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 88 patients with SARS-CoV-2-induced respiratory failure and 211 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 on 10 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, 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-gamma 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 harboring SARS-CoV-2 and T cells form a positive feedback loop that drives persistent alveolar inflammation.

To gain insight into the regulation of expression of peroxisome proliferator-activated receptor (PPAR) isoforms, we have determined the structural organization of the mouse PPAR 'y (mPPARy) gene. This gene extends > 105 kb and gives rise to two mRNAs (mPPARy1 and mPPARy2) that differ at their 5' ends. The mPPARy2 cDNA encodes an additional 30 amino acids N-terminal to the first ATG codon of mPPARyl and reveals a different 5' untranslated sequence.We show that mPPARy1 mRNA is encoded by eight exons, whereas the mPPARy2 mRNA is encoded by seven exons.Most of the 5' untranslated sequence of mPPARy1 mRNA is encoded by two exons, whereas the 5' untranslated sequence and the extra 30 N-terminal amino acids of mPPARy2 are encoded by one exon, which is located between the second and third exons coding for mPPARyl. The last six exons of mPPARy gene code for identical sequences in mPPARyl and mPPARy2 isoforms. The mPPARyl and mPPARy2 isoforms are transcribed from different promoters. The mPPARy gene has been mapped to chromosome 6 E3-F1 by in situ hybridization using a biotin-labeled probe. These results establish that at least one of the PPAR genes yields more than one protein product, similar to that encountered with retinoid X receptor and retinoic acid receptor genes. The existence of multiple PPAR isoforms transcribed from different promoters could increase the diversity of ligand and tissue-specific transcriptional responses.

Fasting causes lipolysis in adipose tissue leading to the release of large quantities of free fatty acids into circulation that reach the liver where they are metabolized to generate ketone bodies to serve as fuels for other tissues. Since fatty acid-metabolizing enzymes in the liver are transcriptionally regulated by peroxisome proliferator-activated receptor ␣ (PPAR␣), we investigated the role of PPAR␣ in the induction of these enzymes in response to fasting and their relationship to the development of hepatic steatosis in mice deficient in PPAR␣ (PPAR␣ ؊/؊ ), peroxisomal fatty acyl-CoA oxidase (AOX ؊/؊ ), and in both PPAR␣ and AOX (double knockout (DKO)). Fasting for 48 -72 h caused profound impairment of fatty acid oxidation in both PPAR␣؊/؊ and DKO mice, and DKO mice revealed a greater degree of hepatic steatosis when compared with PPAR␣ ؊/؊ mice. The absence of PPAR␣ in both PPAR␣ ؊/؊ and DKO mice impairs the induction of mitochondrial ␤-oxidation in liver following fasting which contributes to hypoketonemia and hepatic steatosis. Pronounced steatosis in DKO mouse livers is due to the added deficiency of peroxisomal ␤-oxidation system in these animals due to the absence of AOX. In mice deficient in AOX alone, the sustained hyperactivation of PPAR␣ and up-regulation of mitochondrial ␤-oxidation and microsomal -oxidation systems as well as the regenerative nature of a majority of hepatocytes containing numerous spontaneously proliferated peroxisomes, which appear refractory to store triglycerides, blunt the steatotic response to fasting. Starvation for 72 h caused a decrease in PPAR␣ hepatic mRNA levels in wild type mice, with no perceptible compensatory increases in PPAR␥ and PPAR␦ mRNA levels. PPAR␥ and PPAR␦ hepatic mRNA levels were lower in fed PPAR␣ ؊/؊ and DKO mice when compared with wild type mice, and fasting caused a slight increase only in PPAR␥ levels and a decrease in PPAR␦ levels. Fasting did not change the PPAR isoform levels in AOX ؊/؊ mouse liver. These observations point to the critical importance of PPAR␣ in the transcriptional regulatory responses to fasting and in determining the severity of hepatic steatosis.

In an attempt to identify cofactors that could possibly influence the transcriptional activity of peroxisome proliferator-activated receptors (PPARs), we used a yeast two-hybrid system with Gal4-PPAR␥ as bait to screen a mouse liver cDNA library and have identified steroid receptor coactivator-1 (SRC-1) as a PPAR transcriptional coactivator. We now report the isolation of a cDNA encoding a 165-kDa PPAR␥-binding protein, designated PBP which also serves as a coactivator. PBP also binds to PPAR␣, RAR␣, RXR, and TR␤1, and this binding is increased in the presence of specific ligands. Deletion of the last 12 amino acids from the carboxyl terminus of PPAR␥ results in the abolition of interaction between PBP and PPAR␥. PBP modestly increased the transcriptional activity of PPAR␥, and a truncated form of PBP (amino acids 487-735) acted as a dominantnegative repressor, suggesting that PBP is a genuine coactivator for PPAR. In addition, PBP contains two LXXLL signature motifs considered necessary and sufficient for the binding of several coactivators to nuclear receptors. In situ hybridization and Northern analysis showed that PBP is expressed in many tissues of adult mice, including the germinal epithelium of testis, where it appeared most abundant, and during ontogeny, suggesting a possible role for this cofactor in cellular proliferation and differentiation.The peroxisome proliferator-activated receptors (PPARs) 1 are a group of transcription factors that regulate the expression of target genes, in particular those associated with lipid metabolism (1, 2). PPARs, which derive the designation by virtue of their ability to mediate predictable pleiotropic effects in response to peroxisome proliferators (1,3,4), are members of the nuclear receptor superfamily (5, 6). Three isotypes of PPARs, namely PPAR␣, PPAR␦ (also called ␤ or NUC-1), and PPAR␥ have been identified as products of separate genes from Xenopus, rodents, and humans (1, 7-12). These PPAR isotypes appear to exhibit distinct patterns of tissue distribution and differ considerably in their ligand binding domains, suggesting that they possibly perform different functions in different cell types (7,13,14). Indeed, of the three isotypes, PPAR␣ expression is relatively high in hepatocytes, enterocytes, and the proximal tubular epithelium of kidney when compared with other cell types (13,14), and evidence derived from mice with PPAR␣ gene disruption indicates that this receptor is essential for the pleiotropic responses induced by peroxisome proliferators (15). Several structurally diverse peroxisome proliferators, specific fatty acids, and eicosanoids act as ligands for PPAR␣ (4, 16 -19). Although PPAR␦ isotype is ubiquitously expressed and binds the same ligands as PPAR␣ (18,19), its functional significance remains largely elusive. PPAR␥ exists as two isoforms, PPAR␥1 and PPAR␥2, as a consequence of alternate promoter usage in the gene encoding this receptor (8,20,21). While PPAR␥1 isoform expression is restricted to liver and few other organs (8, 14), the PPAR␥2 i...

Fatty acid ␤-oxidation occurs in both mitochondria and peroxisomes. Long chain fatty acids are also metabolized by the cytochrome P450 CYP4A -oxidation enzymes to toxic dicarboxylic acids (DCAs) that serve as substrates for peroxisomal ␤-oxidation. Synthetic peroxisome proliferators interact with peroxisome proliferator activated receptor ␣ (PPAR␣) to transcriptionally activate genes that participate in peroxisomal, microsomal, and mitochondrial fatty acid oxidation. Mice lacking PPAR␣ (PPAR␣ ؊/؊ ) fail to respond to the inductive effects of peroxisome proliferators, whereas those lacking fatty acyl-CoA oxidase (AOX ؊/؊ ), the first enzyme of the peroxisomal ␤-oxidation system, exhibit extensive microvesicular steatohepatitis, leading to hepatocellular regeneration and massive peroxisome proliferation, implying sustained activation of PPAR␣ by natural ligands. We now report that mice nullizygous for both PPAR␣ and AOX (PPAR␣ ؊/؊ AOX ؊/؊ ) failed to exhibit spontaneous peroxisome proliferation and induction of PPAR␣-regulated genes by biological ligands unmetabolized in the absence of AOX. In AOX ؊/؊ mice, the hyperactivity of PPAR␣ enhances the severity of steatosis by inducing CYP4A family proteins that generate DCAs and since they are not metabolized in the absence of peroxisomal ␤-oxidation, they damage mitochondria leading to steatosis. Blunting of microvesicular steatosis, which is restricted to few liver cells in periportal regions in PPAR␣ ؊/؊ AOX ؊/؊ mice, suggests a role for PPAR␣-induced genes, especially members of CYP4A family, in determining the severity of steatosis in livers with defective peroxisomal ␤-oxidation. In agematched PPAR␣ ؊/؊ mice, a decrease in constitutive mitochondrial ␤-oxidation with intact constitutive peroxisomal ␤-oxidation system contributes to large droplet fatty change that is restricted to centrilobular hepatocytes. These data define a critical role for both PPAR␣ and AOX in hepatic lipid metabolism and in the pathogenesis of specific fatty liver phenotype.In animal cells, mitochondria as well as peroxisomes oxidize fatty acids via ␤-oxidation, with long chain and very long chain fatty acids (LCFAs and VLCFAs) 1 being preferentially oxidized by peroxisomes (1-3). Peroxisomal ␤-oxidation is carried out by two distinct groups of enzymes. The classical first group utilizes straight chain saturated fatty acyl-CoAs as substrates, whereas the second group acts on branched chain acyl-CoAs (3, 4). In the classical L-3-hydroxy-specific ␤-oxidation spiral, dehydrogenation of acyl-CoA esters to their corresponding trans-2-enoyl-CoAs is catalyzed by fatty acyl-CoA oxidase (AOX), whereas the second and third reactions, hydration and dehydrogenation of enoyl-CoA esters to 3-ketoacyl-CoA, are catalyzed by a single enzyme, enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase (L-bifunctional enzyme (L-PBE)) (3). The third enzyme of this classical system, 3-ketoacyl-CoA thiolase (PTL), cleaves 3-ketoacyl-CoAs to acetyl-CoA and an acylCoA that is two carbon atoms shorter than the original mo...