Method to improve macrophages functions

Abstract

The present invention relates to the treatment of inflammatory diseases. Here, the inventors analyzed the immune activation triggered by HRV16 in macrophages to elucidate the cellular pathways involved in ARL5b upregulation. They demonstrated that HRV16 does not replicate in macrophages but induces interferon and pro-inflammatory responses. Conversely, in HeLa OHIO cells where the virus replicates, no induction of immune responses is observed. They identify the ICAM1-PKR-ATF2 axis as the primary regulator of HRV16-mediated ARL5b induction in macrophages. By contrast, HRV16-mediated ARL5b decrease in HeLa OHIO is dependent on ICAM-1 / viral entry in the cells but not on PKR signalling. Finally, ARL5b is regulated at the epigenetic level in both HeLa OHIO and human macrophages. An increase in the repressive mark H3K27Me3 is observed in HeLa OHIO, while the activating mark H3K27Ac is increased on the ARL5b promoter in human macrophages. Thus, the invention relates to relates to an inhibitor of the ICAM1 / PKR / ATF2 signalling pathway for use in the improvement of phagocytosis in a subject in need thereof.

Description

[0001] METHOD TO IMPROVE MACROPHAGES FUNCTIONS

[0002] FIELD OF THE INVENTION:

[0003] The present invention relates to relates to an inhibitor of the ICAM1 / PKR / ATF2 signalling pathway for use in the improvement of phagocytosis in a subject in need thereof.

[0004] BACKGROUND OF THE INVENTION:

[0005] Human Rhinoviruses (HRV) are the causative agents of the common cold (1). These non-enveloped viruses possess an icosahedral capsid containing a single-stranded positivesense RNA genome (1). Over 100 serotypes, categorized into three species (HRV-A, HRV-B, and HRV-C), have been identified to date. These can be further classified into three groups based on the receptor used for cellular entry. HRV-A and HRV-B are divided into a major group, utilizing intercellular adhesion molecule- 1 (ICAM-1) for entry, and a minor group, utilizing the low-density lipoprotein receptor (LDLR) (1). HRV-C serotypes enter cells through Cadherin Related Family Member 3 (CDHR3) (2). Notably, HRV16, a commonly studied strain, belongs to the major group of the HRV-A species.

[0006] While HRV typically infects the upper respiratory tract, in patients with chronic inflammatory respiratory diseases like chronic obstructive pulmonary disease (COPD) or asthma, HRV can also infect the lower respiratory tract (3). Infections in the lower respiratory tract have been linked to disease exacerbations and bacterial superinfections (4-6). Studies, including our own, have demonstrated that bacterial superinfections are associated with impaired phagocytosis in macrophages (7-13).

[0007] Alveolar macrophages, as the first line of defence in the lungs, can phagocytose material and cell debris through specific surface receptors (14,15). Our previous results demonstrated that HRV16 affects phagocytosis in macrophages during bacterial uptake in an Arpin- dependent manner (9) and bacterial clearance in an ADP Ribosylation Factor Like GTPase 5B (ARL5b)-dependent manner (13). Interestingly, in macrophages, ARL5b expression was increased upon viral contact, while it was decreased in HeLa OHIO cells, a model used to replicate HRV16. In HeLa OHIO cells, depletion of ARL5b increased the secretion of infectious virions, while ectopic expression of ARL5b led to a decrease in viral production, suggesting ARL5b acts as a restriction factor (13). The mechanisms governing this dual regulation of ARL5b in HeLa OHIO and human macrophages are yet to be defined. Previous studies have reported that HRV16 triggers an immune response in human macrophages, but the extent of this response and the cellular pathways involved remain unclear (16-18). Furthermore, while HRV has been shown to replicate in epithelial cells (13,19), its potential replication in macrophages has yet to be fully elucidated (9,20).

[0008] Upon detecting pathogens, cells can be activated, leading to the production of cytokines and interferons (21). The activated protein kinase R (PKR) plays a crucial role in various signalling pathways, responding to bacterial or viral infections and cytokine detection (21-23). PKR can phosphorylate different effectors, including Mitogen-activated protein kinases (MAPK) and IKB kinase (IKK) (23), leading to the activation of transcription factors such as c- Jun, ATF2, and the NF-KB pathway (23). Activated transcription factors translocate to the nucleus, influencing gene expression through interactions with the epigenetic status of gene promoters and enhancers, and subsequent DNA compaction (24,25). Various epigenetic marks have been described. Among others, the acetylation of lysine 27 of histone 3 (H3K27Ac) is an activating mark whereas the trimethylation of lysine 27 of histone 3 (H3K27Me3) dependent on the methyltransferase Enhancer of zeste homolog 2 (EZH2) is a repressive mark (26).

[0009] SUMMARY OF THE INVENTION:

[0010] In this study, the inventors analyzed the immune activation triggered by HRV16 in macrophages to elucidate the cellular pathways involved in ARL5b upregulation. They demonstrated that HRV16 does not replicate in macrophages but induces interferon and pro- inflammatory responses. Conversely, in HeLa OHIO cells where the virus replicates, no induction of immune responses is observed. They identify the ICA 1-PKR-ATF2 axis as the primary regulator of HRV16-mediated ARL5b induction in macrophages. By contrast, HRV16- mediated ARL5b decrease in HeLa OHIO is dependent on ICAM-l / viral entry in the cells but not on PKR signalling. Finally, ARL5b is regulated at the epigenetic level in both HeLa OHIO and human macrophages. An increase in the repressive mark H3K27Me3 is observed in HeLa OHIO, while the activating mark H3K27Ac is increased on the ARL5b promoter in human macrophages.

[0011] Thus, the invention relates to relates to an inhibitor of the ICA 1 / PKR / ATF2 signalling pathway for use in the improvement of phagocytosis in a subject in need thereof.

[0012] Particularly, the invention is defined by its claims. DETAILED DESCRIPTION OF THE INVENTION:

[0013] Accordingly, the present invention relates to an inhibitor of the ICAM1 / PKR / ATF2 signalling pathway for use in the improvement of phagocytosis in a subject in need thereof.

[0014] Particularly, the invention relates to an inhibitor of the ICAM1 for use in the improvement of phagocytosis in a subject in need thereof.

[0015] Particularly, the invention relates to an inhibitor of the PKR for use in the improvement of phagocytosis in a subject in need thereof.

[0016] Particularly, the invention relates to an inhibitor of the ATF2 for use in the improvement of phagocytosis in a subject in need thereof.

[0017] As used herein, the term “phagocytosis” refers to a process of the immune system by which a cell uses its plasma membrane to engulf a large particle giving rise to an internal compartment called the phagosome. Phagocytosis is a major mechanism used to remove pathogens and cell debris. The ingested material is then digested in the phagosome. In particular, “improvement of phagocytosis” refers to a reactivation of macrophage function to phagocytose bacteria infecting a subject.

[0018] As used herein the “ICAM1” for “Intercellular Adhesion Molecule” also known as “CD54” (“Cluster of Differentiation 54”) denotes a protein that in humans is encoded by the ICAM1 gene. This gene encodes a cell surface glycoprotein which is typically expressed on endothelial cells and cells of the immune system. It binds to integrins of type CDl la / CD 18, or CD 11b / CD 18 and is also exploited by rhinovirus type A as a receptor for entry into respiratory epithelium. Its Entrez accession number is 3383 and its UniProtKB / Swiss-Prot accession number is P05362.

[0019] As used herein, the term “PKR” for “Protein kinase RNA-activated” also known as “protein kinase R” (PKR) denotes an enzyme that in humans is encoded by the EIF2AK2 gene on chromosome 2. PKR is a serine / tyrosine kinase that is 551 amino acids long. PKR is inducible by various mechanisms of stress and protects against viral infections. It also has a role in several signaling pathways. Its Entrez accession number is 5610 and its UniProtKB / Swiss-Prot accession number is P19525.

[0020] As used herein, the term “ATF2” for “Activating transcription factor 2” denotes a protein that, in humans, is encoded by the ATF2 gene. This gene encodes a transcription factor that is a member of the leucine zipper family of DNA-binding proteins. This protein binds to the cAMP -responsive element (CRE), an octameric palindrome. The protein forms a homodimer or heterodimer with c-Jun. The protein is also a histone acetyltransferase (HAT) that specifically acetylates histones H2B and H4 in vitro., thus, it may represent a class of sequence-specific factors that activate transcription by direct effects on chromatin components. Additional transcript variants have been identified but their biological validity has not been determined. Its Entrez accession number is 1386 and its UniProtKB / Swiss-Prot accession number is P15336.

[0021] As used herein, the term “inhibitor of the ICAM1 / PKR / ATF2 signalling pathway” denotes all molecules which inhibit the activity and the expression of ICAM1, PKR or ATF2. This term also denotes all molecules which inhibit the interaction between ICAM1, PKR and ATF2 with their ligands. The term “inhibitor of the ICAM1 / PKR / ATF2 signalling pathway” also denotes inhibitors of the expression of the gene coding for ICAM1, PKR ad ATF2.

[0022] As used herein, the term ICAM, PKR and ATF2 are designated by the term “the targets of the invention”.

[0023] Inhibitor of the the ICAM 1 / PKR / ATF2 signalling pathway may be determined by any competing assays well known in the art. For example, the assay may consist in assessing the activation of immune genes by reverse transcription-quantitative polymerase chain reaction in presence or absence of the inhibitor and after stimulation with double stranded RNA (Poly I:C (HMW)) which activates PKR and therefore ATF2. The assay may also consist in assessing the capacity of the ICAM 1 / PKR / ATF2 inhibitor to prevent HRV16 entry in cells as determined by formation of lysis plaque in Hela Ohio cells. The assay may also consist in a phagocytic and clearance assay using for instance beads to measure intraphagosomal degradation.

[0024] Thus, the invention also relates to an inhibitor of the ICAM 1 / PKR / ATF2 signalling pathway for use in the improvement of phagocytosis in a subject afflicted with an inflammatory disease. In other word, the invention also related to an inhibitor of the ICAM 1 / PKR / ATF2 signalling pathway for use in the treatment of an inflammatory disease in a subject in need thereof. Particularly, the inflammatory diseases are chronic obstructive pulmonary disease (COPD) or asthma.

[0025] Particularly, the invention relates to an inhibitor of the ICAM 1 / PKR / ATF2 signalling pathway for use in the treatment of chronic obstructive pulmonary disease (COPD) or asthma in a subject in need thereof. More particularly, the invention relates to an inhibitor of the ICAM1 / PKR / ATF2 signalling pathway for use in the treatment of chronic obstructive pulmonary disease (COPD) or asthma induced by a respiratory virus in a subject in need thereof.

[0026] As used herein, the term “subject” denotes a mammal. Typically, a subject according to the invention refers to any subject (preferably human) afflicted with or susceptible to be afflicted with inflammatory diseases. Typically, a subject according to the invention refers to any subject (preferably human) afflicted with or susceptible to be afflicted with like chronic obstructive pulmonary disease (COPD) or asthma.

[0027] As used herein, the term “asthma” has its general meaning in the art and refers to a condition of the airways of the lung. Inflammation and tightening of the muscles around the small airways cause asthma symptoms such as cough, wheeze, shortness of breath and chest tightness. Asthma exacerbation, also called an asthma attack, is defined as a worsening asthma symptoms and lung function, i.e as a respiratory attack that requires emergency treatment. Respiratory infections are the main culprits of asthma exacerbations, and rhinovirus being the most common agent.

[0028] As used herein, the term “respiratory virus” has its general meaning in the art and refers to a virus inducing upper and lower respiratory tract infections. These respiratory viruses include members of the Pneumoviridae family, including human respiratory syncytial virus (hRSV) type A and B, and human metapneumovirus (hMPV) type A and B; members of the Paramyxoviridae family, including parainfluenza virus type 3 (PIV-3) and measles virus; and members of the Coronaviridae family, including endemic human coronaviruses (HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKUl); severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle-East respiratory syndrome coronavirus (MERS-CoV).

[0029] Particularly, the respiratory virus is the human rhinovirus (HRV) or the Human respiratory syncytial virus (HRSV).

[0030] More particularly, the invention relates to an inhibitor of ARL5b for use in the treatment of chronic obstructive pulmonary disease (COPD) or asthma exacerbations induced by a respiratory virus.

[0031] As used herein, the term "Chronic obstructive pulmonary disease (COPD)" has its general meaning in the art and is a type of obstructive lung disease characterized by long-term breathing problems and poor airflow. The main symptoms include shortness of breath and cough with sputum production. COPD is a progressive disease, meaning it typically worsens over time. Eventually, everyday activities such as walking or getting dressed become difficult. Chronic bronchitis and emphysema are older terms used for different types of COPD. The term "chronic bronchitis" is still used to define a productive cough that is present for at least three months each year for two years. Those with such a cough are at a greater risk of developing COPD. The term "emphysema" is also used for the abnormal presence of air or other gas within tissues.

[0032] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease-modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

[0033] In some embodiments, the inhibitor of the ICAM1 / PKR / ATF2 signalling pathway according to the invention targets specifically macrophages and particularly primary human monocyte-derived macrophages (hMDMs) and the alveolar macrophages. In other words, in some embodiments, the inhibitor of the ICAM1 / PKR / ATF2 signalling pathway according to the invention is designed to target macrophages. For example, antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence may be under the control of a macrophage-specific promoters.

[0034] Macrophage-specific promoters are well known in the art (e.g see for example Kang W S, et al. Gene Ther. 2014; Ellett F, et al. Blood. 2017; Walton E M, et al. PLoS One. 2015; Luo Y-L, et al. ACS Nano. 2018; Greaves D R, et al. Int J Hemato. 2002 ; He W, et al. Hum Gene Ther. 2006)

[0035] In some embodiment, the inhibitor of the ICAM1 / PKR / ATF2 signalling pathway is combined with a macrophage-targeted drug delivery system.

[0036] Macrophage-targeted drug delivery systems and methods for designing and producing drug delivery system to target macrophage are well known in the art (e.g. see for example Hu G, et al. Front. Immunol. 2019; Mukhtar M, et al. Expert Opin Drug Deliv. 2020; He W, et al. Advanced Drug Delivery Reviews. 2019; Chono S. Yakugaku Zasshi. 2007).

[0037] In one embodiment, the inhibitor of the ICAM1 / PKR / ATF2 signalling pathway according to the invention may be a low molecular weight compound, e. g. a small organic molecule (natural or not).

[0038] The term "small organic molecule" refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 10,000 Da, more preferably up to 5,000 Da, more preferably up to 2,000 Da and most preferably up to about 1,000 Da.

[0039] Particularly, an inhibitor of PKR can be the molecule C16 (Merck 527450-5MG) (see for example Watanabe Takao et al. Scientific Reports, 2020).

[0040] In one embodiment, the inhibitor of the ICAM1 / PKR / ATF2 signalling pathway according to the invention is an antibody.

[0041] Antibodies directed against ICAM1, PKR or ATF2 can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from camels, pigs, cows, horses, rabbits, goats, sheep, and mice, among others, or organotypic cultures of primary human cells from tonsils, lymph nodes or peripheral blood. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against ICAM1, PKR or ATF2 can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985). Alternatively, techniques described for the production of single chain antibodies (see e.g., U.S. Pat. No. 4,946,778) can be adapted to produce anti- ICAM1, PKR or ATF2 single chain antibodies. Compounds useful in practicing the present invention also include anti- ICAM1, PKR or ATF2 antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and / or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to ICAM1, PKR or ATF2.

[0042] Humanized or human anti- ICAM1, PKR or ATF2 antibodies and antibody fragments therefrom can also be prepared according to known techniques. "Humanized antibodies" are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for making humanized antibodies are described, for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No. 4,816,397). Human antibodies can be generated by organotypic cultures of primary human cells from tonsils, lymph nodes or peripheral blood (see Wagar Lisa E. et al. Nature Medicine volume 27, pagesl25-135 (2021)). In some embodiment, for this invention, neutralizing antibodies of ICAM1, PKR or ATF2 are selected.

[0043] In another embodiment, the antibody according to the invention is a single domain antibody against ICAM1, PKR or ATF2. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb. The term “VHH” refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3. The term “complementarity determining region” or “CDR” refers to the hypervariable amino acid sequences which define the binding affinity and specificity of the VHH.

[0044] The VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation. The VHH variants and modified form thereof may be produced under any known technique in the art such as in-vitro maturation.

[0045] VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2. Antigen-specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells. However, such VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations. The high affinity of VHHs from immune libraries is attributed to the natural selection of variant VHHs during clonal expansion of B-cells in the lymphoid organs of immunized animals. The affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations). VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies. VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells. For example, the “Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695). The “Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).

[0046] In one embodiment, the compound according to the invention is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).

[0047] Then, for this invention, neutralizing aptamers of ICAM1, PKR or ATF2 are selected.

[0048] In one embodiment, the compound according to the invention is a polypeptide.

[0049] In a particular embodiment the polypeptide is an antagonist of ICAM1, PKR or ATF2 and is capable to prevent the function of ICAM1, PKR or ATF2. Particularly, the polypeptide can be a mutated ICAM1, PKR or ATF2 or a similar protein without the function of ICAM1, PKR or ATF2.

[0050] In one embodiment, the polypeptide of the invention may be linked to a cell-penetrating peptide” to allow the penetration of the polypeptide in the cell.

[0051] The term “cell-penetrating peptides” are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012).

[0052] The polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptide or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. Preferably, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known.

[0053] When expressed in recombinant form, the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli.

[0054] In specific embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. In example adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.

[0055] A strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water- soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.

[0056] Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications. Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri -functional monomers such as lysine have been used by VectraMed (Plainsboro, N. J.). The PEG chains (typically 2000 daltons or less) are linked to the a- and e-amino groups of lysine through stable urethane linkages. Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain. The reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules. These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the drug / linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading). In general, increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory halflife of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomular filtration (e.g., less than 60 kDa).

[0057] In addition, to the polymer backbone being important in maintaining circulatory halflife, and biodistribution, linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue. For example, this type of tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology. Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.

[0058] In another embodiment, the inhibitor of the ICAM1 / PKR / ATF2 signalling pathway according to the invention is an inhibitor of ICAM1, PKR or ATF2 gene expression.

[0059] Small inhibitory RNAs (siRNAs) can also function as inhibitors of ICAM1, PKR or ATF2 expression for use in the present invention. ICAM1, PKR or ATF2 gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that ICAM1, PKR or ATF2 gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01 / 36646, WO 99 / 32619, and WO 01 / 68836).

[0060] In a particular embodiment, an siRNA against ATF2 according to the invention can have the following sequences:

[0061] SEQ ID NO:1: GCUUCAGAAGAUGACAUUA

[0062] SEQ ID NO:2: GGAAGUACCAUUGGCACAA

[0063] In a particular embodiment, an siRNA against PKR according to the invention can have the following sequences:

[0064] SEQ ID NO:3: GCGAGAAACUAGACAAAGU

[0065] Ribozymes can also function as inhibitors of ICAM1, PKR or ATF2 gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of ICAM1, PKR or ATF2 mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

[0066] Both antisense oligonucleotides and ribozymes useful as inhibitors of ICAM1, PKR or ATF2 gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and / or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

[0067] Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing Notch receptors or Notch ligands. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

[0068] Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, 1990 and in Murry, 1991). Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wildtype adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

[0069] Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigenencoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC / CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and mi croencap sul ati on .

[0070] In a particular embodiment, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter may be specific for macrophages. Thus, in preferred embodiments, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a macrophage-specific promoter. For example, a specific expression in myeoloid cells (monocytes, macrophages) may be obtained through the promoter of the lysozyme 2 gene (Lyz2) is suitable. The promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.

[0071] In a further aspect, the present invention relates to an inhibitor of the ICAM1 / PKR / ATF2 signalling pathway according to the invention in combination with one or more anti-COPD compound for use in the treatment of COPD in a subject in need thereof.

[0072] The term “anti-COPD” has its general meaning in the art and refers to compounds and therapeutic active agent used which can be used to treat the symptoms and the progression of the disease. Anti-COPD compounds can be bronchodilators like P2 agonists and anticholinergics or corticosteroids.

[0073] In a further aspect, the present invention relates to an inhibitor of ARL5b according to the invention in combination with one or more anti-asthma compound for use in the treatment of asthma in a subject in need thereof.

[0074] The term “anti-asthma” has its general meaning in the art and refers to compounds and therapeutic active agent used which can be used to treat asthma. Anti-asthma compounds can be beta2-adrenoceptor agonists like salbutamol, anticholinergic like ipratropium bromide or adrenergic agonists like epinephrine.

[0075] In some embodiments, the inhibitor of the ICAM1 / PKR / ATF2 signalling pathway of the present invention is administered sequentially or concomitantly with one or more therapeutic active agent.

[0076] In one embodiment, said additional active compounds may be contained in the same composition or administrated separately.

[0077] Typically an inhibitor of the ICAM1 / PKR / ATF2 signalling pathway according to the invention as described above are administered to the subject in a therapeutically effective amount.

[0078] By a "therapeutically effective amount" of the inhibitor of the ICAM1 / PKR / ATF2 signalling pathway of the present invention as above described is meant a sufficient amount of the inhibitor of ICAM, PKR or ATF2 for treating COPD and / or asthma at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the inhibitor of ICAM, PKR or ATF2 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 inhibitor of ICAM, PKR or ATF2 employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific inhibitor of ICAM, PKR or ATF2 employed; the duration of the treatment; drugs used in combination or coincidental with the specific inhibitor of ICAM, PKR or ATF2 employed; 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 inhibitor of ICAM, PKR or ATF2 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 inhibitor of ICAM, PKR or ATF2 of the present invention for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the inhibitor of ICAM, PKR or ATF2 of the present invention, preferably from 1 mg to about 100 mg of the inhibitor of ICAM, PKR or ATF2 of the present invention. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0079] In a particular embodiment, the inhibitor of ICAM, PKR or ATF2 according to the invention may be used in a concentration between 0.01 pM and 20 pM, particularly, the inhibitor of ICAM, PKR or ATF2 of the invention may be used in a concentration of 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 20.0 pM.

[0080] In some embodiments, the inhibitor of the ICAM 1 / PKR / ATF2 signalling pathway is administered to the respiratory tract (e.g. lungs).

[0081] According to the invention, the inhibitor of the ICAM1 / PKR / ATF2 signalling pathway of the present invention is administered to the subject in the form of a pharmaceutical composition. Thus, the invention also relates to a therapeutic composition comprising an inhibitor of the ICAM 1 / PKR / ATF2 signalling pathway for use in the improvement of phagocytosis in a subject in need thereof.

[0082] Typically, the inhibitor of the ICAM 1 / PKR / ATF2 signalling pathway of the present invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0083] In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. 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.

[0084] Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising the inhibitor of the ICAM1 / PKR / ATF2 signalling pathway of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The inhibitor of ARL5b of the present invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. 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. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized agent of the present inventions into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the typical methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the inhibitor of the ICAM1 / PKR / ATF2 signalling pathway of the present invention plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media, which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The pharmaceutical composition of the invention may conveniently be administered by any method that allows administration to the respiratory tract (e.g. lungs). For example, nasal drops can be instilled in the nasal cavity by tilting the head back sufficiently and apply the drops into the nares. The drops may also be inhaled through the nose. Alternatively, a liquid preparation may be placed into an appropriate device so that it may be aerosolized for inhalation through the nasal or buccal cavity. For administration by inhalation the compositions may be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant. Administered spray and drops can be a single dose or multiple doses. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation. It will be appreciated that the form and character of the pharmaceutically acceptable diluent is dictated by the amount of inhibitor of ICAM1 / PKR / ATF2 signalling pathway with which it is to be combined, the route of administration and other well-known variables. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0085] In another embodiment, the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the treatment of COPD and / or asthma in a subject in need thereof.

[0086] In a further aspect, the present invention relates to a method for improving phagocytosis in a subject in need thereof comprising a step of administering to said subject a therapeutically effective amount of an inhibitor of the ICAM1 / PKR / ATF2 signalling pathway.

[0087] The invention also provides kits comprising the inhibitor of the ICAM1 / PKR / ATF2 signalling pathway of the invention.

[0088] 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.

[0089] FIGURES:

[0090] Figure 1: ARL5b regulation in hMDMs and HeLa OHIO is dependent of the viral entry receptor ICAM1. (A-B) HeLa OHIO were treated for Ih with nothing, a control antibody, or an antibody against ICAM1 at 37°C. Afterwards, cells were infected at IxlO7 TCID50 / mL of HRV16 or mock infected (MI) for Ih at room temperature. 24h post infection, cells were lysed, RNA were extracted, and qRT-PCR were performed. (A) HRV, and (B) ARL5b expressions are presented as either (A) relative expression to the house keeping gene or (B) ratio to the Mi-no antibody condition. Data are presented as the mean + / - SD of 3 experiments performed in triplicates. (C) hMDMs were treated for Ih with nothing, or an antibody against ICAM1 at 37°C. Afterwards, cells were exposed to IxlO7TCID50 / mL of HRV16 or mock infected (MI) for Ih at either room temperature or 4°C. After Ih of exposure, cells were washed either with PBS or with an acid buffer. Cells were lysed right away, RNA were extracted, and qRT-PCR were performed. HRV expression is presented as relative expression to the house keeping gene. Data are presented as the mean + / - SD of 3 experiments. (D-F) hMDMs were treated for Ih with nothing, a control antibody, or an antibody against ICAM1 at 37°C. Afterwards, cells were exposed to IxlO7TCID50 / mL of HRV16 or mock infected (MI) for Ih at room temperature. 24h post exposure, cells were lysed, RNA were extracted, and qRT-PCR were performed. (D) HRV, (E) ARL5b and (F) Mxl expressions are presented as either (D) relative expression to the house keeping gene or (E-F) ratio to the Mino antibody condition. Data are presented as the mean + / - SD of 4 experiments performed in triplicates. (A-B, D-F) Two-way ANOVA statistical analyses were performed. **p<0.01; ***p<0.001; ****p<0.0001. ns: not significative.

[0091] Figure 2: ARL5b induction in hMDMs is dependent of PKR. (A-B) hMDMs were treated for Ih with nothing, DMSO, or 50 pM of C16 (PKR inhibitor) at 37°C. Afterwards, cells were exposed to IxlO7TCID50 / mL of HRV16 or mock infected (MI) for Ih at room temperature. 24h post exposure, cells were lysed, RNA were extracted, and qRT-PCR were performed. (A) ARL5b, (B) Mxl, and (C) TNFa expressions are presented as ratio to the corresponding MI conditions. (D) HeLa OHIO were treated for Ih with nothing, DMSO, or 50 pM of C16 (PKR inhibitor) at 37°C. Afterwards, cells were infected at IxlO7TCID50 / mL of HRV16 or mock infected (MI) for Ih at room temperature. 24h post infection, cells were lysed, RNA were extracted, and qRT-PCR were performed. ARL5b expression is presented as ratio to the corresponding MI conditions. (A-D) Data are presented as mean + / - SD of at least 3 experiments performed in triplicates. Two-way ANOVA statistical analyses were performed. *p<0.05; **p<0.01; ****p<0.0001.

[0092] Figure 3: ARL5b induction in hMDMs is dependent of ATF2. (A-E) hMDMs were transfected with 100 nM of si RNA control (si Ctrl) or siRNA against ATF2 (siATF2-l or si ATF2-2), or a combination of siATF2 (each at 50 nM). 72h post transfection, cells were exposed to IxlO7TCID50 / mL of HRV16 or mock infected (MI) for Ih at room temperature. 24h post exposure, cells were lysed, RNA were extracted, and qRT-PCR were performed. (A) ATF2, (B) ARL5b, (C) Mxl, (D) TNFa and (E) HRV expressions are presented as either (A, C-E) ratio to the corresponding Ml-siCtrl condition or (E) relative expression normalised to the house keeping gene. (A,B-E) Data are presented as mean + / - SD of at least 3 experiments performed in triplicates. Two-way ANOVA statistical analyses were performed. *p<0.05; **p<0.01; ****p<0.0001.

[0093] EXAMPLE:

[0094] Material & Methods

[0095] Cell Culture.

[0096] Whole blood of healthy donors was obtained from the Etablissement Frangais du Sang (INSERM agreement #18 / EFS / 030) ensuring that all donors gave a written informed consent and providing anonymized samples. Peripheral blood mononuclear cells (PBMCs) were isolated through a Ficoll-Paque (GE Healthcare) density gradient. Cells were further isolated by a 56% Percoll density gradient (GE Healthcare). PBMCs were plated in the presence of 4 ng / mL GM-CSF and 0.5 ng / mL M-CSF in RIO medium (RPMI 1640 (Life Technologies) supplemented with 10% FBS (Gibco), 10 mM HEPES (Gibco), ImM sodium pyruvate (Gibco), 100 pg / ml penicillin / streptomycin (Gibco) and IX non-essential amino acids (Gibco)). Medium and cytokines were replaced 4 days post plating. 7 days post plating, medium was changed to macrophage medium (RPMI 1640 (Life Technologies) supplemented with 10% FCS (Gibco), 100 pg / ml penicillin / streptomycin (Gibco) and 2 mM L-glutamine (Gibco)). After 10 days of differentiation, primary human monocyte-derived macrophages (hMDMs) were used as described in the figures.

[0097] HeLa OHIO cells were purchased from the European Collection of Authenticated Cell Cultures (ECACC) and were cultured in complete HeLa OHIO medium (DMEM GlutaMax with 25 mM D-glucose (Life Technologies) supplemented with 10% FCS (Gibco), 100 pg / ml penicillin / streptomycin and 2 mM L-glutamine (Gibco)). Cells were passaged 3 times a week.

[0098] Human rhinovirus production.

[0099] Human Rhinovirus 16 (HRV16) (VR-283, strain 11757, lot 62342987) was purchased from the ATCC. To produce viral stocks, HeLa OHIO cells were grown to 80% confluence in 6 well plates and infected in 300 pL of virus medium (DMEM GlutaMax containing 25 mM D- glucose supplemented with 10% FCS and 2 mM L-glutamine) in the presence of 0.25 x 107TCID50 / mL HRV16 or control medium (Mock infected or MI). Infection was performed at room temperature for Ih with agitation. Then, medium was completed to a final volume of 2 mL per well. Once the cytopathic effect reached 90% of the cells in the HRV16 condition, cultures were frozen and thawed 3 times. Supernatants were collected, centrifuged for 15 min at 3900 rpm, and filtered at 0.22 pm. 1 mL stocks were generated and kept at -80°C.

[0100] Quantification of the tissue culture infectious dose 50 (TCID50) of HRV16,

[0101] 15,000 HeLa OHIO cells were plated per well in a 96 well plate. After 48 h, cells were infected with HRV16 or MI. HRV16 and MI were diluted 10-fold from undiluted to 10-9 in virus medium and 100 pL of each dilution was added to the cells in 6 replicate wells for HRV16 and 2 replicate wells for MI. 100 pL of virus medium was added to 8 replicate wells as a control. Cells were cultured at 37°C until a cytopathic effect was observed in 50% of the wells (72h on average). TCID50 was calculated using the Spearman-Karber formula.

[0102] HRV16 infection.

[0103] Macrophages or HeLa OHIO were washed once in virus medium. HRV16, or MI stocks were added to the cells to achieve 1 x 107TCID50 / mL. Alternatively, for the indicated experiments, HRV16 and MI were diluted for the infection as follow: Dose 1 = 1 x 107TCID50 / mL, Dose 2 = 5 x 106TCID50 / mL, Dose 3 = 2.5 x 106TCID50 / mL, Dose 4 = 12.5 x 106TCID50 / mL. Infection was performed at room temperature for 1 h with agitation. Cells were then washed with virus medium and further cultured in macrophage medium or HeLa OHIO medium, respectively, for the indicated amount of time.

[0104] Cytokines, inhibitors, and antibodies treatments.

[0105] Differentiated hMDMs or HeLa OHIO were infected or not with HRV16 to achieve the indicated TCID50. After the infection Ih at room temperature, cells were treated for 24h with four different doses of interferon beta (IFNP; Peprotech, 300-02BC) as follows: Dose 1 = 1 pg / mL, Dose 2 = 100 ng / mL, Dose 3 = 10 ng / mL, Dose 4 = 1 ng / mL.

[0106] Differentiated hMDMs were pre-treated with 50 pM C16 (inhibitor of PKR; Merck 527450-5MG) or with an equivalent amount of DMSO as control for Ih. Then cells were washed with macrophage medium and infected or not as indicated.

[0107] Differentiated hMDMs or HeLa OHIO were incubated with an antibody against human ICAM1 (Biotechne, BBA3) or a control IgG antibody (Abeam, ab 170190) at a final concentration of 10 pg / mL for Ih before and during the infection with HRV. Then cells were washed with macrophage medium. Alternatively, after the infection, the medium containing the virus was removed, then the cells were washed on ice with acidic medium (RPMI 1640 (Life Technologies) supplemented with 50 mM of NaAc, pH 2) for 2 min and further washed with PBS. The two washes were repeated once before cell lysis. siRNA transfection. Differentiated hMDMs were transfected with control siRNA directed against Luciferase, or siRNAs against ATF2 and PKR. Shortly, for a well of 24-wells plate, 0.1 ml of Opti-MEM (Gibco) was mixed with 100 nM of the indicated siRNA (or 50 nM of each siRNA in case of combination of two siRNAs) and with 0.8 pL of Lipofectamine RNAiMax (Invitrogen) in this order. The mix was inverted 5 times and incubated for 20 min at RT. In the meantime, the culture medium of the differentiated hMDMs was replaced with fresh medium. Then, siRNA mix was added drop by drop to the cells. Plate was rocked softly to mix and incubated at 37°C for 72h.

[0108] RNA extraction.

[0109] After the indicated infection time and / or treatment, macrophages or HeLa OHIO were washed once with PBS and lysed in 350 pL of LBP (Macherey-Nagel, 740984.250). Extraction was performed following the manufacturer’s instructions.

[0110] Reverse transcription.

[0111] RNAs were quantified using a NanoDrop 2000 Spectrophotometer. mRNA was reverse transcribed using a high-capacity cDNA reverse transcription kit (ThermoFisher Scientist, 4368813). Reaction was performed using 500 ng of mRNA in 10 pL, 2 pL of 10X RT Buffer, 0.8 pL of 25X dNTP Mic (100 mM), 2 pL of 10X Random Primers, 1 pL of Multi Scribe™ Reverse Transcriptase and 4.2 pL of nuclease-free H2O. Samples were put in a thermocycler and the following steps were applied: 10 min at 25°C, 120 min at 37°C, 5 min at 85°C, and 4°C on hold.

[0112] Chromatin immuno-precipitation.

[0113] First, DNA and proteins were crosslinked as follow: cells were washed once with PBS, then incubated with 1% PF A with agitation for 10 min at RT and washed once with PBS IX. To stop the cross-linking, cells were incubated with agitation 5 min with 0.125 M glycine diluted in PBS before being washed once with PBS IX. Cells were scraped in 1 mL PBS IX, collected and centrifuged at 3,500 rpm for 10 min at 4°C. Pellets were washed once with PBS IX + 1 mM PMSF + IX protease inhibitors cocktail (PIC; Roche Diagnostic, 11836170001). Cells are centrifuged as before and pellets can be kept at -80°C or lysed in lysis buffer (50 mM Tris HCL pH8, 10 mM EDTA, 1% SDS, IX PIC, 1 mM PMSF). Cells are sonicated to obtain fragments between 300 and 500 bp and centrifuged at 13,000 rpm for 10 min at 4°C. Supernatants are pre-cleared by incubating them with Protein A Sepharose beads for 2 h at 4°C with rotation. Protein A Sepharose beads are discarded by a 1 min centrifugation at 1,200 rpm and at 4°C. Antibodies are added to the samples and the samples are rotated at 4°C O / N. The following antibodies are used: anti -Tri -Methyl -Hi stone H3 (Lys27) (diluted 1 :50; Cell Signalling Technologies, 9733S), anti-H3K27Ac (1 pg / sample; Diagenode C15410196), anti- EZH2 (diluted 1 : 100; Cell Signalling Technologies, 5246S), control Rabbit IgG (1 : 100; Cell Signalling Technologies, 2729S). The next day, the samples were incubated with magnetic beads coated with protein A for 2 h at 4°C with rotation. Subsequently, the magnetic beads were washed 4 times with RIPA buffer (10 mM Tris HC1 pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton, 0.1% SDS, 0.1% Na-Deoxycholate, IX PIC, 1 mM PMSF), 1 time with LiCl buffer (0.25 M LiCl, 0.5% NP40, 0.5% Na-Deoxycholate, 10 mM Tris HC1 pH 8, 1 mM EDTa, IX PIC, 1 mM PMSF), and 1 time with TE buffer (10 mM Tris HC1 pH 8, 10 mM EDTA). The cross-linking was reversed in Elution buffer (20 mM Tris HC1 pH 7.5, 5 mM EDTA, 50 mM NaCl, 1% SDS, 50 pg / mL Proteinase K) at 68°C for 2h with 1,300 rpm agitation. DNA is extracted in Phenol:Chloroform:Isoamyl Alcohol (25:24: 1) and resuspended in DNase free water. qPCR. qPCR was performed using SensiFAST SYBRNo-ROX Kit (biotechnofix, BIO-98050) and specific primers detecting the indicated genes. TBP was used as a house-keeping gene. All primers are listed in Table SI.

[0114] Western blots.

[0115] HeLa OHIO were lysed in NP-40 lysis buffer (20 mM Tris HC1, pH 7.5, 150 mM NaCl, 0.5% NP-40, 50 mM NaF and 1 mM sodium orthovanadate) supplemented with PIC for 15 min on ice. Lysates were centrifuged at 13,000 rpm for 10 min at 4°C. The supernatants were collected and stored at -20°C until further notice. Protein concentration was assessed with a Pierce™ BCA protein assay kit (ThermoFisher Scientist, 23225). 30 pg of protein were mixed with loading buffer (final concentrations: glycerol 10%, DTT 0.1%, SDS 2%, Tris pH 6.8 0.062 M) then loaded and run onto a SDS-PAGE gel (Bolt 4-12% Bis-Tris, Invitrogen). Proteins were transferred onto a poly vinylidene difluoride (PVDF) membrane (Merck Millipore, IPVH00010) at 4°C overnight. Membranes were then incubated in blocking solution (TBS IX, 0.1% Tween- 20 supplemented with 5% milk) for 2 h. Membranes were rinsed with TBS IX, 0.1% Tween- 20 and incubated with the EZH2 antibody (Cell Signalling Techonology, 5246S) diluted 1 : 1,000 in the blocking solution overnight with agitation. The membrane was further washed and incubated with the anti-rabbit IgG HRP-coupled secondary antibody (Jackson Immunoresearch, 711-035-152) in blocking buffer for 45 min. Detection was performed using Pierce™ ECL Western Blotting Substrate (ThermoFisher Scientist, 32106) and bands imaged by Fusion (Vilber Lourmat) and quantified in ImageJ. Statistics.

[0116] Statistical tests were performed using Graphpad Prism > version 9 software. All statistical tests are listed in the figure legends and significance is indicated as follow: *: p<0.05; **: p<0.01; ***: p<0.001.

[0117] Results

[0118] HRV16 does not replicate in hMDMs but triggers interferon and pro-inflammatory cytokines responses.

[0119] To evaluate the potential of HRV16 to elicit an innate immune response in human monocyte-derived macrophages (hMDMs), cells were exposed to 1.107TCID50 / mL of HRV16 for 1 hour, and RNA samples were collected at various time points post-infection. HRV RNA was detectable at 0 hour post-infection (h.p.i., the time following the 1-hour exposure to the virus) but rapidly declined (data not shown). Although HRV was still detectable at 120 h.p.i., there was no evidence of replication, as indicated by the absence of an increase in the quantity of viral RNA post-infection. Notably, IFN-a and IFN-P became detectable between 4 h.p.i. and 24 h.p.i., peaking at approximately 25-fold induction at 8 h.p.i., suggestive of an active type I IFN response (data not shown). This IFN induction was followed by the activation of interferon- stimulated genes (ISGs), exemplified by a significant increase in Mxl expression from 8 h.p.i. onwards, reaching a maximum 60-fold induction at 24 and 48 h.p.i. (data not shown). In agreement with our previous study (13), ARL5b exhibited a significant 5-fold induction at 8 h.p.i., gradually returning to basal levels by 72 h.p.i. (data not shown). Additionally, while IL- 6 displayed a steady increase from 8 h.p.i. to 48 h.p.i., TNF-a expression mirrored the patterns of IFN-a and IFN-P, with a peak at 8 h.p.i. (data not shown).

[0120] In our earlier research, we demonstrated that in HRV16-permissive cells, specifically the HeLa OHIO cell line, viral replication was associated with a decrease in ARL5b expression (13). To investigate whether this decrease was linked to an immune response in these cells, we analyzed the type I IFN response. Interestingly, IFN-a and IFN-P mRNAs were not detectable at any of the tested time points (data not shown), and consequently, no induction of Mxl was observed (data not shown).

[0121] Collectively, these findings indicate that whereas HRV16 can enter macrophages, it does not replicate in these cells. However, HRV16 detection induces interferon and pro- inflammatory responses in these cells. ARL5b is not an interferon stimulated gene.

[0122] ARL5b has been previously proposed to be an interferon-stimulated gene (ISG) (27). To investigate whether the induction of ARL5b in macrophages resulted from HRV16- mediated activation of the interferon response, hMDMs were exposed to increasing doses of HRV and / or increasing doses of recombinant IFN-p. Of note, in the condition in which HRV and recombinant IFN-P were combined, both concentrations were gradually increased. In hMDMs, HRV RNA levels were detectable at exposures of IxlO6or IxlO7TCID50 / mL (data not shown). ARL5b induction was modestly observed at IxlO6TCID50 / mL (2-fold induction) but significantly increased at IxlO7TCID50 / mL (5-fold induction), both in the presence and absence of IFN-P (data not shown). Notably, even at the highest dose of recombinant IFN-P (1 pg / mL), no induction of ARL5b was observed, and there was no additional effect of recombinant IFN-P in the presence of HRV16 (data not shown). As a control, Mxl, a well- known ISG, was significantly upregulated with the highest dose of HRV16 (18-fold induction) and recombinant IFN-P (7 -fold induction) (data not shown), showing that the IFN-P stimulation was small but efficient in the experimental conditions used. Similar results as ARL5b were obtained for IL-6 and TNFa (data not shown).

[0123] In HeLa OHIO cells, where no IFN response was detected in the presence of HRV16, recombinant IFN-P had no impact on HRV RNA levels (data not shown). HRV16-mediated inhibition of ARL5b was observed only when cells were infected with IxlO7TCID50 / mL, resulting in a 40% decrease in the gene expression when comparing “MI” to “HRV” or “recombinant IFN-P” to “HRV + recombinant IFN-P” conditions (data not shown). ARL5b decrease was confirmed at the protein level in HRV16 infected cells compared to MI cells (data not shown). Whereas not significant, recombinant IFN-P induced a small increased of ARL5b. Thus, even though the expression of ARL5b was significantly decreased when comparing the “recombinant IFN-P” condition to the “HRV + recombinant IFN-P” condition, no differences were observed between the “MI” and the “HRV + recombinant IFN-P” conditions.

[0124] Collectively, our findings indicate that ARL5b is not an ISG in hMDMs or HeLa OHIO cells. Therefore, the HRV16-mediated upregulation of ARL5b is dependent on one or several signalling pathway(s) activated upon exposure of macrophages to HRV16.

[0125] ARL5b modulation and immune induction by HRV16 is dependent of ICAML

[0126] To elucidate the signaling pathways activated upon exposure to HRV 16, we initially investigated the potential involvement of ICAM1, the cell surface receptor that mediates HRV16 entry in permissive cells. A neutralizing antibody against ICAM1 was used to assess its role in ARL5b modulation.

[0127] In HeLa OHIO cells, the inhibition of ICAM1 fully prevented viral replication, most likely by preventing viral entry (Figure 1A). In that case, the HRV16-mediated inhibition of ARL5b was lost, indicating that viral entry and / or replication is essential for the observed phenotype (Figure IB). Notably, ICAM1 mRNA levels were not modulated by HRV16 infection (data not shown).

[0128] Since HRV16 does not replicate in hMDMs, we first examined whether HRV16 binding to its receptor and / or viral entry, as attested by the presence of viral RNA, was dependent on ICAM1. After 1 hour of contact at room temperature (RT), HRV16 was detected in hMDMs (Figure 1C). To determine if the detected viral genome resulted from the entry of virions into the cells or the binding of viruses to the cell surface, hMDMs were washed with an acid buffer to detach all non-intemalized virions. HRV16 RNA levels decreased roughly by half with the acid wash, indicating that at least part of the virions did enter the cells. Significantly, the anti- ICAM1 antibody prevented HRV16 internalization, indicating that ICAM1 serves as a receptor for HRV16 in hMDMs (Figure 1C). At 4°C, the levels of HRV16 detected were decreased, highlighting endocytosis-dependent entry (Figure 1C). These results were confirmed at 24 h.p.i., as no HRV16 was detected in anti-ICAMl treated cells (Figure ID). Notably, ICAM1 mRNA levels were not modulated by HRV16 (data not shown). Interestingly, ICAM1 inhibition prevented HRV16-mediated induction of ARL5b, Mxl, and TNFcr (Figures 1E-F).

[0129] In summary, our results demonstrate that the modulation of ARL5b is dependent on ICAMl / viral entry in hMDMs and HeLa OHIO cells. Additionally, immune induction in hMDMs was also dependent on ICAMl / viral entry.

[0130] HRV16 activation of ARL5b and immune response in hMDMs is dependent of PKR,

[0131] To further understand the signalling pathway(s) activated by HRV16 following ICAM1 binding / viral entry, we investigated the potential involvement of Protein Kinase R (PKR), which plays a role in various signalling pathways, including cellular responses to pathogen detection and the IFN response (21,28). We assessed whether PKR could be involved in HRV16-mediated immune response induction.

[0132] In our experiments, cells were treated with Cl 6, an inhibitor of PKR, an equivalent amount of DMSO as a control, or left untreated. HRV16-induced a 2 to 8-fold upregulation of ARL5b, which was significantly decreased by Cl 6, indicating the involvement of PKR in its induction (Figure 2A). As expected, PKR inhibition also prevented the HRV16-induced upregulation of Mxl (Figure 2B). Interestingly, in some donors, C16 treatment unexpectedly led to an upregulation of TNF induction by HRV16 (Figure 2C), suggesting a complex interplay of signalling pathways. These results indicate that PKR is implicated in the signalling pathway(s) activated by HRV16 to induce ARL5b expression.

[0133] Similar experiments were conducted on HeLa OHIO cells to assess if PKR could be involved in the inhibition of ARL5b expression in these cells. However, no significant difference was observed between C16-treated cells and the control conditions (Figure 2D). These data suggest that in HeLa OHIO cells, the inhibition of ARL5b does not depend on PKR signalling. The contrasting results between hMDMs and HeLa OHIO cells highlight potential cell-type-specific differences in the signalling pathways activated by HRV16.

[0134] HRV16 activation of ARL5b and Mxl in hMDMs is dependent of ATF2,

[0135] To gain further insight into the signalling induced by HRV16 in hMDMs, we focused on transcription factors known to be activated by PKR, including c-Jun, ATF2, and NF-KB 23. ATF2, previously shown to be activated by HRV16 (16), was of particular interest in deciphering the mechanisms involved in the HRV16-mediated induction of ARL5b.

[0136] ATF2 was selectively depleted using siRNAs in hMDMs and its depletion was assessed by qPCR. Compared to the siRNA control (si Ctrl), siATF2-l reduced ATF2 expression by 60%, as observed in both the siATF2-l and siATF2-l+2 conditions, whereas siATF2-2 depleted ATF2 by 45% (Figure 3A). At the protein level, ATF2 was depleted by 50% using siATF2-l whereas siATF2-2 did not lead to any protein depletion at the tested time point (Data not shown). Whereas in the siCtrl condition a 2.3-fold increase of ARL5b was observed in presence of HRV16, depletion of ATF2 by siRNAs resulted in 1.6-fold increase of ARL5b induction only (Figure 3B). Therefore, ATF2 depletion led to a 30% decrease in ARL5b induction, highlighting a role of ATF2 in HRV16-mediated ARL5b induction (Figure 3B). Similar effects were observed for Mxl, whereas ATF2 played no significant role in TNFa induction (Figures 3C-D). Surprisingly, in some experiments, HRV16 RNA levels increased upon ATF2 depletion (Figure 3E).

[0137] In summary, these results collectively indicate that HRV16 induces ARL5b expression through an ICAM1 / PKR / ATF2 signalling axis in hMDMs.

[0138] HRV16 epigenetically regulates ARL5b promoter.

[0139] Finally, we aimed to investigate whether ARL5b expression is regulated at the epigenetic level. To explore this, HeLa OHIO cells were exposed to the virus, and at various time points post-infection, DNA and proteins were crosslinked and analyzed by chromatin immunoprecipitation (ChIP). Given that ARL5b expression is repressed by HRV16 in these cells, we examined if the repressive epigenetic mark H3K27Me3 exhibited differential enrichment on the ARL5b promoter. At 24 h.p.i., H3K27Me3 was found to be enriched on the ARL5b promoter in HRV16-treated cells compared to Mi-treated cells or the 4 h.p.i. conditions (Data not shown). This increase in H3K27Me3 could potentially explain the observed repression of the gene. The trimethylation of lysine 27 of histone 3 is mediated by the methyltransferase EZH2 (26). Thus, we then assessed if HRV16 infection in HeLa OHIO could modify EZH2 expression. While there was no significant modulation of EZH2 at the mRNA level (Data not shown), protein expression increased by 2-fold, as demonstrated by immunoblotting (Data not shown). Concurrently, EZH2 recruitment to the ARL5b promoter was increased at 24 h.p.i. (Data not shown).

[0140] Next, we examined if similar epigenetic modulations of ARL5b promoter could be observed in macrophages. H3K27Me3 presence on the ARL5b promoter was not different between HRV16 and Mi-challenged hMDMs (Data not shown). As ARL5b expression is increased by HRV16 in hMDMs, we studied the level of the activating epigenetic mark H3K27Ac on the ARL5b promoter. H3K27Ac was twice as much present on the ARL5b promoter in HRV16-challenged cells than in the MI condition (Data not shown). Finally, to investigate if ATF2 activation could be linked to the observed increase in H3K27Ac (29), hMDMs were depleted for ATF2, exposed to HRV16, and analyzed by ChIP. Results showed that ATF2 depletion was associated with a decrease in H3K27Ac on the ARL5b promoter (Data not shown).

[0141] In summary, these data demonstrate that HRV16 induces epigenetic modifications on the promoter of ARL5b, either associated with repression in HeLa OHIO or with activation in hMDMs. The HRV16-mediated increase in H3K27Ac on the ARL5b promoter in hMDMs is dependent on ATF2.

[0142] Conclusion:

[0143] In summary, our findings indicate that HRV16 does not replicate in macrophages, yet it elicits type I interferon and pro-inflammatory responses. We demonstrated that the induction of ARL5b is dependent on a ICAM1 / PKR / ATF2 signalling axis and subsequent epigenetic modifications in macrophages. In HeLa OHIO cells, the inhibition of the restriction factor ARL5b is dependent on ICAM1, but not on PKR. This dual regulation opens avenues for potential targeted treatments to prevent ARL5b induction in macrophages and to reactivate phagocytic functions in these cells, facilitating effective bacterial clearance.

[0144] REFERENCES:

[0145] 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. An inhibitor of the ICAM1 / PKR / ATF2 signalling pathway for use in the improvement of phagocytosis in a subject in need thereof.

2. The inhibitor for use according to the claim 1 wherein the inhibitor is an inhibitor of PKR.

3. The inhibitor for use according to claims 1 or 2 wherein the subject is afflicted with an inflammatory disease.

4. The inhibitor for use according to claim 3 wherein the inflammatory diseases is a chronic obstructive pulmonary disease (COPD) or asthma.

5. The inhibitor for use according to claim 4 wherein the chronic obstructive pulmonary disease (COPD) or asthma is induced by a respiratory virus.

6. The inhibitor for use according to claim 5 wherein the respiratory virus is the human rhinovirus (HRV) or the Human respiratory syncytial virus (HRSV).

7. The inhibitor for use according to any claims 1 to 6 wherein said inhibitor is a siRNA.

8. The inhibitor for use according to any claim 7 wherein said siRNA has a nucleic acid sequence as set for SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:3.

9. A therapeutic composition comprising an inhibitor of ICAM1 / PKR / ATF2 signalling pathway for use in the improvement of phagocytosis in a subject in need thereof.

10. A method for improving phagocytosis in a subject in need thereof comprising a step of administering to said subject a therapeutically effective amount of inhibitor of ICAM1 / PKR / ATF2 signalling pathway.