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The Journal of Immunology, 2008, 180, 4040 -4049
Copyright © 2008 by The American Association of Immunologists, Inc.
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SOCS-1 Protects against Chlamydia pneumoniae-Induced Lethal Inflammation but Hampers Effective Bacterial Clearance1

Tangbin Yang*,{dagger}, Patrik Stark*, Katrin Janik{ddagger}, Hans Wigzell* and Martin E. Rottenberg2,*

* Department of Microbiology, Tumorbiology and Cell Biology, Karolinska Institute, Stockholm, Sweden; {dagger} Laboratory of Space Cellular and Molecular Biology, China Astronaut Research and Training Center, Beijing, China; and {ddagger} Department of Medical Microbiology, Medical School Hannover, Hanover, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Suppressor of cytokine signaling 1 (SOCS1) plays a major role in the inhibition of STAT1-mediated responses. STAT1-dependent responses are critical for resistance against infection with Chlamydia pneumoniae. We studied the regulation of expression of SOCS1 and SOCS3, and the role of SOCS1 during infection with C. pneumoniae in mice. Bone marrow-derived macrophages (BMM) and dendritic cells in vitro or lungs in vivo all showed enhanced STAT1-dependent SOCS1 mRNA accumulation after infection with C. pneumoniae. Infection-increased SOCS1 mRNA levels were dependent on IFN-{alpha}β but not on IFN-{gamma}. T or B cells were not required for SOCS1 mRNA accumulation in vivo. Infection-induced STAT1-phosphorylation occurred more rapidly in SOCS1–/– BMM. In agreement, expression of IFN-{gamma} responsive genes, but not IL-1β, IL-6, or TNF-{alpha} were relatively increased in C. pneumoniae-infected SOCS1–/– BMM. Surprisingly, C. pneumoniae infection-induced IFN-{alpha}, IFN-β, and IFN-{gamma} expression in BMM were attenuated by SOCS1. C. pneumoniae infection of RAG1–/–/SOCS1–/– mice induced a rapid lethal inflammation, accompanied by diminished pulmonary bacterial load and increased levels of iNOS and IDO but not IL-1β, IL-6, or TNF-{alpha} mRNA. In summary, C. pneumoniae infection induces a STAT1, IFN-{alpha}β-dependent and IFN-{gamma} independent SOCS1 mRNA accumulation. Presence of SOCS1 controls the infection-induced lethal inflammatory disease but impairs the bacterial control.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immune and inflammatory systems are controlled by multiple cytokines, including interleukins and IFNs. Many cytokines exert their biological function through Janus kinases and signal transducers and activators of transcription factors. Suppressor of cytokine signaling (SOCS)3 are a family of intracellular proteins, several of which are regulators of cytokine homeostasis (1). There are eight SOCS family members. Gene deletion experiments have demonstrated that SOCS proteins have highly specific functions in vivo. Upon their translation in the cytoplasm, SOCS proteins function in a negative feedback loop to inhibit cytokine signaling by binding to either JAK or the receptor, and either inhibiting JAK activity directly, or targeting the receptor complex for ubiquitination and subsequent proteasome-mediated degradation.

SOCS1 and SOCS3 are among the best described SOCS molecules. Although SOCS1 plays important regulatory roles in many different cytokine pathways, a major role for SOCS1 in the inhibition of STAT1-mediated, IFN-{alpha}, -β, and -{gamma} responses has been reported (2, 3). The vital importance of SOCS1 is stressed by the fact that SOCS1–/– mice die within 3 wk after birth with severe lymphopenia, necrosis of the liver, and mononuclear infiltration of diverse organs (4, 5). The neonatal defects exhibited by SOCS1–/– mice appear to be due to increased production of IFN-{gamma} by T and NK T cells and to uncontrolled IFN-{gamma} signaling in myeloid cells (4, 5, 6). Thus, SOCS1–/– mice show constitutive activation of IFN-{gamma}-inducible genes (4). SOCS1 is also crucial in attenuating IFN-{alpha}β signaling in vivo thus limiting host responses to viral infection (7).

Many cytokines induce expression of both SOCS1 and SOCS3 (8). Among these, IFN-{gamma} induces expression of SOCS3 in STAT1 dependent and independent ways (9). Contrary to STAT1, which activates different immune genes, STAT3 inhibits NF-{kappa}B activation (10). Of interest, STAT3 is triggered by IL-6 and IL-10. However, SOCS3 impair STAT3-mediated IL-6 but not IL-10 responses (11, 12, 13).

The obligate intracellular Gram-negative bacterium Chlamydia pneumoniae is a common cause of high and low respiratory tract diseases and has been associated with development of atherosclerosis (14). After internalization into phagosomes, chlamydia avoid phagolysosomal fusion, and replicate intracellularly. IFN-{gamma} is central in resistance to this pathogen, both in vivo and in vitro (15, 16). CD4+ and CD8+ T cells and myeloid cells are able to secrete IFN-{gamma} and such IFN-{gamma}-secreting cells are all needed for protection (17).

Macrophages infected with C. pneumoniae express IFN-{gamma}, protecting the cells against chlamydial growth (18). Such bacterial infection-induced IFN-{gamma} secretion is IL-12-independent, but requires IFN-{alpha}β and STAT1 (19, 20). A protective role for IFN-{alpha} and IFN-β in protection against C. pneumoniae is also evident during infection in vivo (21).

SOCS1 has been shown to be induced during infection with different mycobacterial species and Borrelia burgdorferi (22, 23, 24). Moreover, in macrophages, SOCS-1 is induced by TLR ligands such as LPS and CpG-DNA (25, 26) and may cause hyporesponsiveness of these cells to cytokines such as IFN-{gamma} after exposure to TLR ligands. More interestingly, absence of SOCS-1 results in hypersensitiveness to LPS shock (27, 28). Although experiments using bacterial TLR ligands suggest the importance of SOCS1 in the immune control of infection, the role of SOCS1 in the outcome of bacterial infections has not been studied.

We tested the hypothesis that SOCS1 may have two different roles: it impairs effective clearance of bacteria but protects the host against dangerous infection-induced inflammatory responses. Because SOCS1 is a main controller of STAT1-mediated responses, we studied the regulation and role of SOCS1 during the infection with C. pneumoniae in vivo and in vitro in bone marrow-derived macrophages (BMM) and bone marrow-derived dendritic cells (BMDC).

We found that C. pneumoniae induced the expression of both SOCS1 and SOCS3 in vivo and in vitro in BMM and BMDC. Both IFN-{alpha} and IFN-{gamma} were able to induce STAT1 dependent, SOCS1, and SOCS3 expression in BMM and BMDC. However, SOCS1 expression during in vitro and in vivo infection with C. pneumoniae required STAT1 and IFN-{alpha}β signaling but did not need IFN-{gamma}.

SOCS1 regulated expression of different IFN-induced genes including IFN-{gamma}, IFN-β, and IFN-{alpha} themselves, but did not affect expression of SOCS3. Importantly, SOCS1 impaired an infection-induced lethal inflammatory process, but at the same time hampered chlamydial control.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Mutant mouse strains with genomic deficiency in STAT1 (29), IFN-{gamma}R (30), IFN-{alpha}βR (31), and RAG1 (32) were generated by homologous recombination in embryonic stem cells. Animals were bred and kept under specific pathogen-free conditions. Mice of the C57BL/6 background were used as controls for all mice except for IFN-{alpha}βR, which were backcrossed into a sv129 genotype.

RAG1–/–/SOCS1–/– mice were obtained by crossing RAG1–/– and SOCS1–/+ mice. The RAG1–/–/SOCS1–/+ progeny was intercrossed and F2 mice were screened for homozygosity of the disrupted SOCS1 gene by PCR analysis of tail DNA lysates. The presence of the introduced neomycin construct and the absence of the wild type gene in both alleles could be detected by amplification with the following primers: Sense SOCS1, 5'-TCTGGAAGGGTTCCGCATACAGGAACG-3; Antisense SOCS1, 5'-ATCGCATTGTCGGCTGCCAC-3'; Sense SOCS1 neomycin, 5'-ATCGCCTTCTATCGCCTTCTTGACGAG-3'; Antisense SOCS1 neomycin, 5'-CAGCCGGTCAGATCTGGAAG-3'.

RAG1–/–/SOCS1–/– mice were healthy and most of them survived for >4 mo after birth, although displaying lower weight (80%) than RAG1–/–/SOCS1+/+ littermates, which were used as controls. However, RAG1–/–/SOCS1–/– mice were sterile and RAG1–/–/SOCS1–/+ mice were used to generate the animals used in this study. RAG1–/–/IFN-{gamma}–/– mice used have been previously described (33).

Generation of mouse BMM

Mouse BMM were obtained from 6- to 10-wk-old mice as described. Mice were euthanized and the femur and tibia of the hind legs were dissected. Bone marrow cavities were flushed with 5 ml cold, sterile PBS. The bone marrow cells were washed and resuspended in DMEM containing glucose and supplemented with 2 mM L-glutamine, 10% FCS, 10 mM HEPES, 100 µg/ml streptomycin, 100 U/ml penicillin (all from Sigma-Aldrich), and 20 to 30% L929 cell-conditioned medium (as a source of macrophage-CSF). Bone marrow cells were passed through a 100-µm cell strainer, plated in 6-well plates (1.2 x 107 cells per well) and incubated for 7 days at 37°C, 5% CO2. Before use, BMM cultures were washed vigorously to remove nonadherent cells. Cells from several wells were also harvested and counted by trypan blue exclusion. Typically, bone marrow cells yielded 2–3 x 106 BMM per well after 7 days in culture. We have previously shown by immunofluorescence staining that these BMM are F4/80+, CD14+, and Mac-3+ (18).

Generation of mouse BMDC

Mouse BMDC were differentiated as previously described (27). In brief, bone marrow was extracted from tibia and femurs and cell suspensions cultured in IMDM (Cambrex) containing 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1 ng/ml GM-CSF (PeproTech). Fresh medium and cytokine were replaced 3 days afterward. After 6 days of culture, loosely adherent cells were harvested and infected with C. pneumoniae as described for BMM.

Infection and infectivity assay

Mycoplasma-free C. pneumoniae isolate Kajaani 6 (34) was propagated in HEp-2 cells. Bacteria were stored in small aliquots in sucrose-phosphate-glutamate solution at –70°C until further use. The infectivity as measured by inclusion forming units (IFU) of bacterial preparation was determined in HEp-2 cells as described below.

BMM were infected with C. pneumoniae by centrifugation for 1 h, 500 x g at 35°C. A multiplicity of infection of one was used. At different time points after infection, cells were washed with PBS and then lysed in sucrose-phosphate-glutamate solution buffer. Assessment of inclusion-forming units in cell lysates was done in HEp-2 cells. Aliquots of cell lysates diluted 10- to 200-fold were used in duplicate to infect overnight cultures of confluent HEp-2 cells. The latter were grown in DMEM containing glucose and supplemented with 2 mM L-glutamine, 5% FCS, 10 mM HEPES, and 25 µg per ml streptomycin (DMEM/Strep) on round 13 mm2 glass cover slides in 24-well plates. Inoculated cells were centrifuged for 1 h, 500 x g at 35°C. Thereafter, supernatant was removed and DMEM/Strep containing 0.5 µg per ml cycloheximide (Sigma-Aldrich) was added. Cells were incubated at 35°C for 72 h, 5% CO2, thereafter washed gently with PBS and fixed in methanol. Glass cover slides were then stained for 30 min at room temperature with a FITC-conjugated Chlamydia genus-specific mAb (1/5 dilution; Pathfinder Chlamydia Confirmation System, Bio-Rad). Cover slides were mounted with fluorescent mounting medium (DakoCytomation) and IFU of C. pneumoniae were quantified by fluorescence microscopy. The infectivity was expressed as IFU of C. pneumoniae per well.

Generation of radiation bone marrow chimeras

Bone marrow cells from uninfected STAT1–/– and wild type (WT) mice were harvested from the tibia and femur by flushing with cold PBS through the bone marrow cavities and RBC were lysed by hypotonic shock. To create bone marrow chimeras, WT and STAT1–/– mice were irradiated with 900 cGy and 4 h later inoculated in the tail vein with 2 x 107 bone marrow cells from WT or STAT1–/– mice. Six weeks after reconstitution, mice were infected with C. pneumoniae. Mice were sacrificed 21 days after infection. Spleen cells from chimeric and control mice were obtained and the presence of total STAT1 was determined by Western blotting.

Real time PCR

Cytokine and hypoxanthine-guanine phosphoribosyltransferase (HPRT) transcripts in BMM at different time points after C. pneumoniae infection were quantified by real time PCR. Total RNA was transcribed to cDNA. The real time PCR was performed in duplicate 25 µl reactions containing Platinum SYBR Green qPCR Supermix-UDG (Invitrogen Life Technologies), 150 nM forward and reverse primers, and 0.5 µl of cDNA on an ABI Prism 7500 sequence detection system (Applied Biosystems). The following primer sequences were used: Sense SOCS1, 5' GCT GTG CCG CAG CAT TAA G 3'; Antisense SOCS1, 5' CCA GAA GTG GGA GGC ATC TC 3'; Sense SOCS3, 5' TTC CCA TGC CGC TCA CA 3'; Antisense SOCS3, 5' CCC ACC CAG CCC CAT AC 3'; Sense IFN-{gamma}, 5' GCT TTG CAG CTC TTC CTC AT 3'; Antisense IFN-{gamma}, 5' CAC ATC TAT GCC ACT TGA GTT AAA ATA GT 3'; Sense IFN-β, 5' CTG GAG CAG CTG AAT GGA AAG 3'; Antisense IFN-β, 5'TCC GTC ATC TCC ATA GGG ATCT 3'; Sense IFN-{alpha}4, 5'TCT GAT GCA GCA GGT GGG 3'; Antisense IFN-{alpha}4, 5'AGG GCT CTC CAG AYT TCT GCT CTG 3'; Sense IL-1β, 5' TGG TGT GTG ACG TTC CCA TT 3'; Antisense IL-1β, 5' CAG CAC GAG GCT TTT TTG TTG 3'; Sense IL-6, 5' ACA AGT CGG AGG CTT AAT TAC ACA T 3'; Antisense IL-6, 5' TTG CCA TTG CAC AAC TCT TTT C 3'; Sense TNF-{alpha}, 5' GGC TGC CCC GAC TAC GT 3'; Antisense TNF-{alpha}, 5'GAC TTT CTC CTG GTA TGA GAT AGC AAA 3'; Sense iNOS, 5' CAG CTG GGC TGT ACA AAC CTT 3'; Antisense iNOS, 5' CAT TGG AAG TGA AGC GTT TCG 3'; Sense IDO, 5' GAG AAA GCC AAG GAA ATT TTT AAG AG 3'; Antisense IDO, 5' TAT GCG GAG AAC GTG GAA AAA C 3'; Sense LRG47, 5' CTG GCA ATG GCA TGT CAT CT 3'; Antisense LRG47, 5' AGC CGA GGC ATC TTC ATC AT 3'; Sense Mig, 5' CTT TTC CTC TTG GGC ATC AT 3'; Antisense Mig, 5' GCA TCG TGC ATT CCT TAT CA 3'; Sense IP-10, 5' GCT GCC GTC ATT TTC TGC 3'; Antisense IP-10, 5' TCT CAC TGG CCC GTC ATC 3'; Sense HPRT, 5' CCC AGC GTC GTG ATT AGC 3'; Antisense HPRT, 5' GGA ATA AAC ACT TTT TCC AAA TCC 3'.

Serial-fold dilutions of a cDNA sample were amplified to control amplification efficiency for each primer pair. Thereafter, the Ct values for all cDNA samples were obtained. HPRT was used as a control gene to calculate the {Delta}Ct values for individual samples. The relative amount of cytokine/HPRT transcripts was calculated using the 2 –{Delta}{Delta}Ct) method as described. These values were then used to calculate the relative expression of cytokine mRNA in uninfected and infected BMM.

Western blotting

Single-cell suspensions from control and C. pneumoniae-infected mice were lysed and separated on 10% separating/5% stacking SDS-polyacrylamide gels as described (15). Samples were then transferred onto nitrocellulose membranes (BioRad) by electroblotting at 100 V, 250 mA for 1 h. Immunostaining was performed using polyclonal rabbit anti-phosphorylated (Tyr 701) STAT1, total STAT1, phosphorylated I{kappa}B (1/1000 dilution; all from Cell Signaling Technology) or anti-actin (1/500 dilution; Sigma-Aldrich). Membranes were then washed and incubated with horseradish peroxidase (HRP)-conjugated polyclonal goat anti-rabbit Ig (1/2000 dilution; DakoCytomation) and developed using ECL-Plus (Amersham Pharmacia Biotech) and photographed using a Fuji intelligent dark box II digital camera.

Nitrite assay

Nitrite concentrations were measured in BMM culture supernatants using the Griess reagent in a previously described colorimetric assay (34). Aliquots (100 µl) of culture medium were mixed in 96-well plates with an equal volume of 0.5% sulfanilamide dihydrochloride and 0.05% naphthylethylenediamide dihydrochloride in 2.5% phosphoric acid and the absorbance (at 540 nm) was determined. Sodium nitrite, dissolved in DMEM, was used to generate a standard concentration curve. The lower limit of detection of the assay was 1 µM NO2.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Expression of SOCS1 and SOCS3 mRNA during infection with C. pneumoniae of BMM and BMDC

We first studied whether expression of SOCS1 and SOCS3 mRNA is increased in C. pneumoniae in vitro infected BMM and BMDC. Infection with C. pneumoniae of BMM or BMDC induced both SOCS1 and SOCS3 mRNA expressions (Fig. 1, A–D).


Figure 1
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  		FIGURE 1. C. pneumoniae infection of BMM and BMDC induces a STAT-1 dependent, IFN-{gamma}-independent accumulation of SOCS1 and SOCS3 mRNA. Total RNA was extracted from WT, STAT1–/– (A–D), or IFN-{gamma}R–/– (E–H) BMM (A, B, E, and F) or BMDC (C, D, G, and H) at the indicated time points after infection with C. pneumoniae. The accumulation of SOCS1 (A, C, E, and G), SOCS3 (B, D, F, and H), and HPRT mRNA were measured by real time PCR. Comparable results were obtained in two separate experiments. The mean fold induction ± SEM of triplicate cultures is depicted. *, Differences with STAT1–/– or IFN-{gamma}R–/– cells are significant (p < 0.05 Student t test).

 
Expression of SOCS1 and SOCS3 mRNA was dependent on STAT1 because no enhanced SOCS1 or SOCS3 mRNA levels were detected in STAT1–/– BMM or BMDC. Because C. pneumoniae infection of BMM induces the expression of IFN-{gamma}, we tested whether IFN-{gamma} is mandatory for SOCS1 and SOCS3 expression. IFN-{gamma} was found nonessential for SOCS1 and SOCS3 expression because increased levels of SOCS1 and SOCS3 mRNA were detected in infected IFN-{gamma}–/– BMM or BMDC (Fig. 1, E–H). Moreover, SOCS1 and SOCS3 mRNA levels in IFN-{gamma}–/– BMM and BMDC were higher than in the WT controls. The mechanisms behind IFN-{gamma}-mediated suppression of SOCS1 expression remain to be studied. Given that STAT1 activation also controls IFN-{alpha}β signaling, the role of IFN-{alpha}β in C. pneumoniae-infection-induced SOCS1 and SOCS3 mRNA expression was studied. IFN-{alpha}β signaling plays a relevant role in SOCS1 mRNA expression because SOCS1 mRNA expression was diminished in C. pneumoniae-infected IFN-{alpha}βR–/– BMM and BMDC compared with controls (Fig. 2, A–D).


Figure 2
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  		FIGURE 2. C. pneumoniae infection of BMM and BMDC induces an IFN-{alpha}β dependent SOCS1 accumulation. Total RNA was extracted from WT, IFN-{alpha}βR–/– BMM (A and B) or BMDC (C and D) at the indicated time points after infection with C. pneumoniae. The accumulation of SOCS1 (A and C) SOCS3 (B and D) and HPRT mRNA were measured by real time PCR. Comparable results were obtained in two separate experiments. The mean fold induction ± SEM of triplicate cultures is depicted. *, Differences with IFN-{alpha}β R–/– cells are significant (p < 0.05 Student’s t test).

 
Because IFN-{gamma} is not required for SOCS1 expression during C. pneumoniae infection, we then tested whether IFN-{gamma} induces SOCS1 mRNA expression in BMM and BMDC. Coincubation of BMM or BMDC with IFN-{gamma} dramatically increased SOCS1 mRNA levels. The IFN-{gamma}-induced SOCS1 mRNA expression is STAT1-dependent because no increase in SOCS1 levels was detected in STAT1–/– BMM or BMDC (Fig. 3, A–B). Incubation of BMM or BMDC with IFN-{alpha} did also induce STAT1-dependent SOCS1 mRNA expression (Fig. 3, C–D). SOCS3 mRNA was also induced by stimulation of BMDC or BMM with IFN-{gamma} or IFN-{alpha} (data not shown).


Figure 3
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  		FIGURE 3. IFN-{alpha} or IFN-{gamma} induce a STAT1-dependent expression of SOCS1 and SOCS3 mRNA in BMM and BMM. Total RNA was extracted from WT, STAT1–/–, or IFN-{gamma}R–/– BMM or BMDC at the indicated time points after stimulation with IFN-{gamma} or IFN-{alpha}. The accumulation of SOCS1 (B) and SOCS3 (C) mRNA were measured by real time PCR. The mean fold induction of SOCS1 or SOCS3 ± SEM of triplicate cultures is depicted. *, Differences with STAT1–/– cells are significant (p < 0.05 Student’s t test).

 
Thus, infection of BMM or BMDC with C. pneumoniae induces the expression of SOCS1 and SOCS3 genes in a STAT1-dependent manner. Infection-induced expression of SOCS1 is IFN-{alpha}β dependent.

Expression of SOCS1 and SOCS3 mRNA levels in lungs from infected mice

We next studied whether SOCS1 mRNA levels increased in lungs of WT mice after intranasal infection with 106 C. pneumoniae. High levels of SOCS1 mRNA were noted in lungs at days 7 and 14 after infection (Fig. 4A), coinciding with high bacterial load and IFN-{gamma} mRNA expression (data not shown) (33). Levels of SOCS1 mRNA were normal at later time points after infection.


Figure 4
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  		FIGURE 4. In vivo infection with C. pneumoniae induces a STAT1-dependent expression of SOCS1 mRNA. Total RNA was extracted from the lungs of WT mice at the indicated time points after infection with C. pneumoniae. The accumulation of SOCS1 mRNA in individual mice (five mice per time point) was measured by real time RT-PCR. Comparable results were obtained in two separate experiments. *, Differences with the noninfected group are significant (p < 0.05 Student’s t test). Total RNA was obtained from individual lungs from STAT1–/– and WT mice (n = 5 per group) 7 days after infection with C. pneumoniae and SOCS1 mRNA were quantified by real time PCR (B). *, Differences with STAT1–/– group are significant (p < 0.05 Student t test). Two x 107 STAT1–/– or WT bone marrow cells were inoculated i.v. into irradiated STAT1–/– or WT mice. Six weeks after cell inoculation mice were infected i.n. with 106 C. pneumoniae. Mice were sacrificed 10 days after infection and the levels of SOCS1 and HPRT mRNA measured (C). The mean fold increase in SOCS1 mRNA ± SEM in each group (n = 5 per group) is depicted.

 
Next, we analyzed whether SOCS1 mRNA expression after infection with C. pneumoniae is dependent of STAT1. Lungs from STAT1–/– mice 7 days after infection with C. pneumoniae indeed showed impaired accumulation of SOCS1 mRNA, indicating that C. pneumoniae-induced SOCS1 mRNA accumulation is STAT1-dependent (Fig. 4B).

We then explored whether the STAT1-dependent expression of SOCS1 is mediated by hemopoietic and/or nonhemopoietic cells. Reciprocal bone marrow radiation chimeras between WT and STAT1–/– mice were generated by inoculation of bone marrow cells into irradiated recipients. STAT1 was detected by Western blotting in spleens from WT (donor)-> STAT1–/– (recipient) mice 8 wk after bone marrow cell transfer confirming repopulation by inoculated stem cells (data not shown). Hemopoietic cells account for STAT1-dependent increased accumulation of SOCS1 mRNA, because WT-> WT chimeras and WT-> STAT1–/– controls showed similar increased levels of SOCS1 mRNA, whereas no induction was detected in STAT1–/– ->STAT1–/– or STAT1–/– ->WT mice (Fig. 4C).

A possible need of T or B cells for SOCS1 mRNA expression in lungs from C. pneumoniae-infected mice was next analyzed. However, similar levels of SOCS1 and SOCS3 mRNA levels were detected in WT and RAG1–/– mice early after infection (Fig. 5, A and B). Lungs from RAG1–/– but not WT mice showed increased levels of these transcripts at late time points after infection (Fig. 5, A and B). Thus, T or B cells are not required for infection-induced increased accumulation of SOCS1 and SOCS3 mRNA.


Figure 5
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  		FIGURE 5. Role of IFN-{alpha}β, IFN-{gamma}, T and B cells in the accumulation of SOCS1 and SOCS3 mRNA during C. pneumoniae infection in vivo. The levels of SOCS1 (A, C, and E), SOCS3 (B, D, and F) and HPRT mRNA were measured in RNA extracted from the lungs of WT (A, B, E, and F), RAG1–/– (A and B), RAG1–/–/IFN-{gamma}–/– (C and D) and IFN-{alpha}βR–/– (E and F) individual mice at the indicated days after intranasal infection with 106 C. pneumoniae. The mean fold increase of SOCS1 and SOCS3 in relation to HPRT of four to five animals per group ± SEM are depicted. Differences with RAG1–/– (A and B), RAG1–/–/IFN-{gamma}–/– (C and D), and IFN-{alpha}βR–/– (E and F) are significant (p < 0.05 Student’s t test).

 
Next, we explored whether IFN-{gamma} signaling will induce expression of SOCS1 and SOCS3 mRNA during in vivo infection with C. pneumoniae. Lower levels of SOCS1 and SOCS3 mRNA in lungs of infected RAG1–/–/IFN-{gamma}–/– were detected early after infection with C. pneumoniae compared with RAG1–/–/IFN-{gamma}+/+ controls. However, later after infection, lungs from RAG1–/–/IFN-{gamma}–/– and control mice showed similar SOCS1 and SOCS3 mRNA titers (Fig. 5, C and D). Thus, and similar to the results obtained in vitro, IFN-{gamma} is not essential for chlamydial infection-induced SOCS1 and SOCS3 mRNA accumulation.

In agreement with results obtained in vitro, IFN-{alpha}β was required for increased expression of SOCS1and SOCS3 mRNA in lungs of C. pneumoniae-infected animals (Fig. 5, E and F).

Thus, infection of mice with C. pneumoniae induces the pulmonary accumulation of SOCS1 mRNA. Hemopoietic cells accounted for the T and B cell independent, infection-induced SOCS1 expression in vivo. IFN-{alpha}β and STAT1 but not IFN-{gamma} were required for SOCS1 expression.

Role of SOCS1 in the control of infection with C. pneumoniae in BMM

The role of SOCS1 in the outcome of infection of BMM with C. pneumoniae was then studied. SOCS1–/– BMM were generated from RAG1–/–SOCS1–/– mice whereas cells from RAG1–/–/SOCS1+/+ mice were used to generate control macrophages. SOCS1–/– BMM showed faster STAT1 phosphorylation than control BMM after infection with C. pneumoniae (Fig. 6A), while an increase in total STAT1 levels was noted in mutant and control BMM 24 and 48 h after infection in agreement with previous reports (21). The expression of IFN-{gamma}-dependent genes involved in bactericidal or bacteriostatic mechanisms such as IDO, iNOS, and LRG47 was also increased in C. pneumoniae-infected BMM SOCS1–/– mice compared with controls (Fig. 6, B–D). The IFN-{alpha}β- and IFN-{gamma}- regulated chemokines CXCL9/monokine induced by IFN-{gamma} (Mig) and CXCL10/IFN-{gamma} -inducible protein 10 (IP-10) also showed higher expression in SOCS1–/– compared with SOCS1+/+ infected BMM (Fig. 6, E and F). SOCS1–/– BMM released higher levels of NO than control cells after infection with C. pneumoniae (Fig. 6J). In contrast, similar levels of IL1, IL6, or TNF-{alpha} were found in SOCS1–/– and control BMM after infection (Fig. 6, G–I). In agreement, mutant and control BMM showed similar kinetics of I{kappa}B phosphorylation after infection with C. pneumoniae (Fig. 6K).


Figure 6
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  		FIGURE 6. Role of SOCS1 in the outcome of infection of BMM with C. pneumoniae. SOCS1–/– and SOCS1+/+ BMM were infected with C. pneumoniae. Protein extracts were prepared from samples at different time points. Samples were separated by SDS-PAGE, electroblotted onto nitrocellulose membranes and immunoblotted with Abs that specifically recognize phosphorylated STAT1 (A), total STAT1 (A), I{kappa}B (K) and actin (A, K). Abs were detected with HRP-conjugated anti-IgG followed by ECL detection. Total RNA was extracted from SOCS1–/– and SOCS1+/+ BMM and BMDC at the indicated time points after infection with C. pneumoniae. The accumulation of iNOS (B), IDO (C), LRG47 (D), IP-10 (E), Mig (F), IL-1β (G), IL-6 (H), and TNF-{alpha} (I) and HPRT mRNA measured by real time PCR. The mean fold accumulation of the transcripts of duplicate cultures per time point in relation to HPRT ± SEM is depicted. The level of NO2–/– ± SEM in the superntant from triplicate cultures of SOCS1–/– and SOCS1+/+ BMM at the indicated time points after infection with C. pneumoniae is depicted (J). *, Differences with SOCS1–/– BMM are significant (p < 0.05 Student’s t test).

 
Because secretion of IFN-{gamma} by C. pneumoniae-infected BMM is STAT1-dependent (21), we studied whether SOCS1 regulates IFN-{gamma} expression in infected BMM. C. pneumoniae-infected SOCS1–/– BMM produced higher titers of IFN-{gamma} than controls (Fig. 7B). Moreover, C. pneumoniae-infected SOCS1–/– BMM showed higher levels of IFN-β and IFN-{alpha}4 mRNA than control cells (Fig. 7, C and D).


Figure 7
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  		FIGURE 7. Role of SOCS1 in the expression of C. pneumoniae-induced IFN-{gamma}, IFN-{alpha}, and IFN-β. Total RNA was extracted from SOCS1–/– and SOCS1+/+ BMM at the indicated time points after infection with C. pneumoniae. The relative accumulation of SOCS3 (A), IFN-{gamma} (B), IFN-β (C), IFN-{alpha}4 (D), and HPRT mRNA was measured by real time PCR. The mean fold increase of the transcript concentration in relation to HPRT is depicted. Triplicate wells of SOCS1–/– and SOCS1+/+ BMM were infected with C. pneumoniae and lysed with SPG buffer at the indicated time points after infection. The number of C. pneumoniae IFU/well ± SEM was quantified by infecting Hep-2 cells with 50 µl of the BMM lysate. A representative from two independent experiments is shown (E). *, Differences with SOCS1–/– BMM are significant (p < 0.05 Student’s t test).

 
We also analyzed whether SOCS1 regulates expression of SOCS3 in C. pneumoniae-infected BMM. Similar levels of SOCS3 were found in infected SOCS1–/– and WT BMM (Fig. 7A). Finally, the role of SOCS1 in the growth control of C. pneumoniae in BMM was investigated. C. pneumoniae levels in SOCS1–/– BMM were lower than that of controls (Fig. 7E).

Thus, SOCS1 regulates the infection-induced BMM expression of IFN-{alpha}, -β and –{gamma} and of different IFN-responsive genes and plays a relevant role in the control of bacterial growth in BMM.

Role of SOCS1 in the outcome of infection with C. pneumoniae in vivo

All RAG1–/–/SOCS1–/– mice died or were moribund seven days after intranasal infection with 106 C. pneumoniae, whereas RAG1–/– survived for >60 days after infection (26). Whereas lungs from RAG1–/– infected mice demonstrated an almost completely normal or slightly affected lung architecture (Fig. 8A), those from SOCS1–/– infected mice showed large areas of lung consolidation, large mononuclear infiltrates and bronchi often filled with inflammatory exudates containing polymorphs (Fig. 8B). However, SOCS1–/– mice showed 10-fold lower bacterial levels than controls at 6 days after infection, whereas similar bacterial levels were noted in lungs 3 days after infection (Fig. 8C). Levels of iNOS and IDO but not LRG47 mRNA were increased in lungs from RAG1–/–/SOCS1–/– infected mice (Fig. 9, A–C). The levels of Mig and IP-10 were also higher in lungs of RAG1–/–/SOCS1–/– compared with RAG1–/–/SOCS1+/+ infected mice (Fig. 9, D and E). On the contrary, similar levels of IL-1β, IL-6, and TNF-{alpha} mRNA were found in lungs from RAG1–/–/SOCS1–/– mice infected with C. pneumoniae (Fig. 9, F–H). Also, similar levels of SOCS3 mRNA were detected in C. pneumoniae-infected RAG1–/–/SOCS1–/– and RAG1–/– mice (Fig. 9I). Different to the finding in BMM, comparable levels of IFN-{gamma} mRNA were detected in RAG1–/–/SOCS1–/– and RAG1–/– infected animals at 5 days after infection (data not shown).


Figure 8
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  		FIGURE 8. Role of SOCS1 in the outcome of infection with C. pneumoniae in vivo. Hematoxylin-eosin staining of paraffin lung sections from RAG1–/–/SOCS1–/– and RAG1–/–/SOCS1+/+ mice infected intranasally with C. pneumoniae 6 days before sacrifice. Original magnification, x200. Note presence of perivascular and peribronchiolar prominant infiltration of inflammatory mononuclear and polymorphonuclear cells in lungs from C. pneumoniae infected RAG1–/–/SOCS1–/– but not RAG-1–/–/SOCS1+/+ mice which showed a mild inflammatory process. Abbreviations: EL, epithelial lining, B, bronchiole, SM, smooth muscle, AS, alveolar sack, PMN, polymorphonuclear cells, MNC, mononuclear cells, RAG1–/–/SOCS1–/– and RAG1–/–/SOCS1+/+ mice were inoculated i.n. with 106 IFU C. pneumoniae. At the indicated time points after infection, six animals per group and time point were sacrificed and lung levels of C. pneumoniae assessed. The mean log10 IFU per lung is depicted. Six mice per group and time point were used. Data are pooled from two experiments in which mice were infected with aliquots of the same bacterial stock. *, Differences with RAG1–/–SOCS1–/– mice are significant (p < 0.05 Student’s t test).

 

Figure 9
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  		FIGURE 9. Role of SOCS1 in regulation of cytokine expression during in vivo infection with C. pneumoniae. Total RNA was extracted from lungs of individual RAG1–/–/SOCS1–/– and RAG1–/–/SOCS1+/+ mice (five individuals per group) 6 days after infection with C. pneumoniae. The accumulation of iNOS (A), IDO (B), LRG47 (C), Mig (D), IP-10 (E), IL-1β (F), IL-6 (G), TNF-{alpha} (H), SOCS3 (I), and HPRT mRNA measured by real time PCR. The mean fold accumulation of the transcripts in relation to HPRT is depicted. *, Differences with RAG1–/–SOCS1–/– mice are significant (p < 0.05 Student’s t test).

 
Thus, SOCS1 protects the host from a lethal inflammatory process but diminishes the efficiency of protective innate-immune mechanims. SOCS1 deficient C. pneumoniae-infected mice have increased expression of IFN-responsive genes controlling microbicidal mechanisms and chemotaxis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We demonstrate that in vitro and in vivo infection with C. pneumoniae induces SOCS1 and SOCS3 mRNA accumulation. SOCS1 hampers a lethal inflammatory response in vivo but diminishes the efficiency of protective innate immune responses.

SOCS-1 is induced by cytokines via the activation of STAT1 and downstream transcription factors such as IRF-1 (35). In addition, SOCS-1 can be induced independently of JAK-STAT pathways, as molecules that do not primarily use JAKs and/or STATs, such as stem cell factor, TGF-β, insulin, and LPS all induce SOCS1 expression (27). However, C. pneumoniae-induced SOCS1 expression in BMM and BMDC was STAT1-dependent (Fig. 1, A and C).

As expected, addition of IFN-{gamma} or IFN-{alpha} dramatically increased expression of SOCS1 mRNA levels in BMDC and BMM in a STAT1-dependent manner. However, IFN-{gamma} controlled SOCS1 mRNA levels early but not late after in vivo infection with C. pneumoniae (Fig. 5C). Moreover, SOCS1 mRNA accumulation in BMM and BMDC was independent of IFN-{gamma} signaling (Fig. 1, E and G). In vitro and in vivo infection with C. pneumoniae induced an IFN-{alpha}β-dependent SOCS1 mRNA accumulation (Fig. 2, A and C and Fig. 5E). Infection of BMM with C. pneumoniae has been shown to induce expression of IFN-{alpha}β which will trigger STAT1-dependent IFN-{gamma} expression (18, 20, 21, 36). Such a pathway could account for the redundancy of IFN-{gamma} and the requirement of IFN-{alpha}β for SOCS1 mRNA expression during infection. The TLR-MyD88 signaling has been shown to be required for IFN-{alpha}β expression in Chlamydia infected cells, and is thus probably involved in infection-induced SOCS1 expression (19, 20, 36).

Although SOCS1 expression in nonhemopoietic cells such as neurons and fibroblasts has been reported (35, 37), SOCS1 deficiency in the hemopoietic compartment is believed to be sufficient to cause a SOCS1–/– disease, as transfer of SOCS1–/– bone marrow into irradiated JAK3-deficient recipients resulted in premature lethality (5). In accordance, SOCS1 expression in C. pneumoniae infected mice was dependent on presence of STAT1 in hemopoietic cells (Fig. 4C).

STAT1 was also required for expression of SOCS3 mRNA accumulation during infection of BMM and BMDC with C. pneumoniae (Fig. 1, B and D). Previous reports have shown that STAT1 is primarily responsible for the induction of SOCS-3 in fibroblasts and macrophages in response to IFN-{gamma} (9, 38). Infection of macrophages with Listeria monocytogenes and Lesihmania donovani has also been shown to activate SOCS3 (39, 40).

We here demonstrated that SOCS1 controls BMM responses to infection with C. pneumoniae. SOCS1–/–-deficient BMM showed higher levels of phosphorylated STAT1 and increased levels of IFN-{gamma} regulated molecules such as inducible NO synthase, IDO, LRG47, Mig, and IP-10 while no effect on the levels of proinflammatory cytokines was observed (Fig. 6). In line with our results, SOCS1–/– macrophages, fibrobasts or dendritic cells produced COX-2, iNOS, IDO, and other IFN-inducible genes more extensively than control cells (4, 41, 42). The RAW macrophage cell line overexpressing SOCS-1 produced little NO in response to IFN-{gamma} (27). Similarly, SOCS1–/– BMM killed intracellular Leishmania major following stimulation with a concentration of IFN-{gamma} two orders of magnitude lower than required by WT cells (4).

Surprisingly, SOCS1 controlled not only expression of IFN-{gamma}-regulated genes but also IFN-{gamma} levels in C. pneumoniae-infected BMM (Fig. 7). The requirement of IFN-{alpha}β-mediated STAT1 activation for IFN-{gamma} expression during infection of BMM with C. pneumoniae probably explains this result (21). Moreover, levels of IFN-β and IFN-{alpha}4 were inhibited by SOCS1 late after infection of BMM (Fig. 7). No differences in levels of IFN-β and IFN-{alpha}4 were observed early after C. pneumoniae infection, suggesting that at later time points SOCS1 regulated IFN-{alpha}β expression is the result of the ability of early-produced IFN-{alpha}β to enhance its own production in a STAT1-dependent manner (43). Together, our data indicate that during macrophage infection with C. pneumoniae SOCS1 expression is induced in a IFN-{alpha}β-dependent manner and is in turn, a main regulator of secretion of and of cellular responses to IFN-{alpha}β and IFN-{gamma} (7, 44) (Fig. 10).


Figure 10
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  		FIGURE 10. Regulation of expression and role SOCS1 expression in BMM infected with C. pneumoniae. During macrophage infection with C. pneumoniae SOCS1 and IFN-{gamma} expression are induced in a STAT1 and IFN-{alpha}β-dependent manner (A). SOCS1 in turn will control the expression of and the cellular responses to IFN-{alpha}β and IFN-{gamma}, inhibiting an effective clearance of C. pneumoniae (B).

 
We found that T and B cells are not needed for SOCS1 expression during infection with C. pneumoniae (Fig. 5A). Moreover, SOCS1 regulates innate immune control of C. pneumoniae, because RAG1–/–/SOCS1–/– mice showed lower pulmonary bacterial load than RAG1–/– controls (Fig. 8). The decreased bacterial load is associated to a higher level of IFN-induced transcripts coding for molecules involved in bacteriocidal mechanisms (Fig. 9). Although these genes have been previously shown to have a role in control of chlamydia (45, 46), IFN-{gamma} regulated P47 GTPases other than LRG47 recently identified in forward genetic experiments (47, 48) might also underlie the increased bacterial control of RAG1–/–/SOCS1–/– mice. However, all RAG1–/–/SOCS1–/– mice died early after infection showing severe pulmonary inflammation. Increased levels of Mig and IP-10 mRNA in lungs from RAG1–/–/SOCS1–/– mice were associated with the enhanced accumulation of pulmonary inflammatory cells and the morbidity recorded (Fig. 9). In line with this, SOCS1–/+ mice show increased severity of cutaneous lesions in mice infected with Leishmania major (49).

The SOCS proteins are indispensable for regulating many biochemical processes, including leukocyte homeostasis, glucose turnover, cell growth, and responses to pathogens. Further understanding of their roles in cytokine signal regulation is essential (50). Our data suggest that SOCS1 expression is induced by infection in a STAT-1 and IFN-{alpha}β-dependent manner and may protect the host from inflammatory disease. However, SOCS1 also decreases the efficiency of protective innate immune mechanisms probably by hampering secretion of, and cellular responses to IFN-{alpha}β and IFN-{gamma}.


   Acknowledgments
 
We are grateful to Prof. Andreas Klos, Department of Medical Microbiology, Medical School Hannover, Hannover, Germany, for comments. We thank Dr. T. Naka and T. Kishimoto for providing us SOCS1+/– mice. We thank Berit Olsson, Karolinska Institute, Stockholm, Sweden, for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 the European Community QLK2-CT-2002-00846 grant, the Karolinska Institute, and by the International Research Training Group 1273, funded by the German Research Foundation and the Swedish Research Council. Back

2 Address correspondence and reprint requests to Dr. Martin E. Rottenberg, Department of Microbiology, Tumorbiology and Cell Biology, Karolinska Institute, 171 77 Stockholm, Sweden. E-mail address: Martin.Rottenberg{at}ki.se Back

3 Abbreviations used in this paper: SOCS, suppressor of cytokine signaling; BMM, bone marrow-derived macrophage; BMDC, bone marrow-derived dendritic cell; WT, wild type. Back

Received for publication October 11, 2007. Accepted for publication January 12, 2008.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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