WO2020094767A1 - Use of nrf2 activators for the treatment of staphylococcus aureus infections - Google Patents

Abstract

Staphylococcus aureus infection affects immune and inflammatory host responses causing severe bacterial infections and complicated antibiotic therapy. As important actors of the innate immune defenses, macrophages are actively involved in the microbial elimination and their active states are sensitive to the microenvironment. In this study, the inventors investigate the impact of Nuclear factor erythroid 2-related factor 2 (Nrf2) activation on the intracellular bacterial load in macrophages and the underlying molecular mechanisms involved in this process. THP1-derived macrophages pretreated with Nrf2 activator sulforaphane (SFN) significantly reduce bacterial internalization and intracellular bacterial survival when challenged with S. aureus. Accordingly, the present invention relates to a method of treating a Staphylococcus aureus infection in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a Nrf2 activator.

Description

USE OF NRF2 ACTIVATORS FOR THE TREATMENT OF STAPHYLOCOCCUS

AUREUS INFECTIONS

FIELD OF THE INVENTION:

The present invention relates to use of Nrf2 activators for the treatment of Staphylococcus aureus infections.

BACKGROUND OF THE INVENTION:

Staphylococcus aureus , a gram-positive human bacterium and facultative intracellular pathogen, is connected with skin and soft tissue infections, osteomyelitis, septic arthritis, pneumonia, and endovascular infections [1] Treatments are increasingly complicated by antibiotic and drug resistance strains of S. aureus , thus resulting in high mortality and morbidity. One pathogenic characteristic of S. aureus is its ability to generate intense local and systemic inflammatory responses. When infection occurs, the innate immune system triggers a rapid and non-specific inflammatory response mechanism to prevent bacteria spreading.

As the first line of defense, macrophages are phagocytic experts and play an important role in clearing foreign particles, including bacteria and cell debris, by phagocytosis, mediating the inflammatory response and restoring cell homeostasis to prevent tissue damage [2, 3]. Following microbial or cell debris recognition, acute inflammatory response is initiated and emphasized by the rapid release of proinflammatory mediators such as IL- 1 b, which will subsequently induce the adaptive immune response [4, 5]. MAPK signaling pathway, which includes the most thoroughly studied members ERK, p38 and JNK, is activated and participates in the expression of inflammatory cytokines and chemokines [6, 7]

Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, is a potent activator of the transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2), a key regulator of the antioxidant and anti-inflammatory responses [8, 9]. Under normal physiological conditions, Nrf2 is sequestered in the cytoplasm by Keap-l and led to ubiquitin-dependent degradation [10]. Treatment with SFN modifies Keapl, allowing Nrf2 release and translocation into the nucleus, where Nrf2 heterodimerizes with one of the small Maf proteins, and binds to the regulatory sequences known as antioxidant response elements (AREs) to initiate transcription of genes involved in the antioxidant and cytoprotective responses, including the phase II detoxifying enzymes NAD(P)H quinone dehydrogenase 1 (NQOl) and heme oxygenase- 1 (HO-l). Nrf2 is an essential factor in the attenuation of the inflammatory response since Nrf2-deficient mice exhibits increased inflammation [11]. In addition, Nrf2 has been shown to inhibit the transcription of the proinflammatory cytokines IL- 1 b and IL-6 in mouse bone marrow derived macrophages [12]. Alveolar macrophages from patients with chronic obstructive pulmonary disease and wild-type mice exposed to cigarette smoke for 6 months were treated with SFN. SFN-mediated increase of Nrf2 up-regulated the transcriptional gene expression coding of the scavenger receptor MARCO, which led to an increase in bacterial clearance and a decrease in inflammation, as opposed to cells treated with vehicle and Nrf2- deficient mice [13]. Furthermore, treatment of nasal epithelial cells with SFN or epigallocatechin gallate significantly decreases influenza virus entry and replication in an Nrf2- dependent manner [14]. Similarly, we have previously demonstrated that SFN ameliorated Mycobacterium abscessus clearance in macrophages through modulation of p38 MAPK signaling pathway and increased cell apoptosis [15] . However, the underlying mechanisms activated by SFN in macrophages infected with S. aureus still remain elusive.

SUMMARY OF THE INVENTION:

The present invention relates to use of Nrf2 activators for the treatment of Staphylococcus aureus infections. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Staphylococcus aureus infection affects immune and inflammatory host responses causing severe bacterial infections and complicated antibiotic therapy. As important actors of the innate immune defenses, macrophages are actively involved in the microbial elimination and their active states are sensitive to the microenvironment. In this study, the inventors investigate the impact of Nuclear factor erythroid 2-related factor 2 (Nrf2) activation on the intracellular bacterial load in macrophages and the underlying molecular mechanisms involved in this process. THP1 -derived macrophages pretreated with Nrf2 activator sulforaphane (SFN) significantly reduce bacterial internalization and intracellular bacterial survival when challenged with S. aureus. Transfecting siRNA targeting Nrf2 in SFN treated macrophages abolishes bactericidal activity demonstrating an essential contribution of the Nrf2 signaling pathway. S. aureus induces inflammation by activation of the MAPK signaling pathway, leading to increased mRNA expression levels of inflammatory markers IL- 1 b, IL-6 and TNFa. In SFN pretreated macrophages, SFN inactivates p38 and JNK, significantly preventing S. aureus induced inflammation, and activates ERK signaling and Nrf2 signaling pathways. Further analyses of cellular signaling mechanisms using specific MAPK inhibitors, PD98059, SB203580, or SP600125, demonstrate that SFN inhibits inflammatory markers through MAPK- dependent and MAPK-independent pathways, triggering a caspase 3-dependent apoptosis. Altogether, these findings provide new molecular insights into the twofold down-regulatory pathways controlled by SFN to inhibit S. aureus- induced inflammation. Modulation of the Nrf2 signaling pathway may be useful to improve bacterial clearance and regulate inflammation.

Accordingly, the first object of the present invention relates to a method of treating a Staphylococcus aureus infection in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a Nrf2 activator.

As used herein, the term“ Staphylococcus aureus” or“5. aureus” has its general meaning in the art and refers to any strain, genotype or isolate of S. aureus , a Gram-positive bacteria. The bacteria are generally harmless, unless they enter the body through a cut or other wound. Typically, infections are minor skin problems in healthy people. Historically, infections were treated by broad-spectrum antibiotics, such as methicillin. Now, though, certain strains have emerged that are resistant to methicillin and other beta-lactam antibiotics, such as penicillin and cephalosporins. They are referred to as methicillin-resistant Staphylococcus aureus (also known as multi-drug resistant Staphylococcus aureus , or“MRSA”). Accordingly, in some embodiments, the method of the present invention is particularly suitable for the treatment of infections by multi-drug resistant Staphylococcus aureus.

In some embodiments, the subject is a human being. The term“subject” does not denote a particular age, and thus encompass adults, children and newborns.

In some embodiments, the subject is immunocompromised. An immunocompromised subject is a subject who is incapable of developing or unlikely to develop a robust immune response, usually as a result of disease, malnutrition, or immunosuppressive therapy. An immunocompromised immune system is an immune system that is functioning below normal. Immunocompromised subjects are more susceptible to mycobacterial infections. Those who can be considered to be immunocompromised include, but are not limited to, subjects with AIDS (or HIV positive), subjects with severe combined immune deficiency (SCID), diabetics, subjects who have had transplants and who are taking immunosuppressives, and those who are receiving chemotherapy for cancer. Immunocompromised individuals also includes subjects with most forms of cancer (other than skin cancer), sickle cell anemia, cystic fibrosis, those who do not have a spleen, subjects with end stage kidney disease (dialysis), and those who have been taking corticosteroids on a frequent basis by pill or injection within the last year. Subjects with severe liver, lung, heart disease, or neurological and muscular disabilities also may be immunocompromised.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

The terms“Nrf2 activator” and“Nuclear factor (erythroid-derived 2)-like 2 activator” as used herein relate to chemical compounds or elements that increase the activity of Nrf2. The term“activity of Nrf2” as used herein relates to the activity of Nrf2 and in particular activation in cell apoptosis. Nrf2 activators are typically classified based on their chemical structures: Diphenols, Michael reaction acceptors, isothiocyanates, thiocarbamates, trivalent arsenicals, l,2-dithiole-3-thiones, hydroperoxides, vicinal dimercaptans, heavy metals, and polyenes.

In some embodiments, the Nrf2 activator of the present invention is selected from the group consisting of wogonin (WG), of Chalcone derivatives as disclosed in J. Med. Chem., 2011, 54 (12), pp 4147-4159, such as 2-trifluoromethyl-2^/ -methoxychalcone, auranofin, ebselen, 1, 2-naphthoquinone, cynnamic aldehyde, caffeic acid and its esters, curcumin, reservatrol, artesunate, tert-butylhydroquinone, and -quinone, (tBHQ, tBQ), vitamins Kl, K2 and K3, preferably menadione, fumaric acid esters, i.e. fumaric acid mono- and/or diester which is preferably selected from the group of monoalkyl hydrogen fumarate and dialkyl fumarate, such as monomethyl hydrogen fumarate, dimethyl fumarate (DMF), monoethyl hydrogen fumarate, and diethyl fumarate, 2-cyclopentenones, ethacrynic acid and its alkyl esters, bardoxolone methyl (methyl 2-cyano-3,l2-dioxooleana-l,9(l l)dien-28-oate) (CDDO-Me, RTA 402), ethyl 2-cyano-3,l2-dioxooleana-l,9(l l)dien-28-oate, 2-cyano-3,l2-dioxooleana- 1,9(1 l)dien-28-oic acid (CDDO), 1 [2-Cyano-3,l2-dioxooleana-l,9(l l)-dien-28- oyl]imidazole (CDDO-Im), (2-cyano-N-methyl-3,l2-dioxooleana-l,9(l l)-dien-28 amide (CDDO-methyl amide, CDDO-MA), l,2-dithiole-3-thione such as oltipraz (OTZ), 3,5-di-tert- butyl-4-hydroxytoluene, 3-hydroxycoumarin, 4-hydroxynonenal, 4-oxononenal, malondialdehyde, (E)-2-hexenal, capsaicin, allicin, allylisothiocyanate, 6-methylthiohexyl isothiocyanate, 7-methylthioheptyl isothiocyanate, sulforaphane (SFN), 8-methylthiooctyl isothiocyanate, corticosteroids, such as dexamethasone, 8-iso prostaglandin A2, alkyl pyruvate, such as methyl and ethyl pyruvate, diethyl or dimethyl oxaloproprionate, 2-acetamidoacrylate, methyl or ethyl-2-acetamidoacrylate, hypoestoxide, parthenolide, eriodictyol, 4-Hydroxy-2- nonenal, 4-oxo-2nonenal, geranial, zerumbone, aurone, isoliquiritigenin, xanthohumol, [10]- Shogaol, eugenol, l' -acetoxychavicol acetate, allyl isothiocyanate, benzyl isothiocyanate, phenethyl isothiocyanate, 4-(Methylthio)-3-butenyl isothiocyanate and 6-Methylsulfinylhexyl isothiocyanate, ferulic acid and its esters, such as ferulic acid ethyl ester, and ferulic acid methyl ester, sofalcone, 4-methyl daphnetin, imperatorin, auraptene, poncimarin, bis [2- hydroxybenzylidene] acetones, alicylcurcuminoid, 4-bromo flavone, b-naphthoflavone, sappanone A, aurones and its corresponding indole derivatives such as benzylidene-indolin-2- ones, perillaldehyde, quercetin, fisetin, koparin, genistein, tanshinone HA, BHA, BHT, PMX- 290, AL-l, avicin D, gedunin, fisetin, andrographolide, tricyclic bis(cyano enone) TBE-31 [(±)- (4bS,8aR,l0aS)-l0a-ethynyl-4-b,8,8-trimethyl-3,7-dioxo-3,4-b,7,8,8a,9,l0,l0a- octahydrophenanthrene-2,6-dicarbonitrile], MCE-l, MCE5, TP-225, ADT as referred to in in Medicinal Research Reviews, 32, No. 4, 687-726, 2012, and the respective quinone or hydroquinone forms of the aforementioned quinone and hydroquinone derivatives and stereoisomers, tautomers or pharmacologically active derivatives of the aforementioned agents.

In some embodiments, the Nrf2 activator of the present invention is selected from the group consisting of fumaric acid derivatives (Joshi and Strebel, WO 2002/055063, US 2006/0205659, and U.S. Pat. No. 7,157,423 (amide compounds and protein-fumarate conjugates); Joshi et a , WO 2002/055066 and Joshi and Strebel, U.S. Pat. No. 6,355,676 (mono and dialkyl esters); Joshi and Strebel, WO 2003/087174 (carbocyclic and oxacarbocylic compounds); Joshi et a , WO 2006/122652 (thiosuccinates); Joshi et a , US 2008/0233185 (dialkyl and diaryl esters) and salts (Nilsson et al., US 2008/0004344) Controlled release pharmaceutical compositions comprising fumaric acid esters are also disclosed by Nilsson and Willer, WO 2007/042034. Prodrugs are described by Nielsen and Bundgaard, J Pharm Sci 1988, 77(4), 285-298 and in WO2010/022177.

Additional examples of Nrf2 activators can be found in US2011/0250300, US 2004/0002463, US 20130172391, US20140275205, W02014100728 the disclosures of each of which are hereby incorporated by reference herein.

By a "therapeutically effective amount" is meant a sufficient amount of the Nrf2 activator of the present invention for treating or reducing the symptoms at reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with the Nrf2 activator of the present inventions; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the Nrf2 activator of the present invention for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the Nrf2 activator of the present invention, typically from 1 mg to about 100 mg of the Nrf2 activator of the present invention. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

Typically the Nrf2 activator of the present invention of the present invention is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. The term "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. In the pharmaceutical compositions of the present invention, the Nrf2 activator of the present inventions of the invention can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: SFN decreases S. aureus internalization and survival in THPl-derived macrophages. (A) THPl-derived macrophages were infected with SYT09 labeled S. aureus for 1 h at 37°C. After washing and PFA fixation, cells were labeled for CD11B (Rhodamine RedX) and bacteria internalization was analyzed by flow cytometry (100 000 events analyzed). Data are presented as mean ± SEM from 4 independent experiments. RFI: relative fluorescence intensity. (B) Intracellular survival of S. aureus in THP-l derived macrophages 24 h after infection. Data are mean ± SEM from 6 independent experiments. * p<0.05, * * p<0.0l.

Figure 2: Involvement of Nrf2 in S. aureus clearance. (A) Twenty-four hours after PM A differentiation, THPl-derived macrophages were transfected with non-targeting and Nrf2 specific siRNA. Forty-eight hours after transfection, cells were pretreated for 3 h with SFN 10 mM prior to infection with S. aureus at a MOI of 10. Densitometric analysis was performed on the Nrf2 bands and normalized to the corresponding GAPDH bands. (B) Intracellular survival of S. aureus in scramble or siNrf2 transfected THP-l derived macrophages treated with SFN and infected with S. aureus for 24 h. Data are mean ± SEM from 3 independent experiments. * p<0.05, * * p<0.0l.

Figure 3: Effects of SFN and S. aureus on Nrf2 signaling pathway. (A) Relative mRNA expression levels of Nrf2 target genes HO- 1 and NQOl were determined by RT-qPCR. Data are mean ± SEM from 7 independent experiments done in triplicate. (B) Densitometry of HO-l and NQOl after 6 h (n=4) and 24 h (n=3) SFN stimulation and S. aureus infection. * p< 0.05, ** p< 0.01.

Figure 4: Effect of SFN and S. aureus on proinflammatory markers (IL-Ib, IL-6 and TNFa). (A-C) THPl-derived macrophages were pretreated with SFN 10 mM for 3 h prior to S. aureus infection. Cells were lyzed and total RNA extracted 3 h post infection. Data are mean ± SEM of 7 independent experiments. * p< 0.05, ** p< 0.01.

Figure 5: SFN promotes caspases 3/7-dependent cell apotosis. THPl-derived macrophages were pretreated with DMSO or SFN 10 mM for 3 h followed by S. aureus infection. After 1 h infection, gentamicin was added to the medium and cells were incubated for an additional 24 h. (A) Cell apoptosis was evaluated using Annexin V-FITC staining (B) Caspases 3/7 assay was assessed using FAM-FLICA caspase assays. (C) Cell necrosis was evaluated by PI staining. PFA fixed cells were analyzed by flow cytometer (h=100 000 events). * p<0.05, * * p O.Ol.

Figure 6: SFN activates ERK signaling pathway but inhibits S. aureus activated JNK and p38 signaling pathways. THPl-derived macrophages were pretreated with SFN 10 mM 3 h prior to S. aureus infection. Total protein lysates were collected after 6 h infection and phosphorylation of ERK, JNK, and p38 were evaluated. Data are presented as mean ± SEM of 3 independent experiments. * p<0.05, * * p<0.0l.

Figure 7: SFN inhibits S. aureus induced transcriptional expressions of proinflammatory genes. THPl-derived macrophages were pretreated with ERK inhibitor PD98059, p38 inhibitor SB203580, or JNK inhibitor SP600125 for 1 h prior to SFN or DMSO treatment and S. aureus infection. After 3 h infection, total RNAs were extracted with Trizol and analysis of IE-1b (A), IF-6 (B) and TNFa (C) mRNA expression levels were determined by RT-qPCR. Data are presented as mean ± SEM from 3 independent experiments. Figure 8: Intracellular survival of S. aureus requires ERK signaling pathway. (A)

Internalization of S. aureus in THP1 derived macrophages pretreated with MAPK inhibitors (ERK inhibitor PD98059, JNK inhibitor SP600125, p38 inhibitor SB203580) and DMSO (white) or SFN (black). (n=l). (B) CFU of S. aureus infection of THP1 pretreated with MAPK inhibitors (ERK inhibitor PD98059, JNK inhibitor SP600125, p38 inhibitor SB203580) and DMSO or SFN. (n=3). (C-D) Cell apoptosis was evaluated by Annexin V-FITC staining and cell necrosis by PI staining. THP1 derived macrophages were pretreated with MAPK inhibitors (PD98059, SB203580, SP600125) for 1 h prior to treatment with DMSO or SFN followed by S. aureus infection (DMSO (white), SFN (black), DMSO+S. aureus (black line), SFN+S. aureus (black dot)). After 1 h infection, gentamicin was added to the medium and cells were incubated for an additional 24 h. Cell apoptosis was evaluated using Annexin V-FITC staining and flow cytometer analysis (h=100 000 events). * p<0.05, * * p<0.0l.

Figure 9: sulforaphane (SFN), wogonin (WG), oltipraz (OTZ), dimethyl fumarate (DMF), bardoxolone-methyl (CDDO-Me) affect differently the bactericidal activity of macrophages. Bacterial viability was assessed using the BacFight bacterial viability kit. Bacteria were stained with SYT09 and dead bacteria with propidium iodide (PI). (A) S. aureus were incubated for 90 minutes with DMSO or each of the selected compound and fluorescent signals were acquired at 45, 60, and 90 minutes with a spectrophotometer (n=3 independent experiments done in triplicate). (B, C) THP-l-derived macrophages (B) and human PBMC- derived macrophages (C) were pretreated with DMSO, SFN, WG, OTZ, DMF, or CDDO-Me for 24 h, infected for 1 h with S. aureus at MOI 10, then gentamycin was added for the remaining incubation time. CFU counts of S. aureus was determined 24 h after infection (n=3 independent experiments). *p< 0.05 **p< 0.01, ***p< 0.001.

EXAMPLE:

Material & Methods

Antibodies and reagents

Sulforaphane (SFN), phorbol l2-myristate l3-acetate (PMA), phalloidin-ATTO 594 were purchased from Sigma- Aldrich. RPMI 1640 medium, fetal bovine serum, FAM-FFICA caspases 3/7 assay kit (ImmunoChemistry Technologies) were obtained from Eurobio-Ingen. Tryptic soy broth and Tryptic soy agar (Conda) were obtained from Dutscher. Rabbit anti- NQOl, Annexin V-FITC apoptosis reagent, PD98059, SB203580, and SP600125 were obtained from Abeam. Rabbit anti-Nrf2 antibody was purchased from Proteintech. Mouse anti- GAPDH, rabbit anti-phospho ERK, rabbit anti-ERK, mouse anti-phospho p38 were obtained from Merck-Millipore. Mouse anti-phospho JNK, mouse anti-JNK, mouse anti-p38 were purchased from BD Biosciences. DC protein assay kit and iTaq SYBRgreen supermix was purchased from BioRad. Goat anti-rabbit IRDye 680RD and goat anti-mouse IRDye 800CW were purchased from LI-COR Biosciences. Maxima First Strand cDNA synthesis kit, Live/dead BacLight bacterial viability kit, CellRox Green flow cytometry assay kit, Oligofectamine transfection reagent were obtained from ThermoFisher Scientific.

Cell culture, cell differentiation and treatments

The human THP-l monocytic cell line (ATCC® TIB-202™) was maintained in RPMI 1640 (Roswell Park Memorial Institute 1640) Glutamax medium supplemented with 10 % heat inactivated fetal bovine serum, 1 mM sodium pyruvate, 10 mM HEPES, and 0.05 mM b- mercaptoethanol in a humidified atmosphere at 37 °C and 5 % C02. Terminal differentiation of THP-l to macrophages was obtained by rinsing the cells twice with sterile phosphate- buffered saline (PBS) prior to treatment with 10 mM PMA for 24 or 48 hours. Depending on the indicated conditions, THP-l -derived macrophages were pretreated with 10 pM SFN or the vehicle DMSO for 3 hours before bacterial infection. When indicated, cells were pretreated with MAPKinhibitors (50 pM PD98059, 25 pM SB203580, or 25 pM SP600125) 1 hour prior to SFN pretreatment and/or S. aureus infection.

RNA interference and transfection

Non-targeting short interfering RNA was purchased from Qiagen and Silencer® Select predesigned siRNA targeting Nrf2 were purchased from Ambion (ThermoFisher Scientific). Twenty-four hours after PMA differentiation, THP-l -derived macrophages were transfected with either 200 nM non-targeting siRNA or 200 nM siRNA targeting Nrf2 using Oligofectamine according to the manufacturer’s instructions. After an additional 24 h incubation, cells were pretreated for 3 h with SFN 10 pM prior to infection with S. aureus at a MOI of 10. Knockdown efficiency was determined by Western blot analysis on cells transfected for 24 h.

Total RNA isolation and RT-qPCR

Total RNA was isolated from treated cells with Trizol and chloroform extraction technique. RNA concentration and purity were determined using the GE NanoVue spectrophotometer (GE Healthcare). One pg of total RNA was reverse transcribed to cDNA using Maxima First strand cDNA synthesis kit (ThermFisher) following the manufacturer’s instructions. cDNA was analyzed using real time quantitative PCR (RT-qPCR), with each sample done in triplicate. RTqPCR was performed using the CFX96 thermocycler (BioRad) and iTaq SYBRgreen qPCR mix. The specific oligonucleotides for HO-l, NQOl, IE-1b, IF-6, TNFa, 18S ribosomal RNA are listed in, and synthetized by Eurogentec. Gene expression levels were normalized to that of the reference gene 18S ribosomal RNA. Data were analyzed on BioRad CFX manager 3.1 using the AACt method.

Bacterial strain, growth culture, and fluorescence labeling

Staphylococcus aureus ATCC 25923™ strain was grown aerobically in Tryptic soy broth to an optical density of 1 at 37°C under agitation. When required, bacteria were seeded on Trypticase soy broth solidified with 1.5 % agar. When required, fluorescent S. aureus were generated, prior to use, by incubating in the dark S. aureus with SYT09 for 15 min (ThermoFisher) with gentle shaking. Fabeled bacteria were washed 3 times in sterile PBS before cell infection.

Bacteria viability

Briefly, in 96-well plates Bacteria viability was determined using the Five/dead Baclight bacterial viability kit, according to the manufacturer’s instructions. Briefly, SYT09 was used to stain all bacteria and propidium iodide to stain membrane damaged bacteria, and stained bacteria were incubated with SFN, DMSO or MAPK inhibitors for up to 90 min. Fluorescent signals were monitored at 60, 75, and 90 min by exciting at 488 nm and measuring the emission signals at 530 nm and 630 nm by spectrophotometer.

Bacteria internalization assay and intracellular survival assay

For phagocytosis assay, 1 x 10⁶ THP1 cells were seeded in 6-well plates and differentiated with PMA for 24 h. After treatment, cells were infected with SYT09 labeled S. aureus at a multiplicity of infection (MOI) of 10. After 1 h infection, cells were washed twice with cold PBS to prevent additional bacteria internalization and to remove extracellular bacteria. Cells were then trypsinized, fixed in paraformaldehyde (PFA) 4% for 15 min, then suspended in 500 mΐ 0.02 % EDTA and quantification of cells infected with SYT09 labeled S. aureus were performed using an FSRFortessa flow cytometer. A forward and side scatter gate was set to exclude dead and aggregated cells. A total of 100 000 events were collected.

For intracellular growth assay, THP1 -derived macrophages seeded in 24- well plates at 2.5 x 10⁵ cells/well were infected with S. aureus at a MOI of 10. After 1 h infection, cells were washed with PBS and extracellular bacteria were eliminated by addition of 20 pg/ml gentamicin in fresh cell culture medium. After 24 h incubation, cells were rinsed with PBS and lyzed in ice-cold sterile water for 20 min at 4°C. Intracellular bacteria were then plated in 5-fold serial dilutions on Trypticase soy agar plates. Colony forming units (CFU) counts were determined 24 h after incubation at 37°C.

Quantification of ROS production CellRox green reagent assay kit (Fisher Scientific) was used to determine ROS levels. Briefly, TF1P-1 -derived macrophages were seeded at 1x105 cells on coverslips in 24-well plates. After treatment, macrophages were stained with 5 mM CellRox green reagent for 30 min at 37°C. Cells were then washed with PBS, fixed in PFA 4% for 15 min and nuclei were stained with DAPI. Analysis of images taken with Leica SP8 confocal microscope (Leica Microsystems) were done using Image J (National Institutes of Health).

Apoptosis and necrosis assays

THP-l -derived macrophages cultured in 6- well plates at 1 x 10⁶ cells/well were trypsinized and stained with Annexin-V FITC apoptosis reagent (Thermofisher) for 10 min in Annexin binding buffer following the manufacturer’s instructions. Cell analysis was performed by flow cytometry, recording 100 000 events for each sample. Staurosporine was used as apoptosis positive control and EtOH 30 % (v/v) was used as necrosis positive control. Caspases 3/7 activities were determined using the Green FAM-FLICA Caspases 3/7 assay kit according to the manufacturer’s instructions. Briefly, the FLICA probe, made of the irreversible caspase inhibitor DEVD-fluoromethyl ketone fused to a carboxyfluorescein, binds specifically and covalently to activated Caspase 3/7 enzymes. THP-l-derived macrophages were seeded at 1 x 10⁶ per well in 6- well plates, treated and infected according to the described conditions. Cells were then incubated at 37°C in the dark for 1 h with the FLICA probe and propidium iodide. After two washes, cells were resuspended in 0.02% EDTA and the enumeration of apoptotic cells was obtained by flow cytometry, recording 100 000 events for each sample.

Western blot and immunofluorescence labeling

For Western blot analysis, THP-l-derived macrophages were rinsed with cold PBS then lyzed with cold RIPA buffer (150 mM NaCl, 1 % Triton X-100, 0.5 % sodium deoxycholate, 0.1 % SDS, 50 mM Tris-HCl, pH 7.5, supplemented with Complete protease inhibitor cocktail mixture). Protein concentrations were determined using DC protein assay kit. Twenty-pg of total proteins were resolved by SDS-PAGE (4-20 % gradient gels) and transferred to polyvinylidene difluoride membrane. Western blot was performed using the IBind Flex Western system (Invitrogen) following the manufacturer’s instructions. Briefly primary antibodies against Nrf2, HO-l, NQOl, GAPDH, phosphorylated and total ERK, phosphorylated and total p38, phosphorylated and total JNK, and IRDye680RD or IRDye800RD conjugated secondary antibodies were diluted in iBind Flex FD Solution. Fluorescent blot imaging was performed using Odyssey CLx Imaging system (LI-COR). Densitometric analysis was done using Image Studio Lite software. Shown Western blots are representative of at least 3 independent experiments. For immunofluorescence labeling, THP1 derived macrophages seeded on coverslips were infected with SYT09-stained S. aureus for 1 h then cells were rinsed with PBS and fixed 10 min in PFA 4%. Images were acquired with the laser scanning confocal fluorescence microscope Leica SP8 (Leica Microsystems) and analyzed using Image J.

Statistical analysis

Results are presented as mean values ± standard errors of the mean (SEM) from at least 3 independent experiments. Imaging flow cytometry results presented are means ± SEM of at least 3 independent experiments of 100 000 events. All statistical comparisons were performed using two-tailed Student’ s t-test and differences were considered significant at a value of p < 0.05.

Example 1:

Results

SFN decreases S. aureus intake and improves bacterial clearance in macrophages

In order to gain a better understanding of the impact of SFN on the intracellular fate of S. aureus , on both bacterial internalization and bactericidal activities in infected macrophages, human THP-l monocytes were differentiated into macrophages using phorbol l2-myristate 13- acetate (PMA) and infected with Staphylococcus aureus. Bacteria internalization of S. aureus by macrophages was first assessed by infecting cells with S. aureus stained with SYT09. Confocal microscopic images were taken of 1 h infected macrophages, and showed internalization of SYT09 labeled S. aureus in macrophages, including labeled S. aureus found enclosed in actin dependent structures reminding of early phagocytosis (data not shown)

[16,17] THP1 -derived macrophages were next pretreated for 3 h with SFN or its vehicle DMSO prior to S. aureus infection to evaluate bacteria intake and bacteria survival in macrophages exposed to SFN treatment. Bacterial invasion assay (1 h incubation post-infection) and survival assay (24 h incubation post- infection) were performed. THPl-derived macrophages challenged with SYT09 labeled S. aureus for 1 h were thoroughly washed, PFA-fixed, and analyzed by flow cytometer. S. aureus internalization decreased 36 % in SFN pretreated THPl-derived macrophages compared to vehicle pretreated macrophages (Figure 1A). In addition, a 50 % decrease in bacteria counts was observed in SFN pretreated macrophages compared to DMSO pretreated macrophages (Figure IB), thus suggesting that SFN contributes to S. aureus clearance in THPl-derived macrophages.

Nr(2 participates in intracellular S. aureus clearance

Since SFN is a well-established activator of the transcription factor Nrf2, we sought to determine whether SFN activated Nrf2 in the process of S. aureus clearance by transfecting THP1 -derived macrophages with non-targeting and Nrf2 specific siRNA. Knockdown efficiency was determined by Western blot analysis of protein lysates extracted from THP1- derived macrophages transfected with non-targeting or Nrf2 targeting siRNA for 24 h then stimulated additionally for 24 h with SFN, since Nrf2 protein level was barely detectable under quiescent conditions. Nrf2 protein level was 33 % down in siNrf2 transfected macrophages compared to non-targeting siRNA transfected cells (Figure 2A). Furthermore, we assessed the impact of Nrf2 depletion on intracellular S. aureus survival 24 h after infection. While THP1- derived macrophages transfected with non-targeting siRNA and pretreated with SFN sustained bactericidal killing properties compared to DMSO pretreated macrophages, THP1 -derived macrophages transfected with Nrf2 targeting siRNA showed comparable levels of bacteria killing in macrophages pretreated with SFN or DMSO (Figure 2B). Thus, these results suggest that Nrf2 is required for SFN to promote S. aureus clearance in macrophages.

S. aureus does not modulate Nrf2 signaling pathway

Since Nrf2 is a major regulator of inducible intracellular defenses in the innate immune system, we first assessed whether S. aureus modulated Nrf2 activity and oxidative stress in infected macrophages. THP1 -derived macrophages were pretreated with DMSO or SFN 3 h prior to infection with S. aureus. One hour after infection, gentamicin was added to the medium and infected macrophages were incubated for an additional 24 h. Western blots showed a SFN dependent increase in Nrf2 protein levels in both non-infected and infected macrophages pretreated with SFN, whereas protein levels in macrophages infected with S. aureus and treated with DMSO were comparable to control macrophages (Data not shown). Heme oxygenase- 1 (HO-l) and NAD(P)H quinone oxidoreductase 1 (NQOl), two target genes of Nrf2, were transcriptionally induced 3.5-fold and 2-fold respectively by SFN in macrophages (Figure 3A). At the protein level, HO-l expression was increased 2-fold by SFN as early as 6 h and was maintained 24 h after SFN stimulation (Figure 3B). NQOl protein levels were not modified 6 h after SFN treatment or S. aureus infection but were increased 2-fold over control after 24 h treatment with SFN. Conversely, S. aureus infection alone of macrophages showed no effect on Nrf2 protein level and Nrf2 activation since expressions of HO-l and NQOl were not increased at the transcription or protein levels (Figure 3A, 3B). CellROX fluorescent probe was used to show that no significant modulation of oxidative stress was detected in THP1- derived macrophages 24 h after pretreatment with SFN and/or infection with S. aureus (data not shown), thus suggesting that S. aureus infection of macrophages did not induce oxidative stress and nor did it activate Nrf2 signaling pathway.

SFN suppresses inflammatory response triggered by S. aureus Since Nrf2 plays an important role in the anti-inflammatory response triggered by infection, we sought to determine the inflammatory response in THP1 -derived macrophages pretreated with SFN or DMSO and infected by S. aureus. Proinflammatory markers IL- 1 b, IL- 6 and TNFa mRNA expression levels were increased 1.77-fold, 22-fold and, 9.9-fold respectively, in macrophages 6 h after S. aureus infection compared to control DMSO treated macrophages (Figures 4A, 4B and 4C). Interestingly, macrophages treated with SFN showed a significant inhibition of IIMb, IL-6 and TNFa mRNA levels compared to DMSO treated macrophages. In SFN-treated macrophages challenged with S. aureus , SFN inhibited mRNA expression levels of IL-6 (5.6-fold) and TNFa (3.9-fold) compared to that seen in S. aureus challenged macrophages. These results suggest SFN strongly inhibits proinflammatory cytokines in quiescent or S. aureus infected macrophages.

SFN induces caspases-3 and -7 dependent apoptosis

Programmed cell death is part of the innate immune response against bacterial infection. We have shown previously that THP1 -derived macrophages pretreated with SFN and challenged with M. abscessus that p38 induced a caspase-dependent cell death [15]. To assess whether cell apoptosis contributed to bacterial clearance in the event of S. aureus infection, THP1 -derived macrophages were pretreated with SFN for 3 h prior to infection with S. aureus. After 1 h incubation, gentamicin was added to the medium. Twenty-four hours after infection, apoptotic and necrotic cells were stained with Annexin V-FITC and propidium iodide respectively and analyzed by flow cytometry. THP1 -derived macrophages, infected or not with S. aureus , presented an approximate 1.6-fold increase in cell apoptosis when pretreated with SFN, while S. aureus infection did not elicit any apoptosis in macrophages as compared to DMSO treated macrophages (Figure 5A). To validate that cell apoptosis was caspase- dependent, caspases-3 and -7 activities were measured using the FAM-FLICA caspase detection probe. Flow cytometry analysis showed a 1.3-fold increase in macrophages pretreated with SFN and 1.7-fold increase in SFN pretreated cells challenged with S. aureus compared to DMSO treated macrophages (Figure 5B). Interestingly, SFN significantly increased cell necrosis in macrophages but not S. aureus (Figure 5C). No modulation in cell apoptosis or cell necrosis was observed in S. aureus infected macrophages compared to control cells. Taken together, these results suggest that SFN impacts on caspase-dependent cell apoptosis and cell necrosis which may participate in bacterial clearance.

SFN inhibits phosphorylation of p38 and JNK in S. aureus infected macrophages

ERK1/2, p38 and JNK, three well-studied isoforms of the MAPK family, are known regulators of the pro-inflammatory response and actively participate in the regulation of the Nrf2 signaling pathway. To determine whether S. aureus challenge activated ERK1/2, p38, and JNK, THP1 -derived macrophages were pretreated with DMSO or SFN followed by S. aureus infection. Six hours after SFN treatment, the phosphorylation state of ERK1/2, p38 and JNK was analyzed by Western blot. Densitometry analysis of total ERK and phosphorylated ERK protein signals showed a 2.5-fold increase in phosphorylated ERK in SFN treated macrophages compared to DMSO treated macrophages (Figure 6). Infection alone with S. aureus showed no effect on the activation of ERK pathway, while it significantly augmented phosphorylation of p38 and JNK. Interestingly, pretreatment of macrophages with SFN notably inhibited activation of p38 and JNK in S. aureus infected and non-infected macrophages.

MAPK-independent inhibition of IL-Ib IL-6 and TNFa mRNA expression levels by

SFN

To better understand the effect of SFN on the crosstalk between Nrf2 signaling pathway and MAPK signaling pathway regulating IL- 1 b, IL-6 and TNFa expressions, THPl-derived macrophages were pretreated with each MAPK inhibitor prior to SFN and/or S. aureus incubation. S. aureus- mediated increase of IL-l expression level is significantly decreased in presence of the ERK inhibitor, and to a lesser extent with p38 and JNK inhibitors (Figure 7A). IL-6 expression level was strongly increased upon S. aureus challenge, but ERK, p38 and JNK inhibitors were able to abolish it (Figure 7B). In addition, S. aureus- mediated increase of TNFa expression levels were decreased 2-fold by ERK inhibitor but not by p38 inhibitor SB203580 or JNK inhibitor SP600125 (Figure 7C). Interestingly, expression levels of IL- 1 b, IL-6 and TNFa were even lower in macrophages pretreated with either MAPK inhibitor when treated with SFN suggesting an alternative inhibitory mechanism that is MAPK-independent. These data suggest that SFN doubly inhibits expressions of IL- 1 b, IL-6 and TNFa, firstly in a MAPK- dependent manner by preventing phosphorylation of p38 and JNK (Figure 6), and secondly by taking a MAPK-independent signaling pathway.

ERK signaling required for S. aureus intracellular survival and p38 and JNK signaling for bacterial clearance by macrophages

To examine the correlation between bacterial uptake, bacterial clearance by macrophages and activations of ERK1/2, p38 and JNK elicited by either S. aureus or SFN, specific MAPK inhibitors were used. THPl-derived macrophages were treated with ERK, p38 and JNK inhibitors, PD98059, SB203580, and SP600125 respectively, prior to treatment with SFN and/or SYT09 stained S. aureus challenge. Flow cytometry analysis 1 h of internalized bacteria showed a loss of SFN-dependent decrease in bacterial uptake in SFN treated THPl- derived macrophages pretreated with either ERK, p38 or JNK inhibitor compared to DMSO treated macrophages (Figure 8A). Bacterial survival was assessed by CFU assay 24 h after pretreatment of THP1 -derived macrophages with a MAPK inhibitor, SFN or DMSO, and S. aureus challenge. In presence of ERK inhibitor PD98059, SFN-dependent decrease in bacterial burden is still significant in infected macrophages compared to DMSO treated cells. This SFN- dependent decrease is abolished when macrophages were pretreated with p38 and JNK, SB203580 and SP600125 respectively (Figure 8B).

Bacteria viability was assessed by staining live bacteria with SYT09 and dead ones with propidium iodide. The ratio of live bacteria was monitored at 60, 75, and 90 min by spectrophotometer and the results showed a significant toxicity of SP600125 on S. aureus , which may account for the lower bacteria counts in the CFU assay when SP600125 was added to the culture medium (data not shown).

In order to better understand the involvement of ERK, p38, and JNK signaling on cell apoptosis, we pretreated THP1 -derived macrophages with MAPK inhibitors prior to SFN/DMSO treatment and S. aureus challenge. Gentamycin was added to the medium 1 h after S. aureus challenge and cells were incubated for 24 h. Apoptotic macrophages were labeled with Annexin V-FITC, and necrotic macrophages were identified with propidium iodide staining. Flow cytometry analysis showed that ERK inhibitor PD98059 decreased SFN- mediated cell apoptosis while increasing S. aureus- mediated apoptosis (Figure 8C). No significant modulation was observed when THPl-derived macrophages were treated with p38 inhibitor SB203580, in contrast to the increase in SFN-mediated apoptosis in macrophages treated with JNK inhibitor SP600125. Flow cytometry analysis of macrophages treated with p38 inhibitor SB203580 showed an abolition of SFN-induced cell necrosis, while cell necrosis was still increased in macrophages treated with ERK inhibitor PD98059 or JNK inhibitor SP600125 (Figure 8D). Taken together, these data suggest that SFN-induced apoptosis in macrophages is ERK-dependent and that JNK signaling pathway has an anti-apoptotic effect. In addition we showed that SFN-mediated necrosis requires activation of p38 signaling pathway.

Example 2:

Inflammation plays an important role in the defense response of the innate immune system against pathogen infection. However, dysregulation of this inflammatory process may be detrimental to the host and lead to chronic disorders. We selected 5 compounds for their potential anti-inflammatory and/or anti-microbial properties to test on our in vitro model of bacteria-infected THP-l-derived macrophages. Those compounds, sulforaphane (SFN) and wogonin (WG), oltipraz (OTZ), dimethyl fumarate (DMF), and bardoxolone-methyl (CDDO- Me) are known activators of the nuclear factor erythroid 2-related factor 2 (Nrf2), a key regulator of the antioxidant, anti-inflammatory response pathways. The five selected compounds were tested for their anti-inflammatory and antioxidant effects on PMA-derived macrophages and LPS-treated macrophages. Moreover, we examined the effect of each compound on the activation state of LPS/IFNy-mediated Ml polarized macrophages. We also demonstrated that each compound affected differently the intracellular bacterial survival of gram-positive S. aureus in PMA-derived macrophages and PBMC-derived macrophages.

Results

Sulforaphane (SFN), wogonin (WG), oltipraz (OTZ), dimethyl fumarate (DMF), and bardoxolone-methyl (CDDO-Me) regulate intracellular survival of S. aureus

To evaluate the effect of each selected compound on the bactericidal activity of macrophages, THP-l -derived macrophages pretreated with each selected compound were infected with gram-positive bacteria S. aureus , and bacterial survival rate was determined. First, we assessed the toxicity of each compound on bacterial viability using the Live/dead bacteria viability kit. Spectrophotometer analysis showed that the cell viability of neither S. aureus were significantly affected by any of the 5 compounds as compared to DMSO treated bacteria after 90 minutes incubation (Figures 9A). Interestingly, the effects of SFN, OTZ, DMF, and CDDO- Me were distinct in macrophages infected with S. aureus. Data showed that SFN, DMF, and CDDO-Me significantly decreased intracellular S. aureus survival, while pretreatment with WG and OTZ showed no effect on S. aureus load (Figure 9B). These effects seen in THP-l- derived macrophages were also replicated in primary macrophages derived from human peripheral blood monocyte cells (Figure 9C). Taken together, these data suggest that pretreatment of WG and OTZ had no effect on the bactericidal activity of infected macrophages and that SFN, DMF, and CDDO-Me have effect on bacterial survival.

Discussion:

In this study, we showed that activation of the Nrf2 signaling pathway with SFN, DMF, or CDDO-Me participates in the decrease of intracellular bacteria burden and an increase in caspase-mediated apoptosis in macrophages treated with SFN and challenged with a non- pathogenic strain of S. aureus. Furthermore, treatment with SFN significantly impedes S. aureus- induced inflammation in challenged macrophages. While SFN anti-inflammatory effect has been previously recognized, the underlying molecular mechanisms involved in the process still remains elusive. For the first time, we highlight the two regulatory mechanism which allows SFN to suppress inflammation. We show here that SFN lessens inflammation in our in vitro model of macrophages in a MAPK-dependent and MAPK- independent manner. S. aureus is considered a facultative intracellular pathogen, as it has been found to proliferate extracellularly and intracellularly within various cell types [18]. Phagocytosed S. aureus strain USA300 successfully replicated in mature phagolysosomes of RAW 264.7-derived macrophages and primary human macrophages derived from peripheral blood monocytes [19]. In chronic and recurrent infections, S. aureus could persist several days within the infected macrophages before proliferation [20] . A recent study has shown that S. aureus a-toxin induced inflammatory cytokines such as IL- 1 b and TNFa in bone-marrow derived macrophages through activation of the acid sphingomyelinase and the rapid release of cathepsins [21]. Upon bacterial recognition by the host innate immune receptors, cascades of highly dynamic signal transduction systems are sequentially activated and amplified resulting in the proinflammatory response needed in the antimicrobial defense mechanism. Hence, it stands to reason that pathogens had developed, among its arsenal of immune evasive strategies, elaborative mechanisms to modulate the innate immune signal transduction pathways [22] MAPK signaling pathway is one of the signaling pathways central to the innate immune response. In human bronchial epithelial cells, concomitant infection of influenza virus and S. aureus synergistically promoted enhanced phosphorylation of p38, ERK and JNK. Treatment of these cells with specific inhibitors of p38 and ERK associated these signaling pathways with the regulation of IL-6 production [23]. In our case, THP-l -derived macrophages infected with S. aureus induced phosphorylation of p38 and JNK. Using RT-qPCR to analyze expression levels of proinflammatory genes in THP-l -derived macrophages infected with S. aureus , our data showed a rapid inflammatory response reflected by the strong increase in IL-6, TNFa, and to a lesser extent IL- 1 b mRNA expression levels in S. aureus infected macrophages. PMb, IL-6 and TNFa are proinflammatory cytokines well-known to initiate and regulate the immune response and inflammation. The use of specific MAPK inhibitors for ERK, p38 and JNK signaling allowed us to better understand the role played by each of the three members of the MAPK family. Based on our results, we determine that transcriptional expressions of PMb, IL-6, and TNFa genes are p38-, ERK-, and JNK-dependent (Figure 7).

Several natural or chemical compounds have been used to modulate the inflammatory response mediated by S. aureus infection by impacting on the intracellular signal transduction pathways. In a mouse model of S. aureus- induced mastitis, mice treated with the natural compound brazilin showed a decrease in S. aureus- induced inflammatory cytokines IE-1b, IL- 6, and TNFa, thus reducing the inflammatory-mediated tissue injury. The authors also showed an inhibition of the S. aureus- induced phosphorylation of p38, JNK and ERK in brazilin-treated mice [24] . Similar regulatory mechanisms of the NF-kB and MAPK signaling pathways were observed in S. aureus infected RAW 264.7-derived macrophages treated with selenium derivatives [25]. The down-regulation of NF-kB and MAPK signaling pathways by selenium was suggested to correlate with the decrease in TNFa, IE-1b, and IF-6 transcriptional expression levels and their cytokine release. Moreover, treatment of a S. aureus- induced peritonitis mouse model with an ephedrine derivative increased the survival rate of infected mice by reducing inflammation through the modulation of PDK/AKT and p38 signaling pathways [26].

In recent years, targeting the antioxidant and anti-inflammatory properties of Nrf2 have emerged as an interesting therapeutic strategy in the treatment of inflammatory diseases, including gastrointestinal, respiratory, cardiovascular and neurodegenerative diseases. As such, various compounds known to activate Nrf2 and its downstream signaling pathway have been tested. Several promising studies have explored the effects of SFN, curcumin, bardoxolone methyl, and dimethyl fumarate in murine model and the promising drugs were tested in clinical trials [27-30]

In agreement with these findings, our data confirm the anti-inflammatory function of SFN on THP-l -derived macrophages infected with S. aureus since SFN effectively abrogated the transcriptional expressions of genes coding for the proinflammatory cytokines IE-1b, IF-6, and TNFa (Figure 4, 7). However, in-depth examination of the molecular mechanisms involved using MAPK inhibitors indicate that SFN specifically inhibits p38 and JNK phosphorylation (Figure 6). We showed here that p38 and JNK signaling actively participate in bacteria phagocytosis, and inhibition of their phosphorylation results in a decrease in bacteria internalization in SFN-treated macrophages (Figure 1, 8A). In addition, expressions of IE-1b, IF-6, and TNFa mRNAs are regulated by p38 and JNK, together with ERK signaling.

We also demonstrated, by bacterial counts of viable intracellular bacteria, that SFN triggers cellular responses leading to the reduction in bacterial burden. We have established that the reduction in intracellular bacteria load is Nrf2-dependent, as inhibition of Nrf2 expression by specific siRNA impairs the macrophage ability to alleviate bacteria load. Therefore, SFN inhibition of p38 and JNK is Nrf2 dependent and impacts directly on the transcriptional regulation of proinflammatory cytokine genes and on the phagocytic activity of macrophages. Interestingly, a recent study described an opposite effect of SFN on phagocytosis. SFN treatment increased the phagocytic activity of human monocyte derived macrophages when challenged with HIV-l viral particles [31]. Up until very recently, the anti-inflammatory contribution of Nrf2 has been thought to be an indirect effect of the up-regulation of genes coding for antioxidant enzymes. Removal of ROS would then decrease inflammation [32] A recent study demonstrated that Nrf2 interferes with the transcriptional regulation of the proinflammatory genes coding for IL- 1 b and IL-6 cytokines [12]. As demonstrated by quantitative PCR done on macrophages pretreated with MAPK inhibitors of ERK, p38 and JNK (Figure 7), mRNA expression levels of IL- 1 b, IL-6 and TNFa were even lower in SFN treated macrophages infected with S. aureus than that seen in S. aureus infected macrophages treated with DMSO, indicative of a MAPK-independent inhibition pathway regulated by SFN. We speculate that this molecular mechanism results from the increase in activated Nrf2, which leads to the down-regulation of the transcriptional expressions of IL- 1 b, IL-6 and TNFa. Taken together, the anti-inflammatory mechanism of SFN resides on a two-level inhibition: first, on the phosphorylation suppression of p38 and JNK signaling, and secondly on the direct Nrf2-mediated transcriptional downregulation of the proinflammatory cytokine genes IL- 1 b, IL-6 and TNFa.

Contrarily to this recent study on keratinocytes, in which intracellular S. aureus activated the complement system and initiated ERK signaling, significantly reducing intracellular bacteria burden [33], our work on THP-l -derived macrophages exhibited no activation of the ERK signaling by S. aureus. We showed that SFN activates ERK signaling, triggering a SFN-mediated increase in caspase-dependent apoptosis and an increase in bactericidal activity in SFN treated macrophages challenged with S. aureus. In agreement with our findings, SFN-mediated apoptosis has been found in various in vitro cancer cell models, even considering the use of SFN as a cancer chemopreventive agent. The authors showed in prostate cancer cells and SV40-transformed MEFs that SFN induced ROS production and activated Bax- and Bak-dependent activation of caspase signaling cascades leading to programmed cell death [34, 35]. Furthermore, SFN-cysteine was found to activate ERK signaling pathway, contributing to the up-regulation of Bax/Bcl-2 and the activation of caspase- 3 and other pro-apoptotic proteins in human glioblastoma U373MG and U87MG [36].

Of note, ROS production was not impacted by S. aureus challenge, which was corroborated by the lack of impact on Nrf2 activation, known for its rapid antioxidant response. Likewise, an unchanged ROS production was observed in S. aureus infected macrophages treated with SFN, indicating a ROS -independent bactericidal activity and elicited by SFN in macrophages. Moreover, the inflammatory response triggered by S. aureus recognition suggests a ROS-independent activation of the MAPK signaling pathway. These new perceptions of the molecular mechanisms regulated by SFN, DMF, or CDDO-Me in the process of fighting infection may help improve bacterial clearance and moderate inflammation.

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Claims

CLAIMS:

1. A method of treating a Staphylococcus aureus infection in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a Nrf2 activator.

2. The method of claim 1 wherein the subject is infected by multi-drug resistant Staphylococcus aureus.

3. The method of claim 1 wherein the subject is immunocompromised.

4. The method of claim 1 wherein the subject is selected from the group consisting of subjects with AIDS (or HIV positive), subjects with severe combined immune deficiency (SCID), diabetics, subjects who have had transplants and who are taking immunosuppressives, subjects who do not have a spleen, subjects with end stage kidney disease (dialysis), and subject who have been taking corticosteroids.

5. The method of claim 1 wherein the subject suffers from a disease selected from the group consisting of sickle cell anemia, cystic fibrosis, severe liver, lung, heart disease, and neurological and muscular disabilities.

6. The method of claim 1 wherein the Nrf2 activator is selected from the group consisting of chalcone derivatives.

7. The method of claim 1 wherein the Nrf2 activator is selected from the group consisting of 2-trifluoromethyl-2'-methoxychalcone, auranofin, ebselen, 1, 2-naphthoquinone, cynnamic aldehyde, caffeic acid and its esters, curcumin, reservatrol, artesunate, tert- butylhydroquinone, and -quinone, (tBHQ, tBQ), vitamins Kl, K2 and K3, preferably menadione, fumaric acid esters, i.e. fumaric acid mono- and/or diester which is preferably selected from the group of monoalkyl hydrogen fumarate and dialkyl fumarate, such as monomethyl hydrogen fumarate, dimethyl fumarate (DMF), monoethyl hydrogen fumarate, and diethyl fumarate, 2-cyclopentenones, ethacrynic acid and its alkyl esters, bardoxolone methyl (methyl 2-cyano-3,l2-dioxooleana- 1,9(1 l)dien-28-oate) (CDDO-Me, RTA 402), ethyl 2-cyano-3,l2-dioxooleana- 1,9(1 l)dien-28-oate, 2-cyano-3,l2-dioxooleana-l,9(l l)dien-28-oic acid (CDDO), 1 [2- Cyano-3,l2-dioxooleana-l,9(l l)-dien-28-oyl]imidazole (CDDO-Im), (2-cyano-N- methyl-3,l2-dioxooleana-l,9(l l)-dien-28 amide (CDDO-methyl amide, CDDO-MA), l,2-dithiole-3-thione, 3,5-di-tert-butyl-4-hydroxytoluene, 3-hydroxycoumarin, 4- hydroxynonenal, 4-oxononenal, malondialdehyde, (E)-2-hexenal, capsaicin, allicin, allylisothiocyanate, 6-methylthiohexyl isothiocyanate, 7-methylthioheptyl isothiocyanate, sulforaphane (SFN), 8-methylthiooctyl isothiocyanate, corticosteroids, such as dexamethasone, 8-iso prostaglandin A2, alkyl pyruvate, such as methyl and ethyl pyruvate, diethyl or dimethyl oxaloproprionate, 2-acetamidoacrylate, methyl or ethyl-2-acetamidoacrylate, hypoestoxide, parthenolide, eriodictyol, 4-Hydroxy-2- nonenal, 4-oxo-2nonenal, geranial, zerumbone, aurone, isoliquiritigenin, xanthohumol, [lO]-Shogaol, eugenol, G-acetoxychavicol acetate, allyl isothiocyanate, benzyl isothiocyanate, phenethyl isothiocyanate, 4-(Methylthio)-3-butenyl isothiocyanate and

6-Methylsulfinylhexyl isothiocyanate, ferulic acid and its esters, such as ferulic acid ethyl ester, and ferulic acid methyl ester, sofalcone, 4-methyl daphnetin, imperatorin, auraptene, poncimarin, bis [2-hydroxybenzylidene] acetones, alicylcurcuminoid, 4- bromo flavone, b-naphthoflavone, sappanone A, aurones and its corresponding indole derivatives such as benzylidene-indolin-2-ones, perillaldehyde, quercetin, fisetin, koparin, genistein, tanshinone HA, BHA, BHT, PMX-290, AL-l, avicin D, gedunin, fisetin, andrographolide, tricyclic bis(cyano enone) TBE-31 [(±)-(4bS,8aR,l0aS)-l0a- ethynyl-4-b,8,8-trimethyl-3,7-dioxo-3,4-b,7,8,8a,9,l0,l0a-octahydrophenanthrene- 2,6-dicarbonitrile], MCE-l, MCE5, and TP-225.

8. The method of claim 1 wherein the Nrf2 activator is sulforaphane.