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).[1], [2], [3] Many of these patients have additional comorbidities including obesity, diabetes, hypertension, and cardiovascular disease, which are emerging as key predictors of COVID-19 severity.4 , 5 Inflammation and T-cell immune dysregulation are also associated with poor COVID-19 outcomes.6 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,7 which has been shown to play a key role in antiviral immune responses against other coronaviruses.8 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 1.
Clinical and immunological characteristics of patients with COVID-19.
| Non-lethal courses of COVID-19 |
Lethal courses of COVID-19 |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| MedStar Georgetown Transplant Institute Liver Patients | SUNY Control Patients | MedStar Georgetown Transplant Institute Liver Patients | SUNY Control Patients | |||||||
| Demographics | ||||||||||
| Age, years | 49 | 55 | 65 | 65 | 48–75 | 70 | 46 | 65 | 61 | 57–77 |
| Sex, M/F | F | M | F | F | 1M, 3F | F | M | F | M | 2M, 2F |
| Race, white (W) or non-white (NW) | NW | NW | W | NW | 1W, 3NW | W | W | NW | NW | 0W, 4NW |
| Key comorbidities, n | 2 | 3 | 3 | 5 | 2–3 | 1 | 2 | 3 | 3 | 1–3 |
| Diabetes | ✓ | 4 of 4 | ✓ | ✓ | ✓ | 1 of 4 | ||||
| Obesity | ✓ | ✓ | ✓ | ✓ | 2 of 4 | ✓ | 2 of 4 | |||
| Hypertension | ✓ | ✓ | 4 of 4 | ✓ | ✓ | 4 of 4 | ||||
| Heart disease | ✓ | ✓ | ✓ | 0 of 4 | ✓ | 2 of 4 | ||||
| Active liver disease | ✓ | ✓ | ✓ | 0 of 4 | ✓ | ✓ | 0 of 4 | |||
| Transplant/liver disease status | ||||||||||
| Transplanted organ type | Liver & Kidney | n.a. | Liver | Liver | Liver & Kidney | n.a. | ||||
| Underlying liver disease | ETOH cirrhosis | ETOH cirrhosis | NAFLD, ALF |
AIH cirrhosis | n.a. | ETOH cirrhosis | HCV, ETOH cirrhosis | PSC cirrhosis | ETOH cirrhosis | n.a. |
| COVID-19 outcome | ||||||||||
| Hospitalization | ✓ | ✓ | ✓ | ✓ | 4 of 4 | ✓ | ✓ | ✓ | ✓ | 4 of 4 |
| Supplemental O2 | ✓ | ✓ | 3 of 4 | ✓ | ✓ | ✓ | ✓ | 4 of 4 | ||
| Intubation/ICU | ✓ | ✓ | 2 of 4 | ✓ | ✓ | ✓ | 4 of 4 | |||
| No discharge by day 14 | ✓ | 3 of 4 | ✓ | ✓ | ✓ | ✓ | 4 of 4 | |||
| Death | 0 of 4 | ✓ | ✓ | ✓ | ✓ | 4 of 4 | ||||
| Liver function tests | ||||||||||
| AST (U/L) [3-34], upon presentation | 45 | 86 | 9654 | 76 | 12–88 | 61 | 99 | 19 | 40 | 17–175 |
| AST, highest value during hospitalization | 45 | 86 | 9654 | 136 | 18–158 | 64 | 233 | 74 | 178 | 117–5147 |
| ALT (U/L) [15-41], upon presentation | 73 | 55 | 3714 | 43 | 21–89 | 27 | 77 | 27 | 35 | 9–126 |
| ALT, highest value during hospitalization | 73 | 55 | 3714 | 79 | 21–197 | 36 | 111 | 89 | 147 | 87–5,658 |
| Total bilirubin (mg/dl) [0.2-1.3], upon presentation | 0.3 | 5.4 | 1.8 | 0.6 | 0.3–0.6 | 0.6 | 26.3 | 0.8 | 0.6 | 0.4–3.7 |
| Total bilirubin, highest value during hospitalization | 0.3 | 5.4 | 1.8 | 0.9 | 0.3–1.3 | 2.1 | 30.8 | 0.8 | 1.4 | 1.0–4.3 |
| ALP (U/L) [45-117], upon presentation | 254 | 324 | 243 | 254 | 63–95 | 182 | 699 | 72 | 80 | 61–143 |
| ALP, highest value during hospitalization | 378 | 324 | 319 | 455 | 63–156 | 182 | 699 | 102 | 633 | 129–195 |
| Inflammatory markers | ||||||||||
| LDH (U/L) [84-246], upon presentation | 246 | 337 | 957 | 388 | 603–685 | 176 | 352 | 411 | 685 | 217–1,919 |
| LDH, highest value during hospitalization | 286 | 368 | 957 | 488 | 723–746 | 190 | 388 | 411 | 685 | 579–1,919 |
| CRP (mg/L) [0.0-3.0], upon presentation | 43.0 | 11.5 | 53.3 | 96.2 | 97–338 | 3.4 | 31.4 | 145 | 79 | 100–271 |
| CRP, highest value during hospitalization | 43.0 | 31.9 | 53.3 | 296.0 | 150–461 | 5.4 | 101.0 | 228.0 | 181.0 | 100–342 |
| Ferritin (ng/ml) [5.0-148.0], upon presentation | 1432 | 56.8 | 211.1 | 258.9 | 410–2,120 | 1,527 | 2,941 | 2,238 | 544.2 | 233–20,956 |
| Ferritin, highest value during hospitalization | 1432 | 113 | 238 | 439 | 1,355–2,120 | 1,527 | 4,585 | 2,754 | 607 | 2,707–20,956 |
| D-dimer (μg/ml FEU) [<0.65], upon presentation | 4.35 | 1.44 | >20 | 1.92 | 0.5–8.0 | 2.43 | 5.71 | 11.55 | 0.5 | n.a. |
| D-dimer, highest value during hospitalization | 4.35 | 1.44 | >20 | 3.56 | 1.0–>8.0 | 2.43 | 5.71 | 16.37 | 7.62 | n.a. |
| Immunomonitoring | ||||||||||
| WBC (K/μl) [4.0-10.8], upon presentation | 9.9 | 6.9 | 17.4 | 3.7 | 5.8–13.4 | 2.7 | 4.2 | 8.0 | 3.6 | 4.6–16.1 |
| WBC (K/μl), lowest value during hospitalization | 2.3 | 5.2 | 5.9 | 3.7 | 5.1–9.2 | 2.7 | 4.2 | 4.8 | 3.6 | 2.3–8.5 |
| Lymphocytes (K/μl) [0.6-4.9], upon presentation | 0.8 | 1.1 | 0.9 | 0.9 | 0.2–2.4 | 0.9 | 0.2 | 0.2 | 0.5 | 0.4–1.1 |
| Lymphocytes, lowest value during hospitalization | 0.4 | 1.1 | 0.9 | 0.4 | 0.2–1.6 | 0.6 | 0.2 | 0.2 | 0.2 | 0.1–0.5 |
| Days of lymphopenia (total days below 1.0 K/μl) | 7 | 0 | 2 | 19 | 0–4 | 8 | 24 | 7 | 17 | 1–13 |
| T-cell counts | ||||||||||
| CD3+ (/μl) [510-2607], upon presentation | 636 | 1012 | 1373 | 1071 | 848–1205 | 592 | 414 | 106 | 273 | 729–1359 |
| CD3+CD4+ (/μl) [302-1779], upon presentation | 415 | 562 | 982 | 668 | 619–774 | 404 | 83 | 56 | 206 | 572–926 |
| CD3+CD8+ (/μl) [101-951], upon presentation | 168 | 401 | 336 | 380 | 199–289 | 176 | 311 | 44 | 59 | 55–343 |
| T-cell phenotype | ||||||||||
| CD4+CD38+HLA-DR+ % [0.30-1.35] | 5.0 | 11.3 | 1.3 | 6.7 | 2.7–4.8 | 4.5 | 13.9 | 5.8 | 16.3 | 3.3–17.8 |
| CD4+CD25+CD127- % [4.64-8.05] | 2.3 | 4.2 | 16.8 | 4.1 | 2.6–4.4 | 6.5 | 3.3 | 0.0 | 8.8 | 2.4–18.1 |
| CD4+ICOS+CXCR5+ % [0.64-2.72] | 1.7 | 3.3 | 1.2 | 1.4 | 0.3–1.6 | 0.1 | 2.6 | 1.3 | 3.8 | 0.6–2.5 |
| CD4+CD45RO+ % [9.9-37.7] | 38.3 | 37.7 | 41.7 | 55.7 | 39.0–50.0 | 35.0 | 18.2 | 15.7 | 68.3 | 15.3–48.1 |
| CD4+CD45RA+ % [3.4-37.9] | 16.7 | 11.5 | 14.5 | 2.3 | 3.5–14.5 | 19.1 | 2.7 | 21.8 | 1.5 | 7.5–48.1 |
| CD8+CD38+HLA-DR+ % [0.13-2.68] | 31.8 | 48.6 | 1.9 | 24.6 | 11.5–33.4 | 35.5 | 27.2 | 21.6 | 20.6 | 14.7–56.6 |
| CD8+CD45RO+ % [1.0-8.3] | 11.0 | 5.5 | 4.3 | 19.5 | 3.1–13.7 | 3.3 | 23.8 | 2.2 | 13.1 | 0.3–7.0 |
| CD8+CD45RA+ % [2.4-23.0] | 8.2 | 18.9 | 3.8 | 5.7 | 6.0–12.3 | 18.9 | 31.3 | 30.2 | 2.2 | 11.3–17.3 |
| Inflammasome activity | ||||||||||
| Caspase-1 CD45+CD3+ % [2.11-4.90] | 22.38 | 26.41 | 21.16 | 19.68 | 19.54–40.11 | 20.21 | 28.44 | 18.36 | 16.80 | 12.79–35.66 |
| Caspase-1 CD45+CD3+CD4+ % [1.87-3.67] | 17.61 | 24.93 | 21.22 | 21.54 | 11.57–38.37 | 15.63 | 30.83 | 16.77 | 17.38 | 11.10–31.11 |
| IL-18 (pg/ml) [60-275] | 400.0 | 989.6 | 304.5 | 439.2 | 1376–4496 | 229.4 | 162.2 | 469.2 | 162.6 | 610–2425 |
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.9 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-1.10 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.
Footnotes
Author names in bold designate shared co-first authorship
Supplementary data to this article can be found online at https://doi.org/10.1016/j.jhep.2020.06.034.
Supplementary data
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