The invention relates to Interleukin 12 (IL12) or derivative thereof for use in the prevention and/or the treatment of secondary disease, in particular nosocomial disease.
The present invention also relates to pharmaceutical composition comprising Interleukin 12 (IL12) or derivative for use in the prevention and/or the treatment of secondary disease, in particular nosocomial disease.
The present invention finds application in the therapeutic and diagnostic medical technical fields.
Pneumonia is the leading cause of death from infectious disease (Mizgerd, 2006 [41]). The risk of developing pneumonia increases following severe primary infections and reaches 30-50% for critically ill patients recovering from a first episode of infection (van Vught et al., 2016a [59]). It is currently accepted that susceptibility to secondary pneumonia increases due to acquired immune defects collectively known as sepsis-induced immunosuppression (Hotchkiss et al., 2013a [26]; Roquilly and Villadangos, 2015 [49]). In-depth understanding of the mechanisms involved is vital to prevent and treat secondary pneumonia in patients recovering from a primary infection.
Healthy lungs are colonized by bacteria whose burden is continuously controlled by mucosal immunity (Charlson et al., 2011 [16]). Infection by pathogenic bacteria disrupts this balance and can induce lung injury through direct damage caused by the pathogen, or through immunopathology elicited by the effector mechanisms of immunity. Therefore, a healthy immune response should maximize the deployment of effector mechanisms against the pathogen while minimizing the damage of self-tissues that may ensue.
It is well known that Nosocomial infections (NI) increase morbidity and mortality. In particular, the most common NI are surgical site infections, infections of the gastrointestinal tract and respiratory tract, urinary tract infections, and primary sepsis. (Ella Ott, Dr. med., et al. “The Prevalence of Nosocomial and Community Acquired Infections in a University Hospital An Observational Study Dtsch Arztebl Int. 2013 August; 110(31-32): 533-540 [69]).
In addition, it is well known that the NI, when due to bacterial infection, are strong infection which are, in most of the time, resistant to the most common antibiotic compound. Thus, these therapies have to be improved since they do not allow to effectively treat the NI and/or are less effective in the treatment than expected.
There is therefore a real need to find a method and/or a compound which allows more efficient treatment and/or effective treatment of Nosocomial Infections (NI). In particular there is a real need to find new strategies, i.e. new targets/pathways, in the treatment Nosocomial infections (NI).
The present invention meets these needs and overcomes the abovementioned drawbacks of the prior art with the use of Interleukin 12 for the prevention and/or treatment of secondary disease, in particular nosocomial disease.
In particular, the macrophages and dendritic cells (DC) orchestrate immunity and tolerance, the inventors have compared their functional properties before, during and after resolution of a first infection, for example pneumonia and demonstrated that both cell types showed profound alterations—which we summarize as ‘paralysis’—in the latter case. Paralysis was caused by excessive release of local mediators of restoration of immune homeostasis. The inventors have supported that DC and macrophage dysfunction is an important contributor to protracted immunosuppression after bacterial or viral primary sepsis and increased susceptibility to secondary infection, for example Nosocomial Infections (NI) such as a secondary pneumonia.
The inventors have also demonstrated that the use of Interleukin 12 allows to treat secondary infection, for example Nosocomial infections whatever is the Nosocomial infection. In other words, the inventors have demonstrated that Interleukin 12 allows to treat Nosocomial infections and also to inhibit protracted immunosuppression after, for example bacterial and/or viral and/or fungus primary sepsis and/or infections.
The inventors have also demonstrated that the use of inhibitor of transforming growth factor-beta allows to treat secondary infection, for example Nosocomial infections whatever is the secondary infection, for example Nosocomial infection. In other words, the inventors have also demonstrated that the use of inhibitor of transforming growth factor-beta inhibitor allows to treat secondary infections, for example Nosocomial infections and also to inhibit the protracted immunosuppression after bacterial and/or viral and/or fungus primary sepsis.
The inventors have also demonstrated that Dendritic Cells (DC), for example DC that develop in the lung after resolution of a first infection, for example pneumonia have diminished capacity to present antigens and to secrete immunostimulatory cytokines, which makes them less capable of initiating adaptive and innate immunity against a secondary infection, for example a bacterial and/or viral and/or fungus infection. In addition, they produce higher levels of TGF-β, which promotes accumulation of Treg cells.
The inventors have also demonstrated that the signals that promote the differentiation of DC to this paralyzed state are not directly associated with the pathogen that caused the primary infection; they are mediated by secondary cytokines acting locally. Accordingly, this effect appears whether the disease and/or the pathogen. Accordingly, the inventors have demonstrated that IL-12 or inhibitor of transforming growth factor-beta allow to treat any secondary infection, for example nosocomial infection.
The inventors have also demonstrated that Interleukin 12 allows to treat secondary infections, nosocomial infections and/or also to inhibit protracted immunosuppression after, for example bacterial and/or viral and/or fungus primary sepsis and/or infections and/or after conditions that could induce primary inflammation, for example trauma, hemorrhage, infection.
In addition, the inventors have also demonstrated that Interleukin 12 (IL-12) allows to prevent secondary infections in a systemic way. In other words, the inventors have demonstrated that after a primary condition, for example bacterial and/or viral and/or fungus primary sepsis and/or infections and/or after conditions that could induce primary inflammation, for example trauma, hemorrhage and/or infection; IL-12 allows to prevent secondary infection whatever the localization and/or the organ infected. In particular, the inventors have demonstrated unexpectedly that IL-12 provides a systemic protection which advantageously would allow to prevent and/or treat a secondary infection which could appear at different localization and/or in different organ with regards to the primary infection.
Moreover, the inventors have demonstrated that the present invention allows surprisingly and unexpectedly to prevent and/or treat secondary infections whatever the cause of the secondary infections. In other words, the origin and/or cause of the secondary infection may be advantageously different from the origin and/or cause of the primary infection.
An object of the present invention is Interleukin 12 (IL12) or derivative thereof for use in the prevention and/or the treatment of secondary infection.
Another object of the invention is interleukin 12 (IL12) or derivative thereof for use as a medicament in the prevention and/or the treatment secondary infection.
In the present interleukin 12 refers to a heterodimeric cytokine encoded by two separate genes, IL-12A (p35) and IL-12B (p40).
In the present interleukin 12 may be any interleukin 12 known from one skilled in the art that can be administered to a patient in need thereof. It may be for example a commercially available Interleukin 12, for example Interleukin 12 commercialized by Abcam, a recombinant human interleukin 12 (rHuIL-12) as disclosed in Gokhale et al. Single low dose rHuIL-12 safely triggers mutyilineage hematopoietic and immune-mediated effects, Experimental Hematology & oncology 2014, 3:11, pages 1-18 [70].
In the present interleukin 12 may be heterodimeric cytokine comprising an IL-12A (p35) amino acid sequence of RVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQT STLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLK MYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVG EADPYRVKMKLCILLHAFSTRWTINRVMGYLSSA (SEQ ID NO 1) and an IL-12A (p40) amino acid sequence of MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGK TLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLK CEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVT LDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIK PDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKE TEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYN SSCSKWACVPCRVRS (SEQ ID NO 2).
In the present, derivative of IL12 may any derivative of IL-12 known to one skilled in the art. For example, derivative of IL12 may be acetylated IL12, for example an acetylated, alkylated, methylated, methylthiolated, biotinylated, glutamylated, glycylated, glycosylated, hydroxylated, isoprenylated, prenylated, myristoylated, farnesylated, geranyl-geranylated, lipoylated, Phosphopantetheinylated, phosphorylated, sulphated, selenated or amidated IL-12.
For example acetylation may be carried out with addition of an acetyl group derived from acetyl-CoA at the N-terminal end; alkylation, or the addition of an alkyl, methyl or ethyl group; methylation may be carried out with addition of a methyl group, generally on the amino acids lysine or arginine; methylthiolation may be carried out with addition of a methylthio group; biotinylation may be carried out with the acylation of a lysine by a biotin group; glutamylation may be carried out with covalent bonding of a glutamic acid residue to tubulin or other protein; glycylation, may be carried out with covalent bond of one or more (up to 40) glycine residues to the C-terminal end; Glycosylation may be carried out with addition of a glycosyl group to an asparagine, hydroxylysine, serine, or threonine residue, hydroxylation, may be carried out with addition of a hydroxyl group to a protein, most often on a proline or lysine residue forming hydroxyproline or hydroxylysine; isoprenylation, may be carried out with addition of an isoprenoid group, for example farnesol or geranylgeraniol; phosphopantetheinylation may be carried out with addition of a 4′-phosphopantetheinyl from coenzyme A, phosphorylation, may be carried out with addition of a phosphate group, typically on an acceptor serine, tyrosine, threonine or histidine; sulphation may be carried out with addition of a sulfate group to a tyrosine.
In the present derivative of IL12 may also encompass pro-drug of IL-12. In the present, pro-drug of IL-12 may any pro-drug of IL-12 known to one skilled in the art. For example prodrug of IL12 may be IL-12 modified with polymers, for example IL-12 conjugated with polyethylene glycol (PEG), IL-12 conjugated with polyoxyethylated glycerol, with polymers.
Interleukin 12 (IL12) or derivative thereof can be administered to humans and other animals orally, rectally, parenterally, intratracheally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments or drops), buccally, as oral or nasal spray, subcutaneously, or the like, depending on the severity of the infection to be treated. For example, the interleukin 12 (IL12) or derivative thereof may be administered, for example subcutaneous at doses of from about 2 to 20 μg, preferably from 5 to 15 μg, preferably equal to 12.5 μg on a single bolus.
For example, the interleukin 12 (IL12) or derivative thereof may be administered, for example subcutaneous at doses of from about 0.1 μg/kg to 1 μg/Kg body weight of the subject per day, one or more times a day, to achieve the desired therapeutic effect.
According to the invention, interleukin 12 (IL12) or derivative thereof may be administered on a single administration or repeated administration, for example one to three time per day, for example for a period up to 21 days.
In the present inhibitors of transforming growth factor-beta (TGF-β) may be any inhibitors known from one skilled in the art. It may be for example comprising antibodies against transforming growth factor-beta, antisense oligo, peptides, mouse antibody, ligand trap, small molecules, pyrrole-imidazole polyamide, inhibitor or TGF-β synthesis, humanized antibody.
In the present antibodies against transforming growth factor-beta may be any corresponding antibody known from one skilled in the art. It may be for example a commercially available antibodies. It may be for example antibody of any mammal origin adapted for the treatment of human being. It may be for example, antibodies obtained according to the process disclosed in Leffleur et al. 2012 [37] comprising administering 0.3 to 8 mg/kg of anti-tgf beta antibody (Trachtman et al. 2012 [55]).
In the present antibodies against transforming growth factor-beta may be mouse antibody, for example any mouse antibody known from one skilled in the art that could inhibit transforming growth factor-beta. It may be for example a commercially available mouse antibody, for example mouse antibody referenced 1D11, 2AR2, X1, 2C6, 8C4.
In the present antibodies against transforming growth factor-beta may be mouse antibody, for example any rat antibody known from one skilled in the art that could inhibit transforming growth factor-beta. It may be for example a commercially available rat antibody, for example rat antibody referenced TB2F.
In the present antibodies against transforming growth factor-beta may be a rabbit antibody, for example any rabbit antibody known from one skilled in the art that could inhibit transforming growth factor-beta. It may be for example a commercially available rabbit antibody, for example rabbit antibody referenced ab92486 commercialized by abcam or aa279)-390 commercialized by antibodies-online.com.
In the present antisense oligo may be any corresponding antisense oligo known from one skilled in the art that could inhibit transforming growth factor-beta. It may be for example a commercially available antisense oligo, for example P144, P17, LSKL commercialized by Trabedersen, Belagen-pumatucel-L.
In the present peptide may be any peptide known from one skilled in the art that could inhibit transforming growth factor-beta. It may be for example a commercially available peptides, for example peptide referenced P144, P17 or LSKL.
In the present ligand trap may be any ligand trap known from one skilled in the art that could inhibit transforming growth factor-beta. It may be for example a commercially available ligand trap, for example ligand trap referenced SR2F and/or soluble TbR2-Fc.
In the present small molecules may be any small molecules known from one skilled in the art that could inhibit transforming growth factor-beta. It may be for example a commercially available small molecules, for example small molecules referenced LY580276, LY550410, LY364947, LY2109761, SB-505124, SB-431542, SD208, SD093, Ki26894, SM16 and/or GW788388.
In the present pyrrole-imidazole polyamide may be any pyrrole-imidazole polyamide known from one skilled in the art that could inhibit transforming growth factor-beta. It may be for example a commercially available pyrrole-imidazole polyamide, for example pyrrole-imidazole polyamide referenced GB1201, GB1203.
In the present inhibitor of TGFb synthesis may be inhibitor of TGFb synthesis known from one skilled in the art. It may be for example a commercially available inhibitor of TGFb synthesis, for inhibitor of TGFb synthesis referenced (Lucanix), a humanized antibody, for example a commercially available humanized antibody selected from the group comprising Lerdelimumab (CAT-152) Metelimumab (CAT-192) Fresolimumab (GC-1008), LY2382770; STX-100, IMC-TR1).
In the present inhibitor of TGFb may also be any inhibitor disclosed in Akhurst et al, targeting the TGFβ signaling pathway in disease, Nature reviews, drug discovery Vol 11, October 2012, p 790-812 [1].
Inhibitors of transforming growth factor-beta (TGF-β) may be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments or drops), buccally, as oral or nasal spray, subcutaneously, or the like, depending on the severity of the infection to be treated.
The way of administration of inhibitors of transforming growth factor-beta (TGF-β) may be adapted with regards to the inhibitor used. One skilled in the art taking into consideration his technical knowledge would adapt the administration way to the used inhibitor.
The doses of inhibitors of transforming growth factor-beta (TGF-β) to be administered may be adapted with regards to the inhibitor used. One skilled in the art taking into consideration his technical knowledge would adapt the administered doses to the used inhibitor. For example, when the inhibitors of transforming growth factor-beta (TGF-β) is small molecules, for example LY2157299, it may be administered, for example at doses around 80 mg. For example, when the inhibitors of transforming growth factor-beta (TGF-β) is recombinant protein, for example Avotermin, it may be administered, for example at doses from 20 ng to 200 ng, preferably from 50 ng to 200 ng, more preferably 100 ng to 200 ng. For example, when the inhibitors of transforming growth factor-beta (TGF-β) is humanized antibody, for example IMC-TR1, it may be administered, for example at doses from 12.5 mg to 1600 mg.
According to the invention, inhibitors of transforming growth factor-beta (TGF-β) may be administered on a single time or repeated administration, for example one to three time per day, for example for a period up to 21 days.
In the present secondary infection means any infection which may occur after a primary infection and/or inflammation and/or postoperatively. It may be for example an infection occurring 1 to 28 days after the beginning of a primary infection, for example 5 to 12 day after the beginning of a primary infection. It may be also for example an infection occurring 1 to 21 days after the end of a primary infection for example 5 to 12 day after the end of the primary infection and/or the absence of any pathological sign and/or symptom.
In the present the secondary infection may be for example the origin and/or cause of the secondary infection may be advantageously different from the origin and/or cause of the primary infection.
In the present the secondary infection may for example affect other organ or another part of the subject compares to the primary infection, and/or inflammation. In other words, the secondary infection may affect an organ and/or part of the body which is different from the organ and/or part of the body infected by the primary infection and/or inflammation.
In the present the secondary infection may be any infection occurring after a primary infection known to one skilled in the art. It may be for example any secondary infection of gastrointestinal tract, respiratory tract, urinary tract infections. It may be for example any secondary infection of organ selected for the group comprising lung, liver, eye, heart, breast, bone, bone marrow, brain, mouth, head & neck, esophageal, tracheal, stomach, colon, pancreatic, cervical, uterine, bladder, prostate, testicular, skin, rectal, and lymphomas.
In the present the secondary infection may be a secondary infection selected from the group comprising pneumonia, pleural infection, urinary infection, peritoneal infection, intra-abdominal abscess, meningitis, mediastinal infection, soft-tissue or skin infection, such as cellulitis). For example it may be a secondary infection selected from the group comprising pneumonia, pleural infection, urinary infection, peritoneal infection, intra-abdominal abscess, meningitis and mediastinal infection.
In the present, the secondary infection may be due to any pathogen known to one skilled in the art. The secondary infection may be due to a bacteria selected from the group comprising Staphylococcus aureus, Methicillin resistant Staphylococcus aureus, Streptococcus pneumonias, Pseudomonas aeruginosa, Enterobacter spp (including E. cloacae), Acinetobacter baumannii, Citrobacter spp (including C. freundii, C. koserii), Klebsiella spp (including K. oxytoca, K. pneumoniae), Stenotrophomonas maltophilia, Clostridium difficile, Escherichia coli, Heamophilus influenza, Tuberculosis, Vancomycin-resistant Enterococcus, Legionella pneumophila. Other types include L. longbeachae, L. feeleii, L. micdadei, and L. anisa.
In the present, the secondary infection may be due to any virus known to one skilled in the art. It may be for example any virus mentioned in CELIA AITKEN et al. “Nosocomial Spread of Viral Disease” Clin Microbiol Rev. 2001 July; 14(3): 528-546 [15]. It may be due to a virus selected from the group comprising RSV, influenza viruses A and B, parainfluenza viruses 1 to 3, rhinoviruses, adenoviruses, measles virus, mumps virus, rubella virus, parvovirus B19, rotavirus, enterovirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus (HSV) types 1 and 2, Varicella-Zoster Virus (VZV), Cytomegalovirus (CMV), Epstein Barr virus (EBV), and human herpesviruses (HHVs) 6, 7, and 8, Ebola virus, Marburg virus, Lassa fever virus, Congo Crimean hemorrhagic fever virus, Rabies virus, Polyomavirus (BK virus).
In the present, the secondary infection may be due to any fungus known to one skilled in the art. It may be for example any fungus disclosed in SCOTT K. FRIDKIN et al. “Epidemiology of Nosocomial Fungal Infection” Clin Microbiol Rev, 1996; 9(4): 499-511 [51]. The secondary infection may be due to a specie of fungus selected from the group comprising Candida spp, Aspergillus spp, Mucor, Adsidia, Rhizopus, Malassezia, Trichosporon, Fusarium spp, Acremonium, Paecilomyces, Pseudallescheria.
In the present the secondary infection may be a nosocomial infection. It may be a nosocomial infection of any organ as previously mentioned. It may be a nosocomial infection due to any pathogen selected from the group comprising virus, bacteria and fungus. It may be a nosocomial infection due to a virus as previously defined. It may be a nosocomial infection due to a bacteria as previously defined. It may be a nosocomial infection due to a fungus as previously defined. It may be nosocomial infection selected from the group comprising pneumonia, pleural infection, urinary infection, peritoneal infection, intra-abdominal abscess, meningitis, mediastinal infection. It may be nosocomial infection selected from the group comprising pneumonia, pleural infection, urinary infection, peritoneal infection, intra-abdominal abscess, meningitis, mediastinal infection, soft-tissue or skin infection (cellulitis), head & neck infection (including otitis).
The secondary infection may be a nosocomial infection, in particular pneumonia
The secondary infection may be a nosocomial infection, for example an infection originated from hospital and/or acquired at the hospital and/or hospital-acquired infection.
In a particular embodiment, the secondary infection may be secondary pneumonia and/or a hospital-acquired pneumonia.
In the present primary infection means an infection due to any pathogen, or sepsis-like syndrome, that could have a negative effect on immune response and/or induce an immunosuppression
In the present primary infection means an infection due to a pathogen selected from the group comprising bacteria, virus or fungus. It may be for example any infection due to pathogen selected from the group comprising bacteria, virus or fungus known from one skilled in the art. It may be for example an infection of gastrointestinal tract, respiratory tract, urinary tract infections, and primary sepsis. It may be for example any infection due to a pathogen selected from the group comprising virus, bacteria and fungus. It may be for example a non-documented infection, for example an infection wherein no pathogens have been searched or found, such as sepsis-like syndrome. In the present the primary infection may be any infection due to a pathogen of at least one organ selected for the group comprising lung, liver, eye, heart, breast, bone, bone marrow, brain, head and neck, esophageal, tracheal, stomach, colon, pancreatic, cervical, uterine, bladder, prostate, testicular, skin, rectal, and lymphomas.
Another object of the present invention is a pharmaceutical composition comprising interleukin 12 (IL12) or derivative thereof and a pharmaceutically acceptable carrier.
The Interleukin 12 (IL12) or derivative thereof is as defined above.
Another object of the present invention is a pharmaceutical composition comprising inhibitor of transforming growth factor-beta and a pharmaceutically acceptable carrier.
The inhibitor transforming growth factor-beta is as defined above.
The pharmaceutical composition may be in any form that can be administered to a human or an animal. The person skilled in the art clearly understands that the term “form” as used herein refers to the pharmaceutical formulation of the medicament for its practical use. For example, the medicament may be in a form selected from the group comprising an injectable form, aerosols forms, an oral suspension, a pellet, a powder, granules or topical form, for example cream, lotion, collyrium, sprayable composition.
As described above, the pharmaceutically acceptable compositions of the present invention further comprise a pharmaceutically acceptable carrier, adjuvant or carrier. The pharmaceutically acceptable carrier may be any known pharmaceutical support used for the administration to a human or animal, depending on the subject to be treated. It may be any solvent, diluent or other liquid carrier, dispersion or suspension, surfactant, isotonic agent, thickening or emulsifying agent, preservative, solid binder, lubricant and the like, adapted to the particular desired dosage form. Remington Pharmaceutical Sciences, sixteenth edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in the formulation of pharmaceutically acceptable compositions and known techniques for their preparation. Except in the case where a conventional carrier medium proves incompatible with the compounds according to the invention, for example by producing any undesirable biological effect or by deleteriously interacting with any other component of the pharmaceutically acceptable composition, Its use is contemplated as falling within the scope of the present invention. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, Buffer substances such as phosphates, glycine, sorbic acid or potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulphate, Disodium phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene polymers, sugars such as lactose, Glucose and sucrose; Starches such as corn and potato starch; Cellulose and derivatives thereof such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; Tragacanth powder; Malt; Gelatin; Talc; Excipients such as cocoa butter and suppository waxes; Oils such as peanut oil, cottonseed oil; Safflower oil; Sesame oil; olive oil; Corn oil and soybean oil; Glycols; Such a propylene glycol or polyethylene glycol; Esters such as ethyl oleate and ethyl laurate; Agar; Agents such as magnesium hydroxide and buffered aluminum hydroxide; Alginic acid; Isotonic saline; Ringer's solution; Ethyl alcohol, and phosphate buffer solutions, as well as other compatible non-toxic lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the composition, according to the judgment of the galenist.
The pharmaceutical form or method of administering a pharmaceutical composition may be selected with regard to the human or animal subject to be treated. For example it may be administered to humans and other animals orally, rectally, parenterally, intratracheally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments or drops), buccally, as oral or nasal spray, subcutaneously, or the like, depending on the severity of the infection to be treated. The pharmaceutical form or method of administering a pharmaceutical composition may be selected with regard to the site of infection and/or infected organ. For example, for an infection of the respiratory tract it may in a form adapted to be administered to humans and other animals as oral or nasal spray or parenteral or intratracheal, for an infection of the gastrointestinal tract it may in a form adapted to be administered to humans and other animals orally, for example a pellet, a capsule, a powder, granules, a syrup or parenteral or intraperitoneal. The pharmaceutical form or method of administering a pharmaceutical composition may be selected with regard to the age of the human to be treated, and/or with regard to comorbidity, associated therapies and/or site of infection. For example, for a child, for example from 1 to 17 years old, or a baby, for example under 1 year old, a syrup or an injection, for example subcutaneous or intravenous may be preferred. Administration may for example be carried out with a weight graduated pipette, a syringe. For example, for an adult over 17 years old, an injection may be preferred. Administration may be carried out with an intravenous weight graduated syringe.
According to the present invention, the pharmaceutical composition may comprise any pharmaceutically acceptable and effective amount of interleukin 12 (IL12) or derivative thereof.
According to the present invention, the pharmaceutical composition may comprise any pharmaceutically acceptable and effective amount of inhibitor of transforming growth factor-beta.
In this document, an “effective amount” of a pharmaceutically acceptable compound or composition according to the invention refers to an amount effective to treat or reduce the severity of nosocomial disease. The compounds and compositions according to the method of treatment of the present invention may be administered using any amount and any route of administration effective to treat or reduce the severity of a nosocomial disease or condition associated with. The exact amount required will vary from one subject to another, depending on the species, age and general condition of the subject, the severity of the infection, the particular compound and its mode of administration.
IL-12 or inhibitor transforming growth factor-beta according to the invention are preferably formulated in unit dosage form to facilitate dosing administration and uniformity. In this document, the term “unit dosage form” refers to a physically distinct unit of compound suitable for the patient to be treated. However, it will be understood that the total daily dosage of the compounds and compositions according to the present invention will be decided by the attending physician. The specific effective dose level for a particular animal or human patient or subject will depend on a variety of factors including the disorder or disease being treated and the severity of the disorder or disease; The activity of the specific compound employed; The specific composition employed; Age, body weight, general health, sex and diet of the patient/subject; The period of administration, the route of administration and the rate of elimination of the specific compound employed; duration of treatment; The drugs used in combination or incidentally with the specific compound used and analogous factors well known in the medical arts. The term “patient” as used herein refers to an animal, preferably a mammal, and preferably a human.
According to the present invention, the pharmaceutical composition may comprise effective amount of Interleukin 12 (IL12) or derivative thereof. For example, the pharmaceutical composition may comprise doses Interleukin 12 (IL12) or derivative thereof adapted with regards to the nosocomial disease to be treated and/or to the subject to be treated. One skilled in the art taking into consideration his technical knowledge would adapt the amount in the pharmaceutical composition with regard to the nosocomial disease to be treated and/or to the subject to be treated. For example the pharmaceutical composition may comprise Interleukin 12 (IL12) at doses about 2 to 20 μg, preferably from 5 to 15 μg, preferably equal to 12.5 μg. For example, the pharmaceutical composition may comprise interleukin 12 (IL12) or derivative thereof in an amount allowing administration of IL-12 at doses of from about 0.1 μg/kg to 1 μg/Kg body weight of the subject.
According to the invention, interleukin 12 (IL12) or derivative thereof may be administered on a single administration or repeated administrations, for example one to three time per day.
According to the invention, interleukin 12 (IL12) or derivative thereof may be administered for example for a period from 1 to 21 days, for example from 1 to 7 days.
According to the present invention, the pharmaceutical composition may comprise any pharmaceutically acceptable and effective amount of inhibitor of transforming growth factor-beta. For example, the pharmaceutical composition may comprise doses of inhibitors of transforming growth factor-beta (TGF-β) adapted with regards to the inhibitor used. One skilled in the art taking into consideration his technical knowledge would adapt the amount in the pharmaceutical composition with regard to the used inhibitor. For example, when the inhibitors of transforming growth factor-beta (TGF-β) is small molecules, for example LY2157299, the pharmaceutical composition may comprise, for example at doses around 80 mg. For example, when the inhibitors of transforming growth factor-beta (TGF-β) is recombinant protein, for example Avotermin, the pharmaceutical composition may comprise, for example at doses from 20 ng to 200 ng, preferably from 50 ng to 200 ng, more preferably 100 ng to 200 ng. For example, when the inhibitors of transforming growth factor-beta (TGF-β) is humanized antibody, for example IMC-TR1, the pharmaceutical composition may comprise, for example at doses from 12.5 mg to 1600 mg.
According to the invention, inhibitors of transforming growth factor-beta (TGF-β) may be administered on a single time or repeated administration, for example one to three time per day.
According to the invention, inhibitors of transforming growth factor-beta (TGF-β) may be administered for example for a period from 1 to 21 days, for example from 1 to 7 days.
According to another aspect, the present invention relates to interleukin 12 (IL12) or derivative thereof, or pharmaceutical composition comprising IL12 or derivative thereof, for its use as a medicament, in particular in the treatment of secondary infection.
The Interleukin 12 (IL12) or derivative thereof is as defined above.
The pharmaceutical composition comprising IL12 or derivative thereof is as defined above.
The secondary infection is as defined above. For example secondary infection may be nosocomial diseases, including pneumonia, pleural infection, urinary infection, peritoneal infection, intra-abdominal abscess, meningitis, mediastinal infection, soft-tissue and/or skin infection, such as cellulitis.
According to another aspect, the present invention relates to an inhibitor of transforming growth factor-beta, or pharmaceutical composition comprising inhibitor of transforming growth factor-beta, for its use as a medicament, in particular in the treatment of secondary infection.
The inhibitor transforming growth factor-beta is as defined above.
The pharmaceutical composition comprising inhibitor transforming growth factor-beta is as defined above.
The secondary infection is as defined above. For example secondary infection may be nosocomial diseases, including pneumonia, pleural infection, urinary infection, peritoneal infection, intra-abdominal abscess, meningitis, mediastinal infection, soft-tissue and/or skin infection, such as cellulitis.
According to another aspect, the present invention relates to a method of treating or preventing secondary diseases comprising administering an effective amount of interleukin 12 (IL12) or derivative thereof or composition comprising interleukin 12 to a subject.
The Interleukin 12 (IL12) or derivative thereof is as defined above.
The composition comprising IL12 or derivative thereof is as defined above.
The secondary infection is as defined above. For example secondary infection may be nosocomial diseases, including pneumonia, pleural infection, urinary infection, peritoneal infection, intra-abdominal abscess, meningitis, mediastinal infection.
The administration of interleukin 12 (IL12) or derivative thereof or composition comprising interleukin 12 (IL12) or derivative thereof may be carried out by any way/routes known to the skilled person. For example it may be administered in any form and/or way/routes as mentioned above.
According to another aspect, the present invention relates to a method of treating or preventing secondary diseases comprising administering an effective amount of inhibitor of transforming growth factor-beta.
The inhibitor transforming growth factor-beta is as defined above.
The secondary infection is as defined above. For example secondary infection may be nosocomial diseases, including pneumonia, pleural infection, urinary infection, peritoneal infection, intra-abdominal abscess, meningitis, mediastinal infection
The administration of inhibitor transforming growth factor-beta or composition comprising inhibitor transforming growth factor-beta may be carried out by any way/routes known to the skilled person. For example it may be administered in any form and/or way/routes as mentioned above.
The medicament may be in any form that can be administered to a human or an animal. It may for example be a pharmaceutical composition as defined above.
The administration of the medicament may be carried out by any way known to one skilled in the art. It may, for example, be carried out directly, i.e. pure or substantially pure, or after mixing of the antibody or antigen-binding portion thereof with a pharmaceutically acceptable carrier and/or medium. According to the present invention, the medicament may be an injectable solution, a medicament for oral administration, for example selected from the group comprising a liquid formulation, a multiparticle system, an orodispersible dosage form. According to the present invention, the medicament may be a medicament for oral administration selected from the group comprising a liquid formulation, an oral effervescent dosage form, an oral powder, a multiparticle system, an orodispersible dosage form.
The interleukin 12 (IL12) or derivative thereof and/or inhibitor of transforming growth factor-beta as described above and pharmaceutically acceptable compositions of the present invention may also be used in combination therapies, i.e., compounds and pharmaceutically acceptable compositions may be administered simultaneously with, before or after one or more other therapeutic agents, or medical procedures. The particular combination of therapies (therapies or procedures) to be employed in an association scheme will take into account the compatibility of the desired therapeutic products and/or procedures and the desired therapeutic effect to be achieved. The therapies used may be directed to the same disease (for example, a compound according to the invention may be administered simultaneously with another agent used to treat the same disease), or may have different therapeutic effects (eg, undesirable).
For example, therapeutic agents known to treat secondary disease, for example nosocomial diseases, for example antibiotics, antifungal and/or antiviral compounds and/or antibacterial antibody and/or interferon therapy. It may be for example any antibiotic known to one skilled in the art. It may be for example antibiotic used for the treatment of pneumonia, pleural infection, urinary infection, peritoneal infection, intra-abdominal abscess, meningitis, mediastinal infection. It may be for example antibiotic selected from the group comprising Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, Streptomycin, Spectinomycin, Geldanamycin, Herbimycin, Rifaximin, Loracarbef, Ertapenem, Doripenem, Imipenem/Cilastatin, Meropenem, Cefadroxil, Cefazolin, Cefalotin or Cefalothin, Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime; Cefdinir; Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil, Ceftobiprole, Ceftolozane, Avibactam, Teicoplanin, Vancomycin, Telavancin, Dalbavancin, Oritavancin, Clindamycin, Lincomycin, Daptomycin, Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, Spiramycin, Aztreonam, Furazolidone, Nitrofurantoin, Linezolid, Tedizolid, Posizolid, Radezolid, Torezolid, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Temocillin, Ticarcillin, Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam, Ticarcillin/clavulanate, Bacitracin, Colistin, Polymyxin B, Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, Temafloxacin, Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole, Sulfonamidochrysoidine, Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, Tetracycline, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol(Bs), Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, Streptomycin, Arsphenamine, Chloramphenicol(Bs), Fosfomycin, Fusidic acid, Metronidazole, Mupirocin, Platensimycin, Quinupristin/Dalfopristin, Thiamphenicol, Tigecycline(Bs), Tinidazole, Trimethoprim(Bs).
It may be for example antifungal compound selected from the group comprising Bifonazole, Butoconazole, Clotrimazole, Econazole, Fenticonazole, Isoconazole, Ketoconazole, Luliconazole, Miconazole, Omoconazole, Oxiconazole, Sertaconazole, Sulconazole, Tioconazole, Amphotericin B, Candicidin, Filipin, Hamycin, Natamycin, Nystatin, Rimocidin, Albaconazole, Efinaconazole, Epoxiconazole, Fluconazole, Isavuconazole, Itraconazole, Posaconazole, Propiconazole, Ravuconazole, Terconazole, Voriconazole, Abafungin, Anidulafungin, Caspofungin, Micafungin, Aurones, Benzoic acid, Ciclopirox, Flucytosine or 5-fluorocytosine, Griseofulvin, Haloprogin, Tolnaftate, Undecylenic acid.
It may be for example antiviral compound selected from the group comprising Abacavir, Acyclovir, Adefovir, Amantadine, Amprenavir, Ampligen, Arbidol, Atazanavir, Atripla, Balavir, Cidofovir, Combivir, Dolutegravir, Darunavir, Delavirdine, Didanosine, Docosanol, Edoxudine, Efavirenz, Emtricitabine, Enfuvirtide, Entecavir, Ecoliever, Famciclovir, Fomivirsen, Fosamprenavir, Foscarnet, Fosfonet, Fusion inhibitor, Ganciclovir, Ibacitabine, Imunovir, Idoxuridine, Imiquimod, Indinavir, Inosine, Integrase inhibitor, Interferon type Ill, Interferon type II, Interferon type I, Interferon, Lamivudine, Lopinavir, Loviride, Maraviroc Moroxydine, Methisazone, Nelfinavir, Nevirapine, Nexavir, Nitazoxanide, Nucleoside analogues, Novir, Oseltamivir, Peginterferon alfa-2a, Penciclovir, Peramivir, Pleconaril, Podophyllotoxin, Protease inhibitor, Raltegravir, Reverse transcriptase inhibitor, Ribavirin, Rimantadine, Ritonavir, Pyramidine, Saquinavir, Sofosbuvir, Stavudine, Telaprevir, Tenofovir, Tenofovir disoproxil, Tipranavir, Trifluridine, Trizivir, Tromantadine, Truvada, Valaciclovir, Valganciclovir, Vicriviroc, Vidarabine, Viramidine, Zalcitabine, Zanamivir, Zidovudine.
The inventors have also demonstrated that the expression of transcription factor Blimp1 is increased in subject susceptible to secondary disease and/or nosocomial disease. In particular, the inventors have demonstrated that the expression of transcription factor Blimp1 is increased in subject with deficient or less reactive immune response to a pathogen.
Another object of the present invention is an ex vivo method for determining the immunity state of a subject comprising
Another object of the present invention is an ex vivo method for determining the susceptibility to a secondary disease of a subject comprising
In the present, “deficiency in immunity” means that the subject may have decreased immunogenic response and/or capacity of initiating adaptive and/or capacity of activating innate immunity with regards to a pathogen and/or a reduction of the activation or efficacy of the immune system.
In the present, “susceptibility to a secondary disease” means a subject having a reduction of the activation or efficacy of the immune system and/or having an increased susceptibility to opportunistic infections and decreased cancer immunosurveillance.
In other words, the method of the invention makes it possible to establish, before any secondary disease and/or nosocomial disease whether a subject may be more susceptible to such disease and whether the condition of a such can be improved by administration of a treatment, in particular a treatment improving and/or restoring the immunity response as the medicament of the invention i.e. IL-12 and/or inhibitor of TGF-β.
According to the invention, “biological sample” means a liquid or solid sample. According to the invention, the sample can be any biological fluid, for example it can be a sample of blood, of plasma, of serum, of cerebrospinal fluid, of respiratory fluid, of vaginal mucus, of nasal mucus, of saliva and/or of urine. Preferably the biological sample is a blood sample.
In the present by Blimp1 transcription factor is a protein that in humans is encoded by the PRDM1 gene.
According to the invention the expression level of transcription factor Blimp1 may be determined by any method or process known from one skilled in the art. It may be for example determined with flow cytomtery or any method disclosed in Marcel Geertz and Sebastian J. Maerkl, Experimental strategies for studying transcription factor—DNA binding specificities, Brief Funct Genomics. 2010 December; 9(5-6): 362-373 [39].
According to the invention the expression level of transcription factor Blimp1 may be determined from any immune cell of the biological sample. For example, the expression level of transcription factor Blimp1 may be determined from immune cell selected from the group comprising lymphocyte cells, phagocytes cells and granulocytes cells. It may be preferably determined from granulocytes selected from the group comprising macrophage, monocyte and dendritic cells. It may be preferably determined from dendritic cells.
According to the invention, the referenced level of expression of transcription factor Blimp1 (Lref) may be the mean expression level of transcription factor Blimp1 (Lref) in subject without any disease and/or which has not been infected with a pathogen at least since two weeks. For example the referenced level of expression of Blimp1 may be between 1000 to 100 000 gMFI in dendritic cells or less than 10% of Blimp1 positive dendritic cells or of B lymphocytes as measured by flow cytometry after intracellular staining.
The term “subject” as used herein refers to an animal, preferably a mammal, and preferably a human.
Other advantages may still be apparent to those skilled in the art by reading the examples below, illustrated by the accompanying figures, given by way of illustration.
*p<0.05, ***p<0.001, # p<0.05 vs all others. Graphs represent mean+/−SD and display data pooled from 2-3 independent experiments.
Graphs illustrate
Macrophages and dendritic cells were depleted in vivo by treating CD11c-DTR mice with diphteria toxin. (a) Number of NK cells (ordinate: number of cells×103) and (b), frequencies of IFN-γ+ Natural Killer cells (ordinate: percentage of IFN-γ+ Natural Killer cells) in uninfected or infected (E. coli intra-tracheal) mice depleted in macrophages and dendritic cells and treated or not with interleukin (IL)-12 (100 ng. ip.) n=4-6 mice per group). (c) Enumeration of colony forming units from bronchoalveolar lavages (ordinate: log 10 of CFU/mL) and (d) weight loss (ordinate: percentage of initial weight) 18 hours after E. coli intra-tracheal administration mice depleted in macrophages and dendritic cells and treated with
interleukin (IL)-12 (n=4-5 mice per group).
The representative examples that follow are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples which follow and the references to the scientific and patent literature cited herein. It should further be appreciated that the contents of those cited references are incorporated herein by reference to help illustrate the state of the art.
The following examples contain important additional information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and the equivalents thereof.
The present invention and its applications can be understood further by the examples that illustrate some of the embodiments by which the inventive medical use may be reduced to practice. It will be appreciated, however, that these examples do not limit the invention. Variations of the invention, now known or further developed, are considered to fall within the scope of the present invention as described herein and as hereinafter claimed.
Mice used were C57BL/6J (B6), B6.SJL-PtprcaPep3b/BoyJ (CD45.1), B6.FVB-Tg(ltgax-DTR/EGFP)57Lan/J (CD11c-DTR mice, Diphteria Toxin Receptor is expressed under the control of ltgax promoter) (Jung et al., 2002 [29]), C57BL/6J-Tlr9M7Btlr/Mmjax (Tlr9−/− mice) (Hemmi et al., 2000 [25]), B6.Cg-Tg(TcraTcrb)425Cbn/J (OT-II mice) (Barnden et al., 1998 [7]), C57/B6.129S2-H2dIAb1-Ea/J (H2 mice) (knock out for MHC-II gene)(Köntgen et al., 1993 [34]), CD11c-OVA (membrane OVA is expressed under the control of Itgax promoter) (Wilson et al., 2006 [66]), C57BL/6-Tg(Foxp3-DTR/EGFP)23.2Spar/Mmjax (Diphteria Toxin Receptor and GFP are expressed under the control of FoxP3 promoter, DEREG)(Lahl et al., 2007 [35]),ID2GFP reporter (GFP is expressed under the control of ID2 promoter) (Jackson et al., 2011 [28]), Blimp1GFP reporter (GFP is expressed under the control of Blimp1 promoter) (Kallies et al., 2004 [30]), IRF8YFP (YFP is expressed under the control of IRF8 promoter) (Chopin et al., 2013 [19]), and Tgfb2rfl/fl (Floxed regions around Tgfb2r gene) (Ramalingam et al., 2012 [44]) crossed to CD11ccre (in which Cre recombinase is expressed under the control of the CD11c promoter) (Caton et al., 2007 [13]).
For technical reasons, mice were used for experiments without taking gender into account. Male and female mice were maintained in specific pathogen-free conditions, group housed, at the Bio21 Institute Animal Facility (Parkville, Australia) following institutional guidelines and were used for experiments between six and fourteen weeks of age. Experimental procedures were approved by the Animal Ethics Committee of the University of Melbourne (protocol #1413066).
Bioresources: IBIS-sepsis (severe septic patients) and IBIS (brain-injured patients), Nantes, France. Patients were enrolled from January 2014 to May 2016 in two French Surgical Intensive Care Units of one university hospital (Nantes, France). The collection of human samples has been declared to the French Ministry of Health (DC-2011-1399), and it has been approved by an institutional review board. Written informed consent from a next-of-kin was required for enrolment. Retrospective consent was obtained from patients, when possible.
For the IBIS-septic study, inclusion criteria were proven bacterial infection, together with a systemic inflammatory response (two signs or more among increased heart rate, abnormal body temperature, increased respiratory rate and abnormal white-cell count) and acute organ dysfunction and/or shock. For the IBIS study, inclusion criteria were brain-injury (Glasgow Coma Scale (GCS) below or equal to 12 and abnormal brain-CT scan) and systemic inflammatory response syndrome. Exclusion criteria were cancer in the previous five years, immunosuppressive drugs and pregnancy. All patients were clinically followed up for 28 days. Control samples were collected from matched healthy blood donors (age±10 years, sex, race), recruited at the Blood Transfusion Center (Etablissement Français du Sang, Nantes, France).
EDTA-anticoagulated blood samples were withdrawn seven days after primary infection in septic patients (IBIS sepsis), or at day 1 and day 7 ICU admission in trauma patients. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation, frozen in liquid nitrogen in a 10% DMSO solution and stored until analysis.
Diphteria toxin (0.1 μg i.p, two injections 24 hours apart, then every 3 days) was administrated to CD11c-DTR or FoxP3GFP-DTR (DEREG) mice to induce depletion of CD11c+ cells or Treg cells respectively. For CD11c-DTR mice, the first DT injection was performed either one day before the primary pneumonia (for outcomes assessed during primary infection or 7 days after), or 1 day before the secondary pneumonia as stated. DEREG mice were treated from day 4 after the primary pneumonia. Efficiency of depletion (number of cells) was controlled during experiments and routinely exceeded 90%.
Primary pneumonia, Escherichia coli (DH5a), grown for 18 hours in luria broth medium at 37° C., was washed twice (1.000× g, 10 min, 37° C.), diluted in sterile isotonic saline and calibrated by nephelometry. As stated, E. coli (75 μL, OD600=0.6-0.7, or OD600=2.0) or Influenza-virus (400 plaque-forming units of influenza, virus strain WSN x31) were injected intra-tracheally or intra-nasally respectively in anesthetized mice to induce a non-lethal acute pneumonia (Broquet et al., 2014; Wakim et al., 2013). For the secondary pneumonia, E. coli (75 μL, OD600=0.6-0.7) or OVA-coated E. coli (see preparation below, 70 μL, OD600=0.6-0.7) or Staphylococcus aureus (ATCC 29213, 70 μL, OD600=0.6-0.7) or Pseudomonas aeruginosa (PAO1, 70 μL, 1/10 dilution of a solution OD600=0.2-0.3) were injected intra-tracheally 7 to 21 days after the primary pneumonia.
CpG 1668 (10 nM) was administrated intra-tracheally under anaesthesia. Mice were kept in a semi-recumbent position for 60 seconds after injection.
Recipient mice were γ-irradiated twice with 550 Gray and were reconstituted with 2.5-5×106 T cell-depleted bone marrow cells of each relevant donor strain at the indicated ratio. Neomycin (50 mg/ml) was added to the drinking water for the next 4 weeks. Chimeras were used for subsequent experiments 6 to 10 weeks after the reconstitution. Percentage of chimerism was tested during the course of the experiments.
DC purification from lungs and spleen, analytical and preparative flow cytometry and DC cultures in vitro were performed as described (Wakim et al., 2015 [64]) and see Table 3. The following conjugated monoclonal antibodies were used: anti-CD11c (N418, made in-house), anti-CD4 (GK1.5; made in-house), anti-CD8a (53-6.7; eBioscience), anti-CD11b (M1/70, in house-made), anti-CD24 (M1/69, made in house), anti-CD172a (Sirp-α, made in-house), anti-MHCII (M5/114, made in-house), anti-CD86 (P03, Biolegend), anti-CD45.1 (A20.1; eBioscience), anti-CD103 (2E7; eBioscience), anti-F4/80 (F4/80, made in-house), anti-Latency Associated Protein/TGFβ1 (TW7-16B4, eBioscience), anti-TCR VαD (620.1; made in-house), anti-IL6 (MP5-20F3, BD Pharmingen), anti-IL12 (C15.6, BD Pharmingen), anti TNFα (MP6-XT22, BD Pharmingen), anti-IFNα (XMG1.2, BD Pharmingen), anti-FoxP3 (FJK-16s, eBioscience), anti-IRF4 (3E4, Invitrogen), Fixable Viability Dye (eBioscience). Samples were acquired on LSR-Fortessa or LSR-II (Becton Dickinson) and analyzed using Flowjo Software (TreeStar Inc, Ashland, Oreg.). For cell culture or analysis of RNA, macrophages and DC obtained from pooled lungs of 4-5 mice and sorted with a FACSAria III (purity>95%).
Circulating DC and monocytes were identified with the following anti-human antibodies: from Biolegend lineage (anti-CD3 (HIT3a), anti-CD14 (63-D3), anti-CD19 (HIB19), anti-CD20 (2H7), anti-CD56 (HCD56)), anti-CD1c (L161), anti-CD11c (3.9), anti-HLA-DR (L243), anti-CD123 (6H6); from BD Biosciences anti-CD141 (1A4) and anti-Blimp-1 (6D3).
Treg cells were identified with CD45-PerCP (clone 2D1), CD25-PC7 (clone 2A3) and CD3-FITC (clone SK7), all from BD Biosciences, and CD127-PE (clone R34.34), CD4-APC (clone 13B8.2) from Beckman Coulter. Treg were identified as CD45+CD3+CD4+CD25highCD127low/−. The number of Treg were deduced from the CD4 T cells number multiplied by the proportion of regulatory T cells in CD4 cells.
For intracellular staining of cytokines in DC or lymphocytes, cell suspensions were obtained by mechanical and collagenase digestion of lungs collected 16 hours after injection of E. coli. Cells were cultured 4 hours in complete media with Golgi Plug, washed twice and then stained for surface markers. Fixation and permeabilization was performed following manufacturer instructions (BD Cytofix/Cytoperm kit, BD Bioscience). Anti-cytokine antibody was incubated overnight at 4° C. Cells were washed twice before FACS analysis.
RNA was extracted with an RNeasy kit (Qiagen, Valencia, Calif.) from alveolar and interstitial macrophages, CD103+ DC and CD11b+ DC cells isolated by flow cytometry from the lungs of uninfected mice or from mice 7 days after E. coli infection. Reverse transcription—PCR was performed with a SuperScript III First-strand synthesis system according to manufacturer's instructions (Invitrogen). Real-time PCR was performed with either RT2 qPCR Primer sets (Qiagen) specific for mouse TGF-β(UniGene Mm.18213), PLAT (UniGene Mm.154660), aldh1a2 (UniGene Mm.42016), Itgb6 (UniGene Mm.98193) and Itgb8 (UniGene Mm.217000) or primers specific for GAPDH (5′-CCAGGTTGTCTCCTGCGACTT-3′ (SEQ ID NO 3) and 5′-CCTGTTGCTGTAGCCGTATTCA-3′ (SEQ ID NO 4)) and LightCycler 480 SYBR Green I master kit according to the supplier's recommendations (Roche). Relative gene expression was calculated by the 2−ΔΔ Ct method using samples from S group as calibrator.
Measure of mRNA levels of IL12 in conventional dendritic cells during S. aureus pneumonia in naive mice (primary pneumonia, 1ary PN) or in trauma-hemorrhage mice (secondary pneumonia, 2ary PN) were carried out according the method disclosed in Roquilly et al. Eur Resp J 2013, p1365-1378 [46]. In particular, Real-time quantitative PCR was performed on CD11c cells sorted with a CD11c cell isolation kit II (Miltenyi Biotec, Paris, France). These procedures routinely yielded cell populations with purity up to 95%. Total RNA was isolated from sorted cells with TRIzol reagent (Invitrogen, Cergy Pontoise, France) and treated for 45 min at 37 uC with 2 U of RQ1 DNase (Promega, Lyon, France). RNA (1 mg) was reverse-transcribed with superscript III reverse transcriptase (Invitrogen). The cDNA (1 mL) was subjected to RT-qPCR in a BioRad iCycler iQ system using the QuantiTect SYBR Green PCR kit (Qiagen, Courtaboeuf, France). GAPDH was used to normalize gene expression. Relative gene expression was calculated by the 2 Ct method using samples from the sham group as calibrator samples.
Frequencies of IL-12+ conventional DC upon in vitro stimulation with LPS of peripheral blood mononuclear cells harvested in healthy controls (HC) and in critically ill patients at the indicated time after acute brain-injury were carried out according the method disclosed in Roquilly et al. PLoS one 2013. In particular, for blood Sample Collection: Venous blood samples were collected in EDTA and heparin vacutainers and processed for analysis within 4 hours on days 2, 5 and 10 after brain-injury. Patient sera were frozen at 280° C. Antibodies and Reagents: DCs were identified using color flow cytometry assay. Briefly, whole blood samples were stained with the following antibodies IL-12-efluor450 (eBiosciences, Paris, France) Abs were used to identify intracellular cytokines after stimulation of peripheral blood mononuclear cells with IL-12. Cytokine Production by Dendritic Cells: Heparinated whole blood samples were incubated for 3h30 at 37 uC under 5% CO2 conditions with IL-12 for peripheral blood mononuclear cells stimulation. GolgiPlug were added during the last 2h30 hours of incubation to inhibit cellular cytokine release. Control conditions included stimulation with medium alone as negative control. Whole blood samples were then incubated with surface mAbs for 15 min, followed by erythrocyte lysis (BD Biosciences). Samples were then fixed, permeabilised with Cytofix/Cytoperm Plus and stained with cytokine-directed mAbs. The percentages of IL12+ dendritic cells was measured by flow cytometry. Data were analyzed with FlowJo.
Survival curves to S. aureus pneumonia induced in naive mice (1ary PN), in trauma-hemorrhage mice (2ary PN), in trauma-hemorrhage mice injected with NK cells treated ex vivo with MPLA (so called 2ary PN+NK(IL12)) or injected with MPLA-treated DCs (producing IL12 and other cytokines so called DC(IL12)) were carried out according the method disclosed in Roquilly et al. Eur Resp J 2013, p1365-1378 [46].
Mice were injected intraperitoneally with 1 mg bromodeoxyuridine (BrdU) (Sigma, St Louis, Mo.) at day 5 and at day 6 after pneumonia. At day 7, macrophages and DC were isolated and analyzed as described (Kamath et al., 2002 [31]).
E. coli (DH5α) was shacken for 2 hours at 37° C. in a solution of OVA diluted in luria broth medium (10 mg/ml, in house-made) and washed 2 times in saline before calibration (OD600=0.6-0.7) and injection.
OT-II T cells were purified from pooled lymph nodes (inguinal, axillary, sacral, cervical and mesenteric) of transgenic Ly5.1+ mice by depletion of non-CD4 T cells and were labeled with Cell Trace Violet as described (Vega-Ramos et al. 2014 [68]). T cell preparations were routinely 85-95% pure, as determined by flow cytometry.
For the assessment of the capacity of antigen presentation in the lungs, mice were injected intra-tracheally with calibrated OVA-coated E. coli or 0.5 μg of anti-DEC205-OVA (clone NLDC-45)(Lahoud et al., 2011 [36]). 1-2.5×106 Violet Cell Tracer-labeled OT-II cells were concomitantly intravenously injected. For the assessment of the capacity of antigen presentation in the spleen, mice were injected i.v. with soluble OVA (0.1 mg) and labeled OT-II cells (1-2.5×106 cells). 60 hours later, cells from the mediastinal lymph node or from the spleen were stained with anti-CD4, CD45.1, anti-TCRVα2 and PI, and resuspended in buffer containing 1-3×104 blank calibration particles (Becton Dickinson). The total number of live dividing OT-II was calculated from the number of dividing cells relative to the number of beads present in each sample.
Mice were treated with IL-12 (100 ng i.p., Abcam) concomitantly to the induction of the secondary pneumonia. Anti-TGFβ monoclonal antibody (1B11, 44 μg i.p. every 3 days) or isotype control IgG1 monoclonal antibody (MG1-45, Biolegend) injections were performed 3 and 6 days after primary pneumonia. TGF-β (1 μg i.p., Thermofisher) was injected once 6 days after primary pneumonia in DT-treated CD11c-DTR chimeric mice
Data were plotted using GraphPad prism (La jolla, CA. United States). Unpaired T-test and Mann-Whitney unpaired test with two-tailed p-values and 95% confidence intervals. One-way ANOVA with Bonferonni correction (post-hoc tests) were used for multiple comparisons. Correlations were investigated by a linear regression test. Correlation between trauma severity (as assessed with Glasgow Coma Scale) and Blimp-1 expression of CD1c DC, or increase of Treg (Delta of Treg Day 7-Day 1) was investigated with a Person test. Statistical details of experiments (exact number of mice per group, exact P-values, dispersion and precision measures) can be found in the figure legends. P<0.05 for statistical significance.
Recovery from Primary Pneumonia is Followed by Increased Susceptibility to Secondary Infection
Escherichia coli (E. coli) is the second most frequent gram negative bacilli involved in both community- and hospital-acquired pneumonia (Roquilly et al., 2016 [47]; van Vught et al., 2016b [60]). Early recurrence of pneumonia to the same pathogens is observed in up to 20% of critically ill patients cured from primary pneumonia (Chastre et al., 2003). To mimic in mice this clinical scenario, we induced secondary pneumonia with E. coli in mice cured from a bacterial (E. coli) or a viral (influenza A virus, IAV) primary pneumonia (
Defective CD4 T cell priming following recovery from primary pneumonia
T cell priming in mice that recovered from bacterial pneumonia by re-infecting them 7-21 days after the primary infection with E. coli associated with the model antigen, ovalbumin (OVA) was assessed. The mice also received MHC II-restricted, OVA-specific OT-II cells, which proliferated in the mediastinal lymph nodes (LN) in response to local presentation of OVA. A severe reduction in OT-II proliferation during secondary pneumonia compared to that observed in mice that received E. coli-OVA as a primary infection (
Tumor growth factor (TGF)-β is critical for tissue healing after injury and is immunosuppressive (Akhurst and Hata, 2012 [1]). To test whether TGF-β released within lung tissue during or after primary pneumonia induced immunosuppression, TGF-β released was neutralized with a mAb injected 3 and 6 days after initiation of primary pneumonia. This treatment did not affect bacterial burden or weight changes during primary infection (
TGF-β induces differentiation of naive CD4 T cells into FoxP3+ T regulatory (Treg) cells (Chen et al., 2003 [18]). The inventors demonstrate a higher proportion of lung Treg cells after recovery from primary bacterial or IAV pneumonia (
The cells that produced TGF-β in the lungs of mice cured from primary infection were next identified. Expression of TGF-β mRNA did not vary in non-hematopoietic cells (CD45neg) in infection-cured mice (
Macrophages and DC Newly Produced after Severe Primary Pneumonia are Paralyzed
DC and macrophages become activated, increase in numbers (
Capture and presentation of pathogen antigens via MHC-II is a hallmark property of DC and macrophages (Guilliams et al., 2013 [23]; Segura and Villadangos, 2009 [53]), and though their numbers during primary and secondary pneumonia were comparable (
Production of immunogenic cytokines by macrophages and DC is as critical, if not more, for the control of infection than antigen presentation, as these cytokines regulate both innate and T cell-dependent immunity (Marchingo et al., 2014 [40]). We identified CD103+ DC, alveolar macrophages and CD11b+ DC as the main sources of interleukin (IL)-12, tumor necrosis factor (TNF)-β and IL-6, respectively, during E. coli primary pneumonia (FIG. 11a). Production of these cytokines during secondary pulmonary infection with E. coli was significantly impaired for up to 30 days in mice recovered from primary E. coli or IAV pneumonia (
In other words, as (IFN)-γ is a marker the induced immunosuppression. The example clearly demonstrate that IL-12 restores (IFN)-γ production and thus allows to treat immunosuppression. Accordingly, this example clearly demonstrates that the present invention allows to prevent and/or treat of secondary infection, in particular by suppressing the primary infection induced immunosuppression.
A comparison of the expression of phenotypic markers and immunoregulatory factors in DC before and after pneumonia was carried out. The inventors demonstrated no significant changes in the expression of characteristic surface markers of DC (CD11c, CD24, MHC-II, DEC205, CD103 and CD11b) and macrophages (F4/80, CD64, Ly6G, CD11c, CD11b) (
To examine whether the signals responsible for reprogramming of DC after pneumonia acted systemically, the phenotype and function of splenic DC 7 days following primary pneumonia were assessed. Neither the expression of transcription factors (
TGF-β Contributes to Program Macrophages and DC after Resolution of Primary Pneumonia
Neutralization of TGF-β with a blocking mAb injected during, or after resolution of, primary pneumonia reduced the defects in DC cytokine production during secondary infection (
In order to investigate the role of TGF-β signaling in macrophages and DC on their ability to prime CD4 T cells, mixed bone marrow chimeras where WT mice were reconstituted with a 1:3 mix of Tgfbr2fl/flCd11ccre and H2−/− (MHC-II-deficient) bone marrow were generated. In these chimeric mice, only the cells derived from the Tgfbr2fl/flCd11ccre bone marrow are able to present antigen to, and prime, CD4 T cells. TGF-βR-deficient CD11c cells retained the ability to elicit effective priming of OT-II cells during secondary pneumonia in vivo (
Finally, reduced cytokine production by DC and macrophages during secondary pneumonia was examined to know whether it was also caused by TGF-β recognition by the cells themselves. This did not appear to be the case because in WT:Tgfbr2fl/flCd11ccre mixed bone marrow chimeras both the WT and the TGF-βR-deficient cells were impaired during secondary pneumonia (
First, PBMC from a prospective cohort of septic patients presenting with E. coli secondary infection [IBIS sepsis, n=5 (Table 1)] was analyzed. Human CD1c DC, the circulating equivalent of murine CD11b DC (Guilliams et al., 2014 [23]), expressed high level of the transcription factor Blimp1 in these patients as compared to matched uninfected donors (
Escherichia coli, yes
The inventors demonstrate that in mice that the reprogramming of DC is not pathogen-driven but induced by secondary mediators of inflammation, the inventors demonstrate that Blimp1+ CD1c DC are also be observed in patients suffering aseptic systemic inflammatory response syndrome (SIRS). Circulating DC from patients suffering from trauma-induced severe inflammation [IBIS cohorts 1 and 2, n-32 and n-29 respectively Table 2] was examined. Circulating DC from these patients have lost their ability to produce TNF-α, IL-6 and IL-12 upon in vitro stimulation reproducing a hallmark of mouse paralyzed DC (
The effector mechanisms deployed by the immune system to fight pathogens can cause tissue damage and have to be tightly controlled to prevent self-harm. Here the example demonstrate a network of regulatory mechanisms that dampen the immune response locally in response to lung infection. It involves multiple cell types and cytokines, with macrophages and DC playing a pivotal role. Importantly, after clearance of the infection the immunosuppression induced by these mechanisms does not restore immune homeostasis to the situation that preceded the infection. It persists locally for weeks after resolution of the infection, increasing the susceptibility to secondary infections.
The examples demonstrate that treatment with IL-12 or inhibitors of TGF-β allows to restore the immunity of a subject after an infection and also to treat secondary infection and/or nosocomial infection.
DC respond quickly to pathogen encounter by presenting antigens to induce T cell responses, and releasing cytokines that promote both innate and adaptive immunity (Banchereau and Steinman, 1998 [6]). During this response they undergo a process of “maturation” that involves multiple genetic, phenotypic and functional changes (Landmann et al., 2001 [37]; Wilson et al., 2006 [66]). DC have a short half-life, both in the steady-state and after infection (Kamath et al., 2002 [31]), being continually replaced by new DC derived from precursors immigrated from the bone marrow (Geissmann et al., 2010 [21]). Final DC differentiation occurs in peripheral tissues under the influence of local cytokines that modulate the responsiveness and functional properties of the newly produced DC (Amit et al., 2015 [2]). The results demonstrate that the DC that develop in the lung after resolution of pneumonia have diminished capacity to present antigens and to secrete immunostimulatory cytokines, which makes them less capable of initiating adaptive and innate immunity against a secondary bacterial infection.
The invention allow to overcome the deficiency of DC cells, for example diminished capacity to present antigens and to secrete immunostimulatory cytokines, with the administration of IL-12 or inhibitors of TGF-β which allows to restore the immunity of a subject after an infection and thus allow to prevent or to treat secondary infection and/or nosocomial infection.
In addition, the inventors are the first who demonstrate that DC cells produce higher levels of TGF-β, which promotes accumulation of Treg cells. They demonstrate that the signals that promote the differentiation of DC to this paralyzed state are not directly associated with the pathogen that caused the primary infection; they are mediated by secondary cytokines acting locally. They also demonstrate that TGF-β plays a prominent role in the differentiation of paralyzed DC, although our results do not discard a role for other cytokines or surface receptors. The source of active TGF-β may be multiple cell types.
As demonstrated in the example, the invention allow to overcome the deficiency of DC cells, for example diminished capacity to present antigens and to secrete immunostimulatory cytokines, with the administration of IL-12 or inhibitors of TGF-β which allows to restore the immunity of a subject after an infection and thus allow to prevent or to treat secondary infection and/or nosocomial infection.
In addition, the inventors have clearly demonstrates that production of IL-12 by dendritic cells and macrophages is critical for the innate immune response and clinical recovery to bacterial pneumonia, the production of IL-12 by macrophages and dendritic cells is drastically decreased during bacterial pneumonia in mice and in humans cured from a primary infection or after non-septic inflammatory response (such as trauma, brain-injury), and that IL-12 treatment restores innate immune response and enhances clinical recovery during bacterial pneumonia in mice cured from infection or from trauma-hemorrhage.
Accordingly, the inventor have clearly demonstrate that the present invention allows to treat secondary infection and/or nosocomial infection, in particular since the treatment is not directly directed to the pathogen or the cause of the disease but improve the defense of the treated subject.
The effects reported can be considered an extension of the phenomenon of “immunological training” induced locally by commensal flora and other environmental stimuli (Carr et al., 2016 [12]). The long term immunosuppression that ensues in mice or humans that survive severe infections can be considered a deleterious consequence of over-adaptation to a challenge that in normal conditions would lead to death but can be overcome in the controlled conditions of the laboratory (mice) or intensive care units (humans). Importantly, the inventors demonstrate that the signals that cause local cell imprinting are non-antigen specific, explaining why recovery from a primary infection can increase the susceptibility to an entirely new pathogen.
The inventors demonstrate that circulating DC of sepsis or trauma patients express characteristic markers of mouse paralyzed DC such as a high level of Blimp1. The presence of Blimp1high DC in critically-ill patients is a prognostic marker of extended immunosuppression, affording an opportunity for early intervention to prevent secondary infections in this high-risk cohort of patients.
Microbiol Rev. 2001 July; 14(3): 528-546
68. Vega-Ramos, J., Roquilly, A., Zhan, Y., Young, L. J., Mintern, J. D., & Villadangos, J. A. (2014). Inflammation conditions mature dendritic cells to retain the capacity to present new antigens but with altered cytokine secretion function. Journal of Immunology (Baltimore, Md.: 1950), 193(8), 3851-3859.
Number | Date | Country | Kind |
---|---|---|---|
17305945.2 | Jul 2017 | EP | regional |
17201074.6 | Nov 2017 | EP | regional |
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/EP2018/069190 | 7/16/2018 | WO | 00 |