Abstract
Group 2 innate lymphoid cells (ILC2s) are rare innate immune cells that accumulate in tissues during allergy and helminth infection, performing critical effector functions that drive type 2 ...inflammation. ILC2s express ST2, the receptor for the cytokine IL-33, and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), a receptor for the bioactive lipid prostaglandin D2 (PGD2). The IL-33–ST2 and the PGD2–CRTH2 pathways have both been implicated in promoting ILC2 accumulation during type 2 inflammation. However, whether these two pathways coordinate to regulate ILC2 population size in the tissue in vivo remains undefined. In this study, we show that ILC2 accumulation in the murine lung in response to systemic IL-33 treatment was partially dependent on CRTH2. This effect was not a result of reduced ILC2 proliferation, increased apoptosis or cell death, or differences in expression of the ST2 receptor in the absence of CRTH2. Rather, data from adoptive transfer studies suggested that defective accumulation of CRTH2-deficient ILC2s in response to IL-33 was due to altered ILC2 migration patterns. Whereas donor wild-type ILC2s preferentially accumulated in the lungs compared with CRTH2-deficient ILC2s following transfer into IL-33–treated recipients, wild-type and CRTH2-deficient ILC2s accumulated equally in the recipient mediastinal lymph node. These data suggest that CRTH2-dependent effects lie downstream of IL-33, directly affecting the migration of ILC2s into inflamed lung tissues. A better understanding of the complex interactions between the IL-33 and PGD2–CRTH2 pathways that regulate ILC2 population size will be useful in understanding how these pathways could be targeted to treat diseases associated with type 2 inflammation.
It is of great interest to understand how invading pathogens are sensed within the brain, a tissue with unique challenges to mounting an immune response. The eukaryotic parasite Toxoplasma gondii ...colonizes the brain of its hosts, and initiates robust immune cell recruitment, but little is known about pattern recognition of T. gondii within brain tissue. The host damage signal IL-33 is one protein that has been implicated in control of chronic T. gondii infection, but, like many other pattern recognition pathways, IL-33 can signal peripherally, and the specific impact of IL-33 signaling within the brain is unclear. Here, we show that IL-33 is expressed by oligodendrocytes and astrocytes during T. gondii infection, is released locally into the cerebrospinal fluid of T. gondii-infected animals, and is required for control of infection. IL-33 signaling promotes chemokine expression within brain tissue and is required for the recruitment and/or maintenance of blood-derived anti-parasitic immune cells, including proliferating, IFN-γ-expressing T cells and iNOS-expressing monocytes. Importantly, we find that the beneficial effects of IL-33 during chronic infection are not a result of signaling on infiltrating immune cells, but rather on radio-resistant responders, and specifically, astrocytes. Mice with IL-33 receptor-deficient astrocytes fail to mount an adequate adaptive immune response in the CNS to control parasite burden-demonstrating, genetically, that astrocytes can directly respond to IL-33 in vivo. Together, these results indicate a brain-specific mechanism by which IL-33 is released locally, and sensed locally, to engage the peripheral immune system in controlling a pathogen.
T cell receptor (TCR) repertoire sequencing has emerged as a powerful tool for understanding the diversity and functionality of T cells within the host immune system. Yet, the chicken TCR repertoire ...remains poorly understood due to incomplete genome annotation of the TCR loci, despite the importance of chickens in agriculture and as an immunological model. Here, we addressed this critical issue by employing 5' rapid amplification of complementary DNA ends (5'RACE) TCR repertoire sequencing with molecular barcoding of complementary DNA (cDNA) molecules. Simultaneously, we enhanced the genome annotation of TCR Variable (V), Diversity (D, only present in β and δ loci) and Joining (J) genes in the chicken genome. To enhance the efficiency of TCR annotations, we developed
, an algorithm designed to extract VJ gene candidates from deoxyribonucleic acid (DNA) sequences. Using this tool, we achieved a comprehensive annotation of all known chicken TCR loci, including the α/δ locus on chromosome 27. Evolutionary analysis revealed that each locus evolved separately by duplication of long homology units. To define the baseline TCR diversity in healthy chickens and to demonstrate the feasibility of the approach, we characterized the splenic α/β/γ/δ TCR repertoire. Analysis of the repertoires revealed preferential usage of specific V and J combinations in all chains, while the overall features were characteristic of unbiased repertoires. We observed moderate levels of shared complementarity-determining region 3 (CDR3) clonotypes among individual birds within the α and γ chain repertoires, including the most frequently occurring clonotypes. However, the β and δ repertoires were predominantly unique to each bird. Taken together, our TCR repertoire analysis allowed us to decipher the composition, diversity, and functionality of T cells in chickens. This work not only represents a significant step towards understanding avian T cell biology, but will also shed light on host-pathogen interactions, vaccine development, and the evolutionary history of avian immunology.
Type 2 inflammation drives the clearance of gastrointestinal helminth parasites, which infect over two billion people worldwide. Basophils are innate immune cells that support host-protective type 2 ...inflammation during murine infection with the helminth
However, the mechanisms required for basophil function and gene expression regulation in this context remain unclear. We show that during
infection, basophils localized to the intestine and up-regulated Notch receptor expression, rendering them sensitive to Notch signals that rapidly regulate gene expression programs. In vitro, Notch inhibition limited basophil cytokine production in response to cytokine stimulation. Basophil-intrinsic Notch signaling was required for
-elicited changes in genome-wide basophil transcriptional programs. Mice lacking basophil-intrinsic functional Notch signaling had impaired worm clearance, decreased intestinal type 2 inflammation, altered basophil localization in the intestine, and decreased CD4
T helper 2 cell responses following infection. These findings demonstrate that Notch is required for basophil gene expression and effector function associated with helminth expulsion during type 2 inflammation.
In chickens, γδ T cells represent a large fraction of peripheral T cells; however, their function remains largely unknown. Here, we describe the selective
expansion of γδ T cells from total ...splenocytes by stimulation with the cytokines IL-2 and IL-12. Under these conditions, γδ T cells proliferated preferentially and reached frequencies of >95% within three weeks. Although IL-2 alone also triggered proliferation, an increased proliferation rate was observed in combination with IL-12. Most of the expanded cells were γδ TCR and CD8 double-positive. Splenocytes sorted into TCR1
CD8
, TCR1
CD8
, and TCR1
CD8
subsets proliferated well upon dual stimulation with IL-2/IL-12, indicating that none of the three γδ T cell subsets require bystander activation for proliferation. TCR1
CD8
cells maintained CD8 surface expression during stimulation, whereas CD8
subpopulations showed varied levels of CD8 upregulation, with the highest upregulation observed in the TCR1
subset. Changes in the γδ T-cell receptor repertoire during cell culture from day 0 to day 21 were analyzed by next-generation sequencing of the γδ variable regions. Overall, long-term culture led to a restricted γ and δ chain repertoire, characterized by a reduced number of unique variable region clonotypes, and specific V genes were enriched at day 21. On day 0, the δ chain repertoire was highly diverse, and the predominant clonotypes differed between animals, while the most frequent γ-chain clonotypes were shared between animals. However, on day 21, the most frequent clonotypes in both the γ and δ chain repertoires were different between animals, indicating that selective expansion of dominant clonotypes during stimulation seems to be an individual outcome. In conclusion, IL-2 and IL-12 were sufficient to stimulate the
outgrowth of γδ T cells. Analyses of the TCR repertoire indicate that the culture leads to an expansion of individual T cell clones, which may reflect previous
activation. This system will be instrumental in studying γδ T cell function.
Distinct types of GABAergic interneurons target different subcellular domains of pyramidal cells, thereby shaping pyramidal cell activity patterns. Whether the presynaptic heterogeneity of GABAergic ...innervation is mirrored by specific postsynaptic factors is largely unexplored. Here we show that dystroglycan, a protein responsible for the majority of congenital muscular dystrophies when dysfunctional, has a function at postsynaptic sites restricted to a subset of GABAergic interneurons. Conditional deletion of Dag1, encoding dystroglycan, in pyramidal cells caused loss of CCK-positive basket cell terminals in hippocampus and neocortex. PV-positive basket cell terminals were unaffected in mutant mice, demonstrating interneuron subtype-specific function of dystroglycan. Loss of dystroglycan in pyramidal cells had little influence on clustering of other GABAergic postsynaptic proteins and of glutamatergic synaptic proteins. CCK-positive terminals were not established at P21 in the absence of dystroglycan and were markedly reduced when dystroglycan was ablated in adult mice, suggesting a role for dystroglycan in both formation and maintenance of CCK-positive terminals. The necessity of neuronal dystroglycan for functional innervation by CCK-positive basket cell axon terminals was confirmed by reduced frequency of inhibitory events in pyramidal cells of dystroglycan-deficient mice and further corroborated by the inefficiency of carbachol to increase IPSC frequency in these cells. Finally, neurexin binding seems dispensable for dystroglycan function because knock-in mice expressing binding-deficient T190M dystroglycan displayed normal CCK-positive terminals. Together, we describe a novel function of dystroglycan in interneuron subtype-specific trans-synaptic signaling, revealing correlation of presynaptic and postsynaptic molecular diversity.
Dystroglycan, an extracellular and transmembrane protein of the dystrophin-glycoprotein complex, is at the center of molecular studies of muscular dystrophies. Although its synaptic distribution in cortical brain regions is long established, function of dystroglycan in the synapse remained obscure. Using mice that selectively lack neuronal dystroglycan, we provide evidence that a subset of GABAergic interneurons requires dystroglycan for formation and maintenance of axonal terminals on pyramidal cells. As such, dystroglycan is the first postsynaptic GABAergic protein for which an interneuron terminal-specific function could be shown. Our findings also offer a new perspective on the mechanisms that lead to intellectual disability in muscular dystrophies without associated brain malformations.
Parasitic helminth infection elicits a type 2 cytokine-mediated inflammatory response. During type 2 inflammation, damaged or stimulated epithelial cells exposed to helminths and their products ...produce alarmins and cytokines including IL-25, IL-33, and thymic stromal lymphopoietin. These factors promote innate immune cell activation that supports the polarization of CD4+ T helper type 2 (Th2) cells. Activated innate and Th2 cells produce the cytokines IL-4, -5, -9, and -13 that perpetuate immune activation and act back on the epithelium to cause goblet cell hyperplasia and increased epithelial cell turnover. Together, these events facilitate worm expulsion and wound healing processes. While the role of Th2 cells in this context has been heavily studied, recent work has revealed that epithelial cell-derived cytokines are drivers of key innate immune responses that are critical for type 2 anti-helminth responses. Cutting-edge studies have begun to fully assess how other factors and pathways, including lipid mediators, chemokines, Fc receptor signaling, danger-associated molecular pattern molecules, and direct cell-cell interactions, also participate in shaping innate cell-mediated type 2 inflammation. In this review, we discuss how these pathways intersect and synergize with pathways controlled by epithelial cell-derived cytokines to coordinate innate immune responses that drive helminth-induced type 2 inflammation.
Type 2 inflammation is associated with epithelial cell responses, including goblet cell hyperplasia, that promote worm expulsion during intestinal helminth infection. How these epithelial responses ...are regulated remains incompletely understood. Here, we show that mice deficient in the prostaglandin D2 (PGD2) receptor CRTH2 and mice with CRTH2 deficiency only in nonhematopoietic cells exhibited enhanced worm clearance and intestinal goblet cell hyperplasia following infection with the helminth Nippostrongylus brasiliensis. Small intestinal stem, goblet, and tuft cells expressed CRTH2. CRTH2-deficient small intestinal organoids showed enhanced budding and terminal differentiation to the goblet cell lineage. During helminth infection or in organoids, PGD2 and CRTH2 down-regulated intestinal epithelial Il13ra1 expression and reversed Type 2 cytokine-mediated suppression of epithelial cell proliferation and promotion of goblet cell accumulation. These data show that the PGD2-CRTH2 pathway negatively regulates the Type 2 cytokine-driven epithelial program, revealing a mechanism that can temper the highly inflammatory effects of the anti-helminth response.
B-cell cloning methods enable the analysis of antibody responses against target antigens and can be used to reveal the host antibody repertoire, antigenic sites (epitopes), and details of protective ...immunity against pathogens. Here, we describe improved methods for isolation of canine peripheral blood B cells producing antibodies against canine parvovirus (CPV) capsids by fluorescence-activated cell sorting, followed by cell cloning. We cultured sorted B cells from an immunized dog in vitro and screened for CPV-specific antibody production. Updated canine-specific primer sets were used to amplify and clone the heavy and light chain immunoglobulin sequences directly from the B cells by reverse transcription and PCR. Monoclonal canine IgGs were produced by cloning heavy and light chain sequences into antibody expression vectors, which were screened for CPV binding. Three different canine monoclonal antibodies were analyzed, including two that shared the same heavy chain, and one that had distinct heavy and light chains. The antibodies showed broad binding to CPV variants, and epitopes were mapped to antigenic sites on the capsid. The methods described here are applicable for the isolation of canine B cells and monoclonal antibodies against many antigens.
•Here, we report the isolation of CPV-VLP binding B cells from peripheral blood cells obtained from a fully vaccinated dog.•We designed new primer sets to amplify the variable regions from rearranged canine Ig genes.•A modified and broadly applicable method for canine B cell culture and the production of cloned canine monoclonal IgGs was developed.•We identified and characterized the binding of three CPV-specific canine mAbs to CPV capsid variants.
In dissociated neuronal cultures the absence of spatial and temporal cues causes the emergence of mismatched synapses, where post‐synaptic proteins of GABAergic synapses are in part apposed to ...glutamatergic pre‐synaptic terminals and vice versa. This mismatch offers an opportunity to study the mechanisms that regulate correct apposition of pre‐ and post‐synaptic elements. We report here that the IQ motif and Sec7 domain‐containing protein 3 (IQSEC3; BRAG3; synArfGEF) specifically regulates the mislocalization of GABAergic post‐synaptic density (PSD) proteins. Over‐expression of IQSEC3 constructs harboring mutations that ablate Sec7 domain or IQ motif function revealed that IQSEC3 catalytic activity is involved in the control of apposition between the GABAergic PSD and glutamatergic terminals. Neurons co‐expressing eGFP‐gephyrin with IQSEC3 Sec7 mutant displayed a drastically increased fraction of mismatched eGFP‐gephyrin clusters compared to other IQSEC3 constructs. Along with eGFP‐gephyrin, endogenous GABAA receptor cluster mismatching was increased by IQSEC3 Sec7 mutant over‐expression. Conversely, GFP‐PSD‐95 clusters were unaffected by over‐expression of any IQSEC3 construct. The GABAergic PSD mismatch phenotype was recapitulated by Arf6 dominant‐negative mutant over‐expression, suggesting that Arf6 activation by IQSEC3 is an essential step in this pathway. In addition, we provide biochemical evidence to confirm gephyrin/IQSEC3 interaction near the IQSEC3 IQ motif, which in turn binds calmodulin at low Ca2+ concentrations. Taken together, our findings identify a post‐synaptic protein which specifically regulates correct apposition of the GABAergic PSD to pre‐synaptic terminals.
During synapse formation, proteins of the post‐synaptic density are properly sorted and aggregated at sites facing the pre‐synaptic terminal releasing a specific neurotransmitter, for example, GABA versus glutamate. This study reports that the guanine nucleotide exchange factor IQSEC3, activating the small GTPase of the ADP‐ribosylation factor family Arf6, regulates the proper matching of the post‐synaptic protein gephyrin in GABAergic versus glutamatergic synapses. IQSEC3 function is modulated by binding to gephyrin and calmodulin and activation of Arf6 by IQSEC3 selectively reduces post‐synaptic clustering of gephyrin in glutamatergic synapses.
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