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Differential outcomes of TLR2 engagement in inflammation‐induced preterm birth

Abstract Preterm birth (PTB) is the leading cause of neonatal mortality worldwide. Infection and inflammation are considered main causes of PTB. Among multiple pathogens, Gram‐positive bacteria are commonly linked with induction of PTB. Although activation of innate immune responses, via TLR2 engagement, by Gram‐positive bacteria is a likely cause, whether induction of PTB depends on the potency of specific microbial components to induce Toll‐like receptor (TLR)2‐driven inflammation has not been elucidated. Here, we show that TLR2 activation by synthetic lipopeptides, Pam2Cys, and Pam3Cys specifically, variably influenced inflammation and subsequent induction of PTB. Pam2Cys challenge, compared to Pam3Cys, induced PTB and promoted significantly higher expression of inflammatory cytokines, specifically IL‐6 and IFN‐β, both in vivo and in vitro. Notably, antibody‐mediated neutralization of IL‐6 or genetic deletion of type I IFN receptor (IFNAR) was sufficient to protect from Pam2Cys‐driven PTB and to temper excessive proinflammatory cytokine production. Conversely, IFN‐β or IL‐6 was not sufficient to promote induction of PTB by Pam3Cys. In summary, our data implies a divergent function of TLR2‐activating lipopeptides in the magnitude and type of ligand‐driven inflammatory vigor in induction of PTB.


INTRODUCTION
Preterm birth (PTB) is a leading cause of infant morbidity and mortality worldwide. 1,2 Although the etiology of PTB is multifactorial and remains largely enigmatic, it is well accepted that maternal inflammation, driven by infectious or noninfectious triggers, can play a critical role in preterm labor. 3 In fact, inflammation and immune activation are paramount for uterine activation and the onset of labor. 2 Pregnant women are more susceptible to infection by multiple pathogens, which increases the risk of severe illness and adverse pregnancy outcome. 2 Infections by Gram-positive bacteria have the most deleterious outcomes during pregnancy. 3,4 Listeria monocytogenes, a Gram-positive bacteria and common contaminant of a variety of raw foods, has tropism for the placenta and is known to cause PTB in humans and mice. 3,5 Moreover, Group B streptococci (GBS), the -hemolytic, Grampositive constituent of the normal vaginal microflora, is frequently ERK), and IRF signaling pathways, leading to proinflammatory (e.g., TNF-, IL-8, IL-6, IFN-, and IFN-), anti-inflammatory (e.g., IL-10), and immunoregulatory (e.g., IL-12) cytokine production. 17,18 TLRs are robustly expressed at the maternal/fetal interface by the pregnant uterus, placenta, amniotic membranes, and trophoblast. [19][20][21][22][23] In mice, challenge with diverse TLR ligands-via intraperitoneal, intrauterine, or intraamniotic routes-increases proinflammatory cytokine release in the uterus 5 and fetal membranes, 24,25 recruits immune cells into the cervix, 26,27 and induces PTB. [27][28][29][30][31] Notably, TLR2 is involved in the recognition of lipoteichoic acid and lipopeptides from Gram-positive bacteria. Specifically, the ability of TLR2 to heterodimerize with either TLR6 or TLR1 results in the recognition of diacylated and triacylated lipopeptides, respectively, hinting at a potential mechanism to discriminate among various microbial ligands and to elicit varied downstream inflammatory responses. However, the contribution of specific TLR2-activating lipopeptide species in the context of PTB has not been examined.
In this study, we hypothesized that induction of PTB depends on the potency of specific lipopeptides to induce TLR2-driven inflammation.
Our data demonstrate that recognition of diacylated lipopeptide (e.g., Pam2Cys) induced PTB and greater proinflammatory cytokine production compared to triacylated lipopeptide (e.g., Pam3Cys). Our data also suggest that sufficiency of inflammatory mediators to induce PTB following Pam2Cys or Pam3Cys challenge is varied. In sum, these data argue that TLR2-driven induction of PTB might depend on the magnitude and milieu of ligand-driven inflammatory vigor and such differences might shed light on why only certain Gram-positive bacterial species are associated with induction of PTB.

Reagents
All cell culture reagents were endotoxin free to the limits of detection of the Limulus amebocyte lysate assay (Lonza, Visp, Switzerland) at the concentrations employed. All TLR ligands (Pam3Cys and Pam2Cys) used were of ultrapure grade (Invivogen, San Diego, CA).

Mice
Animals were housed in a specific pathogen-free animal facility at CCHMC and handled in high-efficiency particulate-filtered laminar flow hoods with free access to food and water. All studies were performed in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals and approved by the CCHMC Institutional Animal Care and Use Committee. Female mice (WT, IFNAR −/− ), on a C57BL/6J background, were mated with fertile male mice of the same strain. The presence of a vaginal plug was considered at day 1 of pregnancy. Parturition events were monitored on days 17-21 of gestation and defined as complete delivery of pups.
PTB was defined as parturition within 24 h after TLR ligand challenge (all pups deceased). Term birth was defined as parturition between days 19 and 21 of gestation (all pups alive).

Cytokine quantification
In vivo systemic IL-6, TNF, and IFN-levels were quantified using in vivo cytokine capture assay. 5,32,33 Briefly, biotinylated capture antibodies (eBioscience) were injected i.p. 3 h prior to Pam3Cys and Pam2Cys administration and serum cytokine levels were determined 4 h later.

Type I IFN activity quantification
Type I IFN activity in mouse serum samples or cell culture supernatants was measured with reference to a recombinant mouse IFN-using an L-929 cell line transfected with an IFN-sensitive luciferase construct as previously described. 5,35 Luciferase activity was measured on a luminometer (SpectraMax L; Molecular Devices, Sunnyvale, CA).

Gene expression
For quantification of mRNA expression in murine samples, cells/tissues were homogenized in TRIzol (Invitrogen, San Diego, CA), RNA was extracted, and cDNA was generated and quantified as previously

Statistical analysis
Data were analyzed by unpaired Student t test in Prism 5a (GraphPad Software Inc., La Jolla, CA) as appropriate. A P value less than 0.05 was considered significant. In vivo serum cytokine values were normalized

Activation of TLR2 by Pam2Cys but not Pam3Cys induces PTB
Gram-positive bacteria (e.g., Listeria, GBS, and Ureaplasma species) are pathogens commonly associated with PTB. 5,6,12-15 However, competency of certain bacteria to trigger PTB remains unclear. Although all experimental models of PTB have limitations compared to human pregnancy and parturition, mice represent the most common animal model of PTB. 2 Specifically, in such experimental models, the doses of inflammatory ligands employed are high. The intent being to enable 100% parturition incidence in fully backcrossed mice. 5,[36][37][38][39][40] Notably, that allows investigators to uncover differences between animal genotypes and/or various ligands in comparison to estab- As inflammation is directly linked with TLR-driven induction of PTB, the capacity of Pam2Cys and Pam3Cys to induce inflammatory responses was examined next. Induction of PTB by Pam2Cys correlated with significantly increased IL-6, IFN-, and IFN-, but similar TNF production compared to Pam3Cys challenge-findings when performed with equivalent doses of these TLR2 ligands ( Figure 1C). The central role of proinflammatory cytokines, and IL-6 in particular, is supported by the correlation between IL-6 levels and PTB in humans 47 and in mouse models of TLR-driven inflammation. 5,38 Pharmacological inhibition of IL-6 (antibody mediated) was sufficient to protect from Pam2Cys-driven PTB, while a single dose of rIL-6 alone or rIL-6 in combination with Pam3Cys was not sufficient to trigger induction of PTB ( Figure 1D)-something supported by a previously published report. 48 Such findings suggest that Pam2Cys and Pam3Cys signaling activate divergent immune mediators that may play a role in induction of PTB.
Further, these data also suggest that IL-6 is not the only proinflamma- Although TLR2 activation was thought to specifically induce NF-kB-driven cytokine production, recently the contribution of endolysosomal TLR2 activation has been shown to promote type I IFN production. 60 However, the contribution of TLR2 homodimerization or dimerization of TLR2 with either TLR1 or TLR6 for induction of type I IFN is not known. 63

IFNAR signaling contributes to Pam2Cys-mediated induction of PTB
Given the robust induction of type I IFN production and signature after Pam2Cys-driven TLR2 activation, the necessity of the common IFN / receptor (IFNAR) in proinflammatory cytokine production and PTB was further investigated. 64 IFNAR is a heteromeric cell surface receptor with 2 subunits, IFNAR1 and IFNAR2. Of note, IFN-specifically interacts with IFNAR1 in an IFNAR2-independent manner. 65 Compared to WT-treated mice, genetic deletion of IFNAR1 tempered Pam2Cys-dependent induction of IL-6 and TNF ( Figure 3A and B) and resulted in a 50% protection from Pam2Cys-driven PTB ( Figure   3C). The lack of IFNAR1 signaling similarly decreased Pam3Cys-driven IL-6 and TNF production. Further Pam2Cys-driven cytokine production was significantly higher compared to Pam3Cys in both WT and IFNAR −/− mice ( Figure 3A and B). These data suggest that both synthetic lipopeptides activate the type I IFN/IFNAR axis and that such activation regulates proinflammatory cytokine production in this context. However, although significant, the protection from Pam2Cysdriven PTB in IFNAR-deficient mice was not complete ( Figure 3C), indicating that additional signaling pathways activated following Pam2Cys challenge are similarly involved. Hence, how IFNAR signaling (e.g., IFNAR1 and IFNAR2) contributes to protection from Pam2Cys-driven PTB warrants further investigation.

IFN--driven enhancement of Pam3Cys-associated inflammation is not sufficient for induction of PTB
A combined recognition of viral and bacterial molecular patterns by TLR, coined a "double hit hypothesis", has been proposed as a central regulator of increased susceptibility to PTB. 40,70,71 We have previously demonstrated that type I IFNs regulates TLR4driven inflammatory vigor, and specifically primes for exacerbated IL-6 and TNF production. 5 As Gram-positive bacteria-derived PAMPs are associated with PTB and robust activation of the type I IFN axis, we asked whether type I IFN priming acts as a universal sensitization mechanism to secondary challenge with all TLRs (including those that alone do not induce PTB). Of note, in vitro IFNadministration was sufficient, although at different levels, to prime for a secondary challenge with Pam2Cys and Pam3Cys ( Figure 4A).
This was recapitulated in vivo as IFN-priming exacerbated IL-6, TNF, and IFN-production by both Pam2Cys and Pam3Cys. However, IFN-priming induced significantly higher levels of proinflammatory cytokines upon secondary challenge with Pam2Cys ( Figure 4B and C).

CONCLUSIONS
Overall, these data suggest that sufficiency of type I IFN priming in augmentation of inflammatory vigor, at least in conditions utilized in this report, is limited to certain secondary challenge. 5

DISCLOSURES
The authors declare no conflict of interest.