For acutely lethal influenza infections, the relative pathogenic contributions of direct viral damage to lung epithelium vs. 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 feed-forward circuit involving pro-inflammatory 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.
Immune cells are thoroughbreds, moving farther and faster and surveying more diverse tissue space than their non-hematopoietic brethren. Intravital 2-photon microscopy has provided insights into the movements and interactions of many immune cell types in diverse tissues, but much more information is needed to link such analyses of dynamic cell behavior to function. Here we describe additional methods whose application promises to extend our vision, allowing more complete, multiscale dissection of how immune cell positioning and movement are linked to system state, host defense, and disease.
2-photon; dynamic; imaging; in vivo; multicolor
Developing T cells express diverse antigen receptors whose specificities are not pre-matched to the foreign antigens they eventually encounter. Past experiments have revealed that thymocytes must productively signal in response to self-antigens to mature and enter the peripheral T cell pool (positive selection), but how this process enhances effective mature T cell responses to foreign antigen is not fully understood. Here we have documented an unsuspected connection between thymic recognition events and foreign antigen-driven T cell responses. We find that the strength of self-reactivity is a clone-specific property unexpectedly directly related to the strength of T cell receptor (TCR) binding to presented foreign antigen. T cells with receptors showing stronger interaction with self dominate in responses to infections and accumulate in aging individuals, revealing that positive selection contributes to effective immunity by skewing the mature TCR repertoire towards highly effective recognition of pathogens that pose a danger to the host.
NLRP3 is a key component of the macromolecular signaling complex called the inflammasome that promotes caspase 1-dependent production of IL-1β. The adapter ASC is necessary for NLRP3-dependent inflammasome function, but it is not known if 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 adapter 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. Since 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.
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 (NK cells, γδ T cells, NKT cells, and innate-like CD8+ T cells) are spatially pre-positioned 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 anti-microbial 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.
Dendritic cells (DCs) regulate T cell function by promoting either tolerance or activation, and in the latter case, by directing response quality. New imaging tools now permit direct visualization of the relevant DC-T cell interactions in vivo and have provided a new perspective on the dynamics of these crucial cellular contacts. Here we discuss the insights generated by these analyses and the controversies/unanswered questions that need to be addressed in future work.
Neutrophil recruitment from blood to extravascular sites of sterile or infectious tissue damage is a hallmark of early innate immune responses, and the molecular events leading to cell exit from the bloodstream have been well defined1,2. Once outside the vessel, individual neutrophils often show extremely coordinated chemotaxis and cluster formation reminiscent of the swarming behaviour of insects3–11. The molecular players that direct this response at the single-cell and population levels within the complexity of an inflamed tissue are unknown. Using two-photon intravital microscopy in mouse models of sterile injury and infection, we show a critical role for intercellular signal relay among neutrophils mediated by the lipid leukotriene B4, which acutely amplifies local cell death signals to enhance the radius of highly directed interstitial neutrophil recruitment. Integrin receptors are dispensable for long-distance migration12, but have a previously unappreciated role in maintaining dense cellular clusters when congregating neutrophils rearrange the collagenous fibre network of the dermis to form a collagen-free zone at the wound centre. In this newly formed environment, integrins, in concert with neutrophil-derived leukotriene B4 and other chemoattractants, promote local neutrophil interaction while forming a tight wound seal. This wound seal has borders that cease to grow in kinetic concert with late recruitment of monocytes and macrophages at the edge of the displaced collagen fibres. Together, these data provide an initial molecular map of the factors that contribute to neutrophil swarming in the extravascular space of a damaged tissue. They reveal how local events are propagated over large-range distances, and how auto-signalling produces coordinated, self-organized neutrophil-swarming behaviour that isolates the wound or infectious site from surrounding viable tissue.
Systems biology is an emerging discipline that combines high-content, multiplexed measurements with informatic and computational modeling methods to better understand biological function at various scales. Here we present a detailed review of the methods used to create computational models and conduct simulations of immune function, We provide descriptions of the key data gathering techniques employed to generate the quantitative and qualitative data required for such modeling and simulation and summarize the progress to date in applying these tools and techniques to questions of immunological interest, including infectious disease. We include comments on what insights modeling can provide that complement information obtained from the more familiar experimental discovery methods used by most investigators and why quantitative methods are needed to eventually produce a better understanding of immune system operation in health and disease.
high-throughput analysis; modeling; genomics; proteomics; RNAi
The immune system is composed of multiple dynamic molecular and cellular networks, the complexity of which has been revealed by decades of exacting reductionist research. However, understanding of the immune system sufficient to anticipate its response to novel perturbations requires a more integrative view, or systems approach, to immunology. While methods for unbiased high-throughput data acquisition and computational integration of the resulting datasets are still relatively new, they have begun to substantially add to our understanding of immunological phenomena. Such approaches have expanded our view of interconnected signaling and transcriptional networks and have highlighted the function of non-linear processes such as spatial regulation and feedback loops. In addition, advances in single cell measurement technology have demonstrated the potential sources and functions of response heterogeneity in system behavior. The success of the studies reviewed here often depended upon integration of one or more systems biology approaches with more traditional methods. We hope these examples will inspire a broader range of immunologists to probe questions in a quantitative and integrated manner, to advance the common efforts to understand the immune “system”.
global analysis; high-throughput; computational modeling; signaling networks; transcriptional networks; single-cell analysis; heterogeneity
After an infection, the immune system generates long-lived memory lymphocytes whose increased frequency and altered state of differentiation enhance host defense against re-infection. Recently, the spatial distribution of memory cells was found to contribute to their protective function. Effector memory CD8+ T cells reside in peripheral tissue sites of initial pathogen encounter, in apparent anticipation of re-infection. Here we show that within lymph nodes (LN), memory CD8+ T cells were concentrated near peripheral entry portals of lymph-borne pathogens, promoting rapid engagement of infected sentinel macrophages. A feed-forward CXCL9-dependent circuit provided additional chemotactic cues that further increase local memory cell density. Memory CD8+ T cells also produced effector responses to local cytokine triggers, but their dynamic behavior differed from that seen following antigen recognition. These data reveal the distinct localization and dynamic behavior of naive vs. memory T cells within LN and how these differences contribute to host defense.
Granulomas play a key role in host protection against mycobacterial pathogens, with their breakdown contributing to exacerbated disease. To better understand the initiation and maintenance of these structures, we employed both high-resolution multiplex static imaging and intravital multiphoton microscopy of Mycobacterium bovis BCG-induced liver granulomas. We found that Kupffer cells directly capture blood-borne bacteria and subsequently nucleate formation of a nascent granuloma by recruiting both uninfected liver-resident macrophages and blood-derived monocytes. Within the mature granuloma, these myeloid cell populations form a relatively immobile cellular matrix that interacts with a highly dynamic effector T cell population. The efficient recruitment of these T cells is highly dependent on TNFα-derived signals, which also maintain the granuloma structure through preferential effects on uninfected macrophage populations. By characterizing the migration of both innate and adaptive immune cells throughout the process of granuloma development, these studies provide a new perspective on the cellular events involved in mycobacterial containment and escape.
Cells lining the gastrointestinal tract serve as both a barrier to and a pathway for infectious agent entry. Dendritic cells (DCs) present in the lamina propria under the columnar villus epithelium of the small bowel extend processes across this epithelium and capture bacteria, but previous studies provided limited information on the nature of the stimuli, receptors, and signaling events involved in promoting this phenomenon. Here, we use immunohistochemical as well as dynamic explant and intravital two-photon imaging to investigate this issue. Analysis of CD11c–enhanced green fluorescent protein (EGFP) or major histocompatibility complex CII-EGFP mice revealed that the number of trans-epithelial DC extensions, many with an unusual “balloon” shape, varies along the length of the small bowel. High numbers of such extensions were found in the proximal jejunum, but only a few were present in the terminal ileum. The extensions in the terminal ileum markedly increased upon the introduction of invasive or noninvasive Salmonella organisms, and chimeric mouse studies revealed the key role of MyD88-dependent Toll-like receptor (TLR) signaling by nonhematopoietic (epithelial) elements in the DC extension response. Collectively, these findings support a model in which epithelial cell TLR signaling upon exposure to microbial stimuli induces active DC sampling of the gut lumen at sites distant from organized lymphoid tissues.
The major histocompatibility complex (MHC)-dependent presentation of processed tissue-specific self-antigens can contribute to either peripheral (extrathymic) tolerance or the differentiation of autoreactive T cells. Here, we have studied the MHC class II molecule presentation of gastric parietal cell (PC)-specific H+/K+-ATPase, which induces a destructive autoimmune gastritis in BALB/c mice lacking CD4+ CD25+ regulatory T cells. Immunofluorescence microscopy showed physical association of CD11c+ dendritic cells (DCs) with PCs in the gastric mucosa. H+/K+-ATPase protein was found within vesicular compartments of a few CD11c+ DCs only in the draining gastric lymph node (LN) and these antigen-containing DCs increased markedly in number with the onset of tissue destruction in autoimmune animals. Both CD8αhi and CD8αlo gastric DCs, but not peripheral or mesenteric DCs, showed evidence of constitutive in vivo processing and presentation of H+/K+-ATPase. These data provide direct support for a widely held model of local tissue antigen uptake and trafficking by DCs in normal animals and demonstrate that DCs in the draining LN can present a tissue-specific self-antigen under noninflammatory conditions without fully deleting autoreactive T cells or inducing active autoimmunity.
antigen-presenting cells; autoimmune disease; gastritis; autoantigen; CD11c antigen
Cutaneous gene (DNA) bombardment results in substantial expression of the encoded antigen in the epidermal layer as well as detectable expression in dendritic cells (DC) in draining lymph nodes (LNs). Under these conditions, two possible modes of DC antigen presentation to naive CD8+ T cells might exist: (a) presentation directly by gene-transfected DC trafficking to local lymph nodes, and (b) cross-presentation by untransfected DC of antigen released from or associated with transfected epidermal cells. The relative contributions of these distinct modes of antigen presentation to priming for cytotoxic T cell (CTL) responses have not been clearly established. Here we show that LN cells directly expressing the DNA-encoded antigen are rare; 24 h after five abdominal skin bombardments, the number of these cells does not exceed 50–100 cells in an individual draining LN. However, over this same time period, the total number of CD11c+ DC increases more than twofold, by an average of 20,000–30,000 DC per major draining node. This augmentation is due to gold bombardment and is independent of the presence of plasmid DNA. Most antigen-bearing cells in the LNs draining the site of DNA delivery appear to be DC and can be depleted by antibodies to an intact surface protein encoded by cotransfected DNA. This finding of predominant antigen presentation by directly transfected cells is also consistent with data from studies on cotransfection with antigen and CD86-encoding DNA, showing that priming of anti-mutant influenza nucleoprotein CTLs with a single immunization is dependent upon coexpression of the DNAs encoding nucleoprotein and B7.2 in the same cells. These observations provide insight into the relative roles of direct gene expression and cross-presentation in CD8+ T cell priming using gene gun immunization, and indicate that augmentation of direct DC gene expression may enhance such priming.
dendritic cells; DNA immunization; cytotoxic T lymphocytes; gene gun; antigen presentation
Intravenous (i.v.) injection of high amounts of soluble proteins often results in the induction of antigen-specific tolerance or deviation to helper rather than inflammatory T cell immunity. It has been proposed that this outcome may be due to antigen presentation to T cells by a large cohort of poorly costimulatory or IL-12–deficient resting B cells lacking specific immunoglobulin receptors for the protein. However, previous studies using T cell activation in vitro to assess antigen display have failed to support this idea, showing evidence of specific peptide–major histocompatibility complex (MHC) class II ligand only on purified dendritic cells (DC) or antigen-specific B cells isolated from protein injected mice. Here we reexamine this question using a recently derived monoclonal antibody specific for the T cell receptor (TCR) ligand formed by the association of the 46-61 determinant of hen egg lysozyme (HEL) and the mouse MHC class II molecule I-Ak. In striking contrast to conclusions drawn from indirect T cell activation studies, this direct method of TCR ligand analysis shows that i.v. administration of HEL protein results in nearly all B cells in lymphoid tissues having substantial levels of HEL 46-61–Ak complexes on their surface. DC readily isolated from spleen also display this TCR ligand on their surface. Although the absolute number of displayed ligands is greater on such DC, the relative specific ligand expression compared to total MHC class II levels is similar or greater on B cells. These results demonstrate that in the absence of activating stimuli, both lymphoid DC and antigen-unspecific B cells present to a similar extent class II–associated peptides derived from soluble proteins in extracellular fluid. The numerical advantage of the TCR ligand–bearing B cells may permit them to interact first or more often with naive antigen-specific T cells, contributing to the induction of high-dose T cell tolerance or immune deviation.
T cell receptor (TCR) recognition of peptide–major histocompatibility complex antigens can elicit a diverse array of effector activities. Here we simultaneously analyze TCR engagement and the production of multiple cytokines by individual cells in a clonal Th1 CD4+ cell population. Low concentrations of TCR ligand elicit only interferon-γ (IFN-γ) production. Increasing ligand recruits more cells into the IFN-γ+ pool, increases IFN-γ produced per cell, and also elicits IL-2, but only from cells already making IFN-γ. Most cells producing only IFN-γ show less TCR downmodulation than cells producing both cytokines, consistent with a requirement for more TCR signaling to elicit IL-2 than to evoke IFN-γ synthesis. These studies emphasize the hierarchical organization of TCR signaling thresholds for induction of distinct cytokine responses, and demonstrate that this threshold phenomenon applies to individual cells. The existence of such thresholds suggests that antigen dose may dictate not only the extent, but also the quality of an immune response, by altering the ratios of the cytokines produced by activated T cells. The quantitative relationships in this response hierarchy change in response to costimulation through CD28 or LFA-1, as well as the differentiation state of the lymphocyte, explaining how variations in these parameters in the face of a fixed antigen load can qualitatively influence immune outcomes. Finally, although the IFN-γ/IL-2 hierarchy is seen with most cells, among cells with the greatest TCR downmodulation, some produce only IFN-γ and not IL-2, and the amount of IFN-γ exceeds that in double producers. Thus, these single cell analyses also provide clear evidence of nonquantitative intraclonal heterogeneity in cytokine production by long-term Th1 cells, indicating additional complexity of T cell function during immune responses.
Leucine-based signals in the cytoplasmic tail of invariant chain (Ii) control targeting of newly synthesized major histocompatibility complex class II molecules to the endocytic pathway for acquisition of antigenic peptides. Some protein determinants, however, do not require Ii for effective class II presentation, although endocytic processing is still necessary. Here we demonstrate that a dileucine-based signal in the cytoplasmic tail of the class II β chain is critical for this Ii-independent presentation. Elimination or mutation of this signal reduces the rate of re-entry of mature surface class II molecules into the endocytic pathway. Antigen presentation controlled by this signal does not require newly synthesized class II molecules and appears to involve determinants requiring only limited proteolysis for exposure, whereas the opposite is true for Ii-dependent determinants. This demonstrates that related leucine-based trafficking signals in Ii and class II control the functional presentation of protein determinants with distinct processing requirements, suggesting that the peptide binding sites of newly synthesized versus mature class II molecules are made available for antigen binding in distinct endocytic compartments under the control of these homologous cytoplasmic signals. This permits capture of protein fragments produced optimally under distinct conditions of pH and proteolytic activity.
One hypothesis seeking to explain the signaling and biological properties of T cell receptor for antigen (TCR) partial agonists and antagonists is the coreceptor density/kinetic model, which proposes that the pharmacologic behavior of a TCR ligand is largely determined by the relative rates of (a) dissociation of ligand from an engaged TCR and (b) recruitment of lck-linked coreceptors to this ligand-engaged receptor. Using several approaches to prevent or reduce the association of CD4 with occupied TCR, we demonstrate that consistent with this hypothesis, the biological and biochemical consequence of limiting this interaction is to convert typical agonists into partial agonist stimuli. Thus, adding anti-CD4 antibody to T cells recognizing a wild-type peptide–MHC class II ligand leads to disproportionate inhibition of interleukin-2 (IL-2) relative to IL-3 production, the same pattern seen using a TCR partial agonist/antagonist. In addition, T cells exposed to wild-type ligand in the presence of anti-CD4 antibodies show a pattern of TCR signaling resembling that seen using partial agonists, with predominant accumulation of the p21 tyrosine-phosphorylated form of TCR-ζ, reduced tyrosine phosphorylation of CD3ε, and no detectable phosphorylation of ZAP-70. Similar results are obtained when the wild-type ligand is presented by mutant class II MHC molecules unable to bind CD4. Likewise, antibody coligation of CD3 and CD4 results in an agonist-like phosphorylation pattern, whereas bivalent engagement of CD3 alone gives a partial agonist-like pattern. Finally, in accord with data showing that partial agonists often induce T cell anergy, CD4 blockade during antigen exposure renders cloned T cells unable to produce IL-2 upon restimulation. These results demonstrate that the biochemical and functional responses to variant TCR ligands with partial agonist properties can be largely reproduced by inhibiting recruitment of CD4 to a TCR binding a wild-type ligand, consistent with the idea that the relative rates of TCR–ligand disengagement and of association of engaged TCR with CD4 may play a key role in determining the pharmacologic properties of peptide–MHC molecule ligands. Beyond this insight into signaling through the TCR, these results have implications for models of thymocyte selection and the use of anti-coreceptor antibodies in vivo for the establishment of immunological tolerance.
Osteoclasts are bone resorbing, multinucleate cells that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursor cells. Although previous studies have revealed important molecular signals, how the bone resorptive functions of such cells are controlled in vivo remains less well characterized. Here, we visualized fluorescently labeled mature osteoclasts in intact mouse bone tissues using intravital multiphoton microscopy. Within this mature population, we observed cells with distinct motility behaviors and function, with the relative proportion of static – bone resorptive (R) to moving – nonresorptive (N) varying in accordance with the pathophysiological conditions of the bone. We also found that rapid application of the osteoclast-activation factor RANKL converted many N osteoclasts to R, suggesting a novel point of action in RANKL-mediated control of mature osteoclast function. Furthermore, we showed that Th17 cells, a subset of RANKL-expressing CD4+ T cells, could induce rapid N-to-R conversion of mature osteoclasts via cell-cell contact. These findings provide new insights into the activities of mature osteoclasts in situ and identify actions of RANKL-expressing Th17 cells in inflammatory bone destruction.
The immune system, like all biological systems, operates based on a set of check and balances that strive for homeostasis of the organism. Because of its potent effector mechanisms, the potential for self-destructive immune responses is especially high and, whether to prevent autoreactivity or chronic inflammation, many negative regulatory modalities exist to prevent excessive tissue damage. This Commentary places such regulatory mechanisms in the larger context of system organization at multiple scales. The sometimes counter-intuitive nature of feedback control is discussed and a case is made for greater attention to quantitative spatiotemporal aspects of regulation, rather than limiting ourselves to the qualitative descriptions of pathways that dominate at present.
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 (LN) 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 micro-anatomical domains, suggesting an unexpected LN demarcation into discrete functional compartments.
To develop and characterize the trafficking of a dual-modal agent that identifies primary draining or sentinel lymph node (LN).
Herein, a dual-reporting silica-coated iron oxide nanoparticle (SCION) is developed. Nude mice were imaged by magnetic resonance (MR) and optical imaging and axillary LNs were harvested for histological analysis. Trafficking through lymphatics was observed with intravital and ex vivo confocal microscopy of popliteal LNs in B6-albino, CD11c-EYFP, and lys-EGFP transgenic mice.
In vivo, SCION allows visualization of LNs. The particle’s size and surface functionality play a role in its passive migration from the intradermal injection site and its minimal uptake by CD11c+ dendritic cells and CD169+ and lys+ macrophages.
After injection, SCION passively migrates to LNs without macrophage uptake and then can be used to image LN(s) by MRI and fluorescence. Thus, SCION can potentially be developed for use in sentinel node resections or for intralymphatic drug delivery.
nanoparticle; molecular imaging; MRI; optical imaging; lymph node; superparamagnetic iron oxide
We present a method for computational reconstruction of the 3-D morphology of biological objects, such as cells, cell conjugates or 3-D arrangements of tissue structures, using data from high-resolution microscopy modalities. The method is based on the iterative optimization of Voronoi representations of the spatial structures. The reconstructions of biological surfaces automatically adapt to morphological features of varying complexity with flexible degrees of resolution. We show how 3-D confocal images of single cells can be used to generate numerical representations of cellular membranes that may serve as the basis for realistic, spatially resolved computational models of membrane processes or intracellular signaling. Another example shows how the protocol can be used to reconstruct tissue boundaries from segmented two-photon image data that facilitate the quantitative analysis of lymphocyte migration behavior in relation to microanatomical structures. Processing time is of the order of minutes depending on data features and reconstruction parameters.
Cellular signaling processes depend on specific spatiotemporal distributions of their molecular components. Multi-color high-resolution microscopy now permits detailed assessment of such distributions, providing the input for fine-grained computational models that explore the mechanisms governing dynamic assembly of multi-molecular complexes and their role in shaping cellular behavior. However, incorporating into such models both complex molecular reaction cascades and the spatial localization of signaling components within dynamic cellular morphologies presents substantial challenges. Here we introduce an approach that addresses these challenges by automatically generating computational representations of complex reaction networks based on simple bi-molecular interaction rules embedded into detailed, adaptive models of cellular morphology. Using examples of receptor-mediated cellular adhesion and signal-induced localized MAPK activation in yeast, we illustrate the capacity of this simulation technique to provide insights into cell biological processes. The modeling algorithms, implemented in a version of the Simmune tool set, are accessible through intuitive graphical interfaces as well as programming libraries.