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.

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.

ANKRD9 is a metabolically-controlled regulator of IMPDH2 abundance and macro-assembly.

Members of a large family of Ankyrin Repeat Domain (ANKRD) proteins regulate numerous cellular processes by binding to specific protein targets and modulating their activity, stability, and other properties. The same ANKRD protein may interact with different targets and regulate distinct cellular pathways. The mechanisms responsible for switches in the ANKRDs' behavior are often unknown. We show that cells' metabolic state can markedly alter interactions of an ANKRD protein with its target and the functional outcomes of this interaction. ANKRD9 facilitates degradation of inosine monophosphate dehydrogenase 2 (IMPDH2), the rate-limiting enzyme in GTP biosynthesis. Under basal conditions ANKRD9 is largely segregated from the cytosolic IMPDH2 in vesicle-like structures. Upon nutrient limitation, ANKRD9 loses its vesicular pattern and assembles with IMPDH2 into rodlike filaments, in which IMPDH2 is stable. Inhibition of IMPDH2 activity with ribavirin favors ANKRD9 binding to IMPDH2 rods. The formation of ANKRD9/IMPDH2 rods is reversed by guanosine, which restores ANKRD9 associations with the vesicle-like structures. The conserved Cys109Cys110 motif in ANKRD9 is required for the vesicle-to-rods transition as well as binding and regulation of IMPDH2. Oppositely to overexpression, ANKRD9 knockdown increases IMPDH2 levels and prevents formation of IMPDH2 rods upon nutrient limitation. Taken together, the results suggest that a guanosine-dependent metabolic switch determines the mode of ANKRD9 action toward IMPDH2.

Practical spectrophotometric assay for the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase, a potential antibiotic target.

A new enzymatic assay for the bacterial enzyme succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18) is described. This assay employs N6-methyl-N2-succinyl-L,L-diaminopimelic acid (N6-methyl-L,L-SDAP) as the substrate with ninhydrin used to detect cleavage of the amide bond of the modified substrate, wherein N6-methylation enables selective detection of the primary amine enzymatic product. Molecular modeling supported preparation of the mono-N6-methylated-L,L-SDAP as an alternate substrate for the assay, given binding in the active site of DapE predicted to be comparable to the endogenous substrate. The alternate substrate for the assay, N6-methyl-L,L-SDAP, was synthesized from the tert-butyl ester of Boc-L-glutamic acid employing a Horner-Wadsworth-Emmons olefination followed by an enantioselective reduction employing Rh(I)(COD)(S,S)-Et-DuPHOS as the chiral catalyst. Validation of the new ninhydrin assay was demonstrated with known inhibitors of DapE from Haemophilus influenza (HiDapE) including captopril (IC50 = 3.4 [± 0.2] µM, 3-mercaptobenzoic acid (IC50 = 21.8 [±2.2] µM, phenylboronic acid (IC50 = 316 [± 23.6] µM, and 2-thiopheneboronic acid (IC50 = 111 [± 16] µM. Based on these data, this assay is simple and robust, and should be amenable to high-throughput screening, which is an important step forward as it opens the door to medicinal chemistry efforts toward the discovery of DapE inhibitors that can function as a new class of antibiotics.