Type I IFNs (IFNIs) have pleiotropic functions in regulating host innate and adaptive immune responses to pathogens. To elucidate the role of IFNIs in host resistance to chlamydial infection in vivo, we compared IFN-α/β receptor knockout (IFNAR−/−) and wild-type control mice in susceptibility to Chlamydia trachomatis mouse pneumonitis (Chlamydia muridarum) lung infection. We found that the IFNAR−/− mice were significantly more resistant to C. muridarum infection showing less bacterial burden and bodyweight loss, and milder pathological changes. However, IFN-γ response, which is believed to be critical in host defense against chlamydial infection, was similar between the wild-type and IFNAR−/− mice. More importantly, TUNEL analysis showed less macrophage apoptosis in IFNAR−/− mice, which was consistent with lower expressions of IFNI-induced apoptotic factors, TRAIL, Daxx, and PKR. Furthermore, depletion of lung macrophages with dichloromethylene diphosphonate-liposome significantly increased the susceptibility of the IFNAR−/− mice to C. muridarum, confirming the importance of macrophages. Overall, the data indicate that IFNIs play a promoting role in C. muridarum lung infection, largely through increase of local macrophage apoptosis.

The group of type I IFNs (IFNIs)3 is a multiple member family, comprising one IFN-β, more than 10 IFN-α subtypes, and several other members such as -ε, -κ, and -ω (1). IFNIs were named after their dominant character of interfering viral replication in mammalian cells, an essential function for host survival from viral infections. IFN-α and IFN-β are the major components of IFNIs in humans and rodents, and they share the same receptor, IFNα/β receptor (IFNAR), for initiating downstream STAT signal transduction (1, 2). The physiological levels of IFNIs are very low, but they are strongly induced upon stimulation. IFNIs are among the earliest cytokines that are induced during infection and play an important regulatory role in both innate and adaptive immune responses (3). IFNIs are able to activate macrophages (4), enhance cytolytic activity of NK cells (5), and promote IFN-γ secretion and Th1 differentiation (5). In particular, IFNIs can activate and modulate the function of dendritic cells (DCs), the most important APCs, via promoting MHC expression, costimulatory molecule expression, and cytokine/chemokine secretion, thus modulating adaptive T cell immune responses (3, 6, 7, 8, 9).

Both viral and bacterial infections can promptly and strongly induce IFNIs secretion through their specific components or metabolic products, but the function of IFNIs in bacterial infections appears complicated, varying in different infection models (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). Studies on Legionella pneumonphila (11), Salmonella typhimurium (12), Shigella flexneri (13), Bacillus anthrasis (14) and Mycobacterium bovis bacillus Calmette-Guérin (15) showed that IFNIs are protective for host, at least partially through enhancing IFN-γ/NO production and suppressing bacterial invasion. In contrast, some studies have shown that IFNIs are detrimental, especially in certain bacterial infections. For example, it was reported that IFNIs could promote Listeria monocytogenes, an intracellular bacterial infection, through increasing listeriolysin O-dependent apoptosis of macrophages and lymphocytes (16). Furthermore, studies using IFNα/β receptor KO (IFNAR−/−) mice showed that the KO mice were more resistant to L. monocytogenes infection than wild-type (WT) mice, which correlated with less lymphocyte apoptosis, and elevated IL-12p70 production in the KO mice (17, 18, 19). It has been also shown that a highly virulent strain of Mycobacterium tuberculosis can promote IFNIs production, which promotes infection in a mouse model (20). Furthermore, the administration of exogenous IFNα enhanced hosts’ susceptibility to M. tuberculosis in vivo (20).

Chlamydiae are obligate intracellular bacteria; some of their species, Chlamydia trachomatis and Chlamydia pneumoniae, cause significant human diseases (21). C. trachomatis can cause trachoma and sexually transmitted diseases, sometimes with serious sequelae, including blindness, pelvic inflammatory diseases, and infertility (22, 23, 24). Type II IFN (IFN-γ) has been shown to be the most important cytokine for host resistance to chlamydial infection (24, 25, 26, 27, 28). However, the role of IFNIs in chlamydial infections is very controversial. Three in vitro studies showed that IFNIs could inhibit chlamydial growth in different cell types (29, 30, 31), and one in vivo study showed that induction of IFNIs through administration of poly(I:C) during C. trachomatis lung infection, at certain conditions, could delay mouse death following infection (32). In the case of C. pneumoniae infection, one study showed that bone marrow-derived macrophages from IFNAR−/− mice, compared with that from WT mice, are more susceptible to C. pneumoniae infection in vitro, which correlated with less IFN-γ and inducible NO synthase production (30), while another study showed that IFNAR−/− and WT mice were similar in susceptibility to C. pneumoniae infection (33). These inconsistent results in chlamydial and other bacterial infection studies indicate a necessity for further study on the role of IFNIs in host resistance/susceptibility to chlamydial infection, especially using in vivo models.

In the present study, using a mouse model of respiratory tract chlamydial infection, we compared the susceptibility of IFNAR−/− and WT mice to infection with C. muridarum, also called C. trachomatis mouse pneumonitis, a well characterized and extensively studied mouse strain of Chlamydia. Our data showed that, following intranasal delivery with C. muridarum, the IFNAR−/− mice displayed significantly higher resistance to the infection, which correlated with higher number of macrophages in the lung. More importantly, WT mice showed more apoptosis of macrophages and higher expression of apoptotic factors in the lungs. In addition, depletion of lung macrophages in the IFNAR−/− mice dramatically increased chlamydial growth, confirming the importance of macrophages in the clearance of infection. These findings suggest a promoting role of IFNIs in chlamydial infection.

Breeding pairs of IFNAR−/− mice (34) were purchased from B&K Universal; the corresponding breeding pairs of WT control mice (129Sv/Ev) were from Taconic. All breeding pairs and the offspring were housed in specific pathogen-free conditions in the Central Animal Care Service of the University of Manitoba. Age-matched mice at 8–12 wk old were used for study. Mice were studied under the guidance of Canada Council for Animal Care, and the protocol was approved by Protocol Management and Review Committee of The University of Manitoba.

The Ab pairs for ELISA tests of IFN-γ, IL-12p70, TNF-α, and IL-10 were bought from eBioscience; Ab pairs for IL-4, -5, and MCP-1 tests were purchased from BD Biosciences. Ab pairs for MIP-1α were from R&D Systems. The biotinylated Abs for mouse serum IgG1, gG2a, and IgM were purchased from Southern Biotechnology Associates. For immunohistochemical testing of cell surface markers, purified rat anti-mouse CD2, and F4/80 Abs and FITC-conjugated rat anti-mouse F4/80 Ab were purchased from eBioscience; FITC- and Texas Red-conjugated goat anti-rat IgG and FITC-conjugated goat anti-rabbit IgG secondary Abs were purchased from Jackson ImmunoResearch Laboratories. Purified rabbit anti-mouse cleaved caspase-3 mAb was from Cell Signaling Technology. For staining of epithelial cells in lung tissues, mouse anti-Pan-Cytokeratin human (mouse) mAb (Calbiochem) was used as primary Ab. Biotin-conjugated rabbit anti-mouse IgG (H+L) (Chemicon International) was used as secondary Ab. Avidin-conjugated Texas Red was purchased from Vector Laboratories. For FACS analyses, FITC-conjugated anti-mouse CD11c, CD8α, CD4, CD40, B220, and I-A, and PE-conjugated anti-mouse CD11c, CD80, and CD86 were from BD Biosciences.

C. muridarum was cultured, purified, and quantified as previously described (35). For infection, isoflurane anesthetized mice were intranasally inoculated with a predetermined dose of C. muridarum in 40 μl of sucrose phosphate glutamic acid buffer. The body weight change was monitored daily following infection. Mice were euthanized at various days following infection and the lungs were aseptically collected. The organs were homogenized in 3 ml of sucrose phosphate glutamic acid buffer. After brief centrifugation, the supernatants were aliquoted into 1 ml aliquots and kept at −80°C until being tested. The chlamydial loads in the homogenates were determined as described (36). Fresh aliquots of the samples were used for repeating tests.

Lung/bronchial draining lymph node cells and spleen cells were cultured at 5 × 106 cells/ml and 7.5 × 106 cells/ml, respectively, in the presence or absence of UV-inactivated C. muridarum elementary bodies (5×105 inclusion forming units (IFUs)/ml) as previously described (35). The 72-h culture supernatants and lung homogenates were subjected for cytokine measurements using a two-Ab sandwich ELISA as previously described (35). Recombinant cytokines were used as standards in all assays.

Serum C. muridarum specific IgM, IgG1, and IgG2a were determined by ELISA as previously described (35). Results are expressed as ELISA titers at 60 min using the end point (cut off OD value of 0.5 at 405 nm) of the titration curves.

Lungs of the mice collected on different days postinfection (p.i.) were subjected to histological analysis as described (36). In brief, formalin fixed tissues were embedded in paraffin and six micron sections were cut and stained with H&E and examined under light microscopy. For bronchioalveolar lavage (BAL) inflammatory cell analysis, total BAL cells were first counted and, after centrifugation, the pellet was spread on slides and stained using leukocyte staining kit (Fisher Scientific) for cell differentials. The examiner was blinded as to the experimental groups.

Lung tissues collected from mice at various days p.i. were snap-frozen by liquid nitrogen and stored at −80°C until staining. In brief, frozen tissues were embedded in frozen tissue embedding media (Fisher Scientific) and ten micron sections were cut. Tissues were fixed with acetone and specific Ags were stained with corresponding Abs and secondary Abs. The tissues were then examined with a fluorescent microscope (Olympus AX70) or a confocal microscope (Olympus IX70).

DC from mouse spleens were isolated with MACS Technology following the manufacturer’s protocol (Miltenyi Biotec). In brief, mouse spleens were treated with collagenase D (1 mg/ml) for 45 min and filtered through 70-μm cell strainers to release single cells. The cells were labeled with CD11c microbeads and passed through magnetic columns. Isolated CD11c-positive cells were used for cell surface marker analysis by flow cytometry. The rest of the cells were cultured in 96-well plates at a concentration of 5 × 105 cells/well. Forty-eight hours later, the supernatants were harvested for cytokine assay by ELISA.

Pulmonary macrophages were depleted by intranasal administration of liposome encapsulated dichloromethylene diphosphonate (clodronate, or CL2MDP), a gift from Roche Diagnostics. Liposome containing CL2MDP and liposome encapsulating PBS were prepared as described previously (37). IFNAR−/− mice were administrated with 90 μl of CL2MDP liposome or PBS liposome, or PBS by intranasal route at 3 days before being infected with C. muridarum. Four days p.i., repeated liposome administration was given intranasally. Mice were euthanized at 7 days p.i.. Chlamydial yields and pathological changes were analyzed as described above.

For intracellular cytokine detection, spleen and draining lymph node cells were cultured as mentioned before. After 72 h, cells were stimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) for 3 h, and with brefeldin A (20 μg/ml) incubation for another 4 h. Cells were harvested, incubated with anti-CD16/32, and stained with anti-CD3e (PE-Cy7), anti-CD4 (PE), and anti-CD8 (FITC) Abs. The cells were then fixed with 2% paraformaldehyde and permeabilized (freshly prepared 0.1% Saponin in flow buffer), and stained with allophycocyanin-labeled anti-IFN-γ, anti-IL-4, or anti-IL-10 Abs. Data were collected using a FACSCalibur flow cytometer (BD Biosciences) and analyzed using CellQuest software (BD Biosciences).

Lung tissues were fixed with 10% neutral buffered formalin, embedded in paraffin, and sectioned. Apoptosis was tested by using TUNEL in situ cell death detection kit-POD (Roche Diagnostics) as described (17). In brief, the tissue sections were first treated with xylene. Hydrated sections were treated with proteinase K (Sigma-Aldrich) and blocked with blocking buffer (Dako). Tissue sections were then incubated with 50 μl TUNEL reaction mixture for 60 min at 37°C in darkness, then labeled with converter-POD in 37°C for 30 min. After thoroughly washing with PBS, tissue sections were treated with 100 μl diaminobenzidine substrate. The reaction was stopped by washing with PBS and counter stained with hemoxylin. Samples were analyzed under a light microscope.

Lung tissues were snap-frozen in liquid nitrogen and stored in −80°C freezer until RNA isolation. Whole lungs were homogenized in 3 ml of cold TRIzol (Invitrogen); total RNA was isolated according to the manufacturer’s instructions. Total RNA was quantified and treated with TURBO DNase (Ambion) to get rid of contaminated DNA and reverse transcribed with Superscript III reverse transcriptase (Invitrogen). Specific genes were amplified with THERMO-START DNA polymerase (ABgene) in a thermo cycler (MJ Research). Semiquantitation of PCR products was done with Scion-Image as described (38). Primers for PCR: β-actin (288bp): sense 5′-TCTTGGGTATGGAATCCTGTGGCA-3′, anti-sense 5′-ACTCCTGCTTGCTGATCCACATCT-3′; GAPDH (191bp): sense 5′-AACGACCCCTTCATTGAC-3′; anti-sense 5′-TCCACGACATACTCAGCAC-3′; IFN-α (524bp): sense 5′-ATGGCTAGGCTCTGTGCTTTCCT-3′; anti-sense 5′-AGGGCTCTCCAGATTTCTGCTCTG-3′; PKR (223bp): sense 5′-GTGGACATCTTTGCTTTGGGCCTT-3′, anti-sense 5′-TGTTCCTCCATTCAGCCAAGGTCT-3′; Daxx (400bp): sense 5′-TGAACTTAGCTCCTGCAGCCTCAA-3′, anti-sense 5′-TAAATGAGCCGTTCAATGCGCCTG-3′; TRAIL (396bp): sense 5′-ACCTCAGCTTCAGTCAGCACTTCA-3′, anti-sense 5′-AAGCTGAGTTGCTTCTCCGAGTGA-3′. MIP-2 (285bp): sense 5′-ACCCTGCCAAGGGTTGACTTC-3′; anti-sense 5′-GGCACATCAGGTACGATTCCAG-3′.

The difference in the cytokine levels and RT-PCR band density were analyzed by Student’s t test. The IFU counts and Ab titers were transformed to base 10 logarithms and analyzed by Student’s t test.

Following intranasal infection, WT control mice displayed continuous body weight loss without signs of recovery when they were infected with either higher (1000 IFUs) or lower doses (200 IFUs) of C. muridarum. In contrast, IFNAR−/− mice showed much less body weight loss at higher dose infection (Fig. 1,A) and very mild body weight loss at lower dose infection compared with the WT mice (Fig. 1,B). In line with the body weight loss, the IFNAR−/− mice showed significantly lower in vivo organism growth at day 6 p.i. when the higher dose was used. The bacterial loads were similar between WT and IFNAR−/− mice at days 2 and 4 p.i. When the lower dose was used, the IFNAR−/− mice showed significantly lower bacterial burden in the lung at both early (day 7) and late (day 14) stages of infection than the WT mice (Fig. 1,D). In particular, at 14 days p.i., while the infection in IFNAR−/− mice was close to being cleared (less than 103 IFUs in the whole lung), the WT mice showed increased bacterial burden, 1000-fold higher than that in the IFNAR−/− mice. To confirm the amount of IFNI response induced by C. muridarum, we examined IFNα mRNA expression in the lung tissues at various days following infection, which showed significant increase at early time points of infection (Fig. 1,E). In agreement with the differences in body weight loss and lung bacterial loads, histological analysis (Fig. 2) showed more intense lung inflammation in WT mice than IFNAR−/− mice starting from a very early time p.i. (day 2). A large proportion of the infiltrating inflammatory cells of WT mice were neutrophils. The heavier inflammation in WT mice became more evident on days 6 and 7 p.i. Most of the interstitial tissues were infiltrated with inflammatory cells, and most alveoli were filled with inflammatory exudates in WT mice. In contrast, IFNAR−/− mice showed only mild inflammation, with minimal cellular infiltration around some bronchi (Fig. 2). At 14 days p.i., the IFNAR−/− mice showed close to normal lung structure while the WT mice still showed extensive lung tissue damage and diffused cellular infiltration, which correlated with continuous bacterial growth (Fig. 1 D). These results indicate that IFNIs play a promoting role in host susceptibility to C. muridarum infection.

FIGURE 1.

WT mice showed more severe disease after infection compared with IFNAR−/− mice. A, Body weight changes of 20 mice in each group (WT or IFNAR−/−) after intranasal infection with 1000 IFUs of C. muridarum. The initial body weights before infection were similar between the groups. B, Body weight changes of 8–10 mice of each group after intranasal infection with 200 IFUs of C. muridarum. The data are representative of three independent experiments that showed similar results. C, Mice were infected intranasally with 1000 IFUs of C. muridarum, and mice were killed at each time point to determine the chlamydial growth in the lung. D, Mice were infected intranasally with 200 IFUs of C. muridarum and killed at days 7 and 14 p.i. (four to five mice for each time point), and the chlamydial growth in the lung was assessed. The data are representative of three independent experiments that showed similar results. E, IFN-α expression in the lungs following C. muridarum infection. WT mice were infected with 1000 IFUs of C. muridarum and killed at various time points (four mice at each time point), and pan-IFN-α expression were tested by RT-PCR and presented as ratio to house-keeping gene (GAPDH) expression. The data are shown as mean ± SEM; ***, p < 0.001.

FIGURE 1.

WT mice showed more severe disease after infection compared with IFNAR−/− mice. A, Body weight changes of 20 mice in each group (WT or IFNAR−/−) after intranasal infection with 1000 IFUs of C. muridarum. The initial body weights before infection were similar between the groups. B, Body weight changes of 8–10 mice of each group after intranasal infection with 200 IFUs of C. muridarum. The data are representative of three independent experiments that showed similar results. C, Mice were infected intranasally with 1000 IFUs of C. muridarum, and mice were killed at each time point to determine the chlamydial growth in the lung. D, Mice were infected intranasally with 200 IFUs of C. muridarum and killed at days 7 and 14 p.i. (four to five mice for each time point), and the chlamydial growth in the lung was assessed. The data are representative of three independent experiments that showed similar results. E, IFN-α expression in the lungs following C. muridarum infection. WT mice were infected with 1000 IFUs of C. muridarum and killed at various time points (four mice at each time point), and pan-IFN-α expression were tested by RT-PCR and presented as ratio to house-keeping gene (GAPDH) expression. The data are shown as mean ± SEM; ***, p < 0.001.

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FIGURE 2.

WT mice showed more severe inflammation after infection compared with IFNAR−/− mice. WT and IFNAR−/− mice were infected intranasally with C. muridarum (200 IFUs). Mice were killed at days 2, 4, 6, 7, and 14 p.i. Lung sections were stained with H&E and analyzed under light microscopy. Slides were photographed at ×400 and ×1000 (inserts) magnifications.

FIGURE 2.

WT mice showed more severe inflammation after infection compared with IFNAR−/− mice. WT and IFNAR−/− mice were infected intranasally with C. muridarum (200 IFUs). Mice were killed at days 2, 4, 6, 7, and 14 p.i. Lung sections were stained with H&E and analyzed under light microscopy. Slides were photographed at ×400 and ×1000 (inserts) magnifications.

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Since cytokine/chemokine patterns have been shown to be important in inflammation formation and host defense against chlamydial infection (24, 39), we examined cytokine production in the lung tissues of the infected mice (Table I). Consistent with heavier inflammation observed in WT mice (Fig. 2), the levels of proinflammatory cytokines, IL-1α, IL-1β, IL-6, and IFN-γ, were significantly higher in the lungs of WT mice than in IFNAR−/− mice at the early stage of infection (Table I). In addition, the WT mice showed significantly higher expression of MCP-1 and MIP-2, the important chemokines for neutrophil infiltration (38, 40, 41), than IFNAR−/− mice. The results suggest that WT mice produce significantly higher local proinflammatory cytokines/chemokines than IFNAR−/− mice, which may be the basis for the heavier inflammation observed in the lungs of WT mice.

Table I.

Cytokine and chemokine production in the lung p.i.a

DayIL-1α (pg/ml)IL-1β (pg/ml)IL-6 (pg/ml)IFN-γ (pg/ml)MCP-1 (pg/ml)MIP-2 (Folds)
      
 WT 444 + 75b 114 + 31b 902 + 246c 650 + 310 2298 + 264 0.54 + 0.04 
 KO 232 + 41 54 + 15 260 + 132 325 + 50 2014 + 566 0.57 + 0.057 
      
 WT 2281 + 300c 426 + 78b 4347 + 1364d 5256 + 1471d 4010 + 966d 0.8 + 0.04b 
 KO 1503 + 213 310 + 58 1918 + 613 1218 + 409 2332 + 226 0.64 + 0.03 
      
 WT 3393 + 320 554 + 89 6390 + 1276b 14000 + 3200b 6449 + 405d 1.45 + 0.25b 
 KO 3942 + 450 662 + 93 4023 + 799.5 5575 + 2076 2218 + 142 0.85 + 0.04 
DayIL-1α (pg/ml)IL-1β (pg/ml)IL-6 (pg/ml)IFN-γ (pg/ml)MCP-1 (pg/ml)MIP-2 (Folds)
      
 WT 444 + 75b 114 + 31b 902 + 246c 650 + 310 2298 + 264 0.54 + 0.04 
 KO 232 + 41 54 + 15 260 + 132 325 + 50 2014 + 566 0.57 + 0.057 
      
 WT 2281 + 300c 426 + 78b 4347 + 1364d 5256 + 1471d 4010 + 966d 0.8 + 0.04b 
 KO 1503 + 213 310 + 58 1918 + 613 1218 + 409 2332 + 226 0.64 + 0.03 
      
 WT 3393 + 320 554 + 89 6390 + 1276b 14000 + 3200b 6449 + 405d 1.45 + 0.25b 
 KO 3942 + 450 662 + 93 4023 + 799.5 5575 + 2076 2218 + 142 0.85 + 0.04 
a

Mice were infected (intranasally) with 1000 IFUs of C. muriarum and euthanized at days 2, 4, and 6 p.i. Whole lungs (4–5 mice/group at each time point) were homogenized and cytokine levels were measured by ELISA (IL-1α, IL-1β, IL-6, IFN-γ, and MCP-1) or RT-PCR (MIP-2, presented as the folds of band density compared with β-actin). The data are shown as mean ± SD.

b

p < 0.05;

c

p < 0.01; and

d

p < 0.001.

Since Chlamydia-specific T cell response is better correlated with host resistance to chlamydial infection, we further investigated Chlamdydia-driven T cell responses in the infected WT and IFNAR−/− mice by examining the cytokine production of ex vivo spleen and draining lymph node cells from infected mice upon organism-specific restimulation. As shown in Table II, the WT and IFNAR−/− mice showed similar levels of Th1 (IFN-γ and TNF-α)- and Th2 (IL-4, IL-5, and IL-10)-related cytokine production by spleen cells at both early (day 7) and late (day 14) stages of infection. Consistently, intracellular IFN-γ analysis for T cells also showed similar results between WT and IFNAR−/− mice (Fig. 3). Analysis of draining lymph nodes showed similar patterns at day 7 p.i. In contrast, at late stage of infection (day 14), the lymph node cells in WT mice produced significantly higher IFN-γ and TNF-α and lower IL-10 than IFNAR−/− mice. The similar Chlamydia-driven Th1- and Th2-related cytokine production by spleen and draining lymph node lymphocytes in the WT and IFNAR−/− mice at the early stage of infection suggest a comparable T cell response in these two groups. The continued high levels of IFN-γ and TNF-α production by WT mice in the late stage of infection may reflect the persistence of infection and inflammation.

Table II.

Cytokine production by spleen, draining lymph node cells, and lung inflammatory cellsa

DayIFN-γIL-12p70TNF-αIL-4IL-5IL-10
LN       
  WT 32420 ± 2930 12 ± 9 129 ± 69 1.6 ± 1.3 148 ± 74 3238 ± 1000 
  KO 31200 ± 4880 12 ± 8 82 ± 50 2.2 ± 1.6 190 ± 140 2641 ± 698 
 14       
  WT 14200 ± 8720b 51 ± 20 148 ± 45b 9.1 ± 8.4 156 ± 71 2401 ± 971b 
  KO 4433 ± 1347 53 ± 23 40 ± 19 8.5 ± 7 207 ± 132 4915 ± 1579 
SP       
  WT 20730 ± 12600 41 ± 29 94 ± 39 47 ± 15 146 ± 90 1742 ± 565 
  KO 20240 ± 12170 29 ± 22 104 ± 15 45 ± 9 121 ± 70 1417 ± 82 
 14       
  WT 5214 ± 2005 66 ± 16 221 ± 63 50 ± 24 100 ± 71 603 ± 75 
  KO 3654 ± 2551 71 ± 32 94 ± 64 18 ± 15 98 ± 77 352 ± 88 
DayIFN-γIL-12p70TNF-αIL-4IL-5IL-10
LN       
  WT 32420 ± 2930 12 ± 9 129 ± 69 1.6 ± 1.3 148 ± 74 3238 ± 1000 
  KO 31200 ± 4880 12 ± 8 82 ± 50 2.2 ± 1.6 190 ± 140 2641 ± 698 
 14       
  WT 14200 ± 8720b 51 ± 20 148 ± 45b 9.1 ± 8.4 156 ± 71 2401 ± 971b 
  KO 4433 ± 1347 53 ± 23 40 ± 19 8.5 ± 7 207 ± 132 4915 ± 1579 
SP       
  WT 20730 ± 12600 41 ± 29 94 ± 39 47 ± 15 146 ± 90 1742 ± 565 
  KO 20240 ± 12170 29 ± 22 104 ± 15 45 ± 9 121 ± 70 1417 ± 82 
 14       
  WT 5214 ± 2005 66 ± 16 221 ± 63 50 ± 24 100 ± 71 603 ± 75 
  KO 3654 ± 2551 71 ± 32 94 ± 64 18 ± 15 98 ± 77 352 ± 88 
a

Mice were infected (intranasally) with 200 IFUs C. muriarum and euthanized at days 7 and 14 p.i. (4–5 mice/group at each time point). The mononuclear cells from draining lymph nodes and the spleen were cultured with UV-killed C. muridarum restimulation. The 72-h culture supernatants were measured for cytokine levels by ELISA. The data (in pg/ml; mean ± SD) from a representative experiment of three independent experiments with similar results are shown.

b

p < 0.05.

FIGURE 3.

Intracellular cytokine staining showed similar patterns of IFN-γ production by T cells from WT and IFNAR−/− mice. Mice were intranasally infected with C. muridarum (200 IFUs), killed at 7 days p.i. and the spleen (A) and draining lymph node (B) cells were harvested and cultured for 3 days with antigenic (UV-killed C. muridarum) restimulation. Cells were then treated with PMA, ionomycin, and Brefeldin A, followed by staining of intracellular IFN-γ and surface markers as described in Materials and Methods. The y-axis (FL4) represents intracellular IFN-γ. C, Graphic summary of intracellular IFN-γ staining patterns of T cells in the lymph nodes and spleen in each group (four mice per group). FL1: FITC-CD8; FL2: PE-CD4; FL3: PE-Cy7-CD3e; and FL4: allophycocyanin-IFN-γ.

FIGURE 3.

Intracellular cytokine staining showed similar patterns of IFN-γ production by T cells from WT and IFNAR−/− mice. Mice were intranasally infected with C. muridarum (200 IFUs), killed at 7 days p.i. and the spleen (A) and draining lymph node (B) cells were harvested and cultured for 3 days with antigenic (UV-killed C. muridarum) restimulation. Cells were then treated with PMA, ionomycin, and Brefeldin A, followed by staining of intracellular IFN-γ and surface markers as described in Materials and Methods. The y-axis (FL4) represents intracellular IFN-γ. C, Graphic summary of intracellular IFN-γ staining patterns of T cells in the lymph nodes and spleen in each group (four mice per group). FL1: FITC-CD8; FL2: PE-CD4; FL3: PE-Cy7-CD3e; and FL4: allophycocyanin-IFN-γ.

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There was no significant difference in the levels of C. muridarum-specific serum IgM, IgG1, and IgG2a between WT and IFNAR−/− mice (data not shown). Both WT and IFNAR−/− mice displayed higher titers of IgG2a than IgG1, which is in line with the cytokine patterns (higher IFN-γ production), indicating that both groups of mice showed Th1 like response following infection. Overall, the analyses of organism-driven cytokine patterns and Ab responses did not show significant difference in adaptive immune responses in the WT and IFNAR−/− mice, suggesting that IFNIs are more important in innate immune responses to C. muridarum infection.

Since DC are among the most important cells linking innate and adaptive immune responses, we assessed changes in the phenotype and cytokine production of DC after C. muridarum infection. As shown in Fig. 4, DC from IFNAR−/− mice showed significantly lower expression of MHC class II and costimulatory molecules including CD40, CD80, and CD86 on cell surface than that in WT mice following C. muridarum infection while the expression of these markers were similar in naive WT and IFNAR−/− mice, although the levels were lower than those in infected mice (data not shown). There was also a lower expansion of CD8α+ DC subpopulation in the IFNAR−/− mice (Fig. 4), whereas the percentages of CD4+ and B220+ DC subpopulation were similar between the IFNAR−/− and WT mice following C. muridarum infection (data not shown). Moreover, the DC from IFNAR−/− mice showed less TNF-α production than the WT mice. The level of IL-12p40 production in IFNAR−/− mice was significantly higher than WT, in line with the previous reports showing a strong inhibitory effect of IFNIs on IL-12 production (42, 43, 44). Taken together, the results suggest a higher degree of DC maturation and a selective expansion of the CD8α+ DC subpopulation in the WT mice following chlamydial infection.

FIGURE 4.

WT mice showed higher surface marker expression and cytokine production than IFNAR−/− mice. Seven days after intranasal infection with 1000 IFUs of C. muridarum, DCs were isolated from the spleens by MACS. A, Surface marker expression was analyzed by flow cytometry as described in Materials and Methods. B, Purified DC were cultured in 96-well plate at 5 × 105cells/well with or without (control) UV-killed C. muridarum stimulation. The 48-h culture supernatants were tested for cytokine levels by ELISA. Shown are the representative data of two independent experiments with similar results. *, p < 0.05; **, p < 0.01.

FIGURE 4.

WT mice showed higher surface marker expression and cytokine production than IFNAR−/− mice. Seven days after intranasal infection with 1000 IFUs of C. muridarum, DCs were isolated from the spleens by MACS. A, Surface marker expression was analyzed by flow cytometry as described in Materials and Methods. B, Purified DC were cultured in 96-well plate at 5 × 105cells/well with or without (control) UV-killed C. muridarum stimulation. The 48-h culture supernatants were tested for cytokine levels by ELISA. Shown are the representative data of two independent experiments with similar results. *, p < 0.05; **, p < 0.01.

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We further analyzed the cellular composition of infiltrating cells in the lung following infection. Analysis of BAL cells showed significantly higher number of macrophages in IFNAR−/− mice, whereas WT mice showed significantly higher neutrophil infiltration (Fig. 5,A). Furthermore, we examined the kinetics of macrophage infiltration in the lungs following C. muridarum infection by immunohistochemical staining analysis using a macrophage-specific Ab. As shown in Fig. 5 B, analyses at days 2, 4, and 7 p.i. showed constantly higher number of macrophages in the lungs of IFNAR−/− mice compared with WT mice starting from day 4 p.i. These results indicate the presence of significantly greater number of macrophages in the lung tissues of IFNAR−/− mice than WT controls, suggesting a higher macrophage infiltration to, and/or survival in, the lung of IFNAR−/− mice although the degree of inflammation was much heavier in WT controls.

FIGURE 5.

Higher macrophage infiltration in the lungs of IFNAR−/− mice. A, Presence of inflammatory cells in BAL. IFNAR−/− and WT mice were infected with 1000 IFUs of C. muridarum and were killed at day 4 p.i. Cells in the BAL were counted and the cell differentials were identified by staining with Fisher Leukostat stain kit. **, p < 0.01, ***, p < 0.001. B, Kinetics of resident macrophages in the infected lungs. IFNAR−/− and WT mice were infected intranasally with 1000 IFUs of C. muridarum and killed at days 2, 4, and 7. Macrophages were assessed using FITC-anti-mouse F4/80 as mentioned in Materials and Methods.

FIGURE 5.

Higher macrophage infiltration in the lungs of IFNAR−/− mice. A, Presence of inflammatory cells in BAL. IFNAR−/− and WT mice were infected with 1000 IFUs of C. muridarum and were killed at day 4 p.i. Cells in the BAL were counted and the cell differentials were identified by staining with Fisher Leukostat stain kit. **, p < 0.01, ***, p < 0.001. B, Kinetics of resident macrophages in the infected lungs. IFNAR−/− and WT mice were infected intranasally with 1000 IFUs of C. muridarum and killed at days 2, 4, and 7. Macrophages were assessed using FITC-anti-mouse F4/80 as mentioned in Materials and Methods.

Close modal

To test whether macrophages contribute to host resistance to C. muridarum infection in this model, we used CL2MDP-liposome treatment to deplete pulmonary macrophages as described (37). CL2MDP-liposome-treated IFNAR−/− mice displayed greater body weight loss (Fig. 6,A), increased organism growth (Fig. 6,B), and more severe lung inflammation (Fig. 6,C) compared with the control mice groups (PBS and PBS-liposome-treated mice). The depletion of macrophages in the lung was confirmed by H&E (Fig. 6,C) and histoimmunochemical staining (not shown). Most of the infiltrating inflammatory cells in CL2MDP-lipsome-treated mice were neutrophils following C. muridarum infection (Fig. 6 C). Notably, PBS-liposome-treated mice also showed slightly higher bacterial load and more pathological changes compared with the PBS-treated mice. This is probably due to a nonspecific suppression of macrophage function by liposome. The macrophage depletion experiments were also performed using another mouse strain, C57BL/6 mice, that are more resistant to C. muridarum infection (35, 36) and the same effect was observed (data not shown). The data suggest that local macrophages are important in host defense against C. muridarum infection.

FIGURE 6.

Depletion of lung macrophages enhanced chlamydial growth in IFNAR−/− mice. Mice (four/group) were treated intranasally with PBS, PBS-liposome, or CL2MDP-liposome, respectively, and infected intranasally with C. muridarum (200 IFUs). Body weight changes (A) were monitored daily, and pulmonary C. muridarum loads (B) and pathological changes (C) were assessed at day 7 p.i. as mentioned in Material and Methods. Shown are the representative data of two independent experiments with similar results. **, p < 0.01; ***, p < 0.001.

FIGURE 6.

Depletion of lung macrophages enhanced chlamydial growth in IFNAR−/− mice. Mice (four/group) were treated intranasally with PBS, PBS-liposome, or CL2MDP-liposome, respectively, and infected intranasally with C. muridarum (200 IFUs). Body weight changes (A) were monitored daily, and pulmonary C. muridarum loads (B) and pathological changes (C) were assessed at day 7 p.i. as mentioned in Material and Methods. Shown are the representative data of two independent experiments with similar results. **, p < 0.01; ***, p < 0.001.

Close modal

It has been suggested that IFNI-dependent apoptosis of lymphocytes and macrophages may be a mechanism contributing to the increased susceptibility to infection in the models of L. monocytogenesis (17, 18). We, therefore, performed TUNEL assay to analyze apoptosis in the lung following C. muridarum infection. As shown in Fig. 7 A, the lung tissues of WT mice contained more apoptotic inflammatory cells, as indicated by TUNEL-positive (diaminobenzidine-stained nuclei) cells, than IFNAR−/− mice at as early as day 4 p.i. However, there was no observed difference in bronchial epithelial cell apoptosis between the WT and IFNAR−/− mice (not shown).

FIGURE 7.

WT mice showed higher apoptosis of pulmonary macrophages. Lungs from WT and IFNAR−/− mice were harvested at day 4 p.i. and analyzed for apoptosis using different methods. A, The lung tissues were analyzed by TUNEL assay. Slides were photographed under light microscope and the pictures represent five mice in each group. Note, more apoptotic inflammatory cells (brown color) in WT than IFNAR−/− mice. B, Immunohistochemical analysis of active caspase 3-positive cells p.i.. lung tissues from WT and IFNAR−/− mice were costained with anti-cleaved caspase 3 and anti-F4/80 (macrophage) or anti-CD2 (lymphocyte) or pan-Cytokeratin (epithelial cell) Abs as mentioned in Materials and Methods. Green: cleaved caspase-3 and Red: F4/80, or CD2 or Cytokeratin.

FIGURE 7.

WT mice showed higher apoptosis of pulmonary macrophages. Lungs from WT and IFNAR−/− mice were harvested at day 4 p.i. and analyzed for apoptosis using different methods. A, The lung tissues were analyzed by TUNEL assay. Slides were photographed under light microscope and the pictures represent five mice in each group. Note, more apoptotic inflammatory cells (brown color) in WT than IFNAR−/− mice. B, Immunohistochemical analysis of active caspase 3-positive cells p.i.. lung tissues from WT and IFNAR−/− mice were costained with anti-cleaved caspase 3 and anti-F4/80 (macrophage) or anti-CD2 (lymphocyte) or pan-Cytokeratin (epithelial cell) Abs as mentioned in Materials and Methods. Green: cleaved caspase-3 and Red: F4/80, or CD2 or Cytokeratin.

Close modal

Using activated caspase 3-specific mAb staining, we found significantly more activated caspase 3-positive inflammatory cells in the lungs of WT mice than in IFNAR−/− mice (Fig. 7,B). Double staining using Abs to cleaved caspase 3 and macrophage (F4/80) or lymphocyte (CD2) or epithelial cell markers showed that most of the cleaved caspase 3-positive cells in the lung tissue of WT mice were F4/80-positive cells, whereas few CD2-positive cells and epithelial cells showed staining of cleaved caspase-3, confirming that macrophages were the major cell type undergone apoptosis. Further tests of the early expression of PKR, Daxx, and TRAIL, the factors related to IFNI-induced apoptosis (17), showed that lung tissue of WT mice expressed significantly higher TRAIL, Daxx, and PKR following chlamydial infection compared with IFNAR−/− mice (Fig. 8). This difference occurred as early as 2 days after infection and still existed at the time when IFNAR−/− and WT mice showed difference in bacterial loads in the lung (day 6). Collectively, these results suggest that IFNIs play a promoting role in macrophage apoptosis following C. muridarum infection.

FIGURE 8.

Higher expression of IFNIs-stimulated apoptotic factors in the lung tissues of WT mice. Lungs from WT and IFNAR−/− mice were harvested at day 4 p.i. and analyzed for IFNIs-stimulated apoptotic factors PKR, Daxx, and TRAIL using RT-PCR. Total RNA were isolated from the lung of each mouse (four mice/group) and were analyzed by RT-PCR for mRNA levels as described in Materials and Methods. The data are presented as mean ± SEM of the band density expressed as folds of that given by β-actin. *, p < 0.05 and ***, p < 0.001: comparison between WT and IFNAR−/− mice at each time point.

FIGURE 8.

Higher expression of IFNIs-stimulated apoptotic factors in the lung tissues of WT mice. Lungs from WT and IFNAR−/− mice were harvested at day 4 p.i. and analyzed for IFNIs-stimulated apoptotic factors PKR, Daxx, and TRAIL using RT-PCR. Total RNA were isolated from the lung of each mouse (four mice/group) and were analyzed by RT-PCR for mRNA levels as described in Materials and Methods. The data are presented as mean ± SEM of the band density expressed as folds of that given by β-actin. *, p < 0.05 and ***, p < 0.001: comparison between WT and IFNAR−/− mice at each time point.

Close modal

Previous reports have shown that IFNIs are beneficial to in vitro cultured host cells in resisting against chlamydial infection through enhancing IFN-γ and NO production (30). We have demonstrated in the present study that IFNIs, instead of being protective, promote C. muridarum infection in vivo. We found that, following infection, IFNAR−/− mice displayed much less body weight loss, lower organism burden, and milder pathological changes in the lung than WT mice following respiratory tract C. muridarum infection. More importantly, we found significantly more infiltration of macrophages to the lung and less apoptosis of the infiltrating macrophages in IFNAR−/− mice than WT mice. However, analysis of Th1- and Th2-related cytokine patterns in the IFNAR−/− and WT mice failed to show a clear correlation between the cytokine pattern and the degree of organism clearance and the severity of the disease, especially in the early stage of infection. The higher production of IFN-γ and TNF-α, and lower IL-10, in WT mice at the later stage (14 days) of infection may not be the result of direct effect of IFNα/β, but rather due to the uncontrolled chlamydial growth and continuous stimulation of host immune systems (Table II). Interestingly, IL-12 was the only cytokine that showed higher levels in the IFNAR−/− mice than the WT mice. DC from IFNAR−/− mice produced higher IL-12 (Fig. 4). Moreover, the IL-12 levels in the lung tissues were also higher in IFNAR−/− mice (data not shown). This is consistent with the previous finding in other model systems (18, 19, 42, 43) and confirms that IFNIs have a selective inhibitory role in IL-12 production. Although IL-12 is important in host defense against chlamydial infection (25), it is unlikely the reason for the higher resistance of IFNAR−/− mice to infection observed in the present study, because IFN-γ production in these mice was not higher than the WT mice. Moreover, although IL-12 has been shown to be able to enhance resistance to microbial infections through an IFN-γ-independent pathway in other models (28), delivery of rIL-12 in vivo failed to provide protection against chlamydial infection (X. Yang and S. Wang, unpublished observations).

One interesting finding in this study is the particularly important role of IFNIs in affecting the efficient functioning of macrophages in response to chlamydial infection. Significant differences between WT and IFNAR−/− mice in susceptibility to C. muridarum was observed starting from the early stage of infection, which indicate that the functional effect of IFNIs operate in the innate immune response phase following infection. The data showed that IFNAR−/− mice had less inflammation at both early and late stages of infection, but the amount of macrophages in the lung of IFNAR−/− mice was more than that in WT mice, which exhibited more neutrophil influx. Notably, the difference in bacterial loads between the WT and IFNAR−/− mice occurred after the time when the difference in macrophage infiltration and survival was observed in these two groups of mice, suggesting a positive correlation between macrophage infiltration and bacterial clearance. The role of macrophages in restricting C. muridarum infection was confirmed by macrophage depletion experiments using liposome encapsulated CL2MDP. This finding is consistent with a previous study that showed an important role of macrophages in host resistance to infection with C. pneumoniae, another chlamydial species, through an adoptive transfer approach (45).

What contributes to the reduction in macrophages in the lungs of WT mice after chlamydial infection? Based on our data, higher apoptosis of macrophages in the lung is likely the major reason. The higher apoptosis of lung macrophages in WT than IFNAR−/− mice was indicated by significantly increased TUNEL-positive cells in WT mice, which correlated with significantly higher expression of IFNI-dependent apoptotic genes TRAIL, PKR, and Daxx. Moreover, we found significantly more cleaved caspase 3-positive macrophages in the lungs of WT mice than in IFNAR−/− mice. Caspase 3 is a central executioner of apoptosis and inactive zymogen caspase 3 must be cleaved into activated caspase 3 (18 kDa) to induce apoptosis (46, 47). Therefore, our finding suggested that the reduction of macrophages in the lungs of WT mice may be due to a combination of reduced recruitment and increased local apoptosis after C. muridarum infection. Since the time for higher macrophage apoptosis in WT mice was observed even before the differential bacterial load being found (day 4), it is reasonable to speculate that the less recruitment and more apoptosis of macrophages contribute significantly to the higher chlamydial growth in the lung of WT mice. This point is supported by the experiment depleting macrophages using CL2MDP-lipsome (Fig. 6). It could be a question why the mice treated with PBS-liposome alone without macrophage depletion also showed more chlamydial growth in vivo than PBS alone-treated mice (Fig. 6). Although the reason is unclear, it is likely that the phagocytosis of liposome by macrophages can nonspecifically suppress, to a certain degree, the function of macrophages to kill bacteria.

It has been shown that, during respiratory tract viral or fungal infections, IFNAR−/− mice produced more Th2 like cytokines and chemokines in the lungs compared with WT mice, which correlated with enhanced eosinophil infiltration (48, 49). However, in our chlamydial infection model, we found less inflammatory cytokines IL-1α, IL-1β, and IL-6 in the lungs of IFNAR−/− mouse, which correlated with less severe inflammatory responses. We also found less chemokines, MCP-1 and MIP-2, in IFNAR−/− mice. Recent studies have demonstrated that MIP-2 is a chemokine selective for neutrophil chemotaxis and that MCP-1 also can promote neutrophil accumulation (40, 41). We and others have shown in previous studies that enhanced neutrophil infiltration, especially in the late stage of infection, contributes significantly to tissue damage and pathology in chlamydial infection (24, 38). We found in the present study that, in addition to the higher apoptosis of macrophages, the WT mice showed higher neutrophil apoptosis. Since the interplay between IL-6 and IFN-γ can enhance neutrophil infiltration and apoptosis (50), it might be the basis for the higher neutrophil apoptosis in WT mice in this model because the WT mice exhibited significantly higher IL-6 and IFN-γ in the lung starting from the early stage of infection (day4) and persisting to the late stage of infection (day 14, data not shown). In theory, the higher apoptosis of neutrophils could contribute to the loss of the protective function of macrophages in WT mice because the more neutrophils are apoptotic in the lung, the more macrophages are needed to clear the apoptotic cells, thus leading to suppressed function of macrophages for clearing bacterial infection.

Our study emphasizes the importance of in vivo experiments in elucidating the comprehensive picture of innate immune responses against infectious agents. Although IFNIs per se may have inhibitory effect on chlamydial growth within different cell types (29, 30, 31), the production of these cytokines in vivo following infection may hinder the function of innate and/or adaptive immune cells, thus resulting in a promoting rather than an inhibitory net effect on the infection. In addition, the study further emphasizes the importance of the coordination of innate and adaptive immunity against chlamydial infection. This point may be useful in explaining why the WT mice mounted high IFN-γ response but fail to control the infection. Although IFN-γ production by T cells have been shown to be critical in host defense against chlamydial infection, the WT mice showed heavier bacterial load and more serious disease even when their T cell IFN-γ response was intact, matching to the level of IFNAR−/− mice (Fig. 3, Table II). These data demonstrate that even when a “normal” T cell response exists, the host cannot defend itself well without the intactness of the innate cells, particularly macrophages. It is likely that Th1 cells are not the major effector cell in controlling chlamydial lung infection, rather they play their role through enhancing the function of macrophages. Therefore, macrophages may play their role in both early and later stages of infection, as a major part of innate immune responses and T cell-activated effector cells, respectively. Notably, the disassociation between IFN-γ production and control of chlamydial infection has been reported in some previous studies (51). In particular, we have reported that C3H mice, although producing high levels of IFN-γ following chlamydial lung infection, failed to control the infection mainly due to reduced NO production by macrophages (36). In line with this theme, a very interesting finding in the present study is the significantly higher degree of DC maturation in WT than in IFNAR−/− mice, however, the former showing more serious infection. Apparently, the results suggest IFNIs can promote DC maturation during chlamydial infection, thus, in theory, being beneficial to the host because DC maturation can promote adaptive immunity. Paradoxically, IFNIs, at the same time, promote macrophage apoptosis, leading to the loss of effector cells in the fight against the infection. In addition, the significant maturation status of DCs in the WT mice may be partially a result of the heavier and persistent bacterial loads in the WT mice, in that the unresolved infection may keep the immune system being stimulated by the continuous activation of DCs. Therefore, although type 1 IFNs could be beneficial to the host defense against chlamydial infection through many ways, their side effect in inducing macrophage apoptosis, unfortunately, lead to a net negative impact.

In summary, our study demonstrated that type 1 IFN responses are detrimental in host defense against primary respiratory tract C. muridarum infection. Our study showed a particular important role played by IFNIs in hindering macrophage function during chlamydial infection. Since type I IFNs are pleotropic in their function in vivo, more in-depth studies are needed to address these immunological responses and complex pathways.

We are grateful for the technical assistance of Dr. Edward Rector in flow cytometry.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by grants (to X.Y.) from Canadian Institutes of Health Research, Manitoba Health Research Council, and Manitoba Institute of Child Health. H.Q. and A.G.J. are trainees in the Canadian Institutes of Health Research/International Centre for Infectious Diseases National Training Program in Infectious Diseases. H.Q. was also a holder of Manitoba Health Research Council Studentship and Health Sciences Foundation of Winnipeg Studentship. X.H. and L.J. were trainees in the Canadian Institutes of Health Research National Training Program in Allergy/asthma. X.Y. is Canada Research Chair in Infection and Immunity.

3

Abbreviations used in this paper: IFNI, IFN type I; IFU, inclusion forming unit; IFNAR−/−, IFN-α/β receptor knockout; WT, wild type; KO, knockout; BAL, bronchioalveolar lavage; DC, dendritic cell; CL2MDP, dichloromethylene diphosphonate; p.i., postinfection.

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