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Department of Biology, Florida Agricultural and Mechanical University, Tallahassee, FL, 32307; firstname.lastname@example.org
Maternal infection during pregnancy is associated with adverse outcomes for the fetus, including postnatal cognitive disorders. However, the underlying mechanisms are obscure. We find that bacterial cell wall peptidoglycan (CW), a universal PAMP for TLR2, traverses across the murine placenta into the developing fetal brain. In contrast to adults, CW-exposed fetal brains did not show any signs of inflammation or neuronal death. Instead, the neuronal transcription factor FoxG1 was induced and neuroproliferation leading to a 50% greater density of neurons in the cortical plate was observed. Bacterial infection of pregnant dams followed by antibiotic treatment, which releases CW, yielded the same result. Neuroproliferation required TLR2 and was recapitulated in vitro with fetal neuronal precursor cells and TLR2/6, but not TLR2/1 ligands. The fetal neuroproliferative response correlated with abnormal cognitive behavior in CW-exposed pups following birth. Thus, the bacterial CW-TLR2 signaling axis affects fetal neurodevelopment and may underlie postnatal cognitive disorders.
Infection and inflammation during pregnancy have been associated with adverse outcomes such as preterm birth, abortion and postnatal cognitive disorders (Lowe et al. 2008, Sorenson et al. 2008, Canetta et al. 2014). Yet, how microbial products circulating in the maternal bloodstream affect fetal development is poorly understood. The disruptive effects of inflammation would be substantial as fetal organs undergo complex waves of proliferation and apoptosis during gestation (Couzin-Frankel, 2013; Roth and D’Sa, 2001). Microbial components display pathogen associated molecular patterns (PAMPs) that are recognized by the innate immune system and serve to trigger inflammation. In Gram positive bacteria, cell wall (CW) is a complex exopolymer that harbors several PAMPs that potently activate inflammation when released into the bloodstream during bacterial infection or when explosively discharged during bacterial lysis by common antibiotics (Tuomanen et al 1985a,b). CW binds to innate immune Toll-like receptors (TLRs), particularly TLR2, and initiates signaling leading to inflammation by a well recognized cascade involving MyD88, NFkB and cytokine secretion (Aliprantis et al., 1999; Yoshimura et al., 1999). This signaling process underlies the pathogenesis of bacterial meningitis where CW/TLR2 ligation provokes a broad spectrum of acute signs and symptoms of brain inflammation (Orihuela et al., 2006; Tuomanen et al., 1985a,b). In children and adults with pneumococcal meningitis, neuronal death predominates (Braun et al., 1999; Braun et al., 2001; Mitchell et al., 2004) and neuroproliferation is insufficient to repair brain injury, leaving survivors with devastating permanent sequelae, such as paralysis, seizures, and memory loss (Gerber et al., 2003; Hoffmann et al., 2007). The degree of damage depends on the pathogen, with pneumococci considered the most severe (Schmidt et al., 2006).
TLR2 is abundant in the embryo (Okun et al, 2010) but little is known of the outcome of activation of this cascade in the fetus. It is unknown if TLR2 ligands, such as CW, cross the placenta. Bacterial CW could be an important participant in microbial-host signaling at the maternal-fetal interface since CW circulates in the maternal bloodstream not only during infection but also from the microbiome in healthy mothers (Clarke et al., 2010). There is an emerging awareness of complex communication ongoing between the microbiome and the brain (Collins et al., 2012; Tracey, 2009) that modulates a variety of complex human behaviors and patterning of neuronal circuitry (Collins et al., 2012; Cryan and Dinan, 2012; Hsiao et al., 2013). Early neurodevelopmental stress and autism spectrum disorders have been linked to gut microbial dysbiosis (Hsiao et al., 2013).
We sought to use concepts of the pathogenesis of bacterial meningitis and CW-induced innate signaling to develop a model to probe the impact of bacterial products on fetal brain development. Since CW components traffic from blood across the blood brain barrier and induce neuroinflammation in the adult (Fillon et al., 2006), we reasoned that such a model, when extended to pregnant mice, could be used to test the ability of bacterial CW to cross the placenta and inform the capacity of the fetal brain to respond directly to bacterial PAMPs. This information would be relevant to general questions concerning the developing brain, such as the nature of the TLR2 response in the fetal brain and the potential for microbial products to affect neurodevelopment.
Our data indicate that the placenta allows CW traffic from the maternal to the fetal circulation. In contrast to neuronal death seen in the postnatal scenario (Tuomanen et al., 1985a,b; Nau and Bruck, 2002), engagement of TLR2 by CW in the fetus results in robust FoxG1-dependent neuroproliferation. This in utero response alters cerebral anatomy and appears have long-term postnatal behavioral consequences.
The peptidoglycan-teichoic acid complex of the CW of Streptococcus pneumoniae was chosen as a prototype component of Gram-positive bacteria that incites inflammation via the innate immune receptor TLR2 (Yoshimura et al., 1999) and whose bioactivities in the brain have been well characterized (Tuomanen et al., 1985a,b; Fillon et al., 2006; Orihuela et al., 2006). To investigate bioactivities of CW in the placenta, this macromolecule was fractionated, purified free of protein adducts, labeled with FITC, and injected intravenously into pregnant dams on embryonic day 15 (E15). FITC-CW was detected in placental tissue within 10 min of maternal administration and accumulated by 24hrs (Fig. 1A).
Bacterial components decorated with phosphorylcholine, a modification present on most oral commensals and respiratory pathogens, bind by molecular mimicry to the G-protein coupled platelet activating factor receptor (PAFr) (Cundell et al., 1995). In the case of pneumococcus, phosphorylcholine covalently modifies the CW, and the interaction between CW and PAFr supports uptake of both free CW and intact pneumococci into eukaryotic cells and across cellular barriers (Fillon et al., 2006). PAFr is expressed extensively in the fetal brain and placenta (Supplementary Fig. 1SA; Okun et al. 2010)). When examined in PAFr−/− mice, significantly fewer FITC-CW particles accumulated in placental tissue (Fig 1A) indicating that this mechanism of translocation across host boundaries by bacterial components operates in the placenta. This was further supported in vitro by robust association of CW with trophoblast cells contrasted by a rare association of EA CW, where ethanolamine was substituted for phosphorylcholine (Supplementary Fig 1SB).
Many organs undergo apoptosis upon postnatal exposure to CW (Braun et al., 1999; Orihuela et al., 2006). In contrast, examination of the placenta after CW administration to wild type mice showed only a modest increase in both TUNEL+ and caspase-3+ cells (Fig. 1B, C). In TLR2−/− mice, CW-induction of death in the placenta was not significantly different from PBS (Fig. 1B, C; Supplementary Fig. 1SC). CW caused no immediate fetal distress, as measured by umbilical vein velocity and fetal heart rate (Fig. 1D, E). All CW and PBS treated embryos were equally viable at birth and had similar weights at the time of weaning (mean ± SD: 10.4g ± 0.4 and 9.8g ± 0.3, CW and PBS respectively; n = 12 per group; p = 0.27). Thus, CW trafficked to placental tissue via PAFr but did not induce strong cell death responses.
In postnatal animal models, CW fragments in the bloodstream distribute across the blood brain barrier and induce signs and symptoms of meningitis (Tuomanen et al., 1985a,b) accompanied by extensive neuronal death (Braun et al., 1999; Braun et al., 2001). To determine if a similar pathology occurred in the developing brain, CW was administered intravenously to pregnant dams and localization of CW was examined in the embryonic cortex. CW injected in a single dose at E10 or E15 was detected in the fetal brain 24hrs later (Fig. 2A). A cell wall density of ~400 pieces/mm2 corresponds to 4 × 105 pieces per entire cortical plate indicating that ~2% of the inoculum into mother trafficked to the fetal brain. CW administered daily from E10-E14 was present in the cortex at E16 at similar levels to that seen in a single E15 injection (Fig. 2A). However, accumulation may be transient since a single dose of CW injected at E10 was not visible in the brain at E16 (Fig. 2A). EA CW that crosses the blood brain barrier poorly (lacks phosphorylcholine), was not detected in the fetal brain (data not shown). TLR2−/− fetal brains showed equivalent accumulation of CW as WT; in contrast, PAFr−/− fetal brains exhibited significantly less CW accumulation (Fig. 2B). In addition to the cortex, pneumococcal CW was found in multiple regions of the developing fetal brain, including the cerebellum, olfactory region, thalamus, and choroid plexus (data not shown). Thus, CW readily enters the developing fetal brain in a PAFr-dependent manner previously well-established in the postnatal brain (Cundell et al., 1995).
Extensive neuronal cell death is evident in brains of humans with meningitis and in postnatal mouse models using living bacteria or intravenous CW components (Fillon et al., 2006; Nau and Bruck, 2002). In the animal models, intravenous challenge with 107 living pneumococci or 107 bacterial equivalents of cell wall induces high levels of neuronal apoptosis by 24 hours (58 ± 6 and 54 ± 7 apoptotic cells/mm2, respectively; Fig. 2C) (Orihuela et al., 2006). However, exposure of the fetus to CW caused only low level TUNEL+ cell death compared to adult brains and no significant increase in caspase-3+ staining (Fig. 2C, D). No increase in cell death was seen in TLR2−/− and PAFr−/− mice (Supplementary Fig. 2S). Microglial activation as indicated by the marker Iba-1 was strong in adult brain post CW while the fetus showed a decrease in this inflammatory marker post CW (Fig. 2E).
Rather than cell death, the response of the fetal brain to CW revealed marked neuronal proliferation. In the cortex, the area and cell density of the cortical plate was measured using stereology with the Cavalieri estimator and the optical fractionator probe (Fig. 3A, B). For embryos exposed to CW at E10 and analyzed at E16, the area of the cortical plate remained stable (1.05×106 ± 0.16 vs 1.09×106 ± 0.14 μm2 PBS) but a 50% increase in density of neurons was observed (Fig. 3B, C left panel). This increased cell density was absent in TLR2−/− or PAFr−/− mice (Fig. 3B, C left panel) or in embryos challenged at E15 and harvested at E16 with wild type CW.
To extend this observation to a more clinically relevant scenario, blood stream infection with S. pneumoniae was established in E9 pregnant dams followed by daily treatment until E16 by ampicillin, known to potently release cell wall and contents by lysis (Tuomanen et al., 1985a,b). Embryos thus exposed to CW debris showed a significant increase in density of cortical plate neurons compared to ampicillin alone (Fig. 3C right panel). Embryos exposed to maternal infection treated with clindamycin, a protein synthesis inhibitor that does not release cell wall, showed no increase in cortical neuronal density (Fig. 3C right panel).
New neurons originate deep in the cortical ventricular zone and migrate out to the superficial cortical plate. To document the effect of CW exposure on immature neurons (Yamaguchi et al., 2000), pregnant Nestin-GFP transgenic dams were treated with CW and the Nestin-GFP signal in the fetal brain, was visualized via fluorescence microscopy. CW exposure strongly enhanced the GFP signal in immature neurons of the ventricular zone of the developing cortex (Fig. 3D; quantitation shown in Supplementary Fig. 3S, p=0.035). PAFr−/− embryos showed no proliferation consistent with the absence of CW entering the brain. Furthermore, expression of TLR2, the innate immune receptor for CW, was required for neuronal proliferation, as TLR2 -deficient embryos did not show a significant increase in Nestin-GFP signal post-CW exposure (Fig. 3D).
To further confirm that CW exposure induced neuronal proliferation in the developing brain, E15 pregnant FUCCI mice, that express genetically encoded markers for the cell cycle transition from G1 to S phase (Sakaue-Sawano et al., 2008), were challenged with CW and dual color intensity of the fetal cortex was analyzed 24hrs later (Fig. 3E). The number of geminin positive dividing cells was significantly increased in the ventricular zone of the fetal cortex post-CW exposure. Immunohistochemical tracking of phosphohistone H3, a general marker of neuronal proliferation, revealed that CW induced a highly significant tripling of proliferating cells in the ventricular zone (Fig. 3F: mean ±SD MFI 319% ± 50 over PBS baseline of 100% ± 62; p= 0.0003). Taken together, results indicate that exposure of embryos to CW from the maternal circulation induces neuroproliferation in the ventricular zone of the cortex that results in an increased cell density in the cortical plate.
To examine the mechanism of the fetal cell response to CW, neuronal precursor cells (NPCs) from fetal brain were harvested and proliferation after CW challenge in vitro was measured either by direct counting by microscopy, by incorporation of BrdU or by loading with CFSE label followed by FACS to quantify dilution of the CFSE signal as cells divided. Using the CSFE assay, CW clearly induced a shift from bright to dim cells compared to PBS treatment indicating active cell division (Fig. 4A), while TLR2−/− and PAFr−/− NPCs failed to proliferate (Fig. 4B). Direct visual enumeration confirmed CFSE assay proliferation (Supplementary Fig. 4SA). Incorporation of BrdU as a proliferation marker also clearly indicated wild type cells proliferated strongly, while PAFr−/− cells showed a low but detectable response and TLR2−/− cells showed no response (Supplementary Fig. 4SB). The degree of proliferation correlated directly with the CW dose but was independent of the timing of the NPC harvest between E12 and E14 (Supplementary Fig. 4SC). Living pneumococci (deficient in the toxin pneumolysin) treated with ampicillin but not clindamycin also induced proliferation (Supplementary Fig. 4SD). Staphylococcal peptidoglycan also induced proliferation (ranging between 20 to 30% of cells above PBS baseline) supporting the general relevance of cell wall/TLR2 signaling. To determine if chemically distinct TLR2 agonists induced proliferation, TLR2/1 agonist Pam3Cys and TLR2/6 agonist MALP2 were compared (Fig. 4C; Supplementary Fig. 4SE). Only Malp2 induced proliferation indicating TLR2/6 heterodimers were likely key to signaling.
The in vitro assay afforded the opportunity to eliminate the role of PAFr in transport of CW across the blood brain barrier and to dissect the role of the pneumococcal teichoic acid component of the cell wall in proliferation. A much stronger decrease in proliferation in PAFr TLR2 double knock out cells compared to single knock outs (Supplementary Fig. 4SA) suggested a role for both receptors in signaling proliferation. The attenuation of proliferation by EA CW or PAFr−/− cells in vitro also suggested at least some role for PAFr in proliferation (Supplementary Fig. 4SE).
Consistent with in vivo findings, CW associated with the neuronal surface and within cells in vitro (Fig. 4D). Neurons also produced significantly longer neurites in response to CW, a response comparable to the positive control VEGF (Fig. 4E). Stimulated NPCs showed increased expression of p-AKT and PI3Kinase (Fig. 4F). CW failed to induce the glial activation marker Iba-1 or secretion of inflammatory cytokines into the NPC supernatant fluid (Fig. 4G). Thus CW failed to induce TLR2/1 inflammatory markers or glial activation but induced mediators characteristic of the alternative TLR2/6 pathway in neuronal cells.
The neuronal nuclear transcription factor FoxG1 maintains progenitor cells (Dastidar et al., 2011; Regad et al., 2007) and is required for patterning of the telencephalon (Hebert and Fishell, 2008), neural cell transit through the cortex (Miyoshi and Fishell, 2012), and regulation of neural precursor proliferation (Martynoga et al., 2005). As these phenotypes were altered by CW, we tested if FoxG1 participated in the effects of CW on neuronal fate. When embryos were exposed to CW at E15, FoxG1 expression was strongly enhanced within 6 hrs of CW exposure, especially in the cortical plate (Fig. 5A). This response was sustained for up to 24 hrs and was similar in E10 embryos (data not shown). In contrast, FoxG1 expression was significantly muted in TLR2−/− or PAFr−/− embryos challenged with CW (Fig. 5A). FoxG1 staining co-localized with the expression of neuronal markers NeuN and tubulin β-III (Fig 5B, Supplementary Fig. 5SA). Adjacent brain sections demonstrated strong induction of expression of PI3kinase, a protein in the FoxG1 signaling cascade (Supplementary Fig. 5SB). Induction of FoxG1 required CW entry into the brain since challenge with EA CW failed to induce enhanced FoxG1 staining (Fig. 5C). In vitro challenge of NPCs indicated that induction of FoxG1 expression was dependent on CW dose (Fig. 5D). Taken together, these data suggest that FoxG1, an important regulatory transcription factor that has central significance in the development of the fetal cortex, is strongly enhanced in the embryonic brain after CW exposure. Furthermore, CW induced signaling via TLR2 played a role in the increased expression of FoxG1 in the fetal brain.
In humans and postnatal mice, pneumococcal meningitis often results in neuronal loss with subsequent impaired memory and cognitive function (Grandgirard et al., 2007; Schmidt et al., 2006; Tauber et al., 2009). In contrast, our data suggest that, within the fetal cortex, CW enhances proliferation of neurons leading to an increase in overall number of neurons in the cortical plate. We sought to determine if this increased proliferation affected postnatal behavior as assessed by three assays of spatial learning and working memory (Supplementary Fig. 6S). In the spatial recognition format, the time spent examining novel arms of the maze was significantly less time for pups exposed to CW at E10 (Fig. 6A). In the delayed non-matched to position Y maze test, pups were tested for working memory sequentially over weeks postnatally (Fig. 6B). Between 6–9 weeks of age, the PBS exposed mice improved memory function as denoted by an increase in the percentage of novel choices while the CW exposed mice made significantly fewer novel choices as they aged. These data suggest that the increased neuronal density in the cortical plate after early exposure to CW in utero (i.e. E10) was associated with a detrimental effect on the memory and cognitive function of postnatal mice.
To determine if the timing of CW challenge during pregnancy affected subsequent cognitive deficits, mothers were challenged with CW at E10 or E15. The pups were carried to term and tested in the Novel Object Recognition test of memory and anxiety at 3 months of age. E10 challenged pups demonstrated significantly less exploration measured as total time, time with an old object or time with a new object (Fig. 6C). E15 challenged pups showed no differences from PBS controls. Thus, although CW challenge at E10 but not E15 induced proliferation in vivo and was associated with subsequent behavioral abnormalities.
The maternal-fetal environment shapes the development and long-term function of the fetal brain, including the consequences of exposure in utero to inflammatory or infectious stimuli. The peptidoglycan-teichoic acid complex of bacterial CW is a potent library of bioactive components that interacts with innate immune receptors to generate inflammation during infection. In the postnatal setting, CW engages PAFr to translocate across endothelial barriers from blood into brain, a process that is also operative at the placental and fetal blood brain barrier. Once in the brain in the postnatal setting, CW interacts with TLR2 engendering an intense acute phase inflammatory response recognized clinically as meningitis. Our data indicate that the TLR2-dependent response to this PAMP in the fetus is fundamentally distinct from the postnatal scenario. Inflammation is strikingly absent in the fetal brain and TLR2-dependent neuronal death is dramatically mitigated. Furthermore, the developing fetal brain responds with neuroproliferation. CW trafficked quickly from the maternal circulation to the fetal brain and then appeared to be cleared over the course of several days. Within 24hrs of CW exposure, proliferation of new neurons was prominent in the ventricular zone of the cortex. Exposure early in gestation (E10) engendered proliferation sufficient to increase neuronal density in the cortical plate at E16. This suggests that immature neurons underwent proliferation in the ventricular zone and then migrated to and accumulated in the cortical plate over time. E15 challenge with E16 histopathology showed no increase in cortical plate neuronal density suggesting either the window for the effect closes late in gestation, or the new neurons did not have time to migrate to the cortex within 24 hrs.
After injury to the postnatal brain, a repair response is limited. Modulation of the brain repair response so as to improve neurogenesis has been sought to improve the outcome of affected patients. For instance, apoptosis can be prevented by increased signaling via fas-apoptotic inhibitory molecule 2 (Reich et al., 2011) or by administration of caspase inhibitors (Braun et al., 1999). Alternatively, some induction of neurogenesis in adult hippocampal cells has been seen by enhancing signaling via TLR2, decreasing signaling by TLR4 (Rolls et al., 2007) or by blockade of IL-6 (Monje et al., 2003). In contrast, TLR2 signaling induced by synthetic ligands in the embryonic brain (E15) has been shown to decrease neuronal proliferation associated with enlarged cerebral ventricles (Okun et al., 2010). Our data indicate that a strong neurogenerative response occurs in the early fetal brain upon challenge with bacterial PAMPs recognized by TLR2 and this response involves a previously unrecognized link between TLR2 and the neuronal transcription factor FoxG1.
FoxG1 is a transcription factor required for development and patterning of the telencephalon in the embryo (Danesin et al., 2009; Hebert and Fishell, 2008; Manuel et al., 2010; Martynoga et al., 2005) and has been referred to as a spatio-temporal hub gene in the brain. Dysregulation of FoxG1 is associated with medulloblastoma (Adesina et al., 2007), impaired neurogenesis (Shen et al., 2006) and autism spectrum disorder (Mariani et al, 2015). Recent studies have also implicated FoxG1 in promoting survival in differentiated, post-mitotic neurons (Dastidar et al., 2011). Our data show that fetal CW exposure greatly enhanced FoxG1 expression in both immature neurons of the ventricular zone and particularly in post-mitotic neurons in the cortical plate. Neuroproliferation could arise by the known ability of FoxG1 to antagonize the cytostatic signal of TGFβ-FoxO-Smad proteins (Chan et al., 2009; Seoane et al., 2004).
CW-induced neuroproliferation and enhanced FoxG1 expression were both dependent on TLR2. TLR2 is a heterodimer with TLR1 or TLR6 (Fukao and Koyasu, 2003). CW/TLR2/1 is known to induce inflammation via MyD88 activation of NFkb but this canonical pathway was not strongly activated in the fetus as evidenced by the striking lack of inflammation in the CW exposed fetal brain and the absence of in vitro neuroproliferation in response to the TLR2/1 agonist. More recently, it has been shown that TLR2/6 down regulates inflammation and induces cell proliferation by signaling via PI3 Kinase and p-AKT (Fukao and Koyasu, 2003). Exposure of NPCs or fetal brains to CW induced PI3 Kinase, p-AKT and neuroproliferation. PI3 Kinase, like FoxG1, antagonizes the cytostatic signal of TGFβ-FoxO-Smad proteins (Chan et al., 2009; Seoane et al., 2004). Thus, FoxG1 and TLR2/6 signaling could converge on TGFβ-FoxO-Smad to alleviate cytostasis. It is also possible that there is an as yet unknown direct link between TLR2 and FoxG1 given the TLR2 dependence of the CW-induced increase in FoxG1 expression in vivo. It is possible that the fetal brain escapes cell death and inflammation while inducing neuroproliferation by TLR2/6 mediated signaling in contrast to the postnatal TLR2/1 scenario.
Neuroproliferation, which occurs variably throughout life (van Praag et al., 2002), is a necessary step toward brain repair. The accumulation of neurons however, may not assure appropriate integration into functioning neuronal circuitry (Scharfman and Hen, 2007). For instance, Bax-deficient mice exhibit excess neurons leading to altered neuroanatomy and behavioral deficits (Buss and Oppenheim, 2004; Kim et al., 2009). Our data indicate a similar outcome in the setting of CW exposure in that expansion of mature neurons in the cortical plate was associated with abnormalities in learning and memory and social behavior between 6 weeks and 5 months after birth. It has been shown that the balance of pro- and anti-inflammatory signals present during fetal development can have an effect on later cognitive function (Fukao and Koyasu, 2003). While a specific mechanism for cognitive and behavioral abnormalities as a result of fetal exposure to CW is unknown, our data suggest that changes in the balance of neuroproliferation and cell death in the developing brain likely contribute to a final outcome of altered brain function in mice exposed to CW in utero.
The similarity of the fetal neuroproliferative response between the CW challenge model and the live infection model raises the possibility that the use of bacteriolytic antibiotics during pregnancy may have significant untoward effects on embryogenesis. On the other hand, there is a potential beneficial clinical relevance in understanding the timing and mechanism by which TLR2 drives neuroproliferation in the fetal brain to efforts to improve brain repair.
Wild type C57BL/6 (The Jackson Laboratory), PAFr−/− (Radin et al., 2005), TLR2−/− (Yoshimura et al., 1999), FUCCI (Sakaue-Sawano et al., 2008), and Nestin promoter GFP-transgenic mice (Yamaguchi et al., 2000) were used in designated experiments. Nestin-GFP mice were backcrossed >6 times with PAFr−/− and TLR2−/− mice for use in neurogenesis experiments. All mice were maintained in the St. Jude Animal Resource Center BSL 1 and 2 housing, and all experiments were carried out in accordance with institutional guidelines and NIH protocols. Timed pregnancies were determined by ultrasound.
Placental health was monitored by pulse-wave doppler measurements were obtained using a VEVO-770 High Resolution Ultrasound System (VisualSonics, Toronto, Canada) equipped with a RMV-706 (40MHz) transducer operating at a transmit frequency of 30 MHz. Pulse-wave (PW) Doppler mode was then used to determine flow within the target umbilical cord prior to treatment and at fixed time-points after intravenous administration of pneumococcal CW. For post-processing of each data set, maternal heart rate was determined from the real-time ECG over a 10-beat period. Umbilical flow was similarly determined over 5 cycles and built-in calibrated software tools were used to determine both fetal heart rate and umbilical vein blood flow velocity.
Working memory of WT CW treated mice was tested as previously described using three assays (Bevins and Besheer, 2006; Crawley, 2007): novel object recognition, spatial recognition and delayed non-matched to position tests. For Novel Object Recognition, mice underwent testing at 3 months of age as previously described. Briefly, mice were acclimated to a clear field in a dimly lit room for 5 minutes on Day 1. On Day 2, mice were presented with two of the same object (red glass diamond) in the boxed field and allowed to explore for 3 minutes. On Day 3, one old object and one new object (white porcelain rectangle) were placed in the field and mice were allowed to explore for 2 minutes. Time spent at each object on Days 2 and 3 was recorded by video. Discrimination indices were determined by the equation: (New Object − Old Object)/Total Time. For spatial recognition, a Y-maze test was conducted in a three-arm maze made from white opaque polyvinyl plastic. The mice were exposed to the start and open arms for 8 minutes. The novel arm was gated. After one hour, each mouse was exposed to all three arms for 5 minutes. Total exploration time in each arm was noted. For delayed non-matched to position, water deprived mice were exposed for 10 minutes to all 3 arms of the Y maze, the open and novel arms containing chocolate milk. The following day mice were allowed access to the start arm and an open arm. Once the mouse reached the open arm, it was returned to the start and the gate for the novel arm was lifted. A correct choice was made if the mouse initially entered the novel arm. This test was repeated randomly switching which arm was gated.
Highly purified CW was prepared as described previously from Streptococcus pneumoniae unencapsulated strain R6 (Tuomanen et al, 1985a and b) and tested endotoxin free by Limulus gel assay (Genscript). This method results in multimeric peptidoglycan with attached teichoic acid that is free of protein adducts and lipid contaminants as determined by HPLC and mass spectrometry of subcomponents (Garcia Bustos et al, 1987). The same procedure was used to purify EA CW from cultures grown in media substituting ethanolamine for choline resulting in modification of the aminoalcohol on the teichoic acid. These preparations have been studied extensively as biologically active and structurally defined TLR2 ligands (Yoshimura et al, 1999). Staphylococcal peptidoglycan was purchased (Sigma). For mouse challenge, CW was suspended in PBS, sonicated, and administered intravenously to pregnant dams as 2×107 bacterial equivalents. For some experiments, CW was directly labeled with 1mg/ml FITC (fluorescein isothiocyanate; Sigma) in carbonate buffer (pH 9.2) for 30min at RT in the dark and washed twice with PBS. Unless otherwise noted, dams were challenged at E10 and fetal brains were harvested at E16.
For live maternal infection and antibiotic treatment, dams at E9 were given 3×106 cfu S. pneumoniae strain TIGR4X pln- intratracheally; pneumolysin negative mutant was chosen to eliminate effects of the toxin on the CW phenotype. The course of systemic infection was monitored over 24 hours by Xenogen imaging and blood titers were obtained to assure consistent bacterial load (mean 5 × 105 cfu/ml). The dams were then treated with either ampicillin (100mg/kg PO every 12 hours) or clindamycin (15mg/kg PO every 12 hours) until E16 when embryos were harvested for analysis of cortical morphology.
Fetal brains and placenta were fixed in 3% paraformaldehyde (PFA) and then rehydrated in 30% sucrose. Cryosections were cut to 20μm and stained for histology by Haematoxylin and Eosin (Sigma) or Vectastain ABC kit (Vector Laboratories). Sections were treated with antibodies to FoxG1 (AbCam), TLR2 (Sigma), PAFr (Santa Cruz Biotech), phosphohistone H3 (Cell Signaling), NeuN (Millipore), Iba1 (Abcam), Tubulin III (Millipore) and fluorescent secondary antibody (Invitrogen). Methyl green (Vector Laboratories), Alexa Fluor 568 (Invitrogen) or Prolong Gold with DAPI (Molecular Probes) were used as counterstain. For TUNEL (Millipore) and Caspase-3 (Cell Signaling) positive cell counts, the area of the cortex was measured and individual positive cells were counted. Immunofluorescent sections were imaged using a Zeiss LSM 510 NLO Meta microscope equipped with HeNe, Argon/2 and Chameleon lasers. Confocal images were analyzed using Zen 2008 software. Histology sections for brightfield imaging were obtained with a Nikon E800 microscope. Image analysis, including MFI determination, was done using ImageJ software.
Calculations of cortical area and cell number per cortex were based on standard stereology methods using the Microbrightfield StereoInvestigator with Cavalieri estimator and the optical fractionator probe, respectively (MBF Biosciences, Williston, VT). Briefly, closely matched paraffin-embedded sagittal sections (three per embryo) of the right cortex were stained with cresyl violet and the average area and cell number were calculated per mouse. Measurements were performed on five to seven animals in each experimental group.
Embryonic NPCs were cultured from WT, PAFr−/− or TLR2−/− dams at E12, E14, or E16 using NB27 medium (Life Technologies). Briefly, brains were digested in 6 mg/mL papain (Sigma) for 30 minutes at 37°C. Tissue was washed twice before sequential trituration in culture media. Dispersed cells were spun, resuspended at 5×105 cells/mL, and plated on poly-D-lysine (Sigma) coated plates. For in vitro proliferation assays, cells were stained while in suspension using CellTrace CFSE Proliferation kit according to manufacturers protocol (Molecular Probes). One day after plating, cells were treated with CW at a MOI of 10. 48 hours later, cells were gently harvested by cell scraper and analyzed by FACS Canto II (Becton Dickinson). For challenge with lytic debris of living bacteria, the pneumolysin deficient mutant TIGR4 pln- was grown to 106 cfu/ml, treated with 10x MIC of ampicillin or clindamycin for 20 min and then applied to NPCs. In some cases, JEG-3 choriocarcinoma cell line ATCC HTB-36 was substituted to represent placental trophoblast cells.
Two assays were used to confirm the CSFE assay. Cell number was quantitated manually by counting in a hemocytometer. Proliferation was also measured by incorporation of BrdU (EMD Millipore). NPC’s were seeded in 96 well plates and treated with CW the next day. Three days later, BrdU was added and, 24 hours later, the cells were fixed and analyzed according to the manufacturer’s protocol.
To visualize CW interactions with neurons, NPCs were exposed to FITC-labeled CW for 48 h, fixed with 3% (v/v) PFA and visualized by confocal microscopy. Extracellular CW was stained with anti-FITC goat sera (Invitrogen) without permeabilization, followed by Alexa Fluor 647-conjugated chicken anti-goat sera (Invitrogen). For identification of neuronal cells, cells were stained with rabbit anti-NeuN sera (Abcam) in the presence of 0.2% (v/v) saponin, followed by Alexa Fluor 568-conjugated donkey anti-rabbit sera (Invitrogen).
To determine CW effects on outgrowth of neurites, NPCs were stimulated with CW (MOI of 10), positive control VEGF (5nM) or PBS. 24 hours post-stimulation, cultures were fixed and immunolabeled with NeuroD1 antibody and an Alexa 488 conjugated secondary antibody. Cultures were imaged with a spinning disc confocal at 40x, and length of individual neurites was measured in Slidebook. Neurons with individually resolveable neurites longer than cell body size were analyzed.
Protein expression in CW treated NPCs was analyzed by western blot. Cells were lysed 5 minutes on ice using NPer neuronal protein extraction buffer (Thermo Scientific), gently scraped and spun. Supernatant was harvested and total protein was quantitated. Undiluted supernatants were analyzed for cytokine levels using mouse IL-6 and CXCL1/KC ELISA kits (R&D Systems) according to manufacturers protocol.
All statistics were done using GraphPad Prism (GraphPad). Two-tailed T-tests with Welch’s correction, Mann-Whitney, and 2-way Anova tests were done as noted.
The authors would like to thank the St. Jude Animal Resource Center and St. Jude Small Animal Imaging Center, especially Dr. Chris Calabrese and Melissa Johnson, RLAT, for help with timed matings and ultrasound data collection and analysis. We thank the St. Jude Cell and Tissue Imaging Center for microscopy assistance (supported by SJCRH and NCI P30 CA021765-35). We thank Drs. Masahiro Yamaguchi and Atushi Miyawaki from the RIKEN Institute, Japan for the FUCCI mice. We thank St Jude colleagues Donnie Eddins for his advice on the animal behavior analysis and Dr. Justin Thornton for review of the manuscript. PM was supported by NCI R25 CA23944; ET was supported by NIAID R0127913 and ALSAC.
The authors have no conflicts of interest to disclose.
Author contributionsConceptualization, E.I.T.; Methodology, J.H., B.M., Y.H., J. R. and R.J.S.; Investigation, J.H., B.M., G.G., P.M., L.N.L, A.F., Y.H., J. R. K.D-E., and S.A.F; Writing – Original Draft, J.H., B.M., and E.I.T.; Writing – Review & Editing, J.H., B.M., L.N.L, and R.J.S.; Funding Acquisition, E.I.T.; Resources, R.J.S.; Supervision, R.J.S. and E.I.T.
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