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.

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.

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.

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.

Induction of Allograft Tolerance While Maintaining Immunity Against Microbial Pathogens: Does Coronin 1 Hold a Key?

Selective suppression of graft rejection while maintaining anti-pathogen responses has been elusive. Thus far, the most successful strategies to induce suppression of graft rejection relies on inhibition of T-cell activation. However, the very same mechanisms that induce allograft-specific T-cell suppression are also important for immunity against microbial pathogens as well as oncogenically transformed cells, resulting in significant immunosuppression-associated comorbidities. Therefore, defining the pathways that differentially regulate anti-graft versus antimicrobial T-cell responses may allow the development of regimen to induce allograft-specific tolerance. Recent work has defined a molecular pathway driven by the immunoregulatory protein coronin 1 that regulates the phosphodiesterase/cyclic adenosine monophosphate pathway and modulates T cell responses. Interestingly, disruption of coronin 1 promotes allograft tolerance while immunity towards a range of pathogenic microbes is maintained. Here, we briefly review the work leading up to these findings as well as their possible implications for transplantation medicine.

Skin Transplantation and Lymphoid Organ Analysis in Mice.

Skin transplantation in mice is an important procedure to evaluate immune responses generated against heterologous grafts, especially given its highly immunogenic nature. In fact, skin is one of the most challenging organs in terms of allograft retention. In this protocol, we provide a detailed procedure for skin grafting using the tail skin as donor organ that is grafted on the dorsal site of thoracic cage in a recipient mouse. We also provide protocols for the systematic analysis of lymphoid organ analysis in transplanted mice. Together these protocols may be valuable for evaluation of parameters that affect skin grafting, including genetic factors, immune cell activation as well as the analysis of compounds that may be useful in allowing graft tolerance.

Interference of Mycobacterium tuberculosis with macrophage responses.

Tuberculosis, caused by Mycobacterium tuberculosis, has become an important health and economic burden, with more than four thousand people succumbing to the disease every day. Thus, there is an urgent need to understand the molecular basis of this pathogen's success in causing disease in humans, in order to develop new drugs superior to conventional drugs available at present. One reason why M. tuberculosis is such a dangerous microbe lies within its ability to survive within infected hosts, thereby efficiently circumventing host immune responses. Over the past few years, a number of mechanisms have been unravelled that are utilized by M. tuberculosis to survive within hosts and to avoid immune defence mechanisms. Several of these mechanisms have been described in this communication that may be useful for the development of novel compounds to treat tuberculosis.

Activation of the cAMP/protein kinase A signalling pathway by coronin 1 is regulated by cyclin-dependent kinase 5 activity.

Coronins constitute a family of conserved proteins expressed in all eukaryotes that have been implicated in the regulation of a wide variety of cellular activities. Recent work showed an essential role for coronin 1 in the modulation of the cAMP/PKA pathway in neurons through the interaction of coronin 1 with the G protein subtype Gαs in a stimulus-dependent manner, but the molecular mechanism regulating coronin 1-Gαs interaction remains unclear. We here show that phosphorylation of coronin 1 on Thr(418/424) by cyclin-dependent kinase (CDK) 5 activity was responsible for coronin 1-Gαs association and the modulation of cAMP production. Together these results show an essential role for CDK5 activity in promoting the coronin 1-dependent cAMP/PKA pathway.

Mechanism of the immunomodulatory activity of glycodelin.

Glycodelin, a progesterone regulated protein synthesized by the endometrium (GdA) has been well documented to inhibit the proliferation of activated T-cells and is an indispensable molecule in the maternal system for the establishment, maintenance and progression of pregnancy. Data from our laboratory have unequivocally shown that the immunosuppression by GdA is via induction of apoptosis in activated T cells. Another isoform of glycodein, GdS, from the male reproductive system, in spite of sharing an identical amino acid sequence as that of GdA has been shown not to harbour the immunosuppressive activity of GdA. As the only difference between the two proteins is glycosylation, we proposed to study the role of the sugars in imparting apoptotic activity to Gd. Using the recombinant baculovirus system, Gd lacking glycosylation was expressed and from the experimental observations we could conclude that the activity of Gd lies in the protein backbone. Recombinant Gd expressed in P. pastoris, and Chinese hamster ovary cells, like the GdS did not exhibit apoptotic activity. A close analyses of the glycans associated with the Gd molecules from various sources suggested that though the apoptogenic activity of Gd lies in the protein backbone, the glycans modulate the activity by masking (as in case of GdS and most recombinant Gd expressed in our laboratory) or unmasking (as in case of GdA and baculovirus expressed Gd), the functional region of the molecule.

Regulation of immune cell homeostasis and function by coronin 1.

Coronin 1 is the most recent candidate in the list of genes causing severe combined immunodeficiency (SCID) in humans. A distinctive feature of the SCID induced by coronin 1 deficiency is selective naïve T cell lymphopenia in the presence of a normal thymus as well as normal B cell and natural killer cell numbers (T(-)B(+)NK(+)). Coronin 1 is a member of the evolutionarily conserved coronin protein family, members of which are widely expressed across the eukaryotic kingdom. Mammals express seven coronin molecules, numbered from coronin 1 to 7. The different coronin proteins have a distinct but overlapping tissue expression and have been reported to be involved in a wide array of cellular functions including calcium homeostasis, cytoskeletal dynamics, immune and inflammatory responses, neuromuscular transmission as well as cognition and behavior. In this minireview, we describe the role of coronin 1 in the maintenance of immune cell diversity and function.

Global 3' UTR shortening has a limited effect on protein abundance in proliferating T cells.

Alternative polyadenylation is a cellular mechanism that generates mRNA isoforms differing in their 3' untranslated regions (3' UTRs). Changes in polyadenylation site usage have been described upon induction of proliferation in resting cells, but the underlying mechanism and functional significance of this phenomenon remain largely unknown. To understand the functional consequences of shortened 3' UTR isoforms in a physiological setting, we used 3' end sequencing and quantitative mass spectrometry to determine polyadenylation site usage, mRNA and protein levels in murine and human naive and activated T cells. Although 3' UTR shortening in proliferating cells is conserved between human and mouse, orthologous genes do not exhibit similar expression of alternative 3' UTR isoforms. We generally find that 3' UTR shortening is not accompanied by a corresponding change in mRNA and protein levels. This suggests that although 3' UTR shortening may lead to changes in the RNA-binding protein interactome, it has limited effects on protein output.

On guard: coronin proteins in innate and adaptive immunity.

Recent work has implicated members of the evolutionarily conserved family of coronin proteins - in particular coronin 1 - in immune homeostasis. Coronins are involved in processes as diverse as pathogen survival in phagocytes and homeostatic T cell signalling. Notably, in both mice and humans, coronin mutations are associated with immune deficiencies and resistance to autoimmunity. In this article, we review what is currently known about these conserved molecules and discuss a potential common mechanism that underlies their diverse activities, which seem to involve cytoskeletal interactions as well as calcium-calcineurin signalling.

Elimination of intracellularly residing Mycobacterium tuberculosis through targeting of host and bacterial signaling mechanisms.

With more than 2 billion latently infected people, TB continues to represent a serious threat to human health. According to the WHO, 1.1 million people died from TB in 2010, which is equal to approximately 3000 deaths per day. The causative agent of the disease, Mycobacterium tuberculosis, is a highly successful pathogen having evolved remarkable strategies to persist within the host. Although normally, upon phagocytosis by macrophages, bacteria are readily eliminated by lysosomes, pathogenic mycobacteria actively prevent destruction within macrophages. The strategies that pathogenic mycobacteria apply range from releasing virulence factors to manipulating host molecules resulting in the modulation of host signal transduction pathways in order to sustain their viability within the infected host. Here, we analyze the current status of how a better understanding of both the bacterial and host factors involved in virulence can be used to develop drugs that may be helpful to curb the TB epidemic.

Exploring prospects of novel drugs for tuberculosis.

Tuberculosis remains a disease with an enormous impact on public health worldwide. With the continuously increasing epidemic of drug-resistant tuberculosis, new drugs are desperately needed. However, even for the treatment of drug-sensitive tuberculosis, new drugs are required to shorten the treatment duration and thereby prevent development of drug resistance. Within the past ten years, major advances in tuberculosis drug research have been made, leading to a considerable number of antimycobacterial compounds which are now in the pipeline. Here we discuss a number of these novel promising tuberculosis drugs, as well as the discovery of two new potential drug targets for the development of novel effective drugs to curb the tuberculosis pandemic, ie, the coronin 1 and protein kinase G pathways. Protein kinase G is secreted by mycobacteria and is responsible for blocking lysosomal delivery within the macrophage. Coronin 1 is responsible for activating the phosphatase, calcineurin, and thereby preventing phagosome-lysosome fusion within the macrophage. Blocking these two pathways may lead to rapid killing of mycobacteria.

Analyzing the interaction of pathogens with the host immune system.

Infectious diseases continue to represent a great burden to human society, being responsible for approximately 14 to 16 million deaths annually. Today, Mycobacterium tuberculosis, the causative agent of tuberculosis, remains one of the most important infectious agents. Moreover, with the emergence of multi drug and extensive drug resistant strains of M. tuberculosis, there is a great need for a better understanding of the virulence strategies utilized by this pathogen. In this manuscript, we discuss some of the strategies that have been followed to unravel virulence mechanisms utilized by M. tuberculosis. Knowledge of these mechanisms is important to reveal potential targets that may be useful for the development of compounds to treat tuberculosis.

RNA interference in J774 macrophages reveals a role for coronin 1 in mycobacterial trafficking but not in actin-dependent processes.

Macrophages are crucial for innate immunity, apoptosis, and tissue remodeling, processes that rely on the capacity of macrophages to internalize and process cargo through phagocytosis. Coronin 1, a member of the WD repeat protein family of coronins specifically expressed in leukocytes, was originally identified as a molecule that is recruited to mycobacterial phagosomes and prevents the delivery of mycobacteria to lysosomes, allowing these to survive within phagosomes. However, a role for coronin 1 in mycobacterial pathogenesis has been disputed in favor for its role in mediating phagocytosis and cell motility. In this study, a role for coronin 1 in actin-mediated cellular processes was addressed using RNA interference in the murine macrophage cell line J774. It is shown that the absence of coronin 1 in J774 macrophages expressing small interfering RNA constructs specific for coronin 1 does not affect phagocytosis, macropinocytosis, cell locomotion, or regulation of NADPH oxidase activity. However, in coronin 1-negative J774 cells, internalized mycobacteria were rapidly transferred to lysosomes and killed. Therefore, these results show that in J774 cells coronin 1 has a specific role in modulating phagosome-lysosome transport upon mycobacterial infection and that it is dispensable for most F-actin-mediated cytoskeletal rearrangements.

Structural basis for the specific inhibition of protein kinase G, a virulence factor of Mycobacterium tuberculosis.

The pathogenicity of mycobacteria such as Mycobacterium tuberculosis is closely associated with their capacity to survive within host macrophages. A crucial virulence factor for intracellular mycobacterial survival is protein kinase G (PknG), a eukaryotic-like serine/threonine protein kinase expressed by pathogenic mycobacteria that blocks the intracellular degradation of mycobacteria in lysosomes. Inhibition of PknG with the highly selective low-molecular-weight inhibitor AX20017 results in mycobacterial transfer to lysosomes and killing of the mycobacteria. Here, we report the 2.4 A x-ray crystal structure of PknG in complex with AX20017. The unique multidomain topology of PknG reveals a central kinase domain that is flanked by N- and C-terminal rubredoxin and tetratrico-peptide repeat domains, respectively. Directed mutagenesis suggests that the rubredoxin domain functions as a regulator of PknG kinase activity. The structure of PknG-AX20017 further reveals that the inhibitor is buried deep within the adenosine-binding site, targeting an active conformation of the kinase domain. Remarkably, although the topology of the kinase domain is reminiscent of eukaryotic kinases, the AX20017-binding pocket is shaped by a unique set of amino acid side chains that are not found in any human kinase. Directed mutagenesis of the unique set of residues resulted in a drastic loss of the compound's inhibitory potency. Our results explain the specific mode of action of AX20017 and demonstrate that virulence factors highly homologous to host molecules can be successfully targeted to block the proliferation of M. tuberculosis.

Survival of mycobacteria in macrophages is mediated by coronin 1-dependent activation of calcineurin.

Pathogenic mycobacteria survive within macrophages by avoiding lysosomal delivery, instead residing in mycobacterial phagosomes. Upon infection, the leukocyte-specific protein coronin 1 is actively recruited to mycobacterial phagosomes, where it blocks lysosomal delivery by an unknown mechanism. Analysis of macrophages from coronin 1-deficient mice showed that coronin 1 is dispensable for F-actin-dependent processes such as phagocytosis, motility, and membrane ruffling. However, upon mycobacterial infection, coronin 1 was required for activation of the Ca(2+)-dependent phosphatase calcineurin, thereby blocking lysosomal delivery of mycobacteria. In the absence of coronin 1, calcineurin activity did not occur, resulting in lysosomal delivery and killing of mycobacteria. Furthermore, blocking calcineurin activation with cyclosporin A or FK506 led to lysosomal delivery and intracellular mycobacterial killing. These results demonstrate a role for coronin 1 in activating Ca(2+) dependent signaling processes in macrophages and reveal a function for calcineurin in the regulation of phagosome-lysosome fusion upon mycobacterial infection.