Interleukin 17 (IL-17) plays critical roles in the pathogenesis of various autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE). How the signals triggered by this powerful inflammatory cytokine are controlled to avoid abnormal inflammatory responses is not well understood. In this study, we report that TRAF3 is a receptor proximal negative regulator of IL-17 receptor (IL-17R) signaling. TRAF3 greatly suppressed IL-17-induced NF-κB and mitogen-activated protein kinase activation and subsequent production of inflammatory cytokines and chemokines. Mechanistically, the binding of TRAF3 to IL-17R interfered with the formation of the receptor signaling activation complex IL-17R-Act1-TRAF6, resulting in suppression of downstream signaling. TRAF3 markedly inhibited IL-17-induced expression of inflammatory cytokine and chemokine genes in vivo and consequently delayed the onset and greatly reduced the incidence and severity of EAE. Thus, TRAF3 is a negative regulator of IL-17R proximal signaling.

We report on the synthesis of TiO2 nanotube (NT) powders using anodic oxidation and ultrasonication. Compared to free-standing NT array films, the powder-type NTs can be easily fabricated in a cost-effective way. Particularly, without the substrate effect arising from underlying Ti metals, highly crystallized NT powders with intact tube structures and pure anatase phase can be obtained using high-temperature heat treatment. The application of NTs with different crystallinity for the photocatalytic decomposition of methylene blue (MB) was then demonstrated. The results showed that with increasing annealing temperature, the photocatalytic decomposition rate was gradually enhanced, and the NT powder electrode annealed at 650°C showed the highest photoactivity. Compared to typical NTs annealed at 450°C, the rate constant increased by 2.7-fold, although the surface area was 21% lower. These findings indicate that the better photocatalytic activity was due to the significantly improved crystallinity of anatase anodic NTs in powder form, resulting in a low density of crystalline defects. This simple and efficient approach is applicable for scaled-up water purification and other light utilization applications.

Schwann cells (SCs) are attractive seed cells in neural tissue engineering, but their application is limited by attenuated biological activities and impaired functions with aging. Therefore, it is important to explore an approach to enhance the viability and biological properties of SCs. In the present study, a magnetic composite made of magnetically responsive magnetic nanoparticles (MNPs) and a biodegradable chitosan-glycerophosphate polymer were prepared and characterized. It was further explored whether such magnetic nanocomposites via applied magnetic fields would regulate SC biological activities. The magnetization of the magnetic nanocomposite was measured by a vibrating sample magnetometer. The compositional characterization of the magnetic nanocomposite was examined by Fourier-transform infrared and X-ray diffraction. The tolerance of SCs to the magnetic fields was tested by flow-cytometry assay. The proliferation of cells was examined by a 5-ethynyl-2-deoxyuridine-labeling assay, a PrestoBlue assay, and a Live/Dead assay. Messenger ribonucleic acid of BDNF, GDNF, NT-3, and VEGF in SCs was assayed by quantitative real-time polymerase chain reaction. The amount of BDNF, GDNF, NT-3, and VEGF secreted from SCs was determined by enzyme-linked immunosorbent assay. It was found that magnetic nanocomposites containing 10% MNPs showed a cross-section diameter of 32.33±1.81 µm, porosity of 80.41%±0.72%, and magnetization of 5.691 emu/g at 8 kOe. The 10% MNP magnetic nanocomposites were able to support cell adhesion and spreading and further promote proliferation of SCs under magnetic field exposure. Interestingly, a magnetic field applied through the 10% MNP magnetic scaffold significantly increased the gene expression and protein secretion of BDNF, GDNF, NT-3, and VEGF. This work is the first stage in our understanding of how to precisely regulate the viability and biological properties of SCs in tissue-engineering grafts, which combined with additional molecular factors may lead to the development of new nerve grafts.

Two kinds of peony roots--white peony root (WPR) and red peony root (RPR)--are used for different remedies in traditional Chinese medicine; however, most of them are derived from the same botanical origin, Paeonia lactiflora. The difference between WPR and RPR has been debated for a long time. This study attempted to clarify the genetic and chemical characteristics of WPR and RPR in order to provide a scientific dataset for their identification and effective use. The nucleotide sequence of nrDNA internal transcribed spacer (ITS) and the contents of 8 main bioactive constituents were analyzed from specimens of P. lactiflora, P. veitchii and two related species as well as crude drug samples of WPR, RPR and peony root produced in Japan. Of the samples derived from P. lactiflora, the WPR produced in the southern parts of China and the RPR produced in the northern parts of China were clearly divided into two subgroups within the P. lactiflora group based on similarity of the ITS sequences. The nucleotides at positions 69, 458 and 523 upstream of the ITS sequence served as molecular markers to discriminate between WPR and RPR. Quantitative analysis indicated that the RPR samples obviously contained a higher content of paeoniflorin and paeonol, but a lower content of albiflorin than the WPR produced in the southern parts of China and peony root produced in Japan. The WPR available from Chinese markets was usually processed by sulfur fumigation, which resulted in an extremely low content of paeoniflorin. This study indicated that WPR and RPR were not only geographically isolated, but also genetically and chemically separated. The ITS sequence provided a genetic index for their identification.

Human noroviruses are the primary cause of severe childhood diarrhea in the United States, and they are of particular clinical importance in pediatric populations in the developing world. A major contributing factor to the general increased severity of infectious diseases in these regions is malnutrition-nutritional status shapes host immune responses and the composition of the host intestinal microbiota, both of which can influence the outcome of pathogenic infections. In terms of enteric norovirus infections, mucosal immunity and intestinal microbes are likely to contribute to the infection outcome in substantial ways. We probed these interactions using a murine model of malnutrition and murine norovirus infection. Our results reveal that malnutrition is associated with more severe norovirus infections as defined by weight loss, impaired control of norovirus infections, reduced antiviral antibody responses, loss of protective immunity, and enhanced viral evolution. Moreover, the microbiota is dramatically altered by malnutrition. Interestingly, murine norovirus infection also causes changes in the host microbial composition within the intestine but only in healthy mice. In fact, the infection-associated microbiota resembles the malnutrition-associated microbiota. Collectively, these findings represent an extensive characterization of a new malnutrition model of norovirus infection that will ultimately facilitate elucidation of the nutritionally regulated host parameters that predispose to more severe infections and impaired memory immune responses. In a broad sense, this model may provide insight into the reduced efficacy of oral vaccines in malnourished hosts and the potential for malnourished individuals to act as reservoirs of emergent virus strains. IMPORTANCE Malnourished children in developing countries are susceptible to more severe infections than their healthy counterparts, in particular enteric infections that cause diarrhea. In order to probe the effects of malnutrition on an enteric infection in a well-controlled system devoid of other environmental and genetic variability, we studied norovirus infection in a mouse model. We have revealed that malnourished mice develop more severe norovirus infections and they fail to mount effective memory immunity to a secondary challenge. This is of particular importance because malnourished children generally mount less effective immune responses to oral vaccines, and we can now use our new model system to probe the immunological basis of this impairment. We have also determined that noroviruses evolve more readily in the face of malnutrition. Finally, both norovirus infection and malnutrition independently alter the composition of the intestinal microbiota in substantial and overlapping ways.

The physiological and pathological functions of angiotensin II are largely mediated through activating the cell surface angiotensin II type 1 receptor (AT1R). However, the molecular mechanisms underlying the transport of newly synthesized AT1R from the endoplasmic reticulum (ER) to the cell surface remain poorly defined. Here we demonstrated that the C-terminus (CT) of AT1R directly and strongly bound to tubulin and the binding domains were mapped to two consecutive Lys residues at positions 310 and 311 in the CT membrane-proximal region of AT1R and the acidic CT of tubulin, suggestive of essentially ionic interactions between AT1R and tubulin. Furthermore, mutation to disrupt tubulin binding dramatically inhibited the cell surface expression of AT1R, arrested AT1R in the ER, and attenuated AT1R-mediated signaling measured as ERK1/2 activation. These data demonstrate for the first time that specific Lys residues in the CT juxtamembrane region regulate the processing of AT1R through interacting with tubulin. These data also suggest an important role of the microtubule network in the cell surface transport of AT1R.

Orf, caused by Orf virus (ORFV), is a globally distributed zoonotic disease responsible for serious economic losses in the agricultural sector. However, the mechanism underlying ORFV infection remains largely unknown. Circular RNAs (circRNAs), a novel type of endogenous non-coding RNAs, play important roles in various pathological processes but their involvement in ORFV infection and host response is unclear. In the current study, whole transcriptome sequencing and small RNA sequencing were performed in ORFV-infected goat skin fibroblast cells and uninfected cells. A total of 151 circRNAs, 341 messenger RNAs (mRNAs), and 56 microRNAs (miRNAs) were differently expressed following ORFV infection. Four circRNAs: circRNA1001, circRNA1684, circRNA3127 and circRNA7880 were validated by qRT-PCR and Sanger sequencing. Gene ontology (GO) analysis indicated that host genes of differently expressed circRNAs were significantly enriched in regulation of inflammatory response, epithelial structure maintenance, positive regulation of cell migration, positive regulation of ubiquitin-protein transferase activity, regulation of ion transmembrane transport, etc. The constructed circRNA-miRNA-mRNA network suggested that circRNAs may function as miRNA sponges indirectly regulating gene expression following ORFV infection. Our study presented the first comprehensive profiles of circRNAs in response to ORFV infection, thus providing new clues for the mechanisms of interactions between ORFV and the host.

Cutaneous squamous cell carcinoma (cSCC) is the second most common non-melanoma skin cancer worldwide. Today, cSCC is diagnosed by visual inspection followed by invasive skin biopsy. There is a need to develop non-invasive diagnostic tools to achieve early and accurate detection. Photoacoustic imaging (PAI) possesses high ultrasonic resolution and strong optical contrast at new depths (<1-5 cm). Together with exogenous contrast agents, PAI has found promising use in various tumors in living subjects. The expression of integrin αvβ6 is significantly up-regulated in cSCC. We fabricated an anti-integrin αvβ6 antibody and labeled it with indocyanine green (ICG) to form an ICG-αvβ6 antibody. The results showed that the ICG-αvβ6 antibody probe could be used to detect cSCC with high specificity (3-fold over the control by PAI) and deep penetration (approximately 1 cm) by PAI. This suggests that the ICG-αvβ6 antibody is a promising probe targeting the integrin αvβ6 for detection of cSCC tumors by PAI and fluorescence imaging. It may find clinical application in the early diagnosis of cSCC as well as in intraoperative navigation.

Quaking (QKI), an RNA-binding protein, has been reported to exhibit numerous biological functions, such as mRNA regulation, cancer suppression, and anti-inflammation. However, little known about the effects of QKI on bone metabolism. In this study, we used a monocyte/macrophage-specific QKI knockout transgenic mouse model to investigate the effects of QKI deficiency on receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis. The loss of QKI promoted the formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts (OCs) from bone marrow macrophages, and upregulated the expression of OC-specific markers, including TRAP (Acp5) and cathepsin K (Ctsk). The pro-osteoclastogenesis effect of QKI deficiency was achieved by amplifying the signaling cascades of the NF-κB and mitogen-activated protein kinase (MAPK) pathways; then, signaling upregulated the activation of nuclear factor of activated T cells c1 (NFATc1), which is considered to be the core transcription factor that regulates OC differentiation. In addition, QKI deficiency could inhibit osteoblast (OB) formation through the inflammatory microenvironment. Taken together, our data suggest that QKI deficiency promoted OC differentiation and disrupted bone metabolic balance, and eventually led to osteopenia under physiological conditions and aggravated the degree of osteoporosis under pathological conditions.

Brucella-caused brucellosis is one of the most widespread worldwide zoonoses. Lipopolysaccharide (LPS) of Brucella, which functions as pathogen-associated molecular patterns (PAMPs), is an important virulence factor that elicits protective antibodies. Per of B. melitensis is involved in the biosynthesis of the O-side chain of LPS. Autophagy is a crucial element of the innate immune response against intracellular pathogens including Brucella. In this study, we observed that autophagy was inhibited in RAW264.7 cells infected with Brucella melitensis ∆per. And, a high-throughput array-based screen and qRT-PCR validation were performed to identify the differentially expressed miRNAs in RAW264.7 cells infected with B. melitensis M5-90 ∆per. The results suggested that mmu-miR-146a-5p, mmu-miR-155-5p, mmu-miR-146b-5p, and mmu-miR-3473a were upregulated and mmu-miR-30c-5p was downregulated. During B. melitensis M5-90 ∆per infection, the increased expression of miR-146b-5p inhibited the autophagy activation in RAW264.7 cells. Using a bioinformatics approach, Tbc1d14 was predicted to be a potential target of miR-146b-5p. The results of a luciferase reporter assay indicated that miR-146b-5p directly targeted the 3'-UTR of Tbc1d14, and the interaction between miR-146b-5p and the 3'-UTR of Tbc1d14 was sequence-specific. High-throughput RNA-Seq-based screening was performed to identify differentially expressed genes in Tbc1d14-expressing RAW264.7 cells, and these were validated by qRT-PCR. Among the differentially expressed genes, four autophagy associated genes, IFNγ-inducible p47 GTPase 1 (IIGP1), nuclear receptor binding protein 2 (Nrbp2), transformation related protein 53 inducible nuclear protein 1 (Trp53inp1), and immunity-related GTPase family M member 1 (Irgm1), were obtained. Our findings provide important insights into the functional mechanism of LPS of B. melitensis.

The structure and growth of a blood clot depend on the localization of tissue factor (TF), which can trigger clotting during the hemostatic process or promote thrombosis when exposed to blood under pathological conditions. We sought to understand how the growth, structure, and mechanical properties of clots under flow are shaped by the simultaneously varying TF surface density and its exposure area. We used an eight-channel microfluidic device equipped with a 20- or 100-μm-long collagen surface patterned with lipidated TF of surface densities ∼0.1 and ∼2 molecules/μm2. Human whole blood was perfused at venous shear, and clot growth was continually measured. Using our recently developed computational model of clot formation, we performed simulations to gain insights into the clot's structure and its resistance to blood flow. An increase in TF exposure area resulted not only in accelerated bulk platelet, thrombin, and fibrin accumulation, but also in increased height of the platelet mass and increased clot resistance to flow. Moreover, increasing the TF surface density or exposure area enhanced platelet deposition by approximately twofold, and thrombin and fibrin generation by greater than threefold, thereby increasing both clot size and its viscous resistance. Finally, TF effects on blood flow occlusion were more pronounced for the longer thrombogenic surface than for the shorter one. Our results suggest that TF surface density and its exposure area can independently enhance both the clot's occlusivity and its resistance to blood flow. These findings provide, to our knowledge, new insights into how TF affects thrombus growth in time and space under flow.

The ongoing global pandemic of COVID-19 disease, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), mainly infect lung epithelial cells, and spread mainly through respiratory droplets. However, recent studies showed potential intestinal infection of SARS-CoV-2, implicated the possibility that the intestinal infection of SARS-CoV-2 may correlate with the dysbiosis of gut microbiota, as well as the severity of COVID-19 symptoms. Here, we investigated the alteration of the gut microbiota in COVID-19 patients, as well as analyzed the correlation between the altered microbes and the levels of intestinal inflammatory cytokine IL-18, which was reported to be elevated in the serum of in COVID-19 patients. Comparing with healthy controls or seasonal flu patients, the gut microbiota showed significantly reduced diversity, with increased opportunistic pathogens in COVID-19 patients. Also, IL-18 level was higher in the fecal samples of COVID-19 patients than in those of either healthy controls or seasonal flu patients. Moreover, the IL-18 levels were even higher in the fecal supernatants obtained from COVID-19 patients that tested positive for SARS-CoV-2 RNA than those that tested negative in fecal samples. These results indicate that changes in gut microbiota composition might contribute to SARS-CoV-2-induced production of inflammatory cytokines in the intestine and potentially also to the onset of a cytokine storm.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with high fatality. Poor prognosis of SFTS has been associated with dysregulated host immunity; however, the immune patterns associated with pathophysiology involving SFTS exacerbation remain unclear. Here, we show that the single-cell landscape of peripheral immune responses is reprogrammed in SFTS and characterized by monocyte shift to an intermediate type along with complement activation, perturbation of plasmablast composition, and highly exhausted T cells, all correlated with lethal consequences. We identify the overexpression of interferon (IFN)-stimulated genes across most immune cell types after SFTSV infection, which are simultaneously related to older age, high viremia, and a hyperinflammatory response. A retrospective clinical study reveals no efficiency of IFN-α in treating SFTS. These data collectively support the intermediate monocytes and IFN-I-inducible plasmablasts to be major targets for SFTS virus infection, and they indicate the pivotal role of the IFN-I response in exacerbating hyperinflammation and lethal SFTS.

Metabolic rewiring is essential for Th17 cells' functional identity to sense and interpret environmental cues. However, the environmental metabolic checkpoints with specific regulation of Th17 cells, manifesting potential therapeutic opportunities to autoimmune diseases, remain largely unknown. Here, by screening more than one hundred compounds derived from intestinal microbes or diet, we found that vitamin B5 (VB5) restrains Th17 cell differentiation as well as related autoimmune diseases such as experimental autoimmune encephalomyelitis and colitis. Mechanistically, VB5 is catabolized into coenzyme A (CoA) in a pantothenate kinase (PANK)-dependent manner, and in turn, CoA binds to pyruvate kinase isoform 2 (PKM2) to impede its phosphorylation and nuclear translocation, thus inhibiting glycolysis and STAT3 phosphorylation. In humans, reduced serum VB5 levels are found in both IBD and MS patients. Collectively, our study demonstrates a role of VB5 in Th17 cell metabolic reprograming, thus providing a potential therapeutic intervention for Th17 cell-associated autoimmune diseases.

Mushroom poisoning is a public health concern worldwide that not only harms the physical and mental health of those who are poisoned but also increases the medical and financial burden on families and society. The present study aimed to describe and analyze the current situations and factors influencing mushroom poisoning outbreaks in Guizhou province, Southwest China, between January 2012 and June 2022, and to predict the future trends of its occurrence. Our study provides a basis for the rational formulation of prevention and control and medical resource allocation policies for mushroom poisoning. The epidemiological characteristics and factors influencing mushroom poisoning incidence were analyzed using descriptive epidemiological methods and the chi-squared test, respectively. Then, future occurrence trends were predicted using the SARIMA and Prophet models. In total, 1577 mushroom poisoning incidents were recorded in Guizhou Province, with 7347 exposures, 5497 cases, 3654 hospitalizations, and 93 fatalities. The mortality rate was 4.45% in 1 ~ 6 years higher than other age groups. There were notable geographic and seasonal characteristics, with the number of occurrences much higher in rural areas (1198) than in cities (379), and poisoning cases were more common during the rainy season (June to September). The mortality rate of household poisoning cases was 1.86%, with the most deaths occurring in households. Statistically significant differences were observed in the incidence across various cities, periods, and poisoning locations (P < 0.05). Both models had advantages and disadvantages for prediction. Nevertheless, the SARIMA model had better overall prediction results than the Prophet model (R > 0.9, the residual plot of the prediction results was randomly distributed, and RMSESARIMA < RMSEProphet). However, the prediction result plot of the Prophet model was more explanatory than the SARIMA model and could visualize overall and seasonal trends. Both models predicted that the prevalence of mushroom poisoning would continue to increase in the future; however, the number of fatalities is generally declining. Seasonal patterns indicated that a high number of deaths from gooseberry mushroom poisoning occurred in October. The epidemiological trends of mushroom poisoning remain severe, and health education on related knowledge must be strengthened in rural areas, with June to October as the key prevention and control phase. Further, medical treatment of mushroom poisoning cases with clinical symptoms should pay attention to inquiries to check whether the mushroom is similar in appearance to the Amanita, particularly in October.

Schwann cell (SC), which plays a key role in peripheral nerve regeneration, is one of the most classic supportive cells in neural tissue engineering. However, the biological activity of SCs seeded in nerve scaffolds decays subsequently due to local hypoxia induced by ischemia. Thus, we aimed to investigate whether a synthetic oxygen carrier-enriched fibrin gel would provide a sustained oxygen release to cultured SCs in vitro for overcoming a temporary (48 h) oxygen deprivation. In this study, perfluorotributylamine (PFTBA)-based oxygen carrying fibrin gel was prepared to provide oxygen for SCs under normoxic or hypoxic conditions. The dissolved oxygen within the culture media was measured by a blood-gas analyzer to quantify the time course of oxygen release from the PFTBA-enriched fibrin gel. SCs were cultured in the presence or absence of PFTBA-enriched fibrin gel under normoxic or hypoxic conditions. The tolerance of SCs to hypoxia was examined by a cell apoptosis assay. The growth of cells was characterized using S-100 staining and a CCK-8 assay. The migration of cells was examined using a Transwell chamber. The mRNA of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial cell derived neurotrophic factor (GDNF), neural cell adhesion molecule (N-CAM) and vascular endothelial growth factor (VEGF) in SCs were assayed by RT-PCR. In addition, SCs cultured in 3D PFTBA-enriched hydrogel were characterized by Live/Dead staining and the mRNA levels of BDNF, NGF, GDNF, N-CAM and VEGF were assayed by RT-PCR. The results showed that the PFTBA-enriched fibrin hydrogel was able to promote cell adhesion, migration, and proliferation under hypoxic conditions. Interestingly, PFTBA applied through the fibrin hydrogel dramatically enhanced the mRNA of BDNF, NGF, GDNF, N-CAM and VEGF under hypoxic condition. These findings highlight the possibility of enhancing nerve regeneration in cellular nerve grafts through PFTBA increased neurotropic secretion in SCs.

Whether or not primary norovirus infections induce protective immunity has become a controversial issue, potentially confounded by the comparison of data from genetically distinct norovirus strains. Early human volunteer studies performed with a norovirus-positive inoculum initially led to the conclusion that primary infection does not generate long-term, protective immunity. More recently though, the epidemiological pattern of norovirus pandemics has led to the extrapolation that primary norovirus infection induces herd immunity. While these are seemingly discordant observations, they may in fact reflect virus strain-, cluster-, or genogroup-specific differences in protective immunity induction. Here, we report that highly genetically related intra-cluster murine norovirus strains differ dramatically in their ability to induce a protective immune response: Primary MNV-3 infection induced robust and cross-reactive protection, whereas primary MNV-1 infection induced modest homotypic and no heterotypic protection. In addition to this fundamental observation that intra-cluster norovirus strains display remarkable differences in protective immunity induction, we report three additional important observations relevant to norovirus:host interactions. First, antibody and CD4⁺ T cells are essential to controlling secondary norovirus infections. Second, the viral minor structural protein VP2 regulates the maturation of antigen presenting cells and protective immunity induction in a virus strain-specific manner, pointing to a mechanism by which MNV-1 may prevent the stimulation of memory immune responses. Third, VF1-mediated regulation of cytokine induction also correlates with protective immunity induction. Thus, two highly genetically-related norovirus strains displayed striking differences in induction of protective immune responses, strongly suggesting that the interpretation of norovirus immunity and vaccine studies must consider potential virus strain-specific effects. Moreover, we have identified immune (antibody and CD4⁺ T cells) and viral (VP2 and possibly VF1) correlates of norovirus protective immunity. These findings have significant implications for our understanding of norovirus immunity during primary infections as well as the development of new norovirus vaccines.

Ischemic stroke is the third most common cause of death and the leading cause of disability worldwide in adults. The antiepileptic drug valproic acid (VPA) was reported to protect cerebral ischemia/reperfusion injury. However, the action mechanism of VPA in cerebral ischemia/reperfusion injury has not been fully understood. We explored the action mechanism of VPA in vivo and in vitro. Gerbils were subjected to transient global cerebral ischemic-reperfusion injury, and hippocampal neuron injury was treated with oxygen-glucose deprivation in vitro. Morris water maze test was performed to evaluate the cognitive dysfunction. Histopathological examinations and western blot were performed to evaluate the pyroptosis of neurons. The results showed that VPA attenuated the cognitive dysfunction, pyroptosis of the gerbils suffer from ischemic-reperfusion injury and decreased hippocampal neurons pyroptosis induced by oxygen-glucose deprivation in vitro. In addition, western blot and real-time PCR analysis revealed that VPA modulated the protein expression of apoptosis repressor with caspase recruitment domain (ARC), caspase-1 and IL-1β/IL-18. Our results suggested that VPA alleviated ischemic/reperfusion injury-mediated neuronal impairment by anti-pyroptotic effects.

Secreted by activated cells or passively released by damaged cells, extracellular HMGB1 is a prototypical damage-associated molecular pattern (DAMP) inflammatory mediator. During the course of developing extracorporeal approaches to treating injury and infection, we inadvertently discovered that haptoglobin, the acute phase protein that binds extracellular hemoglobin and targets cellular uptake through CD163, also binds HMGB1. Haptoglobin-HMGB1 complexes elicit the production of antiinflammatory enzymes (heme oxygenase-1) and cytokines (e.g., IL-10) in WT but not in CD163-deficient macrophages. Genetic disruption of haptoglobin or CD163 expression significantly enhances mortality rates in standardized models of intra-abdominal sepsis in mice. Administration of haptoglobin to WT and to haptoglobin gene-deficient animals confers significant protection. These findings reveal a mechanism for haptoglobin modulation of the inflammatory action of HMGB1, with significant implications for developing experimental strategies targeting HMGB1-dependent inflammatory diseases.

Cytoplasmic membrane-bound connexin 43 (Cx43) proteins oligomerize into hexameric channels (hemichannels) that can sometimes dock with hemichannels on adjacent cells to form gap junctional (GJ) channels. However, the possible role of Cx43 hemichannels in sterile and infectious inflammatory diseases has not been adequately defined due to the lack of selective interventions. Here we report that a proinflammatory mediator, the serum amyloid A (SAA), resembled bacterial endotoxin by stimulating macrophages to up-regulate Cx43 expression and double-stranded RNA-activated protein kinase R (PKR) phosphorylation in a TLR4-dependent fashion. Two well-known Cx43 mimetic peptides, the GAP26 and TAT-GAP19, divergently affected macrophage hemichannel activities in vitro, and differentially altered the outcome of lethal sepsis in vivo. By screening a panel of Cx43 mimetic peptides, we discovered that one cysteine-containing peptide, P5 (ENVCYD), effectively attenuated hemichannel activities, and significantly suppressed endotoxin-induced release of ATP and HMGB1 in vitro. In vivo, the P5 peptide conferred a significant protection against hepatic ischemia/reperfusion injury and lethal microbial infection. Collectively, these findings have suggested a pathogenic role of Cx43 hemichannels in sterile injurious as well as infectious inflammatory diseases possibly through facilitating extracellular ATP efflux to trigger PKR phosphorylation/activation.