Coronavirus Disease 2019 (COVID-19), caused by the novel Severe Acute Respiratory Syndrome–associated Coronavirus 2 (SARS-CoV-2), has become a global threat to public health. COVID-19 is more pathogenic and infectious than the prior 2002 pandemic caused by SARS-CoV-1. The pathogenesis of certain disease manifestations in COVID-19 such as diffuse alveolar damage (DAD) are thought to be similar to SARS-CoV-1. However, the exact pathogenesis of COVID-19 related deaths remains poorly understood. The aim of this article was to systematically summarize the rapidly emerging literature regarding COVID-19 autopsies. A meta-analysis was also conducted based on data accrued from preprint and published articles on COVID-19 (n=241 patients) and the results compared with postmortem findings associated with SARS-CoV-1 deaths (n=91 patients). Both autopsy groups included mostly adults of median age 70 years with COVID-19 and 50 years with SARS-CoV-1. Overall, prevalence of DAD was more common in SARS-CoV-1 (100.0%) than COVID-19 (80.9%) autopsies ( P =0.001). Extrapulmonary findings among both groups were not statistically significant except for hepatic necrosis ( P <0.001), splenic necrosis ( P <0.006) and white pulp depletion ( P <0.001) that were more common with SARS-CoV-1. Remarkable postmortem findings in association with COVID-19 apart from DAD include pulmonary hemorrhage, viral cytopathic effect within pneumocytes, thromboembolism, brain infarction, endotheliitis, acute renal tubular damage, white pulp depletion of the spleen, cardiac myocyte necrosis, megakaryocyte recruitment, and hemophagocytosis.
Primary pulmonary hypertension (PPH) is characterized by increased pulmonary arterial pressure and vascular resistance. We and others have observed that inflammatory cytokines and infiltrates are present in the lung tissue, but the significance is uncertain. Treprostinil (TRE), a prostacyclin analogue with extended half-life and chemical stability, has shown promise in the treatment of PPH. We hypothesize that TRE might exert beneficial effects in PPH by antagonizing inflammatory cytokine production in the lung. Here we show that TRE dose-dependently inhibits inflammatory cytokine (tumor necrosis factor-α, interleukin-1β, interleukin-6, and granulocyte macrophage colony-stimulating factor) secretion and gene expression by human alveolar macrophages. TRE blocks NFκB activation, but IκB-α phosphorylation and degradation are unaffected. Moreover, TRE does not affect the formation of the NFκB·DNA complex but blocks nuclear translocation of p65. These results are the first to illustrate the anti-cytokine actions of TRE in down-regulating NFκB, not through its inhibitory component or by direct binding but by blocking nuclear translocation. These data indicate that inflammatory mechanisms may be important in the pathogenesis of PPH and cytokine antagonism by blocking NFκB may contribute to the efficacy of TRE therapy in PPH. Primary pulmonary hypertension (PPH) is characterized by increased pulmonary arterial pressure and vascular resistance. We and others have observed that inflammatory cytokines and infiltrates are present in the lung tissue, but the significance is uncertain. Treprostinil (TRE), a prostacyclin analogue with extended half-life and chemical stability, has shown promise in the treatment of PPH. We hypothesize that TRE might exert beneficial effects in PPH by antagonizing inflammatory cytokine production in the lung. Here we show that TRE dose-dependently inhibits inflammatory cytokine (tumor necrosis factor-α, interleukin-1β, interleukin-6, and granulocyte macrophage colony-stimulating factor) secretion and gene expression by human alveolar macrophages. TRE blocks NFκB activation, but IκB-α phosphorylation and degradation are unaffected. Moreover, TRE does not affect the formation of the NFκB·DNA complex but blocks nuclear translocation of p65. These results are the first to illustrate the anti-cytokine actions of TRE in down-regulating NFκB, not through its inhibitory component or by direct binding but by blocking nuclear translocation. These data indicate that inflammatory mechanisms may be important in the pathogenesis of PPH and cytokine antagonism by blocking NFκB may contribute to the efficacy of TRE therapy in PPH. primary pulmonary hypertension interleukin regulated on activation normal T cell expressed and secreted treprostinil lipopolysaccharide whole cell extract tumor necrosis factor granulocyte macrophage colony-stimulating factor analysis of variance Primary pulmonary hypertension (PPH)1 is an idiopathic disorder characterized by progressively increasing pulmonary artery pressure and vascular resistance in the absence of secondary causes (1Rubin L.J. N. Engl. J. Med. 1997; 336: 111-117Crossref PubMed Scopus (1171) Google Scholar). The pathogenesis of PPH is not clearly understood. Chronic pulmonary hypertension leads to profound structural alterations in affected vessels, commonly referred to as plexiform lesions (2Voelkel N.F. Tuder R.M. Eur. Respir. J. 1995; 8: 2129-2138Crossref PubMed Scopus (174) Google Scholar). Although the pathogenesis of PPH is not clearly understood, genetic predisposition coupled with inflammation are implicated in the development of PPH. Inflammatory cells (T- and B-lymphocytes and macrophages) are present in PPH plexiform lesions (3Tuder R.M. Groves B. Badesch D.B. Voelkel N.F. Am. J. Pathol. 1994; 144: 275-285PubMed Google Scholar), and several reports describe increased inflammatory cytokines in PPH (4Humbert M. Monti G. Brenot F. Sitbon O. Portier A. Grangeot-Keros L. Duroux P. Galanaud P. Simonneau G. Emilie D. Am. J. Respir. Crit. Care Med. 1995; 151: 1628-1631Crossref PubMed Scopus (645) Google Scholar, 5Fartouk M. Emilie D. LeGall C. Monti G. Simonneau G. Humbert M. Chest. 1998; 114: S50-S51Abstract Full Text Full Text PDF Scopus (69) Google Scholar, 6Chacon M.R. Bishop A.E. Arandilla R.M. Tuder R.M. Voelkel N.F. Yacoub M.H. Polak J.M. Am. J. Respir. Crit. Care Med. 1999; 159: A696Google Scholar, 7Dorfmu¨ller P. Zarka V. Durand-Gasselin I. Monti G. Balabanian K. Garcia G. Capron F. Coulomb-Lhermineá A. Marfaing-Koka A. Simonneau G. Emilie D. Humbert M. Am. J. Respir. Crit. Care Med. 2002; 165: 534-539Crossref PubMed Scopus (233) Google Scholar). Elevated serum levels of interleukin (IL)-1 and IL-6 have been noted in PPH (4Humbert M. Monti G. Brenot F. Sitbon O. Portier A. Grangeot-Keros L. Duroux P. Galanaud P. Simonneau G. Emilie D. Am. J. Respir. Crit. Care Med. 1995; 151: 1628-1631Crossref PubMed Scopus (645) Google Scholar). Increased mRNA levels of macrophage inflammatory protein-1, vascular endothelial growth factor, and RANTES have been detected in PPH lung biopsy specimens (5Fartouk M. Emilie D. LeGall C. Monti G. Simonneau G. Humbert M. Chest. 1998; 114: S50-S51Abstract Full Text Full Text PDF Scopus (69) Google Scholar, 6Chacon M.R. Bishop A.E. Arandilla R.M. Tuder R.M. Voelkel N.F. Yacoub M.H. Polak J.M. Am. J. Respir. Crit. Care Med. 1999; 159: A696Google Scholar, 7Dorfmu¨ller P. Zarka V. Durand-Gasselin I. Monti G. Balabanian K. Garcia G. Capron F. Coulomb-Lhermineá A. Marfaing-Koka A. Simonneau G. Emilie D. Humbert M. Am. J. Respir. Crit. Care Med. 2002; 165: 534-539Crossref PubMed Scopus (233) Google Scholar). Our previous observation of enhanced NFκB activity in alveolar macrophages from PPH patients also demonstrates that PPH alveolar macrophages are activated and suggests a role for inflammatory cytokines in PPH (8Raychaudhuri B. Dweik R.A. Connors M. Buhrow L. Malur A. Drazba J. Arroliga A.C. Erzurum S.C. Kavuru M. Thomassen M.J. Am. J. Respir. Cell Mol. Biol. 1999; 21: 311-316Crossref PubMed Scopus (110) Google Scholar). Increased urinary excretion of prostaglandin D2 metabolites by PPH patients are also suggestive of macrophage activation (9Robbins I.M. Barst R.J. Rubin L.J. Gaine S.P. Price P.V. Morrow J.D. Christman B.W. Chest. 2001; 120: 1639-1644Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). These observations suggest that inflammatory cytokines may participate in the pathogenesis of PPH. Recent studies have demonstrated the therapeutic efficacy of prostacyclin for PPH (10Barst R.J. Rubin L.J. Long W.A. McGoon M.D. Rich S. Badesch D.B. Groves B. Tapson V.F. Bourge R.C. Brundage B.H. Koerner S.K. Langleben D. Keller C.A. Murali S. Uretsky B.F. Clayton L.M. Jobsis M.M. Blackburn S.D. Shortino D. Crow J.W. N. Engl. J. Med. 1996; 334: 296-301Crossref PubMed Scopus (1601) Google Scholar, 11Shapirio S.M. Oudiz R.J. Cao T. Romano M.A. Beckmann J. Georgiou D. Mandayam S. Ginzton L.E. Brundage B.H. J. Am. Coll. Cardiol. 1997; 30: 343-349Crossref PubMed Scopus (271) Google Scholar, 12McLaughlin V.V. Genthner D.E. Panella M.M. Rich S. N. Engl. J. Med. 1998; 338: 273-277Crossref PubMed Scopus (615) Google Scholar, 13Barst R.J. Heart. 1997; 77: 299-301Crossref PubMed Scopus (39) Google Scholar). Prostaglandins comprise a family of lipid compounds derived from arachidonic acid. In 1976, Moncadaet al. (32Moncada S. Gryglewski R. Bunting S. Vane J. Nature. 1976; 263: 663-665Crossref PubMed Scopus (2933) Google Scholar) discovered prostaglandin I2and named it prostacyclin (reviewed in Ref. 14Galie L. Eur. Heart J. 1996; 17: 18-24PubMed Google Scholar). The potent vasodilator activity and capacity for inhibition of platelet aggregation provide the rationale for prostacyclin therapy in PPH. Indeed, continuous intravenous infusion of prostacyclin improves hemodynamics, exercise tolerance, and survival in PPH patients (10Barst R.J. Rubin L.J. Long W.A. McGoon M.D. Rich S. Badesch D.B. Groves B. Tapson V.F. Bourge R.C. Brundage B.H. Koerner S.K. Langleben D. Keller C.A. Murali S. Uretsky B.F. Clayton L.M. Jobsis M.M. Blackburn S.D. Shortino D. Crow J.W. N. Engl. J. Med. 1996; 334: 296-301Crossref PubMed Scopus (1601) Google Scholar, 11Shapirio S.M. Oudiz R.J. Cao T. Romano M.A. Beckmann J. Georgiou D. Mandayam S. Ginzton L.E. Brundage B.H. J. Am. Coll. Cardiol. 1997; 30: 343-349Crossref PubMed Scopus (271) Google Scholar, 12McLaughlin V.V. Genthner D.E. Panella M.M. Rich S. N. Engl. J. Med. 1998; 338: 273-277Crossref PubMed Scopus (615) Google Scholar, 13Barst R.J. Heart. 1997; 77: 299-301Crossref PubMed Scopus (39) Google Scholar). Other properties of prostacyclin may also be important in therapeutic efficacy as suggested by the fact that patients who do not have acute hemodynamic improvement still appear to benefit from chronic therapy (14Galie L. Eur. Heart J. 1996; 17: 18-24PubMed Google Scholar). Interestingly, several studies have demonstrated the inhibitory effects of prostacyclin on cytokine production by blood mononuclear cells (15Luttmann W. Herzog V. Matthys H. Thierauch K.H. Virchow J. Kroegel C. Cytokine. 1999; 11: 127-133Crossref PubMed Scopus (20) Google Scholar, 16Luttmann W. Herzog V. Virchow J.C. Matthys H. Thierauch K.H. Kroegel C. Pulm. Pharmacol. 1996; 9: 43-48Crossref PubMed Scopus (20) Google Scholar, 17Meja K.K. Barnes P.J. Giembycz M.A. Br. J. Pharmacol. 1997; 122: 149-157Crossref PubMed Scopus (92) Google Scholar, 18Eisenhut T. Sinha B. Grottrup-Wolfers E. Semmler J. Siess W. Endres S. Immunopharmacology. 1993; 26: 259-264Crossref PubMed Scopus (111) Google Scholar). However, the effect of such drugs on alveolar macrophages, one of the major sources of cytokines in the lung, is unknown. Treprostinil (TRE) is a more stable benzidine derivative of prostacyclin and has shown success in treating PPH (19Simonneau G. Barst R.J. Galie N. Naeije R. Rich S. Bourge R.C. Keogh A. Oudiz R. Frost A. Blackburn S.D. Crow J.W. Rubin L.J. Am. J. Respir. Crit. Care Med. 2002; 165: 800-804Crossref PubMed Scopus (1240) Google Scholar). We hypothesized that in addition to its direct hemodynamic effects, TRE might also decrease cytokine production and exert anti-inflammatory effect in the lung. To test this hypothesis, we investigated the effect of TRE on inflammatory cytokine secretion and gene expression in human alveolar macrophages. Salmonella typhimuriumlipopolysaccharide (LPS) was obtained from Sigma and used at 0.5 μg/ml for all experiments. TRE was a gift of the United Therapeutic Corporation (Research Triangle Park, NC). Fiberoptic bronchoscopy with bronchoalveolar lavage was performed as described previously (8Raychaudhuri B. Dweik R.A. Connors M. Buhrow L. Malur A. Drazba J. Arroliga A.C. Erzurum S.C. Kavuru M. Thomassen M.J. Am. J. Respir. Cell Mol. Biol. 1999; 21: 311-316Crossref PubMed Scopus (110) Google Scholar). The study population consisted of healthy volunteers 18–65 years of age with no lung disease and on no medication. All of the volunteers provided written informed consent, and the study was approved by the institutional review board of the Cleveland Clinic Foundation. Alveolar macrophages were obtained by adhering cells from bronchoalveolar lavage as described previously (8Raychaudhuri B. Dweik R.A. Connors M. Buhrow L. Malur A. Drazba J. Arroliga A.C. Erzurum S.C. Kavuru M. Thomassen M.J. Am. J. Respir. Cell Mol. Biol. 1999; 21: 311-316Crossref PubMed Scopus (110) Google Scholar, 20Thomassen M.J. Boxerbaum B. Demko C.A. Kuchenbrod P.J. Dearborn D.G. Wood R.E. Pediatr. Res. 1979; 13: 1085-1088Crossref PubMed Scopus (62) Google Scholar). Nonadherent cells were removed by washing. The adherent cell population consisted of greater than 99% macrophages. Alveolar macrophages were cultured overnight prior toin vitro treatment. After overnight incubation, macrophages were treated with LPS ± TRE (2–200 ng/ml) or left untreated for 4 h. TRE did not adversely affect cell viability at any dose tested as measured by trypan blue dye exclusion and cell adherence. Cells were harvested, and WCEs were prepared as described previously (8Raychaudhuri B. Dweik R.A. Connors M. Buhrow L. Malur A. Drazba J. Arroliga A.C. Erzurum S.C. Kavuru M. Thomassen M.J. Am. J. Respir. Cell Mol. Biol. 1999; 21: 311-316Crossref PubMed Scopus (110) Google Scholar). In one group of experiments, TRE or vehicle was added directly to WCE from untreated or LPS-treated cells for 15 min. The protein content of WCEs was measured by BCA protein assay method (Pierce, Rockford, IL). For electrophoretic mobility shift assay, 10 μg of the WCE were incubated in binding buffer (8 mm HEPES, pH 7.0, 10% glycerol, 20 mm KCl, 4 mm MgCl2, 1 mm sodium pyrophosphate) containing 1 μg of poly(dI·dC) and 40,000-cpm probe for 20 min at room temperature as described previously (8Raychaudhuri B. Dweik R.A. Connors M. Buhrow L. Malur A. Drazba J. Arroliga A.C. Erzurum S.C. Kavuru M. Thomassen M.J. Am. J. Respir. Cell Mol. Biol. 1999; 21: 311-316Crossref PubMed Scopus (110) Google Scholar). Supershifts were performed with anti-p65 and anti-p50 (Santa Cruz Biotechnology, Santa Cruz, CA) added prior to the addition of the probe. The specificity of the probe was shown by incubating the extract with a 1000-fold molar excess of cold oligonucleotide. StormImager (Molecular Dynamics, Sunnyvale, CA) and ImageQuant software were used for quantification of autoradiographs. The sequence of the NFκB binding sites was 5′-AACTCCGGGAATTTCCCTGGCCC-3′. Treated cells were washed once with ice-cold phosphate-buffered saline and lysed as described previously (8Raychaudhuri B. Dweik R.A. Connors M. Buhrow L. Malur A. Drazba J. Arroliga A.C. Erzurum S.C. Kavuru M. Thomassen M.J. Am. J. Respir. Cell Mol. Biol. 1999; 21: 311-316Crossref PubMed Scopus (110) Google Scholar). Protein concentrations were measured by BCA assay, and 10 μg of the cell lysate were mixed with 1:1 sample buffer, boiled, and analyzed on a 10% sodium dodecylsulfate-polyacrylamide gel and transferred to Immobilon-P membranes. After blocking membranes, the primary antibody to IκBα (Santa Cruz Biotechnology) or phospho-IκBα (Cell Signaling, Beverly, MA) was applied at 1:1000 dilution for 1 h at room temperature. After secondary antibody application and washing, bands were visualized by enhanced chemiluminescence (AmershamBiosciences). Alveolar macrophages were cultured in chamber slides overnight prior to treatment with LPS ± TRE (200 ng/ml) for 0.5–4 h and then stained for p65 as described previously (21Raychaudhuri B. Fisher C.J. Farver C.F. Malur A. Drazba J. Kavuru M.S. Thomassen M.J. Cytokine. 2000; 12: 1348-1355Crossref PubMed Scopus (56) Google Scholar). The cells were washed gently with 37 °C phosphate-buffered saline and then fixed with 4% paraformaldehyde for 30 min prior to permeabilization with 1% Triton-100. After a blocking step, slides were incubated with primary antibody (Rabbit anti-human p 65, Santa Cruz Biotechnology) (1:1000) overnight at 4 °C. After further washing, slides were exposed to secondary antibody for 45 min. Simultaneous two-color fluorescence images were collected on a Leica TCS-SP laser-scanning confocal microscope. Nuclear p65 was then quantified by the BIOQUANT True Color Windows 95 system, version 2.0 (R & M Biometrics, Inc., Nashville, TN) as described previously (21Raychaudhuri B. Fisher C.J. Farver C.F. Malur A. Drazba J. Kavuru M.S. Thomassen M.J. Cytokine. 2000; 12: 1348-1355Crossref PubMed Scopus (56) Google Scholar). Cytokine products (TNF-α, IL-6, GM-CSF, IL-1β) were analyzed in duplicate samples of cell-free supernatant fluids from 24-h macrophage cultures by enzyme-linked immunosorbent assay (Endogen, Cambridge, MA). Assay sensitivity ranged from 25–1000 pg/ml, and assay coefficient of variation was <10%. Alveolar macrophage RNA was prepared by RNeasy protocol (Qiagen, Valencia, CA). RNase protection assay was performed using a PharMingen kit (BD PharMingen) including a custom-made probe containing IL-10, GM-CSF, IL-6, macrophage inflammatory protein-1α, vascular endothelial growth factor, and two housekeeping genes L32 and glyceraldehyde-3-phosphate dehydrogenase. Quantification of the radioactivity was done using a StormImager and calculated using ImageQuant analysis. Data were analyzed by one-way analysis of variance (ANOVA) using Prism software (GraphPad, Inc., San Diego, CA). Significance was defined as p = 0.05. The means ± S.E. are provided. The effect of TRE (2–400 ng/ml) on LPS-stimulated production of IL-6 was evaluated by enzyme-linked immunosorbent assay (Fig. 1A). Treatment with LPS alone increased cytokine production, whereas the presence of TRE significantly inhibited LPS-mediated increases in a dose-dependent manner (p = 0.0017). To determine whether this result was cytokine-specific, the effect of TRE (200 ng/ml) on TNF, IL-1, and GM-CSF was tested. All of the three cytokines were inhibited by TRE (Fig. 1B). Treatment with LPS also increased alveolar macrophage mRNA expression of TNF-α, IL-1β, IL-6, and GM-CSF in a time-dependent fashion (Fig.2A) when compared with unstimulated cells with a peak for TNF at 8 and 24 h for the other cytokines. Simultaneous treatment with TRE (200 ng/ml) inhibited LPS-induced mRNA expression of all four cytokines. Fig.2B shows the quantification of the same gel by ImageQuant analysis. Because NFκB is a ubiquitous transcription factor involved in the regulation of all four cytokines studied as well as many others, we next determined the effect of TRE on LPS-induced NFκB activation (Fig. 3). LPS treatment increased NFκB activation when compared with unstimulated cells (Fig. 3A). Simultaneous treatment with TRE reduced the formation of the NFκB·DNA complex, suggesting that TRE inhibited NFκB activation. Specificity was confirmed by supershift with p65 and p50 antibodies and competition with cold oligonucleotide. These data were quantified (Fig. 3B by ImageQuant analysis). The possibility that the antagonistic effect of TRE on LPS-induced NFκB was directed against IκBα was next examined (Fig.4). Fig. 4A shows a rapid (within 45 min) disappearance of IκBα upon LPS treatment with or without TRE. Similarly, the phosphorylation of IκBα occurred after LPS treatment in the presence or absence of TRE (Fig. 4B). Data suggest that TRE is not targeting IκBα phosphorylation or degradation. Because TRE had no apparent effect on IκB-α, we investigated whether TRE altered the nuclear translocation of the p65·p50 complex or whether it directly blocked DNA binding. To assess the former mechanism, we employed immunocytochemistry to visualize p65 at different time intervals (30 min, 1, 2, 3, and 4 h) after LPS treatment (Fig.5, 30-min time point). From 30 min to 4 h after LPS treatment, nuclear staining of p65 was prominent. Simultaneous treatment with TRE prevented p65 nuclear translocation as shown by the reduced numbers of intensely stained nuclei. Image quantification confirmed the reduction of p65 translocation (Fig.5E). To assess the second mechanism, we exposed whole cell extracts from LPS-treated cells to TRE (200 ng/ml) in vitro. TRE failed to block NFκB activation as determined by electrophoretic mobility shift assay (data not shown). These results strongly suggest that TRE blocks NFκB activation by impeding nuclear translocation of p65 but not by inhibiting cognate binding. The potent vasodilator activity and capacity for inhibition of platelet aggregation provide the rationale for prostacyclin use in PPH. Other properties of prostacyclin may also be important in therapeutic efficacy as suggested by the fact that patients who do not have acute hemodynamic improvement still appear to benefit from chronic therapy (14Galie L. Eur. Heart J. 1996; 17: 18-24PubMed Google Scholar). Furthermore, the association of PPH with inflammatory cytokines has been well documented (4Humbert M. Monti G. Brenot F. Sitbon O. Portier A. Grangeot-Keros L. Duroux P. Galanaud P. Simonneau G. Emilie D. Am. J. Respir. Crit. Care Med. 1995; 151: 1628-1631Crossref PubMed Scopus (645) Google Scholar, 5Fartouk M. Emilie D. LeGall C. Monti G. Simonneau G. Humbert M. Chest. 1998; 114: S50-S51Abstract Full Text Full Text PDF Scopus (69) Google Scholar, 6Chacon M.R. Bishop A.E. Arandilla R.M. Tuder R.M. Voelkel N.F. Yacoub M.H. Polak J.M. Am. J. Respir. Crit. Care Med. 1999; 159: A696Google Scholar, 7Dorfmu¨ller P. Zarka V. Durand-Gasselin I. Monti G. Balabanian K. Garcia G. Capron F. Coulomb-Lhermineá A. Marfaing-Koka A. Simonneau G. Emilie D. Humbert M. Am. J. Respir. Crit. Care Med. 2002; 165: 534-539Crossref PubMed Scopus (233) Google Scholar). Based on these observations, we hypothesized that prostacyclins might effect inflammatory cytokine production. The current findings show for the first time that the prostacyclin analogue TRE effectively reduces human alveolar macrophage inflammatory cytokine secretion by inhibiting NFκB activation and subsequent transcription of cytokine genes. Furthermore, our data show that the inhibition of NFκB activation by TRE is by blockade of p65 nuclear translocation. The molecular regulation of inflammatory cytokine genes is a complex process in which NFκB constitutes a critical element (22Baldwin A.S., Jr. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5575) Google Scholar, 23Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4598) Google Scholar). In quiescent cells, NFκB is located in the cytosol as a heterodimer or homodimer of protein components bound to an inhibitor IκB. The activation of this transcription factor is controlled by sequential phosphorylation, ubiquitination, and proteasome-mediated degradation of IκB, resulting in the migration of the NFκB complex to the nucleus and binding to the promoter region of many cytokine and growth factor genes (23Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4598) Google Scholar, 24Thanos D. Maniatis T. Cell. 1995; 80: 529-532Abstract Full Text PDF PubMed Scopus (1217) Google Scholar). Most known inhibitors of NFκB activation such as glucocorticosteroids, aspirin, and nitric oxide have been shown to block NFκB activation by interfering with IκB degradation and/or stimulating IκB synthesis (8Raychaudhuri B. Dweik R.A. Connors M. Buhrow L. Malur A. Drazba J. Arroliga A.C. Erzurum S.C. Kavuru M. Thomassen M.J. Am. J. Respir. Cell Mol. Biol. 1999; 21: 311-316Crossref PubMed Scopus (110) Google Scholar, 25Auphan N. DiDonato J.A. Rosette C. Helmberg A. Karin M. Science. 1995; 270: 286-290Crossref PubMed Scopus (2163) Google Scholar, 26Scheinman R.I. Cogswell P.C. Lofquist A.K. Baldwin A.S., Jr. Science. 1995; 270: 283-286Crossref PubMed Scopus (1597) Google Scholar, 27Kopp E. Ghosh S. Science. 1994; 265: 956-959Crossref PubMed Scopus (1617) Google Scholar). TRE prevents NFκB activation without affecting IκB-α degradation by interfering with nuclear translocation of the p65·p60 complex. Other reagents, for example, such as caffeic acid phenethyl ester are known to target nuclear translocation of p65 as a mechanism for anti-NFκB activity (28Natarjan K. Singh S. Burke T.R. Grunberger D. Aggarwal B.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9090-9095Crossref PubMed Scopus (1036) Google Scholar). Several reports have demonstrated that prostacyclin inhibits cytokine secretion from human peripheral blood mononuclear cells and the human monocytic cell lines THP-1 and Mono Mac 6 (15–18;29). Both Luttmann et al. (16Luttmann W. Herzog V. Virchow J.C. Matthys H. Thierauch K.H. Kroegel C. Pulm. Pharmacol. 1996; 9: 43-48Crossref PubMed Scopus (20) Google Scholar) and Crutchley et al. (29Crutchley D.J. Conanan L.B. Que B.G. J. Pharmacol. Exp. Ther. 1994; 271: 446-451PubMed Google Scholar) demonstrated that prostacyclin inhibited gene expression, but whether NFκB is involved was not explored. Surprisingly, the prostacyclin analogue iloprost blocked IκB-α degradation in the murine monocyte/macrophage cell line J774 (30D'Acquisto F. Sautebin L. Iuvone T., Di Rosa M. Carnuccio R. FEBS Lett. 1998; 440: 76-80Crossref PubMed Scopus (96) Google Scholar). Whether this difference is because of the fact that the human alveolar macrophages are primary cells and/or whether there are species-specific differences remains to be investigated. Furthermore, because cytokines promote smooth muscle cell proliferation, the recent study demonstrating that TRE inhibits proliferation of human pulmonary artery cells in vitro may be attributed to anti-cytokine effects (31Clapp L.H. Finney P. Turcato S. Tran S. Rubin L.J. Tinker A. Am. J. Respir. Cell Mol. Biol. 2002; 26: 194-201Crossref PubMed Scopus (203) Google Scholar). Nevertheless, the actions of TRE make it an effective cytokine inhibitor, and this effect is mediated at least in part by blocking NFκB activation, although other mechanism(s) may also be involved in cytokine regulation by TRE. The data presented here show that TRE blocks NFκB translocation, thereby potentially inhibiting inflammatory cytokine production. The association of PPH with inflammatory cytokine production has been well documented (4Humbert M. Monti G. Brenot F. Sitbon O. Portier A. Grangeot-Keros L. Duroux P. Galanaud P. Simonneau G. Emilie D. Am. J. Respir. Crit. Care Med. 1995; 151: 1628-1631Crossref PubMed Scopus (645) Google Scholar, 5Fartouk M. Emilie D. LeGall C. Monti G. Simonneau G. Humbert M. Chest. 1998; 114: S50-S51Abstract Full Text Full Text PDF Scopus (69) Google Scholar, 6Chacon M.R. Bishop A.E. Arandilla R.M. Tuder R.M. Voelkel N.F. Yacoub M.H. Polak J.M. Am. J. Respir. Crit. Care Med. 1999; 159: A696Google Scholar, 7Dorfmu¨ller P. Zarka V. Durand-Gasselin I. Monti G. Balabanian K. Garcia G. Capron F. Coulomb-Lhermineá A. Marfaing-Koka A. Simonneau G. Emilie D. Humbert M. Am. J. Respir. Crit. Care Med. 2002; 165: 534-539Crossref PubMed Scopus (233) Google Scholar). Moreover, our previous studies indicate that PPH alveolar macrophages are endogenously activated as illustrated by activated NFκB (8Raychaudhuri B. Dweik R.A. Connors M. Buhrow L. Malur A. Drazba J. Arroliga A.C. Erzurum S.C. Kavuru M. Thomassen M.J. Am. J. Respir. Cell Mol. Biol. 1999; 21: 311-316Crossref PubMed Scopus (110) Google Scholar). Therefore, it is intriguing to speculate that some of the clinical efficacy shown by TRE in PPH may be attributed to down-regulation of inflammatory cytokine production. We thank Dr. Barbara Barna for helpful suggestions and review of this paper.
In this study, we demonstrate that forkhead box F1 (FOXF1), a mesenchymal transcriptional factor essential for lung development, was retained in a topographically distinct mesenchymal stromal cell population along the bronchovascular space in an adult lung and identify this distinct subset of collagen-expressing cells as key players in lung allograft remodeling and fibrosis. Using Foxf1-tdTomato BAC (Foxf1-tdTomato) and Foxf1-tdTomato Col1a1-GFP mice, we show that Lin-Foxf1+ cells encompassed the stem cell antigen 1+CD34+ (Sca1+CD34+) subset of collagen 1-expressing mesenchymal cells (MCs) with a capacity to generate CFU and lung epithelial organoids. Histologically, FOXF1-expressing MCs formed a 3D network along the conducting airways; FOXF1 was noted to be conspicuously absent in MCs in the alveolar compartment. Bulk and single-cell RNA-Seq confirmed distinct transcriptional signatures of Foxf1+ and Foxf1- MCs, with Foxf1-expressing cells delineated by their high expression of the transcription factor glioma-associated oncogene 1 (Gli1) and low expression of integrin α8 (Itga), versus other collagen-expressing MCs. FOXF1+Gli1+ MCs showed proximity to Sonic hedgehog-expressing (Shh-expressing) bronchial epithelium, and mesenchymal expression of Foxf1 and Gli1 was found to be dependent on paracrine Shh signaling in epithelial organoids. Using a murine lung transplant model, we show dysregulation of epithelial-mesenchymal SHH/GLI1/FOXF1 crosstalk and expansion of this specific peribronchial MC population in chronically rejecting fibrotic lung allografts.
Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a ligand-activated, nuclear transcription factor that regulates genes involved in lipid and glucose metabolism, inflammation, and other pathways. The hematopoietic growth factor, granulocyte macrophage colony-stimulating factor (GM-CSF), is essential for lung homeostasis and is thought to regulate surfactant clearance, but mechanisms involved are unknown. GM-CSF is reported to stimulate PPAR-gamma, but the activation status of PPAR-gamma in human alveolar macrophages has not been defined. In pulmonary alveolar proteinosis (PAP), a rare interstitial lung disease, surfactant accumulates in alveolar airspaces, resident macrophages become engorged with lipoproteinaceous material, and GM-CSF deficiency is strongly implicated in pathogenesis. Here we show that PPAR-gamma mRNA and protein are highly expressed in alveolar macrophages of healthy control subjects but severely deficient in PAP in a cell-specific manner. Further, we show that the PPAR-gamma-regulated lipid scavenger receptor, CD36, is also deficient in PAP. PPAR-gamma and CD36 deficiency are not intrinsic to PAP alveolar macrophages, but can be upregulated by GM-CSF therapy. Moreover, GM-CSF treatment of patients with PAP fully restores PPAR-gamma to healthy control levels. Based upon these novel findings, we hypothesize that GM-CSF regulates lung homeostasis via PPAR-gamma-dependent pathways.
Abstract ARDS due to COVID-19 and other etiologies results from injury to the alveolar epithelial cell (AEC) barrier resulting in noncardiogenic pulmonary edema, which causes acute respiratory failure; clinical recovery requires epithelial regeneration. During physiologic regeneration in mice, AEC2s proliferate, exit the cell cycle, and transiently assume a transitional state before differentiating into AEC1s; persistence of the transitional state is associated with pulmonary fibrosis in humans. It is unknown whether transitional cells emerge and differentiate into AEC1s without fibrosis in human ARDS and why transitional cells differentiate into AEC1s during physiologic regeneration but persist in fibrosis. We hypothesized that incomplete but ongoing AEC1 differentiation from transitional cells without fibrosis may underlie persistent barrier permeability and fatal acute respiratory failure in ARDS. Immunostaining of postmortem ARDS lungs revealed abundant transitional cells in organized monolayers on alveolar septa without fibrosis. They were typically cuboidal or partially spread, sometimes flat, and occasionally expressed AEC1 markers. Immunostaining and/or interrogation of scRNAseq datasets revealed that transitional cells in mouse models of physiologic regeneration, ARDS, and fibrosis express markers of cell cycle exit but only in fibrosis express a specific senescence marker. Thus, in severe, fatal early ARDS, AEC1 differentiation from transitional cells is incomplete, underlying persistent barrier permeability and respiratory failure, but ongoing without fibrosis; senescence of transitional cells may be associated with pulmonary fibrosis.
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