LPA suppresses T cell function by altering the cytoskeleton and disrupting immune synapse formation.

Cancer and chronic infections often increase levels of the bioactive lipid, lysophosphatidic acid (LPA), that we have demonstrated acts as an inhibitory ligand upon binding LPAR5 on CD8 T cells, suppressing cytotoxic activity and tumor control. This study, using human and mouse primary T lymphocytes, reveals how LPA disrupts antigen-specific CD8 T cell:target cell immune synapse (IS) formation and T cell function via competing for cytoskeletal regulation. Specifically, we find upon antigen-specific T cell:target cell formation, IP3R1 localizes to the IS by a process dependent on mDia1 and actin and microtubule polymerization. LPA not only inhibited IP3R1 from reaching the IS but also altered T cell receptor (TCR)­induced localization of RhoA and mDia1 impairing F-actin accumulation and altering the tubulin code. Consequently, LPA impeded calcium store release and IS-directed cytokine secretion. Thus, targeting LPA signaling in chronic inflammatory conditions may rescue T cell function and promote antiviral and antitumor immunity.

NK Cells in a Tug-of-War With Cancer: The Roles of Transcription Factors and Cytoskeleton.

Natural killer (NK) cells are innate immune cells which play a key role in shaping the immune response against cancer. Initially hailed for their potential to recognise and eliminate tumour cells, their application has been greatly hindered by the immunosuppressive tumour microenvironment (TME) which suppresses NK functions (e.g., cytotoxicity). This dysfunctional state that is accompanied by phenotypic changes such as upregulation of inhibitory receptors and downregulation of activating receptors, forms the basis of what many researchers have referred to as 'exhausted' NK cells. However, there is no consensus on whether these phenotypes are sufficient to define an exhausted state of the NK cell. While recent advances in checkpoint inhibition appear to show promise in early-stage pre-clinical studies, much remains to be fully explored and understood in the context of the TME. The TME is where the NK cells are subjected to interaction with various cell types and soluble factors, which could exert an inhibitory effect on NK cytotoxicity. In this review, we provide an overview of the general markers of NK cell exhaustion viz, the surface activating and inhibitory receptors. We also highlight the potential role of T-box transcription factors in characterising such a dysfunctional state and discuss the often-overlooked mechanism of cell cytoskeletal dynamics in regulating NK cell function. These aspects may further contribute to NK exhaustion or NK revival in cancer and may open new avenues to explore cancer treatment strategies.

Inflammasome-Induced Osmotic Pressure and the Mechanical Mechanisms Underlying Astrocytic Swelling and Membrane Blebbing in Pyroptosis.

Cell swelling and membrane blebbing are characteristic of pyroptosis. In the present study, we explored the role of intracellular tension activity in the deformation of pyroptotic astrocytes. Protein nanoparticle-induced osmotic pressure (PN-OP) was found to be involved in cell swelling and membrane blebbing in pyroptotic astrocytes, and was associated closely with inflammasome production and cytoskeleton depolymerization. However, accumulation of protein nanoparticles seemed not to be absolutely required for pyroptotic permeabilization in response to cytoskeleton depolymerization. Gasdermin D activation was observed to be involved in modification of typical pyroptotic features through inflammasome-induced OP upregulation and calcium increment. Blockage of nonselective ion pores can inhibit permeabilization, but not inflammasome production and ion influx in pyroptotic astrocytes. The results suggested that the inflammasomes, as protein nanoparticles, are involved in PN-OP upregulation and control the typical features of pyroptotic astrocytes.

Hypergravity-induced changes in actin response of breast cancer cells to natural killer cells.

Although immunotherapy holds promising cytotoxic activity against lymphoma or leukemia, the immunosuppressive mechanisms of solid tumors remain challenging. In this study, we developed and applied a hypergravity exposure system as a novel strategy to improve the responsiveness of breast cancer cells to natural killer (NK) cells for efficient immunotherapy. Following exposure to hypergravity, either in the presence or absence of NK cells, we investigated for changes in the cell cytoskeletal structure, which is related to the F-actin mediated immune evasion mechanism (referred to as "actin response") of cancer cells. Breast cancer cell line MDA-MB-231 cells were exposed thrice to a 20 min hypergravitational condition (10 × g), with a 20 min rest period between each exposure. The applied hypergravity induces changes in the intracellular cytoskeleton structure without decreasing the cell viability but increasing the cytotoxicity of MDA-MB-231 from 4 to 18% (4.5-fold) at a 3:1 ratio (NK-to-target). Analyses related to F-actin further demonstrate that the applied hypergravity results in rearrangement of the cytoskeleton, leading to inhibition of the actin response of MDA-MB-231. Taken together, our results suggest that the mechanical load increases through application of hypergravity, which potentially improves efficiency of cell-based immunotherapies by sensitizing tumors to immune cell-mediated cytotoxicity.

RhoGTPases and inflammasomes: Guardians of effector-triggered immunity.

Pathogens have evolved smart strategies to invade hosts and hijack their immune responses. One such strategy is the targeting of the host RhoGTPases by toxins or virulence factors to hijack the cytoskeleton dynamic and immune processes. In response to this microbial attack, the host has evolved an elegant strategy to monitor the function of virulence factors and toxins by sensing the abnormal activity of RhoGTPases. This innate immune strategy of sensing bacterial effector targeting RhoGTPase appears to be a bona fide example of effector-triggered immunity (ETI). Here, we review recently discovered mechanisms by which the host can sense the activity of these toxins through NOD and NOD-like receptors (NLRs).

CD317 mediates immunocytolysis resistance by RICH2/cytoskeleton-dependent membrane protection.

Immune evasion is a common hallmark of cancers. Immunotherapies that aim at restoring or increasing the immune response against cancers have revolutionized outcomes for patients, but the mechanisms of resistance remain poorly defined. Here, we report that CD317, a surface molecule with a unique topology that is double anchored into the membrane, protects tumor cells from immunocytolysis. CD317 knockdown in tumor cells renders more severe death in response to NK or chimeric antigen receptor-modified NK cells challenge. Such effects of CD317 silencing might be the results of increasing sensitivity of tumor cells to immune killing rather than strengthening immune response, since neither effector-target cell contact nor the activation of effector cells was affected, and the enhanced cytolysis was also not counteracted by the addition of recombinant CD317 proteins. Mechanistically, CD317 might endow tumor cells with more flexibility to modulate cytoskeleton through its association with RICH2, thereby protects membrane integrity against perforin and consequently promotes survival in response to immunocytolysis. These results reveal a new mechanism of immunocytolysis resistance and suggest CD317 as an attractive target which can be exploited for improving the efficacy of cancer immunotherapies.

Motility and Mechanical Properties of Dendritic Cells Deteriorated by Extracellular Acidosis.

Dendritic cells (DCs) are the most powerful antigen-presenting cells known to date and play an important role in initiating and amplifying both innate and adaptive immune responses. Extracellular acidosis is an important hallmark of a variety of inflammatory processes and solid tumors. However, few studies have focused on the effect of extracellular acidosis on DCs and their functions. Cellular mechanical properties reflect the relationship between cell structure and function, including cytoskeleton (especially F-actin organization), membrane negative charges, membrane fluidity, and osmotic fragility. The study investigated the effects of extracellular acidosis on the DCs functions from the perspective of cellular migration and mechanical properties. The results showed that migration ability, F-actin contents, and membrane negative charges of DCs were reduced by extracellular acidosis no matter whether LPS stimulated its maturation or not. And these functions could not return to normal after removing acidic microenvironment, which revealed that the function impairment induced by extracellular acidosis might be irreversible. In addition, the proliferation capacity of stimulated allogeneic T cells was impaired by extracellular acidosis. Our results suggest extracellular acidosis may play an immunosuppressive role in DCs-mediated immune process.

Regulation and Functions of ROP GTPases in Plant-Microbe Interactions.

Rho proteins of plants (ROPs) form a specific clade of Rho GTPases, which are involved in either plant immunity or susceptibility to diseases. They are intensively studied in grass host plants, in which ROPs are signaling hubs downstream of both cell surface immune receptor kinases and intracellular nucleotide-binding leucine-rich repeat receptors, which activate major branches of plant immune signaling. Additionally, invasive fungal pathogens may co-opt the function of ROPs for manipulation of the cytoskeleton, cell invasion and host cell developmental reprogramming, which promote pathogenic colonization. Strikingly, mammalian bacterial pathogens also initiate both effector-triggered susceptibility for cell invasion and effector-triggered immunity via Rho GTPases. In this review, we summarize central concepts of Rho signaling in disease and immunity of plants and briefly compare them to important findings in the mammalian research field. We focus on Rho activation, downstream signaling and cellular reorganization under control of Rho proteins involved in disease progression and pathogen resistance.

Implications of cellular metabolism for immune cell migration.

Cell migration is an essential, energetically demanding process in immunity. Immune cells navigate the body via chemokines and other immune mediators, which are altered under inflammatory conditions of injury or infection. Several factors determine the migratory abilities of different types of immune cells in diverse contexts, including the precise co-ordination of cytoskeletal remodelling, the expression of specific chemokine receptors and integrins, and environmental conditions. In this review, we present an overview of recent advances in our understanding of the relationship of each of these factors with cellular metabolism, with a focus on the spatial organization of glycolysis and mitochondria, reciprocal regulation of chemokine receptors and the influence of environmental changes.

Cytoskeleton stiffness regulates cellular senescence and innate immune response in Hutchinson-Gilford Progeria Syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is caused by the accumulation of mutant prelamin A (progerin) in the nuclear lamina, resulting in increased nuclear stiffness and abnormal nuclear architecture. Nuclear mechanics are tightly coupled to cytoskeletal mechanics via lamin A/C. However, the role of cytoskeletal/nuclear mechanical properties in mediating cellular senescence and the relationship between cytoskeletal stiffness, nuclear abnormalities, and senescent phenotypes remain largely unknown. Here, using muscle-derived mesenchymal stromal/stem cells (MSCs) from the Zmpste24-/- (Z24-/- ) mouse (a model for HGPS) and human HGPS fibroblasts, we investigated the mechanical mechanism of progerin-induced cellular senescence, involving the role and interaction of mechanical sensors RhoA and Sun1/2 in regulating F-actin cytoskeleton stiffness, nuclear blebbing, micronuclei formation, and the innate immune response. We observed that increased cytoskeletal stiffness and RhoA activation in progeria cells were directly coupled with increased nuclear blebbing, Sun2 expression, and micronuclei-induced cGAS-Sting activation, part of the innate immune response. Expression of constitutively active RhoA promoted, while the inhibition of RhoA/ROCK reduced cytoskeletal stiffness, Sun2 expression, the innate immune response, and cellular senescence. Silencing of Sun2 expression by siRNA also repressed RhoA activation, cytoskeletal stiffness and cellular senescence. Treatment of Zmpste24-/- mice with a RhoA inhibitor repressed cellular senescence and improved muscle regeneration. These results reveal novel mechanical roles and correlation of cytoskeletal/nuclear stiffness, RhoA, Sun2, and the innate immune response in promoting aging and cellular senescence in HGPS progeria.

Human NK Cell Cytoskeletal Dynamics and Cytotoxicity Are Regulated by LIM Kinase.

NK cells provide immune surveillance and host protection against viruses and tumors through their cytotoxic effector function. Cytoskeletal rearrangement is necessary for NK cell lytic granule trafficking and immune synapse formation to trigger apoptosis of targeted cells. LIM kinase (LIMK) regulates F-actin remodeling by phosphorylating cofilin to inhibit actin severing and depolymerization. In this study, in human NK cells, the glucocorticoid dexamethasone downregulated LIMK expression, F-actin accumulation at the immune synapse, lytic granule trafficking, and cytotoxicity. In contrast, the specialized proresolving mediator lipoxin A4 promoted NK cell LIMK expression, lytic granule polarization to the immune synapse and cytotoxicity. Using a LIMK inhibitor, we show that LIMK activity is necessary for NK cell cytotoxicity, including lipoxin A4's proresolving actions. Together, our findings identify LIMK as an important control mechanism for NK cell cytoskeletal rearrangement that is differentially regulated by glucocorticoids and specialized proresolving mediators to influence NK cell cytotoxicity.

Cytoskeletal regulation of dendritic cells: An intricate balance between migration and presentation for tumor therapy.

Dendritic cells (DCs) are the main players in many approaches for cancer therapy. The idea with DC tumor therapy is to promote activation of tumor infiltrating cytotoxic T cells that kill tumor cells. This requires that DCs take up tumor Ag and present peptides on MHC class I molecules in a process called cross-presentation. For this process to be efficient, DCs have to migrate to the tumor draining lymph node and there activate the machinery for cross-presentation. In this review, we will discuss recent progress in understanding the role of actin regulators for control of DC migration and Ag presentation. The potential to target actin regulators for better DC-based tumor therapy will also be discussed.

Molecular mechanisms of dendritic cell migration in immunity and cancer.

Dendritic cells (DCs) are a heterogeneous population of antigen-presenting cells that act to bridge innate and adaptive immunity. DCs are critical in mounting effective immune responses to tissue damage, pathogens and cancer. Immature DCs continuously sample tissues and engulf antigens via endocytic pathways such as phagocytosis or macropinocytosis, which result in DC activation. Activated DCs undergo a maturation process by downregulating endocytosis and upregulating surface proteins controlling migration to lymphoid tissues where DC-mediated antigen presentation initiates adaptive immune responses. To traffic to lymphoid tissues, DCs must adapt their motility mechanisms to migrate within a wide variety of tissue types and cross barriers to enter lymphatics. All steps of DC migration involve cell-cell or cell-substrate interactions. This review discusses DC migration mechanisms in immunity and cancer with a focus on the role of cytoskeletal processes and cell surface proteins, including integrins, lectins and tetraspanins. Understanding the adapting molecular mechanisms controlling DC migration in immunity provides the basis for therapeutic interventions to dampen immune activation in autoimmunity, or to improve anti-tumour immune responses.

Diacylglycerol kinase ζ promotes actin cytoskeleton remodeling and mechanical forces at the B cell immune synapse.

Diacylglycerol kinases (DGKs) limit antigen receptor signaling in immune cells by consuming the second messenger diacylglycerol (DAG) to generate phosphatidic acid (PA). Here, we showed that DGKζ promotes lymphocyte function-associated antigen 1 (LFA-1)-mediated adhesion and F-actin generation at the immune synapse of B cells with antigen-presenting cells (APCs), mostly in a PA-dependent manner. Measurement of single-cell mechanical force generation indicated that DGKζ-deficient B cells exerted lower forces at the immune synapse than did wild-type B cells. Nonmuscle myosin activation and translocation of the microtubule-organizing center (MTOC) to the immune synapse were also impaired in DGKζ-deficient B cells. These functional defects correlated with the decreased ability of B cells to present antigen and activate T cells in vitro. The in vivo germinal center response of DGKζ-deficient B cells was also reduced compared with that of wild-type B cells, indicating that loss of DGKζ in B cells impaired T cell help. Together, our data suggest that DGKζ shapes B cell responses by regulating actin remodeling, force generation, and antigen uptake-related events at the immune synapse. Hence, an appropriate balance in the amounts of DAG and PA is required for optimal B cell function.

NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin decondensation and nuclear envelope rupture.

Neutrophil extracellular traps (NETs) are web-like DNA structures decorated with histones and cytotoxic proteins that are released by activated neutrophils to trap and neutralize pathogens during the innate immune response, but also form in and exacerbate sterile inflammation. Peptidylarginine deiminase 4 (PAD4) citrullinates histones and is required for NET formation (NETosis) in mouse neutrophils. While the in vivo impact of NETs is accumulating, the cellular events driving NETosis and the role of PAD4 in these events are unclear. We performed high-resolution time-lapse microscopy of mouse and human neutrophils and differentiated HL-60 neutrophil-like cells (dHL-60) labeled with fluorescent markers of organelles and stimulated with bacterial toxins or Candida albicans to induce NETosis. Upon stimulation, cells exhibited rapid disassembly of the actin cytoskeleton, followed by shedding of plasma membrane microvesicles, disassembly and remodeling of the microtubule and vimentin cytoskeletons, ER vesiculation, chromatin decondensation and nuclear rounding, progressive plasma membrane and nuclear envelope (NE) permeabilization, nuclear lamin meshwork and then NE rupture to release DNA into the cytoplasm, and finally plasma membrane rupture and discharge of extracellular DNA. Inhibition of actin disassembly blocked NET release. Mouse and dHL-60 cells bearing genetic alteration of PAD4 showed that chromatin decondensation, lamin meshwork and NE rupture and extracellular DNA release required the enzymatic and nuclear localization activities of PAD4. Thus, NETosis proceeds by a stepwise sequence of cellular events culminating in the PAD4-mediated expulsion of DNA.

Moving towards a systems-based classification of innate immune-mediated diseases.

Autoinflammation as a distinct disease category was first reported in 1999 as a group of monogenic disorders characterized by recurrent episodes of systemic and organ-specific inflammation, known as periodic fever syndromes. Since this original description, the focus has shifted considerably to the inclusion of complex multifactorial conditions with an autoinflammatory basis. Furthermore, the boundaries of what are considered to be autoinflammatory disorders are constantly evolving and currently encompass elements of immunodeficiency and autoimmunity. Notable developments in the intervening 20 years include substantial progress in understanding how the different inflammasomes are activated, how infection is sensed by the innate immune system and how intracellular signalling systems are consequently activated and integrated with many different cellular functions in the autoinflammatory process. With these developments, the field of autoinflammation is moving from a gene-centric view of innate immune-mediated disease towards a systems-based concept, which describes how various convergent pathways, including pyrin and the actin cytoskeleton, protein misfolding and cellular stress, NF-κB dysregulation and interferon activation, contribute to the autoinflammatory process. The development and adoption of a systems-based concept of systemic autoinflammatory diseases is anticipated to have implications for the development of treatments that target specific components of the innate immune system.

Multiple actin networks coordinate mechanotransduction at the immunological synapse.

Activation of naive T cells by antigen-presenting cells (APCs) is an essential step in mounting an adaptive immune response. It is known that antigen recognition and T cell receptor (TCR) signaling depend on forces applied by the T cell actin cytoskeleton, but until recently, the underlying mechanisms have been poorly defined. Here, we review recent advances in the field, which show that specific actin-dependent structures contribute to the process in distinct ways. In essence, T cell priming involves a tug-of-war between the cytoskeletons of the T cell and the APC, where the actin cytoskeleton serves as a mechanical intermediate that integrates force-dependent signals. We consider each of the relevant actin-rich T cell structures separately and address how they work together at the topologically and temporally complex cell-cell interface. In addition, we address how this mechanobiology can be incorporated into canonical immunological models to improve how these models explain T cell sensitivity and antigenic specificity.

The Vibrio cholerae MARTX toxin silences the inflammatory response to cytoskeletal damage before inducing actin cytoskeleton collapse.

Multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are pore-forming bacterial toxins that translocate multiple functionally independent effector domains into a target eukaryotic cell. Vibrio cholerae colonizes intestinal epithelial cells (IECs) and uses a MARTX toxin with three effector domains-an actin cross-linking domain (ACD), a Rho inactivation domain (RID), and an α/ß hydrolase domain (ABH)-to suppress innate immunity and enhance colonization. We investigated whether these multiple catalytic enzymes delivered from a single toxin functioned in a coordinated manner to suppress intestinal innate immunity. Using cultured human IECs, we demonstrated that ACD-induced cytoskeletal collapse activated extracellular signal-regulated kinase, p38, and c-Jun amino-terminal kinase mitogen-activated protein kinase (MAPK) signaling to elicit a robust proinflammatory response characterized by the secretion of interleukin-8 (IL-8; also called CXCL8) and the expression of CXCL8, tumor necrosis factor (TNF), and other proinflammatory genes. However, RID and ABH, which are naturally delivered together with ACD, blocked MAPK activation through Rac1 and thus prevented ACD-induced inflammation. RID also abolished IL-8 secretion induced by heat-killed bacteria, TNF, or latrunculin A. Thus, MARTX toxins use enzymatic multifunctionality to silence the host response to bacterial factors and to the damage caused by the toxins. Furthermore, these data show how V. cholerae MARTX toxin suppresses intestinal inflammation and contributes to cholera being classically defined as a noninflammatory diarrheal disease.

Omega-3 polyunsaturated fatty acids impinge on CD4+ T cell motility and adipose tissue distribution via direct and lipid mediator-dependent effects.

AIMS: Adaptive immunity contributes to the pathogenesis of cardiovascular metabolic disorders (CVMD). The omega-3 polyunsaturated fatty acids (n-3PUFA) are beneficial for cardiovascular health, with potential to improve the dysregulated adaptive immune responses associated with metabolic imbalance. We aimed to explore the mechanisms through which n-3PUFA may alter T cell motility and tissue distribution to promote a less inflammatory environment and improve lymphocyte function in CVMD. METHODS AND RESULTS: Using mass spectrometry lipidomics, cellular, biochemical, and in vivo and ex vivo analyses, we investigated how eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the main n-3PUFA, modify the trafficking patterns of activated CD4+ T cells. In mice subjected to allogeneic immunization, a 3-week n-3PUFA-enriched diet reduced the number of effector memory CD4+ T cells found in adipose tissue, and changed the profiles of eicosanoids, octadecanoids, docosanoids, endocannabinoids, 2-monoacylglycerols, N-acyl ethanolamines, and ceramides, in plasma, lymphoid organs, and fat tissues. These bioactive lipids exhibited differing chemotactic properties when tested in chemotaxis assays with activated CD4+ T cells in vitro. Furthermore, CD4+ T cells treated with EPA and DHA showed a significant reduction in chemokinesis, as assessed by trans-endothelial migration assays, and, when implanted in recipient mice, demonstrated less efficient migration to the inflamed peritoneum. Finally, EPA and DHA treatments reduced the number of polarized CD4+ T cells in vitro, altered the phospholipid composition of membrane microdomains and decreased the activity of small Rho GTPases, Rhoα, and Rac1 instrumental in cytoskeletal dynamics. CONCLUSIONS: Our findings suggest that EPA and DHA affect the motility of CD4+ T cells and modify their ability to reach target tissues by interfering with the cytoskeletal rearrangements required for cell migration. This can explain, at least in part, the anti-inflammatory effects of n-3PUFA supporting their potential use in interventions aiming to address adipocyte low-grade inflammation associated with cardiovascular metabolic disease.

Modulation of Immune Responses by Particle Size and Shape.

The immune system has to cope with a wide range of irregularly shaped pathogens that can actively move (e.g., by flagella) and also dynamically remodel their shape (e.g., transition from yeast-shaped to hyphal fungi). The goal of this review is to draw general conclusions of how the size and geometry of a pathogen affect its uptake and processing by phagocytes of the immune system. We compared both theoretical and experimental studies with different cells, model particles, and pathogenic microbes (particularly fungi) showing that particle size, shape, rigidity, and surface roughness are important parameters for cellular uptake and subsequent immune responses, particularly inflammasome activation and T cell activation. Understanding how the physical properties of particles affect immune responses can aid the design of better vaccines.