For acutely lethal influenza infections, the relative pathogenic contributions of direct viral damage to lung epithelium versus dysregulated immunity remain unresolved. Here, we take a top-down ...systems approach to this question. Multigene transcriptional signatures from infected lungs suggested that elevated activation of inflammatory signaling networks distinguished lethal from sublethal infections. Flow cytometry and gene expression analysis involving isolated cell subpopulations from infected lungs showed that neutrophil influx largely accounted for the predictive transcriptional signature. Automated imaging analysis, together with these gene expression and flow data, identified a chemokine-driven feedforward circuit involving proinflammatory neutrophils potently driven by poorly contained lethal viruses. Consistent with these data, attenuation, but not ablation, of the neutrophil-driven response increased survival without changing viral spread. These findings establish the primacy of damaging innate inflammation in at least some forms of influenza-induced lethality and provide a roadmap for the systematic dissection of infection-associated pathology.
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•Early neutrophil infiltration largely accounts for fatal influenza gene signatures•Self-reflexive chemokine feedforward circuits underlie dysregulated inflammation•Fatal interstitial neutrophil activation correlates with unconstrained PAMP encounters•Neutrophil reduction prevents death from self-amplifying damaging inflammation
A systems analysis shows that lethality from influenza infections in mice is associated with early activation of a chemokine circuit that attracts excess neutrophils in the lung, showing that excess inflammation plays a causative role in lethality.
The lymphatic network that transports interstitial fluid and antigens to lymph nodes constitutes a conduit system that can be hijacked by invading pathogens to achieve systemic spread unless ...dissemination is blocked in the lymph node itself. Here, we show that a network of diverse lymphoid cells (natural killer cells, γδ T cells, natural killer T cells, and innate-like CD8+ T cells) are spatially prepositioned close to lymphatic sinus-lining sentinel macrophages where they can rapidly and efficiently receive inflammasome-generated IL-18 and additional cytokine signals from the pathogen-sensing phagocytes. This leads to rapid IFNγ secretion by the strategically positioned innate lymphocytes, fostering antimicrobial resistance in the macrophage population. Interference with this innate immune response loop allows systemic spread of lymph-borne bacteria. These findings extend our understanding of the functional significance of cellular positioning and local intercellular communication within lymph nodes while emphasizing the role of these organs as highly active locations of innate host defense.
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► Spatial and functional organization in lymph nodes prevents systemic pathogen spread ► A macrophage-lymphocyte communication loop promotes innate immune host defense ► Innate effectors are prepositioned close to macrophages to rapidly produce IFNγ ► Inflammasome-generated IL-1 and IL-18 activate and orchestrate the innate defense
A network of diverse lymphoid cells are spatially prepositioned close to sentinel macrophages lining the lymph nodes where they can rapidly and efficiently receive cytokine signals from the pathogen-sensing phagocytes, thus blocking the dissemination of bacteria and viruses that attempt to hijack the lymphatic network.
NLRP3 is a key component of the macromolecular signaling complex called the inflammasome that promotes caspase 1-dependent production of IL-1β. The adaptor ASC is necessary for NLRP3-dependent ...inflammasome function, but it is not known whether ASC is a sufficient partner and whether inflammasome formation occurs in the cytosol or in association with mitochondria is controversial. Here, we show that the mitochondria-associated adaptor molecule, MAVS, is required for optimal NLRP3 inflammasome activity. MAVS mediates recruitment of NLRP3 to mitochondria, promoting production of IL-1β and the pathophysiologic activity of the NLRP3 inflammasome in vivo. Our data support a more complex model of NLRP3 inflammasome activation than previously appreciated, with at least two adapters required for maximal function. Because MAVS is a mitochondria-associated molecule previously considered to be uniquely involved in type 1 interferon production, these findings also reveal unexpected polygamous involvement of PYD/CARD-domain-containing adapters in innate immune signaling events.
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► Inactive NLRP3 is cytosolic and associates with mitochondria upon activation. ► The mitochondrial adaptor MAVS mediates NLRP3 recruitment to mitochondria. ► The N terminus of NLRP3 regulates MAVS association and mitochondrial recruitment. ► MAVS promotes NLRP3-mediated production of mature IL-1β
Optimal NLRP3 inflammasome activity requires recruitment to the mitochondria via the adaptor protein MAVS. The MAVS-NLRP3 interaction plays a critical role in the response to infection and tissue damage and in inflammatory disease.
Flow cytometry allows highly quantitative analysis of complex dissociated populations at the cost of neglecting their tissue localization. In contrast, conventional microscopy methods provide spatial ...information, but visualization and quantification of cellular subsets defined by complex phenotypic marker combinations is challenging. Here, we describe an analytical microscopy method, “histo-cytometry,” for visualizing and quantifying phenotypically complex cell populations directly in tissue sections. This technology is based on multiplexed antibody staining, tiled high-resolution confocal microscopy, voxel gating, volumetric cell rendering, and quantitative analysis. We have tested this technology on various innate and adaptive immune populations in murine lymph nodes (LNs) and were able to identify complex cellular subsets and phenotypes, achieving quantitatively similar results to flow cytometry, while also gathering cellular positional information. Here, we employ histo-cytometry to describe the spatial segregation of resident and migratory dendritic cell subsets into specialized microanatomical domains, suggesting an unexpected LN demarcation into discrete functional compartments.
► Histo-cytometry provides discrete intranodal localization of distinct DC subsets ► Histo-cytometry provides highly multiplex phenotyping of individual cells in tissue sections ► Histo-cytometry provides quantification and spatial fixation of immune cells in situ
Upon infection, adaptive immune responses play catch-up with rapidly replicating pathogens. While mechanisms for efficient humoral responses to lymph-borne antigens have been characterized, the ...current paradigm for T cell responses to infections and particulate vaccines involves delayed migration of peripheral antigen-bearing dendritic cells (DCs) to lymph nodes (LNs), where they elicit effector T cell responses. Utilizing whole LN 3D imaging, histo-cytometry, and intravital 2-photon microscopy, we have identified a specialized population of DCs, enriched in the LN-resident CD11b+ subset, which resides within the lymphatic sinus endothelium and scans lymph with motile dendrites. These DCs capture draining particles and present associated antigens to T lymphocytes, inducing T cell responses much sooner than and independently of migratory DCs. Thus, strategic DC subset positioning in LNs limits a potentially costly delay in generation of T cell responses to lymph-borne antigens, contributing to effective host defense. These findings are also highly relevant to vaccine design.
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•LS-DCs reside within the lymphatic sinus endothelium and sample lymph fluids•LS-DCs capture particulate vaccine antigens and lymph-borne pathogens•LS-DCs generate rapid T cell responses independent of migratory DCs•Strategic DC positioning in LNs promotes optimal cell-mediated immune responses
Gerner and colleagues demonstrate that a specialized dendritic cell (DC) population is strategically positioned within lymph nodes to efficiently capture lymph-borne pathogens and particulate vaccines. These DCs elicit very rapid and robust T cell responses, much sooner and independently of peripheral-tissue migratory DCs.
Polarization of effector CD4+ T cells can be influenced by both antigen-specific signals and by pathogen- or adjuvant-induced cytokines, with current models attributing a dominant role to the latter. ...Here we have examined the relationship between these factors in shaping cell-mediated immunity by using intravital imaging of CD4+ T cell interactions with dendritic cells (DCs) exposed to polarizing adjuvants. These studies revealed a close correspondence between strength of T cell receptor (TCR)-dependent signaling and T helper 1 (Th1) versus Th2 cell fate, with antigen concentration dominating over adjuvant in controlling T cell polarity. Consistent with this finding, at a fixed antigen concentration, adjuvants inducing Th1 cells operated by affecting DC costimulation that amplified TCR signaling. TCR signal strength controlled downstream cytokine receptor expression, linking the two components in a hierarchical fashion. These data reveal how quantitative integration of antigen display and costimulation regulates downstream checkpoints responsible for cytokine-mediated control of effector differentiation.
•Antigen-associated signaling by T cells can be measured by intravital imaging•Strong TCR signals induce Th1 cell differentiation; weak signals induce Th2 cells in vivo•Adjuvants influence TCR signaling and polarization via effects on costimulation•TCR signal duration and intensity regulate expression of cytokine receptors
The roles of antigen receptor versus cytokine signaling in driving CD4+ effector T cell fate is presently unclear. Van Panhuys et al. demonstrate a hierarchical relationship in which quantitative antigen signaling regulates checkpoints responsible for cytokine-mediated control of Th1 versus Th2 cell effector development.
Organ homeostasis, cellular differentiation, signal relay, and in situ function all depend on the spatial organization of cells in complex tissues. For this reason, comprehensive, high-resolution ...mapping of cell positioning, phenotypic identity, and functional state in the context of macroscale tissue structure is critical to a deeper understanding of diverse biological processes. Here we report an easy to use method, clearing-enhanced 3D (Cₑ3D), which generates excellent tissue transparency for most organs, preserves cellular morphology and protein fluorescence, and is robustly compatible with antibody-based immunolabeling. This enhanced signal quality and capacity for extensive probe multiplexing permits quantitative analysis of distinct, highly intermixed cell populations in intact Cₑ3D-treated tissues via 3D histo-cytometry. We use this technology to demonstrate large-volume, high-resolution microscopy of diverse cell types in lymphoid and nonlymphoid organs, as well as to perform quantitative analysis of the composition and tissue distribution of multiple cell populations in lymphoid tissues. Combined with histo-cytometry, Cₑ3D provides a comprehensive strategy for volumetric quantitative imaging and analysis that bridges the gap between conventional section imaging and disassociation-based techniques.
Highly motile and functionally diverse immune cells orchestrate effective immune responses through complex and dynamic cooperative behavior. Multiphoton intravital microscopy (MP-IVM) presents a ...unique and powerful tool to study the coordinated action of immune cell interactions in situ. Here, we review the current state of intravital microscopy in deepening our understanding of the immune system and discuss its fundamental limitations. In addition, we draw insights from recent technical advances in multiplex static tissue-imaging methods and propose an approach that could enable simultaneous visualization of cellular dynamics, deep phenotyping, and transcriptional states through a new type of correlative microscopy that combines these imaging technologies with advances in complex data analysis.
Immune responses are shaped by complex sequences of dynamic interactions between a multitude of functionally diverse immune cell types.Multi-photon intravital microscopy serves as a powerful tool to study immune cell dynamics, but is fundamentally constrained by low plex visualization.Emerging advances in static tissue microscopy deliver high parameter 3D tissue-imaging capability, enabling deep immunophenotyping of immune cell subsets within spatial contexts.A new type of correlative microscopy that combines dynamic intravital microscopy and high-content static 3D tissue imaging may enable deep phenotypic visualization of immune cell interactions at an unprecedented scale.
A defining characteristic of the immune system is the constant movement of many of its constituent cells through the secondary lymphoid tissues, mainly the spleen and lymph nodes, where crucial ...interactions that underlie homeostatic regulation, peripheral tolerance and the effective development of adaptive immune responses take place. What has only recently been recognized is the role that non-haematopoietic stromal elements have in many aspects of immune cell migration, activation and survival. In this Review, we summarize our current understanding of lymphoid compartment stromal cells, examine their possible heterogeneity, discuss how these cells contribute to immune homeostasis and the efficient initiation of adaptive immune responses, and highlight how targeting of these elements by some pathogens can influence the host immune response.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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