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P2X receptors are a class of nonselective cation channels widely distributed in the immune and nervous systems, and their dysfunction is a significant cause of tumors, inflammation, leukemia, and immune diseases. P2X7 is a unique member of the P2X receptor family with many properties that differ from other subtypes in terms of primary sequence, the architecture of N- and C-terminals, and channel function. Here, we suggest that the observed lengthened β2- and β3-sheets and their linker (loop β2,3), encoded by redundant sequences, play an indispensable role in the activation of the P2X7 receptor. We show that deletion of this longer structural element leads to the loss of P2X7 function. Furthermore, by combining mutagenesis, chimera construction, surface expression, and protein stability analysis, we found that the deletion of the longer β2,3-loop affects P2X7 surface expression but, more importantly, that this loop affects channel gating of P2X7. We propose that the longer β2,3-sheets may have a negative regulatory effect on a loop on the head domain and on the structural element formed by E171 and its surrounding regions. Understanding the role of the unique structure of the P2X7 receptor in the gating process will aid in the development of selective drugs targeting this subtype.

The pathologic feature of food allergy (FA) is the aberrant Th2-biased immune response in the intestine. Regulatory T cells (Tregs) play an important role in the suppression of aberrant immune response. The activities of the TLRs regulate multiple cell functions. This study aims to investigate the role of TLR3 activation in the regulation of Th2-biased immune response in the intestine by the generation of inducible Tregs (iTregs). In this study, polyinosinic polycytidylic acid (polyI:C) was used as an activator of TLR3. An FA mouse model was developed to establish the Th2-biased inflammation in the intestine. The effects of TLR3 activation on the generation of iTreg were tested in the culture and in mice. We observed that exposure to polyI:C induced IFN-γ+ Foxp3+ iTregs in mouse intestine and in the culture. The IFN-γ+ Foxp3+ iTregs showed immune suppressive functions. Exposure to polyI:C increased T-bet levels in CD4+ T cells. The T-bet formed a complex with GATA3 to dissociate Foxp3 from GATA3/Foxp3 complex in CD4+ T cells. The Foxp3 thus gained the opportunity to move to TGF-β promoter to generate iTregs. Administration with polyI:C prevented the development of FA and inhibited existing FA. In conclusion, activation of TLR3 induces IFN-γ+ Foxp3+ Tregs, which can prevent FA development and inhibit existing FA in mice.

COVID-19 severity has varied widely, with demographic and cardio-metabolic factors increasing risk of severe reactions to SARS-CoV-2 infection, but the underlying mechanisms for this remain uncertain. We investigated phenotypic and genetic factors associated with subcutaneous adipose tissue expression of Angiotensin I Converting Enzyme 2 (ACE2), which has been shown to act as a receptor for SARS-CoV-2 cellular entry. In a meta-analysis of three independent studies including up to 1,471 participants, lower adipose tissue ACE2 expression was associated with adverse cardio-metabolic health indices including type 2 diabetes (T2D) and obesity status, higher serum fasting insulin and BMI, and lower serum HDL levels (P-4). ACE2 expression levels were also associated with estimated proportions of cell types in adipose tissue; lower ACE2 expression was associated with a lower proportion of microvascular endothelial cells (P=4.25×10-4) and higher macrophage proportion (P=2.74×10-5), suggesting a link to inflammation. Despite an estimated heritability of 32%, we did not identify any proximal or distal genetic variants (eQTLs) associated with adipose tissue ACE2 expression. Our results demonstrate that at-risk individuals have lower background ACE2 levels in this highly relevant tissue. Further studies will be required to establish how this may contribute to increased COVID-19 severity.

Altered lipid metabolism in macrophages is associated with various important inflammatory conditions. Although lipid metabolism is an important target for therapeutic intervention, the metabolic requirement involved in lipid accumulation during pro-inflammatory activation of macrophages remains incompletely characterized. We show here that macrophage activation with IFNγ results in increased aerobic glycolysis, iNOS-dependent inhibition of respiration, and accumulation of triacylglycerol. Surprisingly, metabolite tracing with 13C-labeled glucose revealed that the glucose contributed to the glycerol groups in triacylglycerol (TAG), rather than to de novo synthesis of fatty acids. This is in stark contrast to the otherwise similar metabolism of cancer cells, and previous results obtained in activated macrophages and dendritic cells. Our results establish a novel metabolic pathway whereby glucose provides glycerol to the headgroup of TAG during classical macrophage activation.

Perivascular adipose tissue (PVAT) can regulate vascular homeostasis by secreting various adipokines. This study investigated the effects of PVAT browning on its endocrine function.In the first section of our study, male Sprague-Dawley rats were randomly divided into cold exposure (8°C) and 24°C acclimation groups. After cold exposure for 7 days, interscapular brown adipose tissue (iBAT), subcutaneous white adipose tissue, thoracic aortic PVAT, and abdominal aortic PVAT (aPVAT) were harvested for histological and brown marker gene expression analysis. In the second part, male rats were fed a high fat diet (HFD) for 10 weeks. In the 11th week, the rats were treated with or without cold exposure. After 14-day cold exposure, aPVAT was collected for histological, gene, and protein expression analysis.Cold exposure had a browning effect on aPVAT by increasing UCP-1 and PGC-1α expression levels. After HFD feeding for 10 weeks, 14-day cold exposure was still able to induce aPVAT browning. Compared with thermoneutrality acclimation rats, TNF-α, IL-6, and p-p65 expression levels were significantly lower in aPVAT from HFD-fed rats with cold exposure. In contrast, p-AMPK expression levels were increased in aPVAT from HFD-fed rats with cold exposure.Our study demonstrated that browning of aPVAT in HFD-fed rats lowered the pro-inflammatory adipokine expression levels and activated AMPK.

Summary Dysfunction of the immune regulatory system plays an important role in the pathogenesis of rheumatoid arthritis (RA). Vasoactive intestinal peptide (VIP) has multiple bioactivities. This study aims to investigate the role of VIP in the maintenance of the immune regulatory capacity of monocytes (Mos). Human peripheral blood samples were collected from RA patients and healthy control (HC) subjects. Mos and CD14+ CD71–CD73+CD25+ regulatory Mos (RegMos) were isolated from the blood samples and characterized by flow cytometry. A rat RA model was developed to test the role of VIP in the maintenance of the immune regulatory function of Mos. The results showed that RegMos of HC subjects had immune suppressive functions. RegMos of RA patients expressed less interleukin (IL)-10 and showed an incompetent immune regulatory capacity. Serum levels of VIP were lower in RA patients, which were positively correlated with the expression of IL-10 in RegMos. In-vitro experiments showed that the IL-10 mRNA decayed spontaneously in RegMos, which could be prevented by the presence of VIP in the culture. VIP suppressed the effects of tristetraprolin (TTP) on inducing IL-10 mRNA decay in RegMos. Administration of VIP inhibited experimental RA in rats through restoring the IL-10 expression in RegMos. RegMos have immune suppressive functions. VIP is required in maintaining IL-10 expression in RegMos. The data suggest that VIP has translational potential in the treatment of immune disorders such as RA.

Macrophages are professional phagocytes at the front line of immune defenses against foreign bodies and microbial pathogens. Various bacteria, which are responsible for deadly diseases including tuberculosis and salmonellosis, are capable of hijacking this important immune cell type and thrive intracellularly, either in the cytoplasm or in specialized vacuoles. Tight regulation of cellular metabolism is critical in shaping the macrophage polarization states and immune functions. Lipids, besides being the bulk component of biological membranes, serve as energy sources as well as signaling molecules during infection and inflammation. With the advent of systems-scale analyses of genes, transcripts, proteins, and metabolites, in combination with classical biology, it is increasingly evident that macrophages undergo extensive lipid remodeling during activation and infection. Each bacterium species has evolved its own tactics to manipulate host metabolism toward its own advantage. Furthermore, modulation of host lipid metabolism affects disease susceptibility and outcome of infections, highlighting the critical roles of lipids in infectious diseases. Here, we will review the emerging roles of lipids in the complex host–pathogen relationship and discuss recent methodologies employed to probe these versatile metabolites during the infection process. An improved understanding of the lipid-centric nature of infections can lead to the identification of the Achilles’ heel of the pathogens and host-directed targets for therapeutic interventions. Currently, lipid-moderating drugs are clinically available for a range of non-communicable diseases, which we anticipate can potentially be tapped into for various infections. MOE (Min. of Education, S’pore) Published version

Background During the acute respiratory distress syndrome (ARDS), neutrophils play a central role in the pathogenesis, and their activation requires interaction with the endothelium. Extracellular histones have been recognized as pivotal inflammatory mediators. This study was to investigate the role of pulmonary endothelial activation during the extracellular histone-induced inflammatory response in ARDS. Methods ARDS was induced in male C57BL/6 mice by intravenous injection with lipopolysaccharide (LPS) or exogenous histones. Concurrent with LPS administration, anti-histone H4 antibody (anti-H4) or non-specific IgG was administered to study the role of extracellular histones. The circulating von Willebrand factor (vWF) and soluble thrombomodulin (sTM) were measured with ELISA kits at the preset time points. Myeloperoxidase (MPO) activity in lung tissue was measured with a MPO detection kit. The translocation of P-selectin and neutrophil infiltration were measured by immunohistochemical detection. For in vitro studies, histone H4 in the supernatant of mouse lung vascular endothelial cells (MLVECs) was measured by Western blot. The binding of extracellular histones with endothelial membrane was examined by confocal laser microscopy. Endothelial P-selectin translocation was measured by cell surface ELISA. Adhesion of neutrophils to MLVECs was assessed with a color video digital camera. Results The results showed that during LPS-induced ARDS extracellular histones caused endothelial and neutrophil activation, as seen by P-selectin translocation, release of vWF, an increase of circulating sTM, lung neutrophil infiltration and increased MPO activity. Extracellular histones directly bound and activated MLVECs in a dose-dependent manner. On the contrary, the direct stimulatory effect of exogenous histones on neutrophils was very limited, as measured by neutrophil adhesion and MPO activity. With the contribution of activated endothelium, extracellular histones could effectively activating neutrophils. Both inhibiting the endothelial activation with an anti-toll like receptor (TLR) antibody and inhibiting the interaction of the endothelium with neutrophil using an anti-P-selectin antibody decreased the degree of neutrophil activation. Conclusions Extracellular histones are pro-inflammatory mediators in LPS-induced ARDS in mice. In addition to direct action to neutrophils, extracellular histones promote neutrophil adhesion and subsequent activation by first activating the pulmonary endothelium via TLR signaling. Thus, endothelial activation is important for extracellular histone-induced inflammatory injury.

Background: Systemic inflammation is a key feature in acid aspiration-induced acute respiratory distress syndrome (ARDS), but the factors that trigger inflammation are unclear. The authors hypothesize that extracellular histones, a newly identified inflammatory mediator, play important roles in the pathogenesis of ARDS. Methods: The authors used a hydrochloric acid aspiration-induced ARDS model to investigate whether extracellular histones are pathogenic and whether targeting histones are protective. Exogenous histones and antihistone antibody were administered to mice. Heparin can bind to histones, so the authors studied whether heparin could protect from ARDS using cell and mouse models. Furthermore, the authors analyzed whether extracellular histones are clinically involved in ARDS patients caused by gastric aspiration. Results: Extracellular histones in bronchoalveolar lavage fluid of acid-treated mice were significantly higher (1.832 ± 0.698) at 3 h after injury than in sham-treated group (0.63 ± 0.153; P = 0.0252, n = 5 per group). Elevated histones may originate from damaged lung cells and neutrophil infiltration. Exogenous histones aggravated lung injury, whereas antihistone antibody markedly attenuated the intensity of ARDS. Notably, heparin provided a similar protective effect against ARDS. Analysis of plasma from ARDS patients (n = 21) showed elevated histones were significantly correlated with the degree of ARDS and were higher in nonsurvivors (2.723 ± 0.2933, n = 7) than in survivors (1.725 ± 0.1787, P = 0.006, n = 14). Conclusion: Extracellular histones may play a contributory role toward ARDS by promoting tissue damage and systemic inflammation and may become a novel marker reflecting disease activity. Targeting histones by neutralizing antibody or heparin shows potent protective effects, suggesting a potentially therapeutic strategy.