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Leading researchers at the intersection of infectious disease and systems biology speak about how systems approaches have influenced modern infectious disease research and what these tools can offer for the future of the field.
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Enfermedades Transmisibles , Humanos , Enfermedades Transmisibles/terapia , Biología de SistemasRESUMEN
Background: Angiotensin-converting enzyme inhibitors (ACEi) and angiotensin-II receptor blockers (ARB), the most commonly prescribed antihypertensive medications, counter renin-angiotensin-aldosterone system (RAAS) activation via induction of angiotensin-converting enzyme 2 (ACE2) expression. Considering that ACE2 is the functional receptor for SARS-CoV-2 entry into host cells, the association of ACEi and ARB with COVID-19 outcomes needs thorough evaluation. Methods: We conducted retrospective analyses using both unmatched and propensity score (PS)-matched cohorts on electronic health records (EHRs) to assess the impact of RAAS inhibitors on the risk of receiving invasive mechanical ventilation (IMV) and 30-day mortality among hospitalized COVID-19 patients. Additionally, we investigated the immune cell gene expression profiles of hospitalized COVID-19 patients with prior use of antihypertensive treatments from an observational prospective cohort. Results: The retrospective analysis revealed that there was no increased risk associated with either ACEi or ARB use. In fact, the use of ACEi showed decreased risk for mortality. Survival analyses using PS-matched cohorts suggested no significant relationship between RAAS inhibitors with a hospital stay and in-hospital mortality compared to non-RAAS medications and patients not on antihypertensive medications. From the analysis of gene expression profiles, we observed a noticeable up-regulation in the expression of 1L1R2 (an anti-inflammatory receptor) and RETN (an immunosuppressive marker) genes in monocytes among prior users of ACE inhibitors. Conclusion: Overall, the findings do not support the discontinuation of ACEi or ARB treatment and suggest that ACEi may moderate the COVID-19 hyperinflammatory response.
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The ability to identify the role of a particular gene within a system is dependent on control of the expression of that gene. In this protocol, we describe a method for stable, conditional expression of Nod-Like receptors (NLRs) in THP-1 cells using a lentiviral expression system. This system combines all the necessary components for tetracycline-inducible gene expression in a single lentivector with constitutive co-expression of a selection marker, which is an efficient means for controlling gene expression using a single viral infection of cells. This is done in a third generation lentiviral expression platform that improves the safety of lentiviruses and allows for greater gene expression than previous lentiviral platforms. The lentiviral expression plasmid is first engineered to contain the gene of interest driven by a TRE (tetracycline response element) promoter in a simple gateway cloning step and is then co-transfected into HEK293T cells, along with packaging and envelope plasmids to generate the virus. The virus is used to infect a cell type of interest at a low MOI so that the majority of the transduced cells contain a single viral integration. Infected cells are grown under selection, and viral integration is validated by qPCR. Gene expression in stably transduced cells is induced with doxycycline and validated by qPCR, immunoblot, and flow cytometry. This flexible lentiviral expression platform may be used for stable and robust induction of a gene of interest in a range of cells for multiple applications. Graphic abstract: Schematic overview of lentiviral transduction of THP-1 cells.
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Small, genetically determined differences in transcription [expression quantitative trait loci (eQTLs)] are implicated in complex diseases through unknown molecular mechanisms. Here, we showed that a small, persistent increase in the abundance of the innate pathogen sensor NOD1 precipitated large changes in the transcriptional state of monocytes. A ~1.2- to 1.3-fold increase in NOD1 protein abundance resulting from loss of regulation by the microRNA cluster miR-15b/16 lowered the threshold for ligand-induced activation of the transcription factor NF-κB and the MAPK p38. An additional sustained increase in NOD1 abundance to 1.5-fold over basal amounts bypassed this low ligand concentration requirement, resulting in robust ligand-independent induction of proinflammatory genes and oncogenes. These findings reveal that tight regulation of NOD1 abundance prevents this sensor from exceeding a physiological switching checkpoint that promotes persistent inflammation and oncogene expression. Furthermore, our data provide insight into how a quantitatively small change in protein abundance can produce marked changes in cell state that can serve as the initiator of disease.
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Regulación de la Expresión Génica , Monocitos/metabolismo , Proteína Adaptadora de Señalización NOD1/biosíntesis , Proteínas Oncogénicas/biosíntesis , Transducción de Señal , Transcripción Genética , Humanos , Inflamación/metabolismo , Células THP-1RESUMEN
Inflammasomes have been implicated in the detection and clearance of a variety of bacterial pathogens, but little is known about whether this innate sensing mechanism has any regulatory effect on the expression of stimulatory ligands by the pathogen. During infection with Salmonella and many other pathogens, flagellin is a major activator of NLRC4 inflammasome-mediated macrophage pyroptosis and pathogen eradication. Salmonella switches to a flagellin-low phenotype as infection progresses to avoid this mechanism of clearance by the host. However, the host cues that Salmonella perceives to undergo this switch remain unclear. Here, we report an unexpected role of the NLRC4 inflammasome in promoting expression of its microbial ligand, flagellin, and identify a role for type 1 IFN signaling in switching of Salmonella to a flagellin-low phenotype. Early in infection, activation of NLRC4 by flagellin initiates pyroptosis and concomitant release of lysophospholipids which in turn enhance expression of flagellin by Salmonella thereby amplifying its ability to elicit cell death. TRIF-dependent production of type 1 IFN, however, later represses NLRC4 and the lysophospholipid biosynthetic enzyme iPLA2, causing a decline in intracellular lysophospholipids that results in down-regulation of flagellin expression by Salmonella These findings reveal a previously unrecognized immune-modulating regulatory cross-talk between endosomal TLR signaling and cytosolic NLR activation with significant implications for the establishment of infection with Salmonella.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Unión al Calcio/metabolismo , Flagelina/metabolismo , Fosfolipasas A2 Grupo VI/metabolismo , Interferón Tipo I/metabolismo , Infecciones por Salmonella/inmunología , Salmonella typhimurium/inmunología , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas de Unión al Calcio/genética , Células Cultivadas , Modelos Animales de Enfermedad , Regulación hacia Abajo , Flagelina/inmunología , Fosfolipasas A2 Grupo VI/antagonistas & inhibidores , Humanos , Inmunidad Innata , Inflamasomas/efectos de los fármacos , Inflamasomas/inmunología , Inflamasomas/metabolismo , Cetonas/administración & dosificación , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/inmunología , Lisofosfolípidos/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Naftalenos/administración & dosificación , Cultivo Primario de Células , Piroptosis/inmunología , Infecciones por Salmonella/microbiología , Salmonella typhimurium/aislamiento & purificación , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunologíaRESUMEN
We present an integrated analysis of the clinical measurements, immune cells, and plasma multi-omics of 139 COVID-19 patients representing all levels of disease severity, from serial blood draws collected during the first week of infection following diagnosis. We identify a major shift between mild and moderate disease, at which point elevated inflammatory signaling is accompanied by the loss of specific classes of metabolites and metabolic processes. Within this stressed plasma environment at moderate disease, multiple unusual immune cell phenotypes emerge and amplify with increasing disease severity. We condensed over 120,000 immune features into a single axis to capture how different immune cell classes coordinate in response to SARS-CoV-2. This immune-response axis independently aligns with the major plasma composition changes, with clinical metrics of blood clotting, and with the sharp transition between mild and moderate disease. This study suggests that moderate disease may provide the most effective setting for therapeutic intervention.
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COVID-19 , Genómica , RNA-Seq , SARS-CoV-2 , Análisis de la Célula Individual , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , COVID-19/sangre , COVID-19/inmunología , Femenino , Humanos , Masculino , Persona de Mediana Edad , SARS-CoV-2/inmunología , SARS-CoV-2/metabolismo , Índice de Severidad de la EnfermedadRESUMEN
Host immune responses play central roles in controlling SARS-CoV2 infection, yet remain incompletely characterized and understood. Here, we present a comprehensive immune response map spanning 454 proteins and 847 metabolites in plasma integrated with single-cell multi-omic assays of PBMCs in which whole transcriptome, 192 surface proteins, and T and B cell receptor sequence were co-analyzed within the context of clinical measures from 50 COVID19 patient samples. Our study reveals novel cellular subpopulations, such as proliferative exhausted CD8 + and CD4 + T cells, and cytotoxic CD4 + T cells, that may be features of severe COVID-19 infection. We condensed over 1 million immune features into a single immune response axis that independently aligns with many clinical features and is also strongly associated with disease severity. Our study represents an important resource towards understanding the heterogeneous immune responses of COVID-19 patients and may provide key information for informing therapeutic development.
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Antibiotic resistant infections are projected to cause over 10 million deaths by 2050, yet the development of new antibiotics has slowed. This points to an urgent need for methodologies for the rapid development of antibiotics against emerging drug resistant pathogens. We report on a generalizable combined computational and synthetic approach, called antibody-recruiting protein-catalyzed capture agents (AR-PCCs), to address this challenge. We applied the combinatorial protein catalyzed capture agent (PCC) technology to identify macrocyclic peptide ligands against highly conserved surface protein epitopes of carbapenem-resistant Klebsiella pneumoniae, an opportunistic Gram-negative pathogen with drug resistant strains. Multi-omic data combined with bioinformatic analyses identified epitopes of the highly expressed MrkA surface protein of K. pneumoniae for targeting in PCC screens. The top-performing ligand exhibited high-affinity (EC50 â¼50 nM) to full-length MrkA, and selectively bound to MrkA-expressing K. pneumoniae, but not to other pathogenic bacterial species. AR-PCCs that bear a hapten moiety promoted antibody recruitment to K. pneumoniae, leading to enhanced phagocytosis and phagocytic killing by macrophages. The rapid development of this highly targeted antibiotic implies that the integrated computational and synthetic toolkit described here can be used for the accelerated production of antibiotics against drug resistant bacteria.
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Recognition of peptidoglycan is integral to detection of gram-positive bacterial pathogens. In a recent issue of Cell, Wolf et al. (2016) report that detection of the N-acetylglucosamine component of peptidoglycan by the glycolytic enzyme hexokinase activates the NLRP3 inflammasome, revealing an intriguing interplay between pathogen detection and metabolism.
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Hexoquinasa , Peptidoglicano , Acetilglucosamina , Pared Celular , InflamasomasRESUMEN
The dysregulation of the relationship between gut microbiota and innate immune homeostasis can lead to a range of complex diseases. In this issue, Man et al. reveal that the intracellular innate sensor AIM2 regulates microbial and stem cell homeostasis in the gut to protect against colorectal cancer.
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Proliferación Celular , Neoplasias Colorrectales/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Madre/patología , AnimalesRESUMEN
The mammalian immune system is a dynamic multiscale system composed of a hierarchically organized set of molecular, cellular, and organismal networks that act in concert to promote effective host defense. These networks range from those involving gene regulatory and protein-protein interactions underlying intracellular signaling pathways and single-cell responses to increasingly complex networks of in vivo cellular interaction, positioning, and migration that determine the overall immune response of an organism. Immunity is thus not the product of simple signaling events but rather nonlinear behaviors arising from dynamic, feedback-regulated interactions among many components. One of the major goals of systems immunology is to quantitatively measure these complex multiscale spatial and temporal interactions, permitting development of computational models that can be used to predict responses to perturbation. Recent technological advances permit collection of comprehensive datasets at multiple molecular and cellular levels, while advances in network biology support representation of the relationships of components at each level as physical or functional interaction networks. The latter facilitate effective visualization of patterns and recognition of emergent properties arising from the many interactions of genes, molecules, and cells of the immune system. We illustrate the power of integrating 'omics' and network modeling approaches for unbiased reconstruction of signaling and transcriptional networks with a focus on applications involving the innate immune system. We further discuss future possibilities for reconstruction of increasingly complex cellular- and organism-level networks and development of sophisticated computational tools for prediction of emergent immune behavior arising from the concerted action of these networks.
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Citocinas/inmunología , Sistema Inmunológico/inmunología , Inmunidad Innata/inmunología , Redes y Vías Metabólicas/inmunología , Modelos Inmunológicos , Proteoma/inmunología , Animales , Simulación por Computador , HumanosRESUMEN
NLRP3 is a key component of the macromolecular signaling complex called the inflammasome that promotes caspase 1-dependent production of IL-1ß. 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-1ß 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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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Portadoras/metabolismo , Inflamasomas/metabolismo , Mitocondrias/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Adaptadoras de Señalización CARD , Proteínas Portadoras/química , Línea Celular , Proteínas del Citoesqueleto/metabolismo , Humanos , Inflamasomas/inmunología , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Túbulos Renales/patología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Monocitos/inmunología , Monocitos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR , Necrosis/patología , Dominios y Motivos de Interacción de Proteínas , Alineación de SecuenciaRESUMEN
Mutations in the gene encoding NLRP3 cause a spectrum of autoinflammatory diseases known as cryopyrin-associated periodic syndromes (CAPS). NLRP3 is a key component of one of several distinct cytoplasmic multiprotein complexes (inflammasomes) that mediate the maturation of the proinflammatory cytokine interleukin-1ß (IL-1ß) by activating caspase-1. Although several models for inflammasome activation, such as K(+) efflux, generation of reactive oxygen species and lysosomal destabilization, have been proposed, the precise molecular mechanism of NLRP3 inflammasome activation, as well as the mechanism by which CAPS-associated mutations activate NLRP3, remain to be elucidated. Here we show that the murine calcium-sensing receptor (CASR) activates the NLRP3 inflammasome, mediated by increased intracellular Ca(2+) and decreased cellular cyclic AMP (cAMP). Ca(2+) or other CASR agonists activate the NLRP3 inflammasome in the absence of exogenous ATP, whereas knockdown of CASR reduces inflammasome activation in response to known NLRP3 activators. CASR activates the NLRP3 inflammasome through phospholipase C, which catalyses inositol-1,4,5-trisphosphate production and thereby induces release of Ca(2+) from endoplasmic reticulum stores. The increased cytoplasmic Ca(2+) promotes the assembly of inflammasome components, and intracellular Ca(2+) is required for spontaneous inflammasome activity in cells from patients with CAPS. CASR stimulation also results in reduced intracellular cAMP, which independently activates the NLRP3 inflammasome. cAMP binds to NLRP3 directly to inhibit inflammasome assembly, and downregulation of cAMP relieves this inhibition. The binding affinity of cAMP for CAPS-associated mutant NLRP3 is substantially lower than for wild-type NLRP3, and the uncontrolled mature IL-1ß production from CAPS patients' peripheral blood mononuclear cells is attenuated by increasing cAMP. Taken together, these findings indicate that Ca(2+) and cAMP are two key molecular regulators of the NLRP3 inflammasome that have critical roles in the molecular pathogenesis of CAPS.
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Señalización del Calcio , Calcio/metabolismo , Proteínas Portadoras/metabolismo , AMP Cíclico/metabolismo , Inflamasomas/metabolismo , Receptores Sensibles al Calcio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Proteínas Portadoras/genética , Síndromes Periódicos Asociados a Criopirina/etiología , Síndromes Periódicos Asociados a Criopirina/genética , Síndromes Periódicos Asociados a Criopirina/metabolismo , Retículo Endoplásmico/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Interleucina-1beta/biosíntesis , Interleucina-1beta/metabolismo , Leucocitos Mononucleares/metabolismo , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR , Unión Proteica , Fosfolipasas de Tipo C/metabolismoRESUMEN
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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Infecciones Bacterianas/inmunología , Inmunidad Innata , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Virosis/inmunología , Animales , Interacciones Huésped-Patógeno , Inflamasomas/metabolismo , Interferón gamma/inmunología , Interleucina-18/inmunología , Linfa/microbiología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedades Cutáneas Infecciosas/inmunología , Organismos Libres de Patógenos Específicos , Linfocitos T/inmunologíaRESUMEN
BACKGROUND: Mendelian analysis of disorders of immune regulation can provide insight into molecular pathways associated with host defense and immune tolerance. METHODS: We identified three families with a dominantly inherited complex of cold-induced urticaria, antibody deficiency, and susceptibility to infection and autoimmunity. Immunophenotyping methods included flow cytometry, analysis of serum immunoglobulins and autoantibodies, lymphocyte stimulation, and enzymatic assays. Genetic studies included linkage analysis, targeted Sanger sequencing, and next-generation whole-genome sequencing. RESULTS: Cold urticaria occurred in all affected subjects. Other, variable manifestations included atopy, granulomatous rash, autoimmune thyroiditis, the presence of antinuclear antibodies, sinopulmonary infections, and common variable immunodeficiency. Levels of serum IgM and IgA and circulating natural killer cells and class-switched memory B cells were reduced. Linkage analysis showed a 7-Mb candidate interval on chromosome 16q in one family, overlapping by 3.5 Mb a disease-associated haplotype in a smaller family. This interval includes PLCG2, encoding phospholipase Cγ(2) (PLCγ(2)), a signaling molecule expressed in B cells, natural killer cells, and mast cells. Sequencing of complementary DNA revealed heterozygous transcripts lacking exon 19 in two families and lacking exons 20 through 22 in a third family. Genomic sequencing identified three distinct in-frame deletions that cosegregated with disease. These deletions, located within a region encoding an autoinhibitory domain, result in protein products with constitutive phospholipase activity. PLCG2-expressing cells had diminished cellular signaling at 37°C but enhanced signaling at subphysiologic temperatures. CONCLUSIONS: Genomic deletions in PLCG2 cause gain of PLCγ(2) function, leading to signaling abnormalities in multiple leukocyte subsets and a phenotype encompassing both excessive and deficient immune function. (Funded by the National Institutes of Health Intramural Research Programs and others.).
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Enfermedades Autoinmunes/genética , Síndromes Periódicos Asociados a Criopirina/genética , Síndromes de Inmunodeficiencia/genética , Fosfolipasa C gamma/genética , Eliminación de Secuencia , Frío/efectos adversos , ADN Complementario/análisis , ADN Complementario/aislamiento & purificación , Femenino , Humanos , Masculino , Linaje , Fenotipo , Fosfolipasa C gamma/metabolismo , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADNRESUMEN
Regulatory T cells (Treg) are key players in maintaining immune homeostasis but have also been shown to regulate immune responses against infectious pathogens. Therefore, Treg are a promising target for modulating immune responses to vaccines to improve their efficacy. Using a viral vector system, we found that Treg act on the developing immune response early postinfection by reducing the extent of dendritic cell costimulatory molecule expression. Due to this change and the lower IL-2 production that results, a substantial fraction of CD8(+) effector T cells lose CD25 expression several days after activation. Surprisingly, such Treg-dependent limitations in IL-2 signaling by Ag-activated CD8(+) T cells prevent effector differentiation without interfering with memory cell formation. In this way, Treg fine-tune the numbers of effector T cells generated while preserving the capacity for a rapid recall response upon pathogen re-exposure. This selective effect of Treg on a subpopulation of CD8(+) T cells indicates that although manipulation of the Treg compartment might not be optimal for prophylactic vaccinations, it can be potentially exploited to optimize vaccine efficacy for therapeutic interventions.
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Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica/inmunología , Interleucina-2/inmunología , Linfocitos T Reguladores/inmunología , Vacunas Virales/inmunología , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Subgrupos de Linfocitos T/inmunología , Virus Vaccinia/inmunologíaRESUMEN
Flagellin induces inflammatory and innate immune responses through activation of Toll-like receptor 5. Here we show that proinflammatory monomeric flagellin produced by salmonella during infection of intestinal epithelial cells was not derived from polymeric bacterial cell wall-associated flagellum but instead was synthesized and secreted de novo by the bacterium after direct sensing of host-produced lysophospholipids. Inhibition of lysophospholipid biosynthesis in intestinal epithelial cells reduced flagellin production and release from salmonella. Lysophospholipids induced a cAMP-dependent signaling pathway in salmonella that resulted in production and secretion of active flagellin. The induction of Toll-like receptor ligand synthesis and secretion by a host signal represents a previously unknown regulatory mechanism for inflammation and innate immunity during infection with a bacterial pathogen.