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Inflammasome activation and pyroptosis in lymphopenic liver patients with COVID-19

      Keywords

      To the Editor:
      As has recently been highlighted in the Journal of Hepatology and elsewhere, patients with liver diseases such as non-alcoholic fatty liver disease (NAFLD) or cirrhosis, as well as liver transplant recipients, carry a high risk of morbidity and mortality due to coronavirus disease 2019 (COVID-19).
      • Ji D.
      • Qin E.
      • Xu J.
      • Zhang D.
      • Cheng G.
      • Wang Y.
      • et al.
      Non-alcoholic fatty liver diseases in patients with COVID-19: a retrospective study.
      • Moon A.M.
      • Webb G.J.
      • Aloman C.
      • Armstrong M.J.
      • Cargill T.
      • Dhanasekaran R.
      • et al.
      High mortality rates for SARS-CoV-2 infection in patients with pre-existing chronic liver disease and cirrhosis: preliminary results from an international registry.
      • Webb G.J.
      • Moon A.M.
      • Barnes E.
      • Barritt A.S.
      • Marjot T.
      Determining risk factors for mortality in liver transplant patients with COVID-19.
      Many of these patients have additional comorbidities including obesity, diabetes, hypertension, and cardiovascular disease, which are emerging as key predictors of COVID-19 severity.
      • Richardson S.
      • Hirsch J.S.
      • Narasimhan M.
      • Crawford J.M.
      • McGinn T.
      • Davidson K.W.
      • et al.
      Presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the New York City area.
      ,
      • Onder G.
      • Rezza G.
      • Brusaferro S.
      Case-fatality rate and characteristics of patients dying in relation to COVID-19 in Italy.
      Inflammation and T-cell immune dysregulation are also associated with poor COVID-19 outcomes.
      • Chen G.
      • Wu D.
      • Guo W.
      • Cao Y.
      • Huang D.
      • Wang H.
      • et al.
      Clinical and immunological features of severe and moderate coronavirus disease 2019.
      To what extent these comorbidities cause immune dysregulation in COVID-19 is unknown; but it is known that they are characterized by chronic inflammation involving activation of the inflammasome,
      • Guo H.
      • Callaway J.B.
      • Ting J.P.
      Inflammasomes: mechanism of action, role in disease, and therapeutics.
      which has been shown to play a key role in antiviral immune responses against other coronaviruses.
      • Zhao C.
      • Zhao W.
      NLRP3 inflammasome-A key player in antiviral responses.
      We thus hypothesize that heightened inflammasome activity may drive acute on chronic inflammation, leading to immune dysregulation and ultimately severe disease for these comorbid patients when facing COVID-19. In this letter, we share findings that provide preliminary support for this hypothesis based on data from 8 liver patients with COVID-19 from the MedStar Georgetown Transplant Institute (MGTI), with 8 matched non-liver patients with COVID-19 from SUNY Downstate Medical Center (SUNY).
      This study was IRB approved (MGTI: IRB #STUDY00002359, IRB #2017-0365; SUNY: IRB #269846-8; informed consent was obtained). Retrospective and prospective chart review of all hospitalized liver patients with COVID-19 treated by MGTI consecutively in April and early May of 2020 was conducted. Specifically, we analysed data from 4 patients with chronic liver disease and 4 liver transplant recipients. The 8 control patients from SUNY were matched based on age, sex, race, comorbidities, and COVID-19 outcome during the same period.
      As shown in Table 1, data were collected on demographics, comorbidities, liver disease, and COVID-19-related disease courses as well as from chemistry and immunological laboratory results obtained during hospitalization from the central laboratory of MedStar Georgetown University Hospital (liver patients), SUNY Downstate Medical Center (controls), and Amerimmune, a Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. See supplementary materials & methods for details on methods.
      Table 1Clinical and immunological characteristics of patients with COVID-19.
      Non-lethal courses of COVID-19Lethal courses of COVID-19
      MedStar Georgetown Transplant Institute Liver PatientsSUNY Control PatientsMedStar Georgetown Transplant Institute Liver PatientsSUNY Control Patients
      Demographics
       Age, years4955656548–757046656157–77
       Sex, M/FFMFF1M, 3FFMFM2M, 2F
       Race, white (W) or non-white (NW)NWNWWNW1W, 3NWWWNWNW0W, 4NW
      Key comorbidities, n23352–312331–3
       Diabetes4 of 41 of 4
       Obesity2 of 42 of 4
       Hypertension4 of 44 of 4
       Heart disease0 of 42 of 4
       Active liver disease0 of 40 of 4
      Transplant/liver disease status
       Transplanted organ typeLiver & Kidneyn.a.LiverLiverLiver & Kidneyn.a.
       Underlying liver diseaseETOH cirrhosisETOH cirrhosisNAFLD,

      ALF
      AIH cirrhosisn.a.ETOH cirrhosisHCV, ETOH cirrhosisPSC cirrhosisETOH cirrhosisn.a.
      COVID-19 outcome
       Hospitalization4 of 44 of 4
       Supplemental O23 of 44 of 4
       Intubation/ICU2 of 44 of 4
       No discharge by day 143 of 44 of 4
       Death0 of 44 of 4
      Liver function tests
       AST (U/L) [3-34], upon presentation458696547612–886199194017–175
       AST, highest value during hospitalization4586965413618–1586423374178117–5147
       ALT (U/L) [15-41], upon presentation735537144321–89277727359–126
       ALT, highest value during hospitalization735537147921–197361118914787–5,658
       Total bilirubin (mg/dl) [0.2-1.3], upon presentation0.35.41.80.60.3–0.60.626.30.80.60.4–3.7
       Total bilirubin, highest value during hospitalization0.35.41.80.90.3–1.32.130.80.81.41.0–4.3
       ALP (U/L) [45-117], upon presentation25432424325463–95182699728061–143
       ALP, highest value during hospitalization37832431945563–156182699102633129–195
      Inflammatory markers
       LDH (U/L) [84-246], upon presentation246337957388603–685176352411685217–1,919
       LDH, highest value during hospitalization286368957488723–746190388411685579–1,919
       CRP (mg/L) [0.0-3.0], upon presentation43.011.553.396.297–3383.431.414579100–271
       CRP, highest value during hospitalization43.031.953.3296.0150–4615.4101.0228.0181.0100–342
       Ferritin (ng/ml) [5.0-148.0], upon presentation143256.8211.1258.9410–2,1201,5272,9412,238544.2233–20,956
       Ferritin, highest value during hospitalization14321132384391,355–2,1201,5274,5852,7546072,707–20,956
       D-dimer (μg/ml FEU) [<0.65], upon presentation4.351.44>201.920.5–8.02.435.7111.550.5n.a.
       D-dimer, highest value during hospitalization4.351.44>203.561.0–>8.02.435.7116.377.62n.a.
      Immunomonitoring
       WBC (K/μl) [4.0-10.8], upon presentation9.96.917.43.75.8–13.42.74.28.03.64.6–16.1
       WBC (K/μl), lowest value during hospitalization2.35.25.93.75.1–9.22.74.24.83.62.3–8.5
       Lymphocytes (K/μl) [0.6-4.9], upon presentation0.81.10.90.90.2–2.40.90.20.20.50.4–1.1
       Lymphocytes, lowest value during hospitalization0.41.10.90.40.2–1.60.60.20.20.20.1–0.5
       Days of lymphopenia (total days below 1.0 K/μl)702190–48247171–13
      T-cell counts
       CD3+ (/μl) [510-2607], upon presentation636101213731071848–1205592414106273729–1359
       CD3+CD4+ (/μl) [302-1779], upon presentation415562982668619–7744048356206572–926
       CD3+CD8+ (/μl) [101-951], upon presentation168401336380199–289176311445955–343
      T-cell phenotype
       CD4+CD38+HLA-DR+ % [0.30-1.35]5.011.31.36.72.7–4.84.513.95.816.33.3–17.8
       CD4+CD25+CD127- % [4.64-8.05]2.34.216.84.12.6–4.46.53.30.08.82.4–18.1
       CD4+ICOS+CXCR5+ % [0.64-2.72]1.73.31.21.40.3–1.60.12.61.33.80.6–2.5
       CD4+CD45RO+ % [9.9-37.7]38.337.741.755.739.0–50.035.018.215.768.315.3–48.1
       CD4+CD45RA+ % [3.4-37.9]16.711.514.52.33.5–14.519.12.721.81.57.5–48.1
       CD8+CD38+HLA-DR+ % [0.13-2.68]31.848.61.924.611.5–33.435.527.221.620.614.7–56.6
       CD8+CD45RO+ % [1.0-8.3]11.05.54.319.53.1–13.73.323.82.213.10.3–7.0
       CD8+CD45RA+ % [2.4-23.0]8.218.93.85.76.0–12.318.931.330.22.211.3–17.3
      Inflammasome activity
       Caspase-1 CD45+CD3+ % [2.11-4.90]22.3826.4121.1619.6819.54–40.1120.2128.4418.3616.8012.79–35.66
       Caspase-1 CD45+CD3+CD4+ % [1.87-3.67]17.6124.9321.2221.5411.57–38.3715.6330.8316.7717.3811.10–31.11
       IL-18 (pg/ml) [60-275]400.0989.6304.5439.21376–4496229.4162.2469.2162.6610–2425
      For the 8 MedStar Georgetown Transplant Institute liver patients, individual patient data are shown. For the 8 SUNY Downstate Medical Center (SUNY) non-liver control patients, summaries or ranges of high and low patient data are shown for the 4 non-lethal and 4 lethal cases, respectively. “Upon presentation” refers to the first data point available upon COVID-19 presentation; “during hospitalization” refers to data points collected during COVID-19 related hospitalization. Numbers in square brackets represent normal reference ranges from MedStar Georgetown Transplant Institute and CLIA-certified tests. Normal reference ranges for SUNY are listed in supplementary materials & methods. AIH, autoimmune hepatitis; ALF, acute liver failure; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; COVID-19, coronavirus disease 2019; CD, cluster of differentiation; CD4+CD25+CD127-, T-regulatory cell; CD4+ICOS+CXCR5+, T follicular helper cell; CLIA, Clinical Laboratory Improvement Amendments; CRP, C-reactive protein; ETOH, ethyl alcohol; HCV, hepatitis C virus; HLA, human leukocyte antigen; ICU, intensive care unit; IL, interleukin; LDH, lactate dehydrogenase; NAFLD, non-alcoholic fatty liver disease; O2, oxygen; PSC, primary sclerosing cholangitis; WBC, white blood cell count.
      Demographically, the liver patient cohort included patients between the ages of 46 and 70, 5 females and 3 males, and 5 non-white and 3 white patients. Seven patients had liver cirrhosis and 1 had NAFLD. Four had received liver transplantation and 4 were under our care for liver disease. All patients had significant comorbidities that would imply a chronic inflammatory profile. Six patients had severe COVID-19 courses requiring supplemental oxygen and/or ventilator support, and 4 of those patients died, 3 of whom were liver transplant recipients.
      Over the course of their COVID-19 hospitalization, all liver patients had elevated levels of transaminases and alkaline phosphatase – consistent with previous reports on liver function in patients with COVID-19 – and high levels of inflammatory markers including CRP, ferritin, and D-dimer, as well as profound lymphopenia (mean length of 7 days and 14 days for the non-lethal and lethal cases, respectively). Specifically, the mean lowest lymphocyte count across the liver cohort was 0.5 K/μl, and for the non-lethal and lethal cases it was 0.7 and 0.3 K/μl, respectively, in line with the controls (overall mean of 0.6, with 0.8 and 0.3 K/μl for non-lethal and lethal, respectively). The mean absolute T-cell count across the liver cohort was low, with 685 /μl, and strikingly lower for lethal cases when compared to non-lethal cases (346 and 1,023 /μl, respectively). In the control cohort, the mean absolute T-cell count was 988 /μl with similar levels in the non-lethal and lethal cases. The relatively higher levels in the control cohort lethal cases vs. the liver cohort lethal cases could be explained by the fact that 3 of the 4 lethal liver patients were on post-transplant immunosuppression, indicating that immunosuppression could exacerbate COVID-19 induced T-cell lymphopenia. Moreover, overexpression of both CD38+ and HLA-DR+ in CD4+ and 8+ T cells across the cohorts, with mean values of 8.1% and 26.5% in the liver and 6.2% and 26.7% in the control cohorts, respectively, points to virally induced T-cell activation.
      The T-cell lymphopenia caused us to hypothesize that inflammasome activation might be a driver of pyroptosis-induced T-cell death. This led us to study caspase-1 activation, as its upregulation is the hallmark feature of inflammasome activation. Indeed, we found a global increase in caspase-1 activity levels in T cells, with mean values of 20.7% and 21.7% for CD45+CD3+CD4+ and CD45+CD3+, respectively in the liver cohort and 24.9% and 28.0% in the control cohort. Corroborating this finding across both cohorts were high levels of lactate dehydrogenase (LDH) and IL-18, both of which are known to be released upon pyroptotic cell death (LDH mean values of 472 and 968 u/L and IL-18 mean values of 395 and 1,856 pg/mL in liver and control patients, respectively). The relatively higher IL-18 and LDH levels in control patients may be explained by a priori reduced lymphocytes in immunocompromised liver disease and transplant patients. Of note, elevated LDH, IL-18, and caspase-1 findings were consistent in the liver cohort irrespective of transplant or liver disease status.
      Our patient data corroborate poor COVID-19 outcomes for liver patients, especially for those with inflammatory comorbidities. Furthermore, analysis of lethal cases shows that patients with the lowest T cell counts are more prone to morbid outcomes. The common link between these findings may be the inflammasome, which plays a role in both comorbidities and viral infections. Specifically, our findings suggest that comorbid patients – independent of liver disease – may already have a constitutive chronic activation of the inflammasome, which is further exacerbated by viruses that activate the inflammasome, as has been shown in monocytes and other immune cells in the face of coronaviruses.
      • Chen I.Y.
      • Moriyama M.
      • Chang M.F.
      • Ichinohe T.
      Severe acute respiratory syndrome coronavirus viroporin 3a activates the NLRP3 inflammasome.
      Strong upregulation of caspase-1 activity in T cells as well as high IL-18 and LDH levels across all patients suggests pyroptosis-mediated programmed cell death occurring downstream of inflammasome activation as a cause of lymphopenia and driver of heightened inflammation in COVID-19, similar to SARS-CoV-1.
      • Shi C.S.
      • Nabar N.R.
      • Huang N.N.
      • Kehrl J.H.
      SARS-coronavirus open reading frame-8b triggers intracellular stress pathways and activates NLRP3 inflammasomes.
      Pyroptotic T-cell depletion in patients with COVID-19 may not only prevent the adaptive immune system from mounting an effective antiviral immune response but also fuel a lethal inflammatory response through release of proinflammatory cytokines such as IL-1β and IL-18. Our insights into the potentially critical role of the inflammasome in COVID-19 have therapeutic implications beyond liver diseases, as they imply that upstream prevention of pyroptosis in COVID-19 may be beneficial.

      Financial support

      The authors received no financial support to produce this manuscript.

      Authors' contributions

      AK, KK, and TMF designed the study. AK and KK performed clinical data analysis at MedStar Georgetown Transplant Institute. MAH and RG performed clinical data analysis at SUNY Downstate Medical Center. MP and OA conducted the clinical immunology studies done at Amerimmune. AK, KK, and TMF wrote the manuscript.

      Conflicts of interest

      The authors declare no conflicts of interest that pertain to this work.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

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