and responses DOX may such operate as TNF by preventingα produc- tion.a sequence Coherentof signalingwith this events view, inblocking which glucoseNADPH promotes oxidaseNADPH activity oxidase-mediatedwith APO prevented ROS TNFproductionα release and after subsequent exposure inflammatoryto LPS or αSa. responses We may suchassume as TNFthat αtheproduction. inhibitory Coherenteffect of COLwith-3 this on view,TNFα blocking release resulted NADPH from oxidase the activity inhibition with of APO the redox prevented-sensitive TNF αnuclearrelease tran- after scriptionexposure nuclear to LPS orfactorαSa.-κB, We maya master assume regulator that the of inhibitory cytokine effectproduction of COL-3 in onmicroglial TNFα release cells [31,44]resulted. As from a matter the inhibition of fact, DOX of the was redox-sensitive reported previou nuclearsly transcriptionto inhibit NF nuclear-kB activation factor- κinB, differenta master model regulator systems of cytokine [8,45]. production in microglial cells [31,44]. As a matter of fact, DOXThe was efficacy reported of previouslyCOL-3 and to DOX inhibit to NF-kB restrain activation glucose in-dependent different model NADPH systems synthesis [8,45]. suggestsThe that efficacy both oftetracyclines COL-3 and mitigate DOX to microglial restrain glucose-dependent inflammatory responses NADPH by reducing synthesis thesuggests amount that of both NADPH tetracyclines available mitigate as a cofactor microglial for inflammatoryNADPH oxidase. responses Pointing by reducing to the im- the portanceamount of of NADPH such a available mechanism, as a cofactor we found for NADPH that restraining oxidase. glucosePointing metabolismto the importance and NADPHof such a synthesis mechanism, with we the found non that-metabolizable restraining glucose metabolismanalogue 2- andDG was NADPH sufficient synthesis per sewith to reproduce the non-metabolizable the suppressive glucose effects analogue of COL-3 2-DG and DOX was sufficienton TNF-α perrelease. se to Unexpect- reproduce edly,the suppressive Fodelianaki effectsand colleagues of COL-3 reported and DOX that on 2 TNF--DGα wasrelease. unable Unexpectedly, to prevent LPS Fodelianaki-induced TNFand-α colleagues release in reported microglial that cell 2-DG cultures was unable[39]. The to reason prevent for LPS-induced such a discrepancy TNF-α releasebetween in themicroglial two studies cell culturesis rather [unclear39]. The at reason this stage. for such a discrepancy between the two studies is ratherIt is unclear also worth at this noting stage. that NADPH oxidase inhibition by APO caused a drop in glu- cose uptakeIt is also in activated worth noting microglia, that NADPH indicating oxidase that ROS inhibition might represent by APO an caused activation a drop sig- in nalglucose for microglial uptake in glucose activated accumulation microglia, indicatingvia a specific that transport ROS might system represent. As a matter an activation of fact, ROSsignal production for microglial by NADPH glucose oxidase accumulation was reported via a specific to stimulate transport glucose system. accumulation As a matter via of afact, mechanism ROS production implicating by NADPH glucose oxidasetransporter was-4 reported in muscle to stimulatecells [46]. glucoseThis means accumulation that the inhibitoryvia a mechanism effects of implicating COL-3 and glucose DOX on transporter-4 glucose uptake in muscle may also cells partly [46]. Thisdepend means on thatthe intrinsicthe inhibitory antioxidant effects properties of COL-3 of and these DOX two on compounds glucose uptake [47,48 may]. Therefore, also partly one depend may as- on sumethe intrinsic that the antioxidantcapacity of propertiesCOL-3 and of DOX these to twoinhibit compounds glucose-dependent [47,48]. Therefore, ROS production one may viaassume a suppressive that the capacity effect on of COL-3 NADPH and synthesis DOX to inhibit is strengthened glucose-dependent by intrinsic ROS antioxidant production propertiesvia a suppressive of the two effect tetracyclines on NADPH in a synthesis self-reinforcing is strengthened manner. A by schematic intrinsic drawing antioxidant il- lustratingproperties how of theCOL two-3 and tetracyclines DOX may inlimit a self-reinforcingmicroglial inflammatory manner. responses A schematic is provided drawing inillustrating Figure 7. how COL-3 and DOX may limit microglial inflammatory responses is provided in Figure7.

FigureFigure 7. 7. SchematicSchematic drawing drawing illustrating illustrating how how the the non non-antibiotic-antibiotic tetracycline tetracycline COL COL-3-3 and and its its parent parent compoundcompound DOX DOX may may limit limit microglial microglial inflammatory inflammatory responses. responses. LPS LPS and and ααSaSa activate activate TLR4 TLR4 [33] [33] and and TLR2/P2X7TLR2/P2X7 [24,30] [24,30] signaling signaling pathways, pathways, respectively. respectively. This This results results in in the the act activationivation of of the the superoxide superoxide producingproducing enzyme enzyme NADPH NADPH oxidase. oxidase. ROS ROS produced produced through through NADPH NADPH oxidase oxidase activation activation stimulate stimulate the release of the pro-inflammatory cytokine TNF-α and enhance the uptake of glucose through a mechanism that is preventable by the NADPH oxidase inhibitor APO. The rise in microglial glucose

Cells 2021, 10, 2163 13 of 16

the release of the pro-inflammatory cytokine TNF-α and enhance the uptake of glucose through a mechanism that is preventable by the NADPH oxidase inhibitor APO. The rise in microglial glucose stimulates the synthesis of NADPH through the activation of the pentose phosphate shunt [31]. Increased levels of NADPH, the required substrate for NADPH oxidase [42,43], stimulate ROS production by this enzyme. The inhibition of glucose uptake by COL-3 and DOX leads to a reduction in glucose-derived synthesis of NADPH, which limits ROS production by NADPH oxidase. These effects may be strengthened by intrinsic antioxidant properties of COL-3 and DOX [47] in a self- reinforcing manner. Limiting glucose-dependent NADPH synthesis with the non-metabolizable analogue of glucose, 2-DG restrains microglial inflammatory reactions, indicating that the control of glucose metabolism by COL-3 and DOX is pivotal for their immunosuppressive effects. Note that DEX prevents efficiently TNF-α release but has no inhibitory effect on glucose uptake, suggesting that the glucocorticoid operates through a mechanism of action that is distinct from that of COL-3 or DOX. Inhibitory and activation signals are represented in green and red, respectively. Overall, the present data confirm that the non-antibiotic tetracycline COL-3 and its parent compound DOX can efficiently mitigate microglial inflammatory responses. The anti-inflammatory effects of COL-3 and DOX appear to primarily result from their capacity to restrain glucose-dependent NADPH synthesis and, as a consequence, ROS production via NADPH oxidase activation. Present data provide further confirmation that COL-3 and DOX may have therapeutic utility in chronic CNS pathological conditions such as PD, where long-term neuroinflammatory processes play a contributive role. COL-3 might have, however, an advantage over DOX, as it does not possess antibiotic activity.

Author Contributions: Conceptualization, B.F., N.C.F.J., E.D.B., M.d.S.P., P.P.M. and R.R.-V.; Method- ology, M.d.S.P. and P.P.M.; Investigation, M.d.S.P., N.C.F.J., N.F., P.R. and P.M.; Provision of reagents; F.G.-L. and R.C.; Writing—original draft preparation, E.D.B., F.G.-L., M.d.S.P., P.P.M., R.C. and R.R.-V.; Manuscript revision: P.P.M.; Funding acquisition, B.F., E.D.B., P.P.M. and R.R.-V. All authors have read and agreed to the published version of the manuscript. Funding: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior: #Me928/19, Investisse- ments d’avenir: ANR-10-IAIHU-06, Translational Research Infrastructure dor Biotherapies in Neuro- sciences: ANR-11-INBS-0011-NeurATRIS, Fundação de Amparo à Pesquisa do Estado de São Paulo: 2017/14207-7, France Parkinson: DOXYPARK (Grand Appel d’Offre, 2019). Institutional Review Board Statement: This work followed European Union Council Directives (2010/63/EU). The Committee on the Ethics of Animal Experiments Charles Darwin no. 5 approved experimental protocols. Informed Consent Statement: Not applicable. Data Availability Statement: Data will be available when requested. Acknowledgments: This project was carried out in the framework of a Capes-Cofecub program between the French and Brazilian research institutions [project #Me928/19]. Nilson Carlos Ferreira Junior was supported by Capes Program. This research work benefited from equipment and excellent services from the Celis and ICMQuant facilities, at ICM. In particular, we wish to acknowledge expert assistance from David Akbar for automated inverted fluorescence microscopy and Asha Baskaran and Dominique Langui for TEM studies. We gratefully acknowledge financial support from France Parkinson [DOXYPARK, Grand Appel d’Offre, 2019]. This work also benefited from program Investissements d’Avenir [ANR-10-IAIHU-06] and Translational Research Infrastructure for Biotherapies in Neurosciences [ANR-11-INBS-0011-NeurATRIS]. Conflicts of Interest: No conflict of interest declared. Cells 2021, 10, 2163 14 of 16

Abbreviations αS α-synuclein αSa αS aggregates αSm αS monomers APO Apocynin CMT-3 Chemically modified tetracycline COL-3 4-dedimethylaminosancycline or incyclinide 2-DG 2-deoxy-D-glucose DMEM Dulbecco’s modified Eagle’s medium DOX Doxycycline FCS Fetal calf serum Iba-1 ionized calcium-binding adaptor molecule-1 LPS Lipopolysaccharide PD Parkinson’s disease PEI Polyethyleneimine ROS Reactive oxygen species TLR Toll-like receptor TROL Trolox

References 1. Pioletti, M.; Schluenzen, F.; Harms, J.; Zarivach, R.; Glühmann, M.; Avila, H.; Bashan, A.; Bartels, H.; Auerbach, T.; Jacobi, C.; et al. Crystal structures of complexes of the small ribosomal subunit with tetracycline, edeine and IF3. EMBO J. 2001, 20, 1829–1839. [CrossRef][PubMed] 2. Mortison, J.D.; Schenone, M.; Myers, J.A.; Zhang, Z.; Chen, L.; Ciarlo, C.; Comer, E.; Natchiar, S.K.; Carr, S.A.; Klaholz, B.P.; et al. Tetracyclines Modify by Targeting Key Human rRNA Substructures. Cell Chem. Biol. 2018, 25, 1506–1518. [CrossRef][PubMed] 3. Sapadin, A.N.; Fleischmajer, R. Tetracyclines: Nonantibiotic properties and their clinical implications. J. Am. Acad. Dermatol. 2006, 54, 258–265. [CrossRef][PubMed] 4. Bortolanza, M.; Nascimento, G.C.; Socias, S.B.; Ploper, D.; Chehín, R.N.; Raisman-Vozari, R.; Del-Bel, E. Tetracycline repurposing in neurodegeneration: Focus on Parkinson’s disease. J. Neural. Transm. 2018, 125, 1403–1415. [CrossRef][PubMed] 5. Orsucci, D.; Calsolaro, V.; Mancuso, M.; Siciliano, G. Neuroprotective effects of tetracyclines: Molecular targets, animal models and human disease. CNS Neurol. Disord. Drug Targets 2009, 8, 222–231. [CrossRef][PubMed] 6. Sánchez, J.L.; Valentin, S.; Morales, A.; Rivera, A. Safety and efficacy of doxycycline in the treatment of . Clin. Cosmet. Investig. Dermatol. 2009, 2, 129–140. [CrossRef] 7. Zhong, W.; Chen, S.; Zhang, Q.; Xiao, T.; Qin, Y.; Gu, J.; , B.; Liu, Y.; Jing, X.; Hu, X.; et al. Doxycycline directly targets PAR1 to suppress tumor progression. Oncotarget 2017, 8, 16829–16842. [CrossRef] 8. Santa-Cecília, F.V.; Socias, B.; Ouidja, M.O.; Sepulveda-Diaz, J.E.; Acuña, L.; Silva, R.; Michel, P.P.; Del Bel, E.; Cunha, T.; Raisman-Vozari, R. Doxycycline Suppresses Microglial Activation by Inhibiting the p38 MAPK and NF-kB Signaling Pathways. Neurotox. Res. 2016, 29, 447–459. [CrossRef][PubMed] 9. Cho, Y.; Son, H.J.; Kim, E.-M.; Choi, J.H.; Kim, S.T.; Ji, I.J.; Choi, D.H.; Joh, T.H.; Kim, Y.S.; Hwang, O. Doxycycline is Neuroprotective Against Nigral Dopaminergic Degeneration by a Dual Mechanism Involving MMP-3. Neurotox. Res. 2009, 16, 361–371. [CrossRef] 10. Lazzarini, M.; Martin, S.; Mitkovski, M.; Vozari, R.R.; Stühmer, W.; Del Bel, E. Doxycycline restrains glia and confers neuroprotec- tion in a 6-OHDA Parkinson model. Glia 2013, 61, 1084–1100. [CrossRef] 11. Bortolanza, M.; Nascimento, G.C.D.; Raisman-Vozari, R.; Del-Bel, E. Doxycycline and its derivative, COL-3, decrease dyskinesia induced by l -DOPA in hemiparkinsonian rats. Br. J. Pharmacol. 2021, 178, 2595–2616. [CrossRef] 12. González-Lizárraga, F.; Ploper, D.; Ávila, C.L.; Socías, S.B.; Dos-Santos-Pereira, M.; Machín, B.; Del-Bel, E.; Michel, P.P.; Pietrasanta, L.I.; Raisman-Vozari, R.; et al. CMT-3 targets different α-synuclein aggregates mitigating their toxic and inflammo- genic effects. Sci. Rep. 2020, 10, 20258. [CrossRef] 13. González-Lizárraga, F.; Socías, S.B.; Avila, C.L.; Torres-Bugeau, C.M.; Barbosa, L.R.S.; Binolfi, A.; Sepulveda-Diaz, J.E.; Del-Bel, E.; Fernandez, C.O.; Papy-Garcia, D.; et al. Repurposing doxycycline for synucleinopathies: Remodelling of α-synuclein oligomers towards non-toxic parallel beta-sheet structured species. Sci. Rep. 2017, 7, 41755. [CrossRef] 14. Medina, L.; González-Lizárraga, F.; Dominguez-Meijide, A.; Ploper, D.; Parrales, V.; Sequeira, S.; Cima-Omori, M.-S.; Zweckstetter, M.; Del Bel, E.; Michel, P.P.; et al. Doxycycline Interferes With Tau Aggregation and Reduces Its Neuronal Toxicity. Front. Aging Neurosci. 2021, 13, 635760. [CrossRef][PubMed] 15. Golub, L.M.; Lee, H.-M.; Ryan, M.E.; Giannobile, W.V.; Payne, J.; Sorsa, T. Tetracyclines Inhibit Connective Tissue Breakdown by Multiple Non-Antimicrobial Mechanisms. Adv. Dent. Res. 1998, 12, 12–26. [CrossRef] Cells 2021, 10, 2163 15 of 16

16. Golub, L.M.; Ramamurthy, N.S.; Llavaneras, A.; Ryan, M.E.; Lee, H.M.; Liu, Y.; Bain, S.; Sorsa, T. A chemically modified nonantimicrobial tetracycline (CMT-8) inhibits gingival matrix metalloproteinases, periodontal breakdown, and extra-oral bone loss in ovariectomized rats. Ann. N. Y. Acad. Sci. 1999, 878, 290–310. [CrossRef][PubMed] 17. Syed, S.; Takimoto, C.; Hidalgo, M.; Rizzo, J.; Kuhn, J.G.; Hammond, L.A.; Schwartz, G.; Tolcher, A.; Patnaik, A.; Eckhardt, S.G.; et al. A Phase I and Pharmacokinetic Study of Col-3 (Metastat), an Oral Tetracycline Derivative with Po- tent Matrix Metalloproteinase and Antitumor Properties. Clin. Cancer Res. 2004, 10, 6512–6521. [CrossRef][PubMed] 18. Rudek, M.A.; Figg, W.D.; Dyer, V.; Dahut, W.; Turner, M.L.; Steinberg, S.M.; Liewehr, D.J.; Kohler, D.R.; Pluda, J.M.; Reed, E. Phase I of Oral COL-3, a Matrix Metalloproteinase Inhibitor, in Patients with Refractory Metastatic Cancer. J. Clin. Oncol. 2001, 19, 584–592. [CrossRef] 19. Chu, Q.S.C.; Forouzesh, B.; Syed, S.; Mita, M.; Schwartz, G.; Copper, J.; Curtright, J.; Rowinsky, E.K. A phase II and pharmacologi- cal study of the matrix metalloproteinase inhibitor (MMPI) COL-3 in patients with advanced soft tissue sarcomas. Investig. New Drugs 2007, 25, 359–367. [CrossRef] 20. Dezube, B.J.; Krown, S.E.; Lee, J.Y.; Bauer, K.S.; Aboulafia, D.M. Randomized Phase II Trial of Matrix Metalloproteinase Inhibitor COL-3 in AIDS-Related Kaposi’s Sarcoma: An AIDS Malignancy Consortium Study. J. Clin. Oncol. 2006, 24, 1389–1394. [CrossRef] 21. Zhang, H.; Chang, M.; Hansen, C.N.; Basso, D.M.; Noble-Haeusslein, L.J. Role of Matrix Metalloproteinases and Therapeutic Benefits of Their Inhibition in Spinal Cord Injury. Neurotherapeutics 2011, 8, 206–220. [CrossRef][PubMed] 22. Edan, R.A.; Luqmani, Y.A.; Masocha, W. COL-3, a Chemically Modified Tetracycline, Inhibits Lipopolysaccharide-Induced Microglia Activation and Cytokine Expression in the Brain. PLoS ONE 2013, 8, e57827. [CrossRef] 23. Zhang, W.; Wang, T.; Pei, Z.; Miller, D.S.; Wu, X.; Block, M.L.; Wilson, B.; Zhang, W.; Zhou, Y.; Hong, J.-S.; et al. Aggre- gated α-synuclein activates microglia: A process leading to disease progression in Parkinson’s disease. FASEB J. 2005, 19, 533–542. [CrossRef][PubMed] 24. Dos-Santos-Pereira, M.; Acuña, L.; Hamadat, S.; Rocca, J.; González-Lizárraga, F.; Chehin, R.; Sepulveda-Diaz, J.E.; Del-Bel, E.; Raisman-Vozari, R.; Michel, P.P. Microglial glutamate release evoked by α-synuclein aggregates is prevented by dopamine. Glia 2018, 66, 2353–2365. [CrossRef] 25. Deng, I.; Wiese, M.D.; Zhou, X.-F.; Bobrovskaya, L. The efficacy of systemic administration of lipopolysaccharide in modelling pre-motor Parkinson’s disease in C57BL/6 mice. NeuroToxicology 2021, 85, 254–264. [CrossRef] 26. Sepulveda-Diaz, J.E.; Ouidja, M.O.; Socias, S.B.; Hamadat, S.; Guerreiro, S.; Raisman-Vozari, R.; Michel, P.P. A simplified approach for efficient isolation of functional microglial cells: Application for modeling neuroinflammatory responsesin vitro. Glia 2016, 64, 1912–1924. [CrossRef] 27. Rousseau, E.; Michel, P.P.; Hirsch, E. The -Binding Protein Protects Vulnerable Dopamine Neurons from Degeneration by Preserving Mitochondrial Calcium Homeostasis. Mol. Pharmacol. 2013, 84, 888–898. [CrossRef][PubMed] 28. Pereira, M.D.S.; Abreu, G.H.D.; Rocca, J.; Hamadat, S.; Raisman-Vozari, R.; Michel, P.P.; Del Bel, E. Contributive Role of TNF-α to L-DOPA-Induced Dyskinesia in a Unilateral 6-OHDA Lesion Model of Parkinson’s Disease. Front. Pharmacol. 2021, 11, 617085. [CrossRef] 29. Hoyer, W.; Antony, T.; Cherny, D.; Heim, G.; Jovin, T.M.; Subramaniam, V. Dependence of alpha-synuclein ag-gregate morphology on solution conditions. J. Mol. Biol. 2002, 322, 383–393. [CrossRef] 30. Acuña, L.; Hamadat, S.; Corbalán, N.S.; González-Lizárraga, F.; Dos-Santos-Pereira, M.; Rocca, J.; Díaz, J.S.; Del-Bel, E.; Papy-García, D.; Chehín, R.N.; et al. and Its Derivative Rifampicin Quinone Reduce Microglial Inflammatory Responses and Neurodegeneration Induced In Vitro by α-Synuclein Fibrillary Aggregates. Cells 2019, 8, 776. [CrossRef][PubMed] 31. Dos-Santos-Pereira, M.; Guimarães, F.S.; Del-Bel, E.; Raisman-Vozari, R.; Michel, P.P. Cannabidiol prevents LPS-induced microglial inflammation by inhibiting ROS/NF-κB-dependent signaling and glucose consumption. Glia 2020, 68, 561–573. [CrossRef] 32. Döring, C.; Regen, T.; Gertig, U.; Van Rossum, D.; Winkler, A.; Saiepour, N.; Brück, W.; Hanisch, U.-K.; Janova, H. A presumed antagonistic LPS identifies distinct functional organization of TLR4 in mouse microglia. Glia 2017, 65, 1176–1185. [CrossRef] 33. Qin, L.; Li, G.; Qian, X.; Liu, Y.; Wu, X.; Liu, B.; Hong, J.-S.; Block, M.L. Interactive role of the toll-like receptor 4 and reactive oxygen species in LPS-induced microglia activation. Glia 2005, 52, 78–84. [CrossRef] 34. Ghosh, S.; Castillo, E.; Frias, E.S.; Swanson, R.A. Bioenergetic regulation of microglia. Glia 2018, 66, 1200–1212. [CrossRef] 35. Hsieh, C.-F.; Liu, C.-K.; Lee, C.-T.; Yu, L.-E.; Wang, J.-Y. Acute glucose fluctuation impacts microglial activity, leading to inflammatory activation or self-degradation. Sci. Rep. 2019, 9, 1–16. [CrossRef][PubMed] 36. Parvathy, S.S.; Masocha, W. Matrix Metalloproteinase Inhibitor COL-3 Prevents the Development of Paclitaxel-Induced Hyperal- gesia in Mice. Med. Princ. Pract. 2013, 22, 35–41. [CrossRef] 37. Meesawatsom, P.; Hathway, G.; Bennett, A.; Constantin-Teodosiu, D.; Chapman, V. Spinal neuronal excitability and neuroin- flammation in a model of chemotherapeutic neuropathic pain: Targeting the resolution pathways. J. Neuroinflamm. 2020, 17, 1–17. [CrossRef][PubMed] 38. Cazalis, J.; Tanabe, S.-I.; Gagnon, G.; Sorsa, T.; Grenier, D. Tetracyclines and Chemically Modified Tetracycline-3 (CMT-3) Modulate Cytokine Secretion by Lipopolysaccharide-Stimulated Whole Blood. Inflammation 2009, 32, 130–137. [CrossRef] 39. Fodelianaki, G.; Lansing, F.; Bhattarai, P.; Troullinaki, M.; Zeballos, M.A.; Charalampopoulos, I.; Gravanis, A.; Mirtschink, P.; Chavakis, T.; Alexaki, V.I. Nerve Growth Factor modulates LPS—induced microglial glycolysis and inflammatory responses. Exp. Cell Res. 2019, 377, 10–16. [CrossRef][PubMed] Cells 2021, 10, 2163 16 of 16

40. Gong, H.; Liu, L.; Ni, C.-X.; Zhang, Y.; Su, W.-J.; Lian, Y.-J.; Peng, W.; Zhang, J.-P.; Jiang, C.-L. Dexamethasone rapidly inhibits glucose uptake via non-genomic mechanisms in contracting myotubes. Arch. Biochem. Biophys. 2016, 603, 102–109. [CrossRef][PubMed] 41. Patil, R.H.; Kumar, M.N.; Kumar, K.K.; Nagesh, R.; Kavya, K.; Babu, R.; Ramesh, G.T.; Sharma, S.C. Dexamethasone in- hibits inflammatory response via down regulation of AP-1 transcription factor in human lung epithelial cells. 2018, 645, 85–94. [CrossRef] 42. Vander Heiden, M.G.; Cantley, L.C.; Thompson, C.B. Understanding the Warburg effect: The metabolic requirements of cell proliferation. Science 2009, 324, 1029–1033. [CrossRef] 43. Bedard, K.; Krause, K.H. The NOX Family of ROS-Generating NADPH : Physiology and Pathophysiology. Physiol. Rev. 2007, 87, 245–313. [CrossRef][PubMed] 44. Taetzsch, T.; Levesque, S.; McGraw, C.; Brookins, S.; Luqa, R.; Bonini, M.G.; Mason, R.P.; Oh, U.; Block, M.L. Redox regulation of NF-κB p50 and M1 polarization in microglia. Glia 2015, 63, 423–440. [CrossRef] 45. Tang, X.; Wang, X.; Zhao, Y.Y.; Curtis, J.M.; Brindley, D.N. Doxycycline attenuates breast cancer related inflammation by decreasing plasma lysophosphatidate concentrations and inhibiting NF-κB activation. Mol. Cancer 2017, 16, 1–13. [CrossRef][PubMed] 46. Henríquez-Olguin, C.; Knudsen, J.R.; Raun, S.H.; Li, Z.; Dalbram, E.; Treebak, J.T.; Sylow, L.; Holmdahl, R.; Richter, E.A.; Jaimovich, E.; et al. Cytosolic ROS production by NADPH oxidase 2 regulates muscle glucose uptake during exercise. Nat. Commun. 2019, 10, 1–11. [CrossRef][PubMed] 47. Kładna, A.; Michalska, T.; Berczy´nski,P.; Kruk, I.; Aboul-Enein, H.Y. Evaluation of the antioxidant activity of in vitro. Luminescence 2011, 27, 249–255. [CrossRef][PubMed] 48. Clemens, D.L.; Duryee, M.J.; Sarmiento, C.; Chiou, A.; McGowan, J.D.; Hunter, C.D.; Schlichte, S.L.; Tian, J.; Klassen, L.W.; O’Dell, J.R.; et al. Novel Antioxidant Properties of Doxycycline. Int. J. Mol. Sci. 2018, 19, 4078. [CrossRef][PubMed]