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Acinetobacter baumannii is an emerging nosocomial, opportunistic pathogen with growing clinical significance globally. A . baumannii has an exceptional ability to rapidly develop drug resistance. It is frequently responsible for ventilator-associated pneumonia in clinical settings and inflammation resulting in severe sepsis. The inflammatory response is mediated by host pattern-recognition receptors and the inflammasomes. Inflammasome activation triggers inflammatory responses, including the secretion of the pro-inflammatory cytokines IL-1β and IL-18, the recruitment of innate immune effectors against A . baumannii infection, and the induction programmed cell death by pyroptosis. An important knowledge gap is how variation among clinical isolates affects the host’s innate response and activation of the inflammasome during A . baumannii infection. In this study, we compared nine A . baumannii strains, including clinical locally-acquired isolates, in their ability to induce activation of the inflammasome and programmed cell death in primary macrophages, epithelial lung cell line and mice. We found a variation in survival outcomes of mice and bacterial dissemination in organs among three commercially available A . baumannii strains, likely due to the differences in virulence between strains. Interestingly, we found variability among A . baumannii strains in activation of the NLRP3 inflammasome, non-canonical Caspase-11 pathway, plasmatic secretion of the pro-inflammatory cytokine IL-1β and programmed cell death. Our study highlights the importance of utilising multiple bacterial strains and clinical isolates with different virulence to investigate the innate immune response to A . baumannii infection.

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells in pancreatic islets are progressively destroyed. Clinical trials of immunotherapies in recently diagnosed T1D patients have only transiently and partially impacted the disease course, suggesting that other approaches are required. Our previous studies have demonstrated that heparan sulfate (HS), a glycosaminoglycan conventionally expressed in extracellular matrix, is present at high levels inside normal mouse beta cells. Intracellular HS was shown to be critical for beta cell survival and protection from oxidative damage. T1D development in Non-Obese Diabetic (NOD) mice correlated with loss of islet HS and was prevented by inhibiting HS degradation by the endoglycosidase, heparanase. In this study we investigated the distribution of HS and heparan sulfate proteoglycan (HSPG) core proteins in normal human islets, a role for HS in human beta cell viability and the clinical relevance of intra-islet HS and HSPG levels, compared to insulin, in human T1D. In normal human islets, HS (identified by 10E4 mAb) co-localized with insulin but not glucagon and correlated with the HSPG core proteins for collagen type XVIII (Col18) and syndecan-1 (Sdc1). Insulin-positive islets of T1D pancreases showed significant loss of HS, Col18 and Sdc1 and heparanase was strongly expressed by islet-infiltrating leukocytes. Human beta cells cultured with HS mimetics showed significantly improved survival and protection against hydrogen peroxide-induced death, suggesting that loss of HS could contribute to beta cell death in T1D. We conclude that HS depletion in beta cells, possibly due to heparanase produced by insulitis leukocytes, may function as an important mechanism in the pathogenesis of human T1D. Our findings raise the possibility that intervention therapy with dual activity HS replacers/heparanase inhibitors could help to protect the residual beta cell mass in patients recently diagnosed with T1D.

The morbidity and mortality caused by the globally prevalent human respiratory pathogen respiratory syncytial virus (RSV) approaches that world-wide of influenza. We previously demonstrated that the RSV matrix (M) protein shuttles, in signal-dependent fashion, between host cell nucleus and cytoplasm, and that this trafficking is central to RSV replication and assembly. Here we analyze in detail the nuclear role of M for the first time using a range of novel approaches, including quantitative analysis of de novo cell transcription in situ in the presence or absence of RSV infection or M ectopic expression, as well as in situ DNA binding. We show that M, dependent on amino acids 110–183, inhibits host cell transcription in RSV-infected cells as well as cells transfected to express M, with a clear correlation between nuclear levels of M and the degree of transcriptional inhibition. Analysis of bacterially expressed M protein and derivatives thereof mutated in key residues within M’s RNA binding domain indicates that M can bind to DNA as well as RNA in a cell-free system. Parallel results for point-mutated M derivatives implicate arginine 170 and lysine 172, in contrast to other basic residues such as lysine 121 and 130, as critically important residues for inhibition of transcription and DNA binding both in situ and in vitro. Importantly, recombinant RSV carrying arginine 170/lysine 172 mutations shows attenuated infectivity in cultured cells and in an animal model, concomitant with altered inflammatory responses. These findings define an RSV M-chromatin interface critical for host transcriptional inhibition in infection, with important implications for anti-RSV therapeutic development.

Multidrug-resistant (MDR) Acinetobacter baumannii are of major concern worldwide due to their resistance to last resort carbapenem and polymyxin antibiotics. To develop an effective treatment strategy, it is critical to better understand how an A. baumannii MDR bacterium interacts with its mammalian host. Pattern-recognition receptors sense microbes, and activate the inflammasome pathway, leading to pro-inflammatory cytokine production and programmed cell death. Here, we examined the effects of a systemic MDR A. baumannii infection and found that MDR A. baumannii activate the NLRP3 inflammasome complex predominantly via the non-canonical caspase-11-dependent pathway. We show that caspase-1 and caspase-11-deficient mice are protected from a virulent MDR A. baumannii strain by maintaining a balance between protective and deleterious inflammation. Caspase-11-deficient mice also compromise between effector cell recruitment, phagocytosis, and programmed cell death in the lung during infection. Importantly, we found that cytosolic immunity - mediated by guanylate-binding protein 1 (GBP1) and type I interferon signalling - orchestrates caspase-11-dependent inflammasome activation. Together, our results suggest that non-canonical inflammasome activation via the (Interferon) IFN pathway plays a critical role in the host response against MDR A. baumannii infection. Genetic and Immunological analysis reveal that non-canonical inflammasome activation via type I Interferon pathway plays a critical role in the host response against a multidrug-resistant Acinetobacter baumannii infection in mice.

Airway remodeling is an important process in response to repetitive inflammatory‐mediated airway wall injuries. This is characterized by profound changes and reorganizations at the cellular and molecular levels of the lung tissue. It is of particular importance to understand the mechanisms involved in airway remodeling, as this is strongly associated with severe asthma leading to devastating airway dysfunction. In this study, we have investigated the transforming growth factor‐β (TGFβ, a proinflammatory mediator)‐activated fibroblast to myofibroblast transdifferentiation pathway, which plays a key role in asthma‐related airway remodeling. We show that TGFβ induces fibroblast to myofibroblast transdifferentiation by the expression of αSMA, a specific myofibroblast marker. Furthermore, Smad2/Smad3 gene and protein expression patterns are different between fibroblasts and myofibroblasts. Such a change in expression patterns reveals an important role of these proteins in the cellular phenotype as well as their regulation by TGFβ during cellular transdifferentiation. Interestingly, our data show a myofibroblastic TGFβ‐mediated increase in glucocorticoid receptor (GR) expression and a preferential localization of GR in the nucleus, compared to in fibroblasts. Furthermore, the GRβ (nonfunctional GR isoform) is increased relative to GRα (functional isoform) in myofibroblasts. These results are interesting as they support the idea of a GRβ‐mediated glucocorticoid resistance observed in the severe asthmatic population. All together, we provide evidence that key players are involved in the TGFβ‐mediated fibroblast to myofibroblast transdifferentiation pathway in a human lung fibroblast cell line. These players could be the targets of new treatments to limit airway remodeling and reverse glucocorticoid resistance in severe asthma. Myofibroblasts have an important role in airway remodeling. We show a myofibroblastic transforming growth factor‐β (TGFβ)‐mediated increase in glucocorticoid receptor (GR) expression and a preferential localization of GR in the nucleus, compared to fibroblasts. Furthermore, the GRβ subunit (nonfunctional isoform) is increased relative to GRα (functional isoform) in myofibroblasts. These results support the idea of GRβ mediated glucocorticoid resistance as observed in severe asthmatic remodeled airways.

Plasmodium falciparum malaria causes half a million deaths per year, with up to 9% of this mortality caused by cerebral malaria (CM). One of the major processes contributing to the development of CM is an excess of host inflammatory cytokines. Recently K+ signaling has emerged as an important mediator of the inflammatory response to infection; we therefore investigated whether mice carrying an ENU induced activation of the electroneutral K+ channel KCC1 had an altered response to Plasmodium berghei . Here we show that Kcc1 M935K/M935K mice are protected from the development of experimental cerebral malaria, and that this protection is associated with an increased CD4+ and TNFa response. This is the first description of a K+ channel affecting the development of experimental cerebral malaria.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An important component in host resistance to malaria infection are inherited mutations that give rise to abnormalities and deficiencies in erythrocyte proteins and enzymes. Understanding how such mutations confer protection against the disease may be useful for developing new treatment strategies. A mouse ENU-induced mutagenesis screen for novel malaria resistance-conferring mutations identified a novel non-sense mutation in the gene encoding porphobilinogen deaminase (PBGD) in mice, denoted here as . Heterozygote mice exhibited ~50% reduction in cellular PBGD activity in both mature erythrocytes and reticulocytes, although enzyme activity was ~10 times higher in reticulocytes than erythrocytes. When challenged with blood-stage , which preferentially infects erythrocytes, heterozygote mice showed a modest but significant resistance to infection, including reduced parasite growth. A series of assays conducted to investigate the mechanism of resistance indicated that mutant erythrocyte invasion by was normal, but that following intraerythrocytic establishment a significantly greater proportions of parasites died and therefore, affected their ability to propagate. The resistance phenotype was not recapitulated in -deficient mice infected with , which prefers reticulocytes, or when was cultured in erythrocytes from patients with acute intermittent porphyria (AIP), which had modest (20-50%) reduced levels of PBGD. Furthermore, the growth of -null and -null parasites, which grew at the same rate as their wild-type counterparts in normal cells, were not affected by the PBGD-deficient background of the AIP erythrocytes or -deficient mice. Our results confirm the dispensability of parasite PBGD for infection and intraerythrocytic growth of , but for the first time identify a requirement for host erythrocyte PBGD by during blood stage infection.

Acinetobacter baumannii is an emerging nosocomial, opportunistic pathogen with growing clinical significance. Acinetobacter baumannii has an exceptional ability to rapidly develop drug resistance and to adhere to abiotic surfaces, including medical equipment, significantly promoting bacterial spread and also limiting our ability to control A. baumannii infections. Consequently, A. baumannii is frequently responsible for ventilator-associated pneumonia in clinical settings. In order to develop an effective treatment strategy, understanding host-pathogen interactions during A. baumannii infection is crucial. Various A. baumannii virulence factors have been identified as targets of host innate pattern-recognition receptors, which leads to activation of downstream inflammasomes to develop inflammatory responses, and the recruitment of innate immune effectors against A. baumannii infection. To counteract host immune attack, A. baumannii regulates its expression of different virulence factors. This review summarizes the significance of mechanisms of host-bacteria interaction, as well as different bacteria and host defense mechanisms during A. baumannii infection.

Staphylococcus aureus small-colony variants (SCVs) are associated with unusually chronic and persistent infections despite active antibiotic treatment. The molecular basis for this clinically important phenomenon is poorly understood, hampered by the instability of the SCV phenotype. Here we investigated the genetic basis for an unstable S. aureus SCV that arose spontaneously while studying rifampicin resistance. This SCV showed no nucleotide differences across its genome compared with a normal-colony variant (NCV) revertant, yet the SCV presented the hallmarks of S. aureus linked to persistent infection: down-regulation of virulence genes and reduced hemolysis and neutrophil chemotaxis, while exhibiting increased survival in blood and ability to invade host cells. Further genome analysis revealed chromosome structural variation uniquely associated with the SCV. These variations included an asymmetric inversion across half of the S. aureus chromosome via recombination between type I restriction modification system (T1RMS) genes, and the activation of a conserved prophage harboring the immune evasion cluster (IEC). Phenotypic reversion to the wild-type–like NCV state correlated with reversal of the chromosomal inversion (CI) and with prophage stabilization. Further analysis of 29 complete S. aureus genomes showed strong signatures of recombination between hsdMS genes, suggesting that analogous CI has repeatedly occurred during S. aureus evolution. Using qPCR and long-read amplicon deep sequencing, we detected subpopulations with T1RMS rearrangements causing CIs and prophage activation across major S. aureus lineages. Here, we have discovered a previously unrecognized and widespread mechanism of reversible genomic instability in S. aureus associated with SCV generation and persistent infections.