Obesity alters pathology and treatment response in inflammatory disease.

Decades of work have elucidated cytokine signalling and transcriptional pathways that control T cell differentiation and have led the way to targeted biologic therapies that are effective in a range of autoimmune, allergic and inflammatory diseases. Recent evidence indicates that obesity and metabolic disease can also influence the immune system1-7, although the mechanisms and effects on immunotherapy outcomes remain largely unknown. Here, using two models of atopic dermatitis, we show that lean and obese mice mount markedly different immune responses. Obesity converted the classical type 2 T helper (TH2)-predominant disease associated with atopic dermatitis to a more severe disease with prominent TH17 inflammation. We also observed divergent responses to biologic therapies targeting TH2 cytokines, which robustly protected lean mice but exacerbated disease in obese mice. Single-cell RNA sequencing coupled with genome-wide binding analyses revealed decreased activity of nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) in TH2 cells from obese mice relative to lean mice. Conditional ablation of PPARγ in T cells revealed that PPARγ is required to focus the in vivo TH response towards a TH2-predominant state and prevent aberrant non-TH2 inflammation. Treatment of obese mice with a small-molecule PPARγ agonist limited development of TH17 pathology and unlocked therapeutic responsiveness to targeted anti-TH2 biologic therapies. These studies reveal the effects of obesity on immunological disease and suggest a precision medicine approach to target the immune dysregulation caused by obesity.

Global absence and targeting of protective immune states in severe COVID-19.

Although infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has pleiotropic and systemic effects in some individuals1-3, many others experience milder symptoms. Here, to gain a more comprehensive understanding of the distinction between severe and mild phenotypes in the pathology of coronavirus disease 2019 (COVID-19) and its origins, we performed a whole-blood-preserving single-cell analysis protocol to integrate contributions from all major immune cell types of the blood-including neutrophils, monocytes, platelets, lymphocytes and the contents of the serum. Patients with mild COVID-19 exhibit a coordinated pattern of expression of interferon-stimulated genes (ISGs)3 across every cell population, whereas these ISG-expressing cells are systemically absent in patients with severe disease. Paradoxically, individuals with severe COVID-19 produce very high titres of anti-SARS-CoV-2 antibodies and have a lower viral load compared to individuals with mild disease. Examination of the serum from patients with severe COVID-19 shows that these patients uniquely produce antibodies that functionally block the production of the ISG-expressing cells associated with mild disease, by activating conserved signalling circuits that dampen cellular responses to interferons. Overzealous antibody responses pit the immune system against itself in many patients with COVID-19, and perhaps also in individuals with other viral infections. Our findings reveal potential targets for immunotherapies in patients with severe COVID-19 to re-engage viral defence.

RNA circuits and RNA-binding proteins in T cells.

RNA is integral to the regulatory circuits that control cell identity and behavior. Cis-regulatory elements in mRNAs interact with RNA-binding proteins (RBPs) that can alter RNA sequence, stability, and translation into protein. Similarly, long noncoding RNAs (lncRNAs) scaffold ribonucleoprotein complexes that mediate transcriptional and post-transcriptional regulation of gene expression. Indeed, cell programming is fundamental to multicellular life and, in this era of cellular therapies, it is of particular interest in T cells. Here, we review key concepts and recent advances in our understanding of the RNA circuits and RBPs that govern mammalian T cell differentiation and immune function.

Stimulation of ectopically expressed muscarinic receptors induces IFN-γ but suppresses IL-2 production by inhibiting activation of pAKT pathways in primary T cells.

T cell antigen receptor stimulation induces tyrosine phosphorylation of downstream signaling molecules and the phosphatidylinositol, Ras, MAPK, and PI3 kinase pathways, leading to T cell activation. Previously, we reported that the G-protein-coupled human muscarinic receptor could bypass tyrosine kinases to activate the phosphatidylinositol pathway and induce interleukin-2 production in Jurkat leukemic T cells. Here, we demonstrate that stimulating G-protein-coupled muscarinic receptors (M1 and synthetic hM3Dq) can activate primary mouse T cells if PLCß1 is coexpressed. Resting peripheral hM3Dq+PLCß1 (hM3Dq/ß1) T cells did not respond to clozapine, an hM3Dq agonist, unless they were preactivated by TCR and CD28 stimulation which increased hM3Dq and PLCß1 expression. This permitted large calcium and phosphorylated ERK responses to clozapine. Clozapine treatment induced high IFN-γ, CD69, and CD25 expression, but surprisingly did not induce substantial IL-2 in hM3Dq/ß1 T cells. Importantly, costimulation of both muscarinic receptors plus the TCR even led to reduced IL-2 expression, suggesting a selective inhibitory effect of muscarinic receptor costimulation. Stimulation of muscarinic receptors induced strong nuclear translocation of NFAT and NFκB and activated AP-1. However, stimulation of hM3Dq led to reduced IL-2 mRNA stability which correlated with an effect on the IL-2 3'UTR activity. Interestingly, stimulation of hM3Dq resulted in reduced pAKT and its downstream pathway. This may explain the inhibitory impact on IL-2 production in hM3Dq/ß1T cells. Moreover, an inhibitor of PI3K reduced IL-2 production in TCR-stimulated hM3Dq/ß1 CD4 T cells, suggesting that activating the pAKT pathway is critical for IL-2 production in T cells.

Stromal STAT5-Mediated Trophic Activity Regulates Hematopoietic Niche Factors.

Signal transducer and activator of transcription 5 (STAT5a and STAT5b) are intrinsically critical for normal hematopoiesis but are also expressed in stromal cells. Here, STAT5ab knockout (KO) was generated with a variety of bone marrow hematopoietic and stromal Cre transgenic mouse strains. Vav1-Cre/+STAT5abfl/fl, the positive control for loss of multipotent hematopoietic function, surprisingly dysregulated niche factor mRNA expression, and deleted STAT5ab in CD45neg cells. Single-cell transcriptome analysis of bone marrow from Vav1-Cre/+ wild-type or Vav1-Cre/+STAT5abfl/fl mice showed hematopoietic stem cell (HSC) myeloid commitment priming. Nes+ cells were detected in both CD45neg and CD45+ clusters and deletion of STAT5ab with Nes-Cre caused hematopoietic repopulating defects. To follow up on these promiscuous Cre promoter deletions in CD45neg and CD45+ bone marrow cell populations, more stroma-specific Cre strains were generated and demonstrated a reduction in multipotent hematopoietic progenitors. Functional support for niche-supporting activity was assessed using STAT5-deficient mesenchymal stem cells (MSCs). With Lepr-Cre/+STAT5abfl/fl, niche factor mRNAs were downregulated with validation of reduced IGF-1 and CXCL12 proteins. Furthermore, advanced computational analyses revealed a key role for STAT5ab/Cish balance with Cish strongly co-expressed in MSCs and HSCs primed for differentiation. Therefore, STAT5ab-associated gene regulation supports the bone marrow microenvironment.

Modeling of large-scale hoxbb cluster deletions in zebrafish uncovers a role for segmentation pathways in atrioventricular boundary specification.

Hox genes orchestrate the segmental specification of the muscular circulatory system in invertebrates but it has not proven straightforward to decipher segmental parallels in the vertebrate heart. Recently, patients with HOXB gene cluster deletion were found to exhibit abnormalities including atrioventricular canal defects. Using CRISPR, we established a mutant with the orthologous hoxbb cluster deletion in zebrafish. The mutant exhibited heart failure and atrioventricular regurgitation at 5 days. Analyzing the four genes in the hoxbb cluster, isolated deletion of hoxb1b-/- recapitulated the cardiac abnormalities, supporting hoxb1b as the causal gene. Both in situ and in vitro data indicated that hoxb1b regulates gata5 to inhibit hand2 expression and ultimately is required to pattern the vertebrate atrioventricular boundary. Together, these data reveal a role for segmental specification in vertebrate cardiac development and highlight the utility of CRISPR techniques for efficiently exploring the function of large structural genomic lesions.

ISCOMs/MPLA-Adjuvanted SDAD Protein Nanoparticles Induce Improved Mucosal Immune Responses and Cross-Protection in Mice.

The epidemics caused by the influenza virus are a serious threat to public health and the economy. Adding appropriate adjuvants to improve immunogenicity and finding effective mucosal vaccines to combat respiratory infection at the portal of virus entry are important strategies to boost protection. In this study, a novel type of core/shell protein nanoparticle consisting of influenza nucleoprotein (NP) as the core and NA1-M2e or NA2-M2e fusion proteins as the coating antigens by SDAD hetero-bifunctional crosslinking is exploited. Immune-stimulating complexes (ISCOMs)/monophosphoryl lipid A (MPLA) adjuvants further boost the NP/NA-M2e SDAD protein nanoparticle-induced immune responses when administered intramuscularly. The ISCOMs/MPLA-adjuvanted protein nanoparticles are delivered through the intranasal route to validate the application as mucosal vaccines. ISCOMs/MPLA-adjuvanted nanoparticles induce significantly strengthened antigen-specific antibody responses, cytokine-secreting splenocytes in the systemic compartment, and higher levels of antigen-specific IgA and IgG in the local mucosa. Meanwhile, significantly expanded lung resident memory (RM) T and B cells (TRM /BRM ) and alveolar macrophages population are observed in ISCOMs/MPLA-adjuvanted nanoparticle-immunized mice with a 100% survival rate after homogeneous and heterogeneous H3N2 viral challenges. Taken together, ISCOMs/MPLA-adjuvanted protein nanoparticles could improve strong systemic and mucosal immune responses conferring protection in different immunization routes.

Chinese herbal medicine for the treatment of cardiovascular diseases ─ Targeting cardiac ion channels.

Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality, imposing an increasing global health burden. Cardiac ion channels (voltage-gated NaV, CaV, KVs, and others) synergistically shape the cardiac action potential (AP) and control the heartbeat. Dysfunction of these channels, due to genetic mutations, transcriptional or post-translational modifications, may disturb the AP and lead to arrhythmia, a major risk for CVD patients. Although there are five classes of anti-arrhythmic drugs available, they can have varying levels of efficacies and side effects on patients, possibly due to the complex pathogenesis of arrhythmias. As an alternative treatment option, Chinese herbal remedies have shown promise in regulating cardiac ion channels and providing anti-arrhythmic effects. In this review, we first discuss the role of cardiac ion channels in maintaining normal heart function and the pathogenesis of CVD, then summarize the classification of Chinese herbal compounds, and elaborate detailed mechanisms of their efficacy in regulating cardiac ion channels and in alleviating arrhythmia and CVD. We also address current limitations and opportunities for developing new anti-CVD drugs based on Chinese herbal medicines.

Monophosphoryl lipid A-adjuvanted nucleoprotein-neuraminidase nanoparticles improve immune protection against divergent influenza viruses.

Universal influenza vaccines are urgently needed to prevent recurrent influenza epidemics and inevitable pandemics. We generated double-layered protein nanoparticles incorporating two conserved influenza antigens-nucleoprotein and neuraminidase-through a two-step desolvation-crosslinking method. These protein nanoparticles displayed immunostimulatory properties to antigen-presenting cells by promoting inflammatory cytokine (IL-6 and TNF-α) secretion from JAWS II dendric cells. The nanoparticle immunization induced significant antigen-specific humoral and cellular responses, including antigen-binding and neutralizing antibodies, antibody- and cytokine (IFN-γ and IL-4)-secreting cells, and NP147-155 tetramer-specific cytotoxic T lymphocyte (CTL) responses. Co-administration of monophosphoryl lipid A (MPLA, a toll-like receptor 4 agonist) with the protein nanoparticles further improved immune responses and conferred heterologous and heterosubtypic influenza protection. The MPLA-adjuvanted nanoparticles reduced lung inflammation post-infection. The results demonstrated that the combination of MPLA and conserved protein nanoparticles could be developed into an improved universal influenza vaccine strategy.

SARS-CoV-2 Spike Stem Protein Nanoparticles Elicited Broad ADCC and Robust Neutralization against Variants in Mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global pandemic. The virus is rapidly evolving, characterized by the emergence of several major variants. Stable prefusion spike protein (Pre) is the immunogen in current vaccines but is limited in protecting against different variants. Here, the immune responses induced by the relatively conserved stem subunit (S2) of spike protein versus Pre are investigated. Pre generates the most robust neutralization responses against SARS-CoV-2 variants in vesicular stomatitis virus pseudovirus-based assessment but elicits less antibody-dependent cellular cytotoxicity (ADCC) activity than S2. By contrast, S2 induces the most balanced immunoglobulin G (IgG) antibodies with potent and broad ADCC activity although produces weaker neutralization. The immunogenicity of S2 and Pre improves by incorporating the two proteins into double-layered protein nanoparticles. The resulting protein nanoparticles Pre/S2 elicit higher neutralizing antibodies than Pre alone, and stronger ADCC than S2 alone. Moreover, nanoparticles produce more potent and balanced serum IgG antibodies than the corresponding soluble protein mixture, and the immune responses are sustained for at least four months after the immunization. Thus, the double-layered protein nanoparticles have the potential to be developed into broader SARS-CoV-2 vaccines with excellent safety profiles.

Influenza NP core and HA or M2e shell double-layered protein nanoparticles induce broad protection against divergent influenza A viruses.

Influenza viral infection causes acute upper respiratory diseases in humans, posing severe risks to global public health. However, current vaccines provide limited protection against mismatched circulating influenza A viruses. Here, the immune responses induced in mice by novel double-layered protein nanoparticles were investigated. The nanoparticles were composed of influenza nucleoprotein (NP) cores and hemagglutinin (HA) or matrix 2 protein ectodomain (M2e) shells. Vaccination with the nanoparticles significantly enhanced M2e-specific serum antibody titers and concomitant ADCC responses. Robust NP-specific T cell responses and robust HA neutralization were also detected. Moreover, vaccination with a trivalent nanoparticle combination containing two routinely circulated HA, conserved M2e, and NP reduced lung virus titers, pulmonary pathologies, and weight loss after homologous virus challenge. This combination also improved survival rates against heterologous and heterosubtypic influenza virus challenges. Our results demonstrate that the trivalent combination elicited potent and long-lasting immune responses conferring influenza viral cross-protection.

Predicting Patient Response to the Antiarrhythmic Mexiletine Based on Genetic Variation.

RATIONALE: Mutations in the SCN5A gene, encoding the α subunit of the Nav1.5 channel, cause a life-threatening form of cardiac arrhythmia, long QT syndrome type 3 (LQT3). Mexiletine, which is structurally related to the Na+ channel-blocking anesthetic lidocaine, is used to treat LQT3 patients. However, the patient response is variable, depending on the genetic mutation in SCN5A. OBJECTIVE: The goal of this study is to understand the molecular basis of patients' variable responses and build a predictive statistical model that can be used to personalize mexiletine treatment based on patient's genetic variant. METHODS AND RESULTS: We monitored the cardiac Na+ channel voltage-sensing domain (VSD) conformational dynamics simultaneously with other gating properties for the LQT3 variants. To systematically identify the relationship between mexiletine block and channel biophysical properties, we used a system-based statistical modeling approach to connect the multivariate properties to patient phenotype. We found that mexiletine altered the conformation of the Domain III VSD, which is the same VSD that many tested LQT3 mutations affect. Analysis of 15 LQT3 variants showed a strong correlation between the activation of the Domain III-VSD and the strength of the inhibition of the channel by mexiletine. Based on this improved molecular-level understanding, we generated a systems-based model based on a dataset of 32 LQT3 patients, which then successfully predicted the response of 7 out of 8 patients to mexiletine in a blinded, retrospective trial. CONCLUSIONS: Our results imply that the modulated receptor theory of local anesthetic action, which confines local anesthetic binding effects to the channel pore, should be revised to include drug interaction with the Domain III-VSD. Using an algorithm that incorporates this mode of action, we can predict patient-specific responses to mexiletine, improving therapeutic decision making.

Gating control of the cardiac sodium channel Nav1.5 by its ß3-subunit involves distinct roles for a transmembrane glutamic acid and the extracellular domain.

The auxiliary ß3-subunit is an important functional regulator of the cardiac sodium channel Nav1.5, and some ß3 mutations predispose individuals to cardiac arrhythmias. The ß3-subunit uses its transmembrane α-helix and extracellular domain to bind to Nav1.5. Here, we investigated the role of an unusually located and highly conserved glutamic acid (Glu-176) within the ß3 transmembrane region and its potential for functionally synergizing with the ß3 extracellular domain (ECD). We substituted Glu-176 with lysine (E176K) in the WT ß3-subunit and in a ß3-subunit lacking the ECD. Patch-clamp experiments indicated that the E176K substitution does not affect the previously observed ß3-dependent depolarizing shift of V½ of steady-state inactivation but does attenuate the accelerated recovery from inactivation conferred by the WT ß3-subunit. Removal of the ß3-ECD abrogated both the depolarizing shift of steady-state inactivation and the accelerated recovery, irrespective of the presence or absence of the Glu-176 residue. We found that steady-state inactivation and recovery from inactivation involve movements of the S4 helices within the DIII and DIV voltage sensors in response to membrane potential changes. Voltage-clamp fluorometry revealed that the E176K substitution alters DIII voltage sensor dynamics without affecting DIV. In contrast, removal of the ECD significantly altered the dynamics of both DIII and DIV. These results imply distinct roles for the ß3-Glu-176 residue and the ß3-ECD in regulating the conformational changes of the voltage sensors that determine channel inactivation and recovery from inactivation.

Gold nanoparticles conjugating recombinant influenza hemagglutinin trimers and flagellin enhanced mucosal cellular immunity.

The immunogenicity of subunit vaccines can be augmented by formulating them into nanoparticles. We conjugated recombinant trimetric influenza A/Aichi/2/68(H3N2) hemagglutinin (HA) onto functionalized gold nanoparticle (AuNP) surfaces in a repetitive, oriented configuration. To further improve the immunogenicity, we generated Toll-like receptor 5 (TLR5) agonist flagellin (FliC)-coupled AuNPs as particulate adjuvants. Intranasal immunizations with an AuNP-HA and AuNP-FliC particle mixture elicited strong mucosal and systemic immune responses that protected hosts against lethal influenza challenges. Compared with the AuNP-HA alone group, the addition of AuNP-FliC improved mucosal B cell responses as characterized by elevated influenza specific IgA and IgG levels in nasal, tracheal, and lung washes. AuNP-HA/AuNP-FliC also stimulated antigen-specific interferon-γ (IFN-γ)-secreting CD4+ cell proliferation and induced strong effector CD8+ T cell activation. Our results indicate that intranasal co-delivery of antigen and adjuvant-displaying AuNPs enhanced vaccine efficacy by inducing potent cellular immune responses.

From Variation of Influenza Viral Proteins to Vaccine Development.

Recurrent influenza epidemics and occasional pandemics are one of the most important global public health concerns and are major causes of human morbidity and mortality. Influenza viruses can evolve through antigen drift and shift to overcome the barriers of human immunity, leading to host adaption and transmission. Mechanisms underlying this viral evolution are gradually being elucidated. Vaccination is an effective method for the prevention of influenza virus infection. However, the emergence of novel viruses, including the 2009 pandemic influenza A (H1N1), the avian influenza A virus (H7N9), and the highly pathogenic avian influenza A virus (HPAI H5N1), that have infected human populations frequently in recent years reveals the tremendous challenges to the current influenza vaccine strategy. A better vaccine that provides protection against a wide spectrum of various influenza viruses and long-lasting immunity is urgently required. Here, we review the evolutionary changes of several important influenza proteins and the influence of these changes on viral antigenicity, host adaption, and viral pathogenicity. Furthermore, we discuss the development of a potent universal influenza vaccine based on this knowledge.

A novel NaV1.5 voltage sensor mutation associated with severe atrial and ventricular arrhythmias.

BACKGROUND: Inherited autosomal dominant mutations in cardiac sodium channels (NaV1.5) cause various arrhythmias, such as long QT syndrome and Brugada syndrome. Although dozens of mutations throughout the protein have been reported, there are few reported mutations within a voltage sensor S4 transmembrane segment and few that are homozygous. Here we report analysis of a novel lidocaine-sensitive recessive mutation, p.R1309H, in the NaV1.5 DIII/S4 voltage sensor in a patient with a complex arrhythmia syndrome. METHODS AND RESULTS: We expressed the wild type or mutant NaV1.5 heterologously for analysis with the patch-clamp and voltage clamp fluorometry (VCF) techniques. p.R1309H depolarized the voltage-dependence of activation, hyperpolarized the voltage-dependence of inactivation, and slowed recovery from inactivation, thereby reducing the channel availability at physiologic membrane potentials. Additionally, p.R1309H increased the "late" Na(+) current. The location of the mutation in DIIIS4 prompted testing for a gating pore current. We observed an inward current at hyperpolarizing voltages that likely exacerbates the loss-of-function defects at resting membrane potentials. Lidocaine reduced the gating pore current. CONCLUSIONS: The p.R1309H homozygous NaV1.5 mutation conferred both gain-of-function and loss-of-function effects on NaV1.5 channel activity. Reduction of a mutation-induced gating pore current by lidocaine suggested a therapeutic mechanism.

An alternative splicing isoform of MITA antagonizes MITA-mediated induction of type I IFNs.

Mediator of IFN regulatory transcription factor 3 activation (MITA) is an important adaptor protein to mediate the induction of type I IFNs. In this study, we identified an alternatively spliced isoform of MITA lacking exon 7, termed MITA-related protein (MRP). MRP shares the N-terminal portion aa 1-253 with MITA but possesses a unique 30-aa sequence at the carboxyl terminal part, therefore lacking the conserved domains including TANK-binding kinase 1 (TBK1) and cyclic diguanylate binding domain. MRP is expressed in multiple tissues and distinct cell lines. Overexpression of MRP inhibited MITA-mediated activation of IFN-ß promoter by sendai virus infection and cyclic diguanylate treatment but enhanced that in HSV-1 infection. Interestingly, MRP expression was reduced after Sendai virus infection but was upregulated after HSV-1 infection. Overexpression of MRP inhibited MITA-mediated induction of IFN-ß via TBK1-IFN regulatory transcription factor 3 by disrupting the MITA-TBK1 interaction. However, NF-κB pathway was still activated by MRP, as MRP retained the ability to interact with inducible inhibitor of NF-κB (iκB) kinase. Thus, MRP acts as a dominant negative regulator of MITA-mediated induction of IFN production.

Nuclear receptor 4 group A member 1 determines hepatitis C virus entry efficiency through the regulation of cellular receptor and apolipoprotein E expression.

Orphan nuclear receptor subfamily 4 group A member 1 (NR4A1) is a transcription factor stimulated by many factors and plays pivotal roles in metabolism, proliferation and apoptosis. In this study, the expression of NR4A1 in Huh7.5.1 cells was significantly upregulated by hepatitis C virus (HCV) infection. The silencing of NR4A1 inhibited the entry of HCV and reduced the specific infectivity of secreted HCV particles but had only minor or no effect on the genome replication and translation, virion assembly and virus release steps of the virus life cycle. Further experiments demonstrated that the silencing of NR4A1 affected virus entry through pan-downregulation of the expression of HCV receptors scavenger receptor BI, occludin, claudin-1 and epidermal growth factor receptor but not CD81. The reduced specific infectivity of HCV in the knockdown cells was due to decreased apolipoprotein E (ApoE) expression. These results explain the delayed spread of HCV in NR4A1 knockdown Huh7.5.1 cells. Thus, NR4A1 plays a role in HCV replication through regulating the expression of HCV receptors and ApoE, and facilitates HCV entry and spread.

mRNA Vaccine Nanoplatforms and Innate Immunity.

mRNA-based vaccine technology has been significantly developed and enhanced, particularly highlighted by the authorization of mRNA vaccines for addressing the COVID-19 pandemic. Various biomaterials are developed in nano-scales and applied as mRNA vaccine delivery platforms. However, how these mRNA nanoplatforms influence immune responses has not been thoroughly studied. Hence, we have reviewed the current understanding of various mRNA vaccine platforms. We discussed the possible pathways through which these platforms moderate the host's innate immunity and contribute to the development of adaptive immunity. We shed light on their development in reducing biotoxicity and enhancing antigen delivery efficiency. Beyond the built-in adjuvanticity of mRNA vaccines, we propose that supplementary adjuvants may be required to fine-tune and precisely control innate immunity and subsequent adaptive immune responses.