CN106729714A - Polyinosinic acid is administered and the relation between IFNI expression - Google Patents

Abstract

The present invention relates to biomedicine field, the more particularly to relation between polyinosinic acid administration and IFNI expression.Inventor uses polyinosinic acid, GDQ and physiological saline that mouse is administered respectively, and the expression of the administration energy inducing mouse lung IFNI of PolyI:C is found by com-parison and analysis.

Description

Polyinosinic acid is administered and the relation between IFNI expression

Technical field

The present invention relates to biomedicine field, the more particularly to relation between polyinosinic acid administration and IFNI expression.

Background technology

Respiratory virus infection is common, and often shows benign clinical disease course.However, the disease of a quite a lot of part People is developed into property or Secondary bacterial infections, and this complication can cause respiratory failure or death.Although in children, old man In immunity suppression crowd, such complication breaks out rate highest, but virus and bacteria pneumonia is likely to come across health In Adult Groups, and cause heavy Disease Spectrum.As influenza infection is popular, virus-bacterial pneumonia is repeatedly reported.It 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.

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.Stream The mechanism that sense research case has pointed out causes the damage mechanisms of the general airway epithelial cell layer with protectiveness including influenza, The expression of the combination enhancing mechanism of bacteria mediated acceptor, anti-inflammatory factors such as IL-10 abduction mechanisms, and recognition mode molecule is such as The decrease mechanism of Toll-like receptor.Additionally, in the recent period, we and other people find, host is directed to the immune response of influenza, such as I types It is interferon-induced with II types, may be with the congenital antibacterial immunity response key link of suppression.Reason include macrophage and it is thermophilic in Property the impaired prior document of granulocyte response show：The congenital immunity cell of both types has in terms of lung's pathogen removing There is vital effect.However, in view of influenza infection causes the diversity of lung to change, the non-damage induced by influenza The mechanism whether activation of antiviral immunity path 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.Facing On bed, many viruses can form co-infection, including Respiratory Syncytial Virus(RSV) with bacterium（RSV）With nasal cavity virus（Both of which It is RNA virus）.Therefore, studied We conducted this, it is assumed that the viral RNA identification for only activating host respiratory disease is received Body will cause bacterium to remove the decline of power.We are using synthesis compound（Particularly PolyI:C, imiquimod and loud, high-pitched sound De Mote）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, dsRNA in the recognizable core of TLR3 acceptors；Also RIG Similar acceptor（RLRs）Induced by Retinoic Acid gene（RIG-I）To the Differentiation of Human Melanoma Cell Line-related egg of recognizable RNA virus nucleic acid White 5（MDA5）Cytosol receptor.Imiquimod and GDQ（R848）The TLR7 of the recognizable single stranded RNA of activation.Polyinosinic acid With TLR7 activators as treatment or prophylactic, the various respiratory road pathogen of influenza or potential source biomolecule pathogen is resisted, including Influenza, H5N1 avian influenza virus, and Francisella tularensis.They are considered as anti-a kind of effective and safe antiviral immunity " boost motor " answered.Using pulmonary infection mouse as animal model, we in the way of intranasal administration, injection polyinosinic acid and TLR7 activators, then, with common pathogenic organisms of respiratory tract（The pathogen of secondary pneumonia can be caused）Infection experiment mouse, most Whether the activation of the antiviral immunity dredging collateral for determining eventually increases the sensitiveness of host's Secondary bacterial infections.It was found that poly- flesh Thuja acid is administered, and is similar to influenza infection, and host is to streptococcus pneumonia and the removing power of staphylococcus aureus for infringement.Additionally, poly- flesh The illeffects of thuja acid is probably to be mediated by I types interferon.Our result of study shows that polyinosinic acid may not be one Benign molecules of immunization stimulus, the presence doubt of prevention or therapeutic agent when it is as viral prevalence.

The content of the invention

The virus infection of respiratory tract is common breathing problem, and then most patient can develop into property or Person's Secondary bacterial infections, but be that the mechanism for how promoting bacterium infection is still known little about it for virus infection.Although stream Sense research case has been proposed that some mechanism, but in view of caused by influenza infection lung's change be various, lured by influenza The mechanism whether activation of the antiviral immunity path of the non-damage led contributes to bacterium secondary infection is still not clear.This Outward, whether influenza causes the phenomenon of host defense obstacle related to other viral pathogens unclear.In the recent period, we and other people Research find that the immune response that is caused by influenza can suppress the key component of antibiotic property congenital immunity on the contrary, such as by IFNI It is immune with what IFNII caused.

Prior art in view of the above, invention emulates Mice Body inner virus immune model, first aspect provides poly- Effect of the flesh cytidine monophosphate in host lung is removed to bacterium.

Polyinosinic acid and imiquimod are respectively TLR3 activators and TLR7 activators, be all known to us can The medicine that simulation nucleic acid virus influence on immune system.Experimental data shows, through the mouse pair after PolyI:C nasal-cavity administration Substantially reduced in control group in the Scavenging activity of lung streptococcus pneumonia, and TLR7 activator groups are without significant change, while we Also detect that Scavenging activity reduction of the polyinosinic acid Ye Nengshi lungs to staphylococcus aureus.

It is in inverse relationship that second aspect present invention provides bacterium Scavenging activity and the continued administration time of polyinosinic acid.

Generally need to take several days due to influenza, so We conducted related experiment.One is administered through PolyI:C Secondary or three times experiment mices have all been decreased obviously to the Scavenging activity of bacterium, but be administered once through polyinosinic acid The average CFU values of mouse lung bacterium are 8 times of control group, and the mouse being administered three times through polyinosinic acid is 13 times.Therefore Bacterium Scavenging activity is reduced with the increase of polyinosinic acid continued administration time.

Third aspect present invention provides TLR3 and Cardif paths in the decline of polyinosinic acid Induction of bacterial clearance rate Effect.

Polyinosinic acid can simultaneously activate TLR3 and Cardif paths.It was found that TLR3-/- mouse and Cardif-/- After polyinosinic acid administration, host's mouse is increased to the bacteria clearance of lung, and TLR3 and Cardif pairs is struck mouse Then show as significantly improving.TLR3 and Cardif genes without knock out mouse under conditions of being administered without polyinosinic acid, its The clearance rate of the lung of lung and wild mouse group no significant difference.Therefore, TLR3 and Cardif paths each contribute to poly IC Neurological susceptibility of the thuja acid induction host to bacterium.

Fourth aspect present invention provides the effect that IFNI occurs in Pulmonary bacterial removing

It has proven convenient that during influenza, IFNI can cause the clearance rate of streptococcus pneumonia to reduce for we.In the present invention, it has been found that The expression quantity of the mouse lung IFN after being administered through polyinosinic acid is significantly raised, and the IFNAR after being administered through polyinosinic acid lacks The clearance rate of swaged mouse lung bacterium increased.However, we carry out repeating experiment discovery using IFNAR blocking antibodies, Under conditions of virus-free antibody, IFNI paths will not mediate removing of the lung to streptococcus pneumonia.

The fifth aspect of the present invention provides IFNI can increase body under antiviral immunity mechanism activation condition to pneumonia chain The neurological susceptibility of coccus.

Wild-type mice after the polyinosinic acid treatment death rate after bacterium infection 7 days is to reach 100%, and poly IC Ifnar/mouse after thuja acid treatment still has 60% survival rate after bacterium infection 14 days.Cut into slices by mouse lung and sent out It is existing, after Secondary bacterial infections cause early and late bacterium removing obstacles, the dead danger that IFNI causes in polyinosinic acid Played a role in evil.

Brief description of the drawings：

Fig. 1：Polyinosinic acid improves neurological susceptibility of the lung tissue to bacterium.A. experiment mice uses polyinosinic acid-polycytidylic acid respectively daily (50 ug), TLR7 activators, physiological saline treatment continues 2 days.After last time treatment 24h, with streptococcus pneumonia (900 CFU, 30 ul) infecting mouse.The CFU of mouse lung bacterium was detected when the 3rd day.*, p ＜ 0.05, p value is Polyinosinic acid group and physiological saline and TLR7 groups are respectively compared by one-way analysis of variance method.B. experiment mice is daily Processed with polyinosinic acid-polycytidylic acid (50 mg) and physiological saline respectively, continue three days.After last time treatment 24h, golden yellow Portugal is used Grape coccus (MRSA, LAC USA300; 6*10⁶ CFU), after 24h, the CFU of mouse lung bacterium is detected.P ＜ 0.05, p value is led to Cross experimental group and physiological saline group compares acquisition.Experimental result is obtained by being analyzed in 2 groups of representative experimental datas

Fig. 2：The Pulmonary bacterial Scavenging activity of polyinosinic acid is in dosage dependent effect.Mouse daily with polyinosinic acid (pIC, 50ug) or physiological saline carries out intranasal administration, 1 day or 3 days is continued.After last time administration 24h, with streptococcus pneumonia (1* 10⁵ CFU, 30ul) infecting mouse.After 48h, the CFU of mouse lung bacterium is detected.* p ＜ 0.05.Experimental result has generation by 2 groups Analyzed in the experimental data of table and obtained.

Fig. 3：Virus patterns recognize the effect of approach in the immunosupress that polyinosinic acid is mediated indirectly.Use poly IC glycosides Acid treatment age and the Cardif of sex matching^-/-, Tlr3^-/-, Cardif^-/-,/Tlr3^-/-, wild-type mice three days, then Streptococcus pneumoniae infection experiment mice (1*10⁴CFU), the CFU values of Pulmonary bacterial are detected after 48h.*, p ＜ 0.01, experiment Result is obtained by being analyzed in 2 groups of representative experimental datas.

Fig. 4：Experiment mice after being processed through polyinosinic acid is occurring bacterium removing obstacles phenomenon after a while.With poly IC Thuja acid（50ug）Or physiological saline processes experiment mice three days.After last time administration 24h, with streptococcus pneumonia (1*10⁴ CFU, 30ul) infecting mouse.The bacterium CFU values of mouse lung, every group of all one average value were detected when the 1st, 3,5 day respectively. Error bar represents standard deviation.*, p ＜ 0.05, experimental result is obtained by being analyzed in 2 groups of representative experimental datas.

Fig. 5：The detection of INFI in the treated Mice Body of polyinosinic acid.A. polyinosinic acid is used（50ug）, gardiquimod（100ug）, or physiological saline treatment experiment mice three days.Detected with ELISA after the whole LH treatment of mouse IFN-α4.Per group echo average value and standard deviation（n=4）.*, p ＜ 0.05.B. wild type is processed with polyinosinic acid (Ifnar^+/+)Mouse and (Ifnar^-/-) mouse three days, and then use streptococcus pneumonia（7*10⁴CFU）Infection experiment mouse.48h Afterwards, the CFU of mouse lung bacterium is detected.Every group of 5 mouse.*, p ＜ 0.05.C. wild type is processed with polyinosinic acid (Ifnar^+/+)Mouse three days, uses streptococcus pneumonia on the 4th day（2*10⁴CFU）Infection experiment mouse.Experiment mice injection is given simultaneously Anti- IFNAR antibody or the IgG of control（First day 1.5mg, the 3rd day 0.75mg, the 5th day 0.75mg）.Streptococcus pneumonia sense The CFU values of mouse lung are detected after dye 48h.*, p ＜ 0.05.

Fig. 6：Under the administration of lasting PolyI:C and streptococcus pneumoniae infection, Ifnar/and wild group of depositing in the later stage Motility rate and the clearance rate of bacterium.A. the wild-type mice and Ifnar that age and sex are matched are processed with polyinosinic acid^-/-Mouse Three days, and then use streptococcus pneumonia（1*10⁴CFU）Infection experiment mouse.It is small within 14 days after observation streptococcus pneumoniae infection The sign of life of mouse, if Bearing Mice Life sign is critically ill, euthanasia treatment is carried out to it.**, p = 0.001.Every group of mouse 7- 8.Every other comparing is not statistically significant.Experimental result is obtained by being analyzed in 2 groups of representative experimental datas.B, C. in different experiments, the wild-type mice and Ifnar that age and sex are matched are processed with polyinosinic acid^-/-Mouse three My god, used streptococcus pneumonia at the 3rd day and the 4th day respectively（1.5*10⁴CFU）Infection experiment mouse B groups mouse detects mouse Pulmonary bacterial CFU values, C groups mouse detection Bacteria in Blood CFU values.*, p ＜ 0.05.Every group of mouse 4.Experimental result is by 2 groups Analyzed in representative experimental data and obtained.

Fig. 7：Cardif/and Ifnar/experimental animal blood and lung tissue load after streptococcus pneumoniae infection.Age The Cardif/and Ifnar matched with sex/, and wild type experimental animal injection streptococcus pneumonia（1×10⁴ CFU）.After 48 hours, euthanasia is carried out to experimental animal, take lung tissue and blood and calculated for bacterium CFU values.Ns indicate without Meaning compares；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.Age and property The Tlr3- not matched/- and wild type experimental animal injection streptococcus pneumonia（1×10⁴CFU）.It is dynamic to experiment after 48 hours Thing carries out euthanasia, takes lung tissue and blood and is calculated for bacterium CFU values.Ns represents meaningless comparing；Straight line represents detection most Small 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 My god, and then inject 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 main bronchus The description that macrophage and neutrophil leucocyte (polymorphonuclear leukocyte) total amount are calculated in bronchoalveolar lavage fluid.C. wild group and Neutrophil leucocyte amount in Ifnar-/- animal body.

Figure 10：Polyinosinic acid administration animal lung Histological section after streptococcus pneumoniae infection.Age and sex phase The Ifnar for matching somebody with somebody/and wild type experimental animal carry out polyinosinic acid administration daily（50μg）, continue 3 days, and then the 3rd Its injection streptococcus pneumonia（1×10⁴CFU）.Take lung tissue and be made section.Histotomy shows that wild group of lung tissue is presented big The consolidation and inflammatory cell infiltration of area（It is left）.And Ifnar/group lung tissue performance is compared with the inflammation of small area, the big portion of lung tissue Divide normal（It is right）.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, those skilled in the art can be taken off by this specification The content of dew understands other advantages of the invention and effect easily.The present invention can also be by specific embodiment parties different in addition Formula is embodied or practiced, and the various details in this specification can also be based on different viewpoints and application, without departing from this hair Various modifications or alterations are carried out under bright spirit.

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 down State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe Embodiment, rather than in order to limit the scope of the invention；In description of the invention and claims, unless in text Explicitly point out in addition, singulative " one ", " one " and " this " include plural form.

When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two ends of each number range Any one numerical value can select between point and two end points.Unless otherwise defined, in the present invention all technologies for using and Scientific terminology is identical with the meaning that those skilled in the art of the present technique are generally understood that.Except the specific method, equipment used in embodiment, Outside material, grasp and record of the invention according to those skilled in the art to prior art can also be used and this Any method, equipment and material in the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real The existing present invention.

Unless otherwise indicated, disclosed in this invention experimental technique, detection method, preparation method using this technology lead The conventional mouse nasal-cavity administration in domain, bacterium infection, CFU values measure, bronchoalveolar lavage, ELISA, mouse lung tissue is adopted Collection, makes the routine techniques of paraffin section and dyeing, statistical analysis and association area.

The virus infection of respiratory tract is common breathing problem, and often shows benign clinical disease course.However, quite a lot of one Partial patient is developed into property or Secondary bacterial infections, and this complication can cause respiratory failure or death.Although youngster The crowd of child, old man and hypoimmunity often faces the excessive risk for breaking out such complication, but virus and bacteria pneumonia also may be used Normal adults can be come across, and causes heavy Disease Spectrum.As influenza infection is popular, virus-bacterial pneumonia is repeatedly It is reported.It is that the mechanism for how promoting bacterium infection is still known little about it for virus infection.In view of influenza infection causes lung Various change, whether the activation of antiviral immunity path of the non-damage induced by influenza contribute to the secondary sexuality of bacterium The mechanism of dye is also still not clear.Clinically, many viruses can form co-infection, including Respiratory Syncytial Virus(RSV) with bacterium （RSV）With nasal cavity virus（Both of which is RNA virus）, influenza cause host defense obstacle phenomenon whether with other virosis Substance correlation is unclear.

Mouse

In all experiments, the mouse matched using C57BL/6J genetic backgrounds and age and sex.Interferon-' alpha '/beta receptor base Because double knock-out mices are preserved by this laboratory.Toll-like receptor 3 and CARDIF（Also referred to as interferon beta starts simulator 1, line The antiviral signal protein of plastochondria and virus induction signal adapter）Double knockout mice is by the gene knockout of Toll-like receptor 3 Mouse（From Osaka, Japan university, the laboratory of Shizuo Akira）With CARDIF knock out mice（From University of Lausanne, The laboratory of Jurg doctors Tschopp）Obtained by hybridization is produced, both knock out mice have complete C57BL/6J to lose Background is passed, and is stored in our laboratories.The double knock-out mices of interferon-' alpha '/beta receptor gene, the gene knockout of Toll-like receptor 3 are small Mouse, Toll-like receptor 3 and CARDIF Double knockout mices, CARDIF knock out mice and wild-type mice are maintained in U.S. In the same specific sterile facilities of University of California in Los Angeles of state.All of zoopery is all according to National Institutes of Health Carried out on humanistic care and using the regulations of experimental animal in policy, by the animal of Univ California-Los Angeles USA Research institute exercises supervision（OARO agreements 2005-143）, and focus on reducing the pain of experimental animal and torment.

The inoculation of the part and IFNAR antibody of virus-like acceptor

Polyinosinic acid, imiquimod and GDQ are provided by Invivogen companies.Mouse peritoneal injection ketamine and The mixture of xylazine, anaesthetizes it, then uses polyinosinic acid（50 μg）, imiquimod（50 μg）And GDQ （100 μg）It is mixed in 50 μ l SPSSs or physiological saline, intranasal administration.It is administered first day in polyinosinic acid and is noted Penetrate 1.5 mg MAR1-5A3 antibody（Leinco Technologies, I-401）, will produce in Mice Body in IFNAR1 and body. And then, at the 3rd day and the 5th day（With streptococcus pneumoniae infection）1.5 mg MAR1-5A3 antibody, dosage 0.75 are injected again mg.It is control with the IgG expressions of normal mouse.

Bacterium prepares and mouse Intratracheal inoculation

Unless otherwise noted, the otherwise one plant streptococcus pneumonia of serotype 3（ATCC 6303）To be used in all of experimental study. The bacterium that glycerine freezes is cultivated in Todd-Hewitt liquid, and condition of culture is 5% CO2,37 DEG C.Culture 6 hours, carefully Bacterium reaches exponential phase.Bacterium is centrifuged, will be precipitated resuspended with aseptic PBS.Estimated by determining 600 nm absorbances The concentration of bacterium, after bacterium secondly is carried out into 5 times of dilutions, the incubated overnight on agar plate determines bacterium according to clump count Actual concentrations.

In infection of staphylococcus aureus experiment, the staphylococcus aureus strains of methicillin-resistant（USA300 / LAC）There is provided by Frank doctors DeLeo friendship.Staphylococcus aureus is placed in trypticase soya broth（TSB）In, and deposit It is stored in 37 DEG C of shaken cultivation case（200rpm）In, carry out incubated overnight.The bacterium of culture overnight is through 1:200 dilutions, 4 small When Secondary Culture after, in mid-term exponential phase.Bacterial concentration can be estimated by measuring the absorbance of 600 nm.By inciting somebody to action The microbionation of dilution is in TSB agar cultures, incubated overnight, with the CFU values of this determination indigenous bacteria.

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.

The measure of CFU values

At predetermined time point, injected carbon dioxide makes its euthanasia in Mice Body.Heparin-binding anti-coagulants, collects mouse right The blood of ventricle, and carry out 1 with PBS:2 dilutions, and by 10 μ l blood be inoculated in blood agar (for culture of streptococcus pneumonia) and On tryptic soy broth agar flat board（For staphylococcus aureus culture）, the CFU of Bacteria in Blood is determined with this Value.Before mouse pneumonectomy, the 1mL PBS containing 5mM EDTA are injected into right ventricle, perfusion to Pulmonary Vascular.In the predetermined time Point, by whole pneumonectomy and is containing protease inhibitor cocktail（Roche Holding Ag）1mL PBS in be homogenized.Use PBS Homogenate is carried out 1:5 dilutions.10 μ l solution inoculums are in blood agar or TSB agar plates in every kind of dilution, to determine lung The CFU values of bacterium.

Bronchoalveolar lavage

At predetermined time point, make animal euthanasia.Lung tissue and tracheae in careful peel test animal.By 1ml PBS- EDTA irrigates Pulmonary Vascular by injecting right ventricle.Polyethylene pipe is inserted into tracheae, 1mL irrigating solutions are often poured into, until implantation is full 10ml（General 80% can recharge）.A part is calculated for bacterium in the BAL fluid of first time, remaining bronchus Bronchoalveolar lavage fluid is used to cell count.Cell centrifugation in BAL fluid, is entered with RPMI-1640 culture mediums Row is suspended, and cell count is carried out under hemacytometer.Probably there is the cell of 50000-100000 to be used for cell and smear be centrifuged, To compare iuntercellular difference.Cell is centrifuged smear and is dyeed through Diff-Quik（Improved by Wright-Giemsa）；Substantial amounts of macrophage Cell, neutrophil leucocyte, lymphocyte and other cells use double-blind study（In 2 independent position numbers 10 of each slide Cell）Calculate.

Cell factor enzyme linked immunosorbent assay (ELISA)（ELISA）

Verikine TM mouse interferon-' alpha ' enzyme linked immunological kits are from PBL InterferonSource companies（Piscataway, New Jersey）Purchase.According to the specification of manufacturer, using the kit measurement cytokine levels.

Lung tissue is sampled

At specified time point, experimental animal lung tissue is collected.The paraformaldehyde perfusion right ventricle of 1ml 4%, perfusion to lung blood Pipe, is fixed.Polyethylene pipe inserts tracheae, and the paraformaldehyde of about 1ml 4% is poured into, and expands lung, fixes it；Its The secondary ligation by tracheae.Whole thoracic cavity is cut off, and is fixed, it is soaked in paraformaldehyde 6 hours-overnight, then it is passed to Univ California-Los Angeles USA's conversion pathology core laboratory is carried out FFPE, section and is contaminated by hematoxylin-eosin Color method method is dyeed.Section is that by Dr. W.Dean Wallace, a pathology teacher assists what is carried out, and he is to animal Genotype is not known.

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, by multiple appropriate comparison method（Double tail Mann Whitney U tests（For CFU）Or one-way analysis of variance correction Method）Show the importance of statistical analysis.The AP values of P=0.05 or lower are considered to have statistical significance.All calculating make Carried out with the Prism software programs in Windows operating system（Lattice pressgang Pai get software companys）.The error in all charts Line represents SEM and repeat number.

Embodiment 1

The clearance rate of PolyI:C reduction bacterium

We detect after bacterium infection that PolyI:C is administered the influence to bacteria clearance first.Experimental animal is held It is continuous two days（I.e. 2 times）PolyI:C or imiquimod nasal-cavity administration.At the 3rd day（That is last time PolyI:C dosage Afterwards 24 hours）, animal intrathecal injection streptococcus pneumonia.We have found that the animal lung for carrying out nasal cavity PolyI:C administration is thin Bacterium amount substantially increases.It is interesting that compared with saline control group, imiquimod or GDQ（TLR7 activators, such as Figure 1A）Administration group does not have obvious difference in terms of bacterium removing.But two groups are initially being attacked the powerful removing of malicious stage performance Rate.Therefore TLR7 parts be administered alone be not enough to reduce bacterium clearance rate.

Secondly, we detect whether PolyI:C also reduces another and important cause viral secondary bacterial pneumonia Analysis of clinic pathogenic microorganism（Methicillin-resistant staphylococcus aureus（MRSA））Clearance rate.It is similar to class above, in golden yellow Portugal After grape coccus infection 24h, PolyI:C administration will lower its clearance rate（Figure 1B）.Therefore, PolyI:C seems to compromise Host's lung tissue clinically causes viral secondary bacterial pathogens to two kinds（Streptococcus pneumonia and methicillin-resistant gold Staphylococcus aureus）Defense mechanism.

Embodiment 2

PolyI:C improves neurological susceptibility of the lung tissue to bacterium

Secondly, we detect whether PolyI:C also reduces another and important cause facing for viral secondary bacterial pneumonia Bed pathogen（Methicillin-resistant staphylococcus aureus（MRSA））Clearance rate.It is similar to class above, in Staphylococcus aureus After bacterium infection 24h, PolyI:C administration will lower its clearance rate（Figure 1B）.Therefore, PolyI:C seems to compromise host Lung tissue clinically causes viral secondary bacterial pathogens to two kinds（Streptococcus pneumonia and methicillin-resistant staphylococcus Staphylococcus）Defense mechanism.

Embodiment 3

The risk of duration and bacterium infection after PolyI:C administration

Secondly, we observe, after PolyI:C administration, the time point that the bacteria clearance in animal body body declines.By Infected in virus, such as influenza, it usually needs take several days the even more long time, ground We conducted related experiment immediately Study carefully, the effect that PolyI:C carrys out simulated virus infection is taken with 1 dosage or 3 dosage.Through once or 3 PolyI:Cs or After 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.The experimental animal machine of disposable PolyI:C administration Internal bacteria clearance is by the trend for reducing（The CFU value average than saline control group is high 8 times, p=0.05）, while CFU through 3 experimental animal Pulmonary bacterials of PolyI:C administration is higher（Such as Fig. 2, the CFU value average than physiological saline group It 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 4

TLR3 and Cardif paths contribute to PolyI:C to induce host to the neurological susceptibility of bacterium

Because PolyI:C have activated TLR3 and Cardif dependence unwindases RIG-I and MDA5 simultaneously, it is intended to it is determined that It is related whether one or both of which path all removes power to PolyI:C infringement host bacteria.For such research, I By PolyI:C be used for TLR3（Tlr3-/-）Missing, Cardif (Cardif /) are lacked and both lack simultaneously（It is double Knock out）Experimental animal.At the initial stage of antiviral response, Cardif serves as the adapter molecule of RIG-I and MDA5 signals.We It was found that TLR3（Tlr3-/-）Missing or Cardif (Cardif- /) experimental animal PolyI:C administration after, in body Bacteria clearance is through increasing.Compared to physiological saline group, double knockout (Cardif //Tlr3 /) groups are thin in Secondary cases Under bacterium infection environment, PolyI:C administration is carried out, bacteria clearance is significantly improved in body（Fig. 3）.However, both without Tlr3/ Or the animal of Cardif/defect without PolyI:C under the conditions of being administered, its lung's streptococcus pneumonia clearance rate and wild strain Group is compared has obvious difference（Tu7 ＆8）.Therefore, Research of Animal Model for Study experiment display, PolyI:C is removed to bacterium The adverse effect of rate is relevant with the antiviral signal pathway activated of TLR3 and Cardif dependences.

Embodiment 5

PolyI:C removes the influence of power 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 the detrimental effect, or PolyI:C to be in the later stage to the clearance rate of bacterium It is no simply simply to have delayed bacterium reset procedure.Therefore we carry out every administration of polyinosinic acid on the 3rd, and next carries out pneumonia chain The infection of coccus, and assess the 1st, 3 and 5 day Pulmonary bacterial clump count after infection（CFU）.We have found that until the 5th day, life The Pulmonary bacterial amount of saline control group is managed less than detection minimum value, tends to health；And it is thin in the animal body of PolyI:C administration Bacterium amount increases（Fig. 4）, it is intended to morbidity.Therefore, PolyI:C is adversely affected to lung's antimicrobial defenses in long-term.

Embodiment 6

I types interferon is in the mediation active role of PolyI:C

Had proven to before us：During influenza infection, the I types interferon for having illustrated can lead in host's virus infection Cause the clearance rate reduction of streptococcus pneumonia.Therefore, it is intended to determine, whether the negative effect of PolyI:C can also use I Type interferon is explained.First, we are by PolyI:C（50 micrograms）, GDQ（100 micrograms）And physiological saline Nasal-cavity administration three days, and detect the expression of the I type interferon of lung.It was found that PolyI:C（Non- physiological saline or GDQ）Lung's IFN-α level of nasal-cavity administration group is presented significant change（Fig. 5 A）.Therefore, the GDQ under the dosage It e insufficient to cause lung I type IFN significant changes.Secondly, we are by wild type animal and I type interferon receptors The experimental animal of (Ifnar1- /) defect carries out nasal-cavity administration 3 times；Then, streptococcus pneumoniae infection is carried out；Finally, detect thin Bacterium clearance rate.It was found that (Ifnar /) animal can resist the negative effect of PolyI:C, its bacteria clearance has increased Plus（Fig. 5 B）.There is obvious bacteremia in this time point in none group experimental animal.Because the animal of birth defects IFNAR can Can there is delicate compensation mechanism, we carry out identical experiment using the blocking antibody of IFNAR.In PolyI:C administration Afterwards, it has been found that IFNAR blocking antibodies immune group can preferably control pneumococcal infection (Fig. 5 C) 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 poly- Flesh born of the same parents sweet acid is administered lower body for bacterium scavenging action.Wild type and Ifnar/animal are being administered condition without PolyI:C Under, body amount of bacteria does not have significant change.This is implied：Under the conditions of virus-free part, I type interferon paths can't mediate lung Organize the removing to streptococcus pneumonia in portion.

Embodiment 7

The detection of the I type interferon of PolyI:C administration experimental animal

In view of detection inflammatory cell reaction, PolyI:C is administered 3 days for we；Secondly, by wild type and Ifnar/it is dynamic Thing carries out intrathecal streptococcus pneumoniae infection.The 6h after bacterium infection, we carry out bronchoalveolar lavage, to check that inflammation is thin Born of the same parents.It was found that there is significant difference in wild and amount of bacteria of the Ifnar/group in 6h（Fig. 9 A）.Wild group of display is to several times Amount of bacteria CFU（The average CFU of wild type group be 51000, Ifnar/be 740）.Interestingly, although wild group of bacterium Amount is higher, but 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, Ifnar/animal body Interior neutrophil leucocyte amount is in trend more than twice（Wild group is 4.1*10⁵, Ifnar/group neutrophil leucocyte is 8.0*10⁵Carefully Born of the same parents）, but there was no significant difference（Fig. 9 C）.Therefore, in this animal model, through Ifnar/animal groups that PolyI:C is administered Scavenging activity after 6h is infected increases.Compared with PolyI:C is administered wild group, the Ifnar of PolyI:C administration The neutrophil accumulation amount of/animal groups increases, and causing body to remove power to bacterium strengthens.

Embodiment 8

PolyI:C causes 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, by open country Raw type and Ifnar -/animal groups carry out streptococcus pneumoniae infection（About LD50 is measured）.Experimental animal is monitored, directly To they meet standard euthanasia or its 14 days after streptococcus pneumoniae infection.It was found that the open country being administered through PolyI:C The death rate is 100% to raw group experimental animal in 7 days after pneumococcal infection（Fig. 6 A）.In contrast, it is administered through PolyI:C Ifnar/animal in bacterium infection（P=0.01, PolyI:C is administered wild group and compares Ifnar/animal groups）14 days Afterwards, 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 is activated in antiviral immunity mechanism Under the conditions of can increase neurological susceptibility of the animal body to streptococcus pneumonia.

In order to determine host's Death Mechanism, we detect the amount of bacteria of lung and blood before animal carries out euthanasia.Phase Than Ifnar -/group is administered in PolyI:C, PolyI:C is administered the amount of bacteria in wild group of blood and lung tissue It is higher.Conclusions are implied, under conditions of the administration of PolyI:C and streptococcus pneumoniae infection, the missing of I type interferon Cause bacterium to remove power to differ widely（Fig. 6 B and 6C）.Secondly, we detect PolyI:C administration Ifnar/group and it is wild The lung tissue section of group.Wild group of lung tissue is presented the consolidation and inflammatory cell infiltration of large area, the increase with bacterium in tissue Amount is consistent.And Ifnar/group lung tissue shows the inflammation compared with small area, lung tissue is most of normal（Figure 10）.Therefore, I types are done Disturbing element at least mediation PolyI:C causes the body Partial Mechanism that survival rate declines after Secondary bacterial infections, and its is main It is to reach 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.It is any ripe The personage for knowing this technology all can carry out modifications and changes under without prejudice to spirit and scope of the invention to above-described embodiment.Cause This, those of ordinary skill in the art is complete with institute under technological thought without departing from disclosed spirit such as Into all equivalent modifications or change, should be covered by claim of the invention.

Claims (4)

1. the relation between polyinosinic acid administration and IFNI are expressed.

2. the relation between polyinosinic acid administration as claimed in claim 1 and IFNI are expressed, wherein involved animal model It is mouse model.

3. the relation between polyinosinic acid administration as claimed in claim 1 and IFNI are expressed, wherein involved animal tissue Sample is mouse lung tissue.

4. the relation between polyinosinic acid administration and IFNI are expressed as described in claim 2 to 3, wherein polyinosinic acid can be lured The expression for leading IFNI is that, by polyinosinic acid, GDQ and physiological saline carry out mouse administration, com-parison and analysis mouse lung The expression gained of portion IFNI.