CN106729763A - Polyinosinic acid has activation to the I type interferon paths that mediation lung tissue removes to streptococcus pneumonia - Google Patents
Polyinosinic acid has activation to the I type interferon paths that mediation lung tissue removes to streptococcus pneumonia Download PDFInfo
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Abstract
The present invention relates to biomedicine field, more particularly to polyinosinic acid has activation to the I type interferon paths that mediation lung tissue removes to streptococcus pneumonia.Inventor explores I types interferon in the mediation active role of PolyI:C, and PolyI:C can cause the death rate of the Secondary bacterial infections host for depending on I type interferon to increase.It was found that causing the stimulation of TLR3 and Cardif dependent signals paths by the I type interferon that polyinosinic acid is induced, host's lung tissue is set to be directed to two clinically important gram-positive bacteriums（Streptococcus pneumonia and methicillin-resistant staphylococcus aureus）Defence be damaged, increased neurological susceptibility of the animal body to gram-positive bacterium.Confirm that polyinosinic acid has activation to the I type interferon paths that mediation lung tissue removes to streptococcus pneumonia.
Description
Technical field：
The present invention relates to biomedicine field, more particularly to polyinosinic acid has activation to the I type interferon paths that mediation lung tissue removes to streptococcus pneumonia.
Background technology：
The virus infection of respiratory tract is common breathing problem, and often shows benign clinical disease course.However, the patient of a quite a lot of part is developed into property or Secondary bacterial infections[1][2][3], this complication can cause respiratory failure or death.Although children, the crowd of old man and hypoimmunity often faces the excessive risk for breaking out such complication, and virus and bacteria pneumonia is likely to come across normal adults, and causes heavy Disease Spectrum.As influenza infection is popular, virus-bacterial pneumonia is repeatedly reported.It is to cause the Influenza A H1N1 of the influenza pandemic phase in 20th century and 2009 to be very popular the death main cause of phase[4][5][6]。
It is that the mechanism for how promoting bacterium infection is still known little about it for virus infection, it is likely that extremely complex.The mechanism that influenza research case has pointed out causes the damage mechanisms of the general airway epithelial cell layer with protectiveness including influenza; the combination enhancing mechanism of bacteria mediated acceptor; anti-inflammatory factors such as IL-10 abduction mechanisms, and recognition mode molecule expression as Toll-like receptor decrease mechanism[7][8][9].Additionally, in the recent period, we and other people find, host is directed to the immune response of influenza, and such as I types and II types is interferon-induced, may be with the congenital antibacterial immunity response key link of suppression[10][11][12].Reason includes that macrophage and the impaired prior document of neutrophil cell response show：The congenital immunity cell of both types has vital effect in terms of lung's pathogen removing.However, in view of influenza infection causes various change of lung, the mechanism whether activation of the antiviral immunity path of the non-damage induced by influenza contributes to bacterium secondary infection is still not clear.
Additionally, whether influenza causes the phenomenon of host defense obstacle related to other viral pathogens unclear.Clinically, many viruses can form co-infection, including Respiratory Syncytial Virus(RSV) (RSV) and nasal cavity virus with bacterium (both of which is RNA virus)[3][13][14].Therefore, studied We conducted this, it is assumed that only activating the viral RNA identification receptor of host respiratory disease will cause bacterium to remove the decline of power.We using synthesis compound (particularly PolyI:C, imiquimod and GDQ) administration, next, carrying out bacterium infection.Such medicine be it is well-known can simulated virus nucleic acids on immune system influence medicine.Polyinosinic acid is the synthesis compound that can activate TLR3[15], dsRNA in the recognizable core of TLR3 acceptors；Also RIG is similar to Differentiation of Human Melanoma Cell Line-GAP-associated protein GAP 5 (MDA5) cytosol receptor of acceptor (RLRs) Induced by Retinoic Acid gene (RIG-I) and recognizable RNA virus nucleic acid[16].The TLR7 of imiquimod and the recognizable single stranded RNA of GDQ (R848) activation[17][18].Polyinosinic acid and TLR7 activators resist the various respiratory road pathogen of influenza or potential source biomolecule pathogen, including influenza, H5N1 avian influenza virus, and Francisella tularensis as treatment or prophylactic.They are considered as " boost motor " of a kind of effective and safe antiviral immunity reaction[19][20][21][22].Using pulmonary infection mouse as animal model, we are in the way of intranasal administration, injection polyinosinic acid and TLR7 activators, then, with common pathogenic organisms of respiratory tract (can cause the pathogen of secondary pneumonia) infection experiment mouse, whether the activation of the final antiviral immunity dredging collateral for determining increases the sensitiveness of host's Secondary bacterial infections.It was found that polyinosinic acid is administered, influenza infection is similar to, host is to streptococcus pneumonia and the removing power of staphylococcus aureus for infringement.Additionally, the illeffects of polyinosinic acid is probably to be mediated by I types interferon.Our result of study shows that polyinosinic acid may not be a benign molecules of immunization stimulus, the presence doubt of prevention or therapeutic agent when it is as viral prevalence.
The content of the invention：
Because respiratory virus infection is relatively conventional, and the patient of a quite a lot of part is developed into property or Secondary bacterial infections[1][2][3], therefore its research enjoys public concern.So far, it is that the mechanism for how promoting bacterium infection is still known little about it for virus infection.Research confirms immune response of the host for influenza, and such as I types and II types is interferon-induced, may be with the congenital antibacterial immunity response key link of suppression[10][11][12].But the mechanism whether activation of the antiviral immunity path of the non-damage induced by influenza contributes to bacterium secondary infection is still not clear.Whether influenza causes the phenomenon of host defense obstacle related to other viral pathogens unclear.The effect report of the secondary pneumonia sensitiveness caused to gram-positive bacterium on polyinosinic acid is less.
Prior art in view of the above, first aspect present invention provides influence of the PolyI:C administration to bacteria clearance after streptococcus pneumoniae infection.
Related data shows, compared with imiquimod nasal-cavity administration group, the animal lung pneumonia streptococcus bacterium amount of PolyI:C nasal-cavity administration substantially increases.Compared with saline control group, imiquimod or GDQ (TLR parts) administration group do not have obvious difference in terms of bacterium removing.Therefore TLR7 parts be administered alone be not enough to reduce bacterium clearance rate.
Second aspect present invention provides PolyI:C and enhances neurological susceptibility of the lung tissue to bacterium.
Similar to streptococcus pneumonia, after infection of staphylococcus aureus 24h, its clearance rate is lowered in PolyI:C administration.So PolyI:C seems to compromise the defense mechanism that host's lung tissue clinically causes two kinds viral secondary bacterial pathogens (streptococcus pneumonia and methicillin-resistant staphylococcus aureus).
Third aspect present invention provides the proportional relation of the extent of damage and PolyI:C the administration duration that bacterium removes power.
Related data shows, after through 1 time or 3 dosage PolyI:Cs or physiological saline nasal-cavity administration 24h, experimental animal intrathecal injection streptococcus pneumonia.In 48h, the amount of bacteria in logging machine body.Bacteria clearance in the experimental animal body of the disposable PolyI:C administration of related data is by the trend for reducing, (the CFU value average than saline control group is high 8 times, p=0.05), simultaneously through 3 CFU (such as Fig. 2 higher of the experimental animal Pulmonary bacterial of PolyI:C administration, the CFU value average than physiological saline group is high 13 times, p=0.01).Therefore, the extent of damage of bacterium removing power is directly proportional to the PolyI:C duration.
Fourth aspect present invention provides TLR3 and Cardif paths contributes to PolyI:C to induce host to the neurological susceptibility of bacterium.
At the initial stage of antiviral response, Cardif serves as the adapter molecule of RIG-I and MDA5 signals[25][26][27][28].Related data shows, after PolyI:C administration, bacteria clearance is through increasing in body for the experimental animal of TLR3 (Tlr3-/-) missing or Cardif (Cardif-/-).Compared to physiological saline group, double knockouts (Cardif-/-/Tlr3-/-) group under Secondary bacterial infections environment, carries out PolyI:C administration, and bacteria clearance is significantly improved in body.However, both without Tlr3-/- or Cardif-/- defect animal under the conditions of being administered without PolyI:C, there is obvious difference compared with wild strain group in its lung's streptococcus pneumonia clearance rate.Therefore, Research of Animal Model for Study experiment display, PolyI:C is relevant with the antiviral signal pathway activated of TLR3 and Cardif dependences to the adverse effect of bacteria clearance.
Fifth aspect present invention provides the long-term adverse effect that PolyI:C removes power for bacterium.
Related data shows, after pneumococcal infection 48 hours, the experimental animals of polyinosinic acid administration are contained within amount of bacteria higher.And carrying out the administration of every polyinosinic acid on the 3rd, next carries out the infection of streptococcus pneumonia, and assess after infection the 1st, after 3 and 5 days Pulmonary bacterial clump count (CFU), up to the 5th day, the Pulmonary bacterial amount of saline control group tended to health to data display less than detection minimum value；And amount of bacteria increases in the animal body of PolyI:C administration, it is intended to morbidity.Therefore, PolyI:C is adversely affected to lung's antimicrobial defenses in long-term.
Sixth aspect present invention provides I types interferon in the mediation active role of PolyI:C.
Related data shows that lung's IFN-α level of PolyI:C nasal-cavity administration group is presented significant change.(Ifnar-/-) animal can resist the negative effect of PolyI:C, and its bacteria clearance increased.After PolyI:C administration, IFNAR blocking antibodies immune group can preferably control pneumococcal infection compared with IgG treatment groups.Therefore, gene delection or antibody cause IFNAR to block and cause the elimination of I type IFN interferon signals, and this process can be maintained in PolyI:C and lower body is administered for bacterium scavenging action.Under the conditions of being administered without PolyI:C, body amount of bacteria does not have significant change for wild type and Ifnar-/- animal.This is implied：Under the conditions of virus-free part, I type interferon paths can't mediate removing of the lung tissue to streptococcus pneumonia.
Seventh aspect present invention provides PolyI:C administration and causes Ifnar-/- animal groups neutrophil accumulation amount increase
Related data shown, 3 days are administered in PolyI:C, then by wild type and Ifnar-/- animal carry out intrathecal streptococcus pneumoniae infection after, two groups of Bronchoalveolar Lavage Fluid Cells numbers are similar, i.e. total cell number (wild group of 1.7*106Cell, Ifnar-/- group 2.0*106Cell), macrophage (wild group of 1.2*106Cell, Ifnar-/- group 21.3*106Cell).Although compared to wild group, in trend more than twice, (wild group is 4.1*10 to neutrophil leucocyte amount in Ifnar-/- animal body5, Ifnar-/- group neutrophil leucocyte is 8.0*105Cell), but there was no significant difference.Therefore, in this animal model, Scavenging activity of the Ifnar-/- animal groups being administered through PolyI:C after 6h is infected increases.Compared with PolyI:C is administered wild group, the neutrophil accumulation amount of the Ifnar-/- animal groups of PolyI:C administration increases, and causing body to remove power to bacterium strengthens.
Eighth aspect present invention provides PolyI:C and causes the death rate of the Secondary bacterial infections host for depending on I type interferon to increase.
Related data shows, wild group of experimental animal being administered through PolyI:C in pneumococcal infection after in 7 days the death rate be 100%.In contrast, through the Ifnar-/- animal of PolyI:C administration after bacterium infection (p=0.01, PolyI:C is administered wild group and compares Ifnar-/- animal groups) 14 days, survival rate is more than 60%.Saline control group and Ifnar- under streptococcus pneumoniae infection/- animal groups no significant difference.So PolyI:C is induction of I type interferon, and I types interferon can increase neurological susceptibility of the animal body to streptococcus pneumonia under antiviral immunity mechanism activation condition, and then cause the death rate of the Secondary bacterial infections host for depending on I type interferon to increase
In sum, inventor confirms PolyI:C to lung's antimicrobial defenses in long-term adverse effect.It was found that imiquimod or GDQ (TLR parts) are administered alone to be not enough to reduce bacteria clearance, PolyI:C improves neurological susceptibility of the lung tissue to bacterium, the extent of damage that bacterium removes power is directly proportional to the PolyI:C administration duration, TLR3 and Cardif paths increased PolyI:C and induce host to bacterium neurological susceptibility, PolyI:C removes the long-term adverse effect of power for rear bacterium, I types interferon can cause the death rate of the Secondary bacterial infections host for depending on I type interferon to increase in the mediation active role of PolyI:C and PolyI:C.Show that polyinosinic acid has activation to the I type interferon paths that mediation lung tissue removes to streptococcus pneumonia.
Brief description of the drawings：
Fig. 1：PolyI:C enhances neurological susceptibility of the lung tissue to bacterium.A. experimental animal carries out PolyI:C, or TLR7 activators (50 μ g imiquimods or 50 μ g GDQs), or physiological saline nasal-cavity administration respectively daily, continues 2 days.24 hours after last time dosage, animal intrathecal injection streptococcus pneumonia (30 μ l, common 900CFU).At the 3rd day, lung tissue was taken for the calculating of CFU values.* represent, compared with physiological saline and TLR7 agonist administration groups, p ＜ 0.05 are drawn by one-way analysis of variance.Data are coupled by two separate experiments.B. experimental animal carries out polyinosinic acid (50 μ g) or saline administration daily, continues 3 days.And then methicillin-resistant staphylococcus aureus (MRSA) (6 × 10 are injected after 24 hours6CFU).24 hours after bacterial injections, lung tissue was taken for the calculating of bacterium CFU values.
Fig. 2：Influence of the PolyI:C concentration to Pulmonary bacterial clearance rate.Experimental animal carries out PolyI:C (50 μ g), or physiological saline nasal-cavity administration respectively daily, continues 1 or 3 day.24 hours after last time dosage, animal intrathecal injection streptococcus pneumonia (30 μ l, totally 1 × 105CFU).After 48 hours, lung tissue is taken for the calculating of CFU values.* represent, polyinosinic acid administration group (3 kinds of dosage) compared with physiological saline group, p ＜ 0.05 is drawn by one-way analysis of variance.Data are coupled (n=7-8/group) by two separate experiments.
Fig. 3：Virus patterns recognize effect of the path in the immunologic mechanism that mediation PolyI:C causes.TLR3 that age and sex match (Tlr3-/-) missing, Cardif (Cardif-/-) missing and both simultaneously missing (Cardif-/-/Tlr3-/-, double knockouts), and wild type experimental animal carries out the polyinosinic acid administration of lasting 3 days, and then injects streptococcus pneumonia (1 × 104CFU).After 48 hours, lung tissue is taken for the calculating of CFU values.* represents, wild type administration group with Cardif-/-/Tlr3-/- group compared with, p ＜ 0.05 are drawn by one-way analysis of variance.Other compare that there was no significant difference.Data are coupled by two separate experiments.
Fig. 4：In PolyI:C post-drug period, the bacterium of experimental animal is removed power and has damaged.Experimental animal carries out PolyI:C (50 μ g), or physiological saline nasal-cavity administration respectively daily, continues 3 days.24 hours after last time dosage, animal intrathecal injection streptococcus pneumonia (30 μ l, totally 1 × 104CFU).1, behind 3, or 5 days, lung tissue is taken for the calculating of CFU values, then draw every group of data (n=4/group).Error line represents standard deviation.* represent, in each time point polyinosinic acid administration group compared with physiological saline group, p ＜ 0.05 are drawn by one-way analysis of variance.Data are coupled by two separate experiments.
Fig. 5：The detection of the I type interferon of PolyI:C administration experimental animal.A. experimental animal carries out PolyI:C (50 μ g), GDQ (100 μ g), or physiological saline nasal-cavity administration respectively daily, continues 3 days.And then streptococcus pneumonia (7 × 10 is injected4CFU).After 48 hours, lung tissue is taken for ELISA experimental analyses IFN-α 4.Average and standard deviation as shown in the chart, n=4/group.* represent, polyinosinic acid administration group draws p ＜ 0.05 compared with physiological saline group by one-way analysis of variance.B. the experimental animal of wild type animal and I types interferon receptors (Ifnar1-/-) defect carries out polyinosinic acid nasal-cavity administration daily, continues 3 days.Then carry out streptococcus pneumoniae infection (7 × 104CFU).N=4/group.* p ＜ 0.05 are represented.C. wild type experimental animal carries out polyinosinic acid nasal-cavity administration daily, continues 3 days, then injects streptococcus pneumonia (2 × 10 at the 4th day4CFU)。
Injection IFNAR blocking antibodies and IgG (the 1st day 1.5mg, the 3rd day 0.75mg, the 5th day 0.75mg) in experimental animal body.Lung tissue is taken after 48 hours in bacterial injections.* p ＜ 0.05 are represented.
Fig. 6：Under the administration of lasting PolyI:C and streptococcus pneumoniae infection, Ifnar-/- and the wild group of survival rate and the clearance rate of bacterium in the later stage.A. the wild type that the age matches with sex carries out polyinosinic acid administration daily with Ifnar-/- experimental animal, continues 3 days, and then injects streptococcus pneumonia (1 × 104CFU), the tracking of 14 days is then carried out, moribund animals is carried out with euthanasia, record fatal rate.* represents, polyinosinic acid be administered wild group with Ifnar-/- group compared with, p=0.001 is obtained by Log-Rank test.N=7-8/group.Other compare that there was no significant difference.Data are coupled by two separate experiments.In separate experiment, the wild type that the age matches with sex carries out polyinosinic acid administration daily with Ifnar-/- experimental animal, continues 3 days for B, C., and then injects streptococcus pneumonia (1.5 × 104CFU).The 3rd day after bacterial injections and the 4th day, euthanasia is carried out to experimental animal, goes lung tissue and blood to be used for the calculating of bacterium CFU values.* p ＜ 0.05 are represented；N=4/group.Data are coupled by two separate experiments.
Fig. 7：Cardif- after streptococcus pneumoniae infection/- and Ifnar-/- experimental animal blood and lung tissue load.The Cardif- that age matches with sex/- and Ifnar-/-, and wild type experimental animal injection streptococcus pneumonia (1 × 104CFU).After 48 hours, euthanasia is carried out to experimental animal, take lung tissue and blood and calculated for bacterium CFU values.Ns represents meaningless comparing；Straight line represents detection minimum value.Data are obtained by two experiments for separating.N=8/group.
Fig. 8：Tlr3- after streptococcus pneumoniae infection/- and wild type experimental animal blood and lung tissue load.The Tlr3- that age matches with sex/- and wild type experimental animal injection streptococcus pneumonia (1 × 104CFU).After 48 hours, euthanasia is carried out to experimental animal, take lung tissue and blood and calculated for bacterium CFU values.Ns represents meaningless comparing；Straight line represents detection minimum value.Data are obtained by two experiments for separating.N=8-9/group.
Fig. 9：The response of the early stage bacterial load and inflammatory cell of polyinosinic acid administration animal after streptococcus pneumoniae infection.The Ifnar- that age and sex match/- and wild type experimental animal carry out polyinosinic acid administration (50 μ g) daily, continue 3 days, and then injection streptococcus pneumonia (7 × 103CFU).After 6 hours, bronchoalveolar lavage (BAL) is carried out to experimental animal.A. the BAL fluid of first time 1ml dilutes 5 times, is calculated for bacterium CFU values.B. this time point is for macrophage in main bronchus bronchoalveolar lavage fluid and the description of neutrophil leucocyte (polymorphonuclear leukocyte) total amount calculating.C. neutrophil leucocyte amount in wild group and Ifnar-/- animal body.
Figure 10：Polyinosinic acid administration animal lung Histological section after streptococcus pneumoniae infection.The Ifnar- that age and sex match/- and wild type experimental animal carry out polyinosinic acid administration (50 μ g) daily, continue 3 days, and then in the 3rd day injection streptococcus pneumonia (1 × 104CFU).Take lung tissue and be made section.Histotomy shows that wild group of lung tissue is presented the consolidation and inflammatory cell infiltration (left side) of large area.And Ifnar-/- group lung tissue shows the inflammation compared with small area, lung tissue is most of normal (right side).The description of lung tissue section represents each group；N=3-4/group.
Specific embodiment：
Embodiments of the present invention are illustrated below by way of specific instantiation, the content that those skilled in the art can be as disclosed by this specification understands other advantages of the invention and effect easily.The present invention can also be embodied or practiced by way of a different and different embodiment, and without departing from the spirit of the present invention the various details in this specification can also carry out various modifications or alterations based on different viewpoints and application.
Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to following specific specific embodiments；It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiment, rather than in order to limit the scope of the invention；In description of the invention and claims, unless explicitly pointed out in addition in text, singulative " one ", " one " and " this " include plural form.
When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, any one numerical value can select between two end points and two end points of each number range.Unless otherwise defined, all technologies and scientific terminology for being used in the present invention are identical with the meaning that those skilled in the art of the present technique are generally understood that.In addition to the specific method, equipment, the material that are used in embodiment, grasp and record of the invention according to those skilled in the art to prior art, can also use any method in the prior art similar or equivalent with the method described in the embodiment of the present invention, equipment, material, equipment and material to realize the present invention.
Unless otherwise indicated, disclosed in this invention experimental technique, detection method, preparation method using the conventional associated ligands of the art and the intraperitoneal injection inocalation method of antibody, the cultivation of gram-positive bacterium, bacterial injections method, tissue and blood sampling method, CFU value determination methods, cell calculating method (double-blind study), cell factor enzyme linked immunosorbent assay (ELISA) (ELISA) method, the routine techniques of hematoxylin-eosin paraffin section facture, statistical analysis and association area.
The virus infection of respiratory tract is common breathing problem, and often shows benign clinical disease course.However, the patient of a quite a lot of part is developed into property or Secondary bacterial infections[1][2][3]Virus-bacterial pneumonia is repeatedly reported.It is to cause the Influenza A H1N1 of the influenza pandemic phase in 20th century and 2009 to be very popular the death main cause of phase[4][5][6].But the mechanism whether activation of the antiviral immunity path of the non-damage induced by influenza contributes to bacterium secondary infection is still not clear, and whether influenza causes the phenomenon of host defense obstacle related to other viral pathogens unclear.
PolyI:C, imiquimod and GDQ be it is well-known can simulated virus nucleic acids on immune system influence medicine.Using pulmonary infection mouse as animal model,We are in the way of intranasal administration,Injection polyinosinic acid and imiquimod and GDQ,Then,With common pathogenic organisms of respiratory tract (pathogen of secondary pneumonia can be caused) infection experiment mouse,It is final to determine that PolyI:C administration reduces bacteria clearance and polyinosinic acid administration group animal Pulmonary bacterial amount substantially increases,And imiquimod or GDQ are administered alone and are not enough to reduce bacteria clearance,PolyI:C enhances neurological susceptibility of the lung tissue to bacterium,The extent of damage that bacterium removes power is directly proportional to the PolyI:C administration duration,The enhancing of TLR3 and Cardif paths is by the host of PolyI:C induction to bacterium neurological susceptibility,PolyI:C is removed power and is had a negative impact for later stage bacterium,PolyI:C can cause the death rate of the Secondary bacterial infections host for depending on I type interferon to increase.Show that polyinosinic acid has activation to the I type interferon paths that mediation lung tissue removes to streptococcus pneumonia.
The intraperitoneal injection inocalation method of associated ligands and antibody
Polyinosinic acid, imiquimod and GDQ are provided by Invivogen companies.The mixture of mouse peritoneal injection ketamine and xylazine, anaesthetize it, then 50 μ l SPSSs or physiological saline, intranasal administration are mixed in using polyinosinic acid (50 μ g), imiquimod (50 μ g) and GDQ (100 μ g).Injection 1.5mg MAR1-5A3 antibody (Leinco Technologies, I-401) in first day is administered in polyinosinic acid, will produce in Mice Body in IFNAR1 and body.And then, 1.5mg MAR1-5A3 antibody, dosage 0.75mg are injected again the 3rd day and the 5th day (with streptococcus pneumoniae infection).It is control with the IgG expressions of normal mouse.
The cultivation of gram-positive bacterium
In streptococcus pneumoniae infection experiment, the streptococcus pneumonia (ATCC 6303) that glycerine freezes is cultivated in Todd-Hewitt liquid, and condition of culture is 5% CO2,37 DEG C.In culture 6 hours, bacterium reached exponential phase.Bacterium is centrifuged, will be precipitated resuspended with aseptic PBS.The concentration of bacterium is estimated by determining 600nm absorbances.
In infection of staphylococcus aureus experiment, the staphylococcus aureus strains (USA300/LAC) of methicillin-resistant are placed in trypticase soya broth (TSB), and be stored in 37 DEG C of shaken cultivation case (200rpm), carry out incubated overnight.The bacterium of culture overnight is through 1:200 dilutions, after 4 hour Secondary Cultures, in mid-term exponential phase.Bacterial concentration can be estimated by measuring the absorbance of 600nm
Bacterial injections method
Using ketamine and the mixture of xylazine, through intraperitoneal injection experiment mice, narcosis is at；Secondly the tracheae of mouse is exposed, using the inoculum of the aseptic μ l of No. 27 needle injections 30；Finally its skin incision is sutured.
CFU value determination methods
At predetermined time point, injected carbon dioxide makes its euthanasia in Mice Body.Heparin-binding anti-coagulants, collects the blood of mouse right ventricle, and carries out 1 with PBS:2 dilutions, and 10 μ l blood are inoculated in blood agar (for culture of streptococcus pneumonia) and tryptic soy broth agar flat board (for staphylococcus aureus culture), the CFU values of Bacteria in Blood are determined with this.Before mouse pneumonectomy, the 1mL PBS containing 5mM EDTA are injected into right ventricle, perfusion to Pulmonary Vascular.At predetermined time point, it is homogenized by whole pneumonectomy and in the 1mL PBS containing protease inhibitor cocktail (Roche Holding Ag).Homogenate is carried out 1 with PBS:5 dilutions.10 μ l solution inoculums are in blood agar or TSB agar plates in every kind of dilution, to determine the CFU values of Pulmonary bacterial.
Sample of tissue method
At specified time point, experimental animal lung tissue is collected.The paraformaldehyde perfusion right ventricle of 1ml 4%, perfusion is fixed to Pulmonary Vascular.Polyethylene pipe inserts tracheae, and the paraformaldehyde of about 1ml 4% is poured into, and expands lung, fixes it；Secondly by the ligation of tracheae.Whole thoracic cavity is cut off, and is fixed, it is soaked in paraformaldehyde 6 hours-overnight, being then passed to Univ California-Los Angeles USA's conversion pathology core laboratory is carried out FFPE, section and is dyeed by hematoxylin eosin staining method method.
Cell factor enzyme linked immunosorbent assay (ELISA) (ELISA) method
Verikine TM mouse interferon-' alpha ' enzyme linked immunological kits are bought from PBL InterferonSource companies (Piscataway, New Jersey).According to the specification of manufacturer, using the kit measurement cytokine levels.
Cell calculating method (double-blind study)
Hematoxylin-eosin paraffin section facture
Statistical analysis
By Log-Rank Test, we compare survival curve of the animal under dose of radiation size or time length.And for other data, the importance of statistical analysis is shown by multiple appropriate comparison method (double tail Mann Whitney U tests (for CFU) or one-way analysis of variance correction method).The AP values of P=0.05 or lower are considered to have statistical significance.All calculating have used the Prism software programs in Windows operating system to carry out (lattice pressgang Pai get software companys).Error line represents SEM and repeat number in all charts.
Embodiment 1
PolyI:C improves neurological susceptibility of the lung tissue to bacterium
We detect after bacterium infection that PolyI:C is administered the influence to bacteria clearance first.E.g., experimental animal carries out the PolyI:C or imiquimod nasal-cavity administration of lasting two days (2 multiple dose).In the 3rd day (e.g., 24 hours after last time PolyI:C dosage), animal intrathecal injection streptococcus pneumonia.We have found that the animal lung amount of bacteria for carrying out nasal cavity PolyI:C administration substantially increases.It is interesting that compared with saline control group, imiquimod or GDQ (TLR7 activators, such as Figure 1A) administration group do not have obvious difference in terms of bacterium removing.But two groups are initially being attacked the powerful clearance rate of malicious stage performance.Therefore TLR7 parts be administered alone be not enough to reduce bacterium clearance rate.
Secondly, we detect whether PolyI:C also reduces the clearance rate of another important analysis of clinic pathogenic microorganism (methicillin-resistant staphylococcus aureus (MRSA)) for causing viral secondary bacterial pneumonia.Similar to class above, after infection of staphylococcus aureus 24h, PolyI:C administration will lower its clearance rate (Figure 1B).Therefore, PolyI:C seems to compromise the defense mechanism that host's lung tissue clinically causes two kinds viral secondary bacterial pathogens (streptococcus pneumonia and methicillin-resistant staphylococcus aureus).
Embodiment 2
The extent of damage that bacterium removes power is directly proportional to the PolyI:C administration duration
It is observed that after PolyI:C administration, the time point that the bacteria clearance in animal body body declines.Because virus infects, such as influenza, it usually needs take several days the even more long time, immediately We conducted Related Experimental Study, the effect that PolyI:C carrys out simulated virus infection is taken with 1 dosage or 3 dosage.Through once or after 3 PolyI:Cs or physiological saline nasal-cavity administration 24h, experimental animal intrathecal injection streptococcus pneumonia.In 48h, the amount of bacteria in logging machine body.It was found that the dosage of PolyI:C is related to the clearance rate of bacterium.Bacteria clearance in the experimental animal body of disposable PolyI:C administration is by the trend for reducing, (the CFU value average than saline control group is high 8 times, p=0.05), simultaneously through 3 CFU (such as Fig. 2 higher of the experimental animal Pulmonary bacterial of PolyI:C administration, the CFU value average than physiological saline group is high 13 times, p=0.01).Therefore, the extent of damage of bacterium removing power is directly proportional to the PolyI:C duration.
Embodiment 3
The enhancing of TLR3 and Cardif paths is by the host of PolyI:C induction to bacterium neurological susceptibility
Because PolyI:C have activated TLR3 and Cardif dependence unwindases RIG-I and MDA5 simultaneously, it is intended to determine whether that one or both of which path all removes power to PolyI:C infringement host bacteria related.For such research, we by PolyI:C be used for TLR3 (Tlr3-/-) missing, Cardif (Cardif-/-) missing and both lack the experimental animal of (double knockouts) simultaneously.At the initial stage of antiviral response, Cardif serves as the adapter molecule of RIG-I and MDA5 signals[25][26][27][28].We have found that TLR3 the experimental animal of (Tlr3-/-) missing or Cardif (Cardif-/-) after PolyI:C administration, bacteria clearance is through increasing in body.Compared to physiological saline group, double knockouts (Cardif-/-/Tlr3-/-) group under Secondary bacterial infections environment, carries out PolyI:C administration, and bacteria clearance is significantly improved (Fig. 3) in body.However, both without Tlr3-/- or Cardif-/- defect animal under the conditions of being administered without PolyI:C, there is obvious difference compared with wild strain group in its lung's streptococcus pneumonia clearance rate.Therefore, Research of Animal Model for Study experiment display, PolyI:C is relevant with the antiviral signal pathway activated of TLR3 and Cardif dependences to the adverse effect of bacteria clearance.
Embodiment 4
PolyI:C is removed power and is had a negative impact for later stage bacterium
In view of after pneumococcal infection 48 hours, the experimental animals of polyinosinic acid administration are contained within amount of bacteria higher, it is desirable that determining, whether PolyI:C brings whether detrimental effect, or PolyI:C have simply simply delayed bacterium reset procedure to the clearance rate of bacterium in the later stage.Therefore we carry out every administration of polyinosinic acid on the 3rd, and next carries out the infection of streptococcus pneumonia, and assesses the 1st, 3 and 5 day Pulmonary bacterial clump count (CFU) after infection.We have found that up to the 5th day, the Pulmonary bacterial amount of saline control group tended to health less than detection minimum value；And amount of bacteria increases (Fig. 4) in the animal body of PolyI:C administration, it is intended to morbidity.Therefore, PolyI:C is adversely affected to lung's antimicrobial defenses in long-term.
Embodiment 5
PolyI:C administration causes Ifnar-/- animal groups neutrophil accumulation amount increase
In view of detection inflammatory cell reaction, PolyI:C is administered 3 days for we；Secondly, by wild type and Ifnar-/- animal carry out intrathecal streptococcus pneumoniae infection.The 6h after bacterium infection, we carry out bronchoalveolar lavage, to check inflammatory cell.It was found that there is significant difference (Fig. 9 A) in the wild and Ifnar-/amount of bacteria of-group in 6h.Wild group of display to the amount of bacteria CFU of several times (the average CFU of wild type group be 51000, Ifnar-/- for 740).Interestingly, although wild group of amount of bacteria is higher, its inflammatory reaction is in mitigation state compared to Ifnar-/- missing group.On average, two groups of cell numbers are similar, i.e. total cell number (wild group of 1.7*106Cell, Ifnar-/- group 2.0*106Cell), macrophage (wild group of 1.2*106Cell, Ifnar-/- group 21.3*106Cell) (Fig. 9 B and data do not show).Although compared to wild group, in trend more than twice, (wild group is 4.1*10 to neutrophil leucocyte amount in Ifnar-/- animal body5, Ifnar-/- group neutrophil leucocyte is 8.0*105Cell), but there was no significant difference (Fig. 9 C).Therefore, in this animal model, Scavenging activity of the Ifnar-/- animal groups being administered through PolyI:C after 6h is infected increases.Compared with PolyI:C is administered wild group, the neutrophil accumulation amount of the Ifnar-/- animal groups of PolyI:C administration increases, and causing body to remove power to bacterium strengthens.
Embodiment 6
PolyI:C can cause the death rate of the Secondary bacterial infections host for depending on I type interferon to increase
In view of PolyI:C can cause the bacteria clearance to reduce, it is intended that whether detection, the elimination of I type interferon can improve host's survival rate after bacterium infection.Be administered continuously 3 days for PolyI:C and physiological saline nasal-cavity administration by we；Secondly, wild type and Ifnar--/- animal groups are carried out into streptococcus pneumoniae infection (about LD50 amounts).Experimental animal is monitored, until they meet standard euthanasia or its 14 days after streptococcus pneumoniae infection.It was found that through PolyI:C be administered wild group of experimental animal in pneumococcal infection after in 7 days the death rate be 100% (Fig. 6 A).In contrast, through the Ifnar-/- animal of PolyI:C administration after bacterium infection (p=0.01, PolyI:C is administered wild group and compares Ifnar-/- animal groups) 14 days, survival rate is more than 60%.Saline control group and Ifnar- under streptococcus pneumoniae infection/- animal groups no significant difference.In general, test result indicate that, PolyI:C is induction of I type interferon.I types interferon can increase neurological susceptibility of the animal body to streptococcus pneumonia under antiviral immunity mechanism activation condition.
In order to determine host's Death Mechanism, we detect the amount of bacteria of lung and blood before animal carries out euthanasia.Ifnar--/- group is administered compared to PolyI:C, the amount of bacteria that PolyI:C is administered in wild group of blood and lung tissue is higher.Conclusions are implied, under conditions of the administration of PolyI:C and streptococcus pneumoniae infection, the missing of I type interferon causes bacterium removing power to differ widely (Fig. 6 B and 6C).Secondly, we detect PolyI:C administration Ifnar-/- group and wild group of lung tissue section.Wild group of lung tissue is presented the consolidation and inflammatory cell infiltration of large area, and the incrementss with bacterium in tissue are consistent.And Ifnar-/- group lung tissue shows the inflammation compared with small area, lung tissue is most of normal (Figure 10).Therefore, I types interferon at least mediates the PolyI:C to cause the body Partial Mechanism that survival rate declines after Secondary bacterial infections, and it is mainly and reaches this effect by reducing the clearance rate of early stage and later stage body bacterium.
In sum, the present invention effectively overcomes various shortcoming of the prior art and has high industrial utilization.
The above-described embodiments merely illustrate the principles and effects of the present invention, not for the limitation present invention.Any person skilled in the art all can carry out modifications and changes under without prejudice to spirit and scope of the invention to above-described embodiment.Therefore, those of ordinary skill in the art is completed under without departing from disclosed spirit and technological thought such as all equivalent modifications or change, should be covered by claim of the invention.
Claims (4)
1. polyinosinic acid has activation to the I type interferon paths that mediation lung tissue removes to streptococcus pneumonia.
2. polyinosinic acid as claimed in claim 1 has activation to the I type interferon paths that mediation lung tissue removes to streptococcus pneumonia, wherein involved animal model is the mouse matched with C57BL/6J genetic backgrounds and age and sex.
3. polyinosinic acid as claimed in claim 1 has activation to the I type interferon paths that mediation lung tissue removes to streptococcus pneumonia, wherein involved pathogenic bacteria are the streptococcus pneumonias of serotype 3（ATCC 6303）With methicillin-resistant staphylococcus aureus（MRSA）.
4. polyinosinic acid has activation to the I type interferon paths that mediation lung tissue removes to streptococcus pneumonia as described in claim 2 to 3, wherein described attribute is by being administered to mouse polyinosinic acid and after carrying out the infection experiment of streptococcus pneumonia, by cell factor enzyme linked immunosorbent assay (ELISA)（ELISA）Method is measured to I type interferon, and to Pulmonary bacterial clump count（CFU）It is measured, i.e., being inoculated in blood agar or TSB agar plates etc. by lung tissue dilution is able to what is determined.
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