CFTR Modulators Restore Acidification of Autophago-Lysosomes and Bacterial Clearance in Cystic Fibrosis Macrophages.

Cystic fibrosis (CF) human and mouse macrophages are defective in their ability to clear bacteria such as Burkholderia cenocepacia. The autophagy process in CF (F508del) macrophages is halted, and the underlying mechanism remains unclear. Furthermore, the role of CFTR in maintaining the acidification of endosomal and lysosomal compartments in CF cells has been a subject of debate. Using 3D reconstruction of z-stack confocal images, we show that CFTR is recruited to LC3-labeled autophagosomes harboring B. cenocepacia. Using several complementary approaches, we report that CF macrophages display defective lysosomal acidification and degradative function for cargos destined to autophagosomes, whereas non-autophagosomal cargos are effectively degraded within acidic compartments. Notably, treatment of CF macrophages with CFTR modulators (tezacaftor/ivacaftor) improved the autophagy flux, lysosomal acidification and function, and bacterial clearance. In addition, CFTR modulators improved CFTR function as demonstrated by patch-clamp. In conclusion, CFTR regulates the acidification of a specific subset of lysosomes that specifically fuse with autophagosomes. Therefore, our study describes a new biological location and function for CFTR in autophago-lysosomes and clarifies the long-standing discrepancies in the field.

Caspase-11 regulates lung inflammation in response to house dust mites.

Asthma is an inflammatory lung disorder characterized by mucus hypersecretion, cellular infiltration, and bronchial hyper-responsiveness. House dust mites (HDM) are the most prevalent cause of allergic sensitization. Canonical and noncanonical inflammasomes are multiprotein complexes that assemble in response to pathogen or danger-associated molecular patterns (PAMPs or DAMPs). Murine caspase-11 engages the noncanonical inflammasome. We addressed the role of caspase-11 in mediating host responses to HDM and subsequent allergic inflammation using caspase-11-/- mice, which lack caspase-11 while express caspase-1. We found that HDM induce caspase-11 expression in vitro. The presence of IL-4 and IL-13 promote caspase-11 expression. Additionally, caspase-11-/- macrophages show reduced release of IL-6, IL-12, and KC, and express lower levels of costimulatory molecules (e.g., CD40, CD86 and MHCII) in response to HDM stimulation. Notably, HDM sensitization of caspase-11-/- mice resulted in similar levels of IgE responses and hypothermia in response to nasal HDM challenge compared to WT. However, analysis of cell numbers and cytokines in bronchiolar alveolar lavage fluid (BALF) and histopathology of representative lung segments demonstrate altered inflammatory responses and reduced neutrophilia in the airways of the caspase-11-/- mice. These findings indicate that caspase-11 regulates airway inflammation in response to HDM exposure.

Elevated Expression of MiR-17 in Microglia of Alzheimer's Disease Patients Abrogates Autophagy-Mediated Amyloid-ß Degradation.

Autophagy is a proposed route of amyloid-ß (Aß) clearance by microglia that is halted in Alzheimer's Disease (AD), though mechanisms underlying this dysfunction remain elusive. Here, primary microglia from adult AD (5xFAD) mice were utilized to demonstrate that 5xFAD microglia fail to degrade Aß and express low levels of autophagy cargo receptor NBR1. In 5xFAD mouse brains, we show for the first time that AD microglia express elevated levels of microRNA cluster Mirc1/Mir17-92a, which is known to downregulate autophagy proteins. By in situ hybridization in post-mortem AD human tissue sections, we observed that the Mirc1/Mir17-92a cluster member miR-17 is also elevated in human AD microglia, specifically in the vicinity of Aß deposits, compared to non-disease controls. We show that NBR1 expression is negatively correlated with expression of miR-17 in human AD microglia via immunohistopathologic staining in human AD brain tissue sections. We demonstrate in healthy microglia that autophagy cargo receptor NBR1 is required for Aß degradation. Inhibiting elevated miR-17 in 5xFAD mouse microglia improves Aß degradation, autophagy, and NBR1 puncta formation in vitro and improves NBR1 expression in vivo. These findings offer a mechanism behind dysfunctional autophagy in AD microglia which may be useful for therapeutic interventions aiming to improve autophagy function in AD.

Gasdermin D restricts Burkholderia cenocepacia infection in vitro and in vivo.

Burkholderia cenocepacia (B. cenocepacia) is an opportunistic bacterium; causing severe life threatening systemic infections in immunocompromised individuals including cystic fibrosis patients. The lack of gasdermin D (GSDMD) protects mice against endotoxin lipopolysaccharide (LPS) shock. On the other hand, GSDMD promotes mice survival in response to certain bacterial infections. However, the role of GSDMD during B. cenocepacia infection is not yet determined. Our in vitro study shows that GSDMD restricts B. cenocepacia replication within macrophages independent of its role in cell death through promoting mitochondrial reactive oxygen species (mROS) production. mROS is known to stimulate autophagy, hence, the inhibition of mROS or the absence of GSDMD during B. cenocepacia infections reduces autophagy which plays a critical role in the restriction of the pathogen. GSDMD promotes inflammation in response to B. cenocepacia through mediating the release of inflammasome dependent cytokine (IL-1ß) and an independent one (CXCL1) (KC). Additionally, different B. cenocepacia secretory systems (T3SS, T4SS, and T6SS) contribute to inflammasome activation together with bacterial survival within macrophages. In vivo study confirmed the in vitro findings and showed that GSDMD restricts B. cenocepacia infection and dissemination and stimulates autophagy in response to B. cenocepacia. Nevertheless, GSDMD promotes lung inflammation and necrosis in response to B. cenocepacia without altering mice survival. This study describes the double-edged functions of GSDMD in response to B. cenocepacia infection and shows the importance of GSDMD-mediated mROS in restriction of B. cenocepacia.

Pyroptotic and non-pyroptotic effector functions of caspase-11.

Innate immune cells, epithelial cells, and many other cell types are capable of detecting infection or tissue injury, thus mounting regulated immune response. Inflammasomes are highly sophisticated and effective orchestrators of innate immunity. These oligomerized multiprotein complexes are at the center of various innate immune pathways, including modulation of the cytoskeleton, production and maturation of cytokines, and control of bacterial growth and cell death. Inflammasome assembly often results in caspase-1 activation, which is an inflammatory caspase that is involved in pyroptotic cell death and release of inflammatory cytokines in response to pathogen patterns and endogenous danger stimuli. However, the nature of stimuli and inflammasome components are diverse. Caspase-1 activation mediated release of mature IL-1ß and IL-18 in response to canonical stimuli initiated by NOD-like receptor (NLR), and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). On the other hand, caspase-11 delineates a non-canonical inflammasome that promotes pyroptotic cell death and non-pyroptotic functions in response to non-canonical stimuli. Caspase-11 in mice and its homologues in humans (caspase-4/5) belong to caspase-1 family of cysteine proteases, and play a role in inflammation. Knockout mice provided new genetic tools to study inflammatory caspases and revealed the role of caspase-11 in mediating septic shock in response to lethal doses of lipopolysaccharide (LPS). Recognition of LPS mediates caspase-11 activation, which promotes a myriad of downstream effects that include pyroptotic and non-pyroptotic effector functions. Therefore, the physiological functions of caspase-11 are much broader than its previously established roles in apoptosis and cytokine maturation. Inflammation induced by exogenous or endogenous agents can be detrimental and, if excessive, can result in organ and tissue damage. Consequently, the existence of sophisticated mechanisms that tightly regulate the specificity and sensitivity of inflammasome pathways provides a fine-tuning balance between adequate immune response and minimal tissue damage. In this review, we summarize effector functions of caspase-11.

Insights Into Mucosal Innate Immune Responses in House Dust Mite-Mediated Allergic Asthma.

The prevalence of asthma has been rising steadily for several decades, and continues to be a major public health and global economic burden due to both direct and indirect costs. Asthma is defined as chronic heterogeneous inflammatory diseases characterized by airway obstruction, mucus production and bronchospasm. Different endotypes of asthma are being recognized based on the distinct pathophysiology, genetic predisposition, age, prognosis, and response to remedies. Mucosal innate response to environmental triggers such as pollen, cigarette smoke, fragrances, viral infection, and house dust mite (HDM) are now recognized to play an important role in allergic asthma. HDM are the most pervasive allergens that co-habitat with us, as they are ubiquitous in-house dusts, mattress and bedsheets, and feed on a diet of exfoliated human skin flakes. Dermatophagoides pteronyssinus, is one among several HDM identified up to date. During the last decade, extensive studies have been fundamental in elucidating the interactions between HDM allergens, the host immune systems and airways. Moreover, the paradigm in the field of HDM-mediated allergy has been shifted away from being solely a Th2-geared to a complex response orchestrated via extensive crosstalk between the epithelium, professional antigen presenting cells (APCs) and components of the adaptive immunity. In fact, HDM have several lessons to teach us about their allergenicity, the complex interactions that stimulate innate immunity in initiating and perpetuating the lung inflammation. Herein, we review main allergens of Dermatophagoides pteronyssinus and their interactions with immunological sentinels that promote allergic sensitization and activation of innate immunity, which is critical for the development of the Th2 biased adaptive immunity to HDM allergens and development of allergic asthma.

Caspase-11 Mediates Neutrophil Chemotaxis and Extracellular Trap Formation During Acute Gouty Arthritis Through Alteration of Cofilin Phosphorylation.

Gout is characterized by attacks of arthritis with hyperuricemia and monosodium urate (MSU) crystal-induced inflammation within joints. Innate immune responses are the primary drivers for tissue destruction and inflammation in gout. MSU crystals engage the Nlrp3 inflammasome, leading to the activation of caspase-1 and production of IL-1ß and IL-18 within gout-affected joints, promoting the influx of neutrophils and monocytes. Here, we show that caspase-11-/- mice and their derived macrophages produce significantly reduced levels of gout-specific cytokines including IL-1ß, TNFα, IL-6, and KC, while others like IFNγ and IL-12p70 are not altered. IL-1ß induces the expression of caspase-11 in an IL-1 receptor-dependent manner in macrophages contributing to the priming of macrophages during sterile inflammation. The absence of caspase-11 reduced the ability of macrophages and neutrophils to migrate in response to exogenously injected KC in vivo. Notably, in vitro, caspase-11-/- neutrophils displayed random migration in response to a KC gradient when compared to their WT counterparts. This phenotype was associated with altered cofilin phosphorylation. Unlike their wild-type counterparts, caspase-11-/- neutrophils also failed to produce neutrophil extracellular traps (NETs) when treated with MSU. Together, this is the first report demonstrating that caspase-11 promotes neutrophil directional trafficking and function in an acute model of gout. Caspase-11 also governs the production of inflammasome-dependent and -independent cytokines from macrophages. Our results offer new, previously unrecognized functions for caspase-11 in macrophages and neutrophils that may apply to other neutrophil-mediated disease conditions besides gout.

Caspase-11 counteracts mitochondrial ROS-mediated clearance of Staphylococcus aureus in macrophages.

Methicillin-resistant Staphylococcus aureus (MRSA) is a growing health concern due to increasing resistance to antibiotics. As a facultative intracellular pathogen, MRSA is capable of persisting within professional phagocytes including macrophages. Here, we identify a role for CASP11 in facilitating MRSA survival within murine macrophages. We show that MRSA actively prevents the recruitment of mitochondria to the vicinity of the vacuoles they reside in to avoid intracellular demise. This process requires CASP11 since its deficiency allows increased association of MRSA-containing vacuoles with mitochondria. The induction of mitochondrial superoxide by antimycin A (Ant A) improves MRSA eradication in casp11-/- cells, where mitochondria remain in the vicinity of the bacterium. In WT macrophages, Ant A does not affect MRSA persistence. When mitochondrial dissociation is prevented by the actin depolymerizing agent cytochalasin D, Ant A effectively reduces MRSA numbers. Moreover, the absence of CASP11 leads to reduced cleavage of CASP1, IL-1ß, and CASP7, as well as to reduced production of CXCL1/KC. Our study provides a new role for CASP11 in promoting the persistence of Gram-positive bacteria.

Factors Mediating Environmental Biofilm Formation by Legionella pneumophila.

Legionella pneumophila (L. pneumophila) is an opportunistic waterborne pathogen and the causative agent for Legionnaires' disease, which is transmitted to humans via inhalation of contaminated water droplets. The bacterium is able to colonize a variety of man-made water systems such as cooling towers, spas, and dental lines and is widely distributed in multiple niches, including several species of protozoa In addition to survival in planktonic phase, L. pneumophila is able to survive and persist within multi-species biofilms that cover surfaces within water systems. Biofilm formation by L. pneumophila is advantageous for the pathogen as it leads to persistence, spread, resistance to treatments and an increase in virulence of this bacterium. Furthermore, Legionellosis outbreaks have been associated with the presence of L. pneumophila in biofilms, even after the extensive chemical and physical treatments. In the microbial consortium-containing L. pneumophila among other organisms, several factors either positively or negatively regulate the presence and persistence of L. pneumophila in this bacterial community. Biofilm-forming L. pneumophila is of a major importance to public health and have impact on the medical and industrial sectors. Indeed, prevention and removal protocols of L. pneumophila as well as diagnosis and hospitalization of patients infected with this bacteria cost governments billions of dollars. Therefore, understanding the biological and environmental factors that contribute to persistence and physiological adaptation in biofilms can be detrimental to eradicate and prevent the transmission of L. pneumophila. In this review, we focus on various factors that contribute to persistence of L. pneumophila within the biofilm consortium, the advantages that the bacteria gain from surviving in biofilms, genes and gene regulation during biofilm formation and finally challenges related to biofilm resistance to biocides and anti-Legionella treatments.

The Sphingosine-1-Phosphate Lyase (LegS2) Contributes to the Restriction of Legionella pneumophila in Murine Macrophages.

L. pneumophila is the causative agent of Legionnaires' disease, a human illness characterized by severe pneumonia. In contrast to those derived from humans, macrophages derived from most mouse strains restrict L. pneumophila replication. The restriction of L. pneumophila replication has been shown to require bacterial flagellin, a component of the type IV secretion system as well as the cytosolic NOD-like receptor (NLR) Nlrc4/ Ipaf. These events lead to caspase-1 activation which, in turn, activates caspase-7. Following caspase-7 activation, the phagosome-containing L. pneumophila fuses with the lysosome, resulting in the restriction of L. pneumophila growth. The LegS2 effector is injected by the type IV secretion system and functions as a sphingosine 1-phosphate lyase. It is homologous to the eukaryotic sphingosine lyase (SPL), an enzyme required in the terminal steps of sphingolipid metabolism. Herein, we show that mice Bone Marrow-Derived Macrophages (BMDMs) and human Monocyte-Derived Macrophages (hMDMs) are more permissive to L. pneumophila legS2 mutants than wild-type (WT) strains. This permissiveness to L. pneumophila legS2 is neither attributed to abolished caspase-1, caspase-7 or caspase-3 activation, nor due to the impairment of phagosome-lysosome fusion. Instead, an infection with the legS2 mutant resulted in the reduction of some inflammatory cytokines and their corresponding mRNA; this effect is mediated by the inhibition of the nuclear transcription factor kappa-B (NF-κB). Moreover, BMDMs infected with L. pneumophila legS2 mutant showed elongated mitochondria that resembles mitochondrial fusion. Therefore, the absence of LegS2 effector is associated with reduced NF-κB activation and atypical morphology of mitochondria.

Biofilm-derived Legionella pneumophila evades the innate immune response in macrophages.

Legionella pneumophila, the causative agent of Legionnaire's disease, replicates in human alveolar macrophages to establish infection. There is no human-to-human transmission and the main source of infection is L. pneumophila biofilms established in air conditioners, water fountains, and hospital equipments. The biofilm structure provides protection to the organism from disinfectants and antibacterial agents. L. pneumophila infection in humans is characterized by a subtle initial immune response, giving time for the organism to establish infection before the patient succumbs to pneumonia. Planktonic L. pneumophila elicits a strong immune response in murine, but not in human macrophages enabling control of the infection. Interactions between planktonic L. pneumophila and murine or human macrophages have been studied for years, yet the interface between biofilm-derived L. pneumophila and macrophages has not been explored. Here, we demonstrate that biofilm-derived L. pneumophila replicates significantly more in murine macrophages than planktonic bacteria. In contrast to planktonic L. pneumophila, biofilm-derived L. pneumophila lacks flagellin expression, do not activate caspase-1 or -7 and trigger less cell death. In addition, while planktonic L. pneumophila is promptly delivered to lysosomes for degradation, most biofilm-derived bacteria were enclosed in a vacuole that did not fuse with lysosomes in murine macrophages. This study advances our understanding of the innate immune response to biofilm-derived L. pneumophila and closely reproduces the natural mode of infection in human.

Caspase-11 promotes the fusion of phagosomes harboring pathogenic bacteria with lysosomes by modulating actin polymerization.

Inflammasomes are multiprotein complexes that include members of the NLR (nucleotide-binding domain leucine-rich repeat containing) family and caspase-1. Once bacterial molecules are sensed within the macrophage, the inflammasome is assembled, mediating the activation of caspase-1. Caspase-11 mediates caspase-1 activation in response to lipopolysaccharide and bacterial toxins, and yet its role during bacterial infection is unknown. Here, we demonstrated that caspase-11 was dispensable for caspase-1 activation in response to Legionella, Salmonella, Francisella, and Listeria. We also determined that active mouse caspase-11 was required for restriction of L. pneumophila infection. Similarly, human caspase-4 and caspase-5, homologs of mouse caspase-11, cooperated to restrict L. pneumophila infection in human macrophages. Caspase-11 promoted the fusion of the L. pneumophila vacuole with lysosomes by modulating actin polymerization through cofilin. However, caspase-11 was dispensable for the fusion of lysosomes with phagosomes containing nonpathogenic bacteria, uncovering a fundamental difference in the trafficking of phagosomes according to their cargo.

Activation of the pyrin inflammasome by intracellular Burkholderia cenocepacia.

Burkholderia cenocepacia is an opportunistic pathogen that causes chronic infection and induces progressive respiratory inflammation in cystic fibrosis patients. Recognition of bacteria by mononuclear cells generally results in the activation of caspase-1 and processing of IL-1ß, a major proinflammatory cytokine. In this study, we report that human pyrin is required to detect intracellular B. cenocepacia leading to IL-1ß processing and release. This inflammatory response involves the host adapter molecule ASC and the bacterial type VI secretion system (T6SS). Human monocytes and THP-1 cells stably expressing either small interfering RNA against pyrin or YFP-pyrin and ASC (YFP-ASC) were infected with B. cenocepacia and analyzed for inflammasome activation. B. cenocepacia efficiently activates the inflammasome and IL-1ß release in monocytes and THP-1. Suppression of pyrin levels in monocytes and THP-1 cells reduced caspase-1 activation and IL-1ß release in response to B. cenocepacia challenge. In contrast, overexpression of pyrin or ASC induced a robust IL-1ß response to B. cenocepacia, which correlated with enhanced host cell death. Inflammasome activation was significantly reduced in cells infected with T6SS-defective mutants of B. cenocepacia, suggesting that the inflammatory reaction is likely induced by an as yet uncharacterized effector(s) of the T6SS. Together, we show for the first time, to our knowledge, that in human mononuclear cells infected with B. cenocepacia, pyrin associates with caspase-1 and ASC forming an inflammasome that upregulates mononuclear cell IL-1ß processing and release.

Burkholderia cenocepacia O polysaccharide chain contributes to caspase-1-dependent IL-1beta production in macrophages.

Burkholderia cenocepacia infections in CF patients involve heightened inflammation, fatal sepsis, and high antibiotic resistance. Proinflammatory IL-1ß secretion is important in airway inflammation and tissue damage. However, little is known about this pathway in macrophages upon B. cenocepacia infection. We report here that murine macrophages infected with B. cenocepacia K56-2 produce proinflammatory cytokine IL-1ß in a TLR4 and caspase-1-mediated manner. We also determined that the OPS (O antigen) of B. cenocepacia LPS contributes to IL-1ß production and pyroptotic cell death. Furthermore, we showed that the malfunction of the CFTR channel augmented IL-1ß production upon B. cenocepacia infection of murine macrophages. Taken together, we identified eukaryotic and bacterial factors that contribute to inflammation during B. cenocepacia infection, which may aid in the design of novel approaches to control pulmonary inflammation.

Analysis of HmsH and its role in plague biofilm formation.

The Yersinia pestis Hms(+) phenotype is a manifestation of biofilm formation that causes adsorption of Congo red and haemin at 26 degrees C but not at 37 degrees C. This phenotype is required for blockage of the proventricular valve of the oriental rat flea and plays a role in transmission of bubonic plague from fleas to mammals. Genes responsible for this phenotype are located in three separate operons, hmsHFRS, hmsT and hmsP. HmsH and HmsF are outer membrane (OM) proteins, while the other four Hms proteins are located in the inner membrane. According to the Hidden Markov Method-based predictor, HmsH has a large N terminus in the periplasm, a beta-barrel structure with 16 beta-strands that traverse the OM, eight surface-exposed loops, and seven short turns connecting the beta-strands on the periplasmic side. Here, we demonstrate that HmsH is a heat-modifiable protein, a characteristic of other beta-barrel proteins, thereby supporting the bioinformatics analysis. Alanine scanning mutagenesis was used to identify conserved amino acids in the HmsH-like family that are critical for the function of HmsH in biofilm formation. Of 23 conserved amino acids mutated, four residues affected HmsH function and three likely caused protein instability. We used formaldehyde cross-linking to demonstrate that HmsH interacts with HmsF but not with HmsR, HmsS, HmsT or HmsP. Loss-of-function HmsH variants with single alanine substitutions retained their beta-structure and interaction with HmsF. Finally, using a polar hmsH : : mini-kan mutant, we demonstrated that biofilm development is not important for the pathogenesis of bubonic or pneumonic plague in mice.