Responses of perivascular macrophages to circulating lipopolysaccharides in the subfornical organ with special reference to endotoxin tolerance
Circulating endotoxins including lipopolysaccharides (LPS) cause brain responses such as fever and decrease of food and water intake, while pre-injection of endotoxins attenuates these responses. This phenomenon is called endotoxin tolerance, but the mechanisms underlying it remain unclear. The subfornical organ (SFO) rapidly produces proinflammatory cytokines including interleukin-1β (IL-1β) in response to peripherally injected LPS, and repeated LPS injection attenuates IL-1β production in the SFO, indicating that the SFO is involved in endotoxin tolerance. The purpose of this study is to investigate features of the IL-1β source cells in the SFO of LPS-non-tolerant and LPS-tolerant mice.
We first established the endotoxin-tolerant mouse model by injecting LPS into adult male mice (C57BL/6J). Immunohistochemistry was performed to characterize IL-1β-expressing cells, which were perivascular macrophages in the SFO. We depleted perivascular macrophages using clodronate liposomes to confirm the contribution of IL-1β production. To assess the effect of LPS pre-injection on perivascular macrophages, we transferred bone marrow-derived cells obtained from male mice (C57BL/6-Tg (CAG-EGFP)) to male recipient mice (C57BL/6N). Finally, we examined the effect of a second LPS injection on IL-1β expression in the SFO perivascular macrophages.
We report that perivascular macrophages but not parenchymal microglia rapidly produced the proinflammatory cytokine IL-1β in response to LPS. We found that peripherally injected LPS localized in the SFO perivascular space. Depletion of macrophages by injection of clodronate liposomes attenuated LPS-induced IL-1β expression in the SFO. When tolerance developed to LPS-induced sickness behavior in mice, the SFO perivascular macrophages ceased producing IL-1β, although bone marrow-derived perivascular macrophages increased in number in the SFO and peripherally injected LPS reached the SFO perivascular space.
The current data indicate that perivascular macrophages enable the SFO to produce IL-1β in response to circulating LPS and that its hyporesponsiveness may be the cause of endotoxin tolerance.
KeywordsBone marrow-derived cells Endotoxin tolerance Interleukin-1β Macrophage depletion Sickness behavior
Enhanced green fluorescent protein
Glial fibrillary acidic protein
Ionized calcium-binding adaptor molecule 1
Circulating endotoxins, including lipopolysaccharides (LPS), induce fever, reduce locomotor activity, and decrease food and water intake . These behavioral changes are commonly referred to as sickness behavior, which is uncomfortable but important to fight infection. However, pre-injection of LPS attenuates sickness behavior after subsequent challenge with LPS [2, 3]. Since this phenomenon, known as endotoxin tolerance, can occur naturally in humans and is an important system for host protection against excessive response to endotoxin and endotoxin shock, clarifying the mechanisms for development of tolerance to endotoxins is needed.
Sickness behavior is due to the effects of proinflammatory cytokines on the brain . Although circulating immune cells produce proinflammatory cytokines following infection, endotoxin-induced sickness behavior sometimes precedes the increase in plasma cytokine concentration (for reviews, see [4, 5]). These data raise the possibility that the brain communicates directly with circulating endotoxins. However, the blood-brain barrier (BBB) prevents circulating endotoxins from freely entering the brain.
Accumulating evidence indicates that the subfornical organ (SFO) is a brain area that directly senses circulating LPS to produce proinflammatory cytokines. A sublethal dose of LPS causes a rapid production of proinflammatory cytokines including interleukin-1β (IL-1β), interleukin-6, and tumor necrosis factor-α in the SFO [6, 7]. Among these cytokines, IL-1β is important for the induction of sickness behavior (for a review, see ). Local injection of an IL-1 receptor antagonist into the SFO attenuates LPS-induced fever . These data suggest that the SFO directly senses circulating LPS and produces IL-1β to induce sickness behavior. Moreover, LPS-induced IL-1β production disappeared in the SFO of LPS-pre-injected rabbits . The attenuation of IL-1β expression suggests that the SFO is involved in endotoxin tolerance. However, what cells produce IL-1β in response to circulating LPS and why the SFO ceases to produce IL-1β in LPS-pre-injected animals remain unclear.
Circulating molecules including LPS are thought to diffuse freely into the SFO, since the SFO is a specialized brain area that lacks a typical BBB. However, we have recently shown that peripherally injected tracers (MW > 10,000) accumulate around the SFO vasculature [10, 11]. In the SFO, the perivascular basement membranes are separated into the parenchymal cell-derived outer basement membrane and the endothelial cell-derived inner basement membrane and create a large perivascular space outside the brain parenchyma . Our peripherally injected tracers localized in the perivascular space in the SFO. Since LPS tend to form micelles (MW ~ 300,000–1000,000), with hydrophilic polysaccharides facing outward , it is unlikely that circulating LPS simply diffuse into the SFO. In addition, macrophages around the SFO vasculature were proposed as IL-1β source cells . These data raise the possibility that macrophages in the SFO perivascular space are responsible for IL-1β production in response to circulating LPS and participate in endotoxin tolerance.
Although microglia are brain-resident myeloid cells, macrophages also exist in the perivascular space, meninges, choroid plexus, and circumventricular organs including the SFO . The resident microglia arise from the yolk sac and populate the neuroepithelium in early embryogenesis , whereas the SFO macrophages are replenished by bone marrow-derived cells [16, 17].
Here, we investigated features of the SFO macrophages and their production of LPS-induced IL-1β in LPS-non-tolerant and LPS-tolerant mice, using macrophage depletion and bone marrow transplantation methods.
Eight-week-old male mice (C57BL/6J) were purchased from CLEA Japan. (Tokyo, Japan). Male C57BL/6-Tg (CAG-EGFP) mice and C57BL/6N mice were purchased from Japan SLC (Hamamatsu, Japan). In C57BL/6-Tg (CAG-EGFP) mice, ubiquitously active cytomegalovirus immediate early enhancer-chicken β-actin hybrid (CAG) promoter drives enhanced green fluorescent protein (EGFP) expression. The animals were housed in a colony room with a 12-h light/12-h dark cycle, given access to commercial chow (CE-2, CLEA Japan) and tap water ad libitum, and maintained under pathogen-free conditions. We measured food intake by subtracting the weight of the food container and taking into account any spillage, water consumption by measuring the weight of the water bottle (Shin Factory, Fukuoka, Japan), and body weight daily between 09:00 and 10:00. Animal care and the experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Guidelines for Proper Conduct of Animal Experiments of the Science Council of Japan. The Animal Care Committee of Nara Medical University approved the experimental protocol.
Administration of LPS
Mice were intraperitoneally injected with either LPS (1 mg/kg) from Escherichia coli (serotype O55:B5, Sigma-Aldrich Japan, Tokyo, Japan) or vehicle (pyrogen-free physiological saline, Ohtsuka Chemical, Tokushima, Japan) as previously described . To examine the effect of LPS pre-injection, LPS were again injected 2 or 4 days after the first LPS injection.
Administration of fluorescent tracers
Mice were subjected to intravenous (caudal vein) injection (100 μl) of Texas Red-conjugated lysine-fixable Dextran 70,000 (MW: 70,000, Molecular Probes, 20 mg/ml) in phosphate-buffered saline (PBS; pH 7.4). Animals were sacrificed at 30 min after this injection for immunohistochemical analysis.
Administration of clodronate liposomes
To label control empty liposomes, liposomes were preincubated with 0.125 mg/ml of DiI (Wako, Osaka, Japan) for 10 min and centrifuged (20,000g) for 10 min three times with resuspension in sterile PBS to wash out unlabeled DiI. Mice were subjected to intravenous (caudal vein) injection (200 μl) of DiI-labeled control empty liposomes in PBS. Animals were sacrificed at 1 h after this injection for immunohistochemical analysis.
To investigate the contribution of perivascular macrophages to LPS-induced IL-1β expression in the SFO, mice were subjected to intravenous (caudal vein) injection (200 μl) of clodronate liposomes or control empty liposomes (Macrokiller V100, Cosmo Bio, Tokyo, Japan) in PBS. Three days after the liposome injection, mice were intraperitoneally injected with LPS (1 mg/kg). Animals were sacrificed at 1 h after LPS injection for immunohistochemical analysis.
Preparation of bone marrow chimeras
We employed two different immunosuppression methods: (1) irradiation with head shielding or (2) busulfan treatment, as previously reported [15, 16, 17]. Previously, bone marrow-derived cells have been found in the SFO following bone marrow transplantation into whole-body-irradiated mice [16, 17]. Although most bone marrow transplantation experiments have used whole-body irradiation, this method may perturb the integrity of the BBB . These two immunosuppression methods were selected to avoid the artificial effects of whole-body irradiation. (1) Mice received irradiation (6 Gy × 2 with a 4-h interval) 24 h prior to bone marrow reconstitution using an X-ray irradiator (Model MBR-1520R, Hitachi Power Solutions, Ibaraki, Japan). Mice were exposed to X-rays with 4-mm-thick individual lead shields positioned to protect the head and to irradiate only the lower part of the body. (2) Mice were intraperitoneally injected with the chemotherapeutic agent busulfan (30 μg/g body weight; B2635, Sigma-Aldrich Japan) in a 1:4 solution of dimethyl sulfoxide and PBS 7, 5, and 3 days prior to bone marrow transfer.
All mice were treated with antibiotics (trimethoprim/sulfamethoxazole) for 14 days after irradiation or busulfan treatment. Bone marrow-derived cells were obtained from the femur and tibia of 5-week-old male C57BL/6-Tg (CAG-EGFP) mice and resuspended in PBS with 2% fetal bovine serum. Bone marrow-derived cells (1 × 106) were transferred to 8-week-old male C57BL/6N recipient mice by tail vein injection (100 μl). Mice were left undisturbed for 1 month to allow engraftment. For quantitative analysis, engraftment was verified by determining the percentage of EGFP-expressing cells in the blood. We counted the numbers of EGFP+ cells in peripheral blood by flow cytometry and confirmed efficient chimerism as demonstrated by the large proportions of circulating blood leukocytes expressing EGFP (range between 62.7 and 73.7%).
We found EGFP+ Iba1+ perivascular macrophages in the SFO both of irradiated and busulfan-treated mice (Additional file 1: Figure S1). There were many more EGFP+ Iba1+ cells in the SFO of busulfan-treated mice than of irradiated mice. We therefore employed busulfan for immunosuppression in Fig. 4.
Light microscopic immunohistochemistry
Mice were transcardially perfused with PBS followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) under deep pentobarbital anesthesia. The dissected brains were postfixed for 6 h, cryo-protected with 30% sucrose in PBS, and frozen at − 80 °C in Tissue-Tek OCT compound (Sakura Finetechnical, Tokyo, Japan). Coronal sections were cut at a thickness of 30 μm with a cryostat (Leica, Heidelberg, Germany) at − 15 °C. For immunofluorescence detection, we processed free-floating sections as described previously . In brief, sections were washed with PBS and treated with 25 mM glycine in PBS for 20 min. When mouse primary antibodies were used, the sections were further treated with unlabeled goat Fab against mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA; 1:400) for 2 h to mask endogenous mouse IgG-like proteins. They were then incubated with 5% normal donkey serum in PBS containing 0.3% Triton X-100 for 1 h and then with mouse IgG against desmin (clone D33, DAKO, Glostrup, Denmark; 1:800), against glial fibrillary acidic protein (GFAP; clone GA5, Cell Signaling Technology, Danvers, MA, USA; 1:1000), and against LPS (clone 2D7/1, abcam, Cambridge, UK; 1:200); goat IgG against CD206 (R&D Systems, Minneapolis, MN, USA; 1:1000) and against IL-1β (AF-401-NA, R&D Systems; 1:1000); and rabbit IgG against ionized calcium-binding adaptor molecule 1 (Iba1, Wako; 1:500) for 24 h at 4 °C; and with guinea pig IgG against laminin-111 (the antigen was laminin-111 from Engellbreth-Holm-Swarm murine sarcoma basement membrane; kindly gifted by S. Miyata, Kyoto Institute of Technology, Kyoto, Japan ; 1:200) for 2 h at 37 °C). The primary antibodies were visualized by incubating the sections in Alexa 405-, 488-, 594-, and 633-conjugated donkey IgG against goat, rabbit, rat, and guinea pig (Jackson ImmunoResearch Laboratories; 1:1000) in PBS containing 0.3% Triton X-100 for 1 h. When we used mouse IgG primary antibodies, Alexa 488- and 594-conjugated goat F(ab)2 against mouse IgG (Jackson ImmunoResearch Laboratories; 1:500) was used to avoid nonspecific binding of endogenous mouse Fc receptors. Nuclei were counterstained with 4,6-diamidino-2-phenylindole.
For LPS immunohistochemistry, we performed antigen retrieval by incubating sections in citrate-ethylenediaminetetraacetic acid buffer (10 mM citric acid, 2 mM ethylenediaminetetraacetic acid, 0.05% Tween 20, pH 6.2) for 20 min at 95 °C. For CD206 immunohistochemistry, the sections were pretreated with chilled acetone-methanol for 15 min.
In order to determine IL-1β antibody specificity, the primary antibody was preincubated with 4 μg/ml of the mouse IL-1β recombinant protein (401-ML, R&D Systems) for 24 h at 4 °C. IL-1β immunoreactivity was abolished by primary antibody preadsorption in the SFO of both vehicle- and LPS-injected mice (Additional file 1: Figure S2).
Confocal microscopic observation
Labeled sections were mounted on glass slides, and coverslips were sealed with Vectashield (Vector Laboratories, Burlingame, CA, USA). We observed stained sections using a laser-scanning confocal microscope (C2, Nikon, Tokyo, Japan) by changing the fine focus of the microscope for quantitative analysis. Single-plane images were acquired to make figures using a × 20 objective with 0.75 NA, × 40 objective with 0.95 NA, or × 60 water immersion objective with 1.20 NA at a 1024 × 1024 pixel resolution. We saved images as TIF files using Nikon Nis-Elements AR software and arranged them using Photoshop CS6.
All statistical analyses were carried out in SPSS (Version 23.0, IBM SPSS Statistics, Ehningen, Germany).
Statistical comparisons for food intake, water intake, and body weight were performed using one-way ANOVA with Scheffé’s post hoc test. Difference was assessed at a significance level of P < 0.05. Data are shown as mean ± SEM.
For quantitative analyses of cell numbers, analysis of all images was performed such that the experimenter was blind to the treatment group. We used four animals from each group for quantification, and at least eight sections per animal were chosen from the SFO region (between bregma − 0.23 and − 0.71 mm) according to the mouse brain atlas . Area measurement was performed by delineating manually each SFO using ImageJ software (National Institutes of Health). Independent groups were compared with Student’s t test or one-way ANOVA with Scheffé’s post hoc test. Difference was assessed at a significance level of P < 0.05. Data are shown as mean ± SEM.
Mice develop endotoxin tolerance
Perivascular macrophages are the source of IL-1β in the SFO after the first LPS injection
We then investigated the Iba1+ cells in the perivascular space. Typical perivascular macrophages are elongated or ameboid-like, express mannose receptor CD206 and low levels of Iba1, and are endowed with phagocytic activity . In the SFO, morphologies of perivascular Iba1+ cells were elongated or ameboid-like, whereas parenchymal Iba1+ microglia were ramified (Additional file 1: Figure S3a). Both the SFO perivascular Iba1+ cells and typical perivascular macrophages expressed CD206 (Additional file 1: Figure S3b). The SFO perivascular Iba1+ cells strongly expressed Iba1, whereas typical brain perivascular macrophages expressed lower levels of Iba1 than microglia, consistent with a previous report . The fluorescence of peripherally injected tracer Dextran 70,000 was often localized in the SFO perivascular Iba1+ cells as numerous puncta (Additional file 1: Figure S3c, d), meaning that these cells phagocytose peripherally injected molecules. Since brain pericytes can express Iba1 following ischemia , we examined the expression patterns of Iba1 and the pericyte marker desmin. However, perivascular Iba1+ cells did not overlap with desmin+ pericytes in the SFO (Additional file 1: Figure S3e). These data indicate that perivascular Iba1+ cells in the SFO were perivascular macrophages.
To explore why perivascular macrophages expressed IL-β but parenchymal microglia did not, we examined the distribution of peripherally injected LPS in the SFO. We found that immunoreactivity of peripherally injected LPS localized inside the laminin-111+ outer basement membrane in the SFO, sometimes co-localizing with immunoreactivity of Iba1 (Fig. 2e). The majority of LPS+ spots were observed in the perivascular space (Fig. 2f; 97.5%).
Bone marrow-derived perivascular macrophages in the SFO increase in number in endotoxin-tolerant mice
Next, we examined whether the additional perivascular macrophages were bone marrow-derived cells using bone marrow chimeras. As described in the “Methods” section, mice were immunosuppressed by busulfan treatment, transplanted with bone marrow-derived cells from transgenic mice expressing EGFP ubiquitously, and then injected with vehicle or LPS (Fig. 4d). The number of SFO EGFP+ cells increased 4 days after a single LPS injection (Fig. 4e). In the perivascular space, the number of EGFP+ cells (64.6 ± 5.6 cells in vehicle-injected mice and 151.7 ± 24.0 cells in LPS-injected mice, F3,3 = 9.261, P = 0.012) significantly increased after the single LPS injection (Fig. 4f). On the other hand, we did not observe a significant change in the number of parenchymal EGFP+ cells. These data indicate that the SFO perivascular bone marrow-derived cells increased in number as a result of the first LPS injection.
IL-1β expression is attenuated in SFO perivascular macrophages after the second LPS injection in endotoxin-tolerant mice
Perivascular macrophages were the initial source of LPS-induced IL-1β in the SFO. Macrophages around the SFO vasculature were first proposed as IL-1β source cells , but why they rapidly express IL-1β, and microglia do not, is unknown. In the present study, we distinguished the perivascular space and parenchyma in the SFO by visualizing the outer basement membrane and clarified that perivascular macrophages localized mainly in the perivascular space, i.e., outside the brain parenchyma. Moreover, clodronate liposomes depleted perivascular macrophages and attenuated LPS-induced IL-1β production. Peripherally injected LPS localized in the perivascular space in the SFO. Since intracerebroventricular injection of LPS induces neuronal apoptosis , it is reasonable that the penetration of circulating LPS should be restricted in the SFO, which possesses neurons in its parenchyma like other brain regions. In the SFO, CD45+ immune cells and GFAP+ astrocytes express a LPS receptor, toll-like receptor 4 . Upregulation of IL-1β after LPS injection was mainly observed in Iba1+ perivascular macrophages, whereas IL-1β was constantly expressed in parenchymal cells, which mainly consist of GFAP+ astrocytes. These observations suggest that perivascular macrophages are responsible for IL-1β production in response to circulating LPS. Together, these data indicate that the localization of IL-1β-producing cells enables the SFO to sense circulating LPS and thus to avoid severe neuron damage.
Another key finding is that when mice become endotoxin-tolerant, perivascular macrophages scarcely produced IL-1β in the SFO, although peripherally injected LPS and an increased number of bone marrow-derived cells were observed in the perivascular space. Endotoxin tolerance is not only known as animal hyporesponsiveness, but also known as macrophage hyporesponsiveness to restimulation by endotoxin (for a review, see ). LPS-pre-exposed, endotoxin-tolerant murine macrophages and human leukocytes exhibit suppression of LPS-induced expression of mRNA for IL-1β in vitro [29, 30]. These data suggest that the SFO perivascular macrophages become tolerant by the first LPS injection and may contribute to the development of behavioral endotoxin tolerance. However, the mechanistic details require further investigation, and a direct role for accumulated bone marrow-derived cells in behavioral endotoxin tolerance is yet to be confirmed. Future studies will need to focus on the phenotype of SFO perivascular macrophages to elucidate their temporal changes.
We observed that some perivascular macrophages spanned the outer basement membrane. Similar to LPS, IL-1β is unlikely to diffuse into the SFO parenchyma since it is a large (MW: 17,300) hydrophilic protein. Macrophages that span the outer basement membrane may deliver IL-1β to the SFO parenchyma. Once it enters the brain parenchyma, IL-1β can induce IL-1β production by adjacent microglia . Parenchymal microglia express IL-1β in the SFO at later times after LPS injection . In addition, IL-1β induces a transient depolarization in a subpopulation of isolated SFO neurons . These data suggest that IL-1β propagates its own synthesis to stimulate neurons. Another possibility is that diffusible second messengers are produced downstream of IL-1β. Although IL-1 receptor-expressing vessels exist throughout the brain, especially strong immunoreactivity for the IL-1 receptor is observed in the vasculature of the SFO . IL-1β is able to induce cyclooxygenase-2 activity and the production of prostaglandin E2, which crosses the BBB . Local communication between perivascular macrophages and endothelial cells may thus occur in the SFO perivascular space.
In the present study, we only used male adult mice to simplify the experiment. Because females and offspring are also exposed to endotoxins, it remains for future studies to investigate the effect of LPS on female and developing brains.
SFO perivascular macrophages were the IL-1β source cells in LPS-non-tolerant mice. In contrast, they cease expressing IL-1β in tolerant mice, although bone marrow-derived perivascular macrophages increase in number and peripherally injected LPS reach to the SFO perivascular space. The SFO perivascular macrophages may thus have a role in endotoxin tolerance.
The authors are indebted to Dr. S. Miyata (Kyoto Institute of Technology, Kyoto, Japan) for kindly gifting the polyclonal anti-laminin-111 antibody. We thank Dr. Ian Smith for providing language assistance.
This work was supported by Scientific Research Grants from the Japan Society for the Promotion of Science [grant numbers 16 K18980 to S. Morita-Takemura, and 15 K14354 to A. Wanaka].
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
SMT, SHI, and AW designed and coordinated the study and were responsible for writing the manuscript. SMT, KN, SHI, AI, and MK performed the experiments and analyzed the data. KT, HO, TT, MK, and TI made intellectual contribution, participated in discussions, and helped in writing the manuscript. All the authors have read and approved the final version of the manuscript.
Ethics approval and consent to participate
The Animal Care Committee of Nara Medical University approved the experimental protocol.
Consent for publication
All authors declare that they have no competing interests.
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