Ablation of Siglec-E augments brain inflammation and ischemic injury

Abstract

Sialic acid immunoglobulin-like lectin E (Siglec-E) is a subtype of pattern recognition receptors found on the surface of myeloid cells and functions as a key immunosuppressive checkpoint molecule. The engagement between Siglec-E and the ligand α_2,8-linked disialyl glycans activates the immunoreceptor tyrosine-based inhibitory motif (ITIM) in its intracellular domain, mitigating the potential risk of autoimmunity amid innate immune attacks on parasites, bacteria, and carcinoma. Recent studies suggest that Siglec-E is also expressed in the CNS, particularly microglia, the brain-resident immune cells. However, the functions of Siglec-E in brain inflammation and injuries under many neurological conditions largely remain elusive. In this study, we first revealed an anti-inflammatory role for Siglec-E in lipopolysaccharide (LPS)-triggered microglial activation. We then found that Siglec-E was induced within the brain by systemic treatment with LPS in mice in a dose-dependent manner, while its ablation exacerbated hippocampal reactive microgliosis in LPS-treated animals. The genetic deficiency of Siglec-E also aggravated oxygen-glucose deprivation (OGD)-induced neuronal death in mouse primary cortical cultures containing both neurons and glial cells. Moreover, Siglec-E expression in ipsilateral brain tissues was substantially induced following middle cerebral artery occlusion (MCAO). Lastly, the neurological deficits and brain infarcts were augmented in Siglec-E knockout mice after moderate MCAO when compared to wild-type animals. Collectively, our findings suggest that the endogenous inducible Siglec-E plays crucial anti-inflammatory and neuroprotective roles following ischemic stroke, and thus might underlie an intrinsic mechanism of resolution of inflammation and self-repair in the brain.

Keywords: Cerebral ischemia; DAMPs; Infarct; Innate immunity; LPS; Microgliosis; Neuroprotection; OGD; PAMPs; Sialic acid.

Fig. 1

Siglec-E suppresses prototypical proinflammatory mediators in LPS-activated microglia. A Mouse primary brain microglia from C57BL/6 wild type and Siglec-E knockout pups were prepared in 48-well plates (4 × 10⁴ cells/well). The purity of microglia in these cultures was assessed by immunostaining for the microglial marker Iba-1 (red fluorescence) and compared (n = 9, p = 0.678, Mann–Whitney U test). Note that cell nuclei were stained with DAPI (blue fluorescence) to illustrate all cell types. Scale bar: 50 μm. The cultured cells were then stimulated with LPS (0, 1, 10, or 100 ng/mL) for 16 h. A number of key proinflammatory mediators that were secreted by LPS-activated microglia into the culture medium, such as PGE₂ (B), IL-1β (C), IL-6 (D), and TNF-α (E), were measured by ELISA. Note that all these conventional proinflammatory mediators produced by microglia were induced by LPS treatment in a concentration-dependent manner and were further dramatically increased in the absence of Siglec-E (n = 8–12, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, two-way ANOVA with post hoc Šidák multiple comparisons). All data are presented as mean ± SEM

Fig. 2

Ablation of Siglec-E exacerbates microglial activation in the brain. A C57BL/6 wild type and Siglec-E knockout mice were systemically treated by LPS (0, 3, or 5 mg/kg, i.p.), and 24 h later the mRNA expression of Siglec-E in the hippocampus was measured by qPCR (n = 5–8, *p = 0.01, Kruskal–Wallis test with post hoc Dunn’s multiple comparisons). Data are visualized using box plot. B Reverse transcription PCR was performed to examine the Siglec-E mRNA expression in the hippocampal tissues of wild type and Siglec-E knockout mice treated by LPS, with GAPDH as control. C Immunostaining for Siglec-E (green fluorescence) and Iba1 (red fluorescence) indicating the hippocampal microglial activation in wild type and Siglec-E knockout mice was performed 24 h after LPS treatment (5 mg/kg, i.p.). Representative images are presented here to exemplify the induction of Siglec-E and Iba1 as well as their colocalization in activated microglia. Scale bar: 50 μm. D Iba1-positive (Iba1⁺) microglia in the hippocampus were counted (Left) and their Iba1 expression levels were quantified by measuring the fluorescence intensity (Right) (n = 4–6, *p < 0.05, **p < 0.01, Mann–Whitney U test). Data are shown as mean ± SEM

Fig. 3

Siglec-E regulates the morphology of brain microglia. A The morphological analyses of brain microglia (Iba1⁺) in mice with 26 measurements (Table 1) were performed 24 h after LPS treatment (5 mg/kg, i.p.) using ImageJ/Fiji software. A radar chart was generated to show the morphological changes of brain microglia in mice by LPS treatment and deletion of Siglec-E. The statistical p values less than 0.1 were labeled (n = 4–6, *p < 0.05, **p < 0.01, Mann–Whitney U test). Note that the 26 measurements can be categorized into three clusters indicated by purple, green, and yellow arcs, showing that the measurements in wild-type control mice, when compared to other two groups, were higher, equivalent, and lower, respectively. B Principal component analysis of microglial morphology was performed using IBM SPSS Statistics software with 3000 microglia randomly sampled from each group. The observable core cluster of brain microglia in wild-type control group (black dots) is shown by an ellipse (~ 90%). The remaining cells were scattered either along the positive axis of principal component 1 (~ 6%) or along the negative axis of principal component 2 (~ 4%). Treatment with LPS (blue dots) increased the cells scattered out of the core to ~ 17%, which was further increased in LPS-treated Siglec-E knockout mice (red dots) to ~ 25%

Fig. 4

Induced Siglec-E is neuroprotective after ischemia–reperfusion injuries. A Primary neuron–glia cultures derived from cortices of wild type or Siglec-E knockout mouse embryos were subjected to oxygen–glucose deprivation (OGD) for 1.5, 3, or 4.5 h. Following reoxygenation with full nutrition supply for 16 h, the cell viability in these cultures was measured and compared (n = 8–24, *p = 0.0172, two-way ANOVA with post hoc Šidák multiple comparisons). Data are presented as mean ± SEM. B Intraluminal filament-based middle cerebral artery occlusion (MCAO) model was utilized to examine the effects of genetic ablation of Siglec-E on cerebral ischemia. In this study, adult wild type and Siglec-E knockout male mice (12–14 weeks old) were subjected to transient MCAO for 30 min, which was followed by reperfusion for 72 h. C Siglec-E mRNA expression in the ipsilateral brain tissues of mice subjected to 30-min MCAO and 72-h reperfusion was measured by qPCR and compared to that of sham cohort (n = 7–14, ****p < 0.0001, Mann–Whitney U test). Data are visualized using box plot. D Neurological deficits of wild type and Siglec-E knockout mice after MCAO were evaluated at multiple time points using Bederson’s scale (n = 12–16, ***p < 0.001, two-way ANOVA). Data are presented as mean ± SEM. E Deficiency of Siglec-E in mice exacerbated post-stroke weight loss (n = 12–16, ***p < 0.001, two-way ANOVA with post hoc Šidák multiple comparisons). Data are presented as mean ± SEM. F Triphenyltetrazolium chloride (TTC) staining was performed to measure the brain infarction in wild type and Siglec-E knockout mice 72 h after MCAO. Representative images from each cohort are shown. The viable brain parenchyma appeared reddish, whereas the infarcted areas were pale and highlighted. G The volumes of brain infarcts in wild type and Siglec-E knockout mice were quantified and compared (n = 10–14, *p = 0.022, Mann–Whitney U test). Data are presented as mean ± SEM. H Animal mortality over 72 h following transient MCAO for 30 min (n = 14–17, p = 0.5734, log-rank test)