Proteomics and cytokine analyses distinguish myalgic encephalomyelitis/chronic fatigue syndrome cases from controls.

BACKGROUND: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex, heterogenous disease characterized by unexplained persistent fatigue and other features including cognitive impairment, myalgias, post-exertional malaise, and immune system dysfunction. Cytokines are present in plasma and encapsulated in extracellular vesicles (EVs), but there have been only a few reports of EV characteristics and cargo in ME/CFS. Several small studies have previously described plasma proteins or protein pathways that are associated with ME/CFS. METHODS: We prepared extracellular vesicles (EVs) from frozen plasma samples from a cohort of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) cases and controls with prior published plasma cytokine and plasma proteomics data. The cytokine content of the plasma-derived extracellular vesicles was determined by a multiplex assay and differences between patients and controls were assessed. We then performed multi-omic statistical analyses that considered not only this new data, but extensive clinical data describing the health of the subjects. RESULTS: ME/CFS cases exhibited greater size and concentration of EVs in plasma. Assays of cytokine content in EVs revealed IL2 was significantly higher in cases. We observed numerous correlations among EV cytokines, among plasma cytokines, and among plasma proteins from mass spectrometry proteomics. Significant correlations between clinical data and protein levels suggest roles of particular proteins and pathways in the disease. For example, higher levels of the pro-inflammatory cytokines Granulocyte-Monocyte Colony-Stimulating Factor (CSF2) and Tumor Necrosis Factor (TNFα) were correlated with greater physical and fatigue symptoms in ME/CFS cases. Higher serine protease SERPINA5, which is involved in hemostasis, was correlated with higher SF-36 general health scores in ME/CFS. Machine learning classifiers were able to identify a list of 20 proteins that could discriminate between cases and controls, with XGBoost providing the best classification with 86.1% accuracy and a cross-validated AUROC value of 0.947. Random Forest distinguished cases from controls with 79.1% accuracy and an AUROC value of 0.891 using only 7 proteins. CONCLUSIONS: These findings add to the substantial number of objective differences in biomolecules that have been identified in individuals with ME/CFS. The observed correlations of proteins important in immune responses and hemostasis with clinical data further implicates a disturbance of these functions in ME/CFS.

Novel insight into the underlying dysregulation mechanisms of immune cell-to-cell communication by analyzing multitissue single-cell atlas of two COVID-19 patients.

How does SARS-CoV-2 cause lung microenvironment disturbance and inflammatory storm is still obscure. We here performed the single-cell transcriptome sequencing from lung, blood, and bone marrow of two dead COVID-19 patients and detected the cellular communication among them. Our results demonstrated that SARS-CoV-2 infection increase the frequency of cellular communication between alveolar type I cells (AT1) or alveolar type II cells (AT2) and myeloid cells triggering immune activation and inflammation microenvironment and then induce the disorder of fibroblasts, club, and ciliated cells, which may cause increased pulmonary fibrosis and mucus accumulation. Further study showed that the increase of T cells in the lungs may be mainly recruited by myeloid cells through ligands/receptors (e.g., ANXA1/FPR1, C5AR1/RPS19, and CCL5/CCR1). Interestingly, we also found that certain ligands/receptors (e.g., ANXA1/FPR1, CD74/COPA, CXCLs/CXCRs, ALOX5/ALOX5AP, CCL5/CCR1) are significantly activated and shared among lungs, blood and bone marrow of COVID-19 patients, implying that the dysregulation of ligands/receptors may lead to immune cell's activation, migration, and the inflammatory storm in different tissues of COVID-19 patients. Collectively, our study revealed a possible mechanism by which the disorder of cell communication caused by SARS-CoV-2 infection results in the lung inflammatory microenvironment and systemic immune responses across tissues in COVID-19 patients.

From 2D to 3D Co-Culture Systems: A Review of Co-Culture Models to Study the Neural Cells Interaction.

The central nervous system (CNS) controls and regulates the functional activities of the organ systems and maintains the unity between the body and the external environment. The advent of co-culture systems has made it possible to elucidate the interactions between neural cells in vitro and to reproduce complex neural circuits. Here, we classified the co-culture system as a two-dimensional (2D) co-culture system, a cell-based three-dimensional (3D) co-culture system, a tissue slice-based 3D co-culture system, an organoid-based 3D co-culture system, and a microfluidic platform-based 3D co-culture system. We provide an overview of these different co-culture models and their applications in the study of neural cell interaction. The application of co-culture systems in virus-infected CNS disease models is also discussed here. Finally, the direction of the co-culture system in future research is prospected.

Targeting integrin pathways: mechanisms and advances in therapy.

Integrins are considered the main cell-adhesion transmembrane receptors that play multifaceted roles as extracellular matrix (ECM)-cytoskeletal linkers and transducers in biochemical and mechanical signals between cells and their environment in a wide range of states in health and diseases. Integrin functions are dependable on a delicate balance between active and inactive status via multiple mechanisms, including protein-protein interactions, conformational changes, and trafficking. Due to their exposure on the cell surface and sensitivity to the molecular blockade, integrins have been investigated as pharmacological targets for nearly 40 years, but given the complexity of integrins and sometimes opposite characteristics, targeting integrin therapeutics has been a challenge. To date, only seven drugs targeting integrins have been successfully marketed, including abciximab, eptifibatide, tirofiban, natalizumab, vedolizumab, lifitegrast, and carotegrast. Currently, there are approximately 90 kinds of integrin-based therapeutic drugs or imaging agents in clinical studies, including small molecules, antibodies, synthetic mimic peptides, antibody-drug conjugates (ADCs), chimeric antigen receptor (CAR) T-cell therapy, imaging agents, etc. A serious lesson from past integrin drug discovery and research efforts is that successes rely on both a deep understanding of integrin-regulatory mechanisms and unmet clinical needs. Herein, we provide a systematic and complete review of all integrin family members and integrin-mediated downstream signal transduction to highlight ongoing efforts to develop new therapies/diagnoses from bench to clinic. In addition, we further discuss the trend of drug development, how to improve the success rate of clinical trials targeting integrin therapies, and the key points for clinical research, basic research, and translational research.

Regulatory role of endoplasmic reticulum resident chaperone protein ERp29 in anti-murine ß-coronavirus host cell response.

Gap junctional intercellular communication (GJIC) involving astrocytes is important for proper CNS homeostasis. As determined in our previous studies, trafficking of the predominant astrocyte GJ protein, Connexin43 (Cx43), is disrupted in response to infection with a neurotropic murine ß-coronavirus (MHV-A59). However, how host factors are involved in Cx43 trafficking and the infection response is not clear. Here, we show that Cx43 retention due to MHV-A59 infection was associated with increased ER stress and reduced expression of chaperone protein ERp29. Treatment of MHV-A59-infected astrocytes with the chemical chaperone 4-sodium phenylbutyrate increased ERp29 expression, rescued Cx43 transport to the cell surface, increased GJIC, and reduced ER stress. We obtained similar results using an astrocytoma cell line (delayed brain tumor) upon MHV-A59 infection. Critically, delayed brain tumor cells transfected to express exogenous ERp29 were less susceptible to MHV-A59 infection and showed increased Cx43-mediated GJIC. Treatment with Cx43 mimetic peptides inhibited GJIC and increased viral susceptibility, demonstrating a role for intercellular communication in reducing MHV-A59 infectivity. Taken together, these results support a therapeutically targetable ERp29-dependent mechanism where ß-coronavirus infectivity is modulated by reducing ER stress and rescuing Cx43 trafficking and function.

Impaired cell-cell communication and axon guidance because of pulmonary hypoperfusion during postnatal alveolar development.

BACKGROUND: Pulmonary hypoperfusion is common in children with congenital heart diseases (CHDs) or pulmonary hypertension (PH) and causes adult pulmonary dysplasia. Systematic reviews have shown that some children with CHDs or PH have mitigated clinical outcomes with COVID-19. Understanding the effects of pulmonary hypoperfusion on postnatal alveolar development may aid in the development of methods to improve the pulmonary function of children with CHDs or PH and improve their care during the COVID-19 pandemic, which is characterized by cytokine storm and persistent inflammation. METHODS AND RESULTS: We created a neonatal pulmonary hypoperfusion model through pulmonary artery banding (PAB) surgery at postnatal day 1 (P1). Alveolar dysplasia was confirmed by gross and histological examination at P21. Transcriptomic analysis of pulmonary tissues at P7(alveolar stage 2) and P14(alveolar stage 4) revealed that the postnatal alveolar development track had been changed due to pulmonary hypoperfusion. Under the condition of pulmonary hypoperfusion, the cell-cell communication and axon guidance, which both determine the final number of alveoli, were lost; instead, there was hyperactive cell cycle activity. The transcriptomic results were further confirmed by the examination of axon guidance and cell cycle markers. Because axon guidance controls inflammation and immune cell activation, the loss of axon guidance may explain the lack of severe COVID-19 cases among children with CHDs or PH accompanied by pulmonary hypoperfusion. CONCLUSIONS: This study suggested that promoting cell-cell communication or supplementation with guidance molecules may treat pulmonary hypoperfusion-induced alveolar dysplasia, and that COVID-19 is less likely to cause a cytokine storm in children with CHD or PH accompanied by pulmonary hypoperfusion.

Structural Characteristics of Heparin Binding to SARS-CoV-2 Spike Protein RBD of Omicron Sub-Lineages BA.2.12.1, BA.4 and BA.5.

The now prevalent Omicron variant and its subvariants/sub-lineages have led to a significant increase in COVID-19 cases and raised serious concerns about increased risk of infectivity, immune evasion, and reinfection. Heparan sulfate (HS), located on the surface of host cells, plays an important role as a co-receptor for virus-host cell interaction. The ability of heparin and HS to compete for binding of the SARS-CoV-2 spike (S) protein to cell surface HS illustrates the therapeutic potential of agents targeting protein-glycan interactions. In the current study, phylogenetic tree of variants and mutations in S protein receptor-binding domain (RBD) of Omicron BA.2.12.1, BA.4 and BA.5 were described. The binding affinity of Omicron S protein RBD to heparin was further investigated by surface plasmon resonance (SPR). Solution competition studies on the inhibitory activity of heparin oligosaccharides and desulfated heparins at different sites on S protein RBD-heparin interactions revealed that different sub-lineages tend to bind heparin with different chain lengths and sulfation patterns. Furthermore, blind docking experiments showed the contribution of basic amino acid residues in RBD and sulfo groups and carboxyl groups on heparin to the interaction. Finally, pentosan polysulfate and mucopolysaccharide polysulfate were evaluated for inhibition on the interaction of heparin and S protein RBD of Omicron BA.2.12.1, BA.4/BA.5, and both showed much stronger inhibition than heparin.

SARS-CoV-2 Spike Proteins and Cell-Cell Communication Inhibits TFPI and Induces Thrombogenic Factors in Human Lung Microvascular Endothelial Cells and Neutrophils: Implications for COVID-19 Coagulopathy Pathogenesis.

In SARS-CoV-2-infected humans, disease progression is often associated with acute respiratory distress syndrome involving severe lung injury, coagulopathy, and thrombosis of the alveolar capillaries. The pathogenesis of these pulmonary complications in COVID-19 patients has not been elucidated. Autopsy study of these patients showed SARS-CoV-2 virions in pulmonary vessels and sequestrated leukocytes infiltrates associated with endotheliopathy and microvascular thrombosis. Since SARS-CoV-2 enters and infects target cells by binding its spike (S) protein to cellular angiotensin-converting enzyme 2 (ACE2), and there is evidence that vascular endothelial cells and neutrophils express ACE2, we investigated the effect of S-proteins and cell-cell communication on primary human lung microvascular endothelial cells (HLMEC) and neutrophils expression of thrombogenic factors and the potential mechanisms. Using S-proteins of two different SARS-CoV-2 variants (Wuhan and Delta), we demonstrate that exposure of HLMEC or neutrophils to S-proteins, co-culture of HLMEC exposed to S-proteins with non-exposed neutrophils, or co-culture of neutrophils exposed to S-proteins with non-exposed HLMEC induced transcriptional upregulation of tissue factor (TF), significantly increased the expression and secretion of factor (F)-V, thrombin, and fibrinogen and inhibited tissue factor pathway inhibitor (TFPI), the primary regulator of the extrinsic pathway of blood coagulation, in both cell types. Recombinant (r)TFPI and a thiol blocker (5,5'-dithio-bis-(2-nitrobenzoic acid)) prevented S-protein-induced expression and secretion of Factor-V, thrombin, and fibrinogen. Thrombomodulin blocked S-protein-induced expression and secretion of fibrinogen but had no effect on S-protein-induced expression of Factor-V or thrombin. These results suggests that following SARS-CoV-2 contact with the pulmonary endothelium or neutrophils and endothelial-neutrophil interactions, viral S-proteins induce coagulopathy via the TF pathway and mechanisms involving functional thiol groups. These findings suggest that using rTFPI and/or thiol-based drugs could be a viable therapeutic strategy against SARS-CoV-2-induced coagulopathy and thrombosis.

Scalable workflow for characterization of cell-cell communication in COVID-19 patients.

COVID-19 patients display a wide range of disease severity, ranging from asymptomatic to critical symptoms with high mortality risk. Our ability to understand the interaction of SARS-CoV-2 infected cells within the lung, and of protective or dysfunctional immune responses to the virus, is critical to effectively treat these patients. Currently, our understanding of cell-cell interactions across different disease states, and how such interactions may drive pathogenic outcomes, is incomplete. Here, we developed a generalizable and scalable workflow for identifying cells that are differentially interacting across COVID-19 patients with distinct disease outcomes and use this to examine eight public single-cell RNA-seq datasets (six from peripheral blood mononuclear cells, one from bronchoalveolar lavage and one from nasopharyngeal), with a total of 211 individual samples. By characterizing the cell-cell interaction patterns across epithelial and immune cells in lung tissues for patients with varying disease severity, we illustrate diverse communication patterns across individuals, and discover heterogeneous communication patterns among moderate and severe patients. We further illustrate patterns derived from cell-cell interactions are potential signatures for discriminating between moderate and severe patients. Overall, this workflow can be generalized and scaled to combine multiple scRNA-seq datasets to uncover cell-cell interactions.

Multiple Cell Cultures for MRI Analysis.

Magnetic resonance imaging (MRI) is an imaging method that enables diagnostics. In recent years, this technique has been widely used for research using cell cultures used in pharmaceutical science to understand the distribution of various drugs in a variety of biological samples, from cellular models to tissues. MRI's dynamic development in recent years, in addition to diagnostics, has allowed the method to be implemented to assess response to applied therapies. Conventional MRI imaging provides anatomical and pathological information. Due to advanced technology, MRI provides physiological information. The use of cell cultures is very important in the process of testing new synthesized drugs, cancer research, and stem cell research, among others. Two-dimensional (2D) cell cultures conducted under laboratory conditions, although they provide a lot of information, do not reflect the basic characteristics of the tumor. To replicate the tumor microenvironment in science, a three-dimensional (3D) culture of tumor cells was developed. This makes it possible to reproduce in vivo conditions where, in addition, there is a complex and dynamic process of cell-to-cell communication and cell-matrix interaction. In this work, we reviewed current research in 2D and 3D cultures and their use in MRI studies. Articles for each section were collected from PubMed, ScienceDirect, Web of Science, and Google Scholar.

Translating extracellular vesicle packaging into therapeutic applications.

Extracellular vesicles (EVs) are membrane-bound particles released by cells in various (patho)physiological conditions. EVs can transfer effector molecules and elicit potent responses in recipient cells, making them attractive therapeutic agents and drug delivery platforms. In contrast to their tremendous potential, only a few EV-based therapies and drug delivery have been approved for clinical use, which is largely attributed to limited therapeutic loading technologies and efficiency. As EV cargo has major influence on their functionality, understanding and translating the biology underlying the packaging and transferring of biomolecule cargos (e.g. miRNAs, pathogen antigens, small molecule drugs) into EVs is key in harnessing their therapeutic potential. In this review, through recent insights into EVs' content packaging, we discuss different mechanisms utilized by EVs during cargo packaging, and how one might therapeutically exploit this process. Apart from the well-characterized EVs like exosomes and microvesicles, we also cover the less-studied and other EV subtypes like apoptotic bodies, large oncosomes, bacterial outer membrane vesicles, and migrasomes to highlight therapeutically-diverse opportunities of EV armoury.

Post-COVID-19 Parkinsonism and Parkinson's Disease Pathogenesis: The Exosomal Cargo Hypothesis.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease after Alzheimer's disease, globally. Dopaminergic neuron degeneration in substantia nigra pars compacta and aggregation of misfolded alpha-synuclein are the PD hallmarks, accompanied by motor and non-motor symptoms. Several viruses have been linked to the appearance of a post-infection parkinsonian phenotype. Coronavirus disease 2019 (COVID-19), caused by emerging severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, has evolved from a novel pneumonia to a multifaceted syndrome with multiple clinical manifestations, among which neurological sequalae appear insidious and potentially long-lasting. Exosomes are extracellular nanovesicles bearing a complex cargo of active biomolecules and playing crucial roles in intercellular communication under pathophysiological conditions. Exosomes constitute a reliable route for misfolded protein transmission, contributing to PD pathogenesis and diagnosis. Herein, we summarize recent evidence suggesting that SARS-CoV-2 infection shares numerous clinical manifestations and inflammatory and molecular pathways with PD. We carry on hypothesizing that these similarities may be reflected in exosomal cargo modulated by the virus in correlation with disease severity. Travelling from the periphery to the brain, SARS-CoV-2-related exosomal cargo contains SARS-CoV-2 RNA, viral proteins, inflammatory mediators, and modified host proteins that could operate as promoters of neurodegenerative and neuroinflammatory cascades, potentially leading to a future parkinsonism and PD development.

The Role of Extracellular Vesicles in COVID-19 Pathology.

Extracellular vesicles (EVs) have become a trending topic in recent years; they constitute a new intercellular communication paradigm. Extracellular vesicles are 30-4000 nanometers in diameter particles that are limited by a phospholipid bilayer and contain functional biomolecules, such as proteins, lipids, and nucleic acids. They are released by virtually all types of eukaryotic cells; through their cargoes, EVs are capable of triggering signaling in recipient cells. In addition to their functions in the homeostatic state, EVs have gained attention because of their roles in pathological contexts, eventually contributing to disease progression. In the Coronavirus disease 2019 (COVID-19) pandemic, aside from the scientific race for the development of preventive and therapeutic interventions, it is critical to understand the pathological mechanisms involved in SARS-CoV-2 infection. In this sense, EVs are key players in the main processes of COVID-19. Thus, in this review, we highlight the role of EVs in the establishment of the viral infection and in the procoagulant state, cytokine storm, and immunoregulation of innate and adaptive immune responses.

Context-aware deconvolution of cell-cell communication with Tensor-cell2cell.

Cell interactions determine phenotypes, and intercellular communication is shaped by cellular contexts such as disease state, organismal life stage, and tissue microenvironment. Single-cell technologies measure the molecules mediating cell-cell communication, and emerging computational tools can exploit these data to decipher intercellular communication. However, current methods either disregard cellular context or rely on simple pairwise comparisons between samples, thus limiting the ability to decipher complex cell-cell communication across multiple time points, levels of disease severity, or spatial contexts. Here we present Tensor-cell2cell, an unsupervised method using tensor decomposition, which deciphers context-driven intercellular communication by simultaneously accounting for multiple stages, states, or locations of the cells. To do so, Tensor-cell2cell uncovers context-driven patterns of communication associated with different phenotypic states and determined by unique combinations of cell types and ligand-receptor pairs. As such, Tensor-cell2cell robustly improves upon and extends the analytical capabilities of existing tools. We show Tensor-cell2cell can identify multiple modules associated with distinct communication processes (e.g., participating cell-cell and ligand-receptor pairs) linked to severities of Coronavirus Disease 2019 and to Autism Spectrum Disorder. Thus, we introduce an effective and easy-to-use strategy for understanding complex communication patterns across diverse conditions.

Central Nervous System Pericytes Contribute to Health and Disease.

Successful neuroprotection is only possible with contemporary microvascular protection. The prevention of disease-induced vascular modifications that accelerate brain damage remains largely elusive. An improved understanding of pericyte (PC) signalling could provide important insight into the function of the neurovascular unit (NVU), and into the injury-provoked responses that modify cell-cell interactions and crosstalk. Due to sharing the same basement membrane with endothelial cells, PCs have a crucial role in the control of endothelial, astrocyte, and oligodendrocyte precursor functions and hence blood-brain barrier stability. Both cerebrovascular and neurodegenerative diseases impair oxygen delivery and functionally impair the NVU. In this review, the role of PCs in central nervous system health and disease is discussed, considering their origin, multipotency, functions and also dysfunction, focusing on new possible avenues to modulate neuroprotection. Dysfunctional PC signalling could also be considered as a potential biomarker of NVU pathology, allowing us to individualize therapeutic interventions, monitor responses, or predict outcomes.

Cell transcriptomic atlas of the non-human primate Macaca fascicularis.

Studying tissue composition and function in non-human primates (NHPs) is crucial to understand the nature of our own species. Here we present a large-scale cell transcriptomic atlas that encompasses over 1 million cells from 45 tissues of the adult NHP Macaca fascicularis. This dataset provides a vast annotated resource to study a species phylogenetically close to humans. To demonstrate the utility of the atlas, we have reconstructed the cell-cell interaction networks that drive Wnt signalling across the body, mapped the distribution of receptors and co-receptors for viruses causing human infectious diseases, and intersected our data with human genetic disease orthologues to establish potential clinical associations. Our M. fascicularis cell atlas constitutes an essential reference for future studies in humans and NHPs.

A Human In Vitro Model to Study Adenoviral Receptors and Virus Cell Interactions.

To develop adenoviral cell- or tissue-specific gene delivery, understanding of the infection mechanisms of adenoviruses is crucial. Several adenoviral attachment proteins such as CD46, CAR and sialic acid have been identified and studied. However, most receptor studies were performed on non-human cells. Combining our reporter gene-tagged adenovirus library with an in vitro human gene knockout model, we performed a systematic analysis of receptor usage comparing different adenoviruses side-by-side. The CRISPR/Cas9 system was used to knockout CD46 and CAR in the human lung epithelial carcinoma cell line A549. Knockout cells were infected with 22 luciferase-expressing adenoviruses derived from adenovirus species B, C, D and E. HAdV-B16, -B21 and -B50 from species B1 as well as HAdV-B34 and -B35 were found to be CD46-dependent. HAdV-C5 and HAdV-E4 from species E were found to be CAR-dependent. Regarding cell entry of HAdV-B3 and -B14 and all species D viruses, both CAR and CD46 play a role, and here, other receptors or attachment structures may also be important since transductions were reduced but not completely inhibited. The established human knockout cell model enables the identification of the most applicable adenovirus types for gene therapy and to further understand adenovirus infection biology.

Supermeres are functional extracellular nanoparticles replete with disease biomarkers and therapeutic targets.

Extracellular vesicles and exomere nanoparticles are under intense investigation as sources of clinically relevant cargo. Here we report the discovery of a distinct extracellular nanoparticle, termed supermere. Supermeres are morphologically distinct from exomeres and display a markedly greater uptake in vivo compared with small extracellular vesicles and exomeres. The protein and RNA composition of supermeres differs from small extracellular vesicles and exomeres. Supermeres are highly enriched with cargo involved in multiple cancers (glycolytic enzymes, TGFBI, miR-1246, MET, GPC1 and AGO2), Alzheimer's disease (APP) and cardiovascular disease (ACE2, ACE and PCSK9). The majority of extracellular RNA is associated with supermeres rather than small extracellular vesicles and exomeres. Cancer-derived supermeres increase lactate secretion, transfer cetuximab resistance and decrease hepatic lipids and glycogen in vivo. This study identifies a distinct functional nanoparticle replete with potential circulating biomarkers and therapeutic targets for a host of human diseases.

Cetylpyridinium chloride (CPC) reduces zebrafish mortality from influenza infection: Super-resolution microscopy reveals CPC interference with multiple protein interactions with phosphatidylinositol 4,5-bisphosphate in immune function.

The COVID-19 pandemic raises significance for a potential influenza therapeutic compound, cetylpyridinium chloride (CPC), which has been extensively used in personal care products as a positively-charged quaternary ammonium antibacterial agent. CPC is currently in clinical trials to assess its effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity. Two published studies have provided mouse and human data indicating that CPC may alleviate influenza infection, and here we show that CPC (0.1 µM, 1 h) reduces zebrafish mortality and viral load following influenza infection. However, CPC mechanisms of action upon viral-host cell interaction are currently unknown. We have utilized super-resolution fluorescence photoactivation localization microscopy to probe the mode of CPC action. Reduction in density of influenza viral protein hemagglutinin (HA) clusters is known to reduce influenza infectivity: here, we show that CPC (at non-cytotoxic doses, 5-10 µM) reduces HA density and number of HA molecules per cluster within the plasma membrane of NIH-3T3 mouse fibroblasts. HA is known to colocalize with the negatively-charged mammalian lipid phosphatidylinositol 4,5-bisphosphate (PIP2); here, we show that nanoscale co-localization of HA with the PIP2-binding Pleckstrin homology (PH) reporter in the plasma membrane is diminished by CPC. CPC also dramatically displaces the PIP2-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) from the plasma membrane of rat RBL-2H3 mast cells; this disruption of PIP2 is correlated with inhibition of mast cell degranulation. Together, these findings offer a PIP2-focused mechanism underlying CPC disruption of influenza and suggest potential pharmacological use of this drug as an influenza therapeutic to reduce global deaths from viral disease.