evaluation-of-host-pathogen-responses-and-vaccine-efficacy-in-mice

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

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.

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

Cystic fibrosis (CF) is accompanied with heightened inflammation worsened by drug resistant Burkholderia cenocepacia. Human CF macrophage responses to B. cenocepacia are poorly characterized and variable in the literature. Therefore, we examined human macrophage responses to the epidemic B. cenocepacia J2315 strain in order to identify novel anti-inflammatory targets. Peripheral blood monocyte derived macrophages were obtained from 23 CF and 27 non-CF donors. Macrophages were infected with B. cenocepacia J2315 and analyzed for cytokines, cytotoxicity, and microscopy. CF macrophages demonstrated significant increases in IL-1β, IL-10, MCP-1, and IFN-γ production in comparison to non-CF controls. CF patients on prednisone exhibited globally diminished cytokines compared to controls and other CF patients. CF macrophages also displayed increased bacterial burden and cell death. In conclusion, CF macrophages demonstrate exaggerated IL-1β, IL-10, MCP-1, and IFN-γ production and cell death during B. cenocepacia infection. Treatment with corticosteroids acutely suppressed cytokine responses.

Exaggerated inflammatory responses mediated by Burkholderia cenocepacia in human macrophages derived from Cystic fibrosis patients.

Cystic fibrosis (CF) is the most common inherited lethal disease in Caucasians. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) of which the cftr Î”F508 mutation is the most common. Î”F508 macrophages are intrinsically defective in autophagy due to the sequestration of essential autophagy molecules within unprocessed CFTR aggregates. Defective autophagy allows Burkholderia cenocepacia (B. cepacia) to survive and replicate in Î”F508 macrophages. Infection by B. cepacia poses great risk to CF patients because it causes accelerated lung inflammation and in some cases a lethal necrotizing pneumonia. Autophagy is a cell survival mechanism whereby an autophagosome engulfs non-functional organelles and delivers them to the lysosome for degradation. The ubiquitin binding adaptor protein SQSTM1/p62 is required for the delivery of several ubiquitinated cargos to the autophagosome. In wild-type (WT) macrophages, p62 depletion and overexpression lead to increased and decreased bacterial intracellular survival, respectively. In contrast, depletion of p62 in Î”F508 macrophages results in decreased bacterial survival, whereas overexpression of p62 leads to increased B. cepacia intracellular growth. Interestingly, the depletion of p62 from Î”F508 macrophages results in the release of the autophagy molecule beclin1 (BECN1), from the mutant CFTR aggregates and allows its redistribution and recruitment to the B. cepacia vacuole, mediating the acquisition of the autophagy marker LC3 and bacterial clearance via autophagy. These data demonstrate that p62 differentially dictates the fate of B. cepacia infection in WT and F508 macrophages.

Depletion of the ubiquitin binding adaptor molecule SQSTM1/p62 from macrophages harboring cftr {Delta}F508 mutation improves the delivery of Burkholderia cenocepacia to the autophagic machinery.

Zystische Fibrose (CF) ist die h&auml;ufigste vererbte t&ouml;dliche Krankheit in Caucasians. Es verursacht durch Mutationen in der Cystic Fibrosis transmembrane Conductance Regulator (CFTR), von denen die Mutation des Cftr-&Delta;F508 die h&auml;ufigste ist. &Delta;F508 Makrophagen sind intrinsisch Defekt in Autophagy durch die Beschlagnahme von wesentlichen Autophagy Molek&uuml;le innerhalb unverarbeitete CFTR-Aggregate. Defekte Autophagy erm&ouml;glicht Burkholderia Cenocepacia (B. Cepacia) &uuml;berleben und replizieren in &Delta;F508 Makrophagen. Infektion mit B. Cepacia stellt gro&szlig;en Gefahr CF-Patienten, da beschleunigte Lunge Entz&uuml;ndung verursacht und in einigen F&auml;llen eine t&ouml;dliche nekrotisierende Pneumonie. Autophagy ist ein Zelle &uuml;berleben-Mechanismus, wobei ein Autophagosomen verschlingt nichtfunktionale Organellen und &uuml;bergibt sie an das Lysosom Leistungsabfall. Der Ubiquitin Adapterprotein SQSTM1/p62 Bindung ist f&uuml;r die Lieferung von mehreren Ubiquitinated Ladungen an den Autophagosomen erforderlich. Im Wildtyp (WT) Makrophagen f&uuml;hren p62 Ersch&ouml;pfung und &Uuml;berexpression f&uuml;r erh&ouml;hte und verminderte bakterielle intrazellul&auml;re &uuml;berleben, beziehungsweise. Im Gegensatz dazu f&uuml;hrt Ersch&ouml;pfung der p62 in &Delta;F508 Makrophagen verringerte bakterielle &uuml;berleben, w&auml;hrend eine &Uuml;berexpression von p62 zu erh&ouml;hten B. Cepacia intrazellul&auml;re Wachstum f&uuml;hrt. Interessant ist, die Ersch&ouml;pfung der p62 aus &Delta;F508 Makrophagen in der Ver&ouml;ffentlichung von Autophagy-Molek&uuml;l-beclin1 (BECN1), von den Mutanten CFTR aggregiert und erm&ouml;glicht seine Umverteilung und die Rekrutierung von B. Cepacia-Vakuole, Vermittlung des Erwerbs des Autophagy Markierung LC3 und bakterielle Abfertigung &uuml;ber Autophagy in Verbindung. Diese Daten zeigen, dass p62 differentiell das Schicksal von B. Cepacia Infektion in WT und F508 Makrophagen diktiert.

Ersch&ouml;pfung des Ubiquitin Bindung Adapter Molek&uuml;ls SQSTM1/p62 aus Makrophagen beherbergen Cftr {Delta} F508 Mutation verbessert die Bereitstellung von Burkholderia Cenocepacia f&uuml;r die autophagic Maschinen.

Legionella pneumophila (L. pneumophila) is an intracellular bacterium of human alveolar macrophages that causes Legionnaires' disease. In contrast to humans, most inbred mouse strains are restrictive to L. pneumophila replication. We demonstrate that autophagy targets L. pneumophila vacuoles to lysosomes and that this process requires ubiquitination of L. pneumophila vacuoles and the subsequent binding of the autophagic adaptor p62/SQSTM1 to ubiquitinated vacuoles. The L. pneumophila legA9 encodes for an ankyrin-containing protein with unknown role. We show that the legA9 mutant replicate in WT mice and their bone marrow-derived macrophages. This is the first L. pneumophila mutant to be found to replicate in WT bone marrow-derived macrophages other than the Fla mutant. Less legA9 mutant-containing vacuoles acquired ubiquitin labeling and p62/SQSTM1 staining, evading autophagy uptake and avoiding lysosomal fusion. Thus, we describe a bacterial protein that targets the L. pneumophila-containing vacuole for autophagy uptake.

A bacterial protein promotes the recognition of the Legionella pneumophila vacuole by autophagy.

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.

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

Inflammasomes are multiprotein complexes that include members of the NOD-like receptor family and caspase-1. Caspase-1 is required for the fusion of the Legionella vacuole with lysosomes. Caspase-11, independently of the inflammasome, also promotes phagolysosomal fusion. However, it is unclear how these proteases alter intracellular trafficking. Here, we show that caspase-11 and caspase-1 function in opposing manners to phosphorylate and dephosphorylate cofilin, respectively upon infection with Legionella. Caspase-11 targets cofilin via the RhoA GTPase, whereas caspase-1 engages the Slingshot phosphatase. The absence of either caspase-11 or caspase-1 maintains actin in the polymerized or depolymerized form, respectively and averts the fusion of pathogen-containing vacuoles with lysosomes. Therefore, caspase-11 and caspase-1 converge on the actin machinery with opposing effects to promote vesicular trafficking. 

Caspase-11 and caspase-1 differentially modulate actin polymerization via RhoA and Slingshot proteins to promote bacterial clearance.

Autophagy is a biological process characterized by self-digestion and involves induction of autophagosome formation, leading to degradation of autophagic cargo. Aging is associated with the reduction of autophagy activity leading to neurodegenerative disorders, chronic inflammation, and susceptibility to infection; however, the underlying mechanism is unclear. DNA methylation by DNA methyltransferases reduces the expression of corresponding genes. Since macrophages are major players in inflammation and defense against infection we determined the differences in methylation of autophagy genes in macrophages derived from young and aged mice. We found that promoter regions of Atg5 and LC3B are hypermethylated in macrophages from aged mice and this is accompanied by low gene expression. Treatment of aged mice and their derived macrophages with methyltransferase inhibitor (2)-epigallocatechin-3-gallate (EGCG) or specific DNA methyltransferase 2 (DNMT2) siRNA restored the expression of Atg5 and LC3 in vivo and in vitro. Our study builds a foundation for the development of novel therapeutics aimed to improve autophagy in the elderly population and suggests a role for DNMT2 in DNA methylation activities.

Aging is associated with hypermethylation of autophagy genes in macrophages.

Cystic fibrosis (CF) is a fatal, genetic disorder that critically affects the lungs and is directly caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective CFTR function. Macroautophagy/autophagy is a highly regulated biological process that provides energy during periods of stress and starvation. Autophagy clears pathogens and dysfunctional protein aggregates within macrophages. However, this process is impaired in CF patients and CF mice, as their macrophages exhibit limited autophagy activity. The study of microRNAs (Mirs), and other noncoding RNAs, continues to offer new therapeutic targets. The objective of this study was to elucidate the role of Mirs in dysregulated autophagy-related genes in CF macrophages, and then target them to restore this host-defense function and improve CFTR channel function. We identified the Mirc1/Mir17-92 cluster as a potential negative regulator of autophagy as CF macrophages exhibit decreased autophagy protein expression and increased cluster expression when compared to wild-type (WT) counterparts. The absence or reduced expression of the cluster increases autophagy protein expression, suggesting the canonical inverse relationship between Mirc1/Mir17-92 and autophagy gene expression. An in silico study for targets of Mirs that comprise the cluster suggested that the majority of the Mirs target autophagy mRNAs. Those targets were validated by luciferase assays. Notably, the ability of macrophages expressing mutant F508del CFTR to transport halide through their membranes is compromised and can be restored by downregulation of these inherently elevated Mirs, via restoration of autophagy. In vivo, downregulation of Mir17 and Mir20a partially restored autophagy expression and hence improved the clearance of Burkholderia cenocepacia. Thus, these data advance our understanding of mechanisms underlying the pathobiology of CF and provide a new therapeutic platform for restoring CFTR function and autophagy in patients with CF.

Elevated Mirc1/Mir17-92 cluster expression negatively regulates autophagy and CFTR (cystic fibrosis transmembrane conductance regulator) function in CF macrophages.

Cystic fibrosis (CF) is a multi-organ disorder characterized by chronic sino-pulmonary infections and inflammation. Many patients with CF suffer from repeated pulmonary exacerbations that are predictors of worsened long-term morbidity and mortality. There are no reliable markers that associate with the onset or progression of an exacerbation or pulmonary deterioration. Previously, we found that the Mirc1/Mir17-92a cluster which is comprised of 6 microRNAs (Mirs) is highly expressed in CF mice and negatively regulates autophagy which in turn improves CF transmembrane conductance regulator (CFTR) function. Therefore, here we sought to examine the expression of individual Mirs within the Mirc1/Mir17-92 cluster in human cells and biological fluids and determine their role as biomarkers of pulmonary exacerbations and response to treatment.

The expression of Mirc1/Mir17-92 cluster in sputum samples correlates with pulmonary exacerbations in cystic fibrosis patients.

CASP4/caspase-11-dependent inflammasome activation is important for the clearance of various Gram-negative bacteria entering the host cytosol. Additionally, CASP4 modulates the actin cytoskeleton to promote the maturation of phagosomes harboring intracellular pathogens such as Legionella pneumophila but not those enclosing nonpathogenic bacteria. Nevertheless, this non-inflammatory role of CASP4 regarding the trafficking of vacuolar bacteria remains poorly understood. Macroautophagy/autophagy, a catabolic process within eukaryotic cells, is also implicated in the elimination of intracellular pathogens such as Burkholderia cenocepacia. Here we show that CASP4-deficient macrophages exhibit a defect in autophagosome formation in response to B. cenocepacia infection. The absence of CASP4 causes an accumulation of the small GTPase RAB7, reduced colocalization of B. cenocepacia with LC3 and acidic compartments accompanied by increased bacterial replication in vitro and in vivo. Together, our data reveal a novel role of CASP4 in regulating autophagy in response to B. cenocepacia infection.

CASP4/caspase-11 promotes autophagosome formation in response to bacterial infection.

Autophagy is a highly regulated, biological process that provides energy during periods of stress and starvation. This conserved process also acts as a defense mechanism and clears microbes from the host cell. Autophagy is impaired in Cystic Fibrosis (CF) patients and CF mice, as their cells exhibit low expression levels of essential autophagy molecules. The genetic disorder in CF is due to mutations in the cystic fibrosis transmembrane conductance regulator (cftr) gene that encodes for a chloride channel. CF patients are particularly prone to infection by pathogens that are otherwise cleared by autophagy in healthy immune cells including Burkholderia cenocepacia (B. cenocepacia). The objective of this study is to determine the mechanism underlying weak autophagic activity in CF macrophages and find therapeutic targets to correct it. Using reduced representation bisulfite sequencing (RRBS) to determine DNA methylation profile, we found that the promoter regions of Atg12 in CF macrophages are significantly more methylated than in the wild-type (WT) immune cells, accompanied by low protein expression. The natural product epigallocatechin-3-gallate (EGCG) significantly reduced the methylation of Atg12 promoter improving its expression. Accordingly, EGCG restricted B. cenocepacia replication within CF mice and their derived macrophages by improving autophagy and preventing dissemination. In addition, EGCG improved the function of CFTR protein. Altogether, utilizing RRBS for the first time in the CF field revealed a previously unrecognized mechanism for reduced autophagic activity in CF. Our data also offers a mechanism by which EGCG exerts its positive effects in CF.

Article	Year
Caspase-11 promotes the fusion of phagosomes harboring pathogenic bacteria with lysosomes by modulating actin polymerization. Immunity\| PubMed ID: 22658523	2012
Exaggerated inflammatory responses mediated by Burkholderia cenocepacia in human macrophages derived from Cystic fibrosis patients. Biochemical and biophysical research communications\| PubMed ID: 22728038	2012
Depletion of the ubiquitin binding adaptor molecule SQSTM1/p62 from macrophages harboring cftr {Delta}F508 mutation improves the delivery of Burkholderia cenocepacia to the autophagic machin... The Journal of biological chemistry\| PubMed ID: 23148214	2012
A bacterial protein promotes the recognition of the Legionella pneumophila vacuole by autophagy. European journal of immunology\| PubMed ID: 23420491	2013
Biofilm-derived Legionella pneumophila evades the innate immune response in macrophages. Frontiers in cellular and infection microbiology\| PubMed ID: 23750338	2013
Caspase-11 and caspase-1 differentially modulate actin polymerization via RhoA and Slingshot proteins to promote bacterial clearance. Scientific reports\| PubMed ID: 26686473	2015
Aging is associated with hypermethylation of autophagy genes in macrophages. Epigenetics\| PubMed ID: 26909551	2016
Elevated Mirc1/Mir17-92 cluster expression negatively regulates autophagy and CFTR (cystic fibrosis transmembrane conductance regulator) function in CF macrophages. Autophagy\| PubMed ID: 27541364	2016
The expression of Mirc1/Mir17-92 cluster in sputum samples correlates with pulmonary exacerbations in cystic fibrosis patients. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society\| PubMed ID: 29241629	2018
CASP4/caspase-11 promotes autophagosome formation in response to bacterial infection. Autophagy\| PubMed ID: 30165781	2018
Methylomic correlates of autophagy activity in cystic fibrosis. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society\| PubMed ID: 30737168	2019

Kyle Caution

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