key: cord-0286897-d6rpndgq
authors: Adamik, B.; Ambrozek-Latecka, M.; Dragan, B.; Jeznach, A.; Smiechowicz, J.; Gozdzik, W.; Skirecki, T.
title: Heterogenous pattern of the inflammasome-related markers in the critically ill COVID-19 patients at the ICU admission.
date: 2021-11-01
journal: nan
DOI: 10.1101/2021.10.30.21265662
sha: 7af4daaae584cc5a622a2c88d01e4e0d21204600
doc_id: 286897
cord_uid: d6rpndgq

Purpose: Development of targeted biological therapies to COVID-19 requires reliable biomarkers that could help to indicate the responding patients. Hyperactivation of the inflammasome by SARS-CoV2 virus is hypothesized to contribute to severe course of the COVID-19 disease. Therefore, we aimed to evaluate the prognostic value of several inflammasome-related cytokines and proteins at the admission to the intensive care unit. Patients and methods: Plasma samples were obtained from 45 critically-COVID-19 patients and from 10 patients with severe craniocerebral traumatic brain injury (TBI) at the admission to the ICU. The concentration of IL-1, IL-1, IL-18, IL-1RA, galectin-1, ASC, LDH, ferritin and gasdermin D were analyzed. A novel cell-free caspase-1 plasma assay was developed by inhibitor-based immunoprecipitation followed by Western Blot. Demographic and clinical characteristics were recorded. Results: The inflammasome-related biomarkers were in similar concentration in COVID-19 and TBI patients except for galectin-1 being lower in the former. None of the tested markers was related to the outcome, length of stay or development of secondary infections. Patients with SOFA score of >9 at admission who were at high risk of death had significantly higher galectin-1 but lower IL-1RA in comparison to low-risk patients. Weak but significant correlations were observed for IL-1 and IL-1 and platelets and also for ferritin and INR. Activated caspase-1 p35 was detectable in 12/22 COVID-19 patients and was related with higher fibrinogen and lower D-dimers. It was also significantly higher in patients with SOFA>9. Conclusion: Our results indicate that the activation of the inflammasome in critically ill COVID-19 patients is a heterogenous process and is not directly related with outcome. Therefore, potential interventions aimed at this pathway in this group of patients can be limited and should be biomarker-guided.

Infection with the respiratory SARS-CoV2 virus causes the coronavirus disease 2019 57 which ranges from asymptomatic infection through mild and severe disease 58 (pneumonia) to critical disease which has a form of the acute respiratory distress syndrome 59 (ARDS) or sepsis and septic shock 1 . This range of severity is reflected by the mortality ranges 60 which reaches approx. 40% in critically ill patients 2 . Although other coronavirues can infect 61 humans, the COVID-19 is characterized by unique pathogenesis making it a distinct disease 3 . 62

Aside from respiratory support, the only widely accepted treatment for COVID-19 is 63 dexamethasone and recently tocilizumab 1 . 64

There is an urgent need to develop efficient therapies for COVID-19 and it becomes 65 apparent that as in the case of bacterial sepsis, a biomarker-guided personalized approach is a 66 prerequisite for successful biological therapies 4 . Since early pandemics we among others have 67

proposed that the unbalanced inflammasome signaling may be a key process in the pathogenesis 68 of COVID-19 5,6 . Briefly, inflammasomes are multiprotein complexes activated by various stimuli 69 such as dsDNA (Absent in Melanoma, AIM2), potassium efflux and reactive oxygen species (NLR 70 Family Pyrin Domain Containing 3, NLRP3) 7 . Upon activation, the receptor proteins oligomerize 71 and recruit adaptor proteins (apoptosis-associated speck-like protein, ASC) and pro-caspase-1. 72

Then pro-caspase-1 undergoes autoproteolysis to its active form which then can cleave pro-73 interleukin-1 , , -18 and gasdermin D (GSDMD). N-terminal GSDMD can form pores in the cell 74 membrane which leads to the release of the cleaved cytokines as well as other molecules (such 75 as lactate dehydrogenase; alarmins) and lytic cell death called pyroptosis 7 . Indeed, it has been 76 demonstrated that the inflammasome is activated in the lungs of COVID-19 patients 8, 9 and can be 77 activated in the circulating monocytes 10 . However, the status of inflammasome reactivity in the 78 blood myeloid cells appears complexed 11 . Although some studies showed an increase in 79 concentrations of pro-inflammatory cytokines with increasing severity of COVID-19 12 , there is a 80 lack of studies focused on critically ill COVID-19 patients who are at the highest risk of death. 81

Nevertheless, there are ongoing clinical trials with direct (Dapansutrile, Colchicine) inflammasome 82 inhibitors and indirect (Disulfiram, a GSDMD inhibitor). Also, recently two studies on the IL-1R 83 antagonist in COVID-19 were published with opposite results 13,14 . 84 In this study, we aimed to evaluate the prognostic utility of selected plasma proteins 85 related to inflammasome activation in critically ill COVID-19 patients. Considering the high 86 mortality and lack of effective therapies we focused on the critically ill COVID-19 patients at the 87 admission to intensive care unit (ICU). We hypothesized that biomarkers of good accuracy could 88 serve to guide immunomodulatory therapies targeting the inflammasomes. with severe traumatic brain injury (TBI) were used to compare markers of inflammasome 104 activation between cases with SARS-CoV2 infection and cases without any signs of infection. In 105 all TBI cases written informed consent was obtained from the patient or a legally authorized 106

representative (consent No.KB-391/2015) . The clinical status of the patients was determined with 107 the Acute Physiology and Chronic Health Evaluation (APACHE) II score and Sequential Organ 108 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted November 1, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 Failure Assessment (SOFA) score on admission to the ICU. The APACHE II score it is routinely 109 used as a prediction tool for ICU patients and includes 12 physiological variables (the fraction of 110 inspired oxygen, partial pressure of oxygen, body temperature, mean arterial pressure, blood pH, 111 heart rate, respiratory rate, serum sodium, serum potassium, serum creatinine, hematocrit, white 112 blood cell count, and the Glasgow Coma Scale) and 2 disease-related variables (the history of 113 severe organ failure or immunocompromised and the type of ICU admission). The SOFA score is 114 routinely used in the ICU for monitoring the severity of patient's clinical condition based on the 115 status of the following systems: respiratory (PaO2/FiO2 index), cardiovascular (mean arterial 116 pressure and the dose of vasopressors), hepatic (bilirubin level), coagulation (platelets level), 117 renal (creatinine level/urine output), and neurological (Glasgow coma scale). biotin-streptavidin bead complexes were centrifuged for 5 min at 2300 x g and washed 5 times 135 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 1, 2021. 

CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 1, 2021. ; https://doi.org/10. 1101 /2021 We have enrolled forty-five critically ill COVID-19 patients with RT-PCR confirmed SARS-CoV2 161 infection. The clinical and demographic characteristic of the group is presented in Tab. 1. This 162 group included 24 males (53%) and 21 females and the median age was 58 years (IQ: 43-68). At 163 admission, the patients' median APACHE II score was 14 (IQ: 11-22) and SOFA score was 9 164 (IQ:7-10). The hospital mortality reached 62% and was higher than 28-day mortality which was 165 53%. The patients who did not survive were significantly older ( proteins could differentiate between these groups (Fig. 2) . Moreover, LDH and ferritin which can 185 serve as surrogate markers of cell death and IL-1 signaling, respectively, were not increased in 186 patients with unfavorable outcome (Tab. 2). Other routinely monitored markers of inflammation as 187 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 1, 2021. ; https://doi.org/10.1101/2021.10.30.21265662 doi: medRxiv preprint white blood count, IL-6 or CRP were also similar in these two groups. Of the analyzed 188 parameters, only the international normalized ratio (INR) was significantly higher in the non-189 surviving COVID-19 patients. However, logistic regression analysis did not reveal the relationship 190 between INR either other analyzed parameters and mortality (p>0.05). Also, there were no 191 difference in the concentrations of the analyzed biomarkers of the inflammasome response 192 between patients who were admitted with co-infections and those who were not. These results 193 indicate that inflammasome-related proteins are less elevated in critically ill COVID-19 patients at 194 ICU admission than in TBI patients and are not different between survivors and non-survivors. was not significantly different between these groups, however chronic comorbidities such as 213 coronary artery disease, heart failure and renal disease were significantly more common in the 214 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 1, 2021. ; https://doi.org/10.1101/2021.10.30.21265662 doi: medRxiv preprint high-risk patients. Of interest, patients who developed secondary infections had lower level of 215 galectin-1 than those who did not (14908.51 (IQ: 8394.48 -21,911.48) pg/ml vs. 23,839.62 (IQ: 216 15,628.85 -29,846 .66) pg/ml; p=0.039, however logistic regression analysis did not confirm this 217 relationship (OR=1.00, p=0.042). There were no differences among analyzed inflammasome-218 related biomarkers either standard inflammatory parameters (IL-6, PCT, WBC) between patients 219 who developed secondary infections during ICU stay and those who did not. Altogether, these 220 results suggest that the threshold of 9 SOFA score points indicated low-and high-risk critically ill 221 COVID-19 patients who differed in the inflammasome and inflammatory profile. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 1, 2021. ; https://doi.org/10. 1101 /2021 patients by development of inhibitor-based immunoprecipitation of active caspase-1 followed by 240

Western Blot detection of the specific fragments. Interestingly, the dominant form of caspase-1 241 present in the plasma was the intermediate form p35 found in 12 of 22 COVID -19 patients (Fig.  242   3A) while it was undetectable in TBI patients. In six of 22 COVID-19 patients also mature p20 243 form could be detected (Fig. 3A) . Presence of active caspase-1 was not related with outcome 244 neither severity of the disease. There was also no difference in caspase-1 between patients with 245 coinfections. However, patients at high-risk of death defined by SOFA score >9 had significantly 246 higher amount of p35 in densitometric analysis (Fig. 3B) Of interest, patients with detectable p35 247 had higher fibrinogen level (7.30 g/L (IQ: 5.50 -8.70) vs. 5.45 g/L (IQ: 4.10 -6.30); p=0.020) and 248 lower d-dimers (1.35 μg/mL (IQ: 1.20 -4.35) vs. 10.25 μg/mL (IQ: 6.30 -20.90); p=0.019). 249

Our study is the first to our best knowledge that have investigated comprehensively the 251 plasma biomarkers related with inflammasome regulation in a well-defined group of critically ill 252 COVID-19 patients at the ICU admission. We found that the marked activation of inflammasome 253 defined as presence of active capsase-1 in the patient's plasma is heterogenous and not related 254 with outcome. Also, other quantitative inflammasome-related proteins and cytokines are not 255 related with outcome and only galectin-1 and IL-1RA differed in patients with low-and high-risk of 256 death. 257

In this study we have focused on the critically ill COVID-19 patients at admission to ICU 258 as this group is at high risk of death (reflected by 28-day mortality of 53% outreached by in-259 hospital mortality of 62% in our group). Although many groups have tried to identify new potential 260 biomarkers, we have selected a panel of proteins related with different stages of the 261 inflammasome activity. It included the structural component of inflammasome, the ASC protein; 262 effector proteins cleaved by caspase-1 (IL-1 , IL-1 , IL-18), their antagonist IL-1RA and 263 gasdermin D which forms pyroptotic pores in cell membrane. Also, we measured galectin-1 and 264 LDH which are markers of inflammatory cell death 17 and ferritin which is induced by the 265 inflammasome activity 18 . Moreover, we have established a new method to analyze active 266 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 1, 2021. ; https://doi.org/10.1101/2021.10.30.21265662 doi: medRxiv preprint caspase-1 from human plasma which is based on the immunoprecipitation with biotinylated FAD-267 FMK, a pan-caspase inhibitor followed by Western Blot to specifically detected caspase-1 forms. 268

The concentration of the measures cytokines was not different between critical COVID-19 269 and TBI patients. We choose the TBI patients for comparison as it relatively homogenous group 270 with sterile injury, age-and sex-matched. ASC, IL-1 and capase-1 were already shown to be 271 upregulated in plasma of TBI patients 19 . Although increased galectin-1 was already described in 272 COVID-19 patients 20 it was compared to healthy controls only. Here, we found higher galectin-1 273 in TBI patients than COVID-19 which suggests that the latter is not characterized by extreme Neither of the analyzed protein was related with mortality in critically ill patients. Although some of these cytokines like IL-18 or IL-1RA were already linked with the 279 severity of COVID-19 9,25,26 it should be highlighted that our study is focused on a defined 280 population of critical COVID-19 in contrast to most studies which compare cytokine levels in early 281 COVID-19 of various severity. As the analyzed cytokines can be produced in inflammasome-282 independent manner, we assessed the presence of active caspase-1. We have detected the 283 intermediate p35 form of caspase-1 in 54% cases and mature p20 in 27% patients. These finding 284 clearly indicate that inflammasome activation is heterogenous in critical COVID-19 and complete 285 maturation of caspase-1 is not common in these patients. The status of circulating caspase-1 was 286 not related with outcome similarly to the findings by the group of D. Zamboni who utilized 287 capsase-1 ELISA 9 . Even less frequently we were able to detect the ASC and GSDMD proteins 288 which also indicates infrequent activation of inflammasome in critical COVID-19. Our results stay 289 in accordance with recent findings that showed only small percentage of NLRP3 and AIM2 290 inflammasome activation in the circulating monocytes from COVID-19 patients 10,27 . Moreover, 291 current data point at monocytes and macrophages as the major lung cell types showing activation 292 of the inflammasome in COVID-19 9,10 . Additionally, we compared inflammasome-related proteins 293 in the patients divided by their SOFA score at admission with the most accurate threshold to 294 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 1, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 predict mortality (low-and high-risk groups). Notably, galectin-1 and caspae-1 p35 was increased 295 in the high-risk group, while IL-1RA was decreased. Such pattern may suggest indeed loss of 296 control of this inflammatory pathway in the high-risk group of patients. The high-risk patients had 297 also higher leukocyte count, IL-6, procalcitonin and INR however, neither of these parameters 298 was significantly related with outcome in logistic regression model. 299

As inflammasomes are reported to play role in the susceptibility to secondary 300

infections 28,29 we tested for the relation between inflammasome activation markers and co-301 infections or development of secondary infections however, there were no significant correlations 302 between these variables. We observed some weak but significant correlations between IL-1 and 303 IL-1 , ASC and GSDMD, ferritin and LDH which suggests coordinated regulation of these 304 mediators. Interestingly, we observed links between the inflammasome activity and dysregulated 305 coagulation system. Patients with unfavorable prognosis had marks of coagulopathy indicated by 306

higher INR values. In addition, there was a correlation between ferritin and INR and inversed 307 correlation between IL-1 and IL-1 and platelets. Altogether, these observations suggest 308 mechanistic link between inflammasome activation and coagulopathy and possibly features of 309 secondary hemophagocytic lymphohistiocytosis 3 . 310

This study has several limitations related with small patients' group and lack of 311 mechanistic investigations. Noteworthy, our study group is well-defined and limited to the ARDS 312 and sepsis forms of COVID-19. Although we analyzed several proteins related with activation of 313 the inflammasome we did not measure other relevant mediators that can interact with this 314 pathway (e.g. interferons). The present study would also benefit from the flow cytometry data of 315 the inflammasome activation in circulating blood cells. Moreover, we analyzed only plasma 316 proteins and it should be noted that they may not fully reflect the tissue response. Finally, we 317

were not able to explain the heterogenic pattern of the inflammasome activity in these patients. 318

Our findings are of important clinical relevance as inflammasome-targeted therapies are 320 widely testes in COVID-19, mostly without the use of biomarker guidance. Recently, the negative 321 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 1, 2021. ; https://doi.org/10. 1101 /2021 results of the collaborative trial on the colchicine (which inhibits inflammasome formation 30 ) were 322 published 31 . Also, trials targeting IL-1 in severe COVID-19 with anakinra was reported to be 323 negative 14 . At the same time, another study that used su-PAR levels to guide the anakinra 324 treatment in early COVID-19 showed significant benefit 13 . The results of these studies are not 325 surprising in the light of our findings showing heterogenous inflammasome activation in critically ill 326 COVID-19 patients and lack of its direct relation with mortality. Yet, it could be speculated that in 327 a selected subpopulation of patients with activated caspase-1 or high galectin-1 level these 328 CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Mortality in patients 349 admitted to intensive care with COVID-19: an updated systematic review and meta-350 analysis of observational studies

The COVID-19 puzzle: deciphering 352 pathophysiology and phenotypes of a new disease entity

Sepsis therapies: learning from 30 years of failure of 355 translational research to propose new leads

SARS-CoV-2/COVID-19: Evolving Reality

Inflammasomes and Pyroptosis as Therapeutic 359 Targets for COVID-19

Emerging Activators and Regulators 361 of Inflammasomes and Pyroptosis

Inflammasome formation in the lungs of patients with 363 fatal COVID-19

Inflammasomes are activated in response 365 to SARS-CoV-2 infection and are associated with COVID-19 severity in patients

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SARS-CoV-2 engages 371 inflammasome and pyroptosis in human primary monocytes

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Cytokine Storm in COVID-19: The Current 396 Evidence and Treatment Strategies

Abbreviations: APTT, activated partial thromboplastin time

ASC, apoptosis-associated speck-436 like containing a CARD

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