A RHIM with a View: FLYing with Functional Amyloids.

Recognition of bacterial peptidoglycan by the Drosophila IMD pathway triggers NF-κB activation and an associated immune response. In this issue of Immunity, Kleino et al. (2017) show that proteins in the IMD pathway form functional amyloids via a cryptic motif resembling the RHIM motif found in mammalian RIPK proteins. Amyloid formation can be negatively regulated, suggesting that it presents a regulatory point in multiple biological processes.

TNF licenses macrophages to undergo rapid caspase-1, -11, and -8-mediated cell death that restricts Legionella pneumophila infection.

The inflammatory cytokine tumor necrosis factor (TNF) is necessary for host defense against many intracellular pathogens, including Legionella pneumophila. Legionella causes the severe pneumonia Legionnaires' disease and predominantly affects individuals with a suppressed immune system, including those receiving therapeutic TNF blockade to treat autoinflammatory disorders. TNF induces pro-inflammatory gene expression, cellular proliferation, and survival signals in certain contexts, but can also trigger programmed cell death in others. It remains unclear, however, which of the pleiotropic functions of TNF mediate control of intracellular bacterial pathogens like Legionella. In this study, we demonstrate that TNF signaling licenses macrophages to die rapidly in response to Legionella infection. We find that TNF-licensed cells undergo rapid gasdermin-dependent, pyroptotic death downstream of inflammasome activation. We also find that TNF signaling upregulates components of the inflammasome response, and that the caspase-11-mediated non-canonical inflammasome is the first inflammasome to be activated, with caspase-1 and caspase-8 mediating delayed pyroptotic death. We find that all three caspases are collectively required for optimal TNF-mediated restriction of bacterial replication in macrophages. Furthermore, caspase-8 is required for control of pulmonary Legionella infection. These findings reveal a TNF-dependent mechanism in macrophages for activating rapid cell death that is collectively mediated by caspases-1, -8, and -11 and subsequent restriction of Legionella infection.

Pseudomonas aeruginosa Lipid A Structural Variants Induce Altered Immune Responses.

Pseudomonas aeruginosa causes chronic lung infection in cystic fibrosis (CF), resulting in structural lung damage and progressive pulmonary decline. P. aeruginosa in the CF lung undergoes numerous changes, adapting to host-specific airway pressures while establishing chronic infection. P. aeruginosa undergoes lipid A structural modification during CF chronic infection, not seen in any other disease state. Lipid A, the membrane anchor of lipopolysaccharide (i.e., endotoxin), comprises the majority of the outer membrane of Gram-negative bacteria and is a potent toll-like receptor (TLR)4 agonist. The structure of P. aeruginosa lipid A is intimately linked with its recognition by TLR4, and subsequent immune response. Prior work has identified P. aeruginosa strains with altered lipid A structures that arise during chronic CF lung infection; however, the impact of P. aeruginosa lipid A structure on airway disease has not been investigated. Here, we show that P. aeruginosa lipid A lacks PagL-mediated deacylation during human airway infection using a direct-from-sample mass spectrometry approach on human bronchoalveolar lavage fluid. This structure triggers increased pro-inflammatory cytokine production by primary human macrophages. Furthermore, alterations in lipid A 2-hydroxylation impact cytokine response in a site-specific manner, independent of CFTR function. Interestingly, there is a CF-specific reduction in IL-8 secretion within the epithelial-cell compartment that only occurs in CF bronchial epithelial cells when infected with CF-adapted P. aeruginosa that lack PagL-mediated lipid A deacylation. Taken together, we show that P. aeruginosa alters its lipid A structure during acute lung infection and that this lipid A structure induces stronger signaling through TLR4.

Human NAIP/NLRC4 and NLRP3 inflammasomes detect Salmonella type III secretion system activities to restrict intracellular bacterial replication.

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that uses two distinct type III secretion systems (T3SSs), termed Salmonella pathogenicity island (SPI)-1 and SPI-2, to deliver virulence factors into the host cell. The SPI-1 T3SS enables Salmonella to invade host cells, while the SPI-2 T3SS facilitates Salmonella's intracellular survival. In mice, a family of cytosolic immune sensors, including NAIP1, NAIP2, and NAIP5/6, recognizes the SPI-1 T3SS needle, inner rod, and flagellin proteins, respectively. Ligand recognition triggers assembly of the NAIP/NLRC4 inflammasome, which mediates caspase-1 activation, IL-1 family cytokine secretion, and pyroptosis of infected cells. In contrast to mice, humans encode a single NAIP that broadly recognizes all three ligands. The role of NAIP/NLRC4 or other inflammasomes during Salmonella infection of human macrophages is unclear. We find that although the NAIP/NLRC4 inflammasome is essential for detecting T3SS ligands in human macrophages, it is partially required for responses to infection, as Salmonella also activated the NLRP3 and CASP4/5 inflammasomes. Importantly, we demonstrate that combinatorial NAIP/NLRC4 and NLRP3 inflammasome activation restricts Salmonella replication in human macrophages. In contrast to SPI-1, the SPI-2 T3SS inner rod is not sensed by human or murine NAIPs, which is thought to allow Salmonella to evade host recognition and replicate intracellularly. Intriguingly, we find that human NAIP detects the SPI-2 T3SS needle protein. Critically, in the absence of both flagellin and the SPI-1 T3SS, the NAIP/NLRC4 inflammasome still controlled intracellular Salmonella burden. These findings reveal that recognition of Salmonella SPI-1 and SPI-2 T3SSs and engagement of both the NAIP/NLRC4 and NLRP3 inflammasomes control Salmonella infection in human macrophages.

Stressed-Out Endoplasmic Reticulum Inflames the Mitochondria.

Bacterial infection induces inflammasome activation and release of interleukin-1 (IL-1) cytokines. Bronner et al. (2015) show that during Brucella abortus infection, an endoplasmic reticulum stress sensor, IRE1α, initiates NLRP3- and caspase-2-mediated mitochondrial damage that potentiates NLRP3 inflammasome assembly.

A Hospital-Based Infant Safe Sleep Intervention and Safe Sleep Practices Among Young Women: A Prospective Longitudinal Study.

INTRODUCTION: The rates of sudden unexpected infant death (SUID) are still high in the U.S. The longitudinal effects of SUID preventive education on infant safe sleep practices are less known. The current study evaluated the effects of a comprehensive hospital-based, SUID preventive intervention on safe infant sleep practices in the first six months of life and to identify factors associated with infant sleep practices. METHODS: Using a one-group pretest and multiple posttest design, the current quantitative study examined the impacts of the infant safe sleep intervention among 411 women recruited at a large, urban, university medical center. Participants were prospectively followed and completed four surveys from childbirth. Linear mixed models were used to evaluate the effects of the SUID prevention program on four sleep practice outcomes, including removing unsafe items from the sleeping environment, bed sharing, room sharing without bed sharing, and placing the infant in a supine sleep position. RESULTS: Compared to the baseline, participants were less likely to use unsafe items (e.g., soft bedding) in infants' sleeping areas over time. However, we found that participants reported more frequent bed sharing at 3-month and 6-month follow-ups, compared to the baseline. CONCLUSIONS: Overall, maternal education and family income were positively related to healthy infant safe sleep practices. A hospital-based preventive intervention pairing an educational initiative with home-visiting services might improve safe sleep practices to remove accidental suffocation risks from the infant sleep environment.

Position-Specific Secondary Acylation Determines Detection of Lipid A by Murine TLR4 and Caspase-11.

Immune sensing of the Gram-negative bacterial membrane glycolipid lipopolysaccharide (LPS) is both a critical component of host defense against bacterial infection and a contributor to the hyperinflammatory response, potentially leading to sepsis and death. Innate immune activation by LPS is due to the lipid A moiety, an acylated di-glucosamine molecule that can activate inflammatory responses via the extracellular sensor Toll-like receptor 4 (TLR4)/myeloid differentiation 2 (MD2) or the cytosolic sensor caspase-11 (Casp11). The number and length of acyl chains present on bacterial lipid A structures vary across bacterial species and strains, which affects the magnitude of TLR4 and Casp11 activation. TLR4 and Casp11 are thought to respond similarly to various lipid A structures, as tetra-acylated lipid A structures do not activate either sensor, whereas hexa-acylated structures activate both sensors. However, the precise features of lipid A that determine the differential activation of each receptor remain poorly defined, as direct analysis of extracellular and cytosolic responses to the same sources and preparations of LPS/lipid A structures have been limited. To address this question, we used rationally engineered lipid A isolated from a series of bacterial acyl-transferase mutants that produce novel, structurally defined molecules. Intriguingly, we found that the location of specific secondary acyl chains on lipid A resulted in differential recognition by TLR4 or Casp11, providing new insight into the structural features of lipid A required to activate either TLR4 or Casp11. Our findings indicate that TLR4 and Casp11 sense nonoverlapping areas of lipid A chemical space, thereby constraining the ability of Gram-negative pathogens to evade innate immunity.

Salmonella enterica Serovar Typhimurium Induces NAIP/NLRC4- and NLRP3/ASC-Independent, Caspase-4-Dependent Inflammasome Activation in Human Intestinal Epithelial Cells.

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that causes diseases ranging from gastroenteritis to systemic infection and sepsis. Salmonella uses type III secretion systems (T3SS) to inject effectors into host cells. While these effectors are necessary for bacterial invasion and intracellular survival, intracellular delivery of T3SS products also enables detection of translocated Salmonella ligands by cytosolic immune sensors. Some of these sensors form multimeric complexes called inflammasomes, which activate caspases that lead to interleukin-1 (IL-1) family cytokine release and pyroptosis. In particular, the Salmonella T3SS needle, inner rod, and flagellin proteins activate the NAIP/NLRC4 inflammasome in murine intestinal epithelial cells (IECs), which leads to restriction of bacterial replication and extrusion of infected IECs into the intestinal lumen, thereby preventing systemic dissemination of Salmonella. While these processes are quite well studied in mice, the role of the NAIP/NLRC4 inflammasome in human IECs remains unknown. Unexpectedly, we found the NAIP/NLRC4 inflammasome is dispensable for early inflammasome responses to Salmonella in both human IEC lines and enteroids. Additionally, NLRP3 and the adaptor protein ASC are not required for inflammasome activation in Caco-2 cells. Instead, we observed a necessity for caspase-4 and gasdermin D pore-forming activity in mediating inflammasome responses to Salmonella in Caco-2 cells. These findings suggest that unlike murine IECs, human IECs do not rely on NAIP/NLRC4 or NLRP3/ASC inflammasomes and instead primarily use caspase-4 to mediate inflammasome responses to Salmonella pathogenicity island 1 (SPI-1)-expressing Salmonella.

Lipid A Variants Activate Human TLR4 and the Noncanonical Inflammasome Differently and Require the Core Oligosaccharide for Inflammasome Activation.

Detection of Gram-negative bacterial lipid A by the extracellular sensor, myeloid differentiation 2 (MD2)/Toll-like receptor 4 (TLR4), or the intracellular inflammasome sensors, CASP4 and CASP5, induces robust inflammatory responses. The chemical structure of lipid A, specifically its phosphorylation and acylation state, varies across and within bacterial species, potentially allowing pathogens to evade or suppress host immunity. Currently, it is not clear how distinct alterations in the phosphorylation or acylation state of lipid A affect both human TLR4 and CASP4/5 activation. Using a panel of engineered lipooligosaccharides (LOS) derived from Yersinia pestis with defined lipid A structures that vary in their acylation or phosphorylation state, we identified that differences in phosphorylation state did not affect TLR4 or CASP4/5 activation. However, the acylation state differentially impacted TLR4 and CASP4/5 activation. Specifically, all tetra-, penta-, and hexa-acylated LOS variants examined activated CASP4/5-dependent responses, whereas TLR4 responded to penta- and hexa-acylated LOS but did not respond to tetra-acylated LOS or penta-acylated LOS lacking the secondary acyl chain at the 3' position. As expected, lipid A alone was sufficient for TLR4 activation. In contrast, both core oligosaccharide and lipid A were required for robust CASP4/5 inflammasome activation in human macrophages, whereas core oligosaccharide was not required to activate mouse macrophages expressing CASP4. Our findings show that human TLR4 and CASP4/5 detect both shared and nonoverlapping LOS/lipid A structures, which enables the innate immune system to recognize a wider range of bacterial LOS/lipid A and would thereby be expected to constrain the ability of pathogens to evade innate immune detection.

An Exploration of Practitioners' Perceptions and Beliefs About Trauma-Informed Youth Drug Prevention Programs: a Qualitative Study.

Adverse childhood experiences (ACEs), such as childhood maltreatment and household dysfunction, have been linked to adolescent substance use. As a result, there exists a pressing need for trauma-informed, substance use preventive intervention for adolescents with a history of ACEs. The primary aim of this qualitative study is to increase our understanding of practitioners' perceptions of substance use among ACE-exposed youth and their views on trauma-informed adolescent substance use prevention programs. The present study conducted six focus groups (N = 32) among current child and adolescent health and human service providers in a mid-Atlantic urban area. The focus groups explored the practitioners' views on the main reasons that youth with a history of ACEs use illicit substances and suggestions on components, constructs, or techniques of trauma-informed substance use prevention programs and perceived barriers in implementing such programs. Transcripts of the focus groups were analyzed using open coding and subsequent axial coding, which was followed by thematic analysis. Thematic analysis identified ten themes within three categories, including the etiology of substance use among ACE-exposed youth, barriers to preventing substance use among ACEs-exposed youth, and suggested program components for trauma-informed prevention programs. These findings provide support for developing a preventive intervention that addresses trauma symptoms and overall skill buildings to prevent substance use among ACE-exposed youth. Teaching skills to cope with trauma symptoms, enhancing knowledge about the signs and symptoms of trauma, and improving key social and emotional learning competencies might be important and effective strategies to curb substance use among ACE-exposed youth.

Age-related differences in IL-1 signaling and capsule serotype affect persistence of Streptococcus pneumoniae colonization.

Young age is a risk factor for prolonged colonization by common pathogens residing in their upper respiratory tract (URT). Why children present with more persistent colonization is unknown and there is relatively little insight into the host-pathogen interactions that contribute to persistent colonization. To identify factors permissive for persistent colonization during infancy, we utilized an infant mouse model of Streptococcus pneumoniae colonization in which clearance from the mucosal surface of the URT requires many weeks to months. Loss of a single bacterial factor, the pore-forming toxin pneumolysin (Ply), and loss of a single host factor, IL-1α, led to more persistent colonization. Exogenous administration of Ply promoted IL-1 responses and clearance, and intranasal treatment with IL-1α was sufficient to reduce colonization density. Major factors known to affect the duration of natural colonization include host age and pneumococcal capsular serotype. qRT-PCR analysis of the uninfected URT mucosa showed reduced baseline expression of genes involved in IL-1 signaling in infant compared to adult mice. In line with this observation, IL-1 signaling was important in initiating clearance in adult mice but had no effect on early colonization of infant mice. In contrast to the effect of age, isogenic constructs of different capsular serotype showed differences in colonization persistence but induced similar IL-1 responses. Altogether, this work underscores the importance of toxin-induced IL-1α responses in determining the outcome of colonization, clearance versus persistence. Our findings about IL-1 signaling as a function of host age may provide an explanation for the increased susceptibility and more prolonged colonization during early childhood.

An autonomous CDR3delta is sufficient for recognition of the nonclassical MHC class I molecules T10 and T22 by gammadelta T cells.

It remains unclear whether gammadelta T cell antigen receptors (TCRs) detect antigens in a way similar to antibodies or alphabeta TCRs. Here we show that reactivity between the G8 and KN6 gammadelta TCRs and the major histocompatibility complex class Ib molecule T22 could be recapitulated, with retention of wild-type ligand affinity, in an alphabeta TCR after grafting of a G8 or KN6 complementarity-determining region 3-delta (CDR3delta) loop in place of the CDR3alpha loop of an alphabeta TCR. We also found that a shared sequence motif in CDR3delta loops of all T22-reactive gammadelta TCRs bound T22 in energetically distinct ways, and that T10(d), which bound G8 with weak affinity, was converted into a high-affinity ligand by a single point mutation. Our results demonstrate unprecedented autonomy of a single CDR3 loop in antigen recognition.

Broad detection of bacterial type III secretion system and flagellin proteins by the human NAIP/NLRC4 inflammasome.

Inflammasomes are cytosolic multiprotein complexes that initiate host defense against bacterial pathogens by activating caspase-1-dependent cytokine secretion and cell death. In mice, specific nucleotide-binding domain, leucine-rich repeat-containing family, apoptosis inhibitory proteins (NAIPs) activate the nucleotide-binding domain, leucine-rich repeat-containing family, CARD domain-containing protein 4 (NLRC4) inflammasome upon sensing components of the type III secretion system (T3SS) and flagellar apparatus. NAIP1 recognizes the T3SS needle protein, NAIP2 recognizes the T3SS inner rod protein, and NAIP5 and NAIP6 recognize flagellin. In contrast, humans encode a single functional NAIP, raising the question of whether human NAIP senses one or multiple bacterial ligands. Previous studies found that human NAIP detects both flagellin and the T3SS needle protein and suggested that the ability to detect both ligands was achieved by multiple isoforms encoded by the single human NAIP gene. Here, we show that human NAIP also senses the Salmonella Typhimurium T3SS inner rod protein PrgJ and that T3SS inner rod proteins from multiple bacterial species are also detected. Furthermore, we show that a single human NAIP isoform is capable of sensing the T3SS inner rod, needle, and flagellin. Our findings indicate that, in contrast to murine NAIPs, promiscuous recognition of multiple bacterial ligands is conferred by a single human NAIP.

Childhood maltreatment and problematic alcohol use in young adulthood: the roles of cognitive vulnerability to depression and depressive symptoms.

BACKGROUND: Childhood maltreatment (CM) has been repeatedly linked to future problem drinking. Depression has been identified as a potential factor contributing to problematic alcohol use in maltreated individuals. However, depression has been operationalized as the presence or number of depression symptoms in the majority of previous studies. The role of other relevant measures of depression, such as depressive implicit associations, is not well understood. OBJECTIVES: The present study addresses this gap in the literature by examining the mediating role of both depression symptoms and depressive implicit associations. METHODS: A community sample of young adults (N = 208, mean age = 19.7, 78.4% females) completed self-report measures of CM, depression symptoms, and problem drinking. Depressive implicit associations were assessed by a computer-based implicit association test (IAT). Structural equation modeling (SEM) was used to examine the direct link between CM and problem drinking as well as indirect links through depression symptoms and depressive implicit associations. RESULTS: CM was significantly associated with both depression symptoms (ß = 0.35, p < .001) and depressive implicit associations (ß = 0.36, p < .001). Additionally, CM was associated with problem drinking indirectly via depression symptoms during young adulthood (ß = .06, p = .019). CONCLUSION: Our study provides evidence for the role of depression symptoms, but not for depressive implicit associations, in linking CM and problem drinking. Treating depression in individuals with a history of CM may help to prevent problem drinking in this vulnerable population.

Increased autophagic sequestration in adaptor protein-3 deficient dendritic cells limits inflammasome activity and impairs antibacterial immunity.

Bacterial pathogens that compromise phagosomal membranes stimulate inflammasome assembly in the cytosol, but the molecular mechanisms by which membrane dynamics regulate inflammasome activity are poorly characterized. We show that in murine dendritic cells (DCs), the endosomal adaptor protein AP-3 -which optimizes toll-like receptor signaling from phagosomes-sustains inflammasome activation by particulate stimuli. AP-3 independently regulates inflammasome positioning and autophagy induction, together resulting in delayed inflammasome inactivation by autophagy in response to Salmonella Typhimurium (STm) and other particulate stimuli specifically in DCs. AP-3-deficient DCs, but not macrophages, hyposecrete IL-1ß and IL-18 in response to particulate stimuli in vitro, but caspase-1 and IL-1ß levels are restored by silencing autophagy. Concomitantly, AP-3-deficient mice exhibit higher mortality and produce less IL-1ß, IL-18, and IL-17 than controls upon oral STm infection. Our data identify a novel link between phagocytosis, inflammasome activity and autophagy in DCs, potentially explaining impaired antibacterial immunity in AP-3-deficient patients.

Neutrophils and Ly6Chi monocytes collaborate in generating an optimal cytokine response that protects against pulmonary Legionella pneumophila infection.

Early responses mounted by both tissue-resident and recruited innate immune cells are essential for host defense against bacterial pathogens. In particular, both neutrophils and Ly6Chi monocytes are rapidly recruited to sites of infection. While neutrophils and monocytes produce bactericidal molecules, such as reactive nitrogen and oxygen species, both cell types are also capable of synthesizing overlapping sets of cytokines important for host defense. Whether neutrophils and monocytes perform redundant or non-redundant functions in the generation of anti-microbial cytokine responses remains elusive. Here, we sought to define the contributions of neutrophils and Ly6Chi monocytes to cytokine production and host defense during pulmonary infection with Legionella pneumophila, responsible for the severe pneumonia Legionnaires' disease. We found that both neutrophils and monocytes are critical for host defense against L. pneumophila. Both monocytes and neutrophils contribute to maximal IL-12 and IFNγ responses, and monocytes are also required for TNF production. Moreover, natural killer (NK) cells, NKT cells, and γδ T cells are sources of IFNγ, and monocytes direct IFNγ production by these cell types. Thus, neutrophils and monocytes cooperate in eliciting an optimal cytokine response that promotes effective control of bacterial infection.

Adverse Childhood Experiences and E-Cigarette Use During Young Adulthood.

BACKGROUND AND OBJECTIVES: E-cigarette use among young people is highly prevalent. Individuals exposed to adverse childhood experiences such as childhood maltreatment (CM) may be at particular risk, as CM has been linked to nicotine dependence. Studies testing the association between CM and e-cigarette use are lacking, including research that examines pathways linking CM to e-cigarette use. METHODS: Using a community sample of young adults (N = 208; ages 18-21), we examined the relationship between CM and e-cigarette use and explored the potential role of impulsivity in linking CM to e-cigarette use via a series of structural equation models controlling for demographic characteristics. RESULTS: CM was significantly associated with lifetime e-cigarette use. Furthermore, CM was associated with negative urgency (NU), whereas NU and sensation seeking were significantly related to lifetime e-cigarette use. NU fully mediated the relationship between CM and lifetime e-cigarette use. CONCLUSIONS AND SCIENTIFIC SIGNIFICANCE: Our results suggest that young adults with a history of CM might be vulnerable to e-cigarette use and that NU played a significant role in linking CM to lifetime e-cigarette use. Addressing NU in young adults with a history of CM might be a useful avenue for preventing e-cigarette use in this population. (Am J Addict 2019;28:303-310).

The inflammasome: Learning from bacterial evasion strategies.

The innate immune system plays a critical role in defense against microbial infection and employs germline-encoded pattern recognition receptors to detect broadly conserved microbial structures or activities. Pattern recognition receptors of the nucleotide binding domain/leucine rich repeat (NLR) family respond to particular microbial products or disruption of cellular physiology, and mediate the activation of an arm of the innate immune response termed the inflammasome. Inflammasomes are multiprotein complexes that are inducibly assembled in response to the contamination of the host cell cytosol by microbial products. Individual NLRs sense the presence of their cognate stimuli, and initiate assembly of inflammasomes via the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and the effector pro-enzyme caspase-1. Inflammasome activation leads to rapid release of pro-inflammatory mediators of the IL-1 family as well as the release of intracellular alarmins due to a lytic form of programmed cell death termed pyroptosis. Over the past 15 years, a great deal has been learned about the mechanisms that drive inflammasome activation in response to infection by diverse pathogens. However, pathogens have also evolved mechanisms to evade or suppress host defenses, and the mechanisms by which pathogens evade inflammasome activation are not well-understood. Here, we will discuss emerging evidence on how diverse pathogens evade inflammasome activation, and what these studies have revealed about inflammasome biology. Deeper understanding of pathogen evasion of inflammasome activation has the potential to lead to development of novel classes of immunomodulatory factors that could be used in the context of human inflammatory diseases.

IL-1R signaling enables bystander cells to overcome bacterial blockade of host protein synthesis.

The innate immune system is critical for host defense against microbial pathogens, yet many pathogens express virulence factors that impair immune function. Here, we used the bacterial pathogen Legionella pneumophila to understand how the immune system successfully overcomes pathogen subversion mechanisms. L. pneumophila replicates within macrophages by using a type IV secretion system to translocate bacterial effectors into the host cell cytosol. As a consequence of effector delivery, host protein synthesis is blocked at several steps, including translation initiation and elongation. Despite this translation block, infected cells robustly produce proinflammatory cytokines, but the basis for this is poorly understood. By using a reporter system that specifically discriminates between infected and uninfected cells within a population, we demonstrate here that infected macrophages produced IL-1α and IL-1ß, but were poor producers of IL-6, TNF, and IL-12, which are critical mediators of host protection. Uninfected bystander cells robustly produced IL-6, TNF, and IL-12, and this bystander response required IL-1 receptor (IL-1R) signaling during early pulmonary infection. Our data demonstrate functional heterogeneity in production of critical protective cytokines and suggest that collaboration between infected and uninfected cells enables the immune system to bypass pathogen-mediated translation inhibition to generate an effective immune response.

Human caspase-4 mediates noncanonical inflammasome activation against gram-negative bacterial pathogens.

Inflammasomes are critical for host defense against bacterial pathogens. In murine macrophages infected by gram-negative bacteria, the canonical inflammasome activates caspase-1 to mediate pyroptotic cell death and release of IL-1 family cytokines. Additionally, a noncanonical inflammasome controlled by caspase-11 induces cell death and IL-1 release. However, humans do not encode caspase-11. Instead, humans encode two putative orthologs: caspase-4 and caspase-5. Whether either ortholog functions similar to caspase-11 is poorly defined. Therefore, we sought to define the inflammatory caspases in primary human macrophages that regulate inflammasome responses to gram-negative bacteria. We find that human macrophages activate inflammasomes specifically in response to diverse gram-negative bacterial pathogens that introduce bacterial products into the host cytosol using specialized secretion systems. In primary human macrophages, IL-1ß secretion requires the caspase-1 inflammasome, whereas IL-1α release and cell death are caspase-1-independent. Instead, caspase-4 mediates IL-1α release and cell death. Our findings implicate human caspase-4 as a critical regulator of noncanonical inflammasome activation that initiates defense against bacterial pathogens in primary human macrophages.