The tempo and mode of gene regulatory programs during bacterial infection.

Innate immune recognition of bacterial pathogens is a key determinant of the ensuing systemic response, and host or pathogen heterogeneity in this early interaction can impact the course of infection. To gain insight into host response heterogeneity, we investigate macrophage inflammatory dynamics using primary human macrophages infected with Group B Streptococcus. Transcriptomic analysis reveals discrete cellular states within responding macrophages, one of which consists of four sub-states, reflecting inflammatory activation. Infection with six additional bacterial species-Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Yersinia pseudotuberculosis, Shigella flexneri, and Salmonella enterica-recapitulates these states, though at different frequencies. We show that modulating the duration of infection and the presence of a toxin impacts inflammatory trajectory dynamics. We provide evidence for this trajectory in infected macrophages in an in vivo model of Staphylococcus aureus infection. Our cell-state analysis defines a framework for understanding inflammatory activation dynamics in response to bacterial infection.

Distinct Features of Human Myeloid Cell Cytokine Response Profiles Identify Neutrophil Activation by Cytokines as a Prognostic Feature during Tuberculosis and Cancer.

Myeloid cells are a vital component of innate immunity and comprise monocytes, macrophages, dendritic cells, and granulocytes. How myeloid cell lineage affects activation states in response to cytokines remains poorly understood. The cytokine environment and cellular infiltrate during an inflammatory response may contain prognostic features that predict disease outcome. In this study, we analyzed the transcriptional responses of human monocytes, macrophages, dendritic cells, and neutrophils in response to stimulation by IFN-γ, IFN-ß, IFN-λ, IL-4, IL-13, and IL-10 cytokines to better understand the heterogeneity of activation states in inflammatory conditions. This generated a myeloid cell-cytokine-specific response matrix that can infer representation of myeloid cells and the cytokine environment they encounter during infection, in tumors and in whole blood. Neutrophils were highly responsive to type 1 and type 2 cytokine stimulation but did not respond to IL-10. We identified transcripts specific to IFN-ß stimulation, whereas other IFN signature genes were upregulated by both IFN-γ and IFN-ß. When we used our matrix to deconvolute blood profiles from tuberculosis patients, the IFN-ß-specific neutrophil signature was reduced in tuberculosis patients with active disease, whereas the shared response to IFN-γ and IFN-ß in neutrophils was increased. When applied to glioma patients, transcripts of neutrophils exposed to IL-4/IL-13 and monocyte responses to IFN-γ or IFN-ß emerged as opposing predictors of patient survival. Hence, by dissecting how different myeloid cells respond to cytokine activation, we can delineate biological roles for myeloid cells in different cytokine environments during disease processes, especially during infection and tumor progression.

An intestinal organoid-based platform that recreates susceptibility to T-cell-mediated tissue injury.

A goal in precision medicine is to use patient-derived material to predict disease course and intervention outcomes. Here, we use mechanistic observations in a preclinical animal model to design an ex vivo platform that recreates genetic susceptibility to T-cell-mediated damage. Intestinal graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation. We found that intestinal GVHD in mice deficient in Atg16L1, an autophagy gene that is polymorphic in humans, is reversed by inhibiting necroptosis. We further show that cocultured allogeneic T cells kill Atg16L1-mutant intestinal organoids from mice, which was associated with an aberrant epithelial interferon signature. Using this information, we demonstrate that pharmacologically inhibiting necroptosis or interferon signaling protects human organoids derived from individuals harboring a common ATG16L1 variant from allogeneic T-cell attack. Our study provides a roadmap for applying findings in animal models to individualized therapy that targets affected tissues.

Analysis of Inflammasome Activation in Response to Yersinia Infection by Fluorescence Microscopy Detection of Active Caspase-1 Puncta.

The type of cell death triggered by a particular environmental stimulus influences the outcome of infection or inflammatory disease processes. The ability to identify the cell death pathway that is activated in response to infection is essential for understanding the pathogenesis and host response to infection. Activation of the cysteine protease caspase-1 in various inflammasome complexes indicates that cells are undergoing pyroptosis, a regulated, proinflammatory cell death. Inflammasome assembly and caspase activation can be measured by various methods ranging from detection of inflammasome-dependent cell death, cytokine secretion, cleavage of caspase-1, or the formation of "puncta" within the cell that contain inflammasome components, such as caspase-1 or the adapter protein ASC. Here we describe a method for detecting caspase-1 activation on a single cell level in the context of infection by the Gram-negative pathogen Yersinia using immunofluorescence microscopy. We previously used this approach to quantify caspase-1 puncta formation in cells containing Yersinia translocon components (Zwack et al., MBio 6:e02095-14, 2015). This is a modification of methods used previously by Broz et al. (Cell Host Microbe 8:471-483, 2010) and Case and Roy (MBio 2:e00117-11, 2011). By taking a microscopy-based approach that allows us to quantify puncta as well as other cell-biological features of infection (i.e., number of bacteria associated with a particular cell; levels of bacterial effector or translocon proteins in caspase-1 puncta-containing cells; or levels or localization of host cellular proteins), we can better quantify the heterogeneity between cells undergoing pyroptosis and cells that are not under the same infection conditions. These approaches have the potential to generate hypotheses that can enable further mechanistic insight into activation of pyroptosis in response to bacterial infection.

Inflammasome activation in response to the Yersinia type III secretion system requires hyperinjection of translocon proteins YopB and YopD.

UNLABELLED: Type III secretion systems (T3SS) translocate effector proteins into target cells in order to disrupt or modulate host cell signaling pathways and establish replicative niches. However, recognition of T3SS activity by cytosolic pattern recognition receptors (PRRs) of the nucleotide-binding domain leucine rich repeat (NLR) family, either through detection of translocated products or membrane disruption, induces assembly of multiprotein complexes known as inflammasomes. Macrophages infected with Yersinia pseudotuberculosis strains lacking all known effectors or lacking the translocation regulator YopK induce rapid activation of both the canonical NLRP3 and noncanonical caspase-11 inflammasomes. While this inflammasome activation requires a functional T3SS, the precise signal that triggers inflammasome activation in response to Yersinia T3SS activity remains unclear. Effectorless strains of Yersinia as well as ΔyopK strains translocate elevated levels of T3SS substrates into infected cells. To dissect the contribution of pore formation and translocation to inflammasome activation, we took advantage of variants of YopD and LcrH that separate these functions of the T3SS. Notably, YopD variants that abrogated translocation but not pore-forming activity failed to induce inflammasome activation. Furthermore, analysis of individual infected cells revealed that inflammasome activation at the single-cell level correlated with translocated levels of YopB and YopD themselves. Intriguingly, LcrH mutants that are fully competent for effector translocation but produce and translocate lower levels of YopB and YopD also fail to trigger inflammasome activation. Our findings therefore suggest that hypertranslocation of YopD and YopB is linked to inflammasome activation in response to the Yersinia T3SS. IMPORTANCE: The innate immune response is critical to effective clearance of pathogens. Recognition of conserved virulence structures and activities by innate immune receptors such as NLRs constitute one of the first steps in mounting the innate immune response. However, pathogens such as Yersinia actively evade or subvert components of host defense, such as inflammasomes. The T3SS-secreted protein YopK is an essential virulence factor that limits translocation of other Yops, thereby limiting T3SS-induced inflammasome activation. However, what triggers inflammasome activation in cells infected by YopK-deficient Yersinia is not clear. Our findings indicate that hypertranslocation of pore complex proteins promotes inflammasome activation and that YopK prevents inflammasome activation by the T3SS by limiting translocation of YopD and YopB themselves.

Caspase-8 mediates caspase-1 processing and innate immune defense in response to bacterial blockade of NF-κB and MAPK signaling.

Toll-like receptor signaling and subsequent activation of NF-κB- and MAPK-dependent genes during infection play an important role in antimicrobial host defense. The YopJ protein of pathogenic Yersinia species inhibits NF-κB and MAPK signaling, resulting in blockade of NF-κB-dependent cytokine production and target cell death. Nevertheless, Yersinia infection induces inflammatory responses in vivo. Moreover, increasing the extent of YopJ-dependent cytotoxicity induced by Yersinia pestis and Yersinia pseudotuberculosis paradoxically leads to decreased virulence in vivo, suggesting that cell death promotes anti-Yersinia host defense. However, the specific pathways responsible for YopJ-induced cell death and how this cell death mediates immune defense against Yersinia remain poorly defined. YopJ activity induces processing of multiple caspases, including caspase-1, independently of inflammasome components or the adaptor protein ASC. Unexpectedly, caspase-1 activation in response to the activity of YopJ required caspase-8, receptor-interacting serine/threonine kinase 1 (RIPK1), and Fas-associated death domain (FADD), but not RIPK3. Furthermore, whereas RIPK3 deficiency did not affect YopJ-induced cell death or caspase-1 activation, deficiency of both RIPK3 and caspase-8 or FADD completely abrogated Yersinia-induced cell death and caspase-1 activation. Mice lacking RIPK3 and caspase-8 in their hematopoietic compartment showed extreme susceptibility to Yersinia and were deficient in monocyte and neutrophil-derived production of proinflammatory cytokines. Our data demonstrate for the first time to our knowledge that RIPK1, FADD, and caspase-8 are required for YopJ-induced cell death and caspase-1 activation and suggest that caspase-8-mediated cell death overrides blockade of immune signaling by YopJ to promote anti-Yersinia immune defense.

Caspase-11 activation in response to bacterial secretion systems that access the host cytosol.

Inflammasome activation is important for antimicrobial defense because it induces cell death and regulates the secretion of IL-1 family cytokines, which play a critical role in inflammatory responses. The inflammasome activates caspase-1 to process and secrete IL-1ß. However, the mechanisms governing IL-1α release are less clear. Recently, a non-canonical inflammasome was described that activates caspase-11 and mediates pyroptosis and release of IL-1α and IL-1ß. Caspase-11 activation in response to Gram-negative bacteria requires Toll-like receptor 4 (TLR4) and TIR-domain-containing adaptor-inducing interferon-ß (TRIF)-dependent interferon production. Whether additional bacterial signals trigger caspase-11 activation is unknown. Many bacterial pathogens use specialized secretion systems to translocate effector proteins into the cytosol of host cells. These secretion systems can also deliver flagellin into the cytosol, which triggers caspase-1 activation and pyroptosis. However, even in the absence of flagellin, these secretion systems induce inflammasome activation and the release of IL-1α and IL-1ß, but the inflammasome pathways that mediate this response are unclear. We observe rapid IL-1α and IL-1ß release and cell death in response to the type IV or type III secretion systems of Legionella pneumophila and Yersinia pseudotuberculosis. Unlike IL-1ß, IL-1α secretion does not require caspase-1. Instead, caspase-11 activation is required for both IL-1α secretion and cell death in response to the activity of these secretion systems. Interestingly, whereas caspase-11 promotes IL-1ß release in response to the type IV secretion system through the NLRP3/ASC inflammasome, caspase-11-dependent release of IL-1α is independent of both the NAIP5/NLRC4 and NLRP3/ASC inflammasomes as well as TRIF and type I interferon signaling. Furthermore, we find both overlapping and non-redundant roles for IL-1α and IL-1ß in mediating neutrophil recruitment and bacterial clearance in response to pulmonary infection by L. pneumophila. Our findings demonstrate that virulent, but not avirulent, bacteria trigger a rapid caspase-11-dependent innate immune response important for host defense.