Induction of brain inflammation triggers macrophage infiltration into the liver.

Whole-body analysis using RNA in situ hybridization shows abnormal localization of macrophages in the liver after brain injection of LPS or bacteria.

In vivo time-lapse imaging shows dynamic movements and processes of infiltrating macrophages in the liver at short- (10 hpi) and long- (48 hpi) term timepoints after brain-LPS injection.

Brain-LPS injection was not found to induce mfap4 expression in the liver.

Double transgenic zebrafish expressing the macrophage mpeg1:GFP and endothelial kdrl:mCherry reporters were used to localize infiltrating macrophages after brain-LPS injection at 8–10 hpi in the four dpf zebrafish. (a–f) Representative images corresponding to Video 3. a, b, c 3D volumetric view of whole liver (demarcated by a dotted line) and surrounding region taken from three timepoints of a confocal time-lapse imaging (see Video 3) of an 80 µm z-stack at 40x. d, e, f High magnification of the boxed region shown in the corresponding left panels. Representative infiltrating macrophages are labeled numerically 1–5 with their relative position to the vasculature: inside (white arrow), associated (blue arrow), or independent of vasculature (yellow arrow).

(a) Representative 3D images from six timepoints corresponding to Video 4 from a four dpf zebrafish which was brain-LPS injected and imaged starting at 12 hpi. Infiltrating macrophages can be classified into three groups (examples shown by arrows of different colors): circulating (present in only a single timepoint), transient (<2 hr) and residing cell (>2 hr). Double transgenic zebrafish labeling macrophages (mpeg1:GFP) and hepatocytes (fabp10a:DsRed) were used to track macrophage infiltration over a 10 hr period. Color overlay of both channels and single GFP channel in grayscale for macrophages are shown. LPS was co-injected with Alexa 568 conjugated dextran to validate brain injections and detect systemic distribution of the injected material based on labeling of the pronephros by the fluorescent dextran. Labeling of pronephros has been previously described after intravenous injection of a fluorescent tracer (Oltrabella et al., 2015), but labeling of proximal kidney tubules can also be observed in transgenic lines expressing a red fluorescent protein. (b) 2D projection of the tracking of infiltrating macrophages in the entire liver over all timepoints of analysis. Each traced cell is represented by a circle for its location at each timepoint if present in the liver, and a line for its movement between the timepoints. Different colors represent different cells. (c) Relative percentage of the different types of infiltrating macrophages based on liver occupation. n = 47 macrophages were tracked and analyzed. (d) Scatter plot shows the total duration of each traced infiltrating macrophage in the ‘transient’ and ‘residing’ groups. (e) Total distance and average speed of each macrophage in the ‘transient’ and ‘residing’ groups. Total distance was not significantly different, but the ‘transient’ macrophages moved faster with a significantly higher average speed than the ‘residing’ cells. sd, standard deviation; ns, not significant.

Brain-LPS microinjection leads to drainage of LPS molecules into circulation, and causes a hepatic response similar to intravenous LPS injection.

Wild-type double transgenic zebrafish at four dpf were microinjected with fluorescently tagged LPS in the tectal brain. To localize the LPS relative to the lymphatic and vasculature structures, two transgenes were used: mrc1a:GFP (Jung et al., 2017) and kdrl:mCherry (Fujita et al., 2011), respectively. Since mrc1a:GFP is known to also be expressed by macrophages (Jung et al., 2017), we generated a new macrophage reporter (mpeg1:BFP) to provide a third channel in blue to distinguish lymphatic from macrophage expressions. Blue arrows point to microglia/macrophage. (a) Three multi-tiled representative frontal plane optical slices from an in vivo confocal z-stack at 1.5 hpi show top, middle, and bottom sections through the head and anterior trunk of the LPS-injected animal. (b) Schematic of the frontal plane optical sections imaged. (c, d) High magnification images corresponding to dotted region in top slice in a). Most superficial layer of the CNS where LPS (red) is concentrated in the hindbrain ventricle and adjacent interstitial parenchymal space (c, asterisk) as well as the most-dorsal junction of the hindbrain-spinal cord (d, asterisk). (e, f) High magnification images of dotted region in middle slice shown in a). LPS is found localized to the lymphatic structures (blec and islv), and abundant in the interstitial space extending from the hindbrain ventricle (asterisk). (g,h) High magnification images corresponding to bottom slice in a). LPS is highly abundant in the spinal canal (sc) as well as tectal and hindbrain lymphatic cells (blec). (i) Top and middle slices of a different LPS-injected animal show the same distribution of LPS at 1.5 hpi. LPS does not appear to localize within the blood vasculature in the CNS or trunk to the same extent as it overlaps with the lymphatic cells. Top slice shows vasculature DLAV surrounded by but not overlap with LPS, arrows. *, interstitial space; DLAV, dorsal longitudinal anastomotic vessels; DLLV, dorsal longitudinal lymphatic vessel; blec, brain lymphatic endothelial cells; islv, intersomitic lymphatic vessels; sc, spinal canal; hv, hindbrain ventricle; hb, hindbrain; lym, lymphatics. Skin is highly auto-fluorescent and labeled by all channels at the border of every tissue. Scale bars all show 50 µm.

(a) Schematic of the sagittal optical planes taken by in vivo multi-tiled z-stack confocal imaging. (b) Whole-mount side view of a representative LPS-injected four dpf larvae carrying the vasculature reporter kdrl:mCherry at 12 hpi showing LPS strongly retained in the hindbrain ventricle (hv) and surrounding brain interstitial space as well as the brain and facial lymphatics (blec and FL, arrows) and the peripheral vasculature (PCV and PHS, arrows), but not in the brain blood vessels (vas). (c) High magnification of dotted region in b) showing uptake of LPS as fluorescent puncta (arrows) by the PCV, but LPS is absent in the hepatic region. (d) Characterization of the same timepoint at 12 hpi as in b–c) using fish carrying the lymphatic reporter clearly show localization of LPS within the facial lymphatics (FL, arrows). By stark contrast, LPS is not localized to the brain vessels, thereby providing evidence for drainage of LPS from brain to peripheral circulation through the lymphatics via the interstitial fluid. (e) Higher magnification of dotted box region in d). FL, facial lymphatics; PHS, primary head sinus; PCV, posterior cardinal vein; blec, brain lymphatic endothelial cells; sc, spinal canal; hv, hindbrain ventricle; vas, brain vessels; mb, midbrain; *, interstitial space. Scale bars all show 50 µm.

(a) Time plot showing LPS level changes in different regions as a ratio of injected fluorescent LPS. Injected LPS level was determined by the relative saturation level of fluorescence in the hindbrain ventricle as it is the immediate site filled in by LPS at time of injection. Both hindbrain and telencephalon ventricles reach 100% maximum LPS level based on fluorescence, while hindbrain interstitial space reaches around 30% of maximum and the periphery based on accumulation in the pronephros as a proxy reaches less than 15% of maximum. This plot illustrates a small fraction of maximum LPS that does get passed onto the periphery. Plot shows average and s.e.m. (standard error of means) of data from three independent LPS-injected animals. See Figure 2 for related analysis of tracing LPS from brain microinjection. (b) Still images of key events from tracing LPS-Alexa 594 after brain injection by high-resolution time-lapse stereomicroscopy, taken from a different LPS-injected animal than the example shown in Figure 2. Injection occurred at the 4s timepoint. Some injected animals such as this one, LPS is detected to also move anteriorly from midbrain/hindbrain ventricle into telencephalon ventricle. By 48 min post injection, we detected broad distribution of LPS in circulation at a low intensity. FL, facial lymphatics; blec, brain lymphatic endothelial cells; hbv, hindbrain ventricle; mb, midbrain; hb, hindbrain; tele, telencephalon; m, minute; s, second.

(a) Representative 3D volumetric and single z-plane images shown for after LPS or water vehicle injection in the brain. Dotted region shows liver in the single channel panels. Transgenes fabp10a:DsRed and mpeg1:GFP were used to label hepatocytes and macrophages, respectively. (b) Quantification of the number of macrophages found in the liver at 8hpi in four dpf zebrafish. No difference was found between the control and LPS injected groups as they both lacked macrophage infiltration. Each symbol represents an independent animal. Statistical test was determined by a two-tailed t-test. ns, not significant; MO, morpholino.

Infiltration of liver by macrophages triggered by brain-LPS injection is dependent on adaptor protein myd88 and cytokine il-34.

(a) Frequency of liver infiltration at 8–10 hr after brain-LPS injection showed that only Bay 11–7082 (1 µM) and dexamethasone (6.5 µM) treatments were effective for preventing macrophages from infiltrating the liver as determined by in situ hybridization. Number of independent animals as shown in the parenthesis. (b) Representative whole-mount RNA hybridization for macrophage marker mfap4 showing liver in the dotted region. White dotted liver indicates infiltration, while black dotted liver shows no infiltration. (c) Quantification of the number of infiltrating macrophages either after water or LPS injection in the brain in the control or drug treated conditions as determined by in vivo imaging. (d) Representative single z-plane images from z-stacks that show abundant infiltrating macrophages (arrows) in control treated animals after brain-LPS injection, but no infiltration in dexamethasone or Bay 11–7082 treated larvae. Student’s t-test was used to determine statistical significance.

Neutrophils and macrophages coordinate to infiltrate the liver during a systemic inflammatory response.

(a) Live ex vivo imaging of dissected liver from juvenile adult zebrafish after microinjection of either water vehicle or LPS in the brain at 16–18 hpi. Top, arrows point to macrophages in liver using the mpeg1:GFP transgene. Bottom, arrows point to neutrophils in the liver using the lyz:mCherry transgene. (b) Quantification of macrophage density (# mpeg1:GFP+ cell per field of view, FOV). (c) Quantification of neutrophil density (# lyz:mCherry+ cell per FOV). Scatter plots show uninjected controls, or animals microinjected with water vehicle or LPS in the brain. Each field of view (FOV) equals 0.045 squared mm. Statistical significance was determined by a two-tailed Student’s t-test.

(a) Schematic illustrating the method. Intravenous injection of clodronate liposome was performed at three dpf to allow time for the clodronate to effectively induce cell death to most macrophages over a 48 hr period. (b) Assessment of macrophage depletion by neutral red staining shows that most animals injected with clodronate liposome indeed resulted in an apparent loss of macrophages in the brain (58%, n = 12). Arrows point to microglia in the ‘no depletion’ category. (c) Scatter plot shows number of neutrophils infiltrating the liver to be even more increased after macrophage ablation in response to brain-LPS injection. Since clodronate based macrophage depletion is effective only in a proportion of the animals injected, we also used the cluster of the largest numbers of infiltrating neutrophils as a subset to show the animals with the most significant increase. (d) 3D volumetric view of the whole liver from imaging transgenic reporters fabp10a:DsRed for hepatocytes and lyz:GFP for neutrophils. Left, merged channels. Middle, DsRed channel. Right, GFP channel. Brain-water sample is shown 60 µm beneath the first surface of the liver, and the brain-LPS sample is 45 µm below the most outer liver surface. Significantly higher number of neutrophils (arrows) is easily visualized in the brain-LPS sample. Dextran-Alexa 488 was co-injected into the brain as a tracer to validate injections, and can be seen labeling the pronephros in the GFP channel. Statistical significance was determined by a two-tailed t-test and with Welch’s correction for unequal variances as determined by a F-test.

(a) csf3r morpholino-injected animals have less neutrophils (arrows) compared with controls as assessed by RNA in situ hybridization for a neutrophil marker myeloid-specific peroxidase mpx. Right bar graph, quantification of the area of mpx expression in the body posterior to the head using ImageJ show an average of a 27% reduction. Statistical significance was determined by a two-tailed t-test with Welch’s correction. (b) Reduction of neutrophils leads to a decrease in macrophage infiltration as assessed by RNA in situ hybridization as an alternative approach in addition to live transgenic imaging as shown in Figure 4. As a control, we also verified the level of neutrophil reduction in csf3r morpholino-injected animals by mpx in situ. Right bar graphs show frequency of liver infiltration by each myeloid cell type. N is shown in parenthesis and represents number of independent animals analyzed. (c) Left, scatter plot representing the effects of csf3r morpholino-mediated knockdown on macrophages and neutrophils showed no significant change to overall macrophage numbers, but a significant reduction in neutrophils in csf3r morpholino-injected animals. In vivo confocal imaging of double transgenic zebrafish carrying the macrophage reporter (mpeg1:GFP) and the neutrophil reporter (lyz:mCherry) was conducted. Right, representative fluorescent maximum projection, multi-tiled images of the transgenic zebrafish body. Dotted region shows area of cell number quantification. Statistical significance was determined by a one-tailed t-test. Each symbol represents an individual larva. Scale bar represents 500 µm. ns, not significant.

(a) Scattered dot and bar plot shows after brain-LPS injection, a relative liver size decrease in baseline and in control-p53-MO injected wild-type animals, whereas a significant increase in relative liver size in myeloid-depleted pu.1 morpholino-injected animals. Liver size was calculated based on changes in whole liver volume relative to the water-injected controls in each group. Each dot represents an individual larva analyzed. (b) Representative 3D rendered volumetric images of the whole liver from all groups and conditions analyzed. Statistical significance was determined by a two-tailed t-test for pair-wise comparison, and one-way ANOVA with Brown-Forsythe correction for the three-way comparisons. All scale bars represent 50 µm.