Effects of total alkaloids from Alstonia scholaris (L.) R. Br. on ovalbumin-induced asthma mice.

ETHNOPHARMACOLOGICAL RELEVANCE: More than 300 million people worldwide suffer from asthma, a chronic respiratory inflammatory disease. Total alkaloids (TA) were extracted from the ethnic medicinal plant Alstonia solaris (L.) R.Br., which is used to treat respiratory diseases. They may be effective drugs for treating asthma, but research is still needed to determine their effectiveness and mechanism in treating asthma. AIM OF THE STUDY: To further understand TA's role in the treatment of asthma and to support the phase II trial of the drug. MATERIALS AND METHODS: In this study, we investigated the effects of TA in a mouse asthma model produced by Ovalbumin (OVA). H&E and PAS staining were used to observe the histopathological features of lung. airway hyperresponsiveness (AHR) was detected by ventilator; The expression of interleukin (IL)-33, suppression of tumorigenicity 2 (ST2) and E-cadherin in the lungs was evaluated by IHC. The concentrations of Mucin5AC (MUC5AC), eotaxin, IL-4, IL-5, IL-9, IL-13, interferon (IFN)-γ, IL-6, IL-8, IL-17A, IL-33, IL-25, thymic stromal lymphopoietin (TSLP), monocyte chemoattractant protein 1 (MCP-1), leukotriene (LT) B4, LTC4, LTD4, LTE4 in bronchoalveolar lavage fluid (BALF) and total IgE (tIgE), OVA-Specific IgE (OVA-IgE) in serum were measured by ELISA. ILC2s and eosinophils were detected in lung tissue by flow cytometry. The gene expression levels of IL-33 and ST2 were detected by RT-qPCR. RESULTS: Administration of TA reduced pulmonary inflammatory symptoms, MUC5AC production in the BALF, and AHR. At the same time, TA inhibited eotaxin production and eosinophil recruitment. Moreover, TA significantly decreased Th2 and Th17 cytokines and increased Th1 cytokines, contributing to restore the balance between Th1 and Th2 and Th17 cytokines. TA may reduce ILC2s numbers by inhibiting IL-33, IL-25, and TSLP levels in BALF and IL-33/ST2 signaling in lung tissue. Finally, TA decreased tIgE, OVA-IgE, and MCP-1 levels and subsequently inhibited mast cell activation and leukotriene release. CONCLUSIONS: These findings imply that TA may be an effective immunoregulatory medication for the management and prevention of asthma.

Monitoring of Inflammasome Activation of Macrophages and Microglia In Vitro, Part 2: Assessing Inflammasome Activation.

Inflammasomes are macromolecular complexes that assemble upon the detection of cytoplasmic pathogen-associated or danger-associated signals and induce a necrotic type of cell death termed pyroptosis, facilitating pro-inflammatory cytokine release. Inflammasomes play a critical role in innate immunity and inflammatory response; however, they have also been associated with multiple diseases, including autoinflammatory and neurodegenerative conditions. In the following chapter, we describe methods to detect inflammasome activation and its downstream effects, including detection of ASC oligomerization, detection of activated caspase-1 and cleaved IL-1ß, as well as read-outs for inflammasome-mediated cell death.

Sex Difference in Amebiasis.

Infection with the protozoan parasite Entamoeba histolytica is much more likely to cause severe, focal liver damage in males than females, although the infection rate is the same in both sexes. The differences in disease susceptibility may be due to modulation of key mechanisms of the innate immune response by sex hormones. Complement-mediated mechanisms and estrogen-dependent activated natural killer T cells lead to early elimination of the parasite in females, whereas a pathological immune axis is triggered in males. Testosterone, which is generally thought to have more immunosuppressive properties on cells of the immune response, leads to overwhelming activation of monocytes and host-dependent destruction of liver tissue in males resulting in worse outcomes.

Egg yolk lipids induce sensitization to egg white proteins in a mouse model without adjuvant and exacerbate Th2 responses to egg white in cells from allergic patients.

This study evaluates the influence of egg lipid fractions in the induction of allergic sensitization to egg white (EW) proteins, using a mouse model of orally adjuvant-free induced allergy. Egg triglycerides (TG) and phospholipids (PL), and to a higher extent the whole egg lipid fraction (EL), induced allergy to EW proteins characterized by increased EW-specific IgG1. EL also increased EW-specific IgE. The administration to mice of a mixture of EW and EL increased the intestinal expression of Il33, Il25, and Tslp, the secretion of IL-33 and IL-6, the expansion of group 2 innate lymphoid cells, the regulation of Gata3, Il4 and Il13, dendritic cell (DC) activation and expression of DC molecules that drive Th2 differentiation. TG promoted the absorption of proteins through the intestinal epithelium, enhancing local Th2 responses, while PL favoured the delivery of antigens to the Peyer's Patches. This differential modulation of the site of absorption of egg proteins determined the different behaviour of TG and PL. Egg yolk lipids also induced activation of Th2-inducing innate responses on intestinal human cells in vitro and enhanced adaptive Th2 functions through the activation of DCs in egg-allergic subjects.

A type 2 immune circuit in the stomach controls mammalian adaptation to dietary chitin.

Dietary fiber improves metabolic health, but host-encoded mechanisms for digesting fibrous polysaccharides are unclear. In this work, we describe a mammalian adaptation to dietary chitin that is coordinated by gastric innate immune activation and acidic mammalian chitinase (AMCase). Chitin consumption causes gastric distension and cytokine production by stomach tuft cells and group 2 innate lymphoid cells (ILC2s) in mice, which drives the expansion of AMCase-expressing zymogenic chief cells that facilitate chitin digestion. Although chitin influences gut microbial composition, ILC2-mediated tissue adaptation and gastrointestinal responses are preserved in germ-free mice. In the absence of AMCase, sustained chitin intake leads to heightened basal type 2 immunity, reduced adiposity, and resistance to obesity. These data define an endogenous metabolic circuit that enables nutrient extraction from an insoluble dietary constituent by enhancing digestive function.

Elevation of Metrnß and Its Association with Disease Activity in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is an auto-immune disease, the pathogenesis of which remains to be fully addressed. Metrnß is a novel cytokine involved in the pathogenesis of inflammatory disease, but its regulatory roles in SLE are unclear. We aimed to comprehensively investigate the clinical value of Metrnß in SLE. A massive elevation of circulating Metrnß levels was observed in SLE, and patients with an active phase displayed higher Metrnß concentrations than those with inactive phases. Additionally, we found that Metrnß expression was positively correlated with clinical indicators of SLE. Longitudinal cytokine and chemokine profiles revealed a disturbed immune response in SLE, with high activity profiles displayed severe pathogenic inflammation, and a positive correlation of the serum Metrnß with CXCL9, IL10, IL18 and IL1RA was observed as well. Moreover, Metrnß expressions exhibited an inverse correlation with Treg and B10. Of note, a significant decrease of ILC2 was found in SLE, and there was a negative correlation of Metrnß with ILC2 as well. Further ROC analysis showed that the area under the curve (AUC) for Metrnß was 0.8250 (95% CI: 0.7379-0.9121), with a cutoff value of 1131 pg/mL to effectively distinguish SLE patients from healthy controls. Our study herein demonstrated for the first time that Metrnß values were increased and were immunologically correlated with SLE activity, which could be utilized as an alternative biomarker for the early identification and predicting of the immuno-response of SLE.

Micro-inflammation related gene signatures are associated with clinical features and immune status of fibromyalgia.

BACKGROUND: Fibromyalgia (FM) is a multifaceted disease. Along with the genetic, environmental and neuro-hormonal factors, inflammation has been assumed to have role in the pathogenesis of FM. The aim of the present study was to explore the differences in clinical features and pathophysiology of FM patients under different inflammatory status. METHODS: The peripheral blood gene expression profile of FM patients in the Gene Expression Omnibus database was downloaded. Differentially expressed inflammatory genes were identified, and two molecular subtypes were constructed according to these genes used unsupervised clustering analysis. The clinical characteristics, immune features and pathways activities were compared further between the two subtypes. Then machine learning was used to perform the feature selection and construct a classification model. RESULTS: The patients with FM were divided into micro-inflammation and non-inflammation subtypes according to 54 differentially expressed inflammatory genes. The micro-inflammation group was characterized by more major depression (p = 0.049), higher BMI (p = 0.021), more active dendritic cells (p = 0.010) and neutrophils. Functional enrichment analysis showed that innate immune response and antibacterial response were significantly enriched in micro-inflammation subtype (p < 0.050). Then 5 hub genes (MMP8, ENPP3, MAP2K3, HGF, YES1) were screened thought three feature selection algorithms, an accurate classifier based on the 5 hub DEIGs and 2 clinical parameters were constructed using support vector machine model. Model scoring indicators such as AUC (0.945), accuracy (0.936), F1 score (0.941), Brier score (0.079) and Hosmer-Lemeshow goodness-of-fit test (χ2 = 4.274, p = 0.832) proved that this SVM-based classifier was highly reliable. CONCLUSION: Micro-inflammation status in FM was significantly associated with the occurrence of depression and activated innate immune response. Our study calls attention to the pathogenesis of different subtypes of FM.

Neutrophils: a subgroup of neglected immune cells in ALS.

Amyotrophic lateral sclerosis (ALS) is a chronic, progressive neurodegenerative disease characterized by the loss of motor neurons. Dysregulated peripheral immunity has been identified as a hallmark of ALS. Neutrophils, as the front-line responders of innate immunity, contribute to host defense through pathogen clearance. However, they can concurrently play a detrimental role in chronic inflammation. With the unveiling of novel functions of neutrophils in neurodegenerative diseases, it becomes essential to review our current understanding of neutrophils and to recognize the gap in our knowledge about their role in ALS. Thus, a detailed comprehension of the biological processes underlying neutrophil-induced pathogenesis in ALS may assist in identifying potential cell-based therapeutic strategies to delay disease progression.

Solar ultraviolet B radiation promotes α-MSH secretion to attenuate the function of ILC2s via the pituitary-lung axis.

The immunomodulatory effects of ultraviolet B (UVB) radiation in human diseases have been described. Whether type 2 lung inflammation is directly affected by solar ultraviolet (UV) radiation is not fully understood. Here, we show a possible negative correlation between solar UVB radiation and asthmatic inflammation in humans and mice. UVB exposure to the eyes induces hypothalamus-pituitary activation and α-melanocyte-stimulating hormone (α-MSH) accumulation in the serum to suppress allergic airway inflammation by targeting group 2 innate lymphoid cells (ILC2) through the MC5R receptor in mice. The α-MSH/MC5R interaction limits ILC2 function through attenuation of JAK/STAT and NF-κB signaling. Consistently, we observe that the plasma α-MSH concentration is negatively correlated with the number and function of ILC2s in the peripheral blood mononuclear cells (PBMC) of patients with asthma. We provide insights into how solar UVB radiation-driven neuroendocrine α-MSH restricts ILC2-mediated lung inflammation and offer a possible strategy for controlling allergic diseases.

Subverted macrophages in the triple-negative breast cancer ecosystem.

Tumor-associated macrophages (TAMs) are the most critical effector cells of innate immunity and the most abundant tumor-infiltrating immune cells. They play a key role in the clearance of apoptotic bodies, regulation of inflammation, and tissue repair to maintain homeostasis in vivo. With the progression of triple-negative breast cancer（TNBC）, TAMs are "subverted" from tumor-promoting immune cells to tumor-promoting immune suppressor cells, which play a significant role in tumor development and are considered potential targets for cancer therapy. Here, we explored how macrophages, as the most important part of the TNBC ecosystem, are "subverted" to drive cancer evolution and the uniqueness of TAMs in TNBC progression and metastasis. Similarly, we discuss the rationale and available evidence for TAMs as potential targets for TNBC therapy.

Antagonism between viral infection and innate immunity at the single-cell level.

When infected with a virus, cells may secrete interferons (IFNs) that prompt nearby cells to prepare for upcoming infection. Reciprocally, viral proteins often interfere with IFN synthesis and IFN-induced signaling. We modeled the crosstalk between the propagating virus and the innate immune response using an agent-based stochastic approach. By analyzing immunofluorescence microscopy images we observed that the mutual antagonism between the respiratory syncytial virus (RSV) and infected A549 cells leads to dichotomous responses at the single-cell level and complex spatial patterns of cell signaling states. Our analysis indicates that RSV blocks innate responses at three levels: by inhibition of IRF3 activation, inhibition of IFN synthesis, and inhibition of STAT1/2 activation. In turn, proteins coded by IFN-stimulated (STAT1/2-activated) genes inhibit the synthesis of viral RNA and viral proteins. The striking consequence of these inhibitions is a lack of coincidence of viral proteins and IFN expression within single cells. The model enables investigation of the impact of immunostimulatory defective viral particles and signaling network perturbations that could potentially facilitate containment or clearance of the viral infection.

Castanea crenata honey reduces influenza infection by activating the innate immune response.

Influenza is an acute respiratory disorder caused by the influenza virus and is associated with prolonged hospitalization and high mortality rates in older individuals and chronically ill patients. Vaccination is the most effective preventive strategy for ameliorating seasonal influenza. However, the vaccine is not fully effective in cases of antigenic mismatch with the viral strains circulating in the community. The emergence of resistance to antiviral drugs aggravates the situation. Therefore, developing new vaccines and antiviral drugs is essential. Castanea crenata honey (CH) is an extensively cultivated food worldwide and has been used as a nutritional supplement or herbal medicine. However, the potential anti-influenza properties of CH remain unexplored. In this study, the in vitro and in vivo antiviral effects of CH were assessed. CH significantly prevented influenza virus infection in mouse Raw264.7 macrophages. CH pretreatment inhibited the expression of the viral proteins M2, PA, and PB1 and enhanced the secretion of proinflammatory cytokines and type-I interferon (IFN)-related proteins in vitro. CH increased the expression of RIG-1, mitochondrial antiviral signaling (MAVS) protein, and IFN-inducible transmembrane protein, which interferes with virus replication. CH reduced body weight loss by 20.9%, increased survival by 60%, and decreased viral replication and inflammatory response in the lungs of influenza A virus-infected mice. Therefore, CH stimulates an antiviral response in murine macrophages and mice by preventing viral infection through the RIG-1-mediated MAVS pathway. Further investigation is warranted to understand the molecular mechanisms involved in the protective effects of CH on influenza virus infection.

An inference model gives insights into innate immune adaptation and repertoire diversity.

The innate immune system is the body's first line of defense against infection. Natural killer (NK) cells, a vital part of the innate immune system, help to control infection and eliminate cancer. Studies have identified a vast array of receptors that NK cells use to discriminate between healthy and unhealthy cells. However, at present, it is difficult to explain how NK cells will respond to novel stimuli in different environments. In addition, the expression of different receptors on individual NK cells is highly stochastic, but the reason for these variegated expression patterns is unclear. Here, we studied the recognition of unhealthy target cells as an inference problem, where NK cells must distinguish between healthy targets with normal variability in ligand expression and ones that are clear "outliers." Our mathematical model fits well with experimental data, including NK cells' adaptation to changing environments and responses to different target cells. Furthermore, we find that stochastic, "sparse" receptor expression profiles are best able to detect a variety of possible threats, in agreement with experimental studies of the NK cell repertoire. While our study was specifically motivated by NK cells, our model is general and could also apply more broadly to explain principles of target recognition for other immune cell types.

Modulation of the immune response by the host defense peptide IDR-1002 in chicken hepatic cell culture.

IDR-1002, a synthetic host defense peptide (HDP), appears to be a potential candidate for the treatment of bacterial infections and the consequent inflammatory response due to its potent immunomodulatory activity. This is of relevance to the emerging issue of antimicrobial resistance in the farming sector. In this study, the effects of IDR-1002 were investigated on a chicken hepatocyte‒non-parenchymal cell co-culture, and the results revealed that IDR-1002 had complex effects on the regulation of the hepatic innate immunity. IDR-1002 increased the levels of both RANTES (Regulated on Activation, Normal T cell Expressed and Secreted) and Macrophage colony stimulating factor (M-CSF), suggesting the peptide plays a role in the modulation of macrophage differentiation, also reflected by the reduced concentrations of interleukin (IL)-6 and IL-10. The pro-inflammatory cytokine release triggered by the bacterial cell wall component lipoteichoic acid (LTA) was ameliorated by the concomitantly applied IDR-1002 based on the levels of IL-6, chicken chemotactic and angiogenic factor (CXCLi2) and interferon (IFN)-γ. Moreover, the production of nuclear factor erythroid 2-related factor 2 (Nrf2), an essential transcription factor in the antioxidant defense pathway, was increased after IDR-1002 exposure, while protein carbonyl (PC) levels were also elevated. These findings suggest that IDR-1002 affects the interplay of the cellular immune response and redox homeostasis, thus the peptide represents a promising tool in the treatment of bacterially induced inflammation in chickens.

Lysine methyltransferase SMYD2 inhibits antiviral innate immunity by promoting IRF3 dephosphorylation.

Phosphorylation of IRF3 is critical to induce type I interferon (IFN-I) production in antiviral innate response. Here we report that lysine methyltransferase SMYD2 inhibits the expressions of IFN-I and proinflammatory cytokines in macrophages upon viral infections. The Smyd2-deficient mice are more resistant to viral infection by producing more IFN-I and proinflammatory cytokines. Mechanistically, SMYD2 inhibits IRF3 phosphorylation in macrophages in response to viral infection independent of its methyltransferase activity. We found that SMYD2 interacts with the DNA-binding domain (DBD) and IRF association domain (IAD) domains of IRF3 by its insertion SET domain (SETi) and could recruit phosphatase PP1α to enhance its interaction with IRF3, which leads to decreased phosphorylation of IRF3 in the antiviral innate response. Our study identifies SMYD2 as a negative regulator of IFN-I production against virus infection. The new way of regulating IRF3 phosphorylation will provide insight into the understanding of IFN-I production in the innate response and possible intervention of the related immune disorders.

Fortifying immunity: PLSCR1 picks battle against SARS-CoV-2.

Interferons (IFNs) and interferon-stimulated genes (ISGs) are the major players in the host innate immunity against viral infection. In a recent Nature paper, Xu et al. identified phospholipid scramblase 1 (PLSCR1) as a novel ISG that restricts severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by blocking virus-cell fusion.

Innate immune responses yield tissue-specific bottlenecks that scale with pathogen dose.

To cause infection, pathogens must overcome bottlenecks imposed by the host immune system. These bottlenecks restrict the inoculum and largely determine whether pathogen exposure results in disease. Infection bottlenecks therefore quantify the effectiveness of immune barriers. Here, using a model of Escherichia coli systemic infection, we identify bottlenecks that tighten or widen with higher inoculum sizes, revealing that the efficacy of innate immune responses can increase or decrease with pathogen dose. We term this concept "dose scaling". During E. coli systemic infection, dose scaling is tissue specific, dependent on the lipopolysaccharide (LPS) receptor TLR4, and can be recapitulated by mimicking high doses with killed bacteria. Scaling therefore depends on sensing of pathogen molecules rather than interactions between the host and live bacteria. We propose that dose scaling quantitatively links innate immunity with infection bottlenecks and is a valuable framework for understanding how the inoculum size governs the outcome of pathogen exposure.

Live-cell imaging reveals single-cell and population-level infection strategies of Listeria monocytogenes in macrophages.

Pathogens have developed intricate strategies to overcome the host's innate immune responses. In this paper we use live-cell microscopy with a single bacterium resolution to follow in real time interactions between the food-borne pathogen L. monocytogenes and host macrophages, a key event controlling the infection in vivo. We demonstrate that infection results in heterogeneous outcomes, with only a subset of bacteria able to establish a replicative invasion of macrophages. The fate of individual bacteria in the same host cell was independent from the host cell and non-cooperative, being independent from co-infecting bacteria. A higher multiplicity of infection resulted in a reduced probability of replication of the overall bacterial population. By use of internalisation assays and conditional probabilities to mathematically describe the two-stage invasion process, we demonstrate that the higher MOI compromises the ability of macrophages to phagocytose bacteria. We found that the rate of phagocytosis is mediated via the secreted Listeriolysin toxin (LLO), while the probability of replication of intracellular bacteria remained constant. Using strains expressing fluorescent reporters to follow transcription of either the LLO-encoding hly or actA genes, we show that replicative bacteria exhibited higher PrfA regulon expression in comparison to those bacteria that did not replicate, however elevated PrfA expression per se was not sufficient to increase the probability of replication. Overall, this demonstrates a new role for the population-level, but not single cell, PrfA-mediated activity to regulate outcomes of host pathogen interactions.

Early expansion of activated adaptive but also exhausted NK cells during acute severe SARS-CoV-2 infection.

The analysis of immunological parameters during the course of a SARS-CoV-2 infection is of great importance, both to identify diagnostic markers for the risk of a severe course of COVID-19, and to better understand the role of the immune system during the infection. By using multicolor flow cytometry we compared the phenotype of Natural Killer (NK) cells from hospitalized COVID-19 patients during early SARS-CoV-2 infection with samples from recovered and SARS-CoV-2 naïve subjects. Unsupervised high-dimensional analysis of 28-color flow cytometric data revealed a strong enrichment of NKG2C expressing NK cells in response to the acute viral infection. In addition, we found an overrepresentation of highly activated NK cell subsets with an exhausted phenotype. Moreover, our data show long-lasting phenotypic changes within the NK cell compartment that did not completely reverse up to 2 months after recovery. This demonstrates that NK cells are involved in the early innate immune response against SARS-CoV-2.