T(H)17 cells promote microbial killing and innate immune sensing of DNA via interleukin 26.

Interleukin 17-producing helper T cells (T(H)17 cells) have a major role in protection against infections and in mediating autoimmune diseases, yet the mechanisms involved are incompletely understood. We found that interleukin 26 (IL-26), a human T(H)17 cell-derived cytokine, is a cationic amphipathic protein that kills extracellular bacteria via membrane-pore formation. Furthermore, T(H)17 cell-derived IL-26 formed complexes with bacterial DNA and self-DNA released by dying bacteria and host cells. The resulting IL-26-DNA complexes triggered the production of type I interferon by plasmacytoid dendritic cells via activation of Toll-like receptor 9, but independently of the IL-26 receptor. These findings provide insights into the potent antimicrobial and proinflammatory function of T(H)17 cells by showing that IL-26 is a natural human antimicrobial that promotes immune sensing of bacterial and host cell death.

Cutting Edge: ATP13A2 Is an Endolysosomal Regulator of TLR9/7 Activation in Human Plasmacytoid Dendritic Cells.

ATPase cation transporting 13A2 (ATP13A2) is an endolysosomal P-type ATPase known to be a polyamine transporter, explored mostly in neurons. As endolysosomal functions are also crucial in innate immune cells, we aimed to explore the potential role of ATP13A2 in the human immunocellular compartment. We found that human plasmacytoid dendritic cells (pDCs), the professional type I IFN-producing immune cells, especially have a prominent enrichment of ATP13A2 expression in endolysosomal compartments. ATP13A2 knockdown in human pDCs interferes with cytokine induction in response to TLR9/7 activation in response to bona fide ligands. ATP13A2 plays this crucial role in TLR9/7 activation in human pDCs by regulating endolysosomal pH and mitochondrial reactive oxygen generation. This (to our knowledge) hitherto unknown regulatory mechanism in pDCs involving ATP13A2 opens up a new avenue of research, given the crucial role of pDC-derived type I IFNs in protective immunity against infections as well as in the immunopathogenesis of myriad contexts of autoreactive inflammation.

Integration of Ligand-Based and Structure-Based Methods for the Design of Small-Molecule TLR7 Antagonists.

Toll-like receptor 7 (TLR7) is activated in response to the binding of single-stranded RNA. Its over-activation has been implicated in several autoimmune disorders, and thus, it is an established therapeutic target in such circumstances. TLR7 small-molecule antagonists are not yet available for therapeutic use. We conducted a ligand-based drug design of new TLR7 antagonists through a concerted effort encompassing 2D-QSAR, 3D-QSAR, and pharmacophore modelling of 54 reported TLR7 antagonists. The developed 2D-QSAR model depicted an excellent correlation coefficient (R2training: 0.86 and R2test: 0.78) between the experimental and estimated activities. The ligand-based drug design approach utilizing the 3D-QSAR model (R2training: 0.95 and R2test: 0.84) demonstrated a significant contribution of electrostatic potential and steric fields towards the TLR7 antagonism. This consolidated approach, along with a pharmacophore model with high correlation (Rtraining: 0.94 and Rtest: 0.92), was used to design quinazoline-core-based hTLR7 antagonists. Subsequently, the newly designed molecules were subjected to molecular docking onto the previously proposed binding model and a molecular dynamics study for a better understanding of their binding pattern. The toxicity profiles and drug-likeness characteristics of the designed compounds were evaluated with in silico ADMET predictions. This ligand-based study contributes towards a better understanding of lead optimization and the future development of potent TLR7 antagonists.

Nature and Dimensions of Systemic Hyperinflammation and its Attenuation by Convalescent Plasma in Severe COVID-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing coronavirus disease 2019 (COVID-19), has led to significant morbidity and mortality. While most suffer from mild symptoms, some patients progress to severe disease with acute respiratory distress syndrome (ARDS) and associated systemic hyperinflammation. METHODS: First, to characterize key cytokines and their dynamics in this hyperinflammatory condition, we assessed abundance and correlative expression of a panel of 48 cytokines in patients progressing to ARDS as compared to patients with mild disease. Then, in an ongoing randomized controlled trial of convalescent plasma therapy (CPT), we analyzed rapid effects of CPT on the systemic cytokine dynamics as a correlate for the level of hypoxia experienced by the patients. RESULTS: We identified an anti-inflammatory role of CPT independent of its neutralizing antibody content. CONCLUSIONS: Neutralizing antibodies, as well as reductions in circulating interleukin-6 and interferon-γ-inducible protein 10, contributed to marked rapid reductions in hypoxia in response to CPT. CLINICAL TRIAL REGISTRY OF INDIA: CTRI/2020/05/025209. http://www.ctri.nic.in/.

Endocannabinoids in immune regulation and immunopathologies.

Endocannabinoids are key bioactive components of the endocannabinoid system, and the profound influence of endocannabinoids on the modulation of the immune system is being increasingly appreciated. The knowledge of endocannabinoid-immune cell crosstalk will pave the way to therapeutic implications of modulators of this pathway in autoimmune and chronic inflammatory disorders. Endocannabinoids seem to exert both anti-inflammatory and pro-inflammatory effects in specific contexts, based on specific receptor engagement and the downstream signalling pathways involved. In this review, we summarized the biosynthesis, signalling and degradation of two well-studied endocannabinoids-anandamide and 2-arachidonylglycerol in immune cells. Then, we discussed the effects of these two endocannabinoids on the functioning of major innate and adaptive immune cells, along with the choice of receptors employed in such interactions. Finally, we outline our current knowledge on the involvement of anandamide and 2-arachidonylglycerol in context of inflammation, allergies, autoimmunity and metabolic disorders.

Do Type I Interferons Link Systemic Autoimmunities and Metabolic Syndrome in a Pathogenetic Continuum?

The central pathogenetic role of type I interferons (IFNs) in several systemic autoimmune diseases is well established. Recent studies have also discovered a similar crucial role of type I IFNs in different components of metabolic disorders. Self nucleic acid-driven Toll-like receptor (TLR) activation in plasmacytoid dendritic cells (pDCs) and type I IFN induction appear to be the key initiating events shared by most of these autoimmune and metabolic diseases. Further strengthening this link, many patients with systemic autoimmunities also present with metabolic disorders. This concurrence of autoimmunities and metabolic disorders may be explained by a single pathogenetic continuum, and suggests shared targets for potential new therapies.

Cutting Edge: Dysregulated Endocannabinoid-Rheostat for Plasmacytoid Dendritic Cell Activation in a Systemic Lupus Endophenotype.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease, characterized by loss of tolerance toward self nuclear Ags. Systemic induction of type I IFNs plays a pivotal role in SLE, a major source of type I IFNs being the plasmacytoid dendritic cells (pDCs). Several genes have been linked with susceptibility to SLE in genome-wide association studies. We aimed at exploring the role of one such gene, α/ß-hydrolase domain-containing 6 (ABHD6), in regulation of IFN-α induction in SLE patients. We discovered a regulatory role of ABHD6 in human pDCs through modulating the local abundance of its substrate, the endocannabinoid 2-arachidonyl glycerol (2-AG), and elucidated a hitherto unknown cannabinoid receptor 2 (CB2)-mediated regulatory role of 2-AG on IFN-α induction by pDCs. We also identified an ABHD6High SLE endophenotype wherein reduced local abundance of 2-AG relieves the CB2-mediated steady-state resistive tuning on IFN-α induction by pDCs, thereby contributing to SLE pathogenesis.

Role of Ca2+ in toll-like receptor 9 activation in human plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) are major producers of type I interferons in response to activation of endosomal toll-like receptors (TLRs), e.g. TLR9. While a number of cell biological and intracellular signaling events associated with TLR9 activation in pDCs have been studied, role of free calcium (Ca2+) is not clear. We found that influx of extracellular Ca2+ is crucial for TLR9 mediated IFNα production by human pDCs. We also unraveled a role of Ca2+ in potentiating cellular uptake of self-DNA in complex with the cathelicidin antimicrobial peptide, LL37, an endogenous ligand for human TLR9 in autoimmune contexts. IFNα in response to TLR9 activation, by CpG oligonucleotides, is tuned within a window of Ca2+ concentration, through a bimodal regulatory switch, by differential engagement of Ca2+/calmodulin-dependent protein kinase II (CAMKII) and calcineurin phosphatase (CALN). Ca2+ signaling for TLR9 activation at physiologic calcium concentrations depends on CAMKII recruitment, while inhibition of TLR9 activation at supraphysiologic calcium concentrations is mediated by CALN. This bimodal regulation was masked in response to physiological peptide-DNA complexes, presumably due to potentiation of complex formation and increased cellular uptake in higher Ca2+ concentrations. Thus infection susceptibility associated with relevant clinical contexts as well as role of Ca2+ signaling in autoimmune diseases warrant further investigations for novel pathogenetic cues involving pDC function.

Mechanical Cues for T Cell Activation: Role of Piezo1 Mechanosensors.

T cells are activated in response to the recognition of antigenic peptides on major histocompatibility complex molecules (pMHC) by the T cell receptors (TCR) and induction of downstream signaling. The strength of interaction between specific TCR with pMHC is a key defining factor for optimal T cell activation. But a number of studies have also suggested a crucial involvement of mechanical cues within the immunological synapse. However, a dedicated mechanosensor molecule that senses and transduces the mechanical cues to optimize TCR activation was, until very recently, not known. The putative candidates included the TCR itself as well as the integrins within the immunological synapse. Recently, the Piezo family of proteins was discovered as professional mechanosensors implicated in a number of physiological processes. One of the two Piezo family members, Piezo1, is expressed in human T cells. Recent data suggest that Piezo1 functions as a professional mechanotransducer at the immunological synapse during TCR-pMHC interaction, and thus play a crucial role in human T cell activation. Thus, the domain of T cell activation has gathered a new mechanoregulatory dimension, which should incite further studies for detailed elucidation of the mechanistic underpinnings and its translational implications.

Cutting Edge: Piezo1 Mechanosensors Optimize Human T Cell Activation.

TCRs recognize peptides on MHC molecules and induce downstream signaling, leading to activation and clonal expansion. In addition to the strength of the interaction of TCRs with peptides on MHC molecules, mechanical forces contribute to optimal T cell activation, as reflected by the superior efficiency of immobilized TCR-cross-linking Abs compared with soluble Abs in TCR triggering, although a dedicated mechanotransduction module is not identified. We found that the professional mechanosensor protein Piezo1 is critically involved in human T cell activation. Although a deficiency in Piezo1 attenuates downstream events on ex vivo TCR triggering, a Piezo1 agonist can obviate the need to immobilize TCR-cross-linking Abs. Piezo1-driven Ca2+ influx, leading to calpain activation and organization of cortical actin scaffold, links this mechanosensor to optimal TCR signaling. Thus, we discovered a hitherto unknown regulatory mechanism for human T cell activation and provide the first evidence, to our knowledge, for the involvement of Piezo1 mechanosensors in immune regulation.

Cationic antimicrobial peptides in psoriatic skin cooperate to break innate tolerance to self-DNA.

Psoriasis is a T-cell-mediated skin autoimmune disease characterized by the aberrant activation of dermal dendritic cells (DCs) and the sustained epidermal expression of antimicrobial peptides. We have previously identified a link between these two events by showing that the cathelicidin antimicrobial peptide LL37 has the ability to trigger self-nucleic acid mediated activation of plasmacytoid DCs (pDCs) in psoriatic skin. Whether other cationic antimicrobial peptides exert similar activities is unknown. By analyzing heparin-binding HPLC fractions of psoriatic scales, we found that human beta-defensin (hBD)2, hBD3, and lysozyme are additional triggers of pDC activation in psoriatic skin lesions. Like LL37, hBD2, hBD3, and lysozyme are able to condense self-DNA into particles that are endocytosed by pDCs, leading to activation of TLR9. In contrast, other antimicrobial peptides expressed in psoriatic skin including elafin, hBD1, and psoriasin (S100A7) did not show similar activities. hBD2, hBD3, and lysozyme were detected in psoriatic skin lesions in the vicinity of pDCs and found to cooperate with LL37 to induce high levels of IFN production by pDCs, suggesting their concerted role in the pathogenesis of psoriasis.

Nucleic acid-containing amyloid fibrils potently induce type I interferon and stimulate systemic autoimmunity.

The immunopathophysiologic development of systemic autoimmunity involves numerous factors through complex mechanisms that are not fully understood. In systemic lupus erythematosus, type I IFN (IFN-I) produced by plasmacytoid dendritic cells (pDCs) critically promotes the autoimmunity through its pleiotropic effects on immune cells. However, the host-derived factors that enable abnormal IFN-I production and initial immune tolerance breakdown are largely unknown. Previously, we found that amyloid precursor proteins form amyloid fibrils in the presence of nucleic acids. Here we report that nucleic acid-containing amyloid fibrils can potently activate pDCs and enable IFN-I production in response to self-DNA, self-RNA, and dead cell debris. pDCs can take up DNA-containing amyloid fibrils, which are retained in the early endosomes to activate TLR9, leading to high IFNα/ß production. In mice treated with DNA-containing amyloid fibrils, a rapid IFN response correlated with pDC infiltration and activation. Immunization of nonautoimmune mice with DNA-containing amyloid fibrils induced antinuclear serology against a panel of self-antigens. The mice exhibited positive proteinuria and deposited antibodies in their kidneys. Intriguingly, pDC depletion obstructed IFN-I response and selectively abolished autoantibody generation. Our study reveals an innate immune function of nucleic acid-containing amyloid fibrils and provides a potential link between compromised protein homeostasis and autoimmunity via a pDC-IFN axis.

Multi-omics studies in interpreting the evolving standard model for immune functions.

A standard model that is able to generalize data on myriad involvement of the immune system in organismal physio-pathology and to provide a unified evolutionary teleology for immune functions in multicellular organisms remains elusive. A number of such 'general theories of immunity' have been proposed based on contemporaneously available data, starting with the usual description of self-nonself discrimination, followed by the 'danger model' and the more recent 'discontinuity theory.' More recent data deluge on involvement of immune mechanisms in a wide variety of clinical contexts, a number of which fail to get readily accommodated into the available teleologic standard models, makes deriving a standard model of immunity more challenging. But technological advances enabling multi-omics investigations into an ongoing immune response, covering genome, epigenome, coding and regulatory transcriptome, proteome, metabolome and tissue-resident microbiome, bring newer opportunities for developing a more integrative insight into immunocellular mechanisms within different clinical contexts. The new ability to map the heterogeneity of composition, trajectory and endpoints of immune responses, in both health and disease, also necessitates incorporation into the potential standard model of immune functions, which again can only be achieved through multi-omics probing of immune responses and integrated analyses of the multi-dimensional data.

Suppressed transcript diversity and immune response in COVID-19 ICU patients: a longitudinal study.

Understanding the dynamic changes in gene expression during Acute Respiratory Distress Syndrome (ARDS) progression in post-acute infection patients is crucial for unraveling the underlying mechanisms. Study investigates the longitudinal changes in gene/transcript expression patterns in hospital-admitted severe COVID-19 patients with ARDS post-acute SARS-CoV-2 infection. Blood samples were collected at three time points and patients were stratified into severe and mild ARDS, based on their oxygenation saturation (SpO2/FiO2) kinetics over 7 d. Decline in transcript diversity was observed over time, particularly in patients with higher severity, indicating dysregulated transcriptional landscape. Comparing gene/transcript-level analyses highlighted a rather limited overlap. With disease progression, a transition towards an inflammatory state was evident. Strong association was found between antibody response and disease severity, characterized by decreased antibody response and activated B cell population in severe cases. Bayesian network analysis identified various factors associated with disease progression and severity, viz. humoral response, TLR signaling, inflammatory response, interferon response, and effector T cell abundance. The findings highlight dynamic gene/transcript expression changes during ARDS progression, impact on tissue oxygenation and elucidate disease pathogenesis.

Exploring the Structural Attributes of Yoda1 for the Development of New-Generation Piezo1 Agonist Yaddle1 as a Vaccine Adjuvant Targeting Optimal T Cell Activation.

Piezo1, a mechano-activated ion channel, has wide-ranging physiological and therapeutic implications, with the ongoing development of specific agonists unveiling cellular responses to mechanical stimuli. In our study, we systematically analyzed the chemical subunits in Piezo1 protein agonist Yoda1 to comprehend the structure-activity relationship and push forward next-generation agonist development. Preliminary screening assays for Piezo1 agonism were performed using the Piezo1-mCherry-transfected HEK293A cell line, keeping Yoda1 as a positive control. We introduce a novel Piezo1 agonist Yaddle1 (34, 0.40 µM), featuring a trifluoromethyl group, with further exploration through in vitro studies and density functional theory calculations, emphasizing its tetrel interactions, to act as an ambidextrous wedge between the domains of Piezo1. In contrast to the poor solubility of the established agonist Yoda1, our results showed that the kinetic solubility of Yaddle1 (26.72 ± 1.8 µM at pH 7.4) is 10-fold better than that of Yoda1 (1.22 ± 0.11 µM at pH 7.4). Yaddle1 (34) induces Ca2+ influx in human CD4+ T cell, suggesting its potential as a vaccine adjuvant for enhanced T cell activation.

Piezo1 mechanosensing regulates integrin-dependent chemotactic migration in human T cells.

T cells are crucial for efficient antigen-specific immune responses and thus their migration within the body, to inflamed tissues from circulating blood or to secondary lymphoid organs, plays a very critical role. T cell extravasation in inflamed tissues depends on chemotactic cues and interaction between endothelial adhesion molecules and cellular integrins. A migrating T cell is expected to sense diverse external and membrane-intrinsic mechano-physical cues, but molecular mechanisms of such mechanosensing in cell migration are not established. We explored if the professional mechanosensor Piezo1 plays any role during integrin-dependent chemotaxis of human T cells. We found that deficiency of Piezo1 in human T cells interfered with integrin-dependent cellular motility on ICAM-1-coated surface. Piezo1 recruitment at the leading edge of moving T cells is dependent on and follows focal adhesion formation at the leading edge and local increase in membrane tension upon chemokine receptor activation. Piezo1 recruitment and activation, followed by calcium influx and calpain activation, in turn, are crucial for the integrin LFA1 (CD11a/CD18) recruitment at the leading edge of the chemotactic human T cells. Thus, we find that Piezo1 activation in response to local mechanical cues constitutes a membrane-intrinsic component of the 'outside-in' signaling in human T cells, migrating in response to chemokines, that mediates integrin recruitment to the leading edge.

Hit-to-lead optimization of 2-aminoquinazolines as anti-microbial agents against Leishmania donovani.

Visceral leishmaniasis is a potentially fatal disease caused by infection by the intracellular protist pathogens Leishmania donovani or Leishmania infantum. Present therapies are ineffective because of high costs, variable efficacy against different species, the requirement for hospitalization, toxicity and drug resistance. Detailed analysis of previously published hit molecules suggested a crucial role of 'guanidine' linkage for their efficacy against L. donovani. Here we report the design of 2-aminoquinazoline heterocycle as a basic pharmacophore-bearing guanidine linkage. The introduction of various groups and functionality at different positions of the quinazoline scaffold results in enhanced antiparasitic potency with modest host cell cytotoxicity using a physiologically relevant THP-1 transformed macrophage infection model. In terms of the ADME profile, the C7 position of quinazoline was identified as a guiding tool for designing better molecules. The good ADME profile of the compounds suggests that they merit further consideration as lead compounds for treating visceral leishmaniasis.

Mitigating hERG Liability of Toll-Like Receptor 9 and 7 Antagonists through Structure-Based Design.

hERG is considered to be a primary anti-target in the drug development process, as the K+ channel encoded by hERG plays an important role in cardiac re-polarization. It is desirable to address the hERG safety liability during early-stage development to avoid the expenses of validating leads that will eventually fail at a later stage. We have previously reported the development of highly potent quinazoline-based TLR7 and TLR9 antagonists for possible application against autoimmune disease. Initial experimental hERG assessment showed that most of the lead TLR7 and TLR9 antagonists suffer from hERG liability rendering them ineffective for further development. The present study herein describes a coordinated strategy to integrate the understanding from structure-based protein-ligand interaction to develop non- hERG binders with IC50 >30 µM with retention of TLR7/9 antagonism through a single point change in the scaffold. This structure-guided strategy can serve as a prototype for abolishing hERG liability during lead optimization.

Mitochondrial sourcing of interferogenic ligands and an autoantigen in human obesity-associated metaflammation.

OBJECTIVE: Visceral adipose tissue (VAT) inflammation contributes to metabolic dysregulation in obesity. VAT recruitment and activation of plasmacytoid dendritic cells (pDCs) through toll-like receptor 9 (TLR9) recognition of self-DNA, leading to induction of type I interferons, are crucial innate triggers for this VAT inflammation. It was hypothesized that mitochondrial DNA (mtDNA) can contribute to TLR9 activation in VAT-recruited pDCs in obesity, and this study aimed to identify the carrier protein for ligand access to TLR9 and to explore whether this also provides for a source of autoantigens in this context. METHODS: VAT samples, used for gene expression studies as well as adipose explant cultures, were collected from patients with obesity (n = 54) and lean patients (n = 10). Supernatants from human pDC cultures, treated with adipose explant culture supernatants, were used for interferon α ELISA. Venous plasma, from patients with (n = 114) and without (n = 45) obesity, was used for an ELISA for autoantibodies. RESULTS: MtDNA from VAT in obesity, in complex with mitochondrial transcription factor A protein (TFAM), acts as interferogenic ligands for pDCs. Humoral autoreactivity against TFAM is also induced in obesity. CONCLUSIONS: Interferogenic ligands and an autoantigen can be sourced from dysfunctional mitochondria in VAT of humans with obesity. Further therapeutic and prognostic potential for this immune mechanism in obesity warrants exploration.

Circulating Interleukin-8 Dynamics Parallels Disease Course and Is Linked to Clinical Outcomes in Severe COVID-19.

Severe COVID-19 frequently features a systemic deluge of cytokines. Circulating cytokines that can stratify risks are useful for more effective triage and management. Here, we ran a machine-learning algorithm on a dataset of 36 plasma cytokines in a cohort of severe COVID-19 to identify cytokine/s useful for describing the dynamic clinical state in multiple regression analysis. We performed RNA-sequencing of circulating blood cells collected at different time-points. From a Bayesian Information Criterion analysis, a combination of interleukin-8 (IL-8), Eotaxin, and Interferon-γ (IFNγ) was found to be significantly linked to blood oxygenation over seven days. Individually testing the cytokines in receiver operator characteristics analyses identified IL-8 as a strong stratifier for clinical outcomes. Circulating IL-8 dynamics paralleled disease course. We also revealed key transitions in immune transcriptome in patients stratified for circulating IL-8 at three time-points. The study identifies plasma IL-8 as a key pathogenic cytokine linking systemic hyper-inflammation to the clinical outcomes in COVID-19.