Composition for treatment or prevention of infectious inflammatory diseases, or composition for immune enhancement, comprising tryptophanyl-tRNA synthetase as an active ingredient

Abstract

The present invention relates to a composition for treatment or prevention of infectious inflammatory diseases comprising tryptophanyl-tRNA synthetase as an active ingredient, and a composition for immune enhancement. More specifically, the present invention relates to a pharmaceutical composition for treatment or prevention of infectious inflammatory diseasess comprising tryptophanyl-tRNA synthetase as an active ingredient, a food composition for preventing or improving, a veterinary composition for preventing or treating, and a composition for immune enhancement comprising a tryptophanyl-tRNA synthetase as an active ingredient, respectively. The composition of the present invention can be effectively used for preventing or treating diseases of humans and animals caused by infection from bacteria, viruses or fungi and the like by inhibiting infections such as bacterial, viral, and fungal infections at an early stage particularly through activating innate immune response.

Claims

1. A method for ameliorating or treating an infectious inflammatory disease caused by a bacterium, viruses or fungi in a subject in need thereof, the method comprising administering to the subject in need thereof a composition comprising a tryptophanyl-tRNA synthetase selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 as an active ingredient in an amount effective for ameliorating or treating an infectious inflammatory disease caused by a bacterium, viruses or fungi, wherein the tryptophanyl-tRNA synthetase increases infiltration of neutrophils in the subject.

2. A method for immune enhancement in a subject in need thereof, the method comprising administering to the subject in need thereof a composition comprising a tryptophanyl-tRNA synthetase selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 as an active ingredient in an amount effective for immune enhancement, wherein the tryptophanyl-tRNA synthetase increases infiltration of neutrophils in the subject.

3. The method of claim 1, wherein the composition is a pharmaceutical, food, or veterinary composition.

4. The method of claim 1, wherein the infectious inflammatory disease is selected from the group consisting of salmonellosis, food poisoning, typhoid, paratyphoid, sepsis, septic shock, systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), pneumonia, pulmonary tuberculosis, tuberculosis, cold, influenza, airway infection, rhinitis, nasopharyngitis, otitis media, bronchitis, lymphadenitis, mumps, adenolymphitis, cheilitis, stomatitis, arthritis, myositis, dermatitis, vasculitis, gingivitis, pericementitis, keratitis, conjunctivitis, wound infection, peritonitis, hepatitis, osteomyelitis, cellulitis, meningitis, encephalitis, brain abscess, encephalomyelitis, cerebral meningitis, osteomyelitis, nephritis, carditis, endocarditis, enteritis, gastritis, esophagitis, duodenitis, colitis, urinary tract infection, cystitis, vaginitis, cervicitis, salpingitis, infectious erythema, bacterial dysentery, abscess and ulcer, bacteremia, diarrhea, dysentery, gastritis, gastroenteritis, genitourinary abscess, open wound or wound infection, purulent inflammation, abscesses, boils, pyoderma, impetigo, folliculitis, cellulitis, wound infection after surgery, scalded skin syndrome, skin burn syndrome, thrombotic thrombocytopenia, hemolytic uremic syndrome, renal failure, pyelonephritis, glomerulonephritis, nervous system abscess, otitis media, sinusitis, pharyngitis, tonsillitis, mastoiditis, soft tissue inflammation, dental infection, dacryocystitis, pleurisy, abdominal abscess, liver abscess, cholecystitis, spleen abscess, pericarditis, myocarditis, placentitis, amniotic fluid infection, mammitis, mastitis, puerperal fever, toxic shock syndrome, lyme disease, gas gangrene, atherosclerosis, mycobacterium avium syndrome (MAC), enterohaemorrhagic Escherichia coli (EHEC) infection, enteropathogenic Escherichia coli (EPEC) infection, enteroinvasive Escherichia coli (EIEC) infection, methicillin-resistant Staphylococcus aureus (MRSA) infections, vancomycin-resistant Staphylococcus aureus (VRSA) infections and listerosis.

5. The method of claim 2, wherein the composition is a pharmaceutical, food, or veterinary composition.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1A shows the result of western blot analysis of TrpRS (WRS) of human peripheral blood mononuclear cell supernatant (SUP) and whole cell lysate (WCL) infected with S. typhimurium, and FIG. 1B shows the result of ELISA for the representative amount of TrpRS, HMGB-1 and HSP70 protein in culture supernatant of human peripheral blood mononuclear cells infected by bacteria and fungi.

(3) FIG. 2A shows the result of western blot analysis showing the secretion pattern of tryptophanyl-tRNA synthetase (TrpRS), tumor necrosis factor alpha (TNF-α), HMGB-1 and HSP70 secreted by S. typhimurium-infected human peripheral blood mononuclear cells over time, and FIG. 2B shows the results of ELISA showing the measurement of TNF-α and TrpRS produced in the culture supernatant of human peripheral blood mononuclear cells infected with S. typhimurium over time.

(4) FIG. 3 shows a result of lactate dehydrogenase (LDH) assay to determine whether pyroptosis occurs in human peripheral blood mononuclear cells infected with S. typhimurium.

(5) FIG. 4 shows real time-PCR results showing the mRNA levels of TNF-α and TrpRS produced in human peripheral blood mononuclear cells infected with S. typhimurium over time.

(6) FIG. 5 shows the results of TrpRS antibody treatment and ELISA test to examine the effect of TrpRS on the secretion of TNF-α in human peripheral blood mononuclear cells infected with S. typhimurium.

(7) FIG. 6A shows the amount of TrpRS (WRS) protein in the culture supernatant of human peripheral blood mononuclear cells infected with respiratory syncytial virus (RSV) and FIG. 6B shows human peripheral blood mononuclear cells infected with PR8 influenza virus, respectively. The results obtained by ELISA are shown.

(8) FIG. 7 shows ELISA results showing the secretion pattern of TNF-α and MIP-1α in TrpRS-treated immune cells.

(9) FIG. 8A shows that the secretion amounts of MIP-1α, TNF-α, MIP-1β, IL-6 and IL-8 were analyzed using image Lab 4.1 software after treating PBMC cells with full length-TrpRS and mini-TrpRS; FIG. 8B shows results of ELISA; and FIG. 8C shows RT-PCR for confirming the expression levels of TNF-α, MIP-1α and MIP-1β in cultured supernatant cultured in PBMC.

(10) FIG. 9 shows the results of a transwell migration assay showing the effect of the culture supernatant of TrpRS-treated bone marrow-derived macrophages (BMDM) on the immune cell fluidity.

(11) FIG. 10A-C shows the results of ELISA for the amount of MIP-1α in PBMC-derived macrophages, THP-1 cells, THP-1 derived macrophages and J774A.1 cells, and the amount of TNF-α, MIP-1α MCP-1 protein as determined by ELISA and RT-PCR, respectively.

(12) FIG. 11A-D shows the result of flow cytometry analysis showing the neutrophil recruitment in the mouse peritoneal cavity injected with TrpRS, the result of ELISA showing the secretion amount of MIP1-α, and the results of flow cytometric analysis showing the number of neutrophil, macrophage, and bone marrow cells.

(13) FIG. 12A-B shows flow cytometric analysis results showing CD40, CD80, and CD86 protein expression patterns of TrpRS-treated bone marrow-derived macrophages.

(14) FIG. 13 shows the results of macrophage experiments using fluorescently-labeled Escherichia coli derived from the phagocytosis index of TrpRS-treated bone marrow-derived macrophages.

(15) FIG. 14A-B shows the results of measurement of the amount of TNF-α and MIP-1α in the full length-TrpRS and mini-TrpRS-treated macrophages by ELISA, and the results of measurement of the number of CD11b.sup.+ F4/80.sup.+ cells in PEC (peritoneal exudate cells) and Ly6G.sup.+ neutrophils by flow cytometry.

(16) FIG. 15A-B shows the images (X20) obtained by injecting the full length-TrpRS and mini-TrpRS (red) labeled with Alexa 647 in the LysM-GFP transgenic mice, and the index of macrophage in the mice infected with S. typhimurium, which were injected with full length-TrpRS and mini-TrpRS.

(17) FIG. 16 schematically shows a process for producing an antibody specifically binding to TrpRS.

(18) FIG. 17A-C shows the amount of TNF-α in THP-1 cells treated with full length-TrpRs and antibodies specific to TrpRS, the results of ELISA showing the effect of antibody (scFv 4G1) on TNF-α production in human PBMC cells, and the expression of recombinant protein binding to full length-TrpRS or mini-TrpRS using antibodies.

(19) FIG. 18A-C shows a schematic diagram of an animal experiment using an antibody; the effect of the antibody on the expression of TNF-α and MIP-1α and the effect on the number of Ly6G.sup.+ neutrophils.

(20) FIG. 19 shows a survival plot showing the effect of antibodies specific to TrpRS on the survival rate of S. typhimurium-infected mice.

(21) FIG. 20 shows the overviews of animal experiments to investigate the effect of TrpRS on the survival rate of mice infected with S. typhimurium (top in FIG. 20) and the results of flow cytometry showing the effect of TrpRS on the degree of S. typhimurium-adsorption or -absorption in infiltrating neutrophils.

(22) FIG. 21A-B shows a schematic diagram of animal experiments and shows the results of measuring IHC and CFU in the liver and spleen of mice, which were administered intraperitoneally with PBS(Con), FL-WRS, or mini-WRS (each 20 mg/mouse) 1 hour before the infection with S. typhimurium or L. monocytogenes.

(23) FIG. 22A-B shows a survival plot showing the effect of TrpRS on the survival rate of S. typhimurium-infected mice.

DETAILED DESCRIPTION OF THE INVENTION

(24) Hereinafter, the present invention will be described in detail.

(25) However, the following examples are illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

Example 1

The Tryptophanyl-tRNA Synthetase (TrpRS) Secreted from Bacterial Infected Peripheral Blood Mononuclear Cells

Example 1-1. Identification of Aminoacyl-tRNA Synthetase Secreted from Bacterial and Fungal Infected Human Peripheral Blood Mononuclear Cells

(26) Immunoblot experiments were conducted to determine the type of aminoacyl-tRNA synthetase (ARS) secreted from human peripheral blood mononuclear cells (PBMC) infected with bacteria (FIG. 1). Bacteria and fungi, Salmonella typhimurium (S. typhimurium, ATCC 14028), Listeria monocytogenes (L. monocytogenes, ATCC 15313), Escherichia coli (E. coli, ATCC 10798), Staphylococcus aureus (S. aureus, ATCC 25923), Candida albicans (C. albicans, ATCC 10231), were obtained from the Korea Microbial Conservation Center and the Microbiological Resource Center, and were cultured periodically in nutrient broth or brain heart infusion (BD biosciences). The bacteria used for the infection experiment were cultured overnight, and were obtained at a density of 1×10.sup.8 CFU. The CFU was estimated using an absorbance at 600 nm and a calibration curve prepared beforehand. In the bacterial infection experiment of this example and another example, the obtained bacteria were washed in PBS and resuspended in serum-free RPMI medium or PBS. Human PBMC were cultured at a density of 1×10.sup.6 cells/well for one hour and infected with 1×10.sup.6 CFU of S. typhimurium (MOI=1) or L. monocytogenes (MOI=1). The supernatants cultured for two hours after infection were precipitated by TCA and immunoblot experiments were performed on various kinds of ARS.

(27) As a result, the only full-length TrpRS, 53 kDa, was detected in the culture supernatant among the ARSs including secretory ARS (TrpRS, GRS, KRS, DRS), AIMP1 (a cofactor of ARS complexes) and ARS (TrpRS, MRS, HRS, GRS) containing the WHEP domain. This shows that the ARS secreted after bacterial infection is TrpRS (WRS) (FIG. 1A). It was also confirmed that full-length WRS was secreted from human peripheral blood mononuclear cells by infection with S. typhimurium and L. monocytogenes as well as by E. coli, S. aureus and C. albicans (FIG. 1B). This indicates that TrpRS (WRS) is more likely to be secreted in response to general bacterial and fungal infections than to certain bacteria.

Example 1-2. Kinetic Analysis of TrpRS and TNF-α Secretion

(28) The secretion patterns of tryptophanyl-tRNA synthetase (TrpRS), tumor necrosis factor alpha (TNF-α), HMGB1, and heat shock protein (HSP70) was analyzed in human peripheral blood mononuclear cells (PBMC) infected with S. typhimurium from time to time right after infection (FIG. 2). Human PBMC were infected with S. typhimurium (MOI=1), and whole cell lysate (WCL) and culture supernatant were obtained at 0, 15, 30, 60, and 120 min, and immunoblot experiments were performed (FIG. 2A).

(29) As a result, TrpRS in cultured supernatants was detected from 15 minutes after infection and increased for up to 120 minutes after infection. Also, the amount of TrpRS in WCL tended to decrease slightly with time after infection. Therefore, TrpRS is secreted to the outside of the cell from the beginning after infection. In addition, in the whole cell lysate, the TNF-α precursor (precursor TNF-α) was generated from 30 minutes after infection and was detected at 120 minutes, TNF-α (Mature TNF-α) was detected in trace amounts up to 120 minutes after infection. TNF-α (Mature TNF-α) was detected in the culture supernatant at 120 min. In other words, TrpRS is secreted to the outside of the cell within a short time after the infection, and it is found that it significantly precedes the secretion of TNF-α in time.

(30) On the other hand, HMGB-1 and HSP70 were not detected in the culture supernatant over time after infection, whereas HMGB-1 and HSP 70 in all cell lysates were constantly detected with time after infection. Thus, HMGB-1 and HSP70 are not affected by bacterial infection.

(31) In addition, the amounts of TrpRS and TNF-α secreted in the culture supernatant after bacterial infection were measured by ELISA at 0, 15, 30, 60, and 120 min after infection (FIG. 2B). As observed in the immunoblot experiment, TrpRS present in the culture supernatant increased significantly from 15 minutes after infection to 60 minutes after infection, but TNF-α present in the culture supernatant started to increase from 60 minutes after infection.

Example 1-3. Determination for Bacterial Infection and Pyroptosis

(32) It was investigated whether TrpRS secretion in human peripheral blood mononuclear cells (PBMC) infected with S. typhimurium observed in Example 1-2 was caused by pyroptosis (FIG. 3). The pyroptosis was confirmed by assay of lactate dehydrogenase (LDH; a signature of cell death). In detail, human PBMCs were infected with S. typhimurium, and then LDH levels in culture supernatants at 0, 5, 10, 20, 40, 60, and 120 min after infection were measured using the LDH assay kit (Takara Bio, Inc.). 1% TX-100 treated PBMC was used as a positive control.

(33) As a result, in the culture supernatant of the positive control (1% TX-100), the amount of LDH, a marker of pyroptosis, increased rapidly with time, whereas the culture supernatant of S. typhimurium-infected PBMC reached 120 minutes after infection with no significant change in the amount of LDH, indicating that LDH was not expressed during this period. Therefore, these results verify that PBMC after bacterial infection excretes TrpRS outside the cell by active secretion rather than pyroptosis (FIG. 3).

Example 1-4. Evaluation on Bacterial Infection and Expression Levels of TNF-α and TrpRS

(34) The expression patterns of TNF-α and TrpRS (WRS) genes in human peripheral blood mononuclear cells (PBMC) infected with S. typhimurium were examined by RT-PCR and ELISA (FIG. 4). Human PBMC were infected with S. typhimurium, and cells were obtained at 0, 15, 30, 60, 120 minutes after the infection, and the mRNA levels of TNF-α and TrpRS were measured. RNA was extracted according to the experimental method of RNeasy kit (Qiagen, Hilden, Germany) and reverse transcribed with M-MLV reverse transcriptase (Invitrogen, Carlsbad, Calif., USA), then quantitative real-time PCR (quantitative RT-PCR) was performed with the following primers. The level of GAPDH mRNA was determined as an internal control.

(35) TABLE-US-00001 hTNF-α Forward: (SEQ ID NO: 9) 5-GGAGAAGGGTGACCGACTCA-3 hTNF-α Reverse: (SEQ ID NO: 10) 5-CTGCCCAGACTCGGCAA-3 hTrpRS Forward: (SEQ ID NO: 11) 5-AAGAATTCATGCCCAACAGTGAGCCC-3 hTrpRS Reverse: (SEQ ID NO: 12) 5-AACTCGAGCTACCCTGGAGGACAGTCAGCCTT-3 GAPDH forward: (SEQ ID NO: 13) 5-CGCTCTCTGCTCCTCCTGTTC-3 GAPDH reverse: (SEQ ID NO: 14) 5-TTGACTCCGACCTTCACCTTCC-3

(36) As a result, it was confirmed that the level of TNF-α mRNA increased from 30 minutes to 120 minutes, while the level of TrpRS mRNA did not significantly change until 120 minutes after infection (FIG. 4). This suggests that TrpRS secreted after infection is one, which is already present in the cell without its gene expression induced after infection. It was also confirmed that mRNA of TNF-α, a major proinflammatory cytokine induced by bacterial infection, increased greatly from 30 minutes to 12 minutes after infection. The TNF-α mRNA was significantly increased up to 60 minutes after 40 minutes of infection and the increase in the amount of TNF-α secretion observed in Example 1-2 was detected from 60 minutes after the infection, indicating that the secretion of TrpRS is already occurring before TNF-α production.

Example 1-5. Relationship Between TrpRS and TNF-α Secreted after Bacterial Infection

(37) In Example 1-2, it was confirmed that TrpRS and TNF-α were secreted with time difference in S. typhimurium-infected human peripheral blood mononuclear cells (PBMC). In addition, it was observed that TNF-α was not secreted in the absence of TrpRS secretion at the early stage of infection in the PBMC infected with Heat-killed S. typhimurium. Thus, the functional relationship between TrpRS secreted after infection and TNF-α was examined (FIG. 5). PBMCs were treated with TrpRS function-neutralizing antibody (antibody concentration 10 μg/mL) and infected with S. typhimurium (MOI=1). Then the secretion pattern of TNF-α was measured at 0, 2, 4, and 8 h, by ELISA.

(38) As a result, in PBMC (S. typhimurium+α-WRS ScFv), which had reduced the function of TrpRS by antibody treatment, it was observed that the amount of TNF-α was significantly decreased compared with PBS-treated control PBMC (S. typhimurium+PBS). This suggests that TrpRS plays an important role in the production and secretion of TNF-α. These results suggest that TrpRS may be the first factor to control TNF-α production, especially in bacterial infections.

Example 1-6 Aminoacyl t-RNA Synthetase Secreted from Virus-Infected Human Peripheral Blood Mononuclear Cells

(39) ELISA experiments were performed to determine the amount of aminoacyl t-RNA synthetase (ARS) secreted by human peripheral blood mononuclear cells (PBMC) over time after viral infection (FIG. 6). The virus was titrated by respiratory syncytial virus A2 (RSV A2) and PR8 influenza virus (Influenza A/Puerto Rico/8/1934 virus) by standard plaque analysis. Human PBMCs were each infected with the human PBMC for 2 hours and incubated for 24 hours. Culture supernatants were harvested at each hour post-infection and levels of TrpRS (WRS) were measured by ELISA.

(40) As a result, the concentration of TrpRS increased with time in RSV-infected cells (FIG. 6A), while in the PR8-infected cells, the TrpRS concentration increased up to 120 minutes after infection but decreased after 120 minutes (FIG. 6B). This shows that TrpRS is secreted in response to viral infection.

Example 2

Increased Secretion of Monocyte/Macrophage-Specific TNF-α and MIP1-α by TrpRS

(41) To investigate the types of immune cells responsive to secreted TrpRS, various immune cells were treated with TrpRS and cells that secrete cytokines such as TNF-α and MIP1-α were examined (FIG. 7). Primary B cells, T cells, neutrophils, natural killer cells (NK), and peripheral blood mononuclear cells (NK) and monocyte of mice were isolated using a mouse microbead isolation kit (Miltenyi, Bergisch Gladbach, Germany) The obtained cells showed more than 95% purity when analyzed by flow cytometry. Bone marrow-derived macrophages (BMDM) were prepared by differentiating for 6 to 7 days in the presence of M-CSF (20 ng/mL). Bone marrow-derived dendritic cells (BMDC) were prepared by differentiating for 6 to 7 days in the presence of GM-CSF (20 ng/ml) and IL-4 (20 ng/ml) Immunocytes were cultured for 18 hours in the presence of the control, full-length TrpRS (TrpRS-full, 100 nM), mini-TrpRS (TrpRS-mini, 100 nM), and LPS (100 ng/ml), respectively, and the amount of TNF-α and MIP1-α protein present in the culture supernatant was measured by ELISA kit (BD Sciences, San Jose, Calif., USA and R & D systems). TrpRS all used a protein having the amino acid sequence of human TrpRS (human TrpRS) in the experiment.

(42) As a result, LPS significantly induced TNF-α secretion in most of the immune cells, whereas full-length TrpRS showed the greatest effect in monocytes and BMDM (Table 1, top of FIG. 7). Full-length TrpRS treated with heat or trypsin had no effect on TNF-α secretion (data not shown). In addition, mini-TrpRS had no effect on TNF-α secretion. For MIP1-α, the increase effect of TrpRS was confirmed only in monocytes and BMDM of mice treated with full-length TrpRS (Table 2, FIG. 7 bottom). These results indicate that TrpRS specifically activates monocytes and macrophages, and monocytes and macrophages secrete TNF-α and MIP1-α as a result of activation.

(43) TABLE-US-00002 TABLE 1 Effect of TrpRS on the secretion of TNF-α in immune cells TNF-α Full-length Mini (pg/mL) Control TrpRS TrpRS LPS B cell 107 ± 91  132 ± 34  79 ± 44 227 ± 38  CD4 + T cell 2 ± 4 99 ± 20 134 ± 125 516 ± 4  Monocyte 69 ± 7  511 ± 43  10 ± 13 975 ± 24  BMDM 17 ± 11 3299 ± 175  44 ± 35 3573 ± 7   BMDC 61 ± 11 1083 ± 105  148 ± 119 3557 ± 66  Neutrophil 21 ± 4  597 ± 38  65 ± 4  3765 ± 130  NK 28 ± 8  259 ± 14  3 ± 8 2373 ± 200 

(44) TABLE-US-00003 TABLE 2 Effect of TrpRS on the secretion of MIP1-α in immune cells MIP1-α Full-length Mini (pg/mL) Control TrpRS TrpRS LPS B cell 57 ± 2  124 ± 2  85 ± 23 391 ± 49  CD4 + T cell 8 ± 1 141 ± 13  9 ± 1 552 ± 7  Monocyte 7 ± 0 533 ± 27 6 ± 1 1246 ± 7   BMDM 7 ± 7 2647 ± 205  8 ± 1 3369 ± 45  BMDC 32 ± 5  34 ± 34 50 ± 16 980 ± 78  Neutrophil 6 ± 3 26 ± 3  7 ± 5 3529 ± 8   NK 6 ± 3 36 ± 1  3 ± 1 1157 ± 41 

Example 3

Activation of Innate Immune Response by TrpRS

Example 3-1. Chemokines Secreted by TrpRS-Stimulated Bone Marrow-Derived Macrophages

(45) The types and amounts of chemokines secreted by BMDM in response to TrpRS were examined (Table 3). BMDM was treated with full-length TrpRS (30, 100 nM) or mini-TrpRS (100 nM) for 18 hours, and the presence of chemokines in the culture supernatant was measured by ELISA.

(46) In addition, human PBMC cells were treated with human full length-TrpRS (FL-WRS) and mini-TrpRS (mini-WRS) for 18 hours and cytokines present in the culture supernatant were measured using ELISA. RT-PCR experiments were used to measure the expression levels of cytokines and chemokines.

(47) TABLE-US-00004 hTNF-α Forward: (SEQ ID NO: 9) 5-GGAGAAGGGTGACCGACTCA-3 hTNF-α Reverse: (SEQ ID NO: 10) 5-CTGCCCAGACTCGGCAA-3 hTrpRS Forward: (SEQ ID NO: 11) 5-AAGAATTCATGCCCAACAGTGAGCCC-3 hTrpRS Reverse: (SEQ ID NO: 12) 5-AACTCGAGCTACCCTGGAGGACAGTCAGCCTT-3 hMIP-1α Forward: (SEQ ID NO: 15) 5-ACCATGGCTCTCTGCAACCA-3 hMIP-1α Reverse: (SEQ ID NO: 16) 5-TTAAGAAGAGTCCCACAGTG-3 hMIP-1β Forward: (SEQ ID NO: 17) 5-AGCCTCACCTCTGAGAAAACC-3 hMIP-1β Reverse: (SEQ ID NO: 18) 5-GCAACAGCAGAGAAACAGTGAC-3 GAPDH forward: (SEQ ID NO: 13) 5-CGCTCTCTGCTCCTCCTGTTC-3 GAPDH reverse: (SEQ ID NO: 14) 5-TTGACTCCGACCTTCACCTTCC-3

(48) As a result, full-length TrpRS induced chemokines such as MIP-1α, MCP-1, and IP-10 and cytokines such as TNF-α and IL-1β in a concentration-dependent manner. On the contrary, mini-TrpRS had no effect on cytokine secretion and chemokine secretion (Table 3). In addition, full-length TrpRS induced the secretion of chemokines and cytokines such as TNFα, MIP-1α, MIP-1β, IL-6 and IL-8, but mini-TrpRS had no effect on cytokine and chemocyte secretion (FIG. 8A). In addition, it was confirmed that the expression levels of TNFα, MIP-1α, MIP-1β, IL-6 and IL-8 were increased in PBMC cells treated with full length-TrpRS (FIGS. 8B & 8C)

(49) Thus, it was confirmed that Full length-TrpRS induces the expression and secretion of cytokines, in contrast to mini-TrpRS.

(50) TABLE-US-00005 TABLE 3 The cytokines and chemokines secreted by TrpRS-stimulated bone marrow-derived macrophages (BMDM) Full-length TrpRS Mini TrpRS Cytokines Control 30 nM 100 nM 100 nM TNF-α 17 ± 11 2159 ± 691  3299 ± 175  44 ± 35 MIP-1α 7 ± 7 1621 ± 49  2647 ± 205  8 ± 1 MCP-1 1 ± 1 793 ± 39  1430 ± 114  14 ± 19 IP-10 43 ± 12 283 ± 15  493 ± 76   0 ± 10 IL-1β 14 ± 4  59 ± 4  135 ± 14  16 ± 11

Example 3-2. Effect of TrpRS on Immune Cell Fluidity

(51) The effect of TrpRS on the fluidity of immune cells was examined using a transwell migration assay (FIG. 9). Bone marrow-derived macrophages (BMDM) were treated with full-length TrpRS (FL-WRS, 100 nM) or mini-TrpRS (mini-WRS, 100 nM) and cultured for 18 hours, then transferred to a 24-well plate. Primary immune cells and BMDM were placed in the upper part of the migration chamber and incubated at 37° C. for 4 hours.

(52) As a result, it was observed that the culture supernatant of full-length TrpRS-treated BMDM (TrpRS-F) greatly increased infiltration of monocytes and neutrophils. In contrast, the TrpRS protein itself (data not shown) or the mini-TrpRS-treated culture supernatant (TrpRS-M) had no effect on cell migration.

Example 3-3. Effect of TrpRS on the Secretion of MIP-1 and TNF-α in Various Immune Cells

(53) Effects of TrpRS on secretion of MIP-1 and TNF-α in various immune cells including PBMC-derived macrophages, THP-1 cells, THP-1 derived macrophages and J774A.1 cells were investigated by ELISA experiments and RT-PCR respectively. Each immune cell was treated with full length-TrpRS (FL-WRS, 100 nM) or mini-TrpRS (mini-WRS, 100 nM) and cultured for 18 hours. The culture was transferred to a 96-well plate and subjected to ELISA. The BMDM cells were treated with full-length TrpRS or mini-TrpRS and mRNA was extracted and RT-PCR was performed.

(54) As a result, it was confirmed that full length-TrpRS in PBMC-derived macrophages, THP-1, THP-1 derived macrophages and J774A.1 induce the secretion of MIP-1α (FIG. 10A). In BMDM cells, it was confirmed that full length-TrpRS, which is not mini-TrpRS induces the production of TNF-α, MIP-1α and MCP-1 proteins (FIG. 10B) and induces mRNA expression (FIG. 10C).

Example 3-4. TrpRS-Induced Neutrophil Accumulation in the Body

(55) To investigate the effect of TrpRS on the innate immune response in the body, intraperitoneal neutrophil recruitment following TrpRS injection was observed (FIG. 11). PBS, full-length TrpRS (3, 10, 30 μg), or mini-TrpRS (30 μg) were injected into the peritoneal cavity of mice (6 to 10 per group) and the peritoneal cavity was drained after 4 hours. The peritoneal effusion was extracted and the ratio of Ly6C+Ly6G+cell group was measured by flow cytometry (FIGS. 11A & 11B), and MIP1-α present in the peritoneal effusion was measured by ELISA (FIG. 11C). The statistical significance of the comparison between the PBS-treated negative control and the results of each experimental condition was verified by one-way Anova test using GraphPad (ver. 4.0) software. *** p<0.001, ** p<0.01

(56) As a result of flow cytometry, concentration-dependent tendency was dramatically observed only in the full-length TrpRS injected mice (TrpRS-F3, TrpRS-F10 and TrpRS-F30), whereas mini-TrpRS (TrpRS-M30) did not achieve this effect (FIG. 11B). That is, this result shows that full-length TrpRS can induce innate immune responses that cause neutrophil concentration in vivo. Similarly, MIP1-α, which was measured in the peritoneal effusion, increased in a concentration-dependent manner only in the full-length TrpRS injected mice (TrpRS-F3, TrpRS-F10, and TrpRS-F30), and did not increase in mini-TrpRS injected mice (TrpRS-M30).

(57) In addition, the number of Ly6G.sup.+ neutrophils, CD11b.sup.+ myeloid cells, and CD11b.sup.+F4/80.sup.+ macrophages increased in the full length-TrpRS (FL-WRS) treated peritoneum as compared to the mini-TrpRS and PBS-infected controls (FIG. 11D).

Example 3-5. Effect of TrpRS on the Activation of Bone Marrow-Derived Macrophages

(58) The effect of TrpRS on bone marrow-derived macrophage (BMDM) activation was analyzed through the expression of activated macrophage markers and flow cytometry analysis (FIG. 12). Flow cytometric analysis using Cd11b and F4/80 of FIG. 11A indicates that bone marrow cells were well differentiated into BMDM for macrophage activation label analysis. Differentiated BMDM were treated with full-length TrpRS (30, 100 nM) or mini-TprRS (100 nM) and cultured for 18 h and CD40, CD80 and CD86 protein expression levels were measured by flow cytometry.

(59) As a result, it was confirmed that the cell surface expression of CD40, CD80 and CD86, which are macrophage activation indicators, was abruptly increased only in the full-length TrpRS-treated BMDM (TrpRS-F) (FIG. 12B). The Mini-TrpRS-treated BMDM (TrpRS-M) was not significantly different from the control group. That is, this result verifies that full-length TrpRS promotes activation of macrophages.

Example 3-6. Effect of TrpRS on Phagocytosis of Bone Marrow-Derived Macrophages (BMDM)

(60) The effect of TrpRS on the phagocytosis of bone marrow-derived macrophages (BMDM) was investigated (FIG. 13). BMDM was cultured in 96-well culture dishes overnight, then replaced with RPMI medium without M-CSF, and cultured for 18 hours with full-length TrpRS (30 nM) or mini-TrpRS (30 nM). After washing the cells, 100 μL of fluorescently labeled E. coli bioparticles (Vybrant phagocytosis assay kit, Invitrogen) was added and cultured for 2 hours. After the culture medium was removed, the cells were treated with trypan blue (100 μL) for 1 minute to inhibit autofluorescence. Fluorescence signals due to macrophage were measured at 485 nm (excitation) and 520 nm (emission) to display the degree of phagocytic action (BMG Labtech, FLUOstar OPTIMA, Ortenberg, Germany). Statistical significance was verified by t-test using GraphPad (ver. 4.0) software. *** p<0.0001

(61) As a result, the phagocytic index of full-length TrpRS-treated BMDM (TrpRS Full) was found to be 3 times higher than that of the control group. The Mini-TrpRS (TrpRS Mini) had no effect. Experimental results of Examples 3-1 to 3-4 show that full-length TrpRS induces innate inflammatory responses through macrophage activation.

Example 3-7. Effects of TrpRS on Macrophages

(62) To investigate the effect of the full length-TrpRS-derived congenital immune response on macrophages, the following experiment was carried out using macrophage-depleted mice.

(63) The splenocytes were removed from the spleen of normal mice and macrophage-nulled mice and the removal of macrophages was confirmed by flow cytometry. Separated splenocytes were prepared, cultured overnight in a 24-well culture dish, and treated with full length-TrpRS, and the secretion of TNF-α and MIP-1α was measured by ELISA.

(64) As a result, it was confirmed that the secretion amount of TNF-α and MIP-1α decreased with the decrease of macrophages (FIG. 14A). The decrease in macrophages in the body indicates that macrophages play a functional role in the immunostimulatory activity of full length-TrpRS, as the number of infiltrated neutrophils in the peritoneum decreases (FIG. 14B). This confirms that full length-TrpRS targets macrophages to activate the innate immune response.

Example 3-8. Effect of TrpRS on Macrophage Activity

(65) The effect of TrpRS on bone marrow-derived macrophage activity was investigated. Alexa 647 labeled full-length TrpRS (FL-WRS) or mini-TrpRS (mini-WRS) were inoculated into the ear of mice and photographed 1 and 4 hours post-infection using in vivo imaging techniques. In addition, S. typhimurium labeled with Alexa 647 combined with Full length-TrpRS or mini-TrpRS was inoculated into the ear of GFP-LysM Tg mice capable of observing neutrophils and macrophages by GFP expression. To observe macrophages in mice, macrophages were photographed using in vivo imaging technology and counted Immuno-cellular dynamics were visualized using a custom-built video-rate laser-scanning confocal microscope imaging system (Choe et al., 2013; Seo et al., 2015). Three consecutive lasers were used as the fluorescent stimulus source. Three fluorescence colors emitted from the mice were detected as highly sensitive optoelectronic layer tubes at 488 nm (MLD, Cobolt), 561 nm (Jive, Cobolt) and 640 nm (MLD, Cobolt). And it was digitized by the 8-bit 3-channel frame grabber.

(66) As a result, it was confirmed that infiltration of neutrophils and macrophages appeared at the site of full-length TrpRS inoculation instead of mini-TrpRS. Such an infiltration started at 1 hour after inoculation, and the amount of infiltration at 4 hours after inoculation was the highest (FIG. 15A). In addition, it was confirmed that phagocytic index of macrophage was increased in mice treated with full length-TrpRS (FL-WRS) as compared to mice treated with mini-TrpRS (mini-WRS) or PBS(Con) (FIG. 15B).

Example 4

TrpRS Inhibition-Devised Effect

Example 4-1. Preparation of Antibodies Specifically Binding to TrpRS

(67) TrpRS was titrated with TrpRS-specific antibodies to further validate the protective role of TrpRS against infection. First, an antibody that specifically binds N154 peptide of human TrpRS was prepared by panning a library of phage display human single chain variable fragments (scFV) (FIG. 16) Immuno tubes were coated with TrpRS 10 and blocked with MPBS (PBS with 3% nonfat dried milk). Phage-displayed scFv library (1×10.sup.13 CFU) was added to 1 ml of mPBST, added to the immune tube, incubated for 2 hours and then washed 3-5 times with PBST. The bound phage was eluted with 1 mL of 100 mM trimethylamine and neutralized with 0.5 mL of 1 M Tris-HCl (pH 7.0). E. coli ER257 was infected with the eluted phage and cultured in LB medium containing 2% glucose and ampicillin for one day, and bacteria were collected from the medium logarithm. The bacteria were then cultured in 20 mL SB (3% Tryptone, 2% yeast extract, 1% MOPS, pH 7.0) supplemented with ampicillin to an OD600 of 0.5. VCSM13 helper phage (1×10.sup.11 PFU) was added and incubated at 37° C. for 1 hour, followed by kanamycin (70 μg/ml) and incubated overnight at 30° C. The supernatant was removed by centrifugation, and phage precipitation solution (4% PEG 8000 and 3% NaCl, final concentration) was added and mixed. After 30 minutes of incubation on ice, the precipitated phage were collected by centrifugation. After panning the library four times, individual scFv clones capable of binding TrpRS were screened. This process is illustrated in FIG. 16.

(68) In order to confirm whether the prepared antibodies specifically bound to TrpRS affects the effect of TrpRS on infection, the following experiment was conducted. PBMC was treated with the prepared antibody (10 μg/mL) and infected with S. typhimurium (MOI=1) as described in Example 1 above. The secretion pattern of TNF-α in cell culture was measured by ELISA. In order to confirm whether scFv 4G1 binds to full length-TrpRS (FL-WRS) by selecting 4G1 clones, PBMC cell lysates were extracted and immunoblotted.

(69) As a result, it was confirmed that the F9, Ell, and 4G1 clones among some clones showed a decrease in the secretion amount of TNF-α of full length-TrpRS in human PBMC cells (FIG. 17A), while both only 4G1 treatment and full length-TrpRS and 4G1 treatment (FIG. 17B) decreased the amount of the secreted TNF-α. In addition, it was confirmed through immunoblotting that scFV 4G1 specifically binds to full length-TrpRS (FIG. 17C).

Example 4-2. Decreased Secretion of TNF-α and MIP1-α by TrpRS-Specific Antibodies

(70) To examine the effect of the scFV 4G1 prepared in Example 5-1 on the innate immune response in the body, an animal experiment using mice was carried out according to the experimental outline of FIG. 18A.

(71) Mice were injected with PBS or scFv 4G1 in infected or uninfected groups by intraperitoneal injection of S. typhimurium. Four hours after the infection, the peritoneal fluid was extracted, and the number of Ly6G.sup.+ cells was measured by flow cytometry. The amount of MIP-la and TNF-α was analyzed by ELISA. The statistical significance of the comparison between the PBS-treated negative control and the results of each experimental condition was verified by one-way Anova test using GraphPad (ver. 4.0) software.*** p<0.001, ** p<0.01

(72) ELISA analysis showed that the amount of MIP-1 alpha and TNF-α decreased in scFv 4G1-treated mice (FIG. 18B). In addition, flow cytometry analysis showed that Ly6G.sup.+ macrophages decreased in scFv 4G1-treated mice (FIG. 18C).

Example 4-3. Effect of TrpRS-Specific Antibodies on Survival of Bacterially Infected Mice

(73) To confirm the effect of the scFv 4G1 prepared in Example 5-1 on the survival rate of the mice, an animal experiment using a mouse was conducted according to the experimental outline of FIG. 18A, and survival analysis was performed. As described above, mice were injected with PBS or scFv 4G1 in infected or uninfected groups by injecting S. typhimurium into the abdominal cavity. The survival rate of mice was 12, 24, 36, 48 hours after infection.

(74) As a result, in the control, ST+PBS (injection of PBS after infection with S. typhimurium) survival rate was 50% at 24 hours and 25% at 48 hours, but in ST+4G1 (scFv 4G1 injection after S. typhimurium infection) survival rate was less than 25%, and 0% at 36 hours. Thus, it was confirmed that the survival rate of mice was decreased in the group injected with scFv 4G1 (FIG. 19).

Example 5

Effect of TrpRS In Vivo Infection Inhibition and Increased Survival Rate after Infection

Example 5-1. Effect of TrpRS on the Absorption of Infecting Bacteria by Invasive Neutrophils

(75) To investigate the effect of TrpRS on the survival rate of mice infected with S. typhimurium, an animal experiment using mice was carried out according to the outline of the experiment of FIG. 20 (FIG. 20, top), and the amount of S. typhimurium absorbed by and adsorbed onto infiltrated neutrophil was confirmed for one hour after infection (FIG. 20, bottom).

(76) PBS or TrpRS (10 μg, 0.5 mg/kg) was injected intraperitoneally into mice (C57BL/6 mouse, 9-12 week old female) and fluorescently labeled S. typhimurium (FITC-labeled S. Typhimurium, 1×10.sup.7 CFU/mouse, 5˜6 per group) was injected into the abdominal cavity 1 hour later. One hour after infection, mice were sacrificed and the peritoneal cells were separated and stained with Ly6G, Ly6C antibody and analyzed by flow cytometry. Statistical significance was verified by t-test. *** p<0.001

(77) As a result, it was confirmed that the number of infiltrating neutrophils (Ly6G.sup.+ Neutrophils) was also significantly increased in the full-length TrpRS-treated group (TrpRS-Full) compared with the PBS-treated control group (PBS), and that S. typhimurium (FITC-S. typhimurium.sup.+/Ly6G.sup.+ neutrophil) adsorbed onto and absorbed by neutrophils was significantly increased.

Example 5-2. Effects of TrpRS on Bacterial Clearance in Spleen and Liver

(78) An animal experiment using a mouse was performed according to the outline of FIG. 15, and the degree of bacterial clearance of the spleen after 4 hours of infection was observed (FIG. 21). After 4 hours of infection with S. typhimurim (FIG. 21A) or L. monocytogenes (FIG. 21B), mice were sacrificed. Spleen and hepatic homogenate were made and cultured in NB agar medium (serial ratios of 4 to 8 mice per group) by 10× serial dilution. After culturing at 37° C. for 24 hours, the bacterial CFU was calculated and analyzed. Statistical significance was then verified by t-test using GraphPad (ver. 4.0) software. ** p<0.01

(79) As a result, it was confirmed that, in S. typhimurium—infected mice, bacterial CFU in the spleen and liver of the group treated with full-length TrpRS was significantly reduced, compared with the group treated with mini-TrpRS (mini-WRS) or PBS (FIG. 21A). In mice infected with L. monocytogenes, the bacterial CFU in the spleen and liver of the group treated with full length-TrpRS (FL-WRS) was significantly lower than that of the group treated with mini-TrpRS (mini-WRS) (FIG. 21B). This shows that in the spleen and liver of TrpRS treated mice, the bacteria were eliminated before they caused cell infections.

Example 5-3. Effect of TrpRS on the Survival Rate of Bacteria-Infected Mice

(80) To investigate the effect of TrpRS on the survival rate of mice infected with S. typhimurim (FIG. 22A) or L. monocytogenes (FIG. 22B), animal experiments with mice were carried out according to the experimental outline of FIG. 21 (FIG. 22). Mice were injected with PBS, full-length TrpRS (FL-WRS) or mini-TrpRS (mini-WRS), followed by infusion of 1×10.sup.7 CFU S. typhimurium or L. monocytogenes into the peritoneal cavity. The survival rate of the mice at 12, 24, 36 and 48 hours after the infection was expressed as a survival plot according to the treatment conditions.

(81) As a result, S. typhimurim-infected mice that were given full length-TrpRS were 100% viable until 36 hours post-infection and 75% survived until 48 hours post-infection, whereas the mice receiving PBS or mini-TrpRS were found to have over 80% mortality at 48 hours post-infection (FIG. 22A). L. monocytogenes-infected mice that received full length-TrpRS also survived 100% until 48 h before infection and 75% survived at 48 h after infection, whereas 75% of the mice receiving PBS or mini-TrpRS died in 24 hours (FIG. 22B). This indicates that the survival time of the TrpRS injected group was significantly longer than that of the control group or the mini-TrpRS treated group by the single injection of TrpRS alone.

(82) The results of the above Examples show that full-length TrpRS lowers the mortality of bacteria-infected mice by activating innate immune responses.

INDUSTRIAL AVAILABILITY

(83) As described above, the composition of the present invention can be effectively used to prevent diseases of humans and animals caused by bacterial, viral or fungal infection by inhibiting bacterial, viral or fungal infection at an early stage, particularly through activating innate immune response. In addition, the composition of the present invention can be used for immune enhancement and is highly industrially applicable.