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WO2025021830A1 - Procédé pour moduler la fonction des macrophages et produits associés - Google Patents

Procédé pour moduler la fonction des macrophages et produits associés Download PDF

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Publication number
WO2025021830A1
WO2025021830A1 PCT/EP2024/070932 EP2024070932W WO2025021830A1 WO 2025021830 A1 WO2025021830 A1 WO 2025021830A1 EP 2024070932 W EP2024070932 W EP 2024070932W WO 2025021830 A1 WO2025021830 A1 WO 2025021830A1
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macrophage
rms
aureus
macrophages
nrf2
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Thérèse DERAMAUDT
Marcel BONAY
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Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Versailles Saint Quentin en Yvelines
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Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Versailles Saint Quentin en Yvelines
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/10—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N2/00—Magnetotherapy
    • A61N2/02—Magnetotherapy using magnetic fields produced by coils, including single turn loops or electromagnets
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2529/00—Culture process characterised by the use of electromagnetic stimulation

Definitions

  • 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.
  • 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.
  • Staphylococcus aureus Staphylococcus aureus
  • 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.
  • 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.
  • 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.
  • 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.
  • the sequence has a total duration between 3 minutes and 7 minutes.
  • the sequence has a total duration equal to 5 minutes 25 seconds.
  • the sequence is applied only once.
  • the method is an ex vivo method.
  • the modulation of the macrophage function comprises reducing intracellular survival of bacteria in macrophages, preferably reducing intracellular survival of S. aureus in macrophages.
  • 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.
  • Nrf2 nuclear factor erythroid 2 - related factor
  • Keapl Kelch like ECH associated protein 1
  • the subject is a mammal, preferably a human being.
  • the sample of the subject is a blood sample.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 1 shows a sample and a device for modulating
  • FIG. 2 represents an example of temporal variation of the applied magnetic field with two sequences of repetitive magnetic stimulations
  • FIG. 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.
  • FIG. 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.
  • 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.
  • 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.
  • 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 .
  • FIG. 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.
  • FIG. 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.
  • FIG. 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.
  • 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.
  • FIG 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.
  • treatment with rMS results in an increase in phosphorylated p62 at Ser349, which results in increase interaction with Keapl and the release of Nrf2.
  • 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.
  • ARE antioxidant response element
  • Nrf2 repetitive magnetic stimulation
  • a sample 10 and a device for modulating 12 are schematically represented on figure 1.
  • sample it is meant a sample of a subject.
  • the sample 10 may be any biological sample that comprises at least one macrophage of a subject.
  • the sample 10 is a blood sample.
  • the sample 10 comprises an area 14 comprising at least one macrophage 16.
  • This area 14 is represented on figure 1 by dotted lines.
  • 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.
  • phagocytosis 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.
  • a macrophage is thus adapted to carry out one or several actions on its environment.
  • the device for modulating 12 is a device adapted to generate a modulation of the macrophage function of the macrophage 16.
  • the device for modulating 12 is adapted to modify the macrophage function of the macrophage 16.
  • the device for modulating 12 is adapted to modify an action comprised in the macrophage function.
  • the macrophage function comprises an action of reducing intracellular survival of bacteria in macrophages.
  • the reduction of intracellular survival of bacteria in macrophages is significant.
  • “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.
  • such reducing action comprises reducing intracellular survival of S. aureus in macrophages.
  • the reduction of intracellular survival of S. aureus in macrophages is significant.
  • “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.
  • the macrophage function comprises an action of activating the nuclear factor erythroid 2 - related factor (Nrf2) signalling pathway.
  • 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.
  • the downstream target of Nrf2 is chosen from heme oxygenase-1 (HO-1), p62 and NAD(P)H:quinone oxidoreductase 1 (NQO1).
  • the macrophage function comprises an action of decreasing Kelch like ECH associated protein 1 (Keapl ) expression.
  • Keapl is the negative regulator of Nrf2.
  • Keapl expression results in activating Nrf2 signalling pathway.
  • the macrophage function comprises an action of increasing p62 protein expression or phosphorylation.
  • the macrophage function can thus here be any combination of the previously described actions.
  • the device for modulating 12 comprises an application unit 18.
  • the application unit 18 is adapted to apply a sequence of repetitive magnetic stimulations on the area.
  • the application unit 18 comprises a controller 20 and a magnetic generator 22.
  • the controller 20 is adapted to control the magnetic generator to have this generator generate a sequence of repetitive magnetic stimulations on the area.
  • the controller 20 is an electrical generator and the magnetic generator 22 is one or several coils.
  • Figure 2 represents the evolution of magnetic field applied by the application unit 18 with time.
  • the present evolution comprises two sequences respectively labelled S1 and S2.
  • a sequence is characterized by a frequency of the magnetic stimulations and a total duration.
  • frequency is the number of pulses per second.
  • the frequency is 10 Hz, corresponding to 10 pulses in one second.
  • 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.
  • the time duration for one session was 5 min 25 s.
  • the first sequence is thus characterized by a first frequency and a first total duration.
  • the first frequency is comprised between 4 Hz and 6 Hz.
  • the first total duration is, for instance, equal to 5 minutes 25 seconds.
  • the second frequency is comprised between 14 Hz and 16 Hz.
  • the second total duration is, for instance, equal to 5 minutes 25 seconds.
  • the frequency of the magnetic stimulations associated to a sequence is comprised between 2 Hz and 20 Hz.
  • the total duration equal to 5 minutes 25 seconds.
  • 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.
  • 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.
  • Such method is an ex vivo method.
  • the modulation of the macrophage function may comprise reducing intracellular survival of bacteria in macrophages, preferably reducing intracellular survival of S. aureus in macrophages.
  • 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.
  • the sequence enables to obtain at least one specific isolated macrophage.
  • 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.
  • the bacterial infection is a S. aureus infection.
  • 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.
  • said macrophage is present in a sample of a subject and the bacterial infection occurs in the same subject.
  • 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.
  • 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.
  • the infection is preferably a S. aureus infection in a subject.
  • 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.
  • 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.
  • said macrophage is present in a sample of a subject and the bacterial infection occurs in the same subject.
  • said macrophage is autologous.
  • 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.
  • 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.
  • a nuclear extraction kit phorbol 12-myristate 13-acetate (PMA), dimethyl sulfoxide (DMSO) >99.7%, - mercaptoethanol, paraformaldehyde, H2O2 30% in H 2 O, 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).
  • 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)
  • 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.
  • 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.
  • S. aureus was incubated with SYTO9 dye for 15 min in the dark following the manufacturer’s instructions.
  • 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.
  • Human monocytic THP-1 cell line (ATCC® TIB-202TM) 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.
  • S. aureus was added to the cell culture at a multiplicity of infection of 10.
  • 10 pg/ml gentamicin was added to the cell culture medium to inhibit growth of extracellular bacteria.
  • 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.
  • 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.
  • THP-1 -derived macrophages seeded in 24-well plates at 2.5 x 10 5 cells/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.
  • a nuclear extraction kit was used according to the manufacturer’s protocol. Briefly, following treatment of THP-1 -derived macrophages, 3 x 10 6 cells 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.
  • THP-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.
  • 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.
  • RT-qPCR realtime quantitative PCR
  • 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.
  • Intracellular ROS was measured in live cells using the CellROXTM green fluorogenic probe. Briefly, 2.5 x 10 5 THP-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.
  • PFA paraformaldehyde
  • THP-1 -derived macrophages were obtained by seeding 5 x 10 4 THP-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).
  • 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).
  • 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
  • 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).
  • THP-1 -derived macrophages were subjected to rMS and incubated for the indicated time before analysis.
  • 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).
  • 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).
  • 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).
  • 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).
  • 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.
  • S. aureus Upon infection, S. aureus induces the expression of genes encoding pro- inflammatory cytokines, including IL-6, IL-1 p, and TNF-a.
  • pro-inflammatory cytokines including IL-6, IL-1 p, and TNF-a.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Nrf2 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.
  • mTORCI mammalian target of rapamycin complex 1
  • 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.
  • bone mesenchymal stromal cells stimulated with high-frequency rTMS demonstrated autophagy flux through the activation of the NMDAR-Ca 2+ -ERK-mTOR signaling pathway.
  • 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.
  • rMS treatment inhibits the p38 MAPK signaling pathway, which is implicated in S. aureus intracellular survival and pro-inflammatory responses.
  • ERK extracellular signal-regulated kinase
  • IL-1 p interleukin-1
  • IL-6 interleukin-6
  • JNK c-Jun N-terminal kinase
  • Keapl Kelch-like ECH-associated protein 1
  • MAPK Mitogen-activated protein kinase
  • NQO1 NAD(P)H quinone dehydrogenase 1
  • Nrf2/NFE2L2 nuclear factor erythroid 2-related factor 2
  • p62/SQSTM1 sequestosome 1
  • PBS phosphate-buffered saline
  • RNA ribonucleic acid
  • RIPA radioimmunoprecipitation assay buffer
  • rMS repetitive magnetic stimulation
  • ROS reactive oxygen species
  • rTMS repetitive transcranial magnetic stimulation
  • siRNA small interfering RNA
  • TNF-cc tumor necrosis factor-a TNF-cc tumor necrosis factor-a

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Abstract

L'invention concerne la modulation de la fonction des macrophages. Les inventeurs ont découvert que cette fonction peut être modifiée par l'application d'une séquence de stimulations magnétiques répétitives présentant une fréquence comprise entre 2 Hz et 20 Hz. Cela ouvre la voie à une maîtrise non invasive du macrophage et, par là même, de toute utilisation qui implique le macrophage.
PCT/EP2024/070932 2023-07-25 2024-07-23 Procédé pour moduler la fonction des macrophages et produits associés Pending WO2025021830A1 (fr)

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Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2011094598A2 (fr) * 2010-01-28 2011-08-04 The Johns Hopkins University Compositions et procédés d'inversion de la résistance aux corticostéroïdes ou de traitement d'infections respiratoires

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011094598A2 (fr) * 2010-01-28 2011-08-04 The Johns Hopkins University Compositions et procédés d'inversion de la résistance aux corticostéroïdes ou de traitement d'infections respiratoires

Non-Patent Citations (4)

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Title
JANA FRAHM ET AL: "Alteration in cellular functions in mouse macrophages after exposure to 50 Hz magnetic fields", JOURNAL OF CELLULAR BIOCHEMISTRY, vol. 99, no. 1, 1 September 2006 (2006-09-01), pages 168 - 177, XP055023046, ISSN: 0730-2312, DOI: 10.1002/jcb.20920 *
LUO JING ET AL: "Repetitive Transcranial Magnetic Stimulation Improves Neurological Function and Promotes the Anti-inflammatory Polarization of Microglia in Ischemic Rats", FRONTIERS IN CELLULAR NEUROSCIENCE, 12 April 2022 (2022-04-12), XP093117724, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9039226/pdf/fncel-16-878345.pdf> [retrieved on 20240110], DOI: 10.3389/fncel.2022.878345 *
ROSS CHRISTINA L, HARRISON BENJAMIN S: "Effect of pulsed electromagnetic field on inflammatory pathway markers in RAW 264.7 murine macrophages", JOURNAL OF INFLAMMATION RESEARCH, vol. 6, 12 March 2013 (2013-03-12), GB, pages 45 - 51, XP093116989, [retrieved on 20240109], DOI: 10.2147/JIR.S40269 *
VINHAS ADRIANA ET AL: "Magnetic Stimulation Drives Macrophage Polarization in Cell to-Cell Communication with IL-1[beta] Primed Tendon Cells", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 30 July 2020 (2020-07-30), XP093115118, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432806/pdf/ijms-21-05441.pdf> [retrieved on 20231222], DOI: 10.3390/ijms21155441 *

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