Intracellular Survival and Persistence of <i>Chlamydia muridarum</i> Is Determined by Macrophage Polarization

<div><p>Macrophages can display a number of distinct phenotypes, known collectively as polarized macrophages. The best defined of these phenotypes are the classically-activated, interferon gamma (IFNγ)/LPS induced (M1) and alternatively-activated, IL-4 induced (M2) macrophages. The goal of this study is to characterize macrophage-<i>Chlamydia</i> interactions in the context of macrophage polarization. Here we use <i>Chlamydia muridarum</i> and murine bone-marrow derived macrophages to show <i>Chlamydia</i> does not induce M2 polarization in macrophages as a survival strategy. Unexpectedly, the infection of macrophages was silent with no upregulation of M1 macrophage-associated genes. We further demonstrate that macrophages polarized prior to infection have a differential capacity to control <i>Chlamydia</i>. M1 macrophages harbor up to 40-fold lower inclusion forming units (IFU) than non-polarized or M2 polarized macrophages. Gene expression analysis showed an increase in <i>16sRNA</i> in M2 macrophages with no change in M1 macrophages. Suppressed <i>Chlamydia</i> growth in M1 macrophages correlated with the induction of a bacterial gene expression profile typical of persistence as evident by increased <i>Euo</i> expression and decreased <i>Omp1</i> and <i>Tal</i> expression. Observations of permissive <i>Chlamydia</i> growth in non-polarized and M2 macrophages and persistence in M1 macrophages were supported through electron microscopy. This work supports the importance of IFNγ in the innate immune response to <i>Chlamydia</i>. However, demonstration that the M1 macrophages, despite an antimicrobial signature, fail to eliminate intracellular <i>Chlamydia</i> supports the notion that host–pathogen co-evolution has yielded a pathogen that can evade cellular defenses against this pathogen, and persist for prolonged periods of time in the host.</p> </div

Electron microscopy of infected polarized macrophages demonstrates productive growth in M0 and M2 macrophages but induction of <i>Chlamydia</i> persistence in M1 macrophages.

<p>Polarized BMDM infected for 24 hr prior to processing for electron microscopy. Mature <i>Chlamydia</i> inclusions were seen in M0 and M2 macrophages (top panels). Bottom left panel shows representative uninfected M1 macrophage. Mature inclusions were not seen in infected M1 macrophages. Small inclusions with enlarged amorphous particles, consistent with <i>Chlamydia</i> in the persistent state, were seen frequently in infected M1 macrophages (arrows, bottom panels).</p

Polarized macrophages are not infected equally.

<p>BMDM plated at 2.5 x10⁵ cells/well in 24 well plate and pre-polarized prior to infection for 6–48 hr. Cells fixed and stained with anti-LPS antibody. Representative images of duplicate wells at 6 hr and 24 hr p.i. By 24 hr p.i. large, visible inclusions (white arrows) where seen in M0 and M2 macrophages, but not M1 macrophages. Scale bar is 50µm.</p

Control of <i>Chlamydia</i> by M1 macrophages involves the induction of persistence as demonstrated by RT-PCR.

<p>Polarized macrophages were infected for the indicated timepoints. <i>Chlamydia</i> gene expression is not altered by growth in M2 macrophages versus growth in M0 macrophages. In M1 macrophages, a gene expression profile characteristic of persistence was seen from 6 hr p.i. infection to 24 hr p.i. whereby <i>Euo</i> was up-regulated and <i>Omp1</i> and <i>Tal</i> were down-regulated. Data is mean ±SEM of three independent experiments and was analyzed using one-sample t-test against hypothetical mean of 1.0.</p

RT-PCR of <i>Chlamydia 16sRNA</i> demonstrates that <i>16sRNA</i> remains stable in M1 macrophages, but increases in M0 and M2.

<p>Polarized BMDM infected with <i>Chlamydia</i> were harvested at times indicated. (A) Pfaffl calculated fold change in <i>16sRNA</i> in polarized macrophages (M1/M2) versus M0 demonstrates significantly lower <i>Chlamydia</i> load in M1 macrophages at all timepoints. Treatments and timepoints analyzed separately by one-sample t-test versus hypothetical mean of 1.0 (B) Difference in ∆Ct between <i>HPRT</i> and <i>16sRNA</i> for all samples demonstrates that decreased fold change in <i>16sRNA</i> in M1 macrophages is due to increased <i>16sRNA</i> expression in M0 macrophages rather than a decrease in M1. Data analyzed by repeated measures two-way ANOVA and Bonferroni’s post test. All data is mean ±SEM of three separate experiments.</p

<i>Chlamydia muridarum</i> does not induce a gene expression profile of polarization in naïve macrophages.

<p>Gene expression profiles determined by RT-PCR of BMDM. (A) Positive controls of polarized BMDM show differential expression in key M1- and M2-associated genes relative to expression seen in M0 macrophages. (∆) denotes detectable <i>Retnla</i> expression in M2 macrophages but no detection in M0 or M1 macrophages. (B) M0 macrophages were infected for 24 hr with 1 MOI <i>Chlamydia</i>. <i>16sRNA</i> detected in infected macrophages by RT-PCR, but not uninfected indicative of bacterial presence. Expression of M1-associated genes and expression of M2-associated genes show no significant alteration with infection as determined by one-sample t-test against hypothetical value of 1.0. (∆∆) denotes no <i>Retnla</i> detected in uninfected or uninfected macrophages. Data are averages ±SEM from three independent experiments.</p