PreImplantation factor promotes neuroprotection by targeting microRNA let-7

Significance

Secreted from viable embryos into the maternal circulation, PreImplantation factor (PIF) has long been implicated in modulating maternal immune tolerance and promoting embryo implantation. Recent evidence also suggests its possible therapeutic use in CNS disorders. However, the molecular signal-transduction pathways driving PIF-mediated effects are unclear. Using murine cell culture combined with a rat hypoxic-ischemic brain injury model, we show that synthetic PIF (sPIF) inhibits microRNA let-7 biogenesis by destabilizing the key microRNA-processing protein, KH-type splicing regulatory protein, in a Toll-like receptor 4–dependent manner. This regulation, together with its induced upregulation of the anti-inflammatory cytokine IL-10, may contribute to sPIF-mediated neuroprotection in vivo and also may underlie PIF's physiological role in maternal immune modulation and embryo implantation.

Abstract

Dysfunction and loss of neurons are the major characteristics of CNS disorders that include stroke, multiple sclerosis, and Alzheimer’s disease. Activation of the Toll-like receptor 7 by extracellular microRNA let-7, a highly expressed microRNA in the CNS, induces neuronal cell death. Let-7 released from injured neurons and immune cells acts on neighboring cells, exacerbating CNS damage. Here we show that a synthetic peptide analogous to the mammalian PreImplantation factor (PIF) secreted by developing embryos and which is present in the maternal circulation during pregnancy inhibits the biogenesis of let-7 in both neuronal and immune cells of the mouse. The synthetic peptide, sPIF, destabilizes KH-type splicing regulatory protein (KSRP), a key microRNA-processing protein, in a Toll-like receptor 4 (TLR4)–dependent manner, leading to decreased production of let-7. Furthermore, s.c. administration of sPIF into neonatal rats following hypoxic-ischemic brain injury robustly rescued cortical volume and number of neurons and decreased the detrimental glial response, as is consistent with diminished levels of KSRP and let-7 in sPIF-treated brains. Our results reveal a previously unexpected mechanism of action of PIF and underscore the potential clinical utility of sPIF in treating hypoxic-ischemic brain damage. The newly identified PIF/TLR4/KSRP/let-7 regulatory axis also may operate during embryo implantation and development.

PreImplantation factor (PIF) is a conserved, embryo-derived peptide that can be detected in human, mouse, and bovine maternal circulation during pregnancy (1, 2). The presence of PIF in the maternal circulation has been correlated with live births in murine and bovine models (2–4). Despite the long-standing understanding that PIF is involved in promoting maternal immune tolerance and embryo implantation (2, 5–8), the mechanism by which PIF does so has remained poorly understood. Recently, a synthetic PIF analog (sPIF) of 15 amino acids (MVRIKPGSANKPSDD) was reported to elicit neuroprotective effects in a murine model of experimental autoimmune encephalomyelitis (9). When administrated s.c., sPIF inhibited neuroinflammation, promoted neural repair, and prevented paralysis (9). Analysis of cytokines combined with genomic and proteomic approaches revealed global alterations in gene expression, suggesting that sPIF induced coordinated central and systemic multitargeted effects (9). However, the molecular signal-transduction pathways underpinning the observed effects were not defined.

Premature birth is a major cause of neonatal morbidity and mortality (10). Depending on the degree of prematurity 15–20% of affected newborns die during the postnatal period, and ∼25% of survivors suffer significant long-term disability including cerebral palsy, epilepsy, increased hyperactivity, and developmental disorders (11). Therapeutic approaches to counteract the cascades of neonatal brain injury have been proposed. Unfortunately, thus far no individual neuroprotective agent has been proven safe and effective in immature infants (12).

The developmentally regulated, imprinted H19 is among the most highly expressed genes in developing embryos (13). The H19 gene encodes a 2.6-kb noncoding RNA H19 known to reduce the bioavailability of let-7 by acting as a microRNA sponge (14). Let-7 regulates target gene expression by binding to imperfectly complementary sequences in the mRNA, inducing translational repression and mRNA destabilization (15). The biogenesis of let-7 involves multiple steps. It is first synthesized as long primary transcripts (pri-let-7), which are cleaved in the nucleus by the Drosha complex, yielding a hairpin precursor (prelet-7) of ∼70 nt. Prelet-7 then is exported to the cytoplasm where it is further processed into mature let-7 of 22 nt by the Dicer complex (16). Regulation of microRNA processing has emerged as a major step in controlling mature microRNA levels (17). The RNA-binding proteins LIN28 and hnRNP A1 act to inhibit let-7 processing (18–20), whereas KH-type splicing regulatory protein (KSRP) promotes it (21).

Because developing embryos secrete PIF (3), and H19 is highly expressed in the embryo (13), we hypothesized that PIF might regulate H19 or let-7 expression. In the current study, we show that sPIF inhibits the biogenesis of let-7 by destabilizing the microRNA-processing protein KSRP in cultured neuronal and immune cells and that this regulation depends on the expression of Toll-like receptor 4 (TLR4). We provide evidence that this novel PIF/TLR4/KSRP/let-7 regulatory pathway, combined with its induced up-regulation of the anti-inflammatory cytokine IL-10, may contribute to sPIF’s neuroprotective effects in vivo. We propose that this regulatory mechanism also may underlie the action of PIF in maternal immune modulation and embryo implantation and development.

Results

sPIF Decreases Let-7 Levels in Both Neuronal and Macrophage Cells.

Given the high expression level of H19 in developing embryos (13), which also secrete PIF (3), we set out to examine whether PIF might regulate H19 gene expression. Thus we performed experiments using murine N2a neuroblastoma cells and RAW 264.7 macrophage cells, because sPIF targets both neuronal and immune cells (2, 9). Although we detected only negligible levels of H19 expression, we unexpectedly noticed increased expression of Hmga2 and Dicer in cells treated with sPIF as compared with cells treated with a control scrambled peptide (PIFscr) (Fig. S1 A and B). Hmga2 and Dicer are validated targets of let-7 (22, 23). Let-7 binds to imperfectly complementary sequences in the respective mRNAs, leading to translational repression and mRNA destabilization (22–24). Thus, sPIF might have a direct effect on let-7 levels. Indeed, let-7 was down-regulated in cells treated with sPIF in comparison with cells treated with PBS or PIFscr at the same concentration (Fig. S1C). Further scrutiny revealed that sPIF in both cell types down-regulated the expression of several let-7 family members in an apparently dose- and time-dependent manner (Fig. 1 A–D). sPIF-induced let-7 suppression also was observed in BV-2 cells, a murine microglia cell line derived from resident macrophages of the CNS (Fig. S2A) (25). These results support the notion that sPIF negatively impacts let-7 levels in both neuronal and macrophage cells.

Fig. 1.

sPIF down-regulates let-7 in neuronal (N2a) and macrophage (RAW) cells in a TLR4-dependent manner. (A and C) Cells were incubated with sPIF or PIFscr at the indicated concentrations. RNAs were extracted 48 h later, and levels were determined by qRT-PCR. Let-7 levels from sPIF-treated cells are presented after normalization against those from PIFscr-treated cells, which were arbitrarily set as 1. (B and D) N2a and RAW cells were incubated with sPIF or PIFscr (at 300 nM for N2a cells and 200 nM for RAW cells). RNAs were harvested at the indicated time points, and levels were determined. (E and F) Cells were transfected with control siRNA (siCon) or siRNA specific for TLR4 (siTLR4). RNAs and proteins were harvested 48 h posttransfection and were analyzed by qRT-PCR (Upper) and Western blot (Lower). (G–J) Cells were transfected with siCon or siTLR4. sPIF (+) or PIFscr (−) was added to the cells (300 nM for N2a cells, 200 nM for RAW cells) 48 h posttransfection, and cells were incubated for an additional 48 h. RNAs were extracted, and levels were determined by qRT-PCR. Results are presented with let-7a (or KSRP mRNA) levels in PIFscr-treated cells arbitrarily set as 1. (K and L) Cells were treated as described in G–J. Proteins were harvested after 48-h incubation with PIFscr (−) or sPIF (+). KSRP protein levels were determined by Western blot analysis using β-tubulin (TUBB) as a loading control. Except in K and L, all numbers are mean ± SD (n = 3), and are representative of three independent transfection results. **P < 0.01. In K and L, the numbers were derived from quantitation of protein bands on the presented gels representative of two independent transfection results. The levels of the KSRP protein (after normalization against β-tubulin) in the PIFscr-treated cells were arbitrarily set as 1.

TLR4 Expression Is Necessary for sPIF-Induced Let-7 Repression.

Let-7 is down-regulated in macrophages and epithelial cells as a host defense mechanism against bacterial pathogens (24). This microRNA alteration occurs by TLR4’s sensing bacterial LPS (24). We hypothesized that TLR4’s sensing sPIF also might mediate let-7 repression. Thus, TLR4 was down-regulated using an siRNA (siTLR4) previously reported to silence TLR4 specifically and efficiently in murine cells (26). Although siTLR4 led to significant suppression of TLR4 in both N2a (Fig. 1E) and RAW (Fig. 1F) cells, sPIF-induced let-7 repression was concomitantly abolished (Fig. 1 G and H, compare the two columns on the left with the two columns on the right). Similar results were observed in BV-2 cells (Fig. S2 B and C). These results suggested that TLR4 expression is required for sPIF-mediated let-7 repression.

KSRP Is Down-Regulated by sPIF in a TLR4-Dependent Manner.

As an integrated component of both Drosha and Dicer complexes, KSRP acts to regulate the biogenesis of a cohort of microRNAs including let-7 (21, 27). KSRP binds with high affinity to the terminal loop of microRNA precursors and promotes their processing. Indeed, siRNA-mediated knockdown of KSRP in cultured human and mouse cells inhibits microRNA processing, leading to decreased let-7 levels (21). Because the activation of TLR4 by LPS in human epithelial cells led to decreased KSRP levels (28), we wished to test whether KSRP is involved in sPIF-induced let-7 repression and whether this regulation is dependent on TLR4 expression. Thus, we analyzed the effects of sPIF in combination with TLR4 knockdown on KSRP expression. Although sPIF did not affect KSRP at the mRNA level, with or without TLR4 knockdown (Fig. 1 I and J), it caused a reduction of at least 50% in the KSRP protein level as compared with PIFscr-treated cells (Fig. 1 K and L, compare lanes 2 with lanes 1). This inhibition was abrogated in the siTLR4-transfected cells (Fig. 1 K and L, compare lanes 4 with lanes 3). Similar observations were obtained with BV-2 cells (Fig. S2D). These results suggested that in both neuronal and immune cells KSRP was down-regulated by sPIF in a TLR4-dependent manner, and this down-regulation appeared to be at the posttranslational level.

sPIF Induces Destabilization of KSRP Protein.

To test the possibility that accelerated protein decay might contribute to the observed decrease in KSRP, we carried out Western blot analyses in the presence of cycloheximide (CHX), an inhibitor of de novo protein synthesis. In both RAW and N2a cells, KSRP showed a faster decay in sPIF- than in PIFscr-treated cells (Fig. 2 A and F, compare lanes 1–5 with lanes 6–10). Based on a rough estimation, the KSRP protein half-life was ∼45 min in PIFscr-treated RAW cells and 180 min in sPIF-treated RAW cells. For N2a cells, these half-lives were ∼60 min and 180 min, respectively. Together, these results are consistent with sPIF-induced destabilization of KSRP protein.

Fig. 2.

sPIF represses let-7 by destabilizing KSRP, which involves PI3K/AKT signaling. (A and F) Cells were incubated with sPIF (+) or PIFscr (−) (200 nM for RAW cells and 300 nM for N2a cells) for 24 h, followed by the addition of CHX. Proteins were harvested at the indicated time points and were analyzed. KSRP protein levels are presented after normalization against β-tubulin, with the KSRP level at the 0 time point arbitrarily set as 1. Results are representative of three independent experiments. (B, C, G, and H) Cells were treated with sPIF (+) or PIFscr (−) in the presence or absence of Akt Inh for 48 h. Proteins and RNAs were isolated, and levels were determined by Western blot and qRT-PCR, respectively. (D, E, I, and J) Cells were transfected with siCon or siRNA specific for KSRP (siKSRP). Proteins and RNAs were extracted 48 h later and were analyzed by Western blot and qRT-PCR, respectively. Except in A and F, all numbers are mean ± SD (n = 3), and are representative of three independent experiments. **P < 0.01. (K) Proposed model for sPIF-induced let-7 repression. PIF activates PI3K/AKT signaling through TLR4, leading to KSRP protein degradation, which in turn decreases the production of mature let-7.

The PI3K/AKT Signaling Pathway Is Involved in sPIF-Induced Destabilization of KSRP Protein.

Given that KSRP is a substrate of PI3K/AKT phosphorylation (29) and that stimulation of TLR4 by LPS activates the PI3K/AKT signaling pathway (30, 31), we hypothesized that PI3K/AKT signaling might be involved in sPIF-induced KSRP protein destabilization. Thus, we inhibited PI3K/AKT signaling in RAW cells using an inhibitor specific for AKT (Akt Inh). Although, as expected, sPIF reduced KSRP (Fig. 2B, Upper, compare lane 2 with lane 1), such reduction was not seen in the presence of Akt Inh (Fig. 2B, Upper, compare lane 3 with lane 2). Importantly, application of the inhibitor also abolished sPIF-induced let-7 repression (Fig. 2C). Similar results were obtained in N2a cells (Fig. 2 G and H). Furthermore, in both RAW (Fig. 2D) and N2a (Fig. 2I) cells, siRNA-mediated KSRP knockdown caused a decrease in let-7 (Fig. 2 E and J). Similar results were obtained in BV-2 cells (Fig. S3). These results suggested that PI3K/AKT signaling is involved in the sPIF-induced destabilization of KSRP protein, which in turn impairs let-7 biogenesis, thereby decreasing let-7 levels (Fig. 2K).

sPIF Promotes Neuroprotection in Vivo.

The role of let-7 in inducing neuronal cell death and hence in the spreading of CNS damage has been firmly established through both in vivo and in vitro studies by multiple research groups. For example, Lehmann et al. (32) showed that let-7 released from injured cells to the extracellular environment during CNS damage activated Toll-like receptor 7 in neuronal cells, leading to neurodegeneration in the mouse. Likewise, intracerebroventricular injection of an antagomir to let-7 significantly reduced both cortical and striatal infarcts in a rat ischemic stroke model (33). Because sPIF decreases let-7 levels (Fig. 1 A–D and Fig. S2 A and C), we surmised that sPIF might function to reduce CNS damage, in part, through decreasing let-7 levels in injured brains. Our previously reported newborn rat hypoxic-ischemic brain injury model recapitulates very preterm human infants who are highly susceptible to brain damage and postnatal infections (34). Such significant damage exerts detrimental effects on both mental and motor development (35–37). Preterm human infants share similarities to postnatal day 3 (P3) rats in terms of cortical neuronal and glial development.

Thus, we induced brain injury at P3, followed by sPIF treatment at P6, and harvested brain samples at P13 (Fig. 3A). We observed a significant loss of cortical volume in the injured animals (Fig. 3B, compare the center and left columns), which was abolished by sPIF treatment (Fig. 3B, compare the right and center columns). The sPIF administered s.c. colocalized with both neurons (Fig. 3C, Upper) and glia (Fig. 3C, Lower). The significant neuronal loss in deep cortical layers (Fig. 3D, compare the center and left columns, and Fig. 3E, compare the center and top right panels) was reduced in sPIF-treated animals (Fig. 3D, compare the center and right columns, and Fig. 3E, compare the bottom and middle right panels). Microglia of macrophage lineage represent both the target and source of injury in CNS (38, 39). Decreased glial activation and a restored number of neurons have been associated with reduced cerebral response to injury (40, 41). We observed increased activation of microglia (Fig. 3F, compare the center and left columns, and Fig. 3G, compare the middle and top right panels), which were abrogated by sPIF treatment (Fig. 3F, compare the right and center columns, and Fig. 3G, compare the bottom and middle right panels). Further, we observed morphological changes in Iba-1⁺ microglia shifting from a predominantly amoeboid to a ramified state in sPIF-treated animals (Fig. 3G, compare the cells indicated by red and blue arrowheads). Thus, our results are consistent with the notion that sPIF reduces inflammation and promotes neural protection by targeting neuronal and glial cells.

Fig. 3.

sPIF rescues cortical volume and neuronal loss while decreasing microglial activity in vivo. (A) Experimental outline. (B, D, and F) Quantification of cortical volume (B), neuronal loss (D), and microglial activation (F). Data are presented as mean ± SEM (n = 4 per group; two-tailed Student's t test). (C) Representative immunohistochemistry images of NeuN and Iba-1 staining and immunofluorescence images of sPIF (red) staining in injury (n = 4) and injury+sPIF (n = 4) cortical sections from the left hemisphere. Merged images are shown. (Scale bars, 25 μm.) (E and G) Representative immunohistochemistry images using cresyl violet with NeuN (for neuronal loss) (E) or Iba-1 (for microglial activation) (G) in cortical sections (n = 4 per group). Iba-1⁺ cells display a predominantly amoeboid state (red arrowheads) and a ramified state (blue arrowheads) in the left hemisphere. (Scale bars, 50 μm.)

Decreasing Let-7 and Increasing IL-10 Together May Contribute to sPIF-Mediated Neuroprotection in Vivo.

sPIF down-regulates let-7 in both neuronal and microglia/macrophage cells (Fig. 1 A–D and Fig. S2 A and C). Therefore, we predicted that sPIF also could decrease let-7 in the injured hemisphere, thereby contributing to the observed significant neuroprotection. Indeed, the levels of the four tested let-7 members were significantly lower in sPIF-treated than in vehicle-treated brains (Fig. 4 A–D). Furthermore, there was a significant decrease in the KSRP protein levels in sPIF- as compared with vehicle-treated brains (Fig. 4 E and F). These results provide compelling evidence that the PIF/TLR4/KSRP/Let-7 regulatory axis is active in vivo.

Fig. 4.

sPIF down-regulates KSRP and let-7 and up-regulates IL-10 in vivo. (A–D) RNAs were extracted from frozen brain tissues of sham, injury, and injury+sPIF rats (n = 5 animals per group). Levels of the indicated let-7 family members were determined by qRT-PCR. Results are presented after normalization against U6 RNA. (E) Proteins were extracted from frozen brain tissue of three animals in each group and were analyzed by Western blot. (F) Quantitation of E, with KSRP protein levels shown after normalization against β-tubulin. (G and H) BV-2 and RAW cells were incubated with sPIF (+) or PIFscr (−) (50 nM for BV-2 cells and 200 nM for RAW cells) for 48 h. RNAs were extracted, and levels were determined by qRT-PCR. IL-10 mRNA levels are presented after normalization against β-tubulin mRNA with the level in PIFscr-treated cells arbitrarily set as 1. Numbers are mean ± SD (n = 3). **P < 0.01. (I) RNAs were extracted from frozen brain tissues of sham, injury, and injury+sPIF rats (n = 5 animals per group). IL-10 mRNA levels were determined by qRT-PCR. Results are presented after normalization against β-tubulin mRNA.

IL-10, an anti-inflammatory cytokine, has a well-established role in neuroprotection (42–45). In addition to acting on glia and endothelium to reduce inflammatory responses following ischemic brain damage, it directly protects cortical neurons by activating the PI3K/AKT pathway via IL-10 receptors on neurons (45). As is consistent with IL-10 being a known target of let-7 (24, 46), we observed up-regulation of IL-10 in both BV-2 and RAW cells in sPIF-treated as compared with scrPIF-treated groups (Fig. 4 G and H). Significant IL-10 up-regulation also was evident in sPIF-treated as compared with vehicle-treated brains (Fig. 4I). Thus, the increasing levels of IL-10 also may contribute to sPIF’s neuroprotective effects in vivo.

Discussion

This report demonstrates that sPIF inhibits the biogenesis of let-7 microRNA in both neuronal and immune cells by downregulating the expression of the key microRNA-processing factor KSRP at the posttranslational level and that this regulation is dependent on TLR4 expression. We also show that short-term s.c. administration of sPIF in a clinically relevant model of newborn brain injury leads to a significant reversal of injury. We provide evidence that this novel PIF/TLR4/KSRP/let-7 signal-transduction pathway, together with its induced IL-10 up-regulation, may contribute to the observed sPIF-promoted neuroprotection in vivo.

Our data suggest that TLR4 has an important role in sPIF-induced let-7 repression (Fig. 1 E–H and Fig. S2 B and C). However, the exact mechanism by which sPIF interacts with TLR4 remains to be completely explained. sPIF may not bind TLR4 directly but rather may act via additional, unidentified cofactors, as is the case for LPS and the TLR4 complex (47). The divergent in vivo effects of sPIF (neuroprotection; see Fig. 3) and LPS (neuroinflammation) (48) suggest an association with distinct cofactors. Although LPS inhibits let-7 in vitro (24), other downstream effects of LPS (likely determined by cofactors) may override this potentially beneficial effect of LPS in vivo. Because in vivo repression of both KSRP and let-7 coincides with sPIF-induced neuroprotection (Figs. 3 and 4), it is conceivable that cofactors of sPIF act in concert to elicit downstream effects that are specific to sPIF but are different from those induced by LPS. Identification of the PIF-interacting cofactors is currently underway.

Both let-7 (49) and miR-27b (28) target the 3′ UTR of KSRP mRNA to induce mRNA degradation and translational suppression, respectively. However, regulation of ksrp gene expression at the posttranslational level via protein destabilization (Fig. 2 A and F) has not been documented previously, nor has the involvement of PI3K/AKT signaling in regulating KSRP protein stability (Fig. 2 B and G). KSRP has important roles in both microRNA biogenesis and the regulation of mRNA stability (29). AKT-mediated KSRP phosphorylation at a unique serine residue (S193) through PI3K/AKT signaling induces KSRP association with protein 14-3-3 and impairs KSRP’s ability to promote target mRNA degradation (50). S193 phosphorylation leads to the unfolding of KSRP, creating a binding site for 14-3-3, which drives the nuclear localization of KSRP and prevents it from destabilizing mRNA in the cytoplasm (51). sPIF-induced destabilization of the KSRP protein involves proteasomes (Fig. S3D), as is the case in metformin-induced c-MYC degradation (52). Metformin treatment of tumor cells activates AMPK, leading to c-MYC phosphorylation at the conserved Thr58, which in turn triggers proteasomal degradation of c-MYC (52). Destabilization of the KSRP protein may involve AKT-mediated phosphorylation at a specific site(s). Mapping the sites and determining the effects of phosphorylation will provide critical mechanistic insight into sPIF-induced KSRP destabilization, which warrants future investigation.

sPIF has a short half-life in circulation. For example, in the mouse, the circulating sPIF level peaked at ∼320 ng/mL 30 min after a single-dose s.c. injection of 40 mg/kg and was mostly cleared from circulation within 2 h (Fig. S4A). This peak concentration of sPIF was close to that of endogenous PIF detected in the pregnant bovine serum (∼227 ng/mL) (4). The dosage we used in the rats was 1.5 mg/kg/d (Materials and Methods), and, as expected, the circulating sPIF was undetectable 12 h after injection (Fig. S4B). Despite the rapid clearance of sPIF from circulation, potent neuroprotection was observed (Fig. 3). This finding suggests that the circulating sPIF concentrations are not a good indicator of the local concentration of sPIF at its site of action, as was noted by our immunofluorescence study using anti-PIF antibody at the same time point in the brain (Fig. 3C). Such a scenario would be consistent with our need to use higher concentrations of sPIF to observe sPIF effects in our cell-culture experiments.

As shown in this report, activation of PI3K/AKT signaling by sPIF leads to the down-regulation of let-7, which in turn increases IL-10 expression. Because IL-10 promotes PI3K/AKT signaling by acting on the IL-10 receptor (45), a possible positive feedback loop involving PI3K/AKT, let-7, and IL-10 may exist. Although highly speculative, such a putative feedback regulatory circuit may partially explain the dramatic increase in IL-10 expression in the sPIF-treated brain (Fig. 4I). Given the broad changes in the gene-expression profile induced by sPIF, as determined by both genomic and proteomic analyses (9), it is almost certain that PIF plays pleiotropic roles in gene regulation. Identification and characterization of the PIF/TLR4/KSRP/let-7 pathway represent critical first steps toward the mechanistic understanding of the multifunctional PIF.

Despite the recent purification and characterization of PIF (2), attempts to identify its gene have been unsuccessful so far. It is possible that the PIF gene resides within a highly complex and structured repetitive chromosomal region that has not yet been sequenced and annotated (53). Indeed, antimicrobial peptides encoded by genes in a region flanked by repetitive elements have been reported previously in bovines (54).

Several aspects of perinatal brain injury are recapitulated using rodent models with LPS-induced inflammation and/or hypoxia-ischemia (48). Activation of microglia and innate immunity is considered a major component contributing to immature brain injury (55, 56). sPIF treatment resulted in reduced microglial activation (Fig. 3 F and G). Neuroprotective effects were detected despite the late starting point (P6) of the therapy. This finding is of particular importance, because in premature infants the precise timing of the injury (whether it occurs before or during labor or in the postnatal period) is often unclear (56). The currently available delayed immune-modulatory treatment options often result in no benefit or even in the development of adverse effects (56). Our results strongly suggest that even a significantly delayed initiation of therapy with sPIF is effective in reversing advanced neural damage. The current and previously reported neuroprotective data (9) combined with the recent Food and Drug Administration’s fast-track approval for clinical trials support sPIF’s potential clinical application.

Let-7 repression in blastocysts facilitates embryo implantation in the mouse (57, 58). Proper regulation of expression of IL-10 in trophoblasts and uterine immune cells has been implicated in placental health and fetal development (59). We propose that our newly discovered PIF/TLR4/KSRP/let-7 signal transduction pathway and its mediated regulation of IL-10 expression may underlie PIF’s mode of action on immune control and embryo development in the maternal–fetal interface. The demonstration that systemic sPIF administration targets the brain directly makes sPIF an attractive, minimally invasive approach to brain-injury therapy.

Materials and Methods

Details of antibodies, siRNAs, inhibitors, peptides, cell culture, siRNA knockdown, Western blot, quantitative RT-PCR, and immunohistochemistry are given in SI Materials and Methods.

sPIF Treatment of Cultured Cells.

See SI Materials and Methods for details.

Protein Stability Analysis.

See SI Materials and Methods for details.

Akt Inhibitor Rescue Experiments.

See SI Materials and Methods for details.

Induction of Hypoxic-Ischemic Brain Injury.

See SI Materials and Methods for details.

Assessment of Cortical Volume, Neuronal Loss, and Glial Activity.

See SI Materials and Methods for details.

Quantification and Statistical Analysis.

See SI Materials and Methods for details.