Method for modulating the macrophage function and associated products

Abstract

The invention concerns the modulation of the macrophage function. The inventors have discovered that this function can be modified by applying a sequence of repetitive magnetic stimulations having a frequency comprised between 2 Hz and 20 Hz. This opens the way to a non-invasive control of the macrophage and, thereby, of any use that implies the macrophage.

Description

[0001] METHOD FOR MODULATING THE MACROPHAGE FUNCTION AND ASSOCIATED PRODUCTS

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The present invention concerns a method for modulating the macrophage function of at least one macrophage in a sample of a subject. The present invention also concerns associated products, namely a device for modulating and an isolated macrophage.

[0004] BACKGROUND OF THE INVENTION

[0005] Macrophages, as sentinel cells of the innate immune system, play a crucial role in innate immunity by recognizing and eliminating foreign invaders. Their ability to respond rapidly to microbial challenges and modulate immune responses is essential for host defense. Macrophages migrate to sites of aggression, phagocyte external particles, including microorganisms and cell debris, and trigger rapid responses for their elimination. While essential for protection against bacterial infection, phagocytosis can also serve as the primary pathway for bacteria to enter macrophages. Some pathogens, including Staphylococcus aureus (S. aureus), have developed evasion mechanisms allowing them to survive, escape and disseminate within the host. Studies in a murine model of airway infection have highlighted the active participation of macrophages in clearing methicillin- resistant S. aureus from lung, as depletion of alveolar macrophages was associated with increased mortality.

[0006] S. aureus belongs to Gram-positive bacteria. As a versatile and commensal opportunistic pathogen, it can cause various infections in humans, including skin and soft tissue infections, pneumonia, osteomyelitis and endocarditis.

[0007] There is thus a need for an efficient therapy against pathogen Gram-positive bacteria, especially S. aureus. Indeed, the prevalence of antibiotic-resistance of S. aureus poses significant challenges, leading to increased morbidity, mortality and healthcare costs worldwide.

[0008] Therefore, the development of novel therapeutic strategies to suppress intracellular load of S. aureus, while minimizing potential side effects to the host, is needed.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention aims to provide for a method for modulating the macrophage function of at least one macrophage in a sample of a subject, which is non-invasive.

[0011] To this end, the specification describes a method for modulating the macrophage function of at least one macrophage in a sample of a subject, said method comprising applying a sequence of repetitive magnetic stimulations on an area of the sample, the area comprising at least one macrophage, the magnetic stimulations having a frequency comprised between 2 Hz and 20 Hz.

[0012] According to further aspects of the method for modulating, which are advantageous but not compulsory, the method for modulating might incorporate one or several of the following features, taken in any technically admissible combination: the frequency is comprised between 4 Hz and 6 Hz. the frequency is comprised between 14 Hz and 16 Hz.

[0013] - the sequence has a total duration between 3 minutes and 7 minutes.

[0014] - the sequence has a total duration equal to 5 minutes 25 seconds.

[0015] - the sequence is applied only once. the method is an ex vivo method.

[0016] - the modulation of the macrophage function comprises reducing intracellular survival of bacteria in macrophages, preferably reducing intracellular survival of S. aureus in macrophages.

[0017] - the modulation of the macrophage function comprises activating the nuclear factor erythroid 2 - related factor (Nrf2) signalling pathway and / or decreasing Kelch like ECH associated protein 1 (Keapl ) expression and / or increasing p62 protein expression or phosphorylation.

[0018] - the subject is a mammal, preferably a human being.

[0019] - the sample of the subject is a blood sample.

[0020] The specification also concerns a device for modulating the macrophage function of at least one macrophage in a sample of a subject, the device for modulating comprising an application unit, the application unit being adapted to apply a sequence of repetitive magnetic stimulations on an an area of the sample, the area comprising at least one macrophage, the magnetic stimulations having a frequency comprised between 2 Hz and 20 Hz.

[0021] The specification also deals with an isolated macrophage previously submitted to a sequence of repetitive magnetic stimulations having a frequency comprised between 2 Hz and 20 Hz, for use for treating and / or preventing a bacterial infection, preferably a S. aureus infection.

[0022] The specification also concerns the isolated macrophage for use as previously describes, wherein said macrophage is present in a sample of a subject and the bacterial infection occurs in the same subject.

[0023] The specification also deals with a method for treating and / or preventing a bacterial infection, preferably a S. aureus infection in a subject, the method comprising applying a sequence of repetitive magnetic stimulations on an area of said subject, the area comprising at least one macrophage, the magnetic stimulations having a frequency comprised between 2 Hz and 20 Hz.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The invention will be better understood on the basis of the following description which is given in correspondence with the annexed figures and as an illustrative example, without restricting the object of the invention. In the annexed figures:

[0026] - figure 1 shows a sample and a device for modulating,

[0027] - figure 2 represents an example of temporal variation of the applied magnetic field with two sequences of repetitive magnetic stimulations, and

[0028] - figures 3 to 12 show experimental results. Especially:

[0029] Figure 3: rMS activates Nrf2 translocation and increases Nrf2 mRNA and protein expressions. rMS decreases Keapl protein expression. (A) THP-1 -derived macrophages subjected to rMS treatment (10Hz). Nrf2 mRNA expression was determined by RT-qPCR. Data were analyzed using a one-way ANOVA followed by Dunnett’s multiple comparisons. (B) Nrf2 protein expression was detected by Western blotting 24 h after rMS treatment and analyzed using densitometric analysis. Statistical analysis was performed using two- tailed unpaired t-test. (C) Immunofluorescence microscopy. Nuclear translocation of Nrf2 1 h after rMS treatment. THP-1 -derived macrophages were immunostained with specific Nrf2 antibody, nuclei were conterstained with DAPI. Bars: 20 pm. (D) Nuclear Nrf2 protein expression was determined at the indicated time point using Western blotting and analyzed using densitometry analysis. Data are expressed as Nrf2 protein expression normalized to lamin A / C control values. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons. (E) Keapl protein expression was detected 3 h after rMS treatment and analyzed using densitometric analysis. Statistical analysis was performed using two-tailed unpaired t-test. Data are shown as mean ± SEM * p<0.05, ** p<0.01.

[0030] Figure 4: rMS activates Nrf2 signaling pathway as shown by increase HO-1 and NQO1 mRNA and protein expressions. (A) Transcriptional expression of HO-1 at the indicated time points were determined by SYBRgreen RT-qPCR. Data shown are mean ± SEM from independent experiments. Statistical analysis were performed using one-way ANOVA followed by Dunnett’s multiple comparisons. HO-1 protein expression was determined at 24 h after rMS treatment. (B) NQO1 mRNA and protein expressions. * p<0.05; ** p<0.01 ; *** p<0.005.

[0031] Figure 5: rMS activates p62 and increases p62 protein expression. (A-B) p62 and phosphorylated p62 protein expressions were determined by Western blotting 3 h after rMS treatment. (B) Immunofluorescence microscopy. (C) Confocal microscopy images of THP-1 -derived macrophages treated with rMS. After 3 h, immunofluorescence labeling with p62 and counterstained with Hoechst33342 was performed. Bars: 10 pm. (D-E) Quantitative analysis of p62 puncta and p62 fluorescence intensity in the puncta using Image J. Mean ± SEM were obtained from analysis of n= 100 cells from 2 independent experiments. *p<0.05, ** p<0.01. Statistical analysis were performed using two-tailed unpaired t-test.

[0032] Figure 6: (A) Cell viability measured by MTT assay 24h after rMS treatment. (B) Cell apoptosis was determined using the Annexin V staining. (C) Inflammation was determined by quantifying IL-6, IL-1 p, and TNF-a mRNA expressions 6 h after rMS treatment using RT-qPCR. (D) Oxidative stress was measured by using CellROX fluorogenic probe in live cells to detect ROS. Quantification of CellROX green fluorescence using Image J. H2O2 treated cells were used as positive control. Scale bar: 50 pm. (E) THP-1 -derived macrophages were infected with SYTO9-labeled S. aureus. After 1 h infection, phagocytosis was determined by FACS. (F) S. aureus intracellular survival using CFU assay. 3h after rMS treatment, THP-1 -derived macrophages were infected with S. aureus. Extracellular bacteria were eliminated by addition of gentamicin 1 h after infection. Cells were incubated for 24h and CFU was determined. Data are mean ± SEM. Statistical analysis was performed using two-tailed unpaired t-test ****p<0.0001 .

[0033] Figure 7: (A) THP-1 derived macrophages were transfected with scramble or Nrf2- targeted siRNA 24 h prior to rMS treatment. 24 h after rMS treatment, protein lysates were analyzed Nrf2 and HO-1 protein expressions were determined by Western blotting. Statistical analysis was performed using two-tailed t-test ***p<0.0003, **** p<0.0001. (B) CFU with transfected THP-1 derived macrophages. (C) Primary alveolar macrophages were isolated from WT and Nrf2 knockout mice and used in bacteria intracellular survival assay. Data are means ± SEM. * p<0.05 as determined by one-way ANOVA with Tukey’s multiple comparisons. Figure 8 : (A) rMS-treated TH P-1 -derived macrophages were infected with S. aureus. Total RNAs were isolated 3 h after infection. IL-6, IL-ip, and TNF-a mRNA expressions were quantified by RT-qPCR. (B) Immunoblot analysis. Protein lysates extracted from THP-1 -derived macrophages after 3 h treatment with rMS and infected with S. aureus for 3 h before cell lysis. Immunoblots using the indicated antibodies are representative of 6 independent experiments. (C) Immunofluorescence microscopy. THP- 1 -derived macrophages treated with rMS then infected with Hoechst 33342-stained S. aureus. Cells were fixed with PFA 4% and were immunostained with p62 antibody. Nucleus was stained with Hoechst 33342. Bar scale: 10 pm. p62 mean fluorescence intensity in puncta by cell was quantified using Image J. * p<0.05, ** p<0.01 . Statistical analysis were performed using two-tailed unpaired t-test.

[0034] Figure 9 : Immunoblot analysis of phospho-p38, JNK, and ERK MAPK. THP-1 - derived macrophages were treated with rMS 3 h prior to infection with S. aureus. Cell lysis was done 3 h after infection using RIPA supplemented with a protease and phosphatase inhibitors cocktail. Shown immunoblots were representative of 4 independent experiments. Densitometric analysis was performed using Image Studio Lite. * p<0.05, **p<0.01 were determined by one-way ANOVA with Tukey’s multiple comparisons.

[0035] Figure 10 : (A) Immunofluorescence confocal microscopy. Confocal images of THP- 1 -derived macrophages treated with rMS and infected with Hoechst-labeled S. aureus. (B) Quantitative analysis of phospho-p38 fluorescence intensity per cell. (C) Quantification of the phospho-p38 localization in the nucleus in THP-1 -derived macrophages treated with rMS and infected with S. aureus. * p<0.05, **p<0.01 , ***p<0.0005, **** p<0.0001 were determined by one-way ANOVA with Tukey’s multiple comparisons.

[0036] Figure 11 : Schematic representation of the non-canonical pathway activated by rMS in THP-1 -derived macrophages infected with S. aureus. THP-1 -derived macrophages infected with S. aureus mediates an activation of the p38 MAPK pathway, which regulates the gene expression of pro-inflammatory cytokines such as IL-6, IL-1 p, and TNF-a, and promote S. aureus intracellular survival. In THP-1 -derived macrophages, treatment with rMS results in an increase in phosphorylated p62 at Ser349, which results in increase interaction with Keapl and the release of Nrf2. The phosphorylated p62-Keap1 complex is identified for degradation by autophagy. Nrf2 is then free to translocation to the nucleus, interact with small Maf proteins, and bind antioxidant response element (ARE), leading to the upregulation of genes coding for antioxidant, detoxifying, and anti-inflammatory proteins, as well as coding for p62.

[0037] Figure 12: THP-1 -derived macrophages were subjected to rMS protocol 5Hz and 15Hz and cells were lysed at the indicated time. Purified mRNA were subjected to RT, and transcriptional expression of Nrf2 target genes HO-1 and NQO1 were determined by SYBRgreen qPCR on a BioRad CFX96 apparatus. Data shown are mean ± SD (n = 4).

[0038] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] As shown in the example, the inventors investigated the impact of repetitive magnetic stimulation (rMS) on macrophages and Nrf2 involvement, and observed a significant increase in Nrf2 transcriptional and protein expression, accompanied by a decrease in Keapl protein expression after rMS treatment. Furthermore, downstream targets of Nrf2 (such as heme oxygenase-1 (HO-1 ) and NAD(P)H:quinone oxidoreductase 1 (NQO1)) were upregulated, indicating the activation of the Nrf2 signaling pathway by rMS. An increase in phosphorylated p62 and total p62 protein expressions in rMS-treated macrophages was also found, suggesting an activation of Nrf2 in a p62-dependent manner. Importantly, rMS treatment resulted in a decrease in the intracellular survival of S. aureus in macrophages.

[0040] Thus, the claimed method is based on these findings.

[0041] A sample 10 and a device for modulating 12 are schematically represented on figure 1.

[0042] By “sample”, it is meant a sample of a subject.

[0043] The subject is a mammal. Preferably, the subject is a human being. Preferably, the subject is afflicted by a bacterial infection, preferably a Gram-positive bacteria infection, more preferably an infection with S. aureus.

[0044] The sample 10 may be any biological sample that comprises at least one macrophage of a subject. Preferably, the sample 10 is a blood sample.

[0045] The sample 10 comprises an area 14 comprising at least one macrophage 16.

[0046] This area 14 is represented on figure 1 by dotted lines.

[0047] By “macrophage”, it is meant a type of white blood cell of the innate immune system that engulfs and digests pathogens, such as cancer cells, microbes, cellular debris and foreign substances. This process is called phagocytosis. It acts to defend the host against infection and injury. A macrophage is capable of cell division, proliferation and tissue motility. Macrophages are professional phagocytes found in essentially all tissues where they take various forms (with various names, e.g., histiocytes, Kupffer cells, alveolar macrophages, microglia and others), but all are part of the mononuclear phagocyte system. Besides phagocytosis, they play a critical role in nonspecific defense (innate immunity) and also help initiate specific defense mechanisms (adaptive immunity) by recruiting other immune cells, such as lymphocytes, and through antigen presentation (e.g. to T lymphocytes). Human macrophages are about 21 micrometers in diameter. They can be identified using flow cytometry or immunohistochemical staining by their specific expression of proteins such as CD14, CD40, CD11b, CD64, F4 / 80 (mice) / EMR1 (human), lysozyme M, MAC-1 / MAC-3 and CD68.

[0048] A macrophage is thus adapted to carry out one or several actions on its environment.

[0049] The all set of such actions is named the “macrophage function” hereinafter.

[0050] The device for modulating 12 is a device adapted to generate a modulation of the macrophage function of the macrophage 16.

[0051] This means that the device for modulating 12 is adapted to modify the macrophage function of the macrophage 16.

[0052] In other words, the device for modulating 12 is adapted to modify an action comprised in the macrophage function.

[0053] As a specific example, the macrophage function comprises an action of reducing intracellular survival of bacteria in macrophages. Preferably, the reduction of intracellular survival of bacteria in macrophages is significant. By “significant”, it is meant that the reduction of intracellular survival of bacteria in macrophages in a treated sample of a subject according to the invention is preferably of at least 10%, preferably at least 20%, as compared to an untreated sample.

[0054] Preferably, such reducing action comprises reducing intracellular survival of S. aureus in macrophages. Preferably, the reduction of intracellular survival of S. aureus in macrophages is significant. By “significant”, it is meant that the reduction of intracellular survival of S. aureus in macrophages in a treated sample of a subject according to the invention is preferably of at least 10%, preferably at least 20%, as compared to an untreated sample.

[0055] In variant or in complement, the macrophage function comprises an action of activating the nuclear factor erythroid 2 - related factor (Nrf2) signalling pathway. By “activating the nuclear factor erythroid 2 - related factor (Nrf2) signalling pathway”, it is meant increasing the expression and / or activity, preferably the expression, of at least one downstream target of Nrf2. Said expression and / or activity is significantly increased, for example of at least 2-times, preferably 3-times. The expression of a downstream target of Nrf2 corresponds to a nucleic acid or a protein that is synthetized in response to Nrf2 signalling pathway activation. The activity of a downstream target of Nrf2 corresponds to protein activity in response to Nrf2 signalling pathway activation. Preferably, the downstream target of Nrf2 is chosen from heme oxygenase-1 (HO-1), p62 and NAD(P)H:quinone oxidoreductase 1 (NQO1).

[0056] In variant or in complement, the macrophage function comprises an action of decreasing Kelch like ECH associated protein 1 (Keapl ) expression. Keapl is the negative regulator of Nrf2. Thus, decreasing Keapl expression results in activating Nrf2 signalling pathway.

[0057] In variant or in complement, the macrophage function comprises an action of increasing p62 protein expression or phosphorylation.

[0058] The macrophage function can thus here be any combination of the previously described actions.

[0059] The device for modulating 12 comprises an application unit 18.

[0060] The application unit 18 is adapted to apply a sequence of repetitive magnetic stimulations on the area.

[0061] The application unit 18 comprises a controller 20 and a magnetic generator 22.

[0062] The controller 20 is adapted to control the magnetic generator to have this generator generate a sequence of repetitive magnetic stimulations on the area.

[0063] For instance, the controller 20 is an electrical generator and the magnetic generator 22 is one or several coils.

[0064] An example of sequence, which can be applied by the application unit, is now more specifically described in reference to figure 2.

[0065] Figure 2 represents the evolution of magnetic field applied by the application unit 18 with time.

[0066] For the sake of exemplification, the present evolution comprises two sequences respectively labelled S1 and S2.

[0067] A sequence is characterized by a frequency of the magnetic stimulations and a total duration.

[0068] The definition of frequency is the number of pulses per second.

[0069] In the experimental section, the frequency is 10 Hz, corresponding to 10 pulses in one second.

[0070] The time duration is defined by the total sequence in one session, consisting of 10 series of 100 biphasic pulses with a 25-second interval between each series.

[0071] In the experimental section, the time duration for one session was 5 min 25 s.

[0072] The first sequence is thus characterized by a first frequency and a first total duration. In this example, the first frequency is comprised between 4 Hz and 6 Hz.

[0073] The first total duration is, for instance, equal to 5 minutes 25 seconds.

[0074] The second sequence is thus characterized by a second frequency and a second total duration.

[0075] In this example, the second frequency is comprised between 14 Hz and 16 Hz.

[0076] The second total duration is, for instance, equal to 5 minutes 25 seconds.

[0077] More generally, the frequency of the magnetic stimulations associated to a sequence is comprised between 2 Hz and 20 Hz.

[0078] Furthermore, the total duration equal to 5 minutes 25 seconds.

[0079] Advantageously, only one sequence is applied.

[0080] As explained in the experimental section, one major advantage of this method is the fact that it provides with the ability of modulating the functioning of a macrophage in a non-invasive way.

[0081] Due to such advantages, such sequence may be used in several contexts.

[0082] As a first example, the sequence can be used in a method for modulating the macrophage function of at least one macrophage in a sample of a subject, said method comprising applying a sequence of repetitive magnetic stimulations on an area of the sample, the area comprising at least one macrophage, the magnetic stimulations having a frequency comprised between 2 Hz and 20 Hz.

[0083] Such method is an ex vivo method.

[0084] In particular, the modulation of the macrophage function may comprise reducing intracellular survival of bacteria in macrophages, preferably reducing intracellular survival of S. aureus in macrophages.

[0085] In variant or in complement, the modulation of the macrophage function comprises activating the Nrf2 signalling pathway and / or decreasing Keapl expression and / or increasing p62 protein expression or phosphorylation.

[0086] As a second example, the sequence enables to obtain at least one specific isolated macrophage.

[0087] In particular, the isolated macrophage is a macrophage previously submitted to a sequence of repetitive magnetic stimulations having a frequency comprised between 2 Hz and 20 Hz, for use for treating and / or preventing a bacterial infection.

[0088] Preferably, the bacterial infection is a S. aureus infection.

[0089] S. aureus (or staphylococcus aureus) is a Gram-positive spherically shaped bacterium, a member of the Bacillota, and is a usual member of the microbiota of the body, frequently found in the upper respiratory tract and on the skin. According to a specific embodiment, said macrophage is present in a sample of a subject and the bacterial infection occurs in the same subject.

[0090] As a third example, the sequence is used in a method for treating a bacterial infection, the method for treating comprising applying a sequence of repetitive magnetic stimulations on an area of said subject, the area comprising at least one macrophage, the magnetic stimulations having a frequency comprised between 2 Hz and 20 Hz.

[0091] As a fourth example, the sequence is used in a method for preventing a bacterial infection, the method for treating comprising applying a sequence of repetitive magnetic stimulations on an area of said subject, the area comprising at least one macrophage, the magnetic stimulations having a frequency comprised between 2 Hz and 20 Hz.

[0092] In both the third and the fourth examples, the infection is preferably a S. aureus infection in a subject.

[0093] In each case, the example benefits from the property that a sequence of repetitive magnetic stimulations with a frequency of the magnetic stimulations comprised between 2 Hz and 20 Hz generates a modulation of the macrophage function of at least one macrophage.

[0094] The present invention also relates to an isolated macrophage previously submitted to a sequence of repetitive magnetic stimulations having a frequency comprised between 2 Hz and 20 Hz, for use for treating and / or preventing a bacterial infection, preferably a S. aureus infection.

[0095] Preferably, said macrophage is present in a sample of a subject and the bacterial infection occurs in the same subject. Thus, preferably, said macrophage is autologous.

[0096] As used herein, the term « treatment » or « treating » refers to an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.

[0097] The isolated macrophage previously submitted to a sequence of repetitive magnetic stimulations having a frequency comprised between 2 Hz and 20 Hz is preferably administered to the subject by injection, preferably by intravenous administration. The present invention is now illustrated by the following example.

[0098] Example

[0099] Materials and methods

[0100] Antibodies and reagents

[0101] A Maxima First Strand cDNA synthesis kit, Lipofectamine® LTX & PLUS™ Reagent, Trizol, CellROX™ Green Oxidative Stress Reagent, Fluoromount-G™ (EMS), RIPA Lysis and Extraction Buffer, sodium pyruvate, Pierce protease and phosphatase inhibitor cocktail, SYTO9™ dye from the LIVE / DEAD BacLight bacterial counting kit, anti-phospho- p62 antibody (pS349, Invitrogen), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay kit (Biotium Inc.), secondary antibodies donkey antimouse Alexa Fluor 488, donkey anti-rabbit Alexa Fluor 594, and iBind Western blot system were purchased from Fisher Scientific (lllkirch, France). A nuclear extraction kit, phorbol 12-myristate 13-acetate (PMA), dimethyl sulfoxide (DMSO) >99.7%, - mercaptoethanol, paraformaldehyde, H2O2 30% in H2O, rabbit anti-phosphorylated ERK (pT185 / pY187) antibody, rabbit anti-ERK antibody, mouse anti-phospho-p38 (pT180 / pY182) antibody, rabbit-anti-p62 antibody, rabbit anti-Keap1 antibody, and mouse anti-GAPDH antibody were obtained from Merck (Saint-Quentin-Fallavier, France). iTaq SYBRgreen qPCR Supermix, a DC protein assay kit, 4-20 % mini-Protean precast protein gels were purchased from BioRad (Marnes-la-Coquette, France). RPMI (Roswell Park Memorial Institute) 1640 culture medium, phosphate-buffered saline (PBS), gentamicin, penicillin / streptomycin mixture, Hoechst 33342, and heat-inactivated fetal bovine serum were obtained from Eurobio Scientific (Les Ulis, France). CO2 s 99.99% was purchased from Messer (France). Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection reagent, mouse anti-HO-1 antibody, rabbit anti-NQO1 antibody, and rabbit anti-Lamin A / C antibody were purchased from Abeam (Paris, France). Mouse anti-phospho-JNK (pT183 / pY185) antibody, mouse anti-JNK antibody, mouse anti-p38 antibody were acquired from BD Biosciences (Le Pont de Claix, France). Anti-Nrf2 antibody and anti-p62 antibody were obtained from ProteinTech (Manchester, UK). Tryptic soy broth (TSB) and agar supplemented TSB (TSB agar) were from Conda laboratories (Dutscher, Bernolsheim, France). IRDye® 680CW goat anti-mouse IgG, and IRDye® 800CW Goat anti-Rabbit IgG secondary antibodies were purchased from LI-COR® Biosciences (Bad Homburg, Germany). Repetitive Magnetic Stimulation (rMS)

[0102] Repetitive magnetic stimulation was delivered using a B65 refrigerated butterfly coil connected to a MagPro R30 magnetic stimulator (Magventure, Denmark) purchased from Mag2Health (Villennes-sur-Seine, France). Cells were placed on the cool coil and were subjected to one session of 10 series of 100 biphasic pulses with a 25 s interval between series, resulting in a total of 1000 pulses (10 Hz at 80 % of maximum output). In parallel, control cells were exposed to the same environment for the same duration as the stimulated cells but without stimulation. rMS at 5 Hz and 15 Hz were also tested and showed no effect on HO-1 and NQO mRNA expressions.

[0103] Bacterial strain, growth culture, and fluorescence labeling

[0104] The gram-positive bacteria Staphylococcus aureus strain (ATCC 25923) was grown aerobically in Trypticase soy broth to the optical density of 1 at 37 °C under agitation. Glycerol stocks of S. aureus were prepared and aliquoted. When required, frozen stocks were thawed and bacteria were diluted in sterile PBS at the appropriate concentration.

[0105] For phagocytosis assay, S. aureus was incubated with SYTO9 dye for 15 min in the dark following the manufacturer’s instructions. For immunofluorescence microscopy, S. aureus was incubated with 10 pg / ml Hoechst 33342 in the dark. After 1 h, Hoechst 33342- stained bacteria were washed twice with PBS, and resuspended in PBS.

[0106] Cell culture, cell differentiation, and cell transfection

[0107] Human monocytic THP-1 cell line (ATCC® TIB-202™) was maintained in RPMI 1640 Glutabio medium supplemented with 10 % heat-inactivated fetal bovine serum, 10 mM HEPES buffer, 1 mM sodium pyruvate, and 50 pM p-mercaptoethanol in a humidified atmosphere at 37 °C and 5 % CO2. Terminal differentiation of THP-1 to macrophages was obtained by rinsing cells twice with PBS prior to incubation with 50 nM PMA in p- mercaptoethanol-free complete RPMI 1640 medium for 48 h.

[0108] S. aureus was added to the cell culture at a multiplicity of infection of 10. For experiments requiring incubation time longer than 3 h, 10 pg / ml gentamicin was added to the cell culture medium to inhibit growth of extracellular bacteria.

[0109] In order to knockdown Nrf2, THP-1 -derived macrophages were transfected with a pre-designed siRNA targeting Nrf2 (s9492; ThermoFisher Scientific) or a universal control siRNA (1027310; Quagen) using Lipofectamine LTX following the manufacturer’s recommended protocol.

[0110] Animals and isolation of primary mouse alveolar macrophages Breeding of the C57BL / 6J Nrf2 knockout (Nrf2- / -) mouse strain, provided by Dr Yamamoto (Tohuki University) and purchased from Riken BRC 19, was conducted according to National and European legislation and institutional guidelines for the care and use of laboratory animals approved by the Animal Ethics Committee CEEA47 PELVIPHARM (2Care animal facility, University of Versailles Saint-Quentin-en-Yvelines, UFR Sante Simone Veil) and the Ministry of Higher Education, Research and Innovation APAFIS#8785-201610191723731v3. Mice were maintained in a standard 12-h light / 12-h dark cycle with access to food and water ad libitum.

[0111] Bronchoalveolar lavage was performed following euthanasia of mice and alveolar macrophages were collected by repeated lavages with 1 ml ice-cold PBS. After cell resuspension, total cell count was obtained using Countess automated Cell counter (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA). Primary alveolar macrophages were maintained in complete RPMI 1640 medium complemented with penicillin / streptomycin mixture overnight. The antibiotics were removed prior to performing ex vivo experiments by washing cells twice with PBS, followed by the addition of antibiotics-free complete RPMI 1640 medium.

[0112] Bacteria intracellular survival assay

[0113] THP-1 -derived macrophages, seeded in 24-well plates at 2.5 x 105cells / well, were treated with rMS for 3 h prior to infection with S. aureus at an MOI of 10. Gentamicin was added to the cell culture medium 1 h after infection to inhibit growth of extracellular bacteria, and macrophages were incubated for a total of 24 h before colony forming unit (CFU) assay. Briefly, macrophages were washed twice with ice-cold PBS followed by 20 min incubation in 1 ml ice-cold sterile water to lyse cells. Numeration of intracellular bacteria was obtained by plating 5-fold serial dilutions on TSB solidified with 1 .5 % agar and incubating at 37°C for 24 h.

[0114] Subcellular fractionation and immunoblotting

[0115] For the subcellular fractionation, a nuclear extraction kit was used according to the manufacturer’s protocol. Briefly, following treatment of THP-1 -derived macrophages, 3 x 106cells per condition were collected and washed with PBS. After centrifugation, cell pellets were incubated for 15 min with ice-cold cytoplasmic lysis buffer supplemented with a protease and phosphatase inhibitor cocktail. Cell membranes were then disrupted via repeated passages through a 27G needle. After 20 min centrifugation at 8,000 x g at 4 °C, the cytoplasmic fractions were collected and the remaining cell pellets were resuspended in ice-cold nuclear extraction buffer supplemented with protease and phosphatase inhibitor cocktail. Nuclear membranes were disrupted by repeated passages through a 27G needle, followed by incubation of the nuclear suspensions at 4 °C on an orbital shaker. After 1 h, nuclear fractions were collected by centrifugation at 16,000 x g for 5 min at 4 °C.

[0116] For extraction of total protein lysates, 1 x 106THP-1 -derived macrophages were rinsed once with PBS and lyzed in ice-cold RIPA buffer supplemented with a protease and phosphatase inhibitor cocktail. After 30 min incubation on ice, cell lysates were centrifuged at 12,000 x g for 5 min at 4 °C. The supernatants containing protein extracts were collected.

[0117] Protein concentrations for total protein extracts and subcellular fractions were measured using DC protein assay kit. Protein extracts were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Immobilon-FL, Merck). Immunoblotting was performed using the iBind flex western system according to the manufacturer’s instructions. Briefly, primary antibodies targeting Nrf2, HO-1 , NQO1 , p62, phospho-p62, p38, phospho-p38, ERK, phospho-ERK, JNK, phospho-JNK, GAPDH, and secondary antibodies, IRDye680RD and IRDye800RD, were diluted in iBind Flex FD solution. Fluorescence signals were acquired using Odyssey CLx imaging system (Ll- COR) and densitometric analysis was achieved using Image Studio Lite v4.0.

[0118] Total RNA extraction and real-time quantitative PCR analysis

[0119] Total RNA was isolated from treated THP-1 -derived macrophages using Trizol reagent and chloroform extraction technique following the manufacturer’s instructions. RNA concentrations were determined using the NanoPhotometer® N120 (Implen; Munchen, Germany). First, 1 pg of total RNA was reverse transcribed to cDNA using Maxima First strand cDNA synthesis kit. Quantitative analysis was achieved using realtime quantitative PCR (RT-qPCR), with each cDNA sample done in triplicate. qPCR was realized using the BioRad CFX384 Touch Real-Time PCR Detection system and iTaq SYBRgreen qPCR mix. The following table lists the specific primers used for qPCR, Nrf2, HO-1 , NQO1 , IL-6, IL-ip, TNF-a, and 18S rRNA, and were synthetized by Eurogentec (Seraing, Belgium):

[0120] The cycling parameters for qPCR were 95°C for 3 min, 40 cycles of 95°C for 5s and 60 °C for 20s, with a melting curve from 65°C to 95 °C. The cycle threshold (Ct) values of each target genes were first normalized to that of the reference gene 18S rRNA (ACt) then the final values (AACt values) were expressed as folds of control. Data were analyzed on the BioRad CFX manager 3.1 using the AACt method.

[0121] Cell phagocytosis and cell apoptosis by flow cytometry

[0122] For the assessment of cell phagocytosis, 1 x 106cells / well THP-1 -derived macrophages were treated with rMS 3 h prior to infection with SYTO9-labeled S. aureus. After 1 h infection, macrophages were scraped, washed with PBS, and collected for flow cytometry analysis.

[0123] To assess cell apoptosis 24 h after rMS treatment, 1 x 106cells / well THP-1 -derived macrophages were stained for 10 min in the dark with Annexin V-FITC reagent. Nonadherent and adherent cells were collected and after 3 washes with PBS, cells were analyzed by flow cytometry.

[0124] All flow cytometric analysis was immediately realized on the BD LSRFortessa™ using FACSDiva 7.0 software (BD Biosciences, Franklin Lakes, NJ, USA).

[0125] Quantification of intracellular ROS production Intracellular ROS was measured in live cells using the CellROX™ green fluorogenic probe. Briefly, 2.5 x 105THP-1 -derived macrophages grown on 12 mm-diameter coverslips were treated with rMS. After 6 h incubation, cellROX reagent was added to the cell culture medium. After an additional 30 min, cells were washed with PBS, then fixed in 4 % paraformaldehyde (PFA) in PBS. Nuclei were counterstained with 4’, 6-diamidino-2- phenylindole (DAPI), and macrophages were mounted on glass slides using Fluoromount- G aqueous mounting medium. Positive control for ROS production consisted in the treatment of macrophages with H2O2. Confocal fluorescent images were taken using Leica SP8 confocal microscope (Leica Microsystems) and quantitative analysis of fluorescent signals were done on 7 fields per treatment using Image J v1.53k (National Institutes of Health).

[0126] Cell viability assay

[0127] Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. Briefly, 5 x 104cells / well THP-1 -derived macrophages seeded in a 96-well plate were treated for 24 h before measuring the cellular metabolic activity following the manufacturer’s protocol. Absorbance values were obtained at 550 nm and 600 nm using the FLUOstar Omega microplate reader (BMG Labtech).

[0128] Immunofluorescence microscopy

[0129] THP-1 -derived macrophages were obtained by seeding 5 x 104THP-1 cells on glass coverslips in 24-well plates and differentiated with PMA for 48 h. After rMS treatment and / or S. aureus infection, macrophages were fixed in 4 % PFA in PBS, permeabilized with 0.1 % Triton X-100 for 5 min, blocked with 1 % BSA in PBS for 1 h, and then incubated overnight at 4 °C with the primary antibodies targeting p62 or phospho-p38 diluted in 1 % BSA in PBS. After washes with PBS, cells were incubated with secondary antibodies diluted in 1 % BSA for 1 h at room temperature. Nuclei were counterstained with Hoechst 33342. Coverslips were mounted on slides using Fluoromount-G. Confocal images of p62 / SQSTM1 punctate structures were acquired using Leica SP8 confocal microscope, 40x magnification. Quantitative analysis of fluorescent signals were done on 10 fields per treatment using Image J v1 ,53k (National Institutes of Health, Bethesda, MD, USA).

[0130] Statistical analyses Values are presented as means ± standard errors of mean (SEM). Imaging flow cytometry results presented are means ± SEM of 3 independent experiments of 100,000 events. Statistical comparisons were performed using a two-tailed unpaired t-test for comparisons between 2 groups, or one-way analysis of variance with Tukey’s multiple group comparison test for multiple comparisons. Data analyses were performed with Graph Pad Prism statistical software (v8.0.2). Differences were considered to be statistically significant at p < 0.05.

[0131] Results

[0132] High-frequency rMS activates Nrf2 in TH P-1 -derived macrophages

[0133] In order to determine the effect of repetitive magnetic stimulation on macrophages, THP-1 -derived macrophages were treated with rMS. After the indicated incubation time, RT-qPCR analysis showed a significant increase in Nrf2 transcriptional expression at 4 h and 6 h after rMS treatment (Figure 3A). At 24 h after rMS treatment, Nrf2 protein expression was measured by Western blot, and was found to be significantly increased compared to control (Figure 3B). Confocal images were taken of THP-1 -derived macrophages treated with rMS and immunolabeled with a specific antibody against Nrf2, with the nucleus counterstained with DAPI. An increasing number of Nrf2 localized within the nucleus was observed compared to control (Figure 3C). To quantify the amount of nuclear Nrf2, nuclear extracts were examined by Western blot. A significant increase in nuclear Nrf2 was observed as early as 30 min after rMS treatment (Figure 3D). Moreover, the Applicant found that the expression of Keapl protein was significantly decreased in THP-1 -derived macrophages 3 h after rMS treatment (Figure 3E). These results suggest that rMS treatment is able to modulate the activation of Nrf2 and Keapl by affecting their expression and / or localization in macrophages. rMS activates Nrf2 signaling pathway

[0134] To determine whether the nuclear translocation of Nrf2 leads to the activation of the Nrf2 signaling pathway, the Applicant examined the transcriptional and protein expressions of HO-1 and NQO1 , two downstream targets of Nrf2. RT-qPCR analyses were performed on total mRNA extracted from THP-1 -derived macrophages at 2, 4 and 6 h after rMS treatment. RT-qPCR showed a significant increase in HO-1 mRNA expression at 6 h (Figure 4A). Immunoblotting analysis conducted on protein lysates extracted from THP-1 -derived macrophages 24 h after rMS treatment revealed a significant increase in HO-1 protein expression (Figure 4A). Additionally, a significant increase in NQO1 mRNA expression was observed at 4 h and 6 h after rMS treatment (Figure 4B). Furthermore, NQO1 protein expression exhibited a significant increase at 24 h after rMS treatment. These results validated the activation of the Nrf2 signaling pathway by rMS. rMS increased phosphorylated p62 and total p62 protein expressions

[0135] Next, the Applicant hypothesized that the observed decrease in Keapl expression and increase in Nrf2 expression levels resulted from rMS modulation of p62 phosphorylation and p62 expression levels. Total protein extracts were collected from THP-1 -derived macrophages 1 h after RMS treatment for phosphorylated p62 immunoblotting analysis and 3 h after rMS treatment for p62 immunoblotting analysis. Data analysis showed a significant increase in p62 protein expression, as well as a significant increase in phosphorylated p62 (Figure 5A-B). Furthermore, confocal images were taken from THP-1 -derived macrophages treated with rMS for 3 h. The macrophages were then immunolabeled with a specific p62 antibody, and the nucleus was counterstained with Hoechst33342 (Figure 5C). Using Image J software, the number of p62 puncta and the fluorescence intensity of p62 in the puncta were quantified. Our data showed a decrease in the number of p62 puncta per cell in rMS-treated macrophages, along with a decrease in p62 fluorescent intensity within the puncta of each cell (Figure 5D-E). These results suggest that rMS induces the phosphorylation of p62, which enables the binding of phosphorylated p62 with Keapl , and consequently allows the released Nrf2 to translocate into the nucleus. The phosphorylated p62-Keap1 complex subsequently undergoes p62-dependent autophagy degradation 20.

[0136] Effects of rMS on cell viability, oxidative stress, inflammation, and Macrophage Bactericidal Function

[0137] To further investigate the potential impact of rMS treatment on cellular processes, THP-1 -derived macrophages were subjected to rMS and incubated for the indicated time before analysis. Firstly, the Applicant assessed the effect of rMS on cell viability using the MTT assay and cell apoptosis by staining with Annexin V. Flow cytometry analysis indicated that rMS treatment of THP-1 -derived macrophages had no significant impact on cell viability or cell apoptosis (Figure 6A-B). Next, the Applicant performed RT-qPCR on mRNA extracted from THP-1 -derived macrophages 6 h after rMS treatment to evaluate the transcriptional expression of genes coding for the inflammatory markers IL-1 p, IL-6, and TNF-a. No significant changes were observed in their mRNA expressions (Figure 6C). Next, the Applicant investigated whether rMS affected oxidative stress by staining rMS-treated THP-1 -derived macrophages with CellROX fluorogenic probe, which detected ROS in live cells. Data analysis showed no significant effect of rMS on oxidative stress (Figure 6D). the Applicant then examined the impact of rMS on macrophage phagocytic activity. THP-1 -derived macrophages were treated with rMS and incubated for 3 h prior to infection with SYTO9-labeled S. aureus for 1 h. Flow cytometry analysis revealed no significant difference in macrophage phagocytosis activity after rMS as compared to control (Figure 6E). Furthermore, the Applicant assessed the bactericidal activity of rMS- treated macrophages by determining the intracellular survival of S. aureus 24 h after infection. Gentamicin was added to the cell culture medium 1 h after infection to eliminate extracellular bacteria. CFU analysis demonstrated a significant decrease in intracellular survival of S. aureus in rMS-treated THP-1 -derived macrophages (Figure 6F). Collectively, these findings indicate that a 5-min magnetic stimulation does not affect the cell viability, cell apoptosis, inflammation, or oxidative stress of THP-1 -derived macrophages. Interestingly, rMS appears to impact the intracellular survival of S. aureus. rMS-Mediated Decrease in S. aureus intracellular survival in an Nrf2-dependent manner

[0138] In order to investigate whether the bactericidal activity of rMS-treated THP-1 -derived macrophages resulted from Nrf2 activity, macrophages were transfected with either scrambled siRNA or siRNA specifically targeting Nrf2. Firstly, Western blot confirmed the reduced expression level of Nrf2 and HO-1 proteins in Nrf2-targeted siRNA-transfected macrophages compared to those transfected with scrambled siRNA (figure 7A). Next, the Applicant examined the intracellular survival of S. aureus in scrambled or Nrf2 siRNA- transfected macrophages. As expected, CFU assay demonstrated a significant decrease in intracellular survival of S. aureus in scrambled siRNA-transfected THP-1 -derived macrophages treated with rMS as compared to untreated cells. Interestingly, THP-1 - derived macrophages transfected with Nrf2-targeted siRNA showed no rMS-mediated decrease in intracellular survival of S. aureus as compared to untreated cells (Figure 7B).

[0139] Additionally, the Applicant evaluated the intracellular survival of S. aureus in primary alveolar macrophages isolated from Nrf2 knockout (KO) mice and their wild-type (WT) littermates (Figure 7C). Alveolar macrophages were isolated from bronchoaleolar lavages, cultured for 24 h, and then subjected to rMS treatment and S. aureus infection. Gentamicin was added to the cell culture medium 1 h after infection to inhibit growth of extracellular bacteria. After 24 h, CFU numeration was determined. Quantitative analysis revealed that the rMS-mediated decrease in intracellular survival of S. aureus observed in alveolar macrophages from WT mice did not persist in rMS-treated alveolar macrophages isolated from Nrf2 KO mice. These results suggest the involvement of Nrf2 in the effect of rMS on the bactericidal activity of macrophages. rMS Decreases IL-13 and TNF-a mRNAs Expression Levels and p62 Protein Expression Level in S. aureus-lnfected THP-1 -Derived Macrophages

[0140] Upon infection, S. aureus induces the expression of genes encoding pro- inflammatory cytokines, including IL-6, IL-1 p, and TNF-a. In THP-1 -derived macrophages treated with rMS for 3 h prior to S. aureus infection, RT-qPCR analysis revealed a significant reduction in the transcriptional expression of IL-i and TNF-a (Figure 8A). However, no significant change was observed for IL-6 mRNA expression.

[0141] Furthermore, THP-1 -derived macrophages pretreated with rMS for 3 h and subsequently infected with S. aureus for an additional 3 h exhibited a decrease in p62 protein expression (Figure 8B). Analysis of confocal images using Image J v1.53k demonstrated a similar decrease in p62 fluorescent intensity in the puncta of cells (Figure 8C). These results suggest the potential involvement of autophagy in the intracellular survival of S. aureus. rMS Decreases S. aureus-Mediated phosphorylation of p38 MAPK and Its Nuclear Localization

[0142] Since previous studies have shown that S. aureus is able to modulate the JNK MAPK signaling pathway in RAW264.7 macrophages 21 , the Applicant’s aim was to investigate the involvement of the MAPK signaling pathway in THP-1 -derived macrophages treated with rMS and subsequently infected with S. aureus. Protein lysates extracted from THP-1 -derived macrophages stimulated with rMS for 3 h and then infected with S. aureus for an additional 1 h were analyzed by Western blot. The results showed that rMS treatment alone had no significant effect on the activation of ip38, JNK, and ERK MAPK signaling (Figure 9). In contrast, infection of THP-1 -derived macrophages with S. aureus strongly induced phosphorylation of p38 and JNK, while ERK phosphorylation remained unchanged. Interestingly, in S. aureus-infected cells, rMS treatment specifically inhibited the phosphorylation of p38 compared to control cells.

[0143] To further validate the findings from the Western blot analysis, the Applicant acquired confocal images of THP-1 -derived macrophages labeled with a specific phospho-p38 MAPK antibody, counterstained the nuclei with Hoechst 33342, and conducted quantitative analysis using Image J software (Figure 10A). rMS-treated THP-1 - derived macrophages exhibited a significantly decrease in the fluorescence intensity of phospho-p38 MAPK compared to control cells or S. aureus-infected cells (Figure 10B). Additionally, analysis of confocal images revealed a reduction in the nuclear localization of phospho-p38 MAPK in rMS-treated macrophages compared to control macrophages or S. aureus- infected macrophages (Figure 10C).

[0144] Discussion

[0145] In this study, the Applicant demonstrated that the use of a single 5-min session of high-frequency repetitive magnetic stimulation is able to decrease the intracellular load of S. aureus in THP-1 -derived macrophages and in primary alveolar macrophages. The Applicant determined that rMS treatment activated the Nrf2 / Keap1 signaling pathway through a non-canonical mechanism. rMS increased p62 protein expression and p62 phosphorylation, which increased its affinity binding to Keapl , leading to the release of Nrf2 and its translocation to the nucleus. The Applicant demonstrated that Nrf2 was necessary to the bactericidal activity of macrophages since Nrf2 knock-down, using siRNA in THP-1 -derived macrophages or alveolar macrophages isolated from Nrf2 knockout mice, led to an abolition of the rMS-mediated bactericidal activity. Furthermore, the Applicant showed that S. aureus- induced activation of p38 MAPK was repressed by rMS treatment. rMS-mediated repression of p38 MAPK may explain the decrease in S. aureus- induced expression of genes coding for the pro-inflammatory cytokines IL-1 p and TNF-a. This study is the first report to link macrophage biological activity and the molecular mechanisms elicited by rMS treatment. rMS has become an increasingly popular non-invasive technique for modulating neural excitability and plasticity in clinical and preclinical models, as well as in cultured neurons. However, the effect of rMS on non-excitable cells should not be overlooked. Recent reports have described the significant role of rMS on various cell types, including microglia, mesenchymal stromal cells, chondrocytes, and endothelial cells despite the discrepancies in the stimulatory parameters, which include variations in intensity, frequency, and duration.

[0146] One recent report conducted on lipopolysaccharide-stimulated microglia isolated from newborn Wistar rat brains and treated twice with 10 Hz rTMS, showed no effect of rTMS on IL-i and TNF-a secretions while increasing the production of IL-4 and IL-10. The authors stated that rTMS treatment could promote the anti-inflammatory polarization of microglia. Similarly, another recent study demonstrated the role played by microglia in inducing synaptic plasticity through the release of cytokines upon rTMS treatment in a mouse model of organotypic brain tissue. Furthermore, in vivo depletion of microglia abolished rTMS-induced synaptic plasticity. Here, the Applicant showed that a 5-min rMS treatment of THP-1 -derived macrophages did not significantly alter transcriptional expression levels of IL-6, IL-1 p, and TNF-a. However, when THP-1 -derived macrophages were pretreated with rMS prior to S. aureus infection, a decrease in the transcriptional expression levels of genes coding for IL-1 and TNF-p was observed. While the effects of rMS may vary depending on the specific cell type and variations in stimulatory parameters such as intensity, duration and frequency of treatment, it is reasonable to postulate that rMS may have a significant effect on the inflammatory processes of innate immune cells. Therefore, considering the diverse and still largely unexplored impact of rMS on cell-cell and cell-microenvironment interactions, the Applicant has decided to investigate the cellular and molecular responses induced by rMS in an in vitro model of S. aureus- infected macrophages.

[0147] The Applicant has previously demonstrated that the anti-inflammatory effect of Nrf2 signaling pathway in the in vitro model of S. aureus- infected THP-1 -derived macrophages was obtained through repression of p38 and JNK MAPK phosphorylation. S. aureus- mediated activation of p38 and JNK MAPK signaling pathways was inhibited by the pretreatment of THP-1 -derived macrophages with sulforaphane, a well-established Nrf2 activator. Repression of p38 and JNK MAPK phosphorylation led to a significant decrease in transcriptional expression levels of pro-inflammatory cytokines. In the present study, the Applicant observed an increase in the phosphorylation of p38 and JNK MAPK in THP-1 - derived macrophages infected with S. aureus. rMS treatment of macrophages specifically repressed S. aureus-induced p38 MAPK phosphorylation and decreased phosphorylated p38 nuclear localization, while showing no effect of rMS on S. aureus- mediated activation of JNK MAPK.

[0148] Interestingly, intracellular bacterial pathogens have evolved various strategies to escape degradation mechanisms. Early activation of the p38 MAPK signaling pathway is one of the strategies employed by S. aureus to survive in the host cell. Indeed, activation of p38 MAPK by S. aureus has been found to block autophagy degradation and ensure bacterial survival within the host cell. Specifically, it has been shown that p38 MAPK can activate the NF-KB, which in turn promotes the expression of p62. rMS treatment of THP-1 -derived macrophages resulted in the activation of the Nrf2 signaling pathway, accompanied by a decrease in Keapl protein level. The lack of any change in oxidative stress observed in rMS-stimulated macrophages suggests that the activation of Nrf2 by rMS does not occur through the canonical mechanism. Instead, data indicate that rMS activates Nrf2 via a non-canonical mechanism involving the upregulation of the autophagy-adaptor protein p62 and its subsequent phosphorylation. Phosphorylation of p62 enhances its binding affinity to Keapl , resulting in Nrf2 accumulation and facilitating its translocation to the nucleus. The phosphorylated p62- Keapl complex is led to autophagic degradation. Notably, Nrf2 also positively regulates the transcriptional expression of p62 due to the presence of ARE sequences in the p62 promoter, establishing a positive feedback loop.

[0149] The molecular mechanism responsible for the activation of p62 in response to rMS treatment in this in vitro cell model remains unclear. Previous studies have suggested that mammalian target of rapamycin complex 1 (mTORCI ) may be involved in the phosphorylation of the Ser349 residue, which is located within the KIR domain of p62 and implicated in Keapl affinity binding. Therefore, it would be valuable to explore whether the modulation of p62 by rMS is dependent on mTORCI activity. Furthermore, studies conducted on differentiated Neuro-2a cells have shown that high-frequency rMS treatment led to the activation of Ca2+-calmodulin-dependent protein kinase-cAMP-response element binding protein (CREB) signaling pathway, resulting in increased brain-derived neurotrophic factor (BDNF) expression and synaptic plasticity. Moreover, rMS has been reported to reduce neuronal cell death and promote cell proliferation while inhibiting apoptosis through activation of the ERK and AKT-signaling pathways. In addition, bone mesenchymal stromal cells stimulated with high-frequency rTMS demonstrated autophagy flux through the activation of the NMDAR-Ca2+-ERK-mTOR signaling pathway.

[0150] Autophagy is a fundamental biological process against S. aureus and has been shown to play a crucial role in the control of S. aureus growth. Previous studies have described a recruitment of p62 to S. aureus in vitro in fibroblasts and epithelial cells. Also, a recent report has shown the interaction of p62 with S. aureus in vivo in neutrophils of zebrafish. The disruption of p62 expression in neutrophils resulted in reduced zebrafish survival after S. aureus infection. Since both the p62 and p38 MAPK pathways are well- known players in autophagy, and here are involved in the bactericidal activity of rMS- treated macrophages, it would be interesting to investigate the potential role of autophagy in modulating macrophage function against pathogens by rMS. Overall, these findings indicate a complex interplay between Nrf2 / Keap1 / p62, S. aureus, and the p38 MAPK pathway in response to rMS treatment. Therefore, it would be interesting to investigate the potential impact of rMS on autophagy. While the effects of rMS have been widely acknowledged in clinical and preclinical models, it remains a challenge to apply rMS on systemic immune cells and elicit specific responses due to the limited area targeted by magnetic stimulation. One potential therapeutic strategy to consider would involve the promising developments in ex vivo activated macrophage-based therapies.

[0151] In summary, the Applicant proposes a molecular signaling pathway activated by rMS in THP-1 -derived macrophages based on the Applicant’s findings (Figure 11 ). rMS treatment of activates the Nrf2 signaling pathway through the Nrf2 / Keap1 / p62 pathway. The p62 / Keap1 complex undergoes autophagy degradation, while unbound Nrf2 translocates to the nucleus and promotes the transcriptional upregulation of genes involved in detoxification, antioxidant defense, and anti-inflammatory mechanisms. In addition, rMS treatment inhibits the p38 MAPK signaling pathway, which is implicated in S. aureus intracellular survival and pro-inflammatory responses.

[0152] Abbreviations cDNA: complementary deoxyribonucleic acid

[0153] CFLI: colony forming unit

[0154] DAPI: 4’, 6-diamidino-2-phenylindole

[0155] DMSO: dimethyl sulfoxide

[0156] ERK: extracellular signal-regulated kinase

[0157] HO-1 : Heme oxygenase-1

[0158] IL-1 p: interleukin-1

[0159] IL-6: interleukin-6

[0160] JNK: c-Jun N-terminal kinase

[0161] Keapl : Kelch-like ECH-associated protein 1

[0162] MAPK: Mitogen-activated protein kinase

[0163] MOI: multiplicity of infection

[0164] MTT : 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

[0165] NQO1 : NAD(P)H quinone dehydrogenase 1

[0166] Nrf2 / NFE2L2: nuclear factor erythroid 2-related factor 2 p62 / SQSTM1 : sequestosome 1

[0167] PBS: phosphate-buffered saline

[0168] PCR: polymerase chain reaction

[0169] PFA: paraformaldehyde

[0170] RNA: ribonucleic acid

[0171] RIPA: radioimmunoprecipitation assay buffer rMS: repetitive magnetic stimulation

[0172] ROS: reactive oxygen species rTMS: repetitive transcranial magnetic stimulation siRNA: small interfering RNA

[0173] TNF-cc tumor necrosis factor-a

Claims

CLAIMS1 A method for modulating the macrophage function of at least one macrophage in a sample of a subject, said method comprising applying a sequence of repetitive magnetic stimulations on an area of the sample, the area comprising at least one macrophage, the magnetic stimulations having a frequency comprised between 2 Hz and 20 Hz.2.- The method for modulating according to claim 1 , wherein the frequency is comprised between 4 Hz and 6 Hz.3.- The method for modulating according to claim 1 , wherein the frequency is comprised between 14 Hz and 16 Hz.4.- The method for modulating according to any one of claims 1 to 3, wherein the sequence has a total duration between 3 minutes and 7 minutes.5.- The method for modulating according to any one of claims 1 to 4, wherein the sequence has a total duration equal to 5 minutes 25 seconds.6.- The method for modulating according to any one of claims 1 to 5, wherein the sequence is applied only once.7.- The method for modulating according to any one of claims 1 to 6, which is an ex vivo method.8.- The method for modulating according to any one of claims 1 to 7, wherein the modulation of the macrophage function comprises reducing intracellular survival of bacteria in macrophages, preferably reducing intracellular survival of S. aureus in macrophages.9.- The method for modulating according to any one of claims 1 to 8, wherein the modulation of the macrophage function comprises activating the nuclear factor erythroid 2 - related factor (Nrf2) signalling pathway and / or decreasing Kelch like ECH associated protein 1 (Keapl ) expression and / or increasing p62 protein expression or phosphorylation.10.- The method for modulating according to any one of claims 1 to 9, wherein the subject is a mammal, preferably a human being.11 .- The method for modulating according to any one of claims 1 to 10, wherein the sample of the subject is a blood sample.12.- A device for modulating (12) the macrophage function of at least one macrophage (16) in a sample (10) of a subject, the device for modulating (12) comprising an application unit (18), the application unit (18) being adapted to apply a sequence of repetitive magnetic stimulations on an an area (14) of the sample (10), the area (14) comprising at least one macrophage (16), the magnetic stimulations having a frequency comprised between 2 Hz and 20 Hz.13.- An isolated macrophage previously submitted to a sequence of repetitive magnetic stimulations having a frequency comprised between 2 Hz and 20 Hz, for use for treating and / or preventing a bacterial infection, preferably a S. aureus infection.14.- The isolated macrophage for use as in claim 13, wherein said macrophage is present in a sample of a subject and the bacterial infection occurs in the same subject.

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