Use of rev-erb antagonists for the treatment of lung infections

Abstract

Circadian rhythms control the diurnal nature of many physiological, metabolic and immune processes. The inventors hypothesized that age-related impairments in circadian rhythms are associated with high susceptibility to bacterial respiratory tract infections. The diurnal control of Streptococcus pneumoniae infection is impaired in elderly mice. A lung circadian transcriptome analysis revealed that aging alters the daily oscillations in the expression of a specific set of genes and that some pathways that are rhythmic in young-adult mice are nonrhythmic or time-shifted in elderly mice. In particular, the circadian expression of the clock components Rev-erb-α, and Rev-erb-β to a lesser extent, altogether with apelin / apelin receptor were altered in elderly mice compared to young mice. In young mice, the inventors discovered that novel interaction between Rev-erb and the apelinergic axis controls host defenses against S. pneumoniae via alveolar macrophages. Pharmacological repression of Rev-erb-α and / or Rev- erb-β in elderly mice resulted in greater resistance to pneumococcal infection. Thus, the present invention relates to the use of Rev-erb antagonists for the treatment of lung infections.

Description

[0001] USE OF REV-ERB ANTAGONISTS FOR THE TREATMENT OF LUNG INFECTIONS

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine, in particular infectiology.

[0004] BACKGROUND OF THE INVENTION:

[0005] Immune function dysregulation has a major impact on age-related diseases, with an elevation in the risk of cancer, cardiovascular, metabolic and neurodegenerative pathologies. Furthermore, age-mediated alterations in immune function increase susceptibility to infections in general and lung infections in particular. Indeed, older adults are more vulnerable than the general population to respiratory pathogens such as Streptococcus pneumoniae - the most common cause of bacterial pneumonia1. As life expectancy increases and the number of older adults grows, a better understanding of the causes and mechanisms involved in this age-related elevation in susceptibility to bacterial pathogens is critical for the development of new treatments. The underlying mechanisms are mainly related to impaired pulmonary functions (poor mucociliary clearance and a weak barrier) and impairments in innate and adaptive immunity in the aging lung2’3.

[0006] Circadian rhythms are daily oscillations in several biological processes. These oscillations allow organisms to anticipate and adapt to environmental changes occurring during a 24-hour period4,5. Circadian rhythms are generated by an endogenous biological clock that has been well conserved over the course of evolution. The central pacemaker that receives light information and synchronizes clocks throughout the body is located in the suprachiasmatic nuclei of the hypothalamus. In addition, all mammalian cells (including immune cells) harbor a functional circadian clock, which generates circadian oscillations in the transcriptome, proteome, and thus cell / tissue functions6. On the molecular level, the circadian clock consists of a complex network of transcription factors organized in interconnected positive and negative feedback loops and that generate rhythms with a 24-hour period7. The positive feedback limb is driven by the heterodimer complex brain and muscle ARNT-like 1 (Bmall) and circadian locomotor output cycle kaput (Clock), which binds to E-boxes in the promoter of its target genes. The latter include the Periodl / 2 / 3 (Per) and Cryptochrome 1 / 2 (Cry) clock genes, which form the negative feedback limb. Once they reach a sufficient threshold, the Per and Cry proteins heterodimerize and the complex translocates into the nucleus, quenches the Bmall / Clock heterodimer, and thus inhibits its transcriptional activity Other transcription factors (including nuclear receptor families Rev-erb and retinoic acid receptor-related orphan receptors (ROR) are strong modulators of the molecular clock and can fine-tune the circadian circuitry8. These nuclear receptor families control the expression of many genes and thereby generate rhythmic oscillations in transcriptional programs and specific tissue functions, including those in the lungs9,10. It is estimated that the expression of approximately 80% of genes oscillates in a daily manner at one body site at leastn. Rev-erb-a is not only important in generating circadian rhythmicity by inhibiting the expression of Bmal 1 and Clock but is also critical in metabolic and inflammation / repair processes12'18. It has been shown that Rev-erb-a and Rev-erb-P in mice are critical in acute and chronic inflammatory diseases, including fulminant hepatitis19and atherosclerosis20.

[0007] The biological clock controls host defenses against pathogens12,21'23, including 5. pneumoniae9,24’25. It regulates many immune functions over the circadian cycle, including immune cell infiltration, pattern recognition receptor expression, cytokine secretion, and phagocytosis6,26'30. A robust circadian system is therefore essential for good health and longevity31. However, circadian rhythms are impaired by several conditions, including shift work, erratic light exposure, changes in feeding behavior, consumption of a high-fat diet, social jetlag, and aging32,33. Clock disruption has been implicated in the pathogenesis of chronic metabolic and inflammatory diseases34,35. In older adults, disruption of normal circadian rhythms is associated with clinically relevant disorders, including neurodegeneration and metabolic / inflammatory diseases14,35,36. Recent research has also demonstrated that age-related changes in circadian rhythmicity have major consequences on innate immunity37. Macrophages are the first line of defense against bacterial infections, and their function is controlled strongly by the circadian clock29,37'40. Interestingly, it has been reported that circadian transcription and rhythmicity of phagocytosis are impaired in aged peritoneal macrophages37.

[0008] SUMMARY OF THE INVENTION:

[0009] The present invention is defined by the claims. In particular, the present invention relates to the use of Rev-erb antagonists for the treatment of lung infections.

[0010] DETAILED DESCRIPTION OF THE INVENTION: Circadian rhythms control the diurnal nature of many physiological, metabolic and immune processes. The inventors hypothesized that age-related impairments in circadian rhythms are associated with high susceptibility to bacterial respiratory tract infections. The diurnal control of Streptococcus pneumoniae infection is impaired in elderly mice. A lung circadian transcriptome analysis revealed that aging alters the daily oscillations in the expression of a specific set of genes and that some pathways that are rhythmic in young-adult mice are nonrhythmic or time-shifted in elderly mice. In particular, the circadian expression of the clock components Rev-erb-a, and Rev-erb- to a lesser extent, altogether with apelin / apelin receptor were altered in elderly mice compared to young mice. In young mice, the inventors discovered that novel interaction between Rev-erb and the apelinergic axis controls host defenses against 5. pneumoniae via alveolar macrophages. Pharmacological repression of Rev-erbs in elderly mice resulted in greater resistance to pneumococcal infection. These data demonstrate the causative role of age-associated impairments in circadian rhythms on respiratory infections and have clinical relevance.

[0011] Accordingly, the first object of the present invention relates to a method of treating a lung infection in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a Rev-erb antagonist.

[0012] In some embodiments, the patient according to the invention is a human. In some embodiments, the patient according to the invention is a girl or a boy. In some embodiments, the patient according to the invention is an adult In some embodiments, the patient according to the invention is a child (human being between the stages of birth and puberty), a teenager (human being between the stages of puberty to adulthood), an adult (human being between the end of puberty / beginning of adulthood to sixty-five years of age) or an elderly person (over sixty-five years of age). In some embodiments, the patient is an elderly patient.

[0013] As used herein, the term “lung infection” or “respiratory infection” has its general meaning in the art and means the invasion of lung tissues of a patient by disease-causing microorganisms, their multiplication and the reaction of lung tissues to these microorganisms and the toxins that they produce.

[0014] In some embodiments, the patient suffers from a chronic lung infection

[0015] In some embodiments, the patient suffers from an acute lung infection As used herein, the term “chronic infection” refers to a long-term infection which may be an apparent, unapparent or latent infection.

[0016] As used herein, the term “acute lung infection” has its general meaning in the art and refers to a disease of the lungs characterized by inflammation and consolidation followed by resolution and caused by infection from viruses, fungi, or bacteria. The term is also known as “pneumonia”. Typically, acute lung infection is associated with lung inflammation that is the rapid onset of progressive malfunction of the lungs, and is usually associated with the malfunction of other organs due to the inability to take up oxygen.

[0017] In some embodiments, the lung infection is a bacterial infection, such as bacterial pneumonia. In some embodiments, the bacterial infection is caused by a bacterium selected from the group consisting of Streptococcus pneumoniae (also referred to as pneumococcus), Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pyogenes, Haemophilus influenzae, Haemophilus parainfluenzae, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Moraxella catarrhalis, Chlamydophila pneumoniae, Mycoplasma pneumoniae, Legionella pneumophila, Serratia marcescens, Burkholderia cepacia, Burkholderia pseudomallei, Bacillus anthracis, Bacillus cereus, Bordatella pertussis, Stenotrophomonas maltophili , a bacterium from the citrobacter family, a bacterium from the ecinetobacter family, and Mycobacterium tuberculosis or Mycobacterium abscessus.

[0018] In some embodiments, the lung infection is a fungal infection. In some embodiments, the fungal infection is caused by a fungus selected from the group consisting of Histoplasma capsulatum, Cryptococcus neoformans, Pneumocystis jiroveci, Coccidioides immitis, Candida albicans, and Pneumocystis jirovecii (which causes pneumocystis pneumonia (PCP), also called pneumocystosis).

[0019] In some embodiments, the lung infection is a viral infection, such viral pneumonia. In some embodiments, the viral infection is caused by a virus selected from the group consisting of influenza virus (e.g., Influenza vims A, Influenza vims B), respiratory syncytial vims, adenovirus, metapneumovims, cytomegalovims, parainfluenza vims (e g., hPIV-1, hPIV-2, hPIV-3, hPIV-4), rhinovirus, coxsackie vims, echo vims, herpes simplex vims, coronavirus (SARS-coronavirus such as SARS-Covl or SARS-Cov2), and smallpox. In some embodiments, the viral lung infection may be due to a member of the Pneumoviridae, Paramyxoviridae and / or Coronaviridae families are in particular selected from the group consisting of upper and lower respiratory tract infections due to: human respiratory syncytial virus (hRSV), type A and type B, human metapneumovirus (hMPV) type A and type B; parainfluenza virus type 3 (PIV-3), measles virus, endemic human coronaviruses (HCoV-229E, -NL63, -OC43, and -HKU1), severe acute respiratory syndrome (SARS) and Middle-East respiratory syndrome (MERS) coronaviruses. In particular, the method of the present invention is suitable for the treatment of Severe Acute Respiratory Syndrome (SARS). More particularly, the method of the present invention is suitable for the treatment of lung inflammation in patients suffering from CO VID- 19.

[0020] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy A therapeutic regimen may include an induction regimen and a maintenance regimen The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g , administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

[0021] In some embodiments, the method of the present invention is particularly suitable for the prophylactic treatment of lung infection.

[0022] In some embodiments, the method of the present invention is particularly suitable for the prophylactic treatment of a bacterial superinfection, more particularly a bacterial superinfection secondary to a lung viral infection, even more particularly a bacterial superinfection postinfluenza.

[0023] As used herein, the term "bacterial superinfection" refers to a secondary bacterial infection that occurs in a person who is already infected with another pathogen, such as a virus Bacterial superinfections can complicate the course of the primary infection and cause more severe symptoms and complications.

[0024] As used herein, the term “bacterial superinfection post-influenza” has its general meaning in the art and refers to a bacterial infection (e.g., bacterial pneumonia) which occurs in a subject who suffers or has suffered from an influenza infection. Typically, the bacterial superinfection occurs within 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days after influenza infection.

[0025] The bacterial superinfection may be caused by numerous bacterial pathogens. For example, they may be mediated by at least one organism selected from the group consisting of: Streptococcus pneumoniae,' Staphylococcus aureus,' Haemophilus influenza, Myoplasma species and Moraxella catarrhalis.

[0026] As used herein, the term "Rev-erb-a" refers to a nuclear receptor protein that is involved in the regulation of circadian rhythm. The term is also known as NR1D1 (nuclear receptor subfamily 1 group D member 1). As used herein, the term "Rev-erb-P" refers to a nuclear receptor protein that is involved in the regulation of circadian rhythm. The term is also known as NR1D2 (nuclear receptor subfamily 1 group D member 2). Rev-erb-a and Rev-erb-P belong to the NR1D subfamily of nuclear receptors and act as a transcriptional repressors of various target genes. Heme binds to Rev-erb-a and Rev-erb-P as a ligand whose activity is modulated by its availability and redox state. Rev-erb-a and Rev-erb-P interact with other transcription factors and co-regulators to modulate gene expression in different tissues and physiological conditions. Rev-erb-a and Rev-erb-P share common target genes and have specific target genes. Their respective activity profile mostly depends on their differential expression level in respective tissues. Rev-erb-a has been implicated in various diseases, such as metabolic syndrome, diabetes, obesity, cardiovascular disease, and cancer.

[0027] As used herein, the term "Rev-erb antagonist" refers to a compound or a composition that reduces or blocks the activity or expression of Rev-erb-a and / or Rev-erb-P, either by interfering with its binding to heme, DNA, or other proteins, or by modulating its expression, stability, or localization. Rev-erb antagonists may act directly on Rev-erb-a and Rev-erb-P or indirectly through other signaling pathways or factors that affect Rev-erb-a and / or Rev-erb-P function. Examples of Rev-erb antagonists include, but are not limited to, synthetic small molecules, peptides, antibodies, antisense oligonucleotides, siRNAs, shRNAs, or gene therapy vectors that target Rev-erb-a and / or Rev-erb-P

[0028] Rev-erb antagonists are well known in the art and include those described in:

[0029] Wang S, Li F, Lin Y, Wu B. Targeting REV-ERBa for therapeutic purposes: promises and challenges. Theranostics. 2020 Mar 4; 10(9) :4168-4182. doi: 10.7150 / thno.43834. PMID: 32226546; PMCID: PMC7086371.

[0030] Kojetin DJ, Burris TP. REV-ERB and ROR nuclear receptors as drug targets. Nat Rev Drug Discov. 2014 Mar;13(3): 197-216. doi: 10.1038 / nrd4100. PMID: 24577401; PMCID: PMC4865262.

[0031] Any small molecule which exerts an inhibitory effect on Rev-erb-a and / or Rev-erb-P activity thus can be used as a Rev-erb antagonist according to the present invention.

[0032] In some embodiments, the Rev-erb antagonist is l,2,3,4-Tetrahydro-2-[[5-(methylthio)-2- thienyl]carbonyl]-3-isoquinolinecarboxylic acid ethyl ester, herein referred to as SR8278 and as described in Kojetin, Douglas, et al. "Identification ofSR8278, a synthetic antagonist of the nuclear heme receptor REV-ERB. "ACS chemical biology 6.2 (2011): 131-134.

[0033] In some embodiments, the Rev-erb antagonist is N-(4-Chloro-2-methylbenzyl)-N-(4- chlorobenzyl)-l-(5-nitrothiophen-2-yl)methanamine Hydrochloride, herein referred to as GSK2945 and as described in Zhang T, et al. REV-ERBa Regulates CYP7A1 Through Repression of Liver Receptor Homolog- 1. Drug Metab Dispos. 2018 Mar; 46(3) :248-258.

[0034] As used herein, the term "therapeutically effective amount" is meant a sufficient amount of the active ingredient for treating or reducing the symptoms at reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with the active ingredients; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0 001 mg / kg to 7 mg / kg of body weight per day.

[0035] Typically, the drugs herein disclosed are combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.

[0036] As used herein, the term “pharmaceutical composition” refers to a composition described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and / or excipients. The pharmaceutical compositions as provided herewith typically include a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

[0037] In some embodiments, the Rev-erb antagonist is encapsulated in nanoparticles, preferably biocompatible nanoparticles. Any nanoparticles which have been described in the art for in vivo delivery of active principles in human may be used Such nanoparticles include for example liposomes and micelles, nanosphere or nanoparticles, nanotubes, nanocrystals, hydrogels, carbon-based nanoparticles and the like Typically, the nanoparticle according to the invention has a mean diameter between 1 to 2000 nm diameter, for example between 10 to 500 nm or between 10 to 200 nm The nanoparticles of the invention may comprise an inorganic core, such as, but not limited to, semiconductor, metal (e.g., gold, silver, copper, titanium, nickel, platinum, palladium and alloys), metal oxide nanoparticles (e.g., CnCh, CO3O4, NiO, MnO, CoFe2C>4, and MnFeC>4). In some embodiments, the nanoparticles comprises at least a core with one or more polymers, or their copolymer, such as, e.g., one or more of dextran, carboxymethyl dextran, chitosan, trimetylchitosan, polyvinylalcohol (PVA), polyanhydrides, polyacylates, polymethacrylates, polyacylamides, cellulose, hydromellose, starch, dendrimers, polyamino acids, polyethyleneglycols, polyethyleneglycol-co-propyleneglycol, aliphatic polyesters, including poly(lactic acid (PLA), poly(glycolic acid), and their copolymers including poly(lactic-co-glycolylic)acid (PLGA), or poly(s-caprolactone). In some embodiments, the surface of the nanoparticles may also be functionalised or coated to produce a desirable physical characteristic such as solubility and biocompatibility. For example, the surface of the nanoparticles can be functionalized by incorporating one or more chemical linkers such as, without limitation: carboxyl groups, amine groups, carb oxy 1 / amine, hydroxyl groups, polymers such as silane, dextran or PEG or their derivatives. In some embodiments, nanoparticle according to the present invention has a core that comprises polymers selected from the group consisting of: polyQactic acid), poly(glycolic acid), or mixtures thereof. More particularly, the nanoparticle comprises poly(lactic)poly(glycolic) acid co-polymers (PLGA). Methods for encapsulating the Rev-erb antagonist into the nanoparticles are known in the art and typically include those described in EXAMPLE. In some embodiments, the nanoparticle comprises the encapsulated Rev-erb antagonist at amounts comprised between 0.01 and 1000 ng per mg of nanoparticle.

[0038] In the pharmaceutical compositions of the present invention, the active ingredients of the invention can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

[0039] In some embodiments, the pharmaceutical composition of the invention, in particular the pharmaceutical composition that comprises the nanoparticles of the present invention, is administered topically (i.e., in the respiratory tract of the subject). Therefore, the compositions can be formulated in the form of a spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art If the method of the invention comprises intranasal administration of a composition, the composition can be formulated in an aerosol form, spray, mist or in the form of drops. In particular, the active ingredients for use according to the present invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (composed of, e.g., gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0040] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0041] FIGURES:

[0042] Figure 1. Role of Rev-erb-a and alveolar macrophages in host defense against S. pneumoniae infection. A, Young Nrldl'1' mice and littermates were infected with S. pneumoniae at ZT12. B and C, Wild-type mice were treated by the intranasal route with the Rev-erb-a agonist SR9009 (25 mg / kg) (B) or with the Rev-erb-a antagonist SR8278 (25 mg / kg) (C) at ZT9 and ZT12 and then infected with S. pneumoniae atZT12. A-C, The number of bacteria was determined 24 hours post-infection. The solid lines correspond to the median values. A-C, A pool of two experiments is depicted (A, n = 13-15; B, n = 13-17; C, n = 15). D, Mice were intranasally treated with empty liposomes or clodronate liposomes (50 pl / mouse). 24 hours later, animals were treated with vehicle (Vh) or SR8278 (25 mg / kg) and then infected (as in panel C). E, Mice were inoculated by the intranasal route with SR8278 vectorized in NPs (3.4 mg / kg) at ZT9 and ZT12. Mice were infected with S. pneumoniae at ZT12. D and E, One representative experiment out of two is shown (n = 8). F and G, MPI cells were synchronized as described in the Materials &Methods. F, Cells were exposed to SR8278 (2 pM) for 1 hour and then to eGFP S. pneumoniae (MOI 10) for 3 hours. Analysis was performed by confocal microscopy (n = 6-11 wells per condition). G, Cells were exposed to SR8278 (2 pM) for 1 hour and then to live S. pneumoniae (MOI 10). The percentage of killed bacteria was calculated 5 hours post-infection (n =6 wells per condition). A-C and E-G. Significant differences were determined using the Mann Whitney U test. D, Significant differences were determined using the one-way ANOVA Kruskal-Wallis test, *.P < 0.05; ** P < 0.01, *** P < 0.001.

[0043] Figure 2. Effect of SR8278 on pulmonary defense elderly mice. Elderly mice were treated with SR8278 (25 mg / kg) at ZT9 and ZT12 and then infected with S. pneumoniae at ZT12. The number of bacteria was determined 24 hours post-infection. The solid lines correspond to the median values. Significant differences were determined using the Mann Whitney U test (n (n= EXAMPLE:

[0044] Methods:

[0045] Cell lines, animals, infections, and ethics

[0046] Alveolar macrophage-like Max Planck Institute (MPI) cells were a gift from Dr Gyorgy Fejer (University of Plymouth, UK)50. Our study exclusively examined male mice. It is unknown whether the findings are relevant for female mice. Male specific pathogen-free C57BL / 6J young-adult mice (2-month-old, 20-25g) and elderly mice (22-month-old, 35-45 g) were purchased from Janvier Labs (Le Genest-St-Isle, France). Nrldl'1' mice were obtained from B. Vennstrom (Karolinska Institute!, Stockholm, Sweden)19and backcrossed for 8 generations with 129S 1 / SvlmJ mice. Apf1' mice78(C57BL / 6J background) were from Deltagen (San Carlos, CA) As controls, littermates were used Mice were fed a standard rodent chow (SAFE A04, SAFE, Augy, France) with water ad libitum and housed in a 12-h light / dark cycle. Mice were anesthetized by intramuscular injection of ketamine (50mg / kg'1) and xylazine (lOmg / kg'1), and intranasally infected with 40 pl of DMEM containing (or not, for mock (control) animals) 1 x 106colony forming units (CFU) of 5. pneumoniae serotype 1 (clinical isolate E1586). Mice were infected at different ZTs. ZTO is defined as the time when the lights are turned on. For tissue collection, animals were euthanized with an intraperitoneal injection of euthasol (40 mg / kg'1). Sacrifice was performed 24 hours after infection. The lungs were homogenized in sterile phosphate-buffered saline (PBS) before being diluted and plated on Trypticase soy agar with 5% sheep blood The plates were incubated at 37°C with 5% CO2 for 24 hours and viable bacteria were counted. All experiments were performed within the biosafety level 2 facility of the Institut Pasteur de Lille. The protocols were validated by the local committee (Comite d’Ethique en Experimentation Animale 75, Nord Pas-de-Calais) for the evaluation of the biological risks and complied with current national and institutional regulations and ethical guidelines (Institut Pasteur de Lille / B59-350009). The animal study was authorized by the “Education, Research and Innovation Ministry” under registration number APAFIS#22304- 201910011647335v3.

[0047] Rev-erb agonist / antagonist treatment, apelin treatment, and alveolar macrophage depletion

[0048] The Rev-erb agonist (SR9009) and antagonist (SR8278) were purchased from Tocris Bioscience (Bristol, UK). Apelin ([Pyrl]-Apelin-13) was purchased from Sigma Aldrich (Saint Quentin-Falavier, France). Clodronate liposomes and empty liposomes were obtained from Liposoma Technology (Amsterdam, The Netherlands) Anesthetized mice were treated via the intranasal route with 20pl of SR9009 (25mg / kg-1) or SR8278 (25mg / kg-1) at ZT9 and ZT12. Mice were then intranasally infected with S. pneumoniae as described above. During the procedure, anesthesia was maintained with isoflurane. To deplete alveolar macrophages, mice were intranasally treated with 50 pL clodronate liposomes. Treatment was performed 24 hours before SR8278 inoculation. To maintain a constant blood level of apelin, ALZET® micro- osmotic pumps (Durect Corporation, Cupertino, CA) (14-day delivery at 0.11 pl / hour rate) were inserted subcutaneously. Briefly, pumps were filled with 100 pl of PBS or apelin (50 pg) and kept overnight at 37°C in PBS. The next day, mice were anesthetized and the pumps were inserted subcutaneously. Mice were treated with 200 pl buprenorphine subcutaneously (0.15 mg / kg'1) two times at 8 hours interval to manage pain. Mice were infected with S. pneumoniae seven days after the implantation of the osmotic pumps For Rev-erb antagonist and apelin cotreatment, elderly mice were implanted with the osmotic pump and seven days later mice were treated with SR8278 as described above.

[0049] SR8278 incorporation into nanoparticles

[0050] The biodegradable acid-terminated poly(lactic-co-glycolic acid) (PLGA) (50:50 lactic acid:glycolic acid, molecular weight = 5-20 kDa) copolymer and acid-terminated poly(lactic acid) (PLA) (18-24 kDa) were purchased from Seqens (Aramon, France) and Sigma-Aldrich, respectively. To encapsulate SR8278 in PLGA-based nanoparticles, 60 mg of PLGA and 10 mg of SR8278 were dissolved in 1 5 mL of dichloromethane (Sigma-Aldrich) and emulsified using 4 mL of a 0.5% w / v poly(vinyl alcohol) (88% hydrolyzed, Sigma-Aldrich) aqueous solution by vortexing for 20 seconds The emulsion was further sonicated for 90 seconds (20% power) and 30 seconds (10% power), using a sonicator probe (Bandelin Sonopuls HD 2070, Berlin, Germany). Di chloromethane was allowed to evaporate overnight under magnetic stirring. Empty nanoparticles were also prepared as controls. SR8278 encapsulation rates were determined by high-performance liquid chromatography (HPLC, Agilent 1100 Series, Les Ulis, France), using a C18 column, a mobile phase consisting of Milli-Q water: acetonitrile (gradient grade for HPLC, Sigma-Aldrich) 10:90, a flow rate of 1 mL / min, and a detection wavelength of 310 nm. The encapsulation rate was 90.8%. The stock solution contains 15mg nanoparticles / mL (2.5mg SR8278 / mL). Fluorescent nanoparticles were prepared by adding 0.2 mg of PLA grafted with rhodamine B (Sigma-Aldrich) into dichloromethane before emulsification as previously described79,80. To perform nanoparticles characterization, dynamic light scattering, nanoparticle tracking analysis, transmission electron microscopy and fluorescent confocal microscopy analyses were performed as previously described81.

[0051] Stimulation of pulmonary mononuclear cells and ELISA

[0052] Lungs were obtained from young-adult and old mice at ZTO and ZT12. Single cell suspensions were prepared from lungs minced with a razor blade before being incubated at 37°C for 1 hour with a mix of DNase I (100 pg / mL, Sigma-Aldrich) and collagenase D (400 U / mL, Roche, Basel, Switzerland) and incomplete RPMI-1640 medium. After incubation, tissue-pieces were passed through a 70 LI nylon cell strainer (Coming, Durham, NC) and the single cell suspensions were collected by centrifugation Pelleted cells were resuspended in 2 mL red blood cell lysis buffer (Life Technologies, Carlsbad, CA) and incubated at room temperature for 5 minutes. To stop the reaction, RPMI-1640 completed with essential and non-essential amino acid, penicillin, streptomycin, HEPES, sodium pyruvate, L-glutamate (all from Sigma Aldrich), and 10% FBS (complete RPMI) was added. Lung mononuclear cells (2 x 105 / well, 96-well plates) were stimulated for 24 hours with R848 (10 pg / mL), CpG ODNs (10 pg / mL) or LPS (1 ng / mL) (all from InvivoGen, San Diego, CA). IL-12p40 concentration was measured by ELISA accordingly to protocol’s manufacturer (Invitrogen, Walthnam, MA).

[0053] RNA extraction, RNA sequencing analysis and statistical analysis

[0054] Lungs from young-adult mice and old mice were harvested at ZTO, ZT4, ZT8, ZT12, ZT16 and ZT20 (n=4-5 / group), incubated in RNA later for 10 min at room temperature and frozen in liquid nitrogen. Total RNA was extracted with Trizol and purified on NucleoSpin RNA column (Macherey Nagel, Duren, Germany). The quality of RNA was evaluated with a Bioanalyzer (Agilent Technologies) and the Agilent RNA 6000 nano kit. Only RNAs with RNA Integrity Number (RIN) over 7 were used. 200ng of RNA were used for library construction followed by 100 bp paired-end sequencing performed by DNBSEQ technology and DNA nanoballs. The primary analysis of raw sequencing datasets was performed using the nf-core / rnaseq v3.6 pipeline (htps: / / doi.org / 10.5281 / zenodo.7998767). Raw FastQ files were quality and adapter trimmed with Trim Galore vO 6.7.

[0055] (https: / / www.bioinformatics.babraham.ac.uk / projects / trim_galore / ). Ribosomal RNA reads were filtered out with SortMeRNA v 4 3 ,482. Cleaned reads were aligned with STAR v2.6. Id83against the GRCh38 genome from the Ensembl 108 database and expression for annotated genes was quantified using Salmon vl.5.284in mapping-based mode. Analysis of circadian genes was performed using R (version 4.1.2). Gene counts were normalized using DESeq2 R package85. Detection of genes with a circadian rhythm was done with RAIN44. Raw -val ues from RAIN were adjusted for multiple testing using the Benjamini -Hochberg procedure to control the FDR Over-representation enrichment analysis was performed with clusterProfiler R package86using Over-Representation Analysis.

[0056] Microarray analysis and statistical analysis

[0057] Lungs from young-adult and elderly mice were harvested at ZTO and ZT12 (n = 4 / group) and RNAs were extracted as described above. 300ng of RNA was amplified with GeneChip WT PLUS Reagent Kit (Affymetrix, Santa Clara, CA), labeled with GeneChip WT Terminal. The resulting single- stranded cDNAs were hybridized on the GeneChip Mouse Gene 2.0 ST Array (Affymetrix) according to the manufacturer’s protocol. The microarray data were analyzed using GIANT (version v0.0.2) tools suite44on a local instance of Galaxy87. Data were normalized by the Robust Multi-Average method Transcripts associated with annotated genes were selected for analysis. Differentially expressed genes were identified by using limma88and Raw P values were adjusted for multiple testing using the Benjamini -Hochberg procedure to control the False Discovery Rate (FDR). To identify genes whose circadian regulation is affected by aging, differential analysis with interaction between age and time-of-day variables was performed. To visualize data, PCA analysis and heatmap were performed with Phantasus89(version vl.25.5) and volcano plot was generated with GraphPad Prism (Boston, MA) (version vlO.1.1).

[0058] Assessment of gene expression by quantitative RT-PCR

[0059] RNAs were reverse-transcribed with the High-Capacity cDNA Archive Kit (Life Technologies). The resulting cDNA was amplified using SYBR Green-based real-time PCR and the QuantStudio 12K Flex Real-Time PCR Systems (Applied Biosystems, Waltham, MA). Specific primers were designed using Primer Express software (Applied Biosystems) and ordered to Eurofms Scientifics (Ebersberg, Germany) (not shown). Relative quantification was performed using the gene encoding glyceraldehyde 3-phosphate dehydrogenase (Gapdh). Relative mRNA levels (2-AACt) were determined by comparing (a) the PCR cycle thresholds (Ct) for the gene of interest and the house keeping gene Gapdh (ACt) and (b)ACt values for treated and control groups (AACt). Data was expressed as a fold-increase over the mean gene expression level in young mice (ZTO). Cosinor analysis was performed using GraphPad prism 10. Chromatin immunoprecipitation assay

[0060] Chromatin immunoprecipitation (ChIP) experiments were performed on young lungs harvested atZTIO90. Briefly, lungs were extracted in a hypotonic buffer containing HEPES-KOH 50 mM pH7.5, Triton-XlOO 0.25%, Glycerol 10%, EGTA 0.5 mM, EDTA 1 mM, NaCl 140 mM, NP- 40 0.5%, protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). After 10 minutes incubation, nuclei were washed with a buffer containing Tris-HCl 10 mM, pH8.0, EGTA 0.5 mM, EDTA 1 mM, NaCl 200 mM and protease inhibitor cocktail. DNA and protein were then cross-linked in paraformaldehyde 1% for 10 minutes at room temperature. PFA was quenched using glycine 200 mM for 10 minutes at room temperature. Nuclei were lysed in Tris-HCl 50 mM pH8.0, EDTA lO mM, SDS 1% and protease inhibitor cocktail and DNA fragment of 150- 200 bp were generated using a Bioruptor (20 cycles 30 secondes On / 30 secondes Off). Immunoprecipitation was performed using the anti -Rev-Erb -a antibody (1 / 50, #13418, Cell Signaling Technology, Danvers, MA). Purified DNA was subjected to 40 cycles of PCR amplification. Crossing threshold (Ct) values were determined for each promoter and normalized to the Ct of input using the following equation: relative values = 2'(Ct IP’ct input). \ Data were expressed as a fold induction compared with the negative region using 2'AACtwith 2" [(Ct IP region of interest - Ct input) - (Ct IP negative region - Ct input)]

[0061] Flow cytometry

[0062] Lung MNCs were prepared as described above. Cells were pelleted and incubated for 20 minutes at 4°C in 2.4G2 hybridoma supernatant (FC gamma blocking antibody) diluted in PBS containing 5% FBS, 0.01% NaN3 (FACS buffer). Cells were washed and resuspended in FACS buffer before being pelleted and stained with the following fluorochrome-conjugated antibodies: PB (Pacific Blue) and BV-510-coupled anti-CD45 (1 / 100, #103138, 30-FII), AF700-coupled anti CD45 (1 / 100, #103128, 30-FII), BV-510-couple anti CD19 (1 / 100, #115546, 6D5), PECy7-coupled anti TCRp (1 / 100, #109222, H57-597), APCCy7-coupled anti- CD4 (1 / 100, #100414, GK1.5), FITC-coupled anti-CD8 (1 / 100, #100706, 53-6 7), PerCpCy5.5-coupled anti-TCR / 5 (1 / 100, #118118, GL3), PECy7-coupled anti-TCRp (1 / 100, #109222, H57-597), PE-coupled anti -NK1.1 (1 / 100, #156504, S17016D), PerCpCy5.5-coupled anti-CDl lb (1 / 100, #101228, MI / 70), APCCy7-coupled anti-Ly6G (1 / 100, #127624, 1A8), APC-coupled anti-CD64 (1 / 100, #161006, S18017D), PECy7-coupled anti CDl lc (1 / 100, #117318, N418), AF700-coupled anti-MHCII (1 / 100, #107622, M5 / 114.15.2), FITC-coupled anti-CD103 (1 / 100, #110908, W19396D), APCCy7-coupled anti-CD3 (1 / 100, #100222, 17A2), FITC-coupled anti-F480 (1 / 100, #157310, QA17A29), AF700-coupled anti-Ly6C (1 / 100, #128024, HK 1.4) (all from BioLegend, San Diego, CA). PE-coupled anti-Siglec F (1 / 100, #562068, E50-2440, BD Biosciences, Franklin Lakes, NJ). Cells were washed by centrifugation and resuspended in FACS buffer prior to being analyzed on a BD LSR II Fortessa cytometer (BD Biosciences). Data was analyzed using FlowJo Software (FlowJo LLC, Ashland, OR). To assess internalization of nanoparticles in vivo, alveolar macrophages (3-5 x 106cells) were labelled with the following antibodies: FITC-coupled anti-CDl lc (1 / 200, #557400, HL3), BV421-coupled anti-Siglec F (1 / 200, #562681, LOU / C), allophycocyanin (APC)-coupled anti-F4 / 80 (1 / 200, #566787, BM8) (all from BD Biosciences). Fixable viability dye aqua (Thermo-Fisher) was used to gate viable cells.

[0063] In vitro phagocytosis and killing assay

[0064] MPI cells were grown in complete RPMI and GM-CSF (PeproTech, Cranbury, NJ) (30 ng / mL) as described91MPI cells were synchronized with 50% horse serum for 2 hours, washed with PBS at 37°C and then maintained in complete RPMI for 10 hours before stimulation. To measure phagocytosis and killing activity, SR8278 (2 pM), apelin (5 Lig / mL) or vehicle was added for 1 hour. Cells were then washed three times with PBS at 37°C to remove antibiotics. Opsonized S. pneumoniae was added to the cells at multiplicity of infection of 10 (MOI 10 and incubated at 4°C for 1 hour to induce adherence, and at 37°C for 2 hours for internalization. Non-internalized bacteria were removed by washing with PBS at 37°C with penicillin and streptomycin (30 U / mL) and cells were then incubated for 30 minutes in RPMI with 10% FBS and penicillin and streptomycin (30 U / mL). For phagocytosis (3 hours post S. pneumoniae), cells were washed to remove remaining antibiotics, lysed with cold sterile water, and plated onto TS A 5% sheep blood agar. For killing (5 hours post-5, pneumoniae. ), cells were incubated for 2.5 hours with penicillin, streptomycin, and vancomycin (0.75 pg / mL), and plated onto Trypticase Soy Agar (TSA) 5% sheep blood agar. Phagocytosis was calculated as an increase percentage of phagocytosis in the SR8278 group over the vehicle group (% of phagocytosis = CFU final x 100 / CFU final average of vehicle group). Macrophage killing activity was calculated as % of killed bacteria = [viable bacteria at killing time point (CFU 3 hours post infection - CFU 5 hours post infection)* 100 / viable bacteria at killing time point average of vehicle group]. Confocal microscopy was performed to quantify the numbers of engulfed bacteria. For this, 5 x 105 / well MPI cells were seeded in 8-well plate, synchronized, SR8278 treated (1 hour) and incubated (4 hours) with eGFP S. pneumoniae (MOI 10), a gift from Dr Jose A. Chabalgoity (Montevideo, Uruguay). Cells were fixed with PFA 4%, washed with PBS- T (0.1% Tween 20), and blocked with 1% BSA for 1 hour, and incubated with phalloidin 547 Life Technologies, Carlsbad, CA) (1:500) for 30 min at room temperature, washed with PBS, and then incubated with Hoechst (1 :1000) for 30 min. Cells were washed and mounted using fluorescence mounting medium (Dako Omnis, Agilent) Images were acquired using Zeiss LSM 880 AiryScan microscopy (Carl Zeiss, Rueil-Malmaison, France) and processing using Imaris 8.0 (Oxford Instruments, Les Ulis, France) and ImageJ software (National Institutes of Health, Wisconsin).

[0065] Western blotting

[0066] Lung extracts were lysed in RIPA buffer (50 mM Tris-HCl pH8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS supplemented with protease inhibitors (Roche Diagnostics), heated at 95°C for 20 min, and centrifuged at 10,000g for 10 minutes. Proteins in supernatants were quantified using the Pierce™ BCA protein assay kit (ThermoFisher Scientific) An equal amount of protein was mixed with Laemmli loading buffer (EcoTech Biotechnology, Istanbul, Turkey). Proteins were then separated using 12% SDS-PAGE and then transferred from the gel to a nitrocellulose membrane. The polyclonal rabbit antibody directed against APJ was from Abeam (1:1000, #ab308018). The detection was made by using the appropriate horseradish peroxidase-conjugated secondary antibody (1:2500, #ab6721, Abeam). Antibodies were detected using chemoluminescence (Pierce), and the signals were quantified by applying the “gel quantification” procedure in Image I software (version 1.1.0) (National Institutes of Health). To normalize, an antibody directed against P-actin was used (#A5441, 1 : 1000, Sigma- Aldrich).

[0067] Statistical analyses and reproducibility

[0068] All experiments were performed at least two times except the transcriptomic analyses. For in vivo experiments, between 6 and 9 mice were analyzed per experiment. No power analyses were used to predetermine sample sizes, but our sample sizes were similar or superior to those reported in the previous publications92,93. Data distribution was assumed to be normal, but this was not formally tested. Data collection and analyses were not performed blind to the conditions of the experiments Excepted for the transcriptomic analyses, all statistical analyses were performed using GraphPad Prism v9.2.0 software. A two tailed Mann-Whitney U test was used to compare two groups unless otherwise stated. Comparisons of more than two groups with each other were analyzed with the one-way ANOVA Kruskal -Wallis test (nonparametric), followed by Dunn’s posttest or using the two-way ANOVA followed by a Sidak’s multiple comparisons post-hoc test. Except for the determination of the bacterial load and for the transcriptomic analyses, all data are expressed as the mean ± standard deviation (s.d.).

[0069] Results:

[0070] Aging alters oscillations in pulmonary immune defenses against pneumococcal infections It is still not known whether time-of-day susceptibility to pulmonary infection is altered in aged individuals. Hence, we sought to determine whether host defenses against 5. pneumoniae in young-adult mice (2-month-old) displayed daily oscillations. To this end, young-adult mice were infected at Zeitgeber time (ZT)0, ZT6, ZT12 and ZT18 (data not shown), and the numbers of live pneumococci in the lungs were determined 24 hours post-infection. The youngadult mice were significantly more susceptible to infection at ZT6 and ZT12 (the middle and end of the rest phase, respectively) than at ZTO and ZT18 For the rest of the study, we focused on the two opposing time points ZTO (when young-adult mice are less susceptible to infection) and ZT12 (when young-adult mice are more susceptible to infection). We next assessed the impact of aging on circadian host defenses in 22-month-old mice (considered to be equivalent to 70-year-old humans41and referred to hereafter as “elderly mice”). Strikingly, the time-of- day difference in susceptibility to infection observed in young-adult mice was not significant in elderly mice (data not shown); relative to young-adult mice, elderly mice were significantly more susceptible to infection at ZTO but not at ZT12.

[0071] We next sought to determine whether perturbations in host defense rhythmicity in elderly mice are associated with altered variations in immune cell counts in the lungs at ZTO and ZT12. Flow cytometry analysis revealed major differences between naive (no infection) young-adult mice and elderly mice. In young-adult mice, the absolute alveolar macrophage and natural killer T (NKT) cell counts were higher at ZT12 than at ZTO, whereas the neutrophil and B cell counts were significantly lower at ZT12 than at ZTO (data not shown). For the other cell types examined, the immune cell number remained stable at ZTO and ZT12 (data not shown). Remarkably, the rhythmicity observed in young-adult mice was abrogated in elderly mice, with the exception of the neutrophil count (which was higher at ZT12 than at ZTO). Rapid cytokine production in response to an infectious challenge is critical for lung defenses. In line with the results previously shown, lung cells from young-adult mice produced high amounts of cytokines at ZTO in response to lipopolysaccharide (LPS), resiquimod (R848) and CpG oligodeoxynucleotides (CpG ODN) (data not shown). In contrast, no significant ZTO vs. ZT12 differences in cytokine release were seen in elderly mice. Overall, impaired circadian defenses in elderly mice were associated with lower daily variations in basal cell counts and cytokine production levels.

[0072] Aging alters the circadian transcriptome, with the clock component Rev-erb-a being the most impaired

[0073] We next looked at whether disruption of the rhythmicity of pulmonary defenses in elderly mice was associated with changes in time-of-day variations in gene expression. To this end, we compared the circadian transcriptome in lungs collected from infection-naive young-adult and elderly mice at ZTs 0, 4, 8, 12, 16 and 20 (data not shown). A rhythmicity analysis incorporating non-parametric methods (RAIN)42revealed that 2818 transcripts were rhythmic in both young and elderly mice (adjusted P value < 0.05) (data not shown). Interestingly, a set of 3740 transcripts was rhythmic in young-adult mice only, while another set of 2895 transcripts was rhythmic in elderly mice only. Enrichment analysis of Gene Ontology (GO) biological processes indicated that pathways found to be rhythmic in young mice (such as “proteostasis”, “leukocyte migration” and “apoptosis”) were not rhythmic in elderly mice (data not shown). In contrast, some GO terms (including “cilium functions” and “hemostasis”) became rhythmic in elderly mice (data not shown). These results suggest that the rhythmic transcriptome in the lungs is reprogrammed in elderly animals. Rhythmically expressed genes in both young-adult and elderly groups were in “matrix remodeling”, “kinase activity” and “epithelial / endothelial functions” GO pathways (data not shown). GO pathways related to immune functions (including “leukocyte migration” and “adaptive immunity”) were specifically enriched in young mice (data not shown). In contrast, no specific GO terms related to immune functions were enriched in elderly mice. Importantly, rhythmic genes involved in immune responses (including macrophage functions like “phagocytosis” and “ROS metabolism”) were observed in the lungs from young-adult and elderly mice (data not shown). We therefore hypothesized that young-adult and elderly mice differ with regard to the ZT for peak expression (i.e. the acrophase) of rhythmically expressed genes (including those involved in immune responses) in the lung. Most circadian genes reached their acrophase at ZT4 and ZT16 in young-adult mice and at ZT0 and ZT8 in elderly mice. This observation suggested that the enriched GO terms might be different at specific time points. Indeed, some GO terms were time-shifted in lungs from elderly mice, relative to lungs from young-adult mice. Importantly, genes involved in the control of the circadian clock were enriched at opposing time points in young-adult mice (ZT4) and elderly mice (ZT16). We reasoned that this shift might contribute to the overall impairment in diurnal gene expression observed in lungs from elderly mice. Furthermore, GO terms related to “phagocytosis” were advanced by 4 hours in lungs from elderly mice, relative to lungs from young-adult mice. Terms related to “macrophage migration and activation” were enriched only in young-adult mice (at ZT4 and ZT8, respectively) (data not shown). In contrast, terms related to “glucocorticoid receptor signaling” (stress-associated pathways involved in the control of inflammation and immune suppression43) were enriched at ZTO and ZT16 in elderly mice.

[0074] We next sought to characterize the most intensely dysregulated circadian genes in lungs from elderly mice. For that purpose and for the reasons mentioned above (data not shown), we compared lung transcript expression at ZTO and ZT12 in lungs from young mice and from elderly mice. A principal component analysis (PCA) revealed four distinct groups, based on age and time-of-day conditions (data not shown). We next performed a differential analysis of the interaction between age and time-of-day variables by using the galaxy-based tool for interactive analysis of transcriptomic data (GIANT)44. We identified 12 genes whose circadian expression between ZTO and ZT12 was influenced by age (P value < 0 01) (data not shown). Strikingly, only Nrldl (encoding the clock component Rev-erb-a) displayed a significant fall in its daily expression with age (false rate discovery (FDR) < 0.1). This finding is particularly interesting because Rev-erb-a is a key transcription factor that controls lung immunity and inflammation6’9 10. To confirm that the circadian expression of Nrldl was altered during aging, we analyzed the level of transcripts around the clock (at ZTs 0, 4, 8, 12, 16 and 20), using quantitative RT-PCR. As expected, a cosinor analysis (data not shown) revealed that Nrldl mRNA levels displayed robust daily rhythms in young lungs In contrast, the amplitude of Nrldl rhythms was dramatically lower in lungs from elderly mice. Taken as a whole, these data show that the impaired oscillation of anti-bacterial lung defenses in elderly mice was associated with changes in the circadian transcriptome. Interestingly, the transcription factor Rev-erb-a was the circadian gene most strongly modified with age.

[0075] Rev-erb-a antagonism activates the bactericidal activity of alveolar macrophages

[0076] Whilst the role of Rev-erb-a in inflammation and innate immunity has been well characterized10’12’17, its potential role in lung defenses against infection is more elusive To address this issue, young Rev-erb-a knock-out mice (NrldN ) and Rev-erb-a-profi cient youngadult mice were infected with S. pneumoniae at ZT12 (i.e., a ZT at which the pulmonary expression of Rev-erb-a protein is high10) (data not shown). Compared with littermate 1 - controls, Nrldl'1' mice were more resistant to pneumococcal infection (Fig. 1A). To determine whether Rev-erb-a activation affects susceptibility to pneumococcal infection, we performed experiments with the Rev-erb-a agonist SR9009; the latter is known to stabilize Rev-erb-a protein and provides sustained inhibition on gene expression. In order to avoid systemic metabolic impairments (e.g., lipolysis and body weight loss45’46), the Rev-erb-a proficient mice were given SR9009 intranasally. We administered SR9009 at ZT9 (i.e., three hours before infection) and at ZT12. The SR9009-treated mice exhibited a significant higher bacterial count in the lungs, compared with vehicle-treated mice (Fig. IB). The mice were then treated with the Rev-erb antagonist SR8278 at ZT9 and ZT12. SR8278 is known to inhibit Rev-erb-a repressive transcriptional activity and produces an increase in Rev-erb-a target gene expression47. Indeed, treatment with SR8278 effectively reduced Nrldl expression and induced Arntl expression in the lungs (data not shown). Interestingly, pharmacological inhibition of Rev-erb-a resulted in greater resistance against pneumococcal infection (Fig. 1C). Alveolar macrophages have a critical role in S. pneumoniae clearance48. Airway administration of clodronate liposomes (known to deplete alveolar macrophages49) completely blocked the protective effects of SR8278 on the pulmonary bacterial load (Fig. ID). To confirm this finding, SR8278 was encapsulated in biocompatible nanoparticles (-200 nm in size) so that alveolar macrophages were specifically targeted. Indeed, in vitro and in vivo exposure to nanoparticles led to potent internalization by alveolar macrophages (data not shown). Compared with vehicle-containing nanoparticles, treatment (at ZT9 and ZT12) with SR8278-containing nanoparticles resulted in a lower bacterial load in the lungs (Fig. IE) Taken as a whole, these data show that alveolar macrophages are critical in SR8278-mediated protection against pneumococcal infection. To functionally assess the effect of Rev-erb-a antagonism on alveolar macrophages, we investigated the potential effect of SR8278 on the phagocytic and intracellular killing abilities of Max Planck Institute (MPI) cells (a cell line that mimics the main features of primary alveolar macrophages50,51). To this end, MPI cells were synchronized by exposure to 50% horse serum for 2 hours and then treated with SR8278 in the middle of the ascending phase of Rev-erb-a expression (10 hours post-synchronization) (data not shown). Interestingly, S. pneumoniae ere internalized more effectively by SR8278-treated macrophages than by vehicle-treated cells (Fig. IF) Indeed, the number of cells harboring intracellular bacteria and the number of bacteria per cell were significantly higher when Rev-erb-a was antagonized. Confocal microscopy using eGPF-expressing A pneumoniae confirmed the higher phagocytic activity of macrophages after SR8278 treatment (data not shown). Lastly, treatment of alveolar macrophages with SR8278 significantly enhanced the killing of S. pneumoniae (Fig. 1G). Overall, these observations show that antagonizing Rev-erb-a activity enhances the phagocytosis and bactericidal activity of alveolar macrophages and protects the host against pneumococcal infection.

[0077] Rev-erb-a / p antagonism protects against pneumococcal infection in elderly mice

[0078] To gain insights into mechanisms potentially involved in SR8278-mediated protection, we sought to characterize Rev-erb-a target genes in general and those having altered circadian rhythmicity during aging in particular. To this end, we performed a clustering of genes based on the Nrldl expression profile (ZTO and ZT12) (data not shown). In line with the GIANT interaction analysis (data not shown), genes with opposing profiles included Apln (coding for apelin) and Apj (coding for the apelin receptor, APJ). The apelin pathway is known to regulate many physiological and pathological mechanisms and is a novel target for the maintenance of health during old age52'57. Interestingly, Apln and Apj were among the most strongly downregulated genes in elderly mice, as illustrated in a volcano plot (data not shown). Quantitative RT-PCRs (ZTs 0, 4, 8, 12, 16 and 20) and cosinor analyses revealed that the daily rhythmicity of Apln and Apj mRNA expression was absent in lungs from elderly mice (data not shown). Given that (i) Rev-erb-a and Apln / APJ display antiphase expression in lungs from young-adult mice and (ii) Rev-erb-a is a transcriptional repressor, we looked at whether Rev- erb-a might control the expression of Apln and Apj by binding to their promoters. An in silico analysis revealed the presence of a putative consensus response element (RevRE) in the Apln and Apj promoters (data not shown) To determine whether Rev-erb-a was recruited to Apln and Apj promoters, we performed a chromatin immunoprecipitation assay on lungs from young mice. Rev-erb-a was recruited at different sites within the Apln promoter (-1668 kb, transcription start site) and the Apj promoter (-3901 kb, -3198 kb, -1057 kb) (data not shown). These results demonstrate that Rev-erb-a binds to the Apln and Apj promoters and highlights the latter as new Rev-erb-a target genes.

[0079] As Rev-erb-a antagonism protects against pneumococcal infection (data not shown), we reasoned that activation of APJ may upregulate pulmonary defenses against S. pneumoniae. We therefore investigated the effect of apelin administration on the bacterial load in the lungs (young-adult mice). Since apelin has a short half-life58, we used osmotic pumps to deliver the peptide. Interestingly, apelin treatment significantly reduced the pneumococcal burden in the lungs (data not shown). Although APJ is known to be functional in peritoneal macrophages59, its potential role in alveolar macrophages has yet to be studied. Pretreatment of synchronized MPI cells with apelin resulted in higher levels of phagocytosis and enhanced bactericidal activity (data not shown). Taken as a whole, these results show that the apelin / APJ signaling pathway is relevant in macrophage-dependent bacterial killing. We then looked at whether the SR8278-mediated protection against pneumococcal infection depended on APJ. To this end, we evaluated the in vivo effect of SR8278 in APJ-competent and APJ knock-out (Apj~ / ~') mice. As expected, SR8278 administration was associated with a significantly lower bacterial load in wild-type mice but not in Apj~ / ~ mice (data not shown). This finding suggested that the effect of the Rev-erb antagonist on bacterial infection is mediated (at least in part) by APJ (data not shown).

[0080] Unbiased transcriptomic analysis and pharmacological studies in young mice suggested that Rev-erb-a and APJ are potential targets for enhancing pulmonary defenses in elderly mice (data not shown). We first sought to establish whether Rev-erb-a antagonism can reduce the bacterial load in elderly mice. Intranasal SR8278 inoculation at ZT9 and ZT12 significantly lowered the bacterial load in elderly mice (Fig. 2). Indeed, the bacterial load was similar in elderly mice and untreated young-adult mice; hence, Rev-erb-a antagonism appeared to have “rejuvenated” the pulmonary defenses in elderly mice. Lastly, we looked at whether APJ signaling is associated with better protection. Apelin treatment failed to enhance the protective effect of SR8278 - perhaps partly because the APJ expression level in the lungs was low (data not shown). In summary, APJ signaling alone is relevant for pulmonary defenses in youngadult mice but Rev-erb-a antagonism is sufficient to protect elderly mice against pneumococcal infections.

[0081] Discussion:

[0082] Even though impaired circadian rhythmicity in older adults has a major impact on many diseases and the body’s immune responses60’61, the potential contribution of age-mediated circadian misalignment to infection has not previously been investigated experimentally. To the best of our knowledge, the present study is the first to have demonstrated the presence of links between aging, circadian rhythms, pulmonary innate immunity, and bacterial respiratory infection. Moreover, our results showed that the pharmacological targeting of Rev-erb-a / p in elderly mice can modify susceptibility to pneumococcal infection and thus indicate that approach might have therapeutic value in humans.

[0083] In line with the literature data9,24,25, we found that susceptibility to pneumococcal infection was diumally regulated in young mice. These animals were most susceptible at ZT6 and ZT12, which correspond respectively to the middle and end of the rest phase. Our data revealed that the diurnal control of pneumococcal infection is disrupted in older lungs In our experimental settings, a change in the time-of-day vulnerability to pulmonary infection in elderly mice was associated with impaired diurnal rhythmicity in the number and functions of immune cells in the lungs. It is noteworthy that the cytokine response to microbial danger components was not diumally controlled in lungs from elderly mice. The circadian transcriptome is known to change with age,62,63and the lung is no exception63. We established a time-domain map of GO terms enriched in the basal circadian transcriptome of lungs from young mice and elderly mice; this map revealed that some GO terms present in lungs from young mice were absent in lungs from elderly mice. Interestingly, the GO term “phagocytosis” was phase-shifted in lungs from elderly mice. This is consistent with Wolff et al.’s suggestion64whereby impaired clock gene expression in lungs from elderly mice might lead to changes in the anticipatory nature of the pulmonary response to environmental insults, including infections. Furthermore, our transcriptomic analysis revealed that processes that are not rhythmic in young adult mice (such as “cilium functions” and “glucocorticoid signaling”) become rhythmic in elderly mice. The potential impact of these changes on the lungs’ defenses against infection has yet to be determined It is noteworthy that the apelin pathway and the glucocorticoid pathway regulate each other: glucocorticoids repress apelin expression (whereas apelin induces glucocorticoid signaling in various contexts; this might account for the deregulation of the glucocorticoid pathway with age)64'66. Furthermore, the glucocorticoid receptor represses the transcription of Rev-erb-a mRNA in vitro and in vivo61’6, while Rev-erb-a physically interacts with the glucocorticoid receptor to modulate its transcriptional activity69. Our results therefore suggest that the glucocorticoids-apelin-Rev-erba triad has an important role in lung homeostasis, an age-mediated imbalance of which may promote lung failure. Our results are also in line with Wolff et al. ’s report70of impaired circadian control of gene expression in lungs from elderly mice, even with conserved oscillation of the core clock genes (e.g. Arntl, Per). In our experiments on lungs from elderly mice, Rev-erb-a and components of the apelinergic axis displayed changes in their circadian rhythmicity. It remains to formally determine whether these transcriptional changes explain the modified time-of-day vulnerability to pneumococcal infection. Nonetheless, these data prompted us to study the potential roles of Rev-erb-a and the apelinergic signaling pathway in lung defenses.

[0084] Although the role of Rev-erb-a in lung function and diseases (inflammation and fibrosis) has been well described10,33’71, the protein’s potential role in the lung defense’s against respiratory pathogens is less clear. Griepentrog et al. identified Rev-erb-a as a key element in host protection against Klebsiella pneumoniae infection after exposure to blue light - a procedure known to synchronize circadian rhythms72. In contrast, our data showed that Rev-erb-a gene knock-out and pharmacological inactivation of the protein (using SR8278) enhanced host defenses against 5. pneumoniae. This apparent discrepancy might be due to differences in the protocols used to stimulate or inactivate Rev-erb-a, and the time-of-day treatment in particular. In our setting, mice were treated locally at the end of the rest phase (ZT9 / ZT12, i.e. the peak of Rev-erb-a expression). In Griepentrog et al.’s study, the mice were treated systemically at ZT672. It is important to note that alveolar macrophages are critical for the protective effects of SR8278. Our in vitro analysis of synchronized macrophages showed that Rev-erb-a inactivation upregulated phagocytosis and bacterial killing, which is in agreement with a report of reduced phagocytosis by microglia upon Rev-erb-a activation73. Our new data are consistent and highlight the negative role of Rev-erb-a in macrophage-dependent lung defenses against bacterial infection.

[0085] Hence, in addition to Rev-erb-a’ s role as a rhythmic regulator of macrophage-induced inflammation12,17,18, this protein has an essential role in phagocytosis and bacterial clearance. Our present results are also in line with a recent study in which (i) circadian transcription and phagocytosis were disrupted in aged macrophages and (ii) the transcription factor Klf4 (encoded by a clock-controlled gene) was critical for this process37. Research on whether the loss of rhythmic Klf4 expression in macrophages is correlated with changes in Rev-erb-a expression / activity is now warranted. More generally, further investigations of the oscillatory nature of the aging program of alveolar macrophages (e.g. gene expression, phagocytosis, and bactericidal activity) are likely to be very informative. Our unbiased transcriptomic analysis of lungs from elderly mice suggested a role for altered circadian apelin signaling (along with Rev- erb-a) in susceptibility to pneumococcal infection. The apelinergic system has been linked to a number of diseases, including chronic heart failure, diabetes, inflammation, obesity, and HIV infection52. Accordingly, researchers have highlighted the therapeutic potential of targeting the apelin / APJ system - notably in the context of respiratory diseases52'54. Importantly, it was shown that the apelinergic system changes with age and that its restoration (by means of apelin treatment) reinforces circadian patterns and increases the mammalian healthspan55The role of the apelinergic system in infection has not yet been established. Our data show that pharmacological activation of APJ signaling upregulates pulmonary antibacterial defenses. It remains to be seen whether APJ signaling is relevant in macrophage functions74and whether it acted in our study as a macrophage-intrinsic circadian regulator of phagocytosis and bacterial killing. It is noteworthy that in young mice at least, APJ was involved in the protection against pneumococcal infection triggered by SR8278, which is known to increase the expression of Rev-erb-a target genes.

[0086] Recent evidence indicates that the age-related misalignment of circadian rhythms can be corrected pharmacologically, for example by stimulating molecular oscillators75,76Pharmacological treatments based on the circadian clock include melatonin and agonists of the main components of the stabilizing loop (namely Rev-erbs and RORs), such as nobiletin75,76. Treating respiratory infections in older adults by targeting the circadian system has not yet been investigated. Importantly, our present results showed that local administration of the Rev-erb- a antagonist SR8278 lowered the bacterial load in elderly mice. Our results thus highlighted the potential therapeutic utility of modulating the clock protein machinery to reduce the likelihood of infection. It is noteworthy that the combination of apelin with SR8278 did not strengthen the latter’s protective effect. This might be due to the low level of APJ expression in lungs from elderly mice and to SR8278’s inability to enhance that expression. The present study had some limitations. Although our results provide insights into therapies that target the circadian system and might improve lung defensed in older individuals, data obtained in mice should be interpreted with caution. Indeed, this model does not reproduce all the characteristics of the circadian shift associated with age. Furthermore, mice (unlike humans) are nocturnal. Another limitation of the present study relates to our focus on alveolar macrophages. Although our results are very much in line with a previous study in which aged macrophages were identified as potential targets of clock-based therapeutics for the control of lung infections37, other cell types might also be affected by age-mediated circadian irregularities. For example, epithelial cells have a strong intrinsic circadian cycle, and the latter can be impaired by stress; this might influence the outcome of lung infections9,77. It remains to be seen whether the treatment of elderly mice with SR8278 restores (at least in part) oscillations in pulmonary defenses. In conclusion, our study revealed a novel pathway involved in respiratory bacterial infections in elderly mice and provide new insights into the role of Rev-erb-a in pulmonary defenses. Lastly, our results suggest that age-related circadian impairments could be targeted in infectious disorders.

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[0088] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. A method of treating a lung infection in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a Rev-erb antagonist.

2. The method according to claim 1 wherein the is a child (human being between the stages of birth and puberty), a teenager (human being between the stages of puberty to adulthood), an adult (human being between the end of puberty / beginning of adulthood to sixty-five years of age) or an elderly person (over sixty-five years of age).

3. The method according to claim 2 wherein the patient is an elderly patient.

4. The method according to any one of claims 1 to 3 wherein the patient suffers from a chronic lung infection or from an acute lung infection.

5. The method according to any one of claims 1 to 4 wherein the lung infection is a bacterial infection.

6. The method according to claim 5 wherein the bacterial infection is a Streptococcus pneumoniae infection7. The method according to any one of claims 1 to 4 wherein the lung infection is a fungal infection.

8. The method according to any one of claims 1 to 4 wherein the lung infection is a viral infection.

9. The method according to any one of claims 1 to 8 for the prophylactic treatment of lung infection.

10. The method according to claim 9 for the prophylactic treatment of a bacterial superinfection, more particularly a bacterial superinfection secondary to a lung viral infection, even more particularly a bacterial superinfection post-influenza.

11. The method according to any one of claims 1 to 10 wherein the Rev-erb antagonist is encapsulated in nanoparticles, preferably biocompatible nanoparticles.

12. The method of claim 11 wherein the nanoparticle has a core that comprises polymers selected from the group consisting of: poly(lactic acid), poly(glycolic acid), or mixturesthereof. More particularly, the nanoparticle comprises poly(lactic)poly(glycolic) acid co-polymers (PLGA).

13. The method according to claim 12 wherein the pharmaceutical composition that comprises the nanoparticles, is administered in the respiratory tract of the subject.

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