Astrocytic TLR4 expression and LPS-induced nuclear translocation of STAT3 in the sensory circumventricular organs of adult mouse brain
Introduction
The reaction to pyrogen lipopolysaccharide (LPS), a component of Gram-negative bacteria, is a most well-characterized example of innate recognition which leads to a robust inflammatory response and fever. The binding of LPS to Toll-like receptor 4 (TLR4) and CD14 leads to the activation of two distinct signaling pathways finally to activate nuclear factor-κB (NF-κB) and activator protein-1 (Takeda and Akira, 2000, Takeda and Akira, 2004, Rivest, 2003). NF-κB translocates into the nucleus and induces the transcription of inflammatory genes such as cyclooxygenase-2 (COX-2), tumor necrosisfactor-α, interleukin-1β (IL-1β), and IL-6 (Brasier, 2010). Activator protein-1 is shown to control the expression of IL-3, -4, -5, and -9, and interferon-α and -γ (Adcock, 1997). IL-6 is shown to be increased in the blood circulation of febrile animals and humans (Cartmell et al., 2000, Nijsten et al., 1987) and IL-6-deficient mice show a reduced fever response against peripheral infection (Chai et al., 1996).
Microglia, the resident macrophages, are the main immune response and effector cells in the brains (Rivest, 2009). Microglia are shown to express TLR4 and release various proinflammatory cytokines by the stimulation of LPS (Olson and Miller, 2004). Cultured brain astrocytes have been reported to express low levels of TLRs under normal condition, but they are capable of expressing high levels of TLRs upon inflammatory stimulation (Kielian, 2009). The stimulation of LPS induces both the upregulation of Tlr4 mRNA and secretion of IL-6 in cultured microglia (Bowman et al., 2003, Carpentier et al., 2005). LPS strongly promotes the expression of COX-2 and prostaglandin-E2 synthase-1 mediated by the MyD88-dependent NF-κB pathway in cultured astrocytes (Font-Nieves et al., 2012). Following inflammatory activation, astrocytes are endowed with the ability to secrete soluble mediators, such as C–X–C motif chemokine 10, chemokine C–C motif ligand 2, and IL-6 and B cell activating factor belonging to the tumor necrosis factor family, which have an impact on both innate and adaptive immune responses (Farina et al., 2007). The responses of astrocytes to LPS are completely dependent on the presence of functional microglia via microglia release of soluble mediators in vitro, indicating glial crosstalk in brain responses to inflammation stimulation (Holm et al., 2012).
The brain vasculature has the blood–brain barrier (BBB) that maintains the chemical composition of neuronal milieu for proper functioning of neuronal circuits by preventing free access of blood-derived substances. The dysfunction of the BBB results in the accumulation of neurotoxic molecules within parenchyma and subsequent serious neuronal damages (Zlokovic, 2011). Nonetheless, brain cells are equipped with the mechanisms to recognize blood-derived information at the sensory circumventricular organs (CVOs) that include the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), and area postrema (AP) (Engelhardt, 2003, Sisó et al., 2010, Miyata and Morita, 2011, Morita and Miyata, 2012). For example, plasma Na+ and osmotic levels are monitored in the sensory CVOs to control salt-intake and/or drinking behaviors (Hiyama et al., 2004) and the release of vasopressin from the neurohypophysis (Miyata and Hatton, 2002). Emetic agents are recognized at chemosensitive receptors in the AP and the nucleus of the solitary tract (Sol) to cause vomiting and nausea (Hornby, 2001).
Although it is not conclusively determined how peripheral LPS induces neuroinflammatory responses, a most probable mechanism is that circulating pathogens and/or cytokines directly stimulate parenchyma cells in the sensory CVOs and the information is transmitted to other brain regions to cause neuroinflammatory responses and fever. The electrolytic lesion of the SFO reduces fever response by the intravenous administration of LPS (Takahashi et al., 1997). The expression of Tlr4 mRNA is shown in the CVOs and ventricular ependymal cells in mouse brains (Laflamme and Rivest, 2001, Chakravarty and Herkenham, 2005). The peripheral and intracerebroventricular (icv) administration of LPS causes a rapid upregulation of CD14 in the sensory CVOs (Lacroix et al., 1998, Nadeau and Rivest, 2000).
The peripheral administration of LPS increases the expression of nuclear factor IL-6 in a time-dependent manner within the sensory CVOs which sustains hypothalamic inflammatory target gene induction (Damm et al., 2011). The peripheral administration of LPS induces nuclear translocation of the signal transducer and activator of transcription factor 3 (STAT3) at astrocytes in the sensory CVOs (Harré et al., 2002, Harré et al., 2003, Gautron et al., 2002, Rummel et al., 2004, Rummel et al., 2005). IL-6 is shown to be increased in the blood circulation of animals and humans after inflammatory stimulation (LeMay et al., 1990, Nijsten et al., 1987, Cartmell et al., 2000). Mice deficient for IL-6 gene reveal a reduced fever response after peripheral infection (Chai et al., 1996). IL-6 triggers a signaling pathway of JAK-signaling transducer and activation of STAT3 (Akira, 1997) and the pathway of COX-2 in the brains most likely via STAT3-dependent pathway (Rummel et al., 2006).
Until now no study has been reported about TLR4-expressing cellular phenotype in the sensory CVOs, although strong expression Tlr4 mRNA is reported in these regions of normal adult animals (Laflamme and Rivest, 2001, Chakravarty and Herkenham, 2005). Moreover, it has not been examined whether glial crosstalk between microglia and astrocytes is responsible for neuroinflammatory cascades in the sensory CVOs. In the present study, therefore, we performed immunohistochemistry for TLR4 and the effects of a microglia inhibitor minocycline on the nuclear translocation of STAT3 at astrocytes in the sensory CVOs of adult mice. The expression of TLR4 was observed at astrocytes in the sensory CVOs, whereas its expression was also seen at microglia in the AP and Sol around the central canal. Both intraperitoneal administration and icv infusion of LPS caused a similar pattern of nuclear translocation of STAT3 in the OVLT, SFO, and AP. Pretreatment of a microglia inhibitor minocycline suppressed the nuclear translocation of STAT3 at astrocytes in the OVLT and AP after intraperitoneal administration and icv infusion of LPS, but its inhibitory effect was less in the SFO, indicating heterogeneous contribution of microglia in activating astrocytic STAT3 among the sensory CVOs.
Section snippets
Animals
Adult male mice (ICR) in 70–84 days old were used in the present experiments. The animals were housed in a colony room, under a pathogen-free condition controlled temperature (25.0 ± 1.0 °C), with a 12-h light/12-h dark cycle and given ad libitum access to commercial chow and tap water. Animal care and experiments were in accordance with the Guidelines laid down by the NIH and the Guideline for Proper Conduct of Animal Experiments Science Council of Japan to minimize the number of animals used and
Expression of TLR4 in the sensory CVOs
It is reported that the expression of Tlr4 mRNA was stronger in the sensory CVOs by using in situ hybridization histochemistry (Laflamme and Rivest, 2001, Chakravarty and Herkenham, 2005). Low magnification view showed that the immunoreactivity of TLR4 was prominent at the OVLT as compared with adjacent hypothalamic nucleus such as medial preoptic area and median preoptic nucleus (Fig. 1A and A′). High magnification view revealed that dense TLR4-immunoreactive cellular processes were seen at
Discussion
The sensory CVOs are the important brain regions to initiate neuroinflammatory responses in the brains, since circulating LPS causes faster transcriptional activation of genes encoding a wide variety of pro-inflammatory molecules in the sensory CVOs as compared with other brain regions (Rivest, 2003). The in situ hybridization study shows that the expression of Tlr4 mRNA is detected in the sensory CVOs, median eminence, ventricular ependymal cells, and meninges in the rodent brain (Laflamme and
Acknowledgments
The hybridoma of anti-CD31 (2H8, Dr. Steven Bogen) was obtained from the DSHB developed under the auspices of the NICHD and maintained by The University of Iowa, Iowa City, IA 52242. This work was supported in part by Scientific Research Grants from the Japan Society for the Promotion of Science (No. 24500411 to S. Miyata).
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