key: cord-0939912-2ldq9ma5
authors: Fernandez, J. J.; Mancebo, C.; Garcinuno, S.; March, G.; Alvarez, Y.; Alonso, S.; Inglada, L.; Blanco, J.; Orduna, A.; Montero, O.; Sandoval, T. A.; Cubillos-Ruiz, J. R.; Bustamante, E.; Fernandez, N.; Sanchez Crespo, M.
title: IRE1α-XBP1 Activation Elicited by Viral Singled Stranded RNA via TLR8 May Modulate Lung Cytokine Induction in SARS-CoV-2 Pneumonia
date: 2022-01-28
journal: nan
DOI: 10.1101/2022.01.26.22269752
sha: 80c6dea9c88bf953f57fb2db3b8c7a3bcdd08437
doc_id: 939912
cord_uid: 2ldq9ma5

Initial symptoms of COVID-19 infection depend on viral replication, while hyperinflammation is a hallmark of critical illness and may drive severe pneumonia and death. Among the mechanisms potentially involved in the hyperinflammatory state, we focused on the unfolded protein response, because the IRE1-XBP1 branch can be activated as result of the endoplasmic reticulum stress produced by the overwhelming synthesis of viral components and synergizes with Toll-like receptor signaling to induce cytokine expression. Viral RNA may trigger the IRE1-XBP1 branch via TLR7/8 activation and like TLR2 and TLR4 may underpin cytokine expression trough XBP1 splicing (sXBP1). The expression of IL1B, IL6, and TNF mRNA in bronchoalveolar aspirates (BAAs) were higher in COVID-19 patients under mechanical ventilation and intubation who showed sXBP1. The scrutiny of monocytic/macrophagic markers during active infection showed a reduction of those involved in antigen presentation and survival, as well as the IFN stimulated gene MX1. These changes reverted after infection tests turned negative. In contrast, the expression of the mRNA of the serine protease TMPRSS2 involved in S protein priming showed a high expression during active infection. TLR8 mRNA showed an overwhelming expression as compared to TLR7 mRNA, which suggests the presence of monocyte-derived dendritic cells (MDDCs). In vitro experiments in MDDCs activated with ssRNA40, a positive-sense, single-stranded RNA (+ssRNA) like SARS-CoV-2 RNA, induced sXBP1 and the expression of IL-1{beta}, IL-6, and TNF at mRNA and protein levels. These responses were blunted by the IRE1 ribonuclease inhibitor MKC8866. Given the analogies between the results observed in BAAs and the effects induced by +ssRNA in MDDCs, IRE1 ribonuclease inhibition might be a druggable target in severe COVID-19 disease.

Introduction prostaglandin synthase 2 [7], the ubiquitin-like modifier ISG15, and the cytokines IFNβ, IL-6, 143 IL-23, and TNFα [8] [9] [10] [11] . 144 Although the goal of the UPR is alleviating ER-stress, the IRE1α-XBP1 branch 145 contributes to the pathogenesis of multiple ailments, including cancer, atherosclerosis, 146 infections, and autoimmune diseases [12] [13] [14] [15] . Recent research has disclosed that the IRE1α- forces aberrant glycosylation and ER-stress [19] . In keeping with notion, specific activation of 158 sXBP1 has been reported in monocytes from COVID-19 patients [20] . 159 The association of sXBP1 with TLR2 and TLR4 signaling [8] together with genuine 160 UPR induced by protein synthesis overload point to sXBP1 involvement in the cytokine storm. and sXBP1 (Fig 1D) . The presence of three bands in some cases is explained by the formation 202 of heteroduplexes [29] . sXBP1 was detected in 17.91% of SARS-CoV-2 negative and 40.32% 203 of SARS-CoV-2 positive patients ( Fig 1E) . Quantitation of sXBP1 showed higher values in 204 COVID-19 positive patients as compared to the negative ones ( Fig 1F) . The incidence of 205 sXBP1 was similar in male and females ( Fig 1G) and increased with age ( Fig 1H) . Mortality 206 was observed in four patients who showed a degree of splicing above 10% of total XBP1 (Fig   207   1I ). These findings show that sXBP1 shows higher frequency and extent in nasopharyngeal 208 exudates of patients with active SARS-CoV-2 infection, particularly in dying patients. Further experiments were carried out using RNA extracted from BAAs of patients under 212 mechanical ventilation in intensive care unit (ICU) (Fig. 2A) . Ventilatory support and 213 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 28, 2022. ; https://doi.org/10.1101/2022.01.26.22269752 doi: medRxiv preprint endotracheal intubation were indicated because of acute hypoxemic respiratory failure despite 214 high-flow nasal oxygen therapy or non-invasive ventilation. COVID-19 patients received a 215 standard and proved useful treatment for the hyperinflammatory state consisting of 6 mg 216 dexamethasone daily or 50 mg of IV hydrocortisone every 8 hours for up to 10 days, while this 217 protocol was not routinely used in non-COVID-19 patients (Fig 2B) . The extent of sXBP1 was 218 higher in SARS2-CoV-2 pneumonia patients than in those with respiratory failure due to other 219 conditions, decreased after COVID-19 tests turned negative, and showed higher values than 220 those observed in nasopharyngeal swabs (Fig 2C) . The PERK-eIF2α-ATF4-CHOP branch of 221 the UPR was explored assaying DDIT3/CHOP gene expression. DDIT3 expression also 222 decreased after SARS-CoV-2 tests turned negative, while there was no significant difference 223 of expression between non-COVID-19 and COVID-19 pneumonia patients ( Fig 2D) . As 224 regards cytokine expression (Fig 2E-2N ), IL1B and IL6 mRNA levels during viral proliferation 225 were significantly lower than those detected in non-COVID-19 patients. A similar trend was 226 observed in TNF, IL23A, and IL8 mRNA expression, although these values did not reach 227 statistical significance. Cytokine mRNA did not show a trend to decrease after SARS-CoV-2 228 tests were negative. IL10 and IFNB mRNA were higher in SARS-CoV-2 infection than in non-229 COVID pneumonia and continued elevated after COVID-19 tests turned negative. IFNG 230 showed a trend to be increased in COVID-19 pneumonia. Overall, these results show that with sXBP1 both during infection and after negativization of the RT-PCR test (Fig 3B) . TNF 242 and IL1B mRNA expression was also higher in patients with sXBP1, even after a negative RT-243 PCR test (Fig 3C and 3D ). IL6 mRNA was increased in patients with sXBP1 and active 244 infection ( Fig 3E) . IL8 mRNA was expressed at much lower levels than those encoding other perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 28, 2022. CoV-2 test (Fig 5H) . TMPRSS2 mRNA, a serine protease involved in the cleavage of viral 288 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 28, 2022. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 28, 2022. ; https://doi.org/10.1101/2022.01.26.22269752 doi: medRxiv preprint

[42] and the S1R agonist fluvoxamine, which has been reported to inhibit XBP1 splicing in 314 bacterial sepsis [14] , lacked any significant effect on those responses (Fig 6G-6J ). Real-time 315 assays of energetic metabolism with the Seahorse technology, showed a reduction of O2 316 consumption rate (OCR) and an increased extracellular acidification rate (ECAR) in response 317 to imiquimod (Fig 6K) , thus mimicking the glycolytic rewiring induced by bacterial PAMPs perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 28, 2022. ; https://doi.org/10.1101/2022.01.26.22269752 doi: medRxiv preprint suggesting a direct effect of sXBP1 on MX1 and OAS1 expression, rather than an indirect effect 341 mediated by IFNs. Notably, ssRNA40 did not modify the energetic pattern of MDDCs (S1C perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in which involved larger cohorts for study and showed a significant reduction of morbidity by 383 fluvoxamine, as deemed from a reduced resort to either retention in a COVID-19 emergency 384 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 28, 2022. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in who first disclosed that the TLR7/TLR8 agonist R848 was 100-fold as potent as imiquimod in perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted January 28, 2022. ; https://doi.org/10.1101/2022.01.26.22269752 doi: medRxiv preprint Bioenergetic assays were carried out using an Agilent Seahorse XF HS Mini Analyzer. 10 5

MDDCs were adhered with Cell-Tak to Seahorse plates and treated with stimuli of TLR3, 515 TLR7, and TLR8, as well as sonicated zymosan to activate the fungal pattern receptor dectin- perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 28, 2022. ; https://doi.org/10.1101/2022.01.26.22269752 doi: medRxiv preprint salt, wash buffer, and elution buffer. Cross-links were reversed by heating at 67°C in a water 539 bath, and the DNA bound to the beads isolated by extraction with 540 phenol/chloroform/isoamylalcohol. Irrelevant Ab was used as control of binding specificity.

The sequences of the primers are shown in Table 1 perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in IL1B, TNF, IL6, IL8, IL10, IL12A, IL12B, 840  841  842  843  844  845  846  847  848  849  850  851  852  853  854  855  856  857  858  859  860  861  862  863  864  865  866  867  868  869  870  871  872  873  874  875  876  877 sample Wilkoxon signed rank test. ¶Unpaired (two-tail) t test. #Welch`s test.

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 28, 2022. decarboxylase. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p<0.005. 930 ‡Kruskal-Wallis U test. †Ordinary one-way ANOVA. ¶Paired or unpaired (two-tail) t test.

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 28, 2022. ; https://doi.org/10.1101/2022.01.26.22269752 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 28, 2022. ; https://doi.org/10.1101/2022.01.26.22269752 doi: medRxiv preprint TMPRSS2. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p<0.005. †Ordinary 940 one-way ANOVA. ‡Kruskal-Wallis U test. ζHolm-Sidak`s multiple comparison test.

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 28, 2022. ; https://doi.org/10.1101/2022.01.26.22269752 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted January 28, 2022. ; https://doi.org/10.1101/2022.01.26.22269752 doi: medRxiv preprint

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