TLR9-Dependent and Independent Pathways Drive Activation of the Immune System by Propionibacterium Acnes

Propionibacterium acnes is usually a relatively harmless commensal. However, under certain, poorly understood conditions it is implicated in the etiology of specific inflammatory diseases. In mice, P. acnes exhibits strong immunomodulatory activity leading to splenomegaly, intrahepatic granuloma formation, hypersensitivity to TLR ligands and endogenous cytokines, and enhanced resistance to infection. All these activities reach a maximum one week after P. acnes priming and require IFN-γ and TLR9. We report here the existence of a markedly delayed (1–2 weeks), but phenotypically similar TLR9-independent immunomodulatory response to P. acnes. This alternative immunomodulation is also IFN-γ dependent and requires functional MyD88. From our experiments, a role for MyD88 in the IFN-γ-mediated P. acnes effects seems unlikely and the participation of the known MyD88-dependent receptors, including TLR5, Unc93B-dependent TLRs, IL-1R and IL-18R in the development of the alternative response has been excluded. However, the crucial role of MyD88 can partly be attributed to TLR2 and TLR4 involvement. Either of these two TLRs, activated by bacteria and/or endogenously generated ligands, can fulfill the required function. Our findings hint at an innate immune sensitizing mechanism, which is potentially operative in both infectious and sterile inflammatory disorders

Beneficial or Deleterious Effects of a Preexisting Hypersensitivity to Bacterial Components on the Course and Outcome of Infection

Priming with heat-killed Propionibacterium acnes enhances the sensitivity of mice to lipopolysaccharide (LPS) and other biologically active bacterial components. We show that P. acnes priming has protective and deleterious effects on a subsequent serovar Typhimurium infection. It may result in a complete protection or prolonged survival, or it may accelerate mortality of the infected mice, depending on the number of serovar Typhimurium bacteria administered and on the degree of LPS hypersensitivity at the time of infection. Both effects of P. acnes-induced hypersensitivity are mediated by gamma interferon (IFN-γ) and are based on a differential activation of the innate immune mechanisms which recognize and react against the LPS present in infecting bacteria. In P. acnes-primed mice null for LPS-binding protein (LBP(−/−) mice), the impaired LPS recognition, due to the absence of LBP, resulted in a higher resistance to serovar Typhimurium infection. A similar P. acnes priming of mice had a protective, but no deleterious effect on a subsequent L. monocytogenes infection. This effect was IFN-γ dependent but independent of LBP

Toll-Like Receptor 2- and 6-Mediated Stimulation by Macrophage-Activating Lipopeptide 2 Induces Lipopolysaccharide (LPS) Cross Tolerance in Mice, Which Results in Protection from Tumor Necrosis Factor Alpha but in Only Partial Protection from Lethal LPS Doses

Patients or experimental animals previously exposed to lipopolysaccharide (LPS) become tolerant to further LPS challenge. We investigated the potential of the macrophage-activating lipopeptide 2 (MALP-2) to induce in vivo cross tolerance to tumor necrosis factor alpha (TNF-α) and LPS. MALP-2-induced tolerance could be of practical interest, as MALP-2 proved much less pyrogenic in rabbits than LPS. Whereas LPS signals via Toll-like receptor 4 (TLR4), MALP-2 uses TLR2 and TLR6. LPS-mediated cytokine release was studied in mice pretreated with intraperitoneal injections of MALP-2. No biologically active TNF-α could be detected in the serum of MALP-2-treated animals when challenged with LPS 24 or 72 h later, whereas suppression of LPS-dependent interleukin (IL)-6 lasted for only 24 h. Protection from lethal TNF-α shock was studied in galactosamine-treated mice. Dose dependently, MALP-2 prevented death from lethal TNF-α doses in TLR4(−/−) but not in TLR2(−/−) mice, with protection lasting from 5 to 24 h. To assay protection from LPS, mice were pretreated with MALP-2 doses of up to 10 μg. Five and 24 h later, the animals were simultaneously sensitized and challenged by intravenous coinjection of galactosamine and a lethal dose of 50 ng of LPS. There was only limited protection (four of seven mice survived) when mice were challenged 5 h after MALP-2 pretreatment, and no protection when mice were challenged at later times. The high effectiveness of MALP-2 in suppressing TNF-α, the known ways of biological inactivation, and low pyrogenicity make MALP-2 a potential candidate for clinical use

A. Induction of LPS hypersensitivity in mice pretreated with murine recombinant IFN-γ.

<p><b>WT and MyD88^−/− mice were treated with recombinant IFN-γ (5000 </b><b>U/0.2 </b><b>ml) i.v.</b> or remained untreated (only LPS). Eight hours after pretreatment the animals were challenged with LPS <i>S.a.e.</i> (1 µg/g b.w.) i.v. and plasma was collected two hours later for determination of IFN-αβ. One representative experiment of three, with four to five mice is shown. <b>B</b>. Enhanced expression of IL-12Rβ1 and IL-12Rβ2 in MyD88^−/− and WT bone marrow derived macrophages and dendritic cells after stimulation with murine recombinant IFN<b>-</b>γ<b>.</b> 1.5×10⁶ bone marrow derived macrophages (BMM) or dendritic cells (BMDC) were stimulated with murine recombinant IFN-γ (rIFN-γ, 3000 U). After 24 hours, the cells were lysed in guanidinium-thiocyanate solution and lysates were used for RNA preparation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039155#s4" target="_blank">materials and methods</a>. The mRNA levels were analyzed by quantitative real-time PCR. Each value is represented as a relative expression, which is evaluated after setting background expression in controls as arbitrary value  = 1. One representative experiment of two is shown.</p

LPS sensitivity of <i>P. acnes</i> primed TLR2/TLR4/TLR9^−/− mice after.

<p>Groups of TLR9^−/− and TLR2/TLR4/TLR9^−/− mice were primed with heat-killed <i>P. acnes</i> (20 µg/g b.w.) i.v. or remained untreated. Each mouse was challenged 21 days after priming with a mixture of murine recombinant IL-12 (25 ng) and murine recombinant IL-18 (100 ng). Four hours after challenge, plasma was collected for determination of IFN-γ. Before challenge, no detectable IFN-γ was found in plasma of <i>P. acnes</i>-treated mice of either one of the groups (not shown). One representative experiment of two is shown. *:p-value<0.05 and **:p-value<0.01 as compared to only rIL-12+rIL-18-treated controls.</p

Delayed development of <i>P. acnes</i> effects in primed TLR9^−/− mice.

<p>Groups of WT and TLR9^−/− mice (15–20 animals/group) were primed with heat-killed <i>P. acnes</i> (20 µg/g b.w.) i.v. or remained untreated (controls; day 0). <b>A. Hypersensitivity to LPS:</b> At indicated time points after priming the animals were challenged with LPS from <i>S.a.e.</i> (0.01 µg/g b.w.), i.v. One hour and 4 h later, plasma was collected for determination of TNF-α and IFN-γ, respectively. Before challenge with LPS no detectable TNF-α or IFN-γ was found in plasma of <i>P. acnes</i>-treated mice of either one of the groups (not shown). Cumulative data of 3 different experiments are shown. *:p-value<0.05 and ***:p-value<0.001 WT compared to TLR9^−/− mice, and <i>P. acnes</i>-treated WT or <i>P. acnes</i>-treated TLR9^−/− at the indicated time point compared to untreated controls. <b>B. Splenomegaly:</b> Spleens were removed and weighted at indicated time points after treatment. Cumulative data from 4–5 experiments are shown. *:p-value<0.05 and **:p-value<0.01. <b>C. Enhanced resistance to serovar Typhimurium infection is delayed in TLR9^−/− mice:</b> 7 or 21 days after priming, the animals were infected with <i>Salmonella</i> serovar Typhimurium (200 CFU/0.2 ml PBS/i.v.). Bacterial counts in liver of unprimed and primed animals were determined 4 days after infection. One representative experiment of three is shown. *:p-value<0.05 and **:p-value<0.01.</p

Absence of hypersensitivity to LPS and splenomegaly in MyD88^−/− mice after <i>P. acnes</i> treatment.

<p>Groups of four to five WT, TLR9^−/− and MyD88^−/− mice were treated with heat-killed <i>P. acnes</i> (20 µg/g b.w.) i.v. or remained untreated (control). 7 and 21 days after priming the animals were challenged with LPS <i>S.a.e.</i> (0.01 µg/g b.w.) i.v. and plasma was collected two hours later for determination of IFN-αβ (<b>A</b>). The spleens were removed and weighted (<b>B</b>). One representative experiment of many is shown. *:p-value<0.05, **:p-value<0.01 and ***:p-value<0.001.</p