Self-oxygenation of engineered living tissues orchestrates osteogenic commitment of mesenchymal stem cells.

Oxygenating biomaterials can alleviate anoxic stress, stimulate vascularization, and improve engraftment of cellularized implants. However, the effects of oxygen-generating materials on tissue formation have remained largely unknown. Here, we investigate the impact of calcium peroxide (CPO)-based oxygen-generating microparticles (OMPs) on the osteogenic fate of human mesenchymal stem cells (hMSCs) under a severely oxygen deficient microenvironment. To this end, CPO is microencapsulated in polycaprolactone to generate OMPs with prolonged oxygen release. Gelatin methacryloyl (GelMA) hydrogels containing osteogenesis-inducing silicate nanoparticles (SNP hydrogels), OMPs (OMP hydrogels), or both SNP and OMP (SNP/OMP hydrogels) are engineered to comparatively study their effect on the osteogenic fate of hMSCs. OMP hydrogels associate with improved osteogenic differentiation under both normoxic and anoxic conditions. Bulk mRNAseq analyses suggest that OMP hydrogels under anoxia regulate osteogenic differentiation pathways more strongly than SNP/OMP or SNP hydrogels under either anoxia or normoxia. Subcutaneous implantations reveal a stronger host cell invasion in SNP hydrogels, resulting in increased vasculogenesis. Furthermore, time-dependent expression of different osteogenic factors reveals progressive differentiation of hMSCs in OMP, SNP, and SNP/OMP hydrogels. Our work demonstrates that endowing hydrogels with OMPs can induce, improve, and steer the formation of functional engineered living tissues, which holds potential for numerous biomedical applications, including tissue regeneration and organ replacement therapy.

IL-33 mediates Pseudomonas induced airway fibrogenesis and is associated with CLAD.

BACKGROUND: Long term outcomes of lung transplantation are impacted by the occurrence of chronic lung allograft dysfunction (CLAD). Recent evidence suggests a role for the lung microbiome in the occurrence of CLAD, but the exact mechanisms are not well defined. We hypothesize that the lung microbiome inhibits epithelial autophagic clearance of pro-fibrotic proteins in an IL-33 dependent manner, thereby augmenting fibrogenesis and risk for CLAD. METHODS: Autopsy derived CLAD and non-CLAD lungs were collected. IL-33, P62 and LC3 immunofluorescence was performed and assessed using confocal microscopy. Pseudomonas aeruginosa (PsA), Streptococcus Pneumoniae (SP), Prevotella Melaninogenica (PM), recombinant IL-33 or PsA-lipopolysaccharide was co-cultured with primary human bronchial epithelial cells (PBEC) and lung fibroblasts in the presence or absence of IL-33 blockade. Western blot analysis and quantitative reverse transcription (qRT) PCR was performed to evaluate IL-33 expression, autophagy, cytokines and fibroblast differentiation markers. These experiments were repeated after siRNA silencing and upregulation (plasmid vector) of Beclin-1. RESULTS: Human CLAD lungs demonstrated markedly increased expression of IL-33 and reduced basal autophagy compared to non-CLAD lungs. Exposure of co-cultured PBECs to PsA, SP induced IL-33, and inhibited PBEC autophagy, while PM elicited no significant response. Further, PsA exposure increased myofibroblast differentiation and collagen formation. IL-33 blockade in these co-cultures recovered Beclin-1, cellular autophagy and attenuated myofibroblast activation in a Beclin-1 dependent manner. CONCLUSION: CLAD is associated with increased airway IL-33 expression and reduced basal autophagy. PsA induces a fibrogenic response by inhibiting airway epithelial autophagy in an IL-33 dependent manner.

A Plasmodium falciparum lysophospholipase regulates host fatty acid flux via parasite lipid storage to enable controlled asexual schizogony.

Phospholipid metabolism is crucial for membrane biogenesis and homeostasis of Plasmodium falciparum. To generate such phospholipids, the parasite extensively scavenges, recycles, and reassembles host lipids. P. falciparum possesses an unusually large number of lysophospholipases, whose roles and importance remain to be elucidated. Here, we functionally characterize one P. falciparum lysophospholipase, PfLPL3, to reveal its key role in parasite propagation during asexual blood stages. PfLPL3 displays a dynamic localization throughout asexual stages, mainly localizing in the host-parasite interface. Inducible knockdown of PfLPL3 disrupts parasite development from trophozoites to schizont, inducing a drastic reduction in merozoite progenies. Detailed lipidomic analyses show that PfLPL3 generates fatty acids from scavenged host lipids to generate neutral lipids. These are then timely mobilized to allow schizogony and merozoite formation. We then identify inhibitors of PfLPL3 from Medicine for Malaria Venture (MMV) with potent antimalarial activity, which could also serve as pertinent chemical tools to study parasite lipid synthesis.

Interferon-dependent signaling is critical for viral clearance in airway neutrophils.

Neutrophilic inflammation characterizes several respiratory viral infections, including COVID-19-related acute respiratory distress syndrome, although its contribution to disease pathogenesis remains poorly understood. Blood and airway immune cells from 52 patients with severe COVID-19 were phenotyped by flow cytometry. Samples and clinical data were collected at 2 separate time points to assess changes during ICU stay. Blockade of type I interferon and interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) signaling was performed in vitro to determine their contribution to viral clearance in A2 neutrophils. We identified 2 neutrophil subpopulations (A1 and A2) in the airway compartment, where loss of the A2 subset correlated with increased viral burden and reduced 30-day survival. A2 neutrophils exhibited a discrete antiviral response with an increased interferon signature. Blockade of type I interferon attenuated viral clearance in A2 neutrophils and downregulated IFIT3 and key catabolic genes, demonstrating direct antiviral neutrophil function. Knockdown of IFIT3 in A2 neutrophils led to loss of IRF3 phosphorylation, with consequent reduced viral catabolism, providing the first discrete mechanism to our knowledge of type I interferon signaling in neutrophils. The identification of this neutrophil phenotype and its association with severe COVID-19 outcomes emphasizes its likely importance in other respiratory viral infections and potential for new therapeutic approaches in viral illness.

Type I interferon-dependent IFIT3 signaling is critical for viral clearance in airway neutrophils.

Neutrophilic inflammation characterizes several respiratory viral infections including COVID-19-related ARDS, although its contribution to disease pathogenesis remains poorly understood. Here, we identified two neutrophil subpopulations (A1 and A2) in the airway compartment of 52 severe COVID-19 subjects, where loss of the A2 subset correlated with increased viral burden and reduced 30-days survival. A2 neutrophils showcased a discrete antiviral response with an increased interferon signature. Blockade of type I interferon attenuated viral clearance in A2 neutrophils and downregulated IFIT3 and key catabolic genes, demonstrating direct antiviral neutrophil function. Knockdown of IFIT3 in A2 neutrophils led to loss of IRF3 phosphorylation with consequent reduced viral catabolism, providing the first discrete mechanism of type I interferon signaling in neutrophils. The identification of this novel neutrophil phenotype and its association with severe COVID-19 outcomes emphasizes its likely importance in other respiratory viral infections and potential for new therapeutic approaches in viral illness.

Aberrant Expression of ACO1 in Vasculatures Parallels Progression of Idiopathic Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is characterized by mitochondrial dysfunction. However, details about the non-mitochondrial enzymes that sustain the proliferative nature of IPF are unclear. Aconitases are a family of enzymes that sustain metabolism inside and outside mitochondria. It is hypothesized that aconitase 1 (ACO1) plays an important role in the pathogenesis of IPF given that ACO1 represents an important metabolic hub in the cytoplasm. Objectives: To determine if ACO1 expression in IPF lungs shows specific patterns that may be important in the pathogenesis of IPF. To determine the similarities and differences in ACO1 expression in IPF, bleomycin-treated, and aging lungs. Methods: ACO1 expression in IPF lungs were characterized and compared to non-IPF controls by western blotting, immunostaining, and enzymatic activity assay. ACO1-expressing cell types were identified by multicolor immunostaining. Using similar methods, the expression profiles of ACO1 in IPF lungs versus bleomycin-treated and aged mice were investigated. Measurements and main results: Lower lobes of IPF lungs, unlike non-IPF controls, exhibit significantly high levels of ACO1. Most of the signals colocalize with von Willebrand factor (vWF), a lineage marker for vascular endothelial cells. Bleomycin-treated lungs also show high ACO1 expressions. However, most of the signals colocalize with E-cadherin and/or prosurfactant protein C, representative epithelial cell markers, in remodeled areas. Conclusions: A characteristic ACO1 expression profile observed in IPF vasculatures may be a promising diagnostic target. It also may give clues as to how de novo angiogenesis contributes to the irreversible nature of IPF.

N-myc-interactor mediates microbiome induced epithelial to mesenchymal transition and is associated with chronic lung allograft dysfunction.

BACKGROUND: Recent evidence suggests a role for lung microbiome in occurrence of chronic lung allograft dysfunction (CLAD). However, the mechanisms linking the microbiome to CLAD are poorly delineated. We investigated a possible mechanism involved in microbial modulation of mucosal response leading to CLAD with the hypothesis that a Proteobacteria dominant lung microbiome would inhibit N-myc-interactor (NMI) expression and induce epithelial to mesenchymal transition (EMT). METHODS: Explant CLAD, non-CLAD, and healthy nontransplant lung tissue were collected, as well as bronchoalveolar lavage from 14 CLAD and matched non-CLAD subjects, which were followed by 16S rRNA amplicon sequencing and quantitative polymerase chain reaction (PCR) analysis. Pseudomonas aeruginosa (PsA) or PsA-lipopolysaccharide was cocultured with primary human bronchial epithelial cells (PBEC). Western blot analysis and quantitative reverse transcription (qRT) PCR was performed to evaluate NMI expression and EMT in explants and in PsA-exposed PBECs. These experiments were repeated after siRNA silencing and upregulation (plasmid vector) of EMT regulator NMI. RESULTS: 16S rRNA amplicon analyses revealed that CLAD patients have a higher abundance of phyla Proteobacteria and reduced abundance of the phyla Bacteroidetes. At the genera level, CLAD subjects had an increased abundance of genera Pseudomonas and reduced Prevotella. Human CLAD airway cells showed a downregulation of the N-myc-interactor gene and presence of EMT. Furthermore, exposure of human primary bronchial epithelial cells to PsA resulted in downregulation of NMI and induction of an EMT phenotype while NMI upregulation resulted in attenuation of this PsA-induced EMT response. CONCLUSIONS: CLAD is associated with increased bacterial biomass and a Proteobacteria enriched airway microbiome and EMT. Proteobacteria such as PsA induces EMT in human bronchial epithelial cells via NMI, demonstrating a newly uncovered mechanism by which the microbiome induces cellular metaplasia.

Recipient Age Impacts Long-Term Survival in Adult Subjects with Cystic Fibrosis after Lung Transplantation.

Rationale: Lung transplant is an effective treatment option providing survival benefit in patients with cystic fibrosis (CF). Several studies have suggested survival benefit in adults compared with pediatric patients with CF undergoing lung transplant. However, it remains unclear whether this age-related disparity persists in adult subjects with CF.Objectives: We investigated the impact of age at transplant on post-transplant outcomes in adult patients with CF.Methods: The United Network of Organ Sharing Registry was queried for all adult patients with CF who underwent lung transplantation between 1992 and 2016. Pertinent baseline characteristics, demographics, clinical parameters, and outcomes were recorded. The patients were divided into two groups based on age at transplant (18-29 yr old and 30 yr or older). The primary endpoint was survival time. Assessment of post-transplant survival was performed using Kaplan-Meier tests and log-rank tests with multivariable Cox proportional hazards analysis to adjust for confounding variables.Results: A total of 3,881 patients with CF underwent lung transplantation between 1992 and 2016; mean age was 31.0 (± 9.3) years. The 18-29-year-old at transplant cohort consisted of 2,002 subjects and the 30 years or older cohort had 1,879 subjects. Survival analysis demonstrated significantly higher survival in subjects in the 30 years or older cohort (9.47 yr; 95% confidence interval [CI], 8.7-10.2) compared with the 18-29-year-old cohort (5.21 yr; 95% CI, 4.6-5.8). After adjusting for confounders, survival remained higher in recipients aged 30 years or older (hazard ratio, 0.44; 95% CI, 0.2-0.9). Mortality due to allograft failure was significantly lower in patients with CF aged 30 years or older (28% vs. 36.5%; odds ratio [OR], 0.7; 95% CI, 0.6-0.8), whereas the incidence of malignancy was higher in the 30 years or older cohort (8% vs. 2.9%; OR, 3.0; 95% CI, 1.9-4.6).Conclusions: Age at transplant influences lung transplant outcomes in recipients with CF. Subjects with CF aged 30 years or older at transplant have superior survival compared with adult subjects with CF transplanted between the ages 18 and 29 years.

Differences in airway microbiome and metabolome of single lung transplant recipients.

BACKGROUND: Recent studies suggest that alterations in lung microbiome are associated with occurrence of chronic lung diseases and transplant rejection. To investigate the host-microbiome interactions, we characterized the airway microbiome and metabolome of the allograft (transplanted lung) and native lung of single lung transplant recipients. METHODS: BAL was collected from the allograft and native lungs of SLTs and healthy controls. 16S rRNA microbiome analysis was performed on BAL bacterial pellets and supernatant used for metabolome, cytokines and acetylated proline-glycine-proline (Ac-PGP) measurement by liquid chromatography-high-resolution mass spectrometry. RESULTS: In our cohort, the allograft airway microbiome was distinct with a significantly higher bacterial burden and relative abundance of genera Acinetobacter & Pseudomonas. Likewise, the expression of the pro-inflammatory cytokine VEGF and the neutrophil chemoattractant matrikine Ac-PGP in the allograft was significantly higher. Airway metabolome distinguished the native lung from the allografts and an increased concentration of sphingosine-like metabolites that negatively correlated with abundance of bacteria from phyla Proteobacteria. CONCLUSIONS: Allograft lungs have a distinct microbiome signature, a higher bacterial biomass and an increased Ac-PGP compared to the native lungs in SLTs compared to the native lungs in SLTs. Airway metabolome distinguishes the allografts from native lungs and is associated with distinct microbial communities, suggesting a functional relationship between the local microbiome and metabolome.

COMMD3:BMI1 Fusion and COMMD3 Protein Regulate C-MYC Transcription: Novel Therapeutic Target for Metastatic Prostate Cancer.

Gene rearrangement is reported to be associated to the aggressive phenotype and poor prognosis in prostate cancer. We identified a gene fusion between a transcription repressor (BMI1) and transcriptional factor (COMMD3) in human prostate cancer. We show that COMMD3:BMI1 fusion expression is significantly increased in prostate cancer disease in an order: normal tissue < primary < metastatic tumors (Mets). Although elevated TMPRSS-ERG/ETV fusion is reported in prostate cancer, we identified a subtype of Mets exhibiting low TMPRSS:ETV and high COMMD3:BMI1 We delineated the mechanism and function of COMMD3 and COMMD3:BMI1 in prostate cancer. We show that COMMD3 level is elevated in prostate cancer cell models, PDX models (adenocarcinoma, NECaP), and Mets. The analysis of TCGA/NIH/GEO clinical data showed a positive correlation between increased COMMD3 expression to the disease recurrence and poor survival in prostate cancer. We show that COMMD3 drives proliferation of normal cells and promotes migration/invasiveness of neoplastic cells. We show that COMMD3:BMI1 and COMMD3 regulate C-MYC transcription and C-MYC downstream pathway. The ChIP analysis showed that COMMD3 protein is recruited at the promoter of C-MYC gene. On the basis of these data, we investigated the relevance of COMMD3:BMI1 and COMMD3 as therapeutic targets using in vitro and xenograft mouse models. We show that siRNA-mediated targeting of COMMD3:BMI1 and COMMD3 significantly decreases (i) C-MYC expression in BRD/BET inhibitor-resistant cells, (ii) proliferation/invasion in vitro, and (iii) growth of prostate cancer cell tumors in mice. The IHC analysis of tumors confirmed the targeting of COMMD3-regulated molecular pathway under in vivo conditions. We conclude that COMMD3:BMI1 and COMMD3 are potential progression biomarkers and therapeutic targets of metastatic prostate cancer.

Atomic resolution crystal structure of glutaredoxin 1 from Plasmodium falciparum and comparison with other glutaredoxins.

Glutaredoxins (Grxs) are redox proteins that use glutathione ((γ)Glu-Cys-Gly; GSH) as a cofactor. Plasmodium falciparum has one classic dithiol (CXXC) glutaredoxin (glutaredoxin 1; PfGrx1) and three monothiol (CXXS) Grx-like proteins (GLPs), which have five residue insertions prior to the active-site Cys. Here, the crystal structure of PfGrx1 has been determined by the sulfur single-wavelength anomalous diffraction (S-SAD) method utilizing intrinsic protein and solvent S atoms. Several residues were modelled with alternate conformations, and an alternate position was refined for the active-site Cys29 owing to radiation damage. The GSH-binding site is occupied by water polygons and buffer molecules. Structural comparison of PfGrx1 with other Grxs and Grx-like proteins revealed that the GSH-binding motifs (CXXC/CXXS, TVP, CDD, Lys26 and Gln/Arg63) are structurally conserved. Both the monothiol and dithiol Grxs possess three conserved water molecules; two of these were located in the GSH-binding site. PfGrx1 has several polar and charged amino-acid substitutions that provide structurally important additional hydrogen bonds and salt bridges missing in other Grxs.

Malaria parasite tyrosyl-tRNA synthetase secretion triggers pro-inflammatory responses.

Malaria infection triggers pro-inflammatory responses in humans that are detrimental to host health. Parasite-induced enhancement in cytokine levels correlate with malaria-associated pathologies. Here we show that parasite tyrosyl-tRNA synthetase (PfTyrRS), a housekeeping protein translation enzyme, induces pro-inflammatory responses from host immune cells. PfTyrRS exits from the parasite cytoplasm into the infected red blood cell (iRBC) cytoplasm, from where it is released into the extracellular medium on iRBC lysis. Using its ELR peptide motif, PfTyrRS specifically binds to and internalizes into host macrophages, leading to enhanced secretion of the pro-inflammatory cytokines TNF-α and IL-6. PfTyrRS-macrophage interaction also augments expression of adherence-linked host endothelial receptors ICAM-1 and VCAM-1. Our description of PfTyrRS as a parasite-secreted protein that triggers pro-inflammatory host responses, along with its atomic resolution crystal structure in complex with tyrosyl-adenylate, provides a novel platform for targeting PfTyrRS in anti-parasitic strategies.