Actin dynamics regulation by TTC7A/PI4KIIIα limits DNA damage and cell death under confinement.

BACKGROUND: The actin cytoskeleton has a crucial role in the maintenance of the immune homeostasis by controlling various cellular processes, including cell migration. Mutations in TTC7A have been described as the cause of a primary immunodeficiency associated to different degrees of gut involvement and alterations in the actin cytoskeleton dynamics. OBJECTIVES: This study investigates the impact of TTC7A deficiency in immune homeostasis. In particular, the role of the TTC7A/phosphatidylinositol 4 kinase type III α pathway in the control of leukocyte migration and actin dynamics. METHODS: Microfabricated devices were leveraged to study cell migration and actin dynamics of murine and patient-derived leukocytes under confinement at the single-cell level. RESULTS: We show that TTC7A-deficient lymphocytes exhibit an altered cell migration and reduced capacity to deform through narrow gaps. Mechanistically, TTC7A-deficient phenotype resulted from impaired phosphoinositide signaling, leading to the downregulation of the phosphoinositide 3-kinase/AKT/RHOA regulatory axis and imbalanced actin cytoskeleton dynamics. TTC7A-associated phenotype resulted in impaired cell motility, accumulation of DNA damage, and increased cell death in dense 3-dimensional gels in the presence of chemokines. CONCLUSIONS: These results highlight a novel role of TTC7A as a critical regulator of lymphocyte migration. Impairment of this cellular function is likely to contribute to the pathophysiology underlying progressive immunodeficiency in patients.

Insulin-regulated aminopeptidase contributes to setting the intensity of FcR-mediated inflammation.

The function of intracellular trafficking in immune-complex triggered inflammation remains poorly understood. Here, we investigated the role of Insulin-Regulated Amino Peptidase (IRAP)-positive endosomal compartments in Fc receptor (FcR)-induced inflammation. Less severe FcγR-triggered arthritis, active systemic anaphylaxis and FcεRI-triggered passive systemic anaphylaxis were observed in IRAP-deficient versus wild-type mice. In mast cells FcεRI stimulation induced rapid plasma membrane recruitment of IRAP-positive endosomes. IRAP-deficient cells exhibited reduced secretory responses, calcium signaling and activating SykY519/520 phosphorylation albeit receptor tyrosine phosphorylation on ß and γ subunits was not different. By contrast, in the absence of IRAP, SHP1-inactivating phosphorylation on Ser591 that controls Syk activity was decreased. Ex-vivo cell profiling after FcγR-triggered anaphylaxis confirmed decreased phosphorylation of both SykY519/520 and SHP-1S591 in IRAP-deficient neutrophils and monocytes. Thus, IRAP-positive endosomal compartments, in promoting inhibition of SHP-1 during FcR signaling, control the extent of phosphorylation events at the plasma membrane and contribute to setting the intensity of immune-complex triggered inflammatory diseases.

Rab44 regulates murine mast cell-driven anaphylaxis through kinesin-1-dependent secretory granule translocation.

BACKGROUND: Mast cells (MCs) are key effectors of the allergic response. Following the cross-linking of IgE receptors (FcεRIs), they release crucial inflammatory mediators through degranulation. Although degranulation depends critically on secretory granule (SG) trafficking toward the plasma membrane, the molecular machinery underlying this transport has not been fully characterized. OBJECTIVES: This study analyzed the function of Rab44, a large, atypical Rab guanosine triphosphatase highly expressed in MC, in the MC degranulation process. METHODS: Murine knockout (KO) mouse models (KORab44 and DKOKif5b/Rab44) were used to perform passive cutaneous anaphylaxis experiments and analyze granule translocation in bone marrow-derived MCs during degranulation. RESULTS: This study demonstrate that mice lacking Rab44 (KORab44) in their bone marrow-derived MCs are impaired in their ability to translocate and degranulate SGs at the plasma membrane on FcεRI stimulation. Accordingly, KORab44 mice were less sensitive to IgE-mediated passive cutaneous anaphylaxis in vivo. A lack of Rab44 did not impair early FcεRI-stimulated signaling pathways, microtubule reorganization, lipid mediator release, or cytokine secretion. Mechanistically, Rab44 appears to interact with and function as part of the previously described kinesin-1-dependent transport pathway. CONCLUSIONS: These results highlight a novel role of Rab44 as a regulator of SG transport during degranulation and anaphylaxis acting through the kinesin-1-dependent microtubule transport machinery. Rab44 can thus be considered a potential target for modulating MC degranulation and inhibiting IgE-mediated allergic reactions.

Cytoskeletal Transport, Reorganization, and Fusion Regulation in Mast Cell-Stimulus Secretion Coupling.

Mast cells are well known for their role in allergies and many chronic inflammatory diseases. They release upon stimulation, e.g., via the IgE receptor, numerous bioactive compounds from cytoplasmic secretory granules. The regulation of granule secretion and its interaction with the cytoskeleton and transport mechanisms has only recently begun to be understood. These studies have provided new insight into the interaction between the secretory machinery and cytoskeletal elements in the regulation of the degranulation process. They suggest a tight coupling of these two systems, implying a series of specific signaling effectors and adaptor molecules. Here we review recent knowledge describing the signaling events regulating cytoskeletal reorganization and secretory granule transport machinery in conjunction with the membrane fusion machinery that occur during mast cell degranulation. The new insight into MC biology offers novel strategies to treat human allergic and inflammatory diseases targeting the late steps that affect harmful release from granular stores leaving regulatory cytokine secretion intact.

Kinesin-1 regulates antigen cross-presentation through the scission of tubulations from early endosomes in dendritic cells.

Dendritic cells (DCs) constitute a specialized population of immune cells that present exogenous antigen (Ag) on major histocompatibility complex (MHC) class I molecules to initiate CD8 + T cell responses against pathogens and tumours. Although cross-presentation depends critically on the trafficking of Ag-containing intracellular vesicular compartments, the molecular machinery that regulates vesicular transport is incompletely understood. Here, we demonstrate that mice lacking Kif5b (the heavy chain of kinesin-1) in their DCs exhibit a major impairment in cross-presentation and thus a poor in vivo anti-tumour response. We find that kinesin-1 critically regulates antigen cross-presentation in DCs, by controlling Ag degradation, the endosomal pH, and MHC-I recycling. Mechanistically, kinesin-1 appears to regulate early endosome maturation by allowing the scission of endosomal tubulations. Our results highlight kinesin-1's role as a molecular checkpoint that modulates the balance between antigen degradation and cross-presentation.

Tomosyn functions as a PKCδ-regulated fusion clamp in mast cell degranulation.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family proteins mediate membrane fusion critical for vesicular transport and cellular secretion. Mast cells rely on SNARE-mediated membrane fusion for degranulation stimulated by crosslinking of immunoglobulin E (IgE) bound to the Fcε receptor (FcεRI). We investigated the mechanisms downstream of receptor activation that control degranulation. We found that the SNARE binding protein tomosyn-1 (also known as STXBP5) inhibited FcεRI-stimulated degranulation of mast cells. After mast cell activation, tomosyn-1 was phosphorylated on serine and threonine residues, dissociated from the SNARE protein syntaxin 4 (STX4), and associated with STX3. We identified PKCδ as the major kinase required for tomosyn-1 threonine phosphorylation and for regulation of the interaction with STXs. Incubation with high IgE concentrations increased tomosyn-1 abundance in cultured mast cells. Similarly, in basophils from allergic patients with high amounts of serum IgE, the abundance of tomosyn-1 was increased as compared to that in patients with normal IgE concentrations. Our findings identified tomosyn-1 as an inhibitor of mast cell degranulation that required PKCδ to switch its interaction with STX partners during fusion. We suggest that the IgE-mediated increase in tomosyn-1 abundance in allergic patients may represent a counterregulatory mechanism to limit disease development.

Kinesin-1 Is a New Actor Involved in Platelet Secretion and Thrombus Stability.

OBJECTIVE: Platelet secretion is crucial for many physiological platelet responses. Even though several regulators of the fusion machinery for secretory granule exocytosis have been identified in platelets, the underlying mechanisms are not yet fully characterized. APPROACH AND RESULTS: By studying a mouse model (cKO [conditional knockout]Kif5b) lacking Kif5b (kinesin-1 heavy chain) in its megakaryocytes and platelets, we evidenced unstable hemostasis characterized by an increase of blood loss associated to a marked tendency to rebleed in a tail-clip assay and thrombus instability in an in vivo thrombosis model. This instability was confirmed in vitro in a whole-blood perfusion assay under blood flow conditions. Aggregations induced by thrombin and collagen were also impaired in cKOKif5b platelets. Furthermore, P-selectin exposure, PF4 (platelet factor 4) secretion, and ATP release after thrombin stimulation were impaired in cKOKif5b platelets, highlighting the role of kinesin-1 in α-granule and dense granule secretion. Importantly, exogenous ADP rescued normal thrombin induced-aggregation in cKOKif5b platelets, which indicates that impaired aggregation was because of defective release of ADP and dense granules. Last, we demonstrated that kinesin-1 interacts with the molecular machinery comprising the granule-associated Rab27 (Ras-related protein Rab-27) protein and the Slp4 (synaptotagmin-like protein 4/SYTL4) adaptor protein. CONCLUSIONS: Our results indicate that a kinesin-1-dependent process plays a role for platelet function by acting into the mechanism underlying α-granule and dense granule secretion.

A RAB27A duplication in several cases of Griscelli syndrome type 2: An explanation for cases lacking a genetic diagnosis.

Griscelli syndrome type 2 (GS2) is a rare and often fatal autosomal recessive, hyperinflammatory disorder. It is associated with hypopigmentation of the skin and the hair, resulting in the characteristic pigment accumulation and clumping in the hair shaft. Loss-of-function mutations in RAB27A, resulting from point mutations, short indel, or large deletions, account for all the cases reported to date. However, several GS2 cases originating from Saudi Arabia lack a genetic diagnosis. Here, we report on a new RAB27A genetic anomaly observed in seven Saudi Arabia families that had remained negative after extensive molecular genomic DNA testing. Linkage analysis and targeted sequencing of the RAB27A genomic region in several of these patients led to the identification of a common homozygous tandem duplication of 38 kb affecting exon 2-5 and resulting in a premature stop codon. The pathogenic effect of this duplication was confirmed by a cDNA analysis and functional assays. The identification of microhomology flanking the breakpoint site suggests a possible underlying mechanism.

Kinesin-1 controls mast cell degranulation and anaphylaxis through PI3K-dependent recruitment to the granular Slp3/Rab27b complex.

Cross-linking of mast cell (MC) IgE receptors (FcεRI) triggers degranulation of secretory granules (SGs) and the release of many allergic and inflammatory mediators. Although degranulation depends crucially on microtubule dynamics, the molecular machinery that couples SGs to microtubule-dependent transport is poorly understood. In this study, we demonstrate that mice lacking Kif5b (the heavy chain of kinesin-1) in hematopoietic cells are less sensitive to IgE-mediated, passive, systemic anaphylaxis. After IgE-induced stimulation, bone marrow-derived MCs from Kif5b knockout mice exhibited a marked reduction in SG translocation toward the secretion site. In contrast, a lack of Kif5b did not affect cytokine secretion, early FcεRI-initiated signaling pathways, or microtubule reorganization upon FcεRI stimulation. We identified Slp3 as the critical effector linking kinesin-1 to Rab27b-associated SGs. Kinesin-1 recruitment to the Slp3/Rab27b effector complex was independent of microtubule reorganization but occurred only upon stimulation requiring phosphatidylinositol 3-kinase (PI3K) activity. Our findings demonstrate that PI3K-dependent formation of a kinesin-1/Slp3/Rab27b complex is critical for the microtubule-dependent movement of SGs required for MC degranulation.

Polygenic mutations in the cytotoxicity pathway increase susceptibility to develop HLH immunopathology in mice.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory disease. Inherited forms of HLH are caused by biallelic mutations in several effectors of granule-dependent lymphocyte-mediated cytotoxicity. A small proportion of patients with a so-called "secondary" form of HLH, which develops in the aftermath of infection, autoimmunity, or cancer, carry a monoallelic mutation in one or more HLH-associated genes. Although this observation suggests that HLH may have a polygenic mode of inheritance, the latter is very difficult to prove in humans. In order to determine whether the accumulation of partial genetic defects in lymphocyte-mediated cytotoxicity can contribute to the development of HLH, we generated mice that were doubly or triply heterozygous for mutations in HLH-associated genes, those coding for perforin, Rab27a, and syntaxin-11. We found that the accumulation of monoallelic mutations did indeed increase the risk of developing HLH immunopathology after lymphocytic choriomeningitis virus infection. In mechanistic terms, the accumulation of heterozygous mutations in the two degranulation genes Rab27a and syntaxin-11, impaired the dynamics and secretion of cytotoxic granules at the immune synapse of T lymphocytes. In addition, the accumulation of heterozygous mutations within the three genes impaired natural killer lymphocyte cytotoxicity in vivo. The genetic defects can be ranked in terms of the severity of the resulting HLH manifestations. Our results form the basis of a polygenic model of the occurrence of secondary HLH.

HDAC6 regulates the dynamics of lytic granules in cytotoxic T lymphocytes.

HDAC6 is a tubulin deacetylase involved in many cellular functions related to cytoskeleton dynamics, including cell migration and autophagy. In addition, HDAC6 affects antigen-dependent CD4(+)T cell activation. In this study, we show that HDAC6 contributes to the cytotoxic function of CD8(+)T cells. Immunization studies revealed defective cytotoxic activity in vivo in the absence of HDAC6. Adoptive transfer of wild-type or Hdac6(-/-)CD8(+)T cells to Rag1(-/-)mice demonstrated specific impairment in CD8(+)T cell responses against vaccinia infection. Mechanistically, HDAC6-deficient cytotoxic T lymphocytes (CTLs) showed defective in vitro cytolytic activity related to altered dynamics of lytic granules, inhibited kinesin-1-dynactin-mediated terminal transport of lytic granules to the immune synapse and deficient exocytosis, but not to target cell recognition, T cell receptor (TCR) activation or interferon (IFN)γ production. Our results establish HDAC6 as an effector of the immune cytotoxic response that acts by affecting the dynamics, transport and secretion of lytic granules by CTLs.

LYST controls the biogenesis of the endosomal compartment required for secretory lysosome function.

Chediak-Higashi syndrome (CHS) is caused by mutations in the gene encoding LYST protein, the function of which remains poorly understood. Prominent features of CHS include defective secretory lysosome exocytosis and the presence of enlarged, lysosome-like organelles in several cell types. In order to get further insight into the role of LYST in the biogenesis and exocytosis of cytotoxic granules, we analyzed cytotoxic T lymphocytes (CTLs) from patients with CHS. Using confocal microscopy and correlative light electron microscopy, we showed that the enlarged organelle in CTLs is a hybrid compartment that contains proteins components from recycling-late endosomes and lysosomes. Enlargement of cytotoxic granules results from the progressive clustering and then fusion of normal-sized endolysosomal organelles. At the immunological synapse (IS) in CHS CTLs, cytotoxic granules have limited motility and appear docked while nevertheless unable to degranulate. By increasing the expression of effectors of lytic granule exocytosis, such as Munc13-4, Rab27a and Slp3, in CHS CTLs, we were able to restore the dynamics and the secretory ability of cytotoxic granules at the IS. Our results indicate that LYST is involved in the trafficking of the effectors involved in exocytosis required for the terminal maturation of perforin-containing vesicles into secretory cytotoxic granules.

A novel immunoregulatory role for NK-cell cytotoxicity in protection from HLH-like immunopathology in mice.

The impairment of cytotoxic activity of lymphocytes disturbs immune surveillance and leads to the development of hemophagocytic lymphohistiocytic syndrome (HLH). Although cytotoxic T lymphocyte (CTL) control of HLH development is well documented, the role for natural killer (NK)-cell effector functions in the pathogenesis of this immune disorder remains unclear. In this study, we specifically targeted a defect in cytotoxicity to either CTL or NK cells in mice so as to dissect the contribution of these lymphocyte subsets to HLH-like disease severity after lymphocytic choriomeningitis virus (LCMV) infection. We found that NK-cell cytotoxicity was sufficient to protect mice from the fatal outcome that characterizes HLH-like disease and was also sufficient to reduce HLH-like manifestations. Mechanistically, NK-cell cytotoxicity reduced tissue infiltration by inflammatory macrophages and downmodulated LCMV-specific T-cell responses by limiting hyperactivation of CTL. Interestingly, the critical protective effect of NK cells on HLH was independent of interferon-γ secretion and changes in viral load. Therefore our findings identify a crucial role of NK-cell cytotoxicity in limiting HLH-like immunopathology, highlighting the important role of NK cytotoxic activity in immune homeostasis.

Distinct severity of HLH in both human and murine mutants with complete loss of cytotoxic effector PRF1, RAB27A, and STX11.

Inherited defects of granule-dependent cytotoxicity led to the life-threatening immune disorder hemophagocytic lymphohistiocytosis (HLH), characterized by uncontrolled CD8 T-cell and macrophage activation. In a cohort of HLH patients with genetic abnormalities expected to result in the complete absence of perforin, Rab27a, or syntaxin-11, we found that disease severity as determined by age at HLH onset differed significantly, with a severity gradient from perforin (early onset) > Rab27a > syntaxin-11 (late onset). In parallel, we have generated a syntaxin-11-deficient (Stx11(-/-)) murine model that faithfully reproduced the manifestations of HLH after lymphocytic choriomeningitis virus (LCMV) infection. Stx11(-/-) murine lymphocytes exhibited a degranulation defect that could be rescued by expression of human syntaxin-11 but not expression of a C-terminal-truncated mutant. Comparison of the characteristics of LCMV infection-induced HLH in the murine counterparts of the 3 human conditions revealed a similar gradient in the phenotypic severity of HLH manifestations. Strikingly, the severity of HLH was not correlated with the LCMV load and not fully with differences in the intensity of cytotoxic activity. The capacity of antigen presentation differed in vivo between Rab27a- and Syntaxin-11-deficient mutants. Our data indicate that cytotoxic effectors may have other immune-regulatory roles in addition to their role in controlling viral replication.

Terminal transport of lytic granules to the immune synapse is mediated by the kinesin-1/Slp3/Rab27a complex.

Cytotoxic T lymphocytes kill target cells via the polarized secretion of cytotoxic granules at the immune synapse. The lytic granules are initially recruited around the polarized microtubule-organizing center. In a dynein-dependent transport process, the granules move along microtubules toward the microtubule-organizing center in the minus-end direction. Here, we found that a kinesin-1-dependent process is required for terminal transport and secretion of polarized lytic granule to the immune synapse. We show that synaptotagmin-like protein 3 (Slp3) is an effector of Rab27a in cytotoxic T lymphocytes and interacts with kinesin-1 through the tetratricopeptide repeat of the kinesin-1 light chain. Inhibition of the Rab27a/Slp3/kinesin-1 transport complex impairs lytic granule secretion. Our data provide further molecular insights into the key functional and regulatory mechanisms underlying the terminal transport of cytotoxic granules and the latter's secretion at the immune synapse.

Inherited defects causing hemophagocytic lymphohistiocytic syndrome.

Hemophagocytic lymphohistiocytosis (HLH) manifests as the uncontrolled activation of T lymphocytes and macrophages infiltrating multiple organs. Molecular studies of individuals with HLH have demonstrated in most of these conditions a critical role of granule-dependent cytotoxic activity in the regulation of lymphocyte homeostasis, and have allowed the characterization of key effectors regulating cytotoxic granule release. The cytolytic process may now be considered a multistep process, including cell activation; the polarization of cytotoxic granules toward the conjugated target cell; the tethering, priming, and fusion of the cytotoxic granules with the plasma membrane; and the release of their contents (perforin and granzymes) into the intercellular cleft, leading to target cell death. Cytolytic cells have a second effector function involving the production of cytokines, principally γ-interferon, which is secreted independently of the exocytosis cytotoxic granule pathway. An analysis of the mechanisms underlying HLH has identified γ-interferon as a key cytokine inducing uncontrolled macrophage activation, and thus represents a potential therapeutic target.

Premature birth, respiratory distress, intracerebral hemorrhage, and silvery-gray hair: differential diagnosis of the 3 types of Griscelli syndrome.

SUMMARY: A preterm neonate, born to consanguineous parents, presented with respiratory distress, intracerebral hemorrhage, and a silvery-gray sheen of the hair and eyelashes. Griscelli syndrome (GS) type 3 was diagnosed after the detection of a novel homozygous mutation of the melanophilin gene. Thus, only the hypopigmentation, but not the patient's other clinical features, were attributable to this form of GS. Differential diagnosis of the various forms of GS must be performed as early as possible as GS2 is associated with a life threatening but curable immune disorder.

Molecular mechanisms of biogenesis and exocytosis of cytotoxic granules.

Cytotoxic T cells and natural killer cells are crucial for immune surveillance against virus-infected cells and tumour cells. Molecular studies of individuals with inherited defects that impair lymphocyte cytotoxic function have also highlighted the importance of cytotoxicity in the regulation and termination of immune responses. As discussed in this Review, characterization of these defects has contributed to our understanding of the key steps that are required for the maturation of cytotoxic granules and the secretion of their contents at the immunological synapse during target cell killing. This has revealed a marked similarity between cytotoxic granule exocytosis at the immunological synapse and synaptic vesicle exocytosis at the neurological synapse. We explore the possibility that comparison of these two kinetically and spatially regulated secretory pathways will provide clues to uncover additional effectors that regulate the cytotoxic function of lymphocytes.

Inherited defects in lymphocyte cytotoxic activity.

The granule-dependent cytotoxic activity of lymphocytes plays a critical role in the defense against virally infected cells and tumor cells. The importance of this cytotoxic pathway in immune regulation is evidenced by the severe and often fatal condition, known as hemophagocytic lymphohistiocytic syndrome (HLH) that occurs in mice and humans with genetically determined impaired lymphocyte cytotoxic function. HLH manifests as the occurrence of uncontrolled activation of T lymphocytes and macrophages infiltrating multiple organs. In this review, we focus on recent advances in the characterization of effectors regulating the release of cytotoxic granules, and on the role of this cytotoxic pathway in lymphocyte homeostasis and immune surveillance. Analysis of the mechanisms leading to the occurrence of hemophagocytic syndrome designates gamma-interferon as an attractive therapeutic target to downregulate uncontrolled macrophage activation, which sustains clinical and biological features of HLH.