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.
By analysing the gene expression profiles of individual peritoneal macrophages from BALB/c and C57BL/6 mice in the absence of any specific antigen stimulation, we found intrinsic cell‐to‐cell variability along the previously reported axes of M1/M2 polarization. The gene expression bias was stronger in C57BL/6 compared to BALB/c macrophages.
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.
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 Å 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.
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.
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 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.
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.
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.
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.
Glycodelin A, also known as placental protein-14, is a multifunctional glycosylated protein secreted by the uterine endometrium during the early phases of pregnancy. It is a known suppressor of T ...cell proliferation, inducer of T cell apoptosis, and inhibitor of sperm zona binding. Unlike in contraceptive activity, where the glycans on the molecule have been shown to play a crucial role, mutagenesis of the asparagines at sites of N-linked glycosylation (Asn28 and Asn63) to glutamine shows that the apoptogenic activity of glycodelin A is executed by the protein backbone. Glycosylation at Asn28 appears to play a role in the extracellular secretion of the molecule, as mutation of Asn28 resulted in a significant decrease in the amount of secreted protein, and loss of both glycosylation sites reduced the secretion drastically. Our results also suggest that the loss of glycosylation does not affect the dimerization status of the molecule.
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