Reactive astrogliosis is a hallmark of neonatal brain injury and many other pathologies of the CNS. Despite this commonality, its beneficial vs. harmful consequences in various contexts are not well understood, and cell non-autonomous effects of astrogliosis on neonatal brain development have never been demonstrated. Although inflammatory insults and hypoxic-ischemic events are considered the main causal factors for neonatal WMI, the multifactorial nature of the pathology has limited the advances of our mechanistic understanding. In this regard, pathological studies on hypoxic-ischemia cases generally show concurrent micro- and astro-gliosis, making it difficult to dissociate the sequence of key factors leading to the white matter pathology. In the current model, we attempted to study the reactive glia component of neonatal WMI in relative isolation, and therefore optimized an inflammation to consistently induce gliosis in mice at a time corresponding to the heightened vulnerability to WMI in humans. We found that a single peripherally-administered dose of LPS was sufficient to induce micro-and astro-gliosis and WMI injury in mice in a pattern consistent with that observed in human postmortem cases. This protocol, along with genetically-engineered mice were then used to probe the complex nature of neonatal brain injury. Importantly, the studies revealed a protective role of astrocytes, and led to the identification of a candidate pathway for disease protection involving modulation of the TGFβ-1 signaling pathway.
Our data in human postmortem tissues of neonatal WMI samples demonstrated that the STAT3 pathway was commonly active in hypertrophic astrocytes, so the potential consequences of STAT3-mediated astrogliosis were investigated in our model using conditional knockout mice. The STAT3 pathway has been demonstrated to be a mediator of reactive astrogliosis, particularly with respect to GFAP upregulation, hypertrophic morphology, and scar formation 24
. By manipulating the STAT3 gene in astrocytes, we demonstrated a protective role of reactive astrocytes in limiting myelin injury. An investigation of potential mechanisms revealed that aberrant TGFβ-1 in microglia might be a key factor in this pathology. In this regard, TGFβ-1 was found to be tightly regulated during development in healthy mice, and was detected exclusively in microglia. Moreover, it was suggested that excess TGFβ-1 had a direct action on developing OL, as evidenced by the fact that higher levels of the signal transducer pSMAD2 were observed in OL lineage cells in vivo under conditions of high TGFβ-1, and that TGFβ-1 inhibited the maturation of purified OL progenitors in culture. The that fact that pharmacological inhibition of pSMAD2 pathway partially reversed the inflammation-induced delayed myelin development in vivo suggest that TGFβ-1 signaling might be a therapeutic target for limiting myelin defects in infants at risk for WMI. However, it must be cautioned that the ability of TGFβ-1 blockade to diminish perinatal white matter injury may be model dependent. For example, other factors, including cytokines IL-6, IL-10, ciliary neurotrophic factor (CNTF), and leukemia inhibitory factor (LIF) in addition to LPS can activate STAT3 pathway in astrocytes, and it is uncertain if they would trigger a similar STAT3-dependent induction of TGFβ-1. In any case, the current study highlights a new role of TGFβ-1 in neonatal brain development, whose dysregulation may mediate pathology associated with a type of WMI.
Interestingly, whereas we showed TGFβ-1 was expressed primarily in microglia of postmortem WMI samples, and exclusively in microglia in the normal and inflamed neonatal mouse brain, TGFβ-1 immunoreactivity has been demonstrated in both macrophages and reactive astrocytes in multiple sclerosis (MS) lesions58
. Zhang et al. recently proposed an alternative mechanism in which astrocytes expressing TGFβ-1 in MS lesions was hypothesized to induce Jagged1/Notch1 signaling in OL, thereby inhibiting their differentiation59
. Being a secreted molecule, TGFβ-1 can be secreted by a variety of cellular sources and may potentially act on multiple cell types with appropriate receptors. However, as discussed, we did not observe expression of TGFβ-1 in astrocytes in the mouse model, and the great majority of cells expressing TGFβ-1 in human postmortem tissues were Iba+
microglia. On the other hand, in the developing mouse brain, Jagged1 expression was found exclusively in callosal axons in discrete locations and rapidly became undetectable during the examined postnatal period (data not shown). Based on these observations, it is unlikely that the Jagged1 expression by reactive astrocytes contributed significantly to the broadly affected pathology we reported in the neonatal mouse model used here.
Another potential mechanism is suggested by the fact that astrocytes are known to secrete cytokines including LIF60
which promote survival and maturation of OL. In addition, reactive astrocytes are known to express factors such as FGF262
that are inhibitory for OL differentiation. Of those, we examined expression levels of LIF in our model and found that it was mildly upregulated by LPS, but was not regulated in astrocytic STAT3-dependent manner (data not shown). How other molecules are regulated in the context of inflammatory insult in the neonatal brain requires further examination.
Why should an inhibitory factor for myelination be highly expressed in newborn brain, at the time when myelin is developing? One potential explanation is that OL progenitors must be maintained at an immature stage until neuronal connections are fully established and/or until other developmental processes are complete. The disruption of such a carefully-timed process by astrogliosis could presumably have pathological effects on subsequent CNS function. Notably, microglial expression of TGFβ-1, as well as nuclear pSTAT3 in reactive astrocytes was observed in the white matter of human neonatal WMI examined here. Using cell culture system, we demonstrated that astrocyte-derived factors (obtained from conditioned media from WT vs. STAT3-deficient astrocytes) had the capacity to differentially regulate TGFβ-1 secretion by cultured microglia. Although the identification of that factor remains unknown, Sarafian et al. recently reported microarray data on STAT3 WT and deficient astrocytes, which may suggest potential candidate genes53
In summary, the clinical evidence presented along with the findings reported here in mouse and tissue culture models suggest that STAT3-mediated reactive astrocytes attempt to protect myelin development against neuroinflammation by constraining microglial TGFβ-1 expression in cell non-autonomous manner. These observations imply a potential benefit of promoting STAT3 pathway activation during inflammatory insult. Collectively, the results presented here support the concept that process of healthy myelination involves intricate and timed communications among multiple glial cell types, and that the temporally-regulated communication can be dysregulated by reactive astrogliosis during a critical time, leading to impairment in myelin development.