Presence of hyaluronan in lung alveoli in severe Covid-19: An opening for new treatment options?

Abstract

Severe coronavirus disease 2019 (Covid-19) is characterized by inflammation of the lungs with increasing respiratory impairment. In fatal Covid-19, lungs at autopsy have been filled with a clear liquid jelly. However, the nature of this finding has not yet been determined. The aim of the study was to demonstrate whether the lungs of fatal Covid-19 contain hyaluronan, as it is associated with inflammation and acute respiratory distress syndrome (ARDS) and may have the appearance of liquid jelly. Lung tissue obtained at autopsy from three deceased Covid-19 patients was processed for hyaluronan histochemistry using a direct staining method and compared with staining in normal lung tissue. Stainings confirmed that hyaluronan is obstructing alveoli with presence in exudate and plugs, as well as in thickened perialveolar interstitium. In contrast, normal lungs only showed hyaluronan in intact alveolar walls and perivascular tissue. This is the first study to confirm prominent hyaluronan exudates in the alveolar spaces of Covid-19 lungs, supporting the notion that the macromolecule is involved in ARDS caused by SARS-CoV-2. The present finding may open up new treatment options in severe Covid-19, aiming at reducing the presence and production of hyaluronan in the lungs. Severe coronavirus disease 2019 (Covid-19) is characterized by inflammation of the lungs with increasing respiratory impairment. In fatal Covid-19, lungs at autopsy have been filled with a clear liquid jelly. However, the nature of this finding has not yet been determined. The aim of the study was to demonstrate whether the lungs of fatal Covid-19 contain hyaluronan, as it is associated with inflammation and acute respiratory distress syndrome (ARDS) and may have the appearance of liquid jelly. Lung tissue obtained at autopsy from three deceased Covid-19 patients was processed for hyaluronan histochemistry using a direct staining method and compared with staining in normal lung tissue. Stainings confirmed that hyaluronan is obstructing alveoli with presence in exudate and plugs, as well as in thickened perialveolar interstitium. In contrast, normal lungs only showed hyaluronan in intact alveolar walls and perivascular tissue. This is the first study to confirm prominent hyaluronan exudates in the alveolar spaces of Covid-19 lungs, supporting the notion that the macromolecule is involved in ARDS caused by SARS-CoV-2. The present finding may open up new treatment options in severe Covid-19, aiming at reducing the presence and production of hyaluronan in the lungs. The ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has hitherto caused more than 730,000 reported deaths globally (by August 10, 2020, according to Johns Hopkins University). In severe Covid-19, there is a pronounced inflammatory response in the lungs resulting in respiratory failure and potential death, especially among individuals with predisposing chronic diseases or advanced age. The glycosaminoglycan hyaluronan (HA) has recently been suggested to be a potential cause of fatalities in Covid-19 lung infection (1Shi Y. Wang Y. Shao C. Huang J. Gan J. Huang X. Bucci E. Piacentini M. Ippolito G. Melino G. COVID-19 infection: the perspectives on immune responses.Cell Death Differ. 2020; 27 (32205856): 1451-145410.1038/s41418-020-0530-3Crossref PubMed Scopus (935) Google Scholar). The authors referred to findings at autopsies of deceased Covid-19 patients where the lungs were filled with a clear liquid jelly, much resembling the lungs of wet drowning (2Xu Z. Shi L. Wang Y. Zhang J. Huang L. Zhang C. Liu S. Zhao P. Liu H. Zhu L. Tai Y. Bai C. Gao T. Song J. Xia P. et al.Pathological findings of COVID-19 associated with acute respiratory distress syndrome.Lancet Respir. Med. 2020; 8 (32085846): 420-42210.1016/S2213-2600(20)30076-XAbstract Full Text Full Text PDF PubMed Scopus (5614) Google Scholar). Although the nature of this liquid jelly has not yet been determined, HA has been proposed, as it is associated with acute respiratory distress syndrome (ARDS) (1Shi Y. Wang Y. Shao C. Huang J. Gan J. Huang X. Bucci E. Piacentini M. Ippolito G. Melino G. COVID-19 infection: the perspectives on immune responses.Cell Death Differ. 2020; 27 (32205856): 1451-145410.1038/s41418-020-0530-3Crossref PubMed Scopus (935) Google Scholar, 3Hällgren R. Samuelsson T. Laurent T.C. Modig J. Accumulation of hyaluronan (hyaluronic acid) in the lung in adult respiratory distress syndrome.Am. Rev. Respir. Dis. 1989; 139 (2923370): 682-68710.1164/ajrccm/139.3.682Crossref PubMed Scopus (126) Google Scholar). Hyaluronan is an important component of all extracellular matrices, and its levels are generally elevated in tissue in response to inflammation and injury (4Nagy N. Kuipers H.F. Frymoyer A.R. Ishak H.D. Bollyky J.B. Wight T.N. Bollyky P.L. 4-Methylumbelliferone treatment and hyaluronan inhibition as a therapeutic strategy in inflammation, autoimmunity, and cancer.Front. Immunol. 2015; 6 (25852691): 12310.3389/fimmu.2015.00123Crossref PubMed Scopus (177) Google Scholar). The HA molecule is highly hygroscopic and has the ability to absorb water up to 1,000 times its molecular weight, which can promote edema (5Laurent T.C. Laurent U.B. Fraser J.R. The structure and function of hyaluronan: an overview.Immunol. Cell Biol. 1996; 74 (8724014): A1-A710.1038/icb.1996.32Crossref PubMed Scopus (396) Google Scholar). Due to its high molecular weight and semiflexible polymer chain, it forms a gel-like fluid with high viscosity causing exclusion properties and barrier functions in tissues (5Laurent T.C. Laurent U.B. Fraser J.R. The structure and function of hyaluronan: an overview.Immunol. Cell Biol. 1996; 74 (8724014): A1-A710.1038/icb.1996.32Crossref PubMed Scopus (396) Google Scholar). There are also a large number of HA-binding proteins that can involve HA in diverse biological processes (6Day A.J. Prestwich G.D. Hyaluronan-binding proteins: tying up the giant.J. Biol. Chem. 2002; 277 (11717315): 4585-458810.1074/jbc.R100036200Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar). High-molecular weight HA predominates in most tissues under healthy conditions, whereas fragmented low-molecular weight HA polymers predominate at sites of active inflammation (4Nagy N. Kuipers H.F. Frymoyer A.R. Ishak H.D. Bollyky J.B. Wight T.N. Bollyky P.L. 4-Methylumbelliferone treatment and hyaluronan inhibition as a therapeutic strategy in inflammation, autoimmunity, and cancer.Front. Immunol. 2015; 6 (25852691): 12310.3389/fimmu.2015.00123Crossref PubMed Scopus (177) Google Scholar). It has been proposed that HA could be responsible for some of the disease manifestations and mortality in the most severe form of Covid-19 (1Shi Y. Wang Y. Shao C. Huang J. Gan J. Huang X. Bucci E. Piacentini M. Ippolito G. Melino G. COVID-19 infection: the perspectives on immune responses.Cell Death Differ. 2020; 27 (32205856): 1451-145410.1038/s41418-020-0530-3Crossref PubMed Scopus (935) Google Scholar). However, arguments that HA is involved in the pathogenesis of Covid-19 must be verified before new treatment options against the formation of HA can be discussed. Accordingly, the aim of our study was to demonstrate that hyaluronan is present in alveolar spaces in lungs of deceased patients with severe Covid-19. Autopsy lung tissue from the three Covid-19 cases is shown in Fig. 1 (magnification ×20). In two patients, histochemical staining revealed intra-alveolar and interstitial HA localization in the exudative phase (Fig. 1, A and B), whereas the third patient showed the proliferative phase of the Covid-19–associated diffuse alveolar damage (Fig. 1C). The alveolar spaces were filled with exudate and alveolar plugs exhibiting pronounced HA staining. The alveolar walls were hyperplastic and damaged, and HA staining was evident in the thickened perialveolar interstitium (Fig. 2, B–D). In contrast, HA staining was only visible in the alveolar walls and in perivascular tissue in normal lung tissue (Figs. 2A and 3A). The specificity of the staining method for HA is shown in Fig. 3 (B and C). Treatment of the section with hyaluronidase from bovine testes (in Fig. 3C) effectively abolished the HA staining. The pathogenesis of Covid-19 is not fully understood, and there is an urgent need for effective treatments to alleviate the respiratory failure and decrease the case fatality rate in severe disease. This is the first report verifying the presence of HA in the alveolar spaces in lung tissue from three lethal Covid-19 cases. In recent publications of post-mortem findings in Covid-19 lungs (2Xu Z. Shi L. Wang Y. Zhang J. Huang L. Zhang C. Liu S. Zhao P. Liu H. Zhu L. Tai Y. Bai C. Gao T. Song J. Xia P. et al.Pathological findings of COVID-19 associated with acute respiratory distress syndrome.Lancet Respir. Med. 2020; 8 (32085846): 420-42210.1016/S2213-2600(20)30076-XAbstract Full Text Full Text PDF PubMed Scopus (5614) Google Scholar, 7Carsana L. Sonzogni A. Nasr A. Rossi R.S. Pellegrinelli A. Zerbi P. Rech R. Colombo R. Antinori S. Corbellino M. Galli M. Catena E. Tosoni A. Gianatti A. Nebuloni M. Pulmonary post-mortem findings in a series of COVID-19 cases from northern Italy: a two-centre descriptive study.Lancet Infect. Dis. 2020; 20 (32526193): 1135-114010.1016/s1473-3099(20)30434-5Abstract Full Text Full Text PDF PubMed Scopus (741) Google Scholar), the authors describe some characteristic hallmarks, including hyaline membranes, intra-alveolar fibrinous exudate, and alveolar plugs. In our material, the intra-alveolar material and perialveolar interstitium showed pronounced staining for HA, supporting the notion that HA plays an important role in the pathogenesis of severe Covid-19. There is accumulating evidence suggesting that suppressed innate antiviral defenses along with a hyperactive and dysregulated innate immune response may contribute to a “cytokine storm” that drives the clinical presentation of acute lung injury in patients with severe Covid-19 (1Shi Y. Wang Y. Shao C. Huang J. Gan J. Huang X. Bucci E. Piacentini M. Ippolito G. Melino G. COVID-19 infection: the perspectives on immune responses.Cell Death Differ. 2020; 27 (32205856): 1451-145410.1038/s41418-020-0530-3Crossref PubMed Scopus (935) Google Scholar, 8Blanco-Melo D. Nilsson-Payant B.E. Liu W.C. Uhl S. Hoagland D. Moller R. Jordan T.X. Oishi K. Panis M. Sachs D. Wang T.T. Schwartz R.E. Lim J.K. Albrecht R.A. tenOever B.R. Imbalanced host response to SARS-CoV-2 drives development of COVID-19.Cell. 2020; 181 (32416070): 1036-1045.e910.1016/j.cell.2020.04.026Abstract Full Text Full Text PDF PubMed Scopus (2374) Google Scholar, 9Jose R.J. Manuel A. COVID-19 cytokine storm: the interplay between inflammation and coagulation.Lancet Respir. Med. 2020; 8 (32353251): e46-e4710.1016/S2213-2600(20)30216-2Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar, 10Huang C. Wang Y. Li X. Ren L. Zhao J. Hu Y. Zhang L. Fan G. Xu J. Gu X. Cheng Z. Yu T. Xia J. Wei Y. Wu W. et al.Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China.Lancet. 2020; 395 (31986264): 497-50610.1016/S0140-6736(20)30183-5Abstract Full Text Full Text PDF PubMed Scopus (28324) Google Scholar, 11Vardhana S.A. Wolchok J.D. The many faces of the anti-COVID immune response.J. Exp. Med. 2020; 217 (32353870): e2020067810.1084/jem.20200678Crossref PubMed Google Scholar). Several important inflammatory cytokines, such as TNFα, IL-1β, and IL-6, are involved in severe Covid-19 (12McElvaney O.J. McEvoy N.L. McElvaney O.F. Carroll T.P. Murphy M.P. Dunlea D.M. Ní Choileáin O. Clarke J. O'Connor E. Hogan G. Ryan D. Sulaiman I. Gunaratnam C. Branagan P. O'Brien M.E. et al.Characterization of the Inflammatory Response to severe COVID-19 illness.Am. J. Respir. Crit. Care Med. 2020; 202 (32584597): 812-82110.1164/rccm.202005-1583OCCrossref PubMed Scopus (352) Google Scholar). The innate immune response, in terms of the antiviral interferon type I and III, has been shown to inhibit SAR-CoV-2 infections in cell lines (13Felgenhauer U. Schoen A. Gad H.H. Hartmann R. Schaubmar A.R. Failing K. Drosten C. Weber F. Inhibition of SARS-CoV-2 by type I and type III interferons.J. Biol. Chem. 2020; 295 (32587093): XXXXX-YYYYY10.1074/jbc.ac120.013788Abstract Full Text Full Text PDF Scopus (134) Google Scholar). Furthermore, it has been shown that the increase in IL-6 in Covid-19–infected patients is associated with the need for mechanical ventilation due to primary respiratory failure and, eventually, with mortality in the disease (12McElvaney O.J. McEvoy N.L. McElvaney O.F. Carroll T.P. Murphy M.P. Dunlea D.M. Ní Choileáin O. Clarke J. O'Connor E. Hogan G. Ryan D. Sulaiman I. Gunaratnam C. Branagan P. O'Brien M.E. et al.Characterization of the Inflammatory Response to severe COVID-19 illness.Am. J. Respir. Crit. Care Med. 2020; 202 (32584597): 812-82110.1164/rccm.202005-1583OCCrossref PubMed Scopus (352) Google Scholar, 14Chen G. Wu D. Guo W. Cao Y. Huang D. Wang H. Wang T. Zhang X. Chen H. Yu H. Zhang X. Zhang M. Wu S. Song J. Chen T. et al.Clinical and immunological features of severe and moderate coronavirus disease 2019.J. Clin. Invest. 2020; 130 (32217835): 2620-262910.1172/JCI137244Crossref PubMed Scopus (2898) Google Scholar). A recent study has suggested that levels of HA in serum at admission could distinguish critically ill patients with Covid-19 infection (15Ding M. Zhang Q. Li Q. Wu T. Huang Y.Z. Correlation analysis of the severity and clinical prognosis of 32 cases of patients with COVID-19.Respir. Med. 2020; 167 (32421546): 10598110.1016/j.rmed.2020.105981Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). However, increased serum HA levels in Covid-19 patients do not necessarily mean that lung alveoli are filled with HA. Also, previous reports have suggested that a decline in peripheral blood lymphocytes, as well as an increase in peripheral inflammatory factors, such as CRP and IL-6, may be early warning indicators of a poor prognosis for Covid-19 patients (16Yang X. Yu Y. Xu J. Shu H. Xia J. Liu H. Wu Y. Zhang L. Yu Z. Fang M. Yu T. Wang Y. Pan S. Zou X. Yuan S. et al.Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in Wuhan, China: a single-centered, retrospective, observational study.Lancet Respir. Med. 2020; 8 (32105632): 475-48110.1016/S2213-2600(20)30079-5Abstract Full Text Full Text PDF PubMed Scopus (6085) Google Scholar). The “cytokine storm,” with high levels of inflammatory cytokines (IL-1 β, IL-6, and TNFα) in the lungs of patients with severe Covid-19, implies a close relation to HA, as these cytokines are strong inducers of HA synthase 2 (HAS2) in endothelium, lung alveolar epithelial cells, and fibroblasts (17Wilkinson T.S. Potter-Perigo S. Tsoi C. Altman L.C. Wight T.N. Pro- and anti-inflammatory factors cooperate to control hyaluronan synthesis in lung fibroblasts.Am. J. Respir. Cell Mol. Biol. 2004; 31 (14764429): 92-9910.1165/rcmb.2003-0380OCCrossref PubMed Scopus (91) Google Scholar). This fits well with the notion that the state of “hyperinflammation” induces the production and accumulation of HA within the alveolar spaces of patients with severe Covid-19. It has also been shown that HA accumulates in the airways during influenza infection in mice and that HA may be a target for treatment (18Bell T.J. Brand O.J. Morgan D.J. Salek-Ardakani S. Jagger C. Fujimori T. Cholewa L. Tilakaratna V. Östling J. Thomas M. Day A.J. Snelgrove R.J. Hussell T. Defective lung function following influenza virus is due to prolonged, reversible hyaluronan synthesis.Matrix Biol. 2019; 80 (29933044): 14-2810.1016/j.matbio.2018.06.006Crossref PubMed Scopus (74) Google Scholar). In a recently published study, it was reported that in patients hospitalized due to severe Covid-19 and receiving either invasive mechanical ventilation or oxygen, anti-inflammatory treatment with dexamethasone resulted in lower 28-day mortality (19Horby P. Lim W.S. Emberson J.R. Mafham M. Bell J.L. Linsell L. Staplin N. Brightling C. Ustianowski A. Elmahi E. Prudon B. Green C. Felton T. Chadwick D. Rege K. et al.Dexamethasone in hospitalized patients with Covid-19—preliminary report.N. Engl. J. Med. 2020; (32678530)10.1056/NEJMoa2021436Google Scholar). Whereas corticosteroids in high doses may be more harmful than helpful, beneficial effects of corticosteroid treatment in Covid-19 are suggested to depend on the right dose, at the right time, and in the right patient (19Horby P. Lim W.S. Emberson J.R. Mafham M. Bell J.L. Linsell L. Staplin N. Brightling C. Ustianowski A. Elmahi E. Prudon B. Green C. Felton T. Chadwick D. Rege K. et al.Dexamethasone in hospitalized patients with Covid-19—preliminary report.N. Engl. J. Med. 2020; (32678530)10.1056/NEJMoa2021436Google Scholar). In connection with this, it is also interesting to note that corticosteroids are known to be effective in reducing HA levels in bronchoalveolar lavage and circulation (20Ernst G. Lompardía S. Cordo Russo R. Gentilini V. Venturiello S. Galíndez F. Grynblat P. Hajos S.E. Corticosteroid administration reduces the concentration of hyaluronan in bronchoalveolar lavage in a murine model of eosinophilic airway inflammation.Inflamm. Res. 2012; 61 (22842766): 1309-131710.1007/s00011-012-0530-yCrossref PubMed Scopus (3) Google Scholar, 21Engstrom-Laurent A. Hallgren R. Circulating hyaluronate in rheumatoid arthritis: relationship to inflammatory activity and the effect of corticosteroid therapy.Ann. Rheum. Dis. 1985; 44 (2579613): 83-8810.1136/ard.44.2.83Crossref PubMed Scopus (185) Google Scholar). The verification of abundant HA in the lungs and, specifically, in alveolar spaces of individuals deceased with the most severe form of Covid-19 thus indicates a possible pathogenetic mechanism behind the hypoxemia and respiratory failure seen in these critically ill patients. A recent synopsis proposed some new possible approaches to the treatment of critically ill Covid-19 patients, among them reducing the presence, or inhibiting the production, of HA in the lungs (1Shi Y. Wang Y. Shao C. Huang J. Gan J. Huang X. Bucci E. Piacentini M. Ippolito G. Melino G. COVID-19 infection: the perspectives on immune responses.Cell Death Differ. 2020; 27 (32205856): 1451-145410.1038/s41418-020-0530-3Crossref PubMed Scopus (935) Google Scholar). However, the article presented no proof at all that HA was present in Covid-19 lungs. Nevertheless, it was suggested that airway inhalation of hyaluronidase would degrade and reduce HA. It is possible that early in the disease, when hypoxemia is developing, inhalation of hyaluronidase could possibly clear the hygroscopic macromolecule from the lungs and facilitate respiration and oxygenation. Experimentally, it has been shown that intranasal administration of exogenous hyaluronidase can reduce lung HA content and restore lung function following influenza infection (18Bell T.J. Brand O.J. Morgan D.J. Salek-Ardakani S. Jagger C. Fujimori T. Cholewa L. Tilakaratna V. Östling J. Thomas M. Day A.J. Snelgrove R.J. Hussell T. Defective lung function following influenza virus is due to prolonged, reversible hyaluronan synthesis.Matrix Biol. 2019; 80 (29933044): 14-2810.1016/j.matbio.2018.06.006Crossref PubMed Scopus (74) Google Scholar). Hyaluronan shows a molecular weight–dependent role in regulating inflammation, and HA fragments generated in the course of inflammation might influence the release of inflammatory cytokines (22Petrey A.C. de la Motte C.A. Hyaluronan, a crucial regulator of inflammation.Front. Immunol. 2014; 5 (24653726): 10110.3389/fimmu.2014.00101Crossref PubMed Scopus (308) Google Scholar). In inflammatory diseases, high-molecular weight HA can also be found in cross-linked complexes (e.g. with inter-α-trypsin inhibitor heavy chain and versican) (22Petrey A.C. de la Motte C.A. Hyaluronan, a crucial regulator of inflammation.Front. Immunol. 2014; 5 (24653726): 10110.3389/fimmu.2014.00101Crossref PubMed Scopus (308) Google Scholar, T.N. I. extracellular regulator of and inflammation.Front. Immunol. 2020; PubMed Scopus Google Scholar). of a of HA by hyaluronidase to polymers could possibly reduce the ongoing inflammatory in Covid-19. suggested is the clinical of (1Shi Y. Wang Y. Shao C. Huang J. Gan J. Huang X. Bucci E. Piacentini M. Ippolito G. Melino G. COVID-19 infection: the perspectives on immune responses.Cell Death Differ. 2020; 27 (32205856): 1451-145410.1038/s41418-020-0530-3Crossref PubMed Scopus (935) Google Scholar), which can inhibit the production of HA in inflammation, autoimmunity, and (4Nagy N. Kuipers H.F. Frymoyer A.R. Ishak H.D. Bollyky J.B. Wight T.N. Bollyky P.L. 4-Methylumbelliferone treatment and hyaluronan inhibition as a therapeutic strategy in inflammation, autoimmunity, and cancer.Front. Immunol. 2015; 6 (25852691): 12310.3389/fimmu.2015.00123Crossref PubMed Scopus (177) Google Scholar). the of two HA and and the in the formation of HA from A. S. M. R. E. 4-Methylumbelliferone hyaluronan synthesis by of and of hyaluronan synthase 2 and Cell Res. PubMed Scopus Google Scholar). This or its is in which may the effects of in some Covid-19 patients (1Shi Y. Wang Y. Shao C. Huang J. Gan J. Huang X. Bucci E. Piacentini M. Ippolito G. Melino G. COVID-19 infection: the perspectives on immune responses.Cell Death Differ. 2020; 27 (32205856): 1451-145410.1038/s41418-020-0530-3Crossref PubMed Scopus (935) Google Scholar). In clinical not including Covid-19 patients, has been during administration in doses (4Nagy N. Kuipers H.F. Frymoyer A.R. Ishak H.D. Bollyky J.B. Wight T.N. Bollyky P.L. 4-Methylumbelliferone treatment and hyaluronan inhibition as a therapeutic strategy in inflammation, autoimmunity, and cancer.Front. Immunol. 2015; 6 (25852691): 12310.3389/fimmu.2015.00123Crossref PubMed Scopus (177) Google Scholar). In have for the first a presence of hyaluronan in alveolar spaces of the lungs in lethal cases of Covid-19. on this novel treatment hyaluronan may be a to reduce mortality in critically ill Covid-19 patients. However, clinical are to the and of these in the case of severe Covid-19. Lung tissue was obtained at autopsy from three adult Covid-19 patients, two and and of the patients been in the and of them showed of normal lung tissue was obtained from patients and the were processed in an as the Covid-19 lung tissue. and of lung tissue were processed for HA histochemistry using a direct and HA staining method M. B. A. Hyaluronan and its in the of and adult A Mol. Cell Biol. PubMed Scopus Google Scholar). of lung tissue were with a by with the HA were in a of The were with were with hyaluronidase from bovine testes a the specificity of the were in the for the study was obtained from the and The only for clinical at the are within the hyaluronan acute respiratory distress syndrome

Topics & Concepts