Exploiting induced pluripotent stem cell-derived macrophages to unravel host factors influencing Chlamydia trachomatis pathogenesis

Abstract

Chlamydia trachomatis remains a leading cause of bacterial sexually transmitted infections and preventable blindness worldwide. There are, however, limited in vitro models to study the role of host genetics in the response of macrophages to this obligate human pathogen. Here, we describe an approach using macrophages derived from human induced pluripotent stem cells (iPSdMs) to study macrophage-Chlamydia interactions in vitro. We show that iPSdMs support the full infectious life cycle of C. trachomatis in a manner that mimics the infection of human blood-derived macrophages. Transcriptomic and proteomic profiling of the macrophage response to chlamydial infection highlighted the role of the type I interferon and interleukin 10-mediated responses. Using CRISPR/Cas9 technology, we generated biallelic knockout mutations in host genes encoding IRF5 and IL-10RA in iPSCs, and confirmed their roles in limiting chlamydial infection in macrophages. This model can potentially be extended to other pathogens and tissue systems to advance our understanding of host-pathogen interactions and the role of human genetics in influencing the outcome of infections.

Figure 1. Differentiation of macrophages from human iPSCs.

(a) Schematic diagram of the method of in vitro differentiation from human iPSCs to macrophages, with accompanying representative light microscopy images for each stage. Scale bars, 200 μm. (b) EM images of a human iPSdM (left) and a human blood monocyte differentiated macrophage (right). (c) Flow cytometry analyses for the expression of macrophage markers (CD11b, CD14, CD16, CD68) and pluripotency markers (SSEA4, OCT3/4) on human iPSdMs. Red lines represent cells stained with control isotype and blue lines represent cells stained with the relevant antibody.

Figure 2. Imaging of human iPSdMs infected with C. trachomatis.

(a) Representative transmission electron microscopy (TEM) image of iPSdM (left) and blood monocyte-derived macrophage (right) infected for 1 h (arrows indicating Chlamydia-containing inclusions formed inside infected macrophages), (b) representative TEM image of iPSdM (left) and blood monocyte-derived macrophage (right) infected for 24 h. (c) Representative scanning electron microscopy image of iPSdM infected for 48 h. (d) Changes in GFP intensity during a 48 h infection of iPSdMs with GFP-tagged C. trachomatis. Results are the average of three independent measurements ±s.d. using the Incucyte imaging system.

Figure 3. In vitro infection of mammalian cells with GFP-tagged C. trachomatisharvested from 48 h infections.

(a) Human iPSdMs. (b) Human blood monocyte-derived macrophages. (c) McCoy epithelial cells. Representative images were taken using the Cellomics CellInsight NXT at × 10 magnification, scale bar, 80 μm.

Figure 4. Overproduction of cytokines in human iPSdMs after infection with C. trachomatisfor 24 h.

Results are the average of three independent measurements±s.d. using the Luminex customised anti-human cytokine Milliplex kit. *Represents statistically significant different (P<0.05) between uninfected and Chlamydia-infected as determined using two-way ANOVA.

Figure 5. Effect of inhibitors on C. trachomatis infection of human iPSdMs.

(a) Anti-IFNAR1 antibody; (b) IDO inhibitor INCB024360. Results are the average of three independent measurements±s.d. using the Cellomics CellInsight NXT. *Represent statistically significant different (P<0.05) between untreated and treated as determined using two-way ANOVA.

Figure 6. Schematic diagram for the generation of biallelic knockout mutants.

Strategy for the generation of biallelic knockouts is to replace a critical exon of one allele of the target gene with a drug selection cassette by homologous recombination and to screen clones for damage to the second allele induced by error-prone non-homologous end-joining (NHEJ). Since only one copy of the target exon will be present in correctly targeted clones, NHEJ-induced damage to the non-targeted allele can be simply assessed by Sanger sequencing of PCR products from the target exon.

Figure 7. C. trachomatis infection of human iPSdM CRISPR/Cas9 mutants.

(a,c) Level of GFP-tagged C. trachomatis infection of human iPSdM CRISPR/Cas9 mutants after 48 h. Results are the averages from three independent measurements±s.d. using the Cellomics CellInsight NXT. (b) Production of cytokines from the IRF5^−/− mutant. Results are the average from three biological replicates assessed using the Luminex Multiplex and shown as fold change relative to expression of each cytokine in KOLF2 parent iPSdMs. *Represent statistically significant different (P<0.05) between parent and mutants as determined using two-way ANOVA. Equal numbers of WT and mutant cells were seeded for each experiment.

Figure 8. Network of expression changes due to infection in the IRF5^−/− mutant compared to parent KOLF2 cells.

Green=decreased expression; Red=increased expression. RNA-Seq data for differentially expressed genes due to infection (that is, differences between IRF5^−/− mutant infected/uninfected compared to parent KOLF2 cells infected/uninfected) were submitted to NetworkAnalyst and interconnected based on known protein:protein interactions (www.innatedb.ca).