Circuits between infected macrophages and T cells in SARS-CoV-2 pneumonia.

Some patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop severe pneumonia and acute respiratory distress syndrome1 (ARDS). Distinct clinical features in these patients have led to speculation that the immune response to virus in the SARS-CoV-2-infected alveolus differs from that in other types of pneumonia2. Here we investigate SARS-CoV-2 pathobiology by characterizing the immune response in the alveoli of patients infected with the virus. We collected bronchoalveolar lavage fluid samples from 88 patients with SARS-CoV-2-induced respiratory failure and 211 patients with known or suspected pneumonia from other pathogens, and analysed them using flow cytometry and bulk transcriptomic profiling. We performed single-cell RNA sequencing on 10 bronchoalveolar lavage fluid samples collected from patients with severe coronavirus disease 2019 (COVID-19) within 48 h of intubation. In the majority of patients with SARS-CoV-2 infection, the alveolar space was persistently enriched in T cells and monocytes. Bulk and single-cell transcriptomic profiling suggested that SARS-CoV-2 infects alveolar macrophages, which in turn respond by producing T cell chemoattractants. These T cells produce interferon-γ to induce inflammatory cytokine release from alveolar macrophages and further promote T cell activation. Collectively, our results suggest that SARS-CoV-2 causes a slowly unfolding, spatially limited alveolitis in which alveolar macrophages containing SARS-CoV-2 and T cells form a positive feedback loop that drives persistent alveolar inflammation.

Resetting proteostasis with ISRIB promotes epithelial differentiation to attenuate pulmonary fibrosis.

Pulmonary fibrosis is a relentlessly progressive and often fatal disease with a paucity of available therapies. Genetic evidence implicates disordered epithelial repair, which is normally achieved by the differentiation of small cuboidal alveolar type 2 (AT2) cells into large, flattened alveolar type 1 (AT1) cells as an initiating event in pulmonary fibrosis pathogenesis. Using models of pulmonary fibrosis in young adult and old mice and a model of adult alveologenesis after pneumonectomy, we show that administration of ISRIB, a small molecule that restores protein translation by EIF2B during activation of the integrated stress response (ISR), accelerated the differentiation of AT2 into AT1 cells. Accelerated epithelial repair reduced the recruitment of profibrotic monocyte-derived alveolar macrophages and ameliorated lung fibrosis. These findings suggest a dysfunctional role for the ISR in regeneration of the alveolar epithelium after injury with implications for therapy.

SIRT3 Overexpression Ameliorates Asbestos-Induced Pulmonary Fibrosis, mt-DNA Damage, and Lung Fibrogenic Monocyte Recruitment.

Alveolar epithelial cell (AEC) mitochondrial (mt) DNA damage and fibrotic monocyte-derived alveolar macrophages (Mo-AMs) are implicated in the pathobiology of pulmonary fibrosis. We showed that sirtuin 3 (SIRT3), a mitochondrial protein regulating cell fate and aging, is deficient in the AECs of idiopathic pulmonary fibrosis (IPF) patients and that asbestos- and bleomycin-induced lung fibrosis is augmented in Sirt3 knockout (Sirt3-/-) mice associated with AEC mtDNA damage and intrinsic apoptosis. We determined whether whole body transgenic SIRT3 overexpression (Sirt3Tg) protects mice from asbestos-induced pulmonary fibrosis by mitigating lung mtDNA damage and Mo-AM recruitment. Crocidolite asbestos (100 µg/50 µL) or control was instilled intratracheally in C57Bl6 (Wild-Type) mice or Sirt3Tg mice, and at 21 d lung fibrosis (histology, fibrosis score, Sircol assay) and lung Mo-AMs (flow cytometry) were assessed. Compared to controls, Sirt3Tg mice were protected from asbestos-induced pulmonary fibrosis and had diminished lung mtDNA damage and Mo-AM recruitment. Further, pharmacologic SIRT3 inducers (i.e., resveratrol, viniferin, and honokiol) each diminish oxidant-induced AEC mtDNA damage in vitro and, in the case of honokiol, protection occurs in a SIRT3-dependent manner. We reason that SIRT3 preservation of AEC mtDNA is a novel therapeutic focus for managing patients with IPF and other types of pulmonary fibrosis.

A spatially restricted fibrotic niche in pulmonary fibrosis is sustained by M-CSF/M-CSFR signalling in monocyte-derived alveolar macrophages.

Ontologically distinct populations of macrophages differentially contribute to organ fibrosis through unknown mechanisms.We applied lineage tracing, single-cell RNA sequencing and single-molecule fluorescence in situ hybridisation to a spatially restricted model of asbestos-induced pulmonary fibrosis.We demonstrate that tissue-resident alveolar macrophages, tissue-resident peribronchial and perivascular interstitial macrophages, and monocyte-derived alveolar macrophages are present in the fibrotic niche. Deletion of monocyte-derived alveolar macrophages but not tissue-resident alveolar macrophages ameliorated asbestos-induced lung fibrosis. Monocyte-derived alveolar macrophages were specifically localised to fibrotic regions in the proximity of fibroblasts where they expressed molecules known to drive fibroblast proliferation, including platelet-derived growth factor subunit A. Using single-cell RNA sequencing and spatial transcriptomics in both humans and mice, we identified macrophage colony-stimulating factor receptor (M-CSFR) signalling as one of the novel druggable targets controlling self-maintenance and persistence of these pathogenic monocyte-derived alveolar macrophages. Pharmacological blockade of M-CSFR signalling led to the disappearance of monocyte-derived alveolar macrophages and ameliorated fibrosis.Our findings suggest that inhibition of M-CSFR signalling during fibrosis disrupts an essential fibrotic niche that includes monocyte-derived alveolar macrophages and fibroblasts during asbestos-induced fibrosis.

Multidimensional Assessment of the Host Response in Mechanically Ventilated Patients with Suspected Pneumonia.

Rationale: The identification of informative elements of the host response to infection may improve the diagnosis and management of bacterial pneumonia. Objectives: To determine whether the absence of alveolar neutrophilia can exclude bacterial pneumonia in critically ill patients with suspected infection and to test whether signatures of bacterial pneumonia can be identified in the alveolar macrophage transcriptome. Methods: We determined the test characteristics of alveolar neutrophilia for the diagnosis of bacterial pneumonia in three cohorts of mechanically ventilated patients. In one cohort, we also isolated macrophages from alveolar lavage fluid and used the transcriptome to identify signatures of bacterial pneumonia. Finally, we developed a humanized mouse model of Pseudomonas aeruginosa pneumonia to determine if pathogen-specific signatures can be identified in human alveolar macrophages. Measurements and Main Results: An alveolar neutrophil percentage less than 50% had a negative predictive value of greater than 90% for bacterial pneumonia in both the retrospective (n = 851) and validation cohorts (n = 76 and n = 79). A transcriptional signature of bacterial pneumonia was present in both resident and recruited macrophages. Gene signatures from both cell types identified patients with bacterial pneumonia with test characteristics similar to alveolar neutrophilia. Conclusions: The absence of alveolar neutrophilia has a high negative predictive value for bacterial pneumonia in critically ill patients with suspected infection. Macrophages can be isolated from alveolar lavage fluid obtained during routine care and used for RNA-Seq analysis. This novel approach may facilitate a longitudinal and multidimensional assessment of the host response to bacterial pneumonia.

The Sphingosine Kinase 1 Inhibitor, PF543, Mitigates Pulmonary Fibrosis by Reducing Lung Epithelial Cell mtDNA Damage and Recruitment of Fibrogenic Monocytes.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease for which novel approaches are urgently required. We reported increased sphingosine kinase 1 (SPHK1) in IPF lungs and that SPHK1 inhibition using genetic and pharmacologic approaches reduces murine bleomycin-induced pulmonary fibrosis. We determined whether PF543, a specific SPHK1 inhibitor post bleomycin or asbestos challenge mitigates lung fibrosis by reducing mitochondrial (mt) DNA damage and pro-fibrotic monocyte recruitment-both are implicated in the pathobiology of pulmonary fibrosis. Bleomycin (1.5 U/kg), crocidolite asbestos (100 µg/50 µL) or controls was intratracheally instilled in Wild-Type (C57Bl6) mice. PF543 (1 mg/kg) or vehicle was intraperitoneally injected once every two days from day 7-21 following bleomycin and day 14-21 or day 30-60 following asbestos. PF543 reduced bleomycin- and asbestos-induced pulmonary fibrosis at both time points as well as lung expression of profibrotic markers, lung mtDNA damage, and fibrogenic monocyte recruitment. In contrast to human lung fibroblasts, asbestos augmented lung epithelial cell (MLE) mtDNA damage and PF543 was protective. Post-exposure PF543 mitigates pulmonary fibrosis in part by reducing lung epithelial cell mtDNA damage and monocyte recruitment. We reason that SPHK1 signaling may be an innovative therapeutic target for managing patients with IPF and other forms of lung fibrosis.

A role for heat shock factor 1 in hypercapnia-induced inhibition of inflammatory cytokine expression.

Hypercapnia, elevated levels of CO2 in the blood, is a known marker for poor clinical prognosis and is associated with increased mortality in patients hospitalized with both bacterial and viral pneumonias. Although studies have established a connection between elevated CO2 levels and poor pneumonia outcomes, a mechanistic basis of this association has not yet been established. We previously reported that hypercapnia inhibits expression of key NF-κB-regulated, innate immune cytokines, TNF-α, and IL-6, in LPS-stimulated macrophages in vitro and in mice during Pseudomonas pneumonia. The transcription factor heat shock factor 1 (HSF1) is important in maintaining proteostasis during stress and has been shown to negatively regulate NF-κB activity. In this study, we tested the hypothesis that HSF1 activation in response to hypercapnia results in attenuated NF-κB-regulated gene expression. We found that hypercapnia induced the protein expression and nuclear accumulation of HSF1 in primary murine alveolar macrophages and in an alveolar macrophage cell line (MH-S). In MH-S cells treated with short interfering RNA targeting Hsf1, LPS-induced IL-6 and TNF-α release were elevated during exposure to hypercapnia. Pseudomonas-infected Hsf1+/+ (wild-type) mice, maintained in a hypercapnic environment, showed lower levels of IL-6 and TNF-α in bronchoalveolar lavage fluid and IL-1ß in lung tissue than did infected mice maintained in room air. In contrast, infected Hsf1+/- mice exposed to either hypercapnia or room air had similarly elevated levels of those cytokines. These results suggest that hypercapnia-mediated inhibition of NF-κB cytokine production is dependent on HSF1 expression and/or activation.-Lu, Z., Casalino-Matsuda, S. M., Nair, A., Buchbinder, A., Budinger, G. R. S., Sporn, P. H. S., Gates, K. L. A role for heat shock factor 1 in hypercapnia-induced inhibition of inflammatory cytokine expression.

Prolonged exposure to lung-derived cytokines is associated with activation of microglia in patients with COVID-19.

BACKGROUNDSurvivors of pneumonia, including SARS-CoV-2 pneumonia, are at increased risk for cognitive dysfunction and dementia. In rodent models, cognitive dysfunction following pneumonia has been linked to the systemic release of lung-derived pro-inflammatory cytokines. Microglia are poised to respond to inflammatory signals from the circulation, and their dysfunction has been linked to cognitive impairment in murine models of dementia and in humans.METHODSWe measured levels of 55 cytokines and chemokines in bronchoalveolar lavage fluid and plasma from 341 patients with respiratory failure and 13 healthy controls, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. We used flow cytometry to sort neuroimmune cells from postmortem brain tissue from 5 patients who died from COVID-19 and 3 patients who died from other causes for single-cell RNA-sequencing.RESULTSMicroglia from patients with COVID-19 exhibited a transcriptomic signature suggestive of their activation by circulating pro-inflammatory cytokines. Peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, but cumulative cytokine exposure was higher in patients with COVID-19. Treatment with corticosteroids reduced expression of COVID-19-specific cytokines.CONCLUSIONProlonged lung inflammation results in sustained elevations in circulating cytokines in patients with SARS-CoV-2 pneumonia compared with those with pneumonia secondary to other pathogens. Microglia from patients with COVID-19 exhibit transcriptional responses to inflammatory cytokines. These findings support data from rodent models causally linking systemic inflammation with cognitive dysfunction in pneumonia and support further investigation into the role of microglia in pneumonia-related cognitive dysfunction.FUNDINGSCRIPT U19AI135964, UL1TR001422, P01AG049665, P01HL154998, R01HL149883, R01LM013337, R01HL153122, R01HL147290, R01HL147575, R01HL158139, R01ES034350, R01ES027574, I01CX001777, U01TR003528, R21AG075423, T32AG020506, F31AG071225, T32HL076139.

Androgen receptor primes prostate cancer cells to apoptosis through down-regulation of basal p21 expression.

The androgen receptor (AR) for the male hormone androgen plays an important role in regulation of cell survival or death depending on the nature of cellular context and extracellular stimuli. The pro-survival function of AR is mediated mainly by transcriptional regulation of its target genes. By contrast, the pro-death function of AR can be transcription-dependent or -independent, although the underlying mechanism of the latter is incompletely understood. Here we report that, in androgen-independent prostate cancer cells, AR promotes UV-induced apoptosis through down-regulation of basal expression of p21 independently of its transcriptional activity. Down-regulation of basal p21 expression depends on AR N-terminal interacting protein PIRH2, an E3 ligase for proteasomal degradation of p53. Silencing of PIRH2 up-regulates p53, which in turn activates p21 transcription. Consistent with this, knockdown of PIRH2 suppresses UV-induced AR-dependent apoptosis. Our data suggest that AR primes androgen-independent prostate cancer cells to DNA damage-induced apoptosis through the PIRH2-p53-p21 axis.

Histone-lysine methyltransferase EHMT2 is involved in proliferation, apoptosis, cell invasion, and DNA methylation of human neuroblastoma cells.

Neuroblastoma (NB), a childhood neoplasm arising from neural crest cells, is characterized by a diversity of clinical behaviors ranging from spontaneous remission to rapid tumor progression and death. In addition to genetic abnormalities, recent studies have indicated that epigenetic aberrations also contribute toward NB pathogenesis. However, the epigenetic mechanisms underlying the pathogenesis of NB are largely unknown. Inhibition of euchromatic histone-lysine N-methyltransferase 2 (EHMT2) was evaluated through the measurement of H3K9Me2 levels. Cell proliferation was examined by cell counting in human NB cell lines (LA1-55n, IMR-5, and NMB). The RNA expression of EHMT2, MYCN, and p21 was measured by real-time PCR. The expression of PCNA, MYCN, p53, cyclinD1, H3, H3K27M2, and H3K9Me2 was examined by western blot analysis. In-vitro invasion and the effects of the EHMT2 inhibitor (BIX-01294) were assessed in the Transwell chamber assay. Caspase 3 and 8 activities were measured using a Caspase-Glo assay kit. The level of overall DNA methylation was measured by liquid chromatography-mass spectroscopy. BIX-01294, a specific inhibitor of EHMT2 (a key enzyme for histone H3 dimethylation at lysine-9), specifically decreases the overall H3K9Me2 level but not H3K27Me2. The inhibition of EHMT2 decreased the proliferation of NB cells and induced apoptosis by increasing caspase 8/caspase 3 activity. BIX-01294 inhibited NB cell mobility and invasion. This was accompanied by a decreased expression of the MYCN oncogene. Inhibition of EHMT2 enhanced a doxorubicin-induced inhibitory effect on cell proliferation. Finally, EHMT2 inhibition modulated overall DNA methylation levels in NB cells. Our results show that histone-lysine methylation is involved in cell proliferation, apoptosis, cell invasion, and overall DNA methylation in human NB cells. Further understanding of this mechanism may provide an insight into the pathogenesis of NB progression and lead to novel treatment strategies.

Prolonged exposure to lung-derived cytokines is associated with inflammatory activation of microglia in patients with COVID-19.

Neurological impairment is the most common finding in patients with post-acute sequelae of COVID-19. Furthermore, survivors of pneumonia from any cause have an elevated risk of dementia1-4. Dysfunction in microglia, the primary immune cell in the brain, has been linked to cognitive impairment in murine models of dementia and in humans5. Here, we report a transcriptional response in human microglia collected from patients who died following COVID-19 suggestive of their activation by TNF-α and other circulating pro-inflammatory cytokines. Consistent with these findings, the levels of 55 alveolar and plasma cytokines were elevated in a cohort of 341 patients with respiratory failure, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. While peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, cumulative cytokine exposure was higher in patients with COVID-19. Corticosteroid treatment, which has been shown to be beneficial in patients with COVID-196, was associated with lower levels of CXCL10, CCL8, and CCL2-molecules that sustain inflammatory circuits between alveolar macrophages harboring SARS-CoV-2 and activated T cells7. These findings suggest that corticosteroids may break this cycle and decrease systemic exposure to lung-derived cytokines and inflammatory activation of microglia in patients with COVID-19.

Pulmonary artery smooth muscle cell proliferation and migration in fetal lambs acclimatized to high-altitude long-term hypoxia: role of histone acetylation.

High-altitude long-term hypoxia (LTH) is known to induce pulmonary arterial smooth muscle cell (PASMC) proliferation in the fetus, leading to pulmonary arterial remodeling and pulmonary hypertension of the newborn. The mechanisms underlying these conditions remain enigmatic however. We hypothesized that epigenetic alterations in fetal PASMC induced by high-altitude LTH may play an important role in modulating their proliferation during pulmonary arterial remodeling. To test this hypothesis, we have analyzed epigenetic alterations in the pulmonary vasculature of fetal lambs exposed to high-altitude LTH [pregnant ewes were kept at 3,801 m altitude from ~40 to 145 days gestation] or to sea level atmosphere. Intrapulmonary arteries were isolated, and fetal PASMC were cultured from both control and LTH fetuses. Compared with controls, in LTH fetus pulmonary arteries measurements of histone acetylation and global DNA methylation demonstrated reduced levels of global histone 4 acetylation and DNA methylation, accompanied by the loss of the cyclin-dependent kinase inhibitor p21. Treatment of LTH fetal PASMCs with histone deacetylase (HDAC) inhibitor trichostatin A decreased their proliferation rate, in part because of altered expression of p21 at both RNA and protein level. In PASMC of LTH fetuses, HDAC inhibition also decreased PDGF-induced cell migration and ERK1/2 activation and modulated global DNA methylation. On the basis of these observations, we propose that epigenetic alterations (reduced histone acetylation and DNA methylation) caused by chronic hypoxia leads to fetal PASMC proliferation and vessel remodeling associated with vascular proliferative disease and that this process is regulated by p21.

Valproic acid shows a potent antitumor effect with alteration of DNA methylation in neuroblastoma.

Epigenetic aberrations and a CpG island methylator phenotype are associated with poor outcome in children with neuroblastoma (NB). Previously, we have shown that valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, exerts antitumor effects in an NB xenograft model. However, the underlying antitumor molecular mechanisms are largely unknown. In this study, we examined the role of HDAC in cell proliferation, cell cycle progression, gene expression patterns, and epigenome in NB. Cell proliferation, cell cycle progression, caspase activity, RNA and protein expression, quantitative methylation, and global DNA methylation were examined in NBL-W-N and LA1-55n NB cell lines. Our studies showed that inhibition of HDAC decreased NB proliferation, and induced caspase activity and G1 growth arrest. Expression patterns of cancer-related genes were modulated by VPA. The expression of THBS1, CASP8, SPARC, CDKN1A, HIC1, CDKN1B, and HIN1 was upregulated, and that of MYCN and TIG1 was downregulated. HDAC inhibition decreased methylation levels of THBS1 and RASSF1A promoters. Inhibition of HDAC increased acetylation of histone 4 and overall DNA methylation levels. Our studies showed that inhibition of HDAC blocked cell proliferation and cell cycle progression in relation to alteration in cancer-related genes, increased overall DNA methylation, and decreased methylation of tumor suppressor genes. Further studies examining the antitumor effects of VPA in NB are warranted.

Antibacterial Potential Analysis of Novel α-Helix Peptides in the Chinese Wolf Spider Lycosa sinensis.

The spider Lycosa sinensis represents a burrowing wolf spider (family Lycosidae) widely distributed in the cotton region of northern China, whose venom is rich in various bioactive peptides. In previous study, we used a combination strategy of peptidomic and transcriptomic analyses to systematically screen and identify potential antimicrobial peptides (AMPs) in Lycosa sinensis venom that matched the α-helix structures. In this work, the three peptides (LS-AMP-E1, LS-AMP-F1, and LS-AMP-G1) were subjected to sequence analysis of the physicochemical properties and helical wheel projection, and then six common clinical pathogenic bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) with multiple drug-resistance were isolated and cultured for the evaluation and analysis of antimicrobial activity of these peptides. The results showed that two peptides (LS-AMP-E1 and LS-AMP-F1) had different inhibitory activity against six clinical drug-resistant bacteria; they can effectively inhibit the formation of biofilm and have no obvious hemolytic effect. Moreover, both LS-AMP-E1 and LS-AMP-F1 exhibited varying degrees of synergistic therapeutic effects with traditional antibiotics (azithromycin, erythromycin, and doxycycline), significantly reducing the working concentration of antibiotics and AMPs. In terms of antimicrobial mechanisms, LS-AMP-E1 and LS-AMP-F1 destroyed the integrity of bacterial cell membranes in a short period of time and completely inhibited bacterial growth within 10 min of action. Meanwhile, high concentrations of Mg2+ effectively reduced the antibacterial activity of LS-AMP-E1 and LS-AMP-F1. Together, it suggested that the two peptides interact directly on bacterial cell membranes. Taken together, bioinformatic and functional analyses in the present work sheds light on the structure-function relationships of LS-AMPs, and facilitates the discovery and clinical application of novel AMPs.

Red mud based passivator reduced Cd accumulation in edible amaranth by influencing root organic matter metabolism and soil aggregate distribution.

Red mud was a highly alkaline hazardous waste, and their resource utilization was a research hotspot. In this study, influencing mechanisms of red mud based passivator on the transformation of Cd fraction in acidic Cd-polluted soil, photosynthetic property, and Cd accumulation in edible amaranth were investigated based on the evaluation of Cd adsorption capacity, root metabolic response, and soil aggregate distribution. Results showed that red mud exhibited good Cd adsorption capacities at about 35 °C and pH 9 in an aqueous solution, and the adsorption behavior of red mud on Cd in rhizosphere soil solution was considered to have some similarity. In the soil-pot trial, red mud application significantly facilitated edible amaranth growth by enhancing the maximum photochemical efficiency and light energy absorption by per unit leaf area by activating more reaction centers. The main mechanisms of rhizosphere soil Cd immobilisation by red mud application included: i) the reduction of mobilized Cd caused by the increasing negative surface charge of soil and precipitation of Cd hydroxides and carbonates at high pH; ii) the increase of organics-Cd complexes caused by the increasing -OH and -COOH amounts adsorbed on the surface of rhizosphere soil after red mud application; and iii) the decrease of available Cd content in soil aggregates caused by the increasing organic matters after red mud application. This study would provide the basis for the safe utilization of red mud remediating acidic Cd-polluted soil.

Nitrogen fertilizer affects rhizosphere Cd re-mobilization by mediating gene AmALM2 and AmALMT7 expression in edible amaranth roots.

In-situ stabilization of Cd-contaminated farmland is a commonly used remediation technology. Yet, rhizosphere metabolites (e.g., organic acids) during crop cultivation may cause Cd re-mobilization and over-accumulation. Here, we identified four pivotal cytomembrane-localized genes underlying Cd accumulation difference between two contrasting edible amaranth cultivars based on root gene expression profile, studied their subcellular localization and functional characteristics, and then investigated effects of nitrogen fertilizer on their expression and rhizosphere Cd re-mobilization. Results showed that more Cd accumulated by edible amaranth was due to rhizosphere Cd mobilization by mediating high expression of AmALMT2 and AmALMT7 genes, not Cd transporters in roots. This was confirmed by heterologous expression of AmALMT2 and AmALMT7 genes in Arabidopsis thaliana, since they mediated malic, fumaric, succinic, and aspartic acids efflux. Furthermore, nitrogen influencing rhizosphere acidification might be closely associated with organic acids efflux genes. Compared with N-NO3- application, N-NH4+ was massively assimilated into glutamates and oxaloacetates through up-regulating glutamine synthetase and alanine-aspartate-glutamate metabolic pathways, thereby enhancing TCA cycle and organic acids efflux dominated by binary carboxylic acids via up-regulating AmALMT2 and AmALMT7 genes, which finally caused Cd re-mobilization. Therefore, N-NO3--dominated nitrogen retarded rhizosphere Cd re-mobilization via inhibiting organic acids efflux function of AmALMT2 and AmALMT7 proteins.

The lung microenvironment shapes a dysfunctional response of alveolar macrophages in aging.

Alveolar macrophages orchestrate the response to viral infections. Age-related changes in these cells may underlie the differential severity of pneumonia in older patients. We performed an integrated analysis of single-cell RNA-Seq data that revealed homogenous age-related changes in the alveolar macrophage transcriptome in humans and mice. Using genetic lineage tracing with sequential injury, heterochronic adoptive transfer, and parabiosis, we found that the lung microenvironment drove an age-related resistance of alveolar macrophages to proliferation that persisted during influenza A viral infection. Ligand-receptor pair analysis localized these changes to the extracellular matrix, where hyaluronan was increased in aged animals and altered the proliferative response of bone marrow-derived macrophages to granulocyte macrophage colony-stimulating factor (GM-CSF). Our findings suggest that strategies targeting the aging lung microenvironment will be necessary to restore alveolar macrophage function in aging.

Phytoremediation of cadmium-polluted soil assisted by D-gluconate-enhanced Enterobacter cloacae colonization in the Solanum nigrum L. rhizosphere.

Microbe-assisted phytoremediation for Cd-polluted soil is being regarded increasingly. However, the availability of microbes that can collaborate with Cd-hyperaccumulators effectively has become one of bottlenecks restricting the remediation efficiency. A siderophore-producing bacterium (Y16; Enterobacter cloacae) isolated from the rhizospheric soil of Cd-hyperaccumulator Solanum nigrum L. was identified by 16S rRNA gene sequencing and biochemical analysis, and then used for analyzing microbial chemotaxis, carbon source utilization, and insoluble P/Cd mobilization capacities. Besides, a soil-pot trial was performed to underlie the phytoremediation mechanism of Cd-polluted soil assisted by D-gluconate-enhanced Enterobacter cloacae colonization (DEYC) in the Solanum nigrum L. rhizosphere. Results displayed that D-gluconate was an effective chemoattractant and carbon source strengthening Y16 colonization, and Y16 exhibited strong abilities to mobilize insoluble P/Cd in shake flask by extracellular acidification (p < 0.05). In the soil-pot trial, DEYC observably enhanced soil Cd phytoextraction by Solanum nigrum L., and increased microbial diversity according to alpha- and beta-diversity analysis (p < 0.05). Taxonomic distribution and co-occurrence network analysis suggested that DEYC increased relative abundances of dominant microbial taxa associated with soil acidification (Acidobacteria-6), indoleacetic acid secretion (Ensifer adhaerens), soil fertility improvement (Flavisolibacter, Bdellovibrio bacteriovorus, and Candidatus nitrososphaera), and insoluble Cd mobilization (Massilia timonae) at different classification levels. Importantly, COGs analysis further shown that DEYC aroused the up-regulation of key genes related to chemotactic motility, carbon fixation, TCA cycle, and propanoate metabolism. These results indicated that DEYC drove the rhizospheric enrichment of pivotal microbial taxa directly or indirectly involved in soil Cd mobilization, meanwhile distinctly promoted plant growth for accumulating more mobilizable Cd. Therefore, Y16 could be used as bio-inoculants for assisting phytoremediation of Cd-polluted soil.

Impaired phagocytic function in CX3CR1+ tissue-resident skeletal muscle macrophages prevents muscle recovery after influenza A virus-induced pneumonia in old mice.

Skeletal muscle dysfunction in survivors of pneumonia disproportionately affects older individuals in whom it causes substantial morbidity. We found that skeletal muscle recovery was impaired in old compared with young mice after influenza A virus-induced pneumonia. In young mice, recovery of muscle loss was associated with expansion of tissue-resident skeletal muscle macrophages and downregulation of MHC II expression, followed by a proliferation of muscle satellite cells. These findings were absent in old mice and in mice deficient in Cx3cr1. Transcriptomic profiling of tissue-resident skeletal muscle macrophages from old compared with young mice showed downregulation of pathways associated with phagocytosis and proteostasis, and persistent upregulation of inflammatory pathways. Consistently, skeletal muscle macrophages from old mice failed to downregulate MHCII expression during recovery from influenza A virus-induced pneumonia and showed impaired phagocytic function in vitro. Like old animals, mice deficient in the phagocytic receptor Mertk showed no macrophage expansion, MHCII downregulation, or satellite cell proliferation and failed to recover skeletal muscle function after influenza A pneumonia. Our data suggest that a loss of phagocytic function in a CX3CR1+ tissue-resident skeletal muscle macrophage population in old mice precludes satellite cell proliferation and recovery of skeletal muscle function after influenza A pneumonia.

Alveolitis in severe SARS-CoV-2 pneumonia is driven by self-sustaining circuits between infected alveolar macrophages and T cells.

Some patients infected with Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) develop severe pneumonia and the acute respiratory distress syndrome (ARDS) [1]. Distinct clinical features in these patients have led to speculation that the immune response to virus in the SARS-CoV-2-infected alveolus differs from other types of pneumonia [2]. We collected bronchoalveolar lavage fluid samples from 86 patients with SARS-CoV-2-induced respiratory failure and 252 patients with known or suspected pneumonia from other pathogens and subjected them to flow cytometry and bulk transcriptomic profiling. We performed single cell RNA-Seq in 5 bronchoalveolar lavage fluid samples collected from patients with severe COVID-19 within 48 hours of intubation. In the majority of patients with SARS-CoV-2 infection at the onset of mechanical ventilation, the alveolar space is persistently enriched in alveolar macrophages and T cells without neutrophilia. Bulk and single cell transcriptomic profiling suggest SARS-CoV-2 infects alveolar macrophages that respond by recruiting T cells. These T cells release interferon-gamma to induce inflammatory cytokine release from alveolar macrophages and further promote T cell recruitment. Our results suggest SARS-CoV-2 causes a slowly unfolding, spatially-limited alveolitis in which alveolar macrophages harboring SARS-CoV-2 transcripts and T cells form a positive feedback loop that drives progressive alveolar inflammation. This manuscript is accompanied by an online resource: https://www.nupulmonary.org/covid-19/. ONE SENTENCE SUMMARY: SARS-CoV-2-infected alveolar macrophages form positive feedback loops with T cells in patients with severe COVID-19.