Search

High-mobility group box 1 (HMGB1) is a nucleoprotein with proinflammatory functions following cellular release during tissue damage. Moreover, antibody-mediated HMGB1 neutralization alleviates lipopolysaccharide (LPS)-induced shock, suggesting a role for HMGB1 as a superordinate therapeutic target for inflammatory and infectious diseases. Recent genetic studies have indicated cell-intrinsic functions of HMGB1 in phagocytes as critical elements of immune responses to infections, yet the role of extracellular HMGB1 signaling in this context remains elusive. We performed antibody-mediated and genetic HMGB1 deletion studies accompanied by in vitro experiments to discern context-dependent cellular sources and functions of extracellular HMGB1 during murine bloodstream infection with Listeria monocytogenes. Antibody-mediated neutralization of extracellular HMGB1 favors bacterial dissemination and hepatic inflammation in mice. Hepatocyte HMGB1, a key driver of postnecrotic inflammation in the liver, does not affect Listeria-induced inflammation or mortality. While we confirm that leukocyte HMGB1 deficiency effectuates disseminated listeriosis, we observed no evidence of dysfunctional autophagy, xenophagy, intracellular bacterial degradation, or inflammatory gene induction in primary HMGB1-deficient phagocytes or altered immune responses to LPS administration. Instead, we demonstrate that mice devoid of leukocyte HMGB1 exhibit impaired hepatic recruitment of inflammatory monocytes early during listeriosis, resulting in alterations of the transcriptional hepatic immune response and insufficient control of bacterial dissemination. Bone marrow chimera indicate that HMGB1 from both liver-resident and circulating immune cells contributes to effective pathogen control. Conclusion: Leukocyte-derived extracellular HMGB1 is a critical cofactor in the immunologic control of bloodstream listeriosis. HMGB1 neutralization strategies preclude an efficient host immune response against Listeria.

Additional file 1: Supplementary Fig. 1 p-IONX induced widespread neuropathic pain, anxiety- like behaviors and HMGB1 upregulation in the mPFC of C57BL/6J mice. a Schedule of experimental procedures. b Paw withdrawal thresholds to mechanical stimulation and noxious heat stimulation at the left hind paw and head withdrawal thresholds to noxious heat stimulation at the left vibrissal pad, respectively. *, ** and *** indicate P < 0.05, 0.01 and 0.001, respectively, compared with baseline (BL), Kruskal-Wallis test with repeated measures and Dunn’s post hoc. ## and ### indicate P < 0.01 and 0.001, respectively, compared with the sham group at the same time points, two-way ANOVA with Bonferroni post hoc. n = 8/group. c Anxiety-like behaviors measured by EPM, LDB and OFT tests. *, ** and *** indicate P < 0.05, 0.01 and 0.001, respectively, compared with the sham group, unpaired t test. n = 8/group. d Representative photomicrographs of HMGB1 immunostaining in the mPFC indicated by the red box in the left panel on D16 PO. Scale bar, 200 μm. e Quantification of HMGB1 fluorescence intensity in the mPFC on D9 and D16 PO. ** indicates P < 0.01, compared with the sham group, unpaired t test. n = 4/group. Supplementary Fig. 2 HMGB1 expression was upregulated in multiple brain regions after p-IONX in MRL/MPJ mice. a Schematic of experimental procedures. b Representative photomicrographs of HMGB1 immunostaining in the cortex, thalamus and amygdala. AI, agranular insular cortex; Au1, primary auditory cortex; AuV, secondary auditory cortex, ventral area; BLP, basolateral amygdaloid nucleus, posterior part; BMP, basomedial amygdaloid nucleus, posterior part; M1, primary motor cortex; MD, mediodorsal thalamic nucleus; PVA, paraventricular thalamic nucleus, anterior part; S1, primary sensory cortex; TeA, temporal association cortex; VPL, ventral posterolateral thalamic nucleus; VPM, ventral posteromedial thalamic nucleus. Scale bar, 200 μm. Supplementary Fig. 3 HMGB1 upregulation in the BLA did not affect anxiety onset and pain sensitization after p-IONX in MRL/MPJ mice. a Schedule of procedures of BLA drug delivery experiments. b Schematic of canula implantation in bilateral BLA (upper) and representative photomicrograph in the left BLA (lower). Dashed lines indicate the trace of implanted canula. Scale bar, 100 μm. c Paw withdrawal thresholds to mechanical stimulation and noxious heat stimulation at the left hind paw and head withdrawal threshold to noxious heat stimulation at the ipsilateral vibrissal pad before and after p-IONX. ** and *** indicate P < 0.01 and 0.001, respectively, compared with the baseline (BL), Kruskal-Wallis test with repeated measures and Dunn’s post hoc. #, ## and ### indicate P < 0.05, 0.01 and 0.001, respectively, compared with the sham + mAb group at the same time points, two-way ANOVA with Bonferroni post hoc. d Anxiety-like behaviors measured by EPM, LDB and OFT tests on D8/9 after p-IONX. n = 8/group. Supplementary Fig. 4 Systemic anti-HMGB1 mAb reduced HMGB1 expression after p-IONX in MRL/MPJ mice. a Schedule of experimental procedures. b-c Representative images of protein bands in western blotting (b) and quantification of HMGB1 (c) in the mPFC, BLA and medulla on D7 after p-IONX. * and ** indicate P < 0.05 and 0.01, respectively, compared with the indicated groups, unpaired t test. n = 4/group. Supplementary Fig. 5 Systemic anti-HMGB1 mAb did not affect the reduced activity of pyramidal neurons in mPFC layer 5/6 after p-IONX in MRL/MPJ mice. a Schedule of experimental procedures. b Example of sEPSCs and sIPSCs in sham + saline, p-IONX+ saline and p-IONX+ anti-HMGB1 mAb groups. c-d Amplitude, frequency and charge transfer of sEPSCs (c) and sIPSCs (d). e Quantification of E/I ratio. f Resting potential. g Rheobase. h-i Representative traces of action potential firing (h) and number of spikes (i) evoked by injecting current at two-fold of rheobase. * and ** indicate P < 0.05 and 0.01, respectively, compared with the indicated groups, unpaired t test or Mann-Whitney test. n = 9-10 neurons from 4 mice/group. Supplementary Fig. 6 Optogenetic inhibition of mPFC pyramidal neurons alleviated anxiety after p-IONX. a Schematic drawing of sites for virus injection and optical fiber placement. b Example photomicrographs showing the Arch-eYFP in mPFC (left) and colocalization of eYFP and CaMKIIα, a marker of glutamatergic neurons (right). c Example neuron in the mPFC that deceased its firing frequency in response to yellow laser stimulation. d Paw withdrawal thresholds to mechanical stimulation and noxious heat stimulation. e Anxiety-like behaviors measured by OFT and EPM tests on D14 after surgery. ** indicates P < 0.01, compared with the laser off group, unpaired t test. n = 8-10/group.

BackgroundSystemic allergic reaction is characterized by vasodilation and vascular leakage, which causes a rapid, precipitous and sustained decrease in arterial blood pressure with a concomitant decrease of cardiac output. Histamine is a major mediator released by mast cells in allergic inflammation and response. It causes a cascade of inflammation and strongly increases vascular permeability within minutes through its four G-protein-coupled receptors (GPCRs) on endothelial cells. High mobility group box-1 (HMGB1), a nonhistone chromatin-binding nuclear protein, can be actively secreted into the extracellular space by endothelial cells. HMGB1 has been reported to exert pro-inflammatory effects on endothelial cells and to increase vascular endothelial permeability. However, the relationship between histamine and HMGB1-mediated signaling in vascular endothelial cells and the role of HMGB1 in anaphylactic-induced hypotension have never been studied.Methods and resultsEA.hy 926 cells were treated with different concentrations of histamine for the indicated periods. The results showed that histamine induced HMGB1 translocation and release from the endothelial cells in a concentration- and time-dependent manner. These effects of histamine were concentration-dependently inhibited by d-chlorpheniramine, a specific H1 receptor antagonist, but not by H2 or H3/4 receptor antagonists. Moreover, an H1-specific agonist, 2-pyridylethylamine, mimicked the effects of histamine, whereas an H2-receptor agonist, 4-methylhistamine, did not. Adrenaline and noradrenaline, which are commonly used in the clinical treatment of anaphylactic shock, also inhibited the histamine-induced HMGB1 translocation in endothelial cells. We therefore established a rat model of allergic shock by i.v. injection of compound 48/80, a potent histamine-releasing agent. The plasma HMGB1 levels in compound 48/80-injected rats were higher than those in controls. Moreover, the treatment with anti-HMGB1 antibody successfully facilitated the recovery from compound 48/80-induced hypotension.ConclusionHistamine induces HMGB1 release from vascular endothelial cells solely through H1 receptor stimulation. Anti-HMGB1 therapy may provide a novel treatment for life-threatening systemic anaphylaxis.

Human adipose-derived stem cells (HASCs) represent pluripotent cells capable of differentiating into the bone tissue. Meanwhile, human amnion-derived mesenchymal stem cells (HAMSCs) could cause mesenchymal stem cells to differentiate into the bone tissue. This work assessed the osteogenic effects exerted by HAMSCs on the potential of HASCs to form bone cells. Cell growth was evaluated flow-cytometrically. Differentiation into osteoblasts and mineral formation were assessed by chromogenic alkaline phosphatase activity substrate assay and Alizarin red S staining. Adiponectin (APN), the adipocytokine secreted by adipocytes, was evaluated by enzyme-linked immunosorbent assay. In this study, HAMSCs concentration-dependently induced growth, osteoblastic differentiation, and APN excretion in HASCs. Mechanistically, immunofluorescence and immunoblot revealed HAMSCs promoted cytosolic translocation of leucine zipper motif (APPL1) from the nucleus and induced extracellular signaling-regulated kinase 1/2 (ERK1/2) phosphorylation in HASCs. Furthermore, HAMSC effects were markedly blunted by pretreatment with APPL1 siRNA and U0126, an ERK1/2 signaling inhibitor with high selectivity. These results suggested that APN excretion is not suppressed by APPL1 knockdown in HASCs, but by ERK1/2 inhibition. These findings collectively indicate that HAMSCs induce the osteogenesis of HASCs by promoting APN excretion through APPL1-ERK1/2 activation.

Effects of anti-high mobility group box 1 (anti-HMGB1) monoclonal antibody (mAb) treatment on the expression of toll-like receptor 4 (TLR4). The results were normalized to the expression of glyceralaldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The basal expression level of normal mice was calibrated as 1.0 (dotted line). Data represent the mean (± SEM) of 5 to 10 mice. The sense and antisense primers used for the analysis of the expression of TLR4 were as follows: 5′-GCACTGTTCTTCTCCTGCC-3′ and 5′-GTTTCCTGTCAGTATCAAG-3′ [GenBank NM_021297]. There was no statistical difference between groups as determined by the Mann–Whitney U test.

Bone volume inadequacy is an emerging clinical problem impairing the feasibility and longevity of dental implants. Human bone marrow mesenchymal stem cells (HBMSCs) have been widely used in bone remodeling and regeneration. This study examined the effect of long noncoding RNAs (lncRNAs)-H19 on the human amnion-derived mesenchymal stem cells (HAMSCs)-droved osteogenesis in HBMSCs. HAMSCs and HBMSCs were isolated from abandoned amniotic membrane samples and bone marrow. The coculture system was conducted using transwells, and H19 level was measured by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). The mechanism was further verified. We here discovered that osteogenesis of HBMSCs was induced by HAMSCs, while H19 level in HAMSCs was increased during coculturing. H19 had no significant effect on the proliferative behaviors of HBMSCs, while its overexpression of H19 in HAMSCs led to the upregulated osteogenesis of HBMSCs in vivo and in vitro; whereas its knockdown reversed these effects. Mechanistically, H19 promoted miR-675 expression and contributed to the competitively bounding of miR-675 and Adenomatous polyposis coli (APC), thus significantly activating the Wnt/β-catenin pathway. The results suggested that HAMSCs promote osteogenic differentiation of HBMSCs via H19/miR-675/APC pathway, and supply a potential target for the therapeutic treatment of bone-destructive diseases.