Chlamydia inhibits interferon gamma-inducible major histocompatibility complex class II expression by degradation of upstream stimulatory factor 1

Abstract

We report that chlamydiae, which are obligate intracellular bacterial pathogens, can inhibit interferon (IFN)-gamma-inducible major histocompatibility complex (MHC) class II expression. However, the IFN-gamma-induced IFN regulatory factor-1 (IRF-1) and intercellular adhesion molecule 1 (ICAM-1) expression is not affected, suggesting that chlamydia may selectively target the IFN-gamma signaling pathways required for MHC class II expression. Chlamydial inhibition of MHC class II expression is correlated with degradation of upstream stimulatory factor (USF)-1, a constitutively and ubiquitously expressed transcription factor required for IFN-gamma induction of class II transactivator (CIITA) but not of IRF-1 and ICAM-1. CIITA is an obligate mediator of IFN-gamma-inducible MHC class II expression. Thus, diminished CIITA expression as a result of USF-1 degradation may account for the suppression of the IFN-gamma-inducible MHC class II in chlamydia-infected cells. These results reveal a novel immune evasion strategy used by the intracellular bacterial pathogen chlamydia that improves our understanding of the molecular basis of pathogenesis.

Figure 1

Chlamydial infection selectively inhibits IFN-γ–inducible MHC class II expression. MCF-7 cells with or without chlamydial infection were stimulated with IFN-γ and collected for flow cytometry (A), Western blot (B), and RT-PCR (C) analysis. (A) Chlamydial infection prevents IFN-γ–inducible HLA-DR but not ICAM-1 surface expression. (B) Chlamydial infection suppresses the total cellular protein level of IFN-γ–inducible HLA-DRα in various human cell lines. MRC-5, 2C4, and HeLa cells were stimulated with IFN-γ at 400 U/ml. HLA-DRα was detected in a Western blot assay. (C) Chlamydial infection inhibits HLA-DRα, DMα, and Ip41 mRNA expression. Gene-specific primers were used to amplify random primer–directed cDNA templates in an RT-PCR reaction as described in Materials and Methods.

Figure 2

Chlamydial infection disrupts IFN-γ intracellular signaling pathways. (A) Chlamydial infection suppresses both HLA-DRα and CIITA but not IRF-1, STAT1α, or USF-1 mRNA expression in MCF-7 cells. The mRNA levels were analyzed by RT-PCR as described in Materials and Methods. (B) USF-1 protein is not detected in chlamydia-infected MCF-7 cells. The protein levels of IFN-γR, JAK-1, STAT1α, tyrosine-phosphorylated STAT1α, IRF-1, and USF-1 were detected using a Western blot assay. (C) Lactacystin prevents USF-1 degradation and preserves HLA-DRα expression in chlamydia-infected MCF-7 cells. 10 h before IFN-γ stimulation, a chlamydia-infected cell sample was treated with lactacystin (Calbiochem) at a final concentration of  75 μM or with an equivalent amount of solvent DMSO and kept in culture during IFN-γ stimulation. HLA-DRα and USF-1 were detected in a Western blot assay. ns, nonspecific binding.

Figure 2

Chlamydial infection disrupts IFN-γ intracellular signaling pathways. (A) Chlamydial infection suppresses both HLA-DRα and CIITA but not IRF-1, STAT1α, or USF-1 mRNA expression in MCF-7 cells. The mRNA levels were analyzed by RT-PCR as described in Materials and Methods. (B) USF-1 protein is not detected in chlamydia-infected MCF-7 cells. The protein levels of IFN-γR, JAK-1, STAT1α, tyrosine-phosphorylated STAT1α, IRF-1, and USF-1 were detected using a Western blot assay. (C) Lactacystin prevents USF-1 degradation and preserves HLA-DRα expression in chlamydia-infected MCF-7 cells. 10 h before IFN-γ stimulation, a chlamydia-infected cell sample was treated with lactacystin (Calbiochem) at a final concentration of  75 μM or with an equivalent amount of solvent DMSO and kept in culture during IFN-γ stimulation. HLA-DRα and USF-1 were detected in a Western blot assay. ns, nonspecific binding.

Figure 3

Chlamydial protein synthesis is required for both USF-1 degradation and inhibition of HLA-DRα expression. (A) Correlation between infection dose and USF-1 degradation. 24 h after chlamydial infection at various MOI, MCF-7 cells were analyzed for chlamydial MOMP and host USF-1 and USF-2 protein levels in a Western blot assay. As the USF-2 protein level was not altered by chlamydial infection, it served as an internal control. (B) Time course relationship between chlamydial growth and USF-1 degradation. At various time points after infection, MCF-7 cells were lysed for the detection of USF-1, STAT1α, and MOMP in a Western blot assay. (C) Inhibition of chlamydial but not host protein synthesis prevents USF-1 degradation and preserves HLA-DRα expression. Rifampin (RF; final concentration, 0.1 μg/ml), chloramphenicol (CH; 60 μg/ml), and penicillin (PG; 100 μg/ml) were added at the beginning of chlamydial infection and maintained throughout the culture. Cycloheximide (CY; 10 μg/ml) was added to the culture 10 h before IFN-γ treatment and maintained during the IFN-γ stimulation. The MCF-7 cells were analyzed for protein levels of HLA-DRα, USF-1, USF-2, and chlamydial MOMP in a Western blot assay.

Figure 4

Model for chlamydial inhibition of IFN-γ–inducible MHC class II expression. IFN-γ binding activates intracellular JAK/STAT pathways. USF-1 is a constitutively and ubiquitously expressed downstream transcription factor required for IFN-γ–inducible CIITA expression. Chlamydial infection degrades USF-1, which results in diminished expression of CIITA in chlamydia-infected cells. As CIITA is an obligate mediator for transcription of MHC class II genes, chlamydial degradation of USF-1 finally leads to suppression of IFN-γ–inducible MHC class II. However, because the upstream pathways of IFN-γ signaling are still intact in chlamydia-infected cells, expression of IRF-1, ICAM-1, and IDO is not affected by chlamydial infection.