Disruption of Coronin 1 Signaling in T Cells Promotes Allograft Tolerance while Maintaining Anti-Pathogen Immunity.

The ability of the immune system to discriminate self from non-self is essential for eradicating microbial pathogens but is also responsible for allograft rejection. Whether it is possible to selectively suppress alloresponses while maintaining anti-pathogen immunity remains unknown. We found that mice deficient in coronin 1, a regulator of naive T cell homeostasis, fully retained allografts while maintaining T cell-specific responses against microbial pathogens. Mechanistically, coronin 1-deficiency increased cyclic adenosine monophosphate (cAMP) concentrations to suppress allo-specific T cell responses. Costimulation induced on microbe-infected antigen presenting cells was able to overcome cAMP-mediated immunosuppression to maintain anti-pathogen immunity. In vivo pharmacological modulation of this pathway or a prior transfer of coronin 1-deficient T cells actively suppressed allograft rejection. These results define a coronin 1-dependent regulatory axis in T cells important for allograft rejection and suggest that modulation of this pathway may be a promising approach to achieve long-term acceptance of mismatched allografts.

Regulation of T cell survival through coronin-1-mediated generation of inositol-1,4,5-trisphosphate and calcium mobilization after T cell receptor triggering.

T cell homeostasis is essential for the functioning of the vertebrate immune system, but the intracellular signals required for T cell homeostasis are largely unknown. We here report that the WD-repeat protein family member coronin-1, encoded by the gene Coro1a, is essential in the mouse for T cell survival through its promotion of Ca2+ mobilization from intracellular stores. Upon T cell receptor triggering, coronin-1 was essential for the generation of inositol-1,4,5-trisphosphate from phosphatidylinositol-4,5-bisphosphate. The absence of coronin-1, although it did not affect T cell development, resulted in a profound defect in Ca2+ mobilization, interleukin-2 production, T cell proliferation and T cell survival. We conclude that coronin-1, through activation of Ca2+ release from intracellular stores, is an essential regulator of peripheral lymphocyte survival.

Interactome and F-Actin Interaction Analysis of Dictyostelium discoideum Coronin A.

Coronin proteins are evolutionary conserved WD repeat containing proteins that have been proposed to carry out different functions. In Dictyostelium, the short coronin isoform, coronin A, has been implicated in cytoskeletal reorganization, chemotaxis, phagocytosis and the initiation of multicellular development. Generally thought of as modulators of F-actin, coronin A and its mammalian homologs have also been shown to mediate cellular processes in an F-actin-independent manner. Therefore, it remains unclear whether or not coronin A carries out its functions through its capacity to interact with F-actin. Moreover, the interacting partners of coronin A are not known. Here, we analyzed the interactome of coronin A as well as its interaction with F-actin within cells and in vitro. Interactome analysis showed the association with a diverse set of interaction partners, including fimbrin, talin and myosin subunits, with only a transient interaction with the minor actin10 isoform, but not the major form of actin, actin8, which was consistent with the absence of a coronin A-actin interaction as analyzed by co-sedimentation from cells and lysates. In vitro, however, purified coronin A co-precipitated with rabbit muscle F-actin in a coiled-coil-dependent manner. Our results suggest that an in vitro interaction of coronin A and rabbit muscle actin may not reflect the cellular interaction state of coronin A with actin, and that coronin A interacts with diverse proteins in a time-dependent manner.

Coronin 1 Is Required for Integrin ß2 Translocation in Platelets.

Remodeling of the actin cytoskeleton is one of the critical events that allows platelets to undergo morphological and functional changes in response to receptor-mediated signaling cascades. Coronins are a family of evolutionarily conserved proteins implicated in the regulation of the actin cytoskeleton, represented by the abundant coronins 1, 2, and 3 and the less abundant coronin 7 in platelets, but their functions in these cells are poorly understood. A recent report revealed impaired agonist-induced actin polymerization and cofilin phosphoregulation and altered thrombus formation in vivo as salient phenotypes in the absence of an overt hemostasis defect in vivo in a knockout mouse model of coronin 1. Here we show that the absence of coronin 1 is associated with impaired translocation of integrin ß2 to the platelet surface upon stimulation with thrombin while morphological and functional alterations, including defects in Arp2/3 complex localization and cAMP-dependent signaling, are absent. Our results suggest a large extent of functional overlap among coronins 1, 2, and 3 in platelets, while aspects like integrin ß2 translocation are specifically or predominantly dependent on coronin 1.

A Coronin 1-Dependent Decision Switch in Juvenile Mice Determines the Population of the Peripheral Naive T Cell Compartment.

Following thymic maturation, T cells egress as recent thymic emigrants to peripheral lymphoid organs where they undergo an additional maturation step to mature naive T cells that circulate through secondary lymphoid organs ready to be activated upon pathogenic challenges. Thymic maturation and peripheral T cell survival depend on several signaling cascades, but whether a dedicated mechanism exists that exclusively regulates homeostasis of mature naive T cells without affecting thymocytes and/or recent thymic emigrants remains unknown. In this article, we provide evidence for a specific and exclusive role of the WD repeat containing protein coronin 1 in the maintenance of naive T cells in peripheral lymphoid organs. We show that coronin 1 is dispensable for thymocyte survival and development, egress from the thymus, and survival of recent thymic emigrants. Importantly, coronin 1-deficient mice possessed comparable levels of peripheral T cells within the first 2 wk after birth but failed to populate the peripheral T cell compartment at later stages. Furthermore, dendritic cell- and IL-2/7-dependent T cell survival was found to be independent of coronin 1. Together, these results suggest the existence of a hitherto unrecognized coronin 1-dependent decision switch early during life that is responsible for peripheral naive T cell survival and homeostasis.

Coronin 1 regulates cognition and behavior through modulation of cAMP/protein kinase A signaling.

Cognitive and behavioral disorders are thought to be a result of neuronal dysfunction, but the underlying molecular defects remain largely unknown. An important signaling pathway involved in the regulation of neuronal function is the cyclic AMP/Protein kinase A pathway. We here show an essential role for coronin 1, which is encoded in a genomic region associated with neurobehavioral dysfunction, in the modulation of cyclic AMP/PKA signaling. We found that coronin 1 is specifically expressed in excitatory but not inhibitory neurons and that coronin 1 deficiency results in loss of excitatory synapses and severe neurobehavioral disabilities, including reduced anxiety, social deficits, increased aggression, and learning defects. Electrophysiological analysis of excitatory synaptic transmission in amygdala revealed that coronin 1 was essential for cyclic-AMP-protein kinase A-dependent presynaptic plasticity. We further show that upon cell surface stimulation, coronin 1 interacted with the G protein subtype Gαs to stimulate the cAMP/PKA pathway. The absence of coronin 1 or expression of coronin 1 mutants unable to interact with Gαs resulted in a marked reduction in cAMP signaling. Strikingly, synaptic plasticity and behavioral defects of coronin 1-deficient mice were restored by in vivo infusion of a membrane-permeable cAMP analogue. Together these results identify coronin 1 as being important for cognition and behavior through its activity in promoting cAMP/PKA-dependent synaptic plasticity and may open novel avenues for the dissection of signal transduction pathways involved in neurobehavioral processes.

Activated group 3 innate lymphoid cells promote T-cell-mediated immune responses.

Group 3 innate lymphoid cells (ILC3s) have emerged as important cellular players in tissue repair and innate immunity. Whether these cells meaningfully regulate adaptive immune responses upon activation has yet to be explored. Here we show that upon IL-1ß stimulation, peripheral ILC3s become activated, secrete cytokines, up-regulate surface MHC class II molecules, and express costimulatory molecules. ILC3s can take up latex beads, process protein antigen, and consequently prime CD4(+) T-cell responses in vitro. The cognate interaction of ILC3s and CD4(+) T cells leads to T-cell proliferation both in vitro and in vivo, whereas its disruption impairs specific T-cell and T-dependent B-cell responses in vivo. In addition, the ILC3-CD4(+) T-cell interaction is bidirectional and leads to the activation of ILC3s. Taken together, our data reveal a novel activation-dependent function of peripheral ILC3s in eliciting cognate CD4(+) T-cell immune responses.

Inflammatory stimuli reprogram macrophage phagocytosis to macropinocytosis for the rapid elimination of pathogens.

Following an infectious challenge, macrophages have to be activated in order to allow efficient clearance of infectious pathogens, but how macrophage activation is coupled to increased clearance remains largely unknown. We here describe that inflammatory stimuli induced the reprogramming of the macrophage endocytic machinery from receptor-mediated phagocytosis to macropinocytosis, allowing the rapid transfer of internalized cargo to lysosomes in a receptor-independent manner. Reprogramming occurred through protein kinase C-mediated phosphorylation of the macrophage protein coronin 1, thereby activating phosphoinositol (PI)-3-kinase activity necessary for macropinocytic uptake. Expression of a phosphomimetic form of coronin 1 was sufficient to induce PI3-kinase activation and macropinocytosis even in the absence of inflammatory stimuli. Together these results suggest a hitherto unknown mechanism to regulate the internalization and degradation of infectious material during inflammation.

Analysis of SecA2-dependent substrates in Mycobacterium marinum identifies protein kinase G (PknG) as a virulence effector.

The pathogenicity of mycobacteria is closely associated with their ability to export virulence factors. For this purpose, mycobacteria possess different protein secretion systems, including the accessory Sec translocation pathway, SecA2. Although this pathway is associated with intracellular survival and virulence, the SecA2-dependent effector proteins remain largely undefined. In this work, we studied a Mycobacterium marinum secA2 mutant with an impaired capacity to initiate granuloma formation in zebrafish embryos. By comparing the proteomic profile of cell envelope fractions from the secA2 mutant with wild type M. marinum, we identified putative SecA2-dependent substrates. Immunoblotting procedures confirmed SecA2-dependent membrane localization for several of these proteins, including the virulence factor protein kinase G (PknG). Interestingly, phenotypical defects of the secA2 mutant are similar to those described for ΔpknG, including phagosomal maturation. Overexpression of PknG in the secA2 mutant restored its localization to the cell envelope. Importantly, PknG-overexpression also partially restored the virulence of the secA2 mutant, as indicated by enhanced infectivity in zebrafish embryos and restored inhibition of phagosomal maturation. These results suggest that SecA2-dependent membrane localization of PknG is an important determinant for M. marinum virulence.

Surviving the macrophage: tools and tricks employed by Mycobacterium tuberculosis.

Mycobacterium tuberculosis has evolved to withstand one of the most inhospitable cells within the human body, namely the macrophage, a cell that is normally geared toward the destruction of any invading microbe. How M. tuberculosis achieves this is still incompletely understood; however, a number of mechanisms are now known that provide advantages to M. tuberculosis for its survival and proliferation inside the macrophage. While some of these mechanisms are mediated by factors released by M. tuberculosis, others rely on host components that are being hijacked to benefit survival of M. tuberculosis within the macrophage as well to avoid the generation of an effective immune response. Here, we describe several of these mechanisms, also pointing out the potential usage of this knowledge toward the development of novel strategies to treat tuberculosis. Furthermore, we attempt to put the 'macrophage niche' into context with other intracellular pathogens and discuss some of the generalities as well as specializations that M. tuberculosis employs to survive.

Coronin 1-dependent cell density sensing and regulation of the peripheral T cell population size.

The establishment and maintenance of peripheral T cells is important to ensure appropriate immunity. In mammals, T cells are produced in the thymus before seeding the periphery early in life, and thereafter progressive thymus involution impairs new T cell production. Yet, peripheral T cells are maintained lifelong at approximately similar cell numbers. The question thus arises: what are the mechanisms that enable the maintenance of the appropriate number of circulating T cells, ensuring that T cell numbers are neither too low nor too high? Here, we highlight recent research suggesting a key role for coronin 1, a member of the evolutionarily conserved family of coronin proteins, in both allowing T cells to reach as well as maintain their appropriate cell population size. This cell population size controlling pathway was found to be conserved in amoeba, mice and human. We propose that coronin 1 is an integral part of a cell-intrinsic pathway that couples cell density information with prosurvival signalling thereby regulating the appropriate number of peripheral T cells.

Migration and homeostasis of naive T cells depends on coronin 1-mediated prosurvival signals and not on coronin 1-dependent filamentous actin modulation.

Coronins are WD repeat-containing proteins highly conserved in the eukaryotic kingdom implicated in the regulation of F-actin. Mammalian coronin 1, one of the most conserved isoforms expressed in leukocytes, regulates survival of T cells, which has been suggested to be due to its role in preventing F-actin-induced apoptosis. In this study, we come to a different conclusion. We show that coronin 1 does not modulate F-actin and that induction of F-actin failed to induce apoptosis. Instead, coronin 1 was required for providing prosurvival signals, in the absence of which T cells rapidly underwent apoptosis. These results argue against a role for coronin 1 in F-actin-mediated T cell apoptosis and establish coronin 1 as an essential regulator of the balance between prosurvival and proapoptotic signals in naive T cells.

Coronin 1-mediated naive T cell survival is essential for the development of autoimmune encephalomyelitis.

Autoimmune encephalomyelitis is a disease of the CNS that can develop when an initial peripheral inflammatory stimulus is followed by infiltration and reactivation of T lymphocytes in the CNS. We report a crucial role for coronin 1, which is essential for maintenance of the naive T cell pool, for the development of murine experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. In the absence of coronin 1, immunization with myelin oligoglycoprotein (MOG(35-55)) peptide largely failed to induce EAE symptoms, despite normal mobilization of leukocyte subsets in the blood, as well as effector cytokine expression comparable with wild-type T cells on polyclonal stimulation. Susceptibility of coronin 1-deficient mice to EAE induction was restored by transfer of wild-type CD4(+) T cells, suggesting that the observed resistance of coronin 1-deficient mice to EAE development is T cell intrinsic. Importantly, although coronin 1-deficient regulatory T cells (Tregs) showed a suppressor activity comparable with wild-type Tregs, Treg depletion failed to restore EAE development in coronin 1-deficient animals. These results suggest a hitherto unrecognized role of naive T cells in the development of autoimmune encephalomyelitis and reveal coronin 1 as a crucial modulator of EAE induction.

A novel approach to single-cell analysis reveals intrinsic differences in immune marker expression in unstimulated BALB/c and C57BL/6 macrophages.

The origin of functional heterogeneity among macrophages, key innate immune system components, is still debated. While mouse strains differ in their immune responses, the range of gene expression variation among their pre-stimulation macrophages is unknown. With a novel approach to scRNA-seq analysis, we reveal the gene expression variation in unstimulated macrophage populations from BALB/c and C57BL/6 mice. We show that intrinsic strain-to-strain differences are detectable before stimulation and we place the unstimulated single cells within the gene expression landscape of stimulated macrophages. C57BL/6 mice show stronger evidence of macrophage polarization than BALB/c mice, which may contribute to their relative resistance to pathogens. Our computational methods can be generally adopted to uncover biological variation between cell populations.

An evolutionarily conserved coronin-dependent pathway defines cell population size.

Maintenance of cell population size is fundamental to the proper functioning of multicellular organisms. Here, we describe a cell-intrinsic cell density-sensing pathway that enabled T cells to reach and maintain an appropriate population size. This pathway operated "kin-to-kin" or between identical or similar T cell populations occupying a niche within a tissue or organ, such as the lymph nodes, spleen, and blood. We showed that this pathway depended on the cell density-dependent abundance of the evolutionarily conserved protein coronin 1, which coordinated prosurvival signaling with the inhibition of cell death until the cell population reached threshold densities. At or above threshold densities, coronin 1 expression peaked and remained stable, thereby resulting in the initiation of apoptosis through kin-to-kin intercellular signaling to return the cell population to the appropriate cell density. This cell population size-controlling pathway was conserved from amoeba to humans, thus providing evidence for the existence of a coronin-regulated, evolutionarily conserved mechanism by which cells are informed of and coordinate their relative population size.

Chemotaxis and phagocytosis in neutrophils is independent of coronin 1.

The WD repeat protein family member coronin 1 is exclusively expressed in leukocytes, where it colocalizes with the cortical cytoskeleton. Although initially coronin 1 was believed to regulate F-actin dynamics such as leukocyte motility, phagocytosis, and membrane ruffling, recent work showed that in macrophages, T cells, and B cells, coronin 1 is dispensable for these F-actin dependent processes, instead being involved in the regulation of calcium dependent signaling reactions. In this study, we show that in mice lacking coronin 1 neutrophil populations developed normally, and that coronin 1-deficient neutrophils are fully functional with respect to adherence, membrane dynamics, migration, phagocytosis and the oxidative burst. Therefore, these data argue against a role for coronin 1 in the modulation of F-actin and suggest that coronin 1 is dispensable for neutrophil functioning.

Coronin 1 is essential for IgM-mediated Ca2+ mobilization in B cells but dispensable for the generation of immune responses in vivo.

Coronin 1 is a leukocyte specific regulator of Ca(2+)-dependent signaling and is essential for the survival of peripheral T lymphocytes, but its role in B cells is unknown. In this study, we show that coronin 1 is essential for intracellular Ca(2+) mobilization and proliferation upon triggering of the BCR. However, the presence of costimulatory signals rendered coronin 1 dispensable for B cell signaling, consistent with the generation of normal immune responses against a variety of Ags in coronin 1-deficient mice. We conclude that coronin 1, while being essential for T cell function and survival, is dispensable for B cell function in vivo.

Suppression of caspase 8 activity by a coronin 1-PI3Kδ pathway promotes T cell survival independently of TCR and IL-7 signaling.

The control of T cell survival is crucial for defense against infectious pathogens or emerging cancers. Although the survival of peripheral naïve T cells has been proposed to be controlled by interleukin-7 (IL-7) signaling and T cell receptor (TCR) activation by peptide-loaded major histocompatibility complexes (pMHC), the essential roles for these pathways in thymic output and T cell proliferation have complicated the analysis of their contributions to T cell survival. Here, we showed that the WD repeat­containing protein coronin 1, which is dispensable for thymic selection and output, promoted naïve T cell survival in the periphery in a manner that was independent of TCR and IL-7 signaling. Coronin 1 was required for the maintenance of the basal activity of phosphoinositide 3-kinase δ (PI3Kδ), thereby suppressing caspase 8­mediated apoptosis. These results therefore reveal a coronin 1­dependent PI3Kδ pathway that is independent of pMHC:TCR and IL-7 signaling and essential for peripheral T cell survival.

Differential expression of a virulence factor in pathogenic and non-pathogenic mycobacteria.

The pathogenicity of mycobacterial infections depends on virulence factors that mediate survival inside host macrophages. These virulence factors are generally believed to be specific for pathogenic species and absent or mutated in non-pathogenic strains. The serine/threonine protein kinase G (PknG) mediates survival of mycobacteria within macrophages by blocking lysosomal delivery. Here we describe a gene of the non-pathogenic species Mycobacterium smegmatis that is 78% identical with pknG of Mycobacterium tuberculosis and M. bovis bacillus Calmette-Guérin (BCG). When cloned into expression vectors, the M. smegmatis pknG orthologue produced an active kinase and performed the same function as its M. bovis BCG counterpart in intracellular survival. In addition, similar levels of pknG transcripts were found in M. bovis BCG and M. smegmatis. However, virtually no translation product of chromosomal pknG could be detected in M. smegmatis both after in vitro growth and after macrophage infection. This lack of efficient translation was shown to be caused by regulatory elements in the upstream region of the M. smegmatis gene. The data reveal dramatically increased translational efficiency of a virulence gene in a pathogenic mycobacterium compared with a non-pathogenic mycobacterium suggesting that changes in expression levels may underlie evolution of pknG and other pathogenicity genes in mycobacterium.

The Trojan horse: survival tactics of pathogenic mycobacteria in macrophages.

Mycobacterium tuberculosis, the causative agent of tuberculosis, has infected billions of people worldwide. A key to the success of M. tuberculosis and related pathogenic mycobacteria lies in their ability to persist within the hostile environment of the host macrophage. After internalization by macrophages, most microbes are rapidly transported to lysosomes in which they are destroyed. By contrast, pathogenic mycobacteria prevent fusion of phagosomes with lysosomes, thereby surviving intracellularly. Recent progress in understanding the molecular biology of host-mycobacteria interactions is providing insights into these survival tactics.