干扰素控制结核菌.pdf

Interferon ab mediates partial control of early pulmonary Mycobacterium bovis bacillus Calmette Gue rin infection Introduction Type I interferon IFN ab is produced by human and mouse dendritic cells and macrophages infected with mycobacteria or exposed to mycobacterial antigens 1 3 However it is not clear whether IFN ab has a protective or exacerbating function in the immune response to mycobacteria In support of a disease exacerbating effect IFN ab can inhibit mycobactericidal activity of human macrophages against Mycobacterium bovis bacillus Calm ette Gue rin BCG 4Also intranasal IFN ab can enhance the growth of a virulent clinical strain of M tuberculosis MTB in murine lungs 2Since MTB inhibits signal trans duction downstream of the IFN ab receptor5and impairs IFN a mediated dendritic cell maturation 6MTB may subvert or repress IFN ab mediated responses to promote its survival within host macrophages In contrast a bene fi cial role for IFN ab has been suggested by enhanced growth of virulent MTB in IFN ab receptor defi cient IFN abR mice7and by clinical improvements obser ved in tuberculosis patients treated with aerosolized IFN a 8 10Since IFN ab activates nitric oxide synthase 2 NOS 2 11 12 benefi cial effects of IFN ab during tubercu losis may involve direct activation of NOS 2 in human alveolar macrophages 13 14which are the initial cellular targets of aerosolized droplets containing MTB John Kuchtey 1 4Scott A Fulton 2 Scott M Reba 2Clifford V Harding1and W Henry Boom2 3 1Institute of Pathology 2Division of Infectious Diseases and 3The Tuberculosis Research Unit Case Western Reserve University and Univer sity Hospitals of Cleveland Cleveland Ohio USA doi 10 1111 j 1365 2567 2006 02337 x Received 24 August 2005 revised 13 December 2005 accepted 4 January 2006 Present address 4Department of Ophthalmology Vanderbilt University Medical Center Light Hall Room 1115B 2215 Garland Avenue Nashville TN 37232 USA John Kuchtey and Scott A Fulton are joint fi rst authors Correspondence Dr Scott A Fulton Case Western Reserve University Division of Infectious Diseases BRB Room 1021 10900 Euclid Avenue Cleveland OH 44106 4984 USA Email sxf24 po cwru edu Senior authors Clifford V Harding email cvh3 po cwru edu and W Henry Boom email whb po cwru edu Summary The role of type I interferon IFN ab in modulating innate or adaptive immune responses against mycobacterial infection in the lung is unclear In this study we investigated the susceptibility of IFN ab receptor defi ci ent IFN abR mice to pulmonary infection with aerosolized Mycobac terium bovis bacillus Calmette Gue rin BCG During early infection 2 3 weeks enhanced growth of BCG was measured in the lungs of IFN abR mice compared to wild type mice However during late infection the burden of BCG was similar in the lungs of IFN abR and wild type mice Although control of BCG growth was delayed recruitment and acti vation of T and natural killer cells production of IFN c and cytokine expression were all similar in wild type and IFN abR mice However decreased expression of nitric oxide in bronchoalveolar lavage fl uids from IFN abR mice correlated with enhanced growth of BCG Bone marrow derived macrophages from IFN abR mice also produced less nitric oxide following infection with BCG in vitro These fi ndings suggest that IFN ab contributes to innate immunity to pulmonary mycobacterial infection by augmenting production of nitric oxide Keywords lung mouse mycobacteria nitric oxide type I interferon Abbreviations BAL bronchoalveolar lavage BCG Mycobacterium bovis bacillus Calmette Gue rin BMM bone marrow derived macrophages IFN abR interferon ab receptor defi cient MTB Mycobacterium tuberculosis NK natural killer NOS 2 nitric oxide synthase 2 PBS phosphate buffered saline 2006 Blackwell Publishing Ltd Immunology 118 39 4939 IMMUNOLOGYORIGINAL ARTICLE Activation of NOS 2 occurs in response to mycobacte rial infection or exposure to mycobacterial antigens13 15 and is important for but may be non essential to myco bactericidal immunity 7 16 17Involvement of NOS 2 is most evident when T cell responses develop and IFN c is expressed 7 17 18During the adaptive phase of antigen specifi cimmunecontrol IFN ccontributestothe activation of NOS 2 and nitric oxide production by macrophages 16IFN ab also directly promotes T cell acti vation 19dendritic cell maturation and IFN c production which are crucial elements of antimycobacterial immu nity 16 20 21However it remains unclear whether IFN ab contributes to the development of protective mycobacteri al antigen specifi c T cell responses During pulmonary MTB infection in mice recruitment and activation of natural killer NK cells may be enhanced by expression of IFN ab 20 22 25Furthermore NK cells are capable of killing human macrophages infec ted with MTB 26Although depletion of NK cells in mice does not appear to affect the control of MTB infection inthelung 24NK celldepletionexacerbatessplenic M avium infection 27 29Thus a precise role for NK cells in host defence against mycobacterial infections has not been well defi ned We investigated the role of IFN ab in protection against aerosolized BCG infection which is characterized by an early phase that limits bacterial growth and a late phase when infection resolves 30In IFN abR mice we observed an initial phase of enhanced growth in the lungs However BCG growth was eventually controlled at bacterial burdens similar to those detected in wild type mice Recruitment and activation of NK and T cells and production of IFN c were essentially normal in the IFN abR mice However despite higher bacterial burdens bronchoalveolar nitrite levels were lower in IFN abR mice compared to controls suggesting a defect in NOS 2 induction We also determined that nitric oxide produc tion was signifi cantly lower in IFN abR bone marrow derived macrophages BMM infected with BCG in vitro While not required for development of protective T cell immunity during BCG infection IFN ab contributes to the production of nitric oxide and early control of BCG growth in the murine lung Materials and methods Mice Wild type male and female mice were housed under spe cifi c pathogen free conditions using microisolator cages Laboratory Products Inc Maywood NJ and fed a standard rodent diet and water ad libitum IFN abR A129 mice on the 129S6 SvEv background H 2b were purchased from B Shandon Pittsburgh PA and stained with Diff Quik Dade Diagnostics Aguada PR for differential cell counts Lung cell homogenates were prepared as previously described 30 Briefl y after BAL lungs all fi ve lobes were perfused with 10 ml PBS injected into the right ventricle and then dissected from mainstem bronchi Lungs were minced with scalpels in 4 ml of RPMI 1640 and digested with 125 units ml of type IV collagenase and 30 units ml DNase C5138 and D4527 Sigma for1 5 hrat 37 Tissuewas further disruptedby sequential passage through an 18G needle clarifi ed by fi ltration through sterile cotton and centrifuged 800 g for 15 min The cells were resuspended in red blood cell lysis buffer containing 10 mMTris HCl and 0 83 ammonium chloride incubated at room temperature for 5 min and pelleted 800 g for 15 min Lung cells were resuspended in PBS and held on ice for staining and fl ow cytometric studies In addition lung cells 4 106 ml were cultured for 72 hr in complete med ium Dulbeccos modifi edEagle sminimalessen tial medium DMEM 10 fetal calf serum 0 05 mM 2 mercaptoethaol 2 mMHEPES 1 mMsodium pyruvate 100 mMnon essential amino acids 100 U ml penicillin and 0 1 mg ml streptomycin Culture supernatants were stored at 70 until analysed for cytokine chemokine and nitrite expression CFU determination Aliquots of lung tissue homogenates were serially diluted in PBS containing 0 05 Tween 20 and plated on Mid dlebrook 7H10 agar enriched with 10 o ADC Difco and 0 5 glycerol and incubated at 37 for 2 3 weeks Colonies were counted and total tissue CFU was calcula ted based on the volume of homogenate obtained from each organ mouse Cell staining and fl ow cytometry For analysis of T cell and NK cell numbers and activa tion the following antibodies were used allophycocya nin anti CD3e clone145 2C11 biotin anti CD49b clone DX5 fl uorescein isothicyanate FITC anti CD94 clone 18d3 APC hamster immunoglobulin G IgG biotin rat IgM and streptavidin APC Cy7 from eBio sciences San Diego CA and phycoerythrin PE Cy7 anti CD69 clone H1 2F3 biotin anti CD25 clone 7D4 FITC anti Ly49D clone 4E5 anti CD16 CD32 clone 2 4G2 and PE anti TRAIL clone N2B2 from BD Bio sciences San Diego CA After BAL lung cell homogen ates were prepared as described above resuspended at 1 107 2 107cells ml in staining buffer PBS contain ing 2 fetal bovine seurm and 0 1 sodium azide and kept in the dark on ice To stain lung T cells and NK cells Fc receptors were blocked for 5 min with anti CD16 CD32 5 lg ml Spe cifi c antibodies and isotype controls were then added at 5 lg ml After 30 min cells were washed in staining buf fer and streptavidin APC Cy7 1 100 was added for 15 min Next cells were washed re suspended in 0 3 ml PBS containing 2 paraformaldehyde stored at 4 and analysed within 3 days Data were acquired using an LSR II system fl ow cytometer BD Biosciences San Diego CA To assess activation markers on NK cell subsets at least 105ungated events were acquired Post acquisition compensation and analysis was performed using FLOWJO software Tree Star Ashland OR Cells were analysed within a lymphocyte gate based on typical forward scatter and side scatter properties Expression of activation mark ers CD69 and CD25 was analysed on CD3 CD4 T cells DX5 CD3 NK cells and DX5 CD3 NK cells expressing the phenotypic markers Ly49D or CD94 were analysed for expression of CD69 and TRAIL To express data as total number of cells lung the per cent of total ungated events was multiplied by the total number of lung cells harvested Interferon c ELISPOT Sterile ELISPOT plates Unifi lter plate 7770 0006 What man Clifton NJ were precoated overnight at 4 with 5 lg ml anti IFN c antibody Pharmingen 551216 dilu ted in PBS and washed fi ve times with PBS Next 1 106 5 105and 2 5 105lung cells from individual mice were cultured in duplicate and either incubated in complete medium alone or with exogenous BCG at a ratio of 1 1 After a 48 hr incubation at 37 cells were washed away four times with PBS containing Tween 20 0 05 and 0 1 ml of biotinylated anti IFN c XMG1 2 Pharmingen 554410 diluted to 0 5 mg ml in PBS con taining 0 05 Tween 20 and 1 bovine serum albumin was added to each well After 4 hr at room temperature plates were washed four times and bound IFN c was detected using streptavidin alkaline phosphatase accord ing to the manufacturer s instructions R Pharmingen 558874 IFN a R Pharmingen 555252 IL 12 p40 R and before the BCG burden had peaked As shown in Fig 2 e h no signifi cant differences in NK cell number or CD69 expression were detected between wild type Fig 2e f and IFN abR Fig 2g h lungs after 21 days We also measured TNF related apop tosis inducing ligand TRAIL expression on NK cells which is induced by IFN ab and may be involved in NK mediated cell killing 38However only a small percentage 1 7 0 1 of NK cells expressed TRAIL in the lungs of both wild type and IFN abR mice during BCG infection data not shown CD69 and TRAIL expression was also the same on DX5 CD3 NK cell subsets further character ized by the phenotypic markers CD94 and Ly49D which were expressed on 30 40 of the DX5 CD3 NK cells data not shown Thus NK cell activation appeared intact suggesting that a NK cell defect did not account for enhanced early growth of BCG in IFN abR mice Since the numbers of T and NK cells were similar in infected wild type and IFN abR mice we further exam ined T and NK cell effector functions using a sensitive IFN c Elispot assay In our hands this assay is capable of detect ing as few as 1 100 000 IFN c secreting cells As shown in Fig 3 after 1 2 weeks of infection the frequency of IFN c secreting lung parenchymal cells which includes both T and NK cells was similar in wild type and IFN abR cells whether they had been cultured in the presence closed and open triangles range 5 50 106cells or absence closed and open circles range 5 10 106cells of additional BCG After 21 days the mean frequency of IFN c expressing lung cells following re stimulation with BCG increased but remained statistically similar P 0 05 in both groups of mice 624 247 106in wild type and 700 70 106in IFN abR mice Thus total lung cell IFN c expression further correlated with fl ow cytometry data revealing sim ilar expression of activation markers on CD4 and NK cells in wild type and IFN abR mice Fig 2a h In addition despite enhanced growth of BCG in IFN abR mice we did not detect signifi cant differences in TNF a IL 10 IL 12 p40 and p70 or IFN c expression in BAL fl uids or lung cell culture supernatants prepared from IFN abR mice data not shown Thus although lung CFUs were higher in IFN abR mice during early infection suffi cient T cell and NK cell activation and IFN c production developed and probably contributed to control of BCG growth similar to that observed in wild type mice Decreased nitric oxide expression is associated with enhanced BCG growth in IFN abR mice Although nitric oxide dependent killing of mycobacteria by macrophages is important for controlling mycobacterial growth after adaptive immunity develops it is unclear 1000 750 500 250 0 0 Days after infection wild type BCG BCG IFN spots 106 lung cells 7142128354249 wild type Figure 3 IFN ab receptor expression is not required for develop ment of antigen specifi c IFN c production by lung cells during M bovis BCG infection Mice were infected by aerosol with BCG 4087 1102 CFU and lung cells prepared 1 6 weeks after infec tion IFN c production by total lung was measured by Elispot Lung cells from wild type mice d m and IFN abR mice s n were incubated without d s or with additional BCG bacilli moi 1 m n for 48 hr Cellular IFN c production was determined by Eli spot Mean spot number per 106lung cells from three mice is shown at each time point for a representative experiment n 3 No signi fi cant differences in IFN c spot forming units were detected using Student s t test in three independent experiments with or without BCG restimulation in vitro Fewer than 5 10 spots per 106lung cells were detected from uninfected mice Positive control responses to concanavalin A 4 lg ml were always too numerous to count 44 2006 Blackwell Publishing Ltd Immunology 118 39 49 J Kuchtey et al if NOS 2 expression is important during early pulmonary mycobacterial infection 7 16 39Since IFN ab induces nitric oxide production we infected wild type and IFN abR mice with BCG and measured nitrite expression in BAL and lung cell culture supernatants during early infection 2 4 weeks As shown in Fig 4 a local bronchoalveolar production of nitrite was signifi cantly lower in IFN abR mice Since signifi cantly higher numbers of bron choalveolarmacrophages monocytesweredetectedin IFN abR mice 178 333 28 915 compared to wild type mice 80 000 10 408 P 0 05 decreased BAL nitrite levels could not be attributed to defective alveolar macrophage monocyte recruitment in BCG infected IFN abR mice However we could not exclude a defect in BAL nitrite expressed by respiratory epithelial cells 40In wild type mice maximal nitrite production occurred when both BAL CFUs and BAL cell numbers were max imal Fig 1b However despite the two to threefold higher CFU burden in IFN abR mice Fig 1 BAL nitrite levels remained low Fig 4a In contrast low lev els of nitrite in lung cell culture supernatants were meas ured at the lower limit of detection 6 10 lMusing the Griess reagent and therefore we were unable to defi nit ively rule out differences in nitrite expression by paren chymal lung cells isolated from wild type and IFN abR mice data not shown The low levels of nitrite in lung cell cultures may also have refl ected the lower proportion of macrophages present in the lung cell homogenates compared to BAL where the bronchoalveolar macropha ges represent the predominant cell type 65 95 during BCG infection 41 Thus high levels of nitrite in BAL fl uids expressedbybronchoalveolarmacrophages recruited monocytes or respiratory epithelial cells40may be unusu ally dependent upon IFN ab receptor coactivation during BCG infection Since the majority of bronchoalveolar cells during aerogenic BCG infection are alveolar macrophages or recruited monocytes41we inferred that macrophages in IFN abR miceproducedlessnitriteduringBCG infection Thus westudiednitricoxideproduction in vitro using BMM isolated from wild type and IFN abR mice BMM were infected with BCG and cumu lative nitrite production in culture supernatants was measured after 72 hr In six independent experiments we observed signifi cantly less nitrite production by IFN abR macrophages as compared to wild type cells As shown in a representative experiment Fig 4b at a MOI of 10 nitrite expression by IFN abR BMM was 40 lower than wild type control levels 64 9 9 9 lM versus 39 1 10 8 lM P 0 05 In three independent experiments a 53 8 18 8 mean SD difference in nitrite was measured In contrast diminished NOS 2 activity in BCG infected IFN abR BMM was not observed at lower MOI These data suggest that at a low MOI i e 1 5 BCG infection induces nitrite pro duction independent of IFN ab expression and that at higher infection ratios MOI 10 BCG induces higher levels of IFN ab expression that could augment nitric a b c Wild type Wild type moi moi BCG bacilli macrophage Days after infection 0 0 25 50 75 100 125 150 175 200 225 0 0 20 40 60 80 4 0714212835 8 12 16 20 24 1510 Nitrite concentration M Nitrite concentration M per 2 106 BMM cells BAL Nitrite M 10101010 000 515IFN ng ml Wild type Figure 4 IFN ab receptor defi ciency is associated with a decrease in nitric oxide expression in response to M bovis BCG infection a Wild type d and IFN abR s mice were infected wit