key: cord-0901401-7bh9p76m
authors: Croci, S.; Venneri, M. A.; Mantovani, S.; Fallerini, C.; Benetti, E.; Picchiotti, N.; Campolo, F.; Imperatore, F.; Palmieri, M.; Daga, S.; Gabbi, C.; Montagnani, F.; Beligni, G.; Farias, T. D. J.; Carriero, M. L.; Di Sarno, L.; Alaverdian, D.; Aslaksen, S.; Cubellis, M. V.; Spiga, O.; Baldassarri, M.; Fava, F.; Norman, P. J.; Frullanti, E.; Isidori, A. M.; Amoroso, A.; Mari, F.; Furini, S.; Mondelli, M.; GEN-COVID Multicenter Study,; Chiariello, M.; Renieri, A.; Meloni, I.
title: The polymorphism L412F in TLR3 inhibits autophagy and is a marker of severe COVID-19 in males.
date: 2021-03-26
journal: nan
DOI: 10.1101/2021.03.23.21254158
sha: 2423d7ee032c10b86e4147f556a90b59a01a1865
doc_id: 901401
cord_uid: 7bh9p76m

The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired autophagy and reduced TNF production was demonstrated in HEK293 cells transfected with TLR3-L412F plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (P=0.038). An increased frequency of autoimmune disorders as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways.

In December 2019, a new virus was isolated in Wuhan, China, which was called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is an enveloped positivesense RNA virus that caused a new pandemic which WHO named COVID-19 (CoronaVirus Disease-2019).

To date, many characteristics of SARS-CoV-2 are still unclear and, although its ability to be transmitted from one person to another has been ascertained, uncertainties remain about the exact modes of transmission and pathogenicity. In addition, a high variability of symptoms in infected patients and between different populations has been reported; one of the possible explanations of such variability is the genetic background of the host that may affect immune responses to the virus. Among host genetic factors that might impact on symptoms severity there are genes involved in virus entry and mediators of innate immunity [1, 2] . Toll-Like Receptors (TLRs) are a class of proteins that play a key role in host innate immunity, causing the production of pro-inflammatory cytokines (TNF-, IL-1, and IL-6) and type I and II Interferons (IFN), that are responsible for innate antiviral responses. Among TLR genes, TLR3

is an interferon inducing dsRNA sensor, whose activation is involved in protection against different RNA viruses [2] [3] [4] . Upon viral infection, TLR3 signalling leads to the activation of two factors, NF-kB and Interferon (IFN)-Regulatory Factor 3 (IRF3), that play an essential role in the immune response. This results in the production of various cytokines, including tumor necrosis factor-(TNF-), activating immune responses. However, increased inflammatory responses can make the patient more susceptible to pneumonia and autoimmune diseases.

Accordingly, a protective effect against fatal pneumonia has been reported in the absence of TLR3 [5] [6] [7] . Among TLR3 variants, the functional L412F polymorphism (rs3775291; c.1234C>T) is known to decrease TLR3 expression on the cell surface [8] . This polymorphism also leads to poor recognition of SARS-CoV-2 dsRNA compared to its wild type counterpart [9] and has been recently associated with SARS-CoV-2 susceptibility and mortality [10] .

There is evidence that TLR3, as other TLRs, acts through autophagy in determining susceptibility to infections [11] . The autophagic pathway is essential during infection and for molecular processes as cell maintenance and homeostasis [11] [12] [13] [14] . Indeed, autophagy is one of the major cell defence mechanisms against pathogens [15] . A role for autophagy is reported in different studies on other Coronaviruses such as the mouse hepatitis virus (MHV) and the transmissible gastroenteritis virus (TGEV) [16, 17] . A role in SARS-CoV-2 infection has also been described [18] [19] [20] . In particular, SARS-CoV-2 can inhibit autophagy resulting in accumulation of autophagosomes and inhibition of viral clearance that, together with immune dysfunction and the activation of numerous inflammatory cytokines, leads to a more severe form of COVID-19 [21] [22] [23] .

To shed light on the mechanisms underlying the diverse susceptibility to COVID-19, we performed a nested-control study within our GEN-COVID cohort, confirming the role of L412F polymorphism in TLR3 gene in susceptibility to SARS-CoV-2 and further defining the potential mechanisms by which this effect is exerted.

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Comparing the extreme phenotypes of SARS-CoV-2 infection, severe COVID-19 patients (cases) versus SARS-CoV-2 PCR-positive oligo-asymptomatic subjects (controls), and using LASSO Logistic regression on common bi-allelic polymorphisms from whole-exome sequencing, we identified the L412F polymorphism (rs3775291; c.1234C>T) in TLR3 as a severity marker (Fig. 1A) . The grid search curve of the cross-validation score (Fig.1B) shows a maximum of the regularization parameter in 10. With this calibration setting, the 10-fold cross-validation provides good performances in terms of accuracy (73%), precision (74%), sensitivity (73%), and specificity (73%) as shown in Fig.1C . The confusion matrix is reported in Fig.1D , whereas the Receiver Operating Characteristic (ROC) curve ( Fig.1E ) provides an Area Under the Curve (AUC) score of 80%.

The L412F polymorphism has an overall allele frequency of about 20%, ranging from 30% in European to 0.88% in African (mainly sub Saharan) populations [8] . It is intriguing that a COVID-19-free population such as sub Saharan has a very low frequency (0.88%) of this polymorphism and that Asian (26.97%) and European (30.01%) have a much higher frequency.

The variant protein with phenylalanine is under-represented on the cell surface, it is not efficiently secreted into the culture medium when expressed as the soluble ectodomain, and it has reduced capability to activate the expression of TLR3-dependent reporter constructs [8] . In order to confirm the role of the polymorphism, we compared individuals showing severe COVID-19 (cases) and those with no sign of the disease (controls). We subdivided patients in two categories, those having the polymorphism in heterozygous or homozygous state and those homozygous for the wild type allele. We found that the prevalence of L412F polymorphism is significantly higher in cases compared to controls (p-value 2.8x10 -2 ) ( Table 1a) . The global allele frequency of L412F in our cohort (cases and controls) is 29.38%, like to the allele frequency of 29.79% reported in the European (non-Finnish) population in the gnomAD database (https://gnomad.broadinstitute.org/). The identified frequencies were in Hardy-Weinberg equilibrium.

Sex-related differences of TLRs activation following stimulation by viral nucleic acid may be involved in the sex-related variability in response to viral infections [24] . Several rare TLR3 loss of function mutations are known linked both to influenza and SARS-CoV-2 virus as well as hyperfunctioning mutations [25, 26] . In agreement with these data, when we stratified by gender, the statistically significant difference increased in the sub-cohort of males giving an OR of 1.94 (95% CI, 1.23 to 3.06; p=3.8x10 -3 ), while it was lost in the sub-cohort of females (p-value 5.8x10 -1 ), ( Table 1) .

We then investigated the prevalence of patients carrying L412F in heterozygous or homozygous states in all 4 categories of COVID-19 clinical severity, considering only male subjects regardless of age (n=665). We found that the prevalence of carriers directly increased with the severity of COVID-19, from a clinical condition not-requiring hospitalization to intratracheal intubation (Fig. 1F) The L412F substitution in TLR3 falls in the ectodomain, in the 14 Leucine-Rich Repeats (LRR) domain, a motif of 22 amino acids in length that folds into a horseshoe shape [27] . Proteins containing LRRs are involved in a variety of biological processes, including signal transduction, cell adhesion, DNA repair, recombination, transcription, RNA processing, disease resistance, apoptosis, and the immune response [slit: an extracellular protein necessary for development of midline glia and commissural axon pathways contains both EGF and LRR domains [28] . The L412F substitution is expected to have a limited structural impact with minimal rearrangement of near hydrophobic amino acids like tryptophan 386 (Fig. 2) .

However, the absence of one of the leucines probably determines a different rearrangement of the motif and consequently of the near glycosylation site ASN414, having an impact on proteinprotein interaction and in signal transduction process [29] . 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 Germline knockout of TLR3 inhibits autophagy and upregulation of TLR3 promotes damage after myocardial infarction mainly because of autophagy rather than inflammatory activation [30] . Interestingly, we could notice a statistically significant (p=3.8x10 -2 ) reduced survival at 28 days in TLR3_L412F COVID-19 patients treated with hydroxychloroquine (HCQ) In order to validate data obtained on transfected HEK cells, we next isolated and cultured skin fibroblasts from healthy donors with different genotypes relative to the TLR3 locus: wild-type (WT/WT) and L412F (L412F/L412F) homozygous and WT/L412F heterozygous. In these 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 . Accordingly, an increased frequency of autoimmune disorders as co-morbidity was found in our cohort in L412F COVID-19 patients with specific HLA class II haplotypes prone to autoantigen presentation. In particular, we analysed a DR3-DQ2 haplotype which predisposes to different types of autoimmune diseases [45, 46] . The frequency of autoimmune disorders is indeed significantly increased in male patients with HLA DR3/DQ2 haplotype and L412F, especially diabetes (25%) (Table 2a and b) . These results suggest that the combination 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 March 26, 2021. ; https://doi.org/10.1101/2021.03.23.21254158 doi: medRxiv preprint of L412F in TLR3 and a specific class II HLA haplotype puts male patients at risk of post-COVID autoimmune exacerbation emphasizing the need for appropriate follow-up.

No association was found between AIRE loss of function variants and COVID-19 outcome, as outlined by the absence of the gene in Figure 1 .

In conclusion, we have identified the second protein-encoding polymorphism that modulates COVID-19 outcome. These results indicate that L412F polymorphism in the TLR3 gene makes males, in whom after puberty testosterone lowers TLR3 expression, at risk of severe COVID-19 in a context of a polygenic model. Moreover, based on impairment of autophagy, these data provide a rationale for reinterpreting clinical trials with HCQ stratifying patients by L412F. 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 

We performed a nested case-control study (NCC). We used a cohort of 1319 subjects (cases and controls) from the Italian GEN-COVID Multicenter study, infected with SARS-CoV-2 diagnosed by RT-PCR on nasopharyngeal swab [47] . Cases were defined as patients needing endotracheal intubation or CPAP/biPAP ventilation. Controls were oligoasymptomatic subjects not requiring hospitalization. regularization [48] term of the loss, has the effect of shrinking the estimated coefficients to 0.

In this way, the weights of the logistic regression algorithm can be interpreted as the feature importances of the subset of the most relevant features for the task [49] . The input features are the common bi-allelic polymorphisms from whole-exome sequencing as well as gender, and the age, the latter as a continuous variable normalized between 0 and 1. Common bi-allelic polymorphisms are defined as combinations of two polymorphisms, each with MAF above 1%, with frequency above 5% in the cohort. 

For the LC3B-positive dot count, we performed intensitometric analysis of fluorescence using the Quantitation Module of Volocity software (PerkinElmer Life Science). Dot count was subjected to statistical analysis. Measures were obtained by analyzing at least 400 cells/sample from 3 different experiments. Significance (P value) was assessed by Student s t test, using GraphPad Prism6 software. Asterisks were attributed for the following significance values: P > 0.05 (ns), P < 0.05 (*), P < 0.01 (**), P < 0.001 (***).

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Total RNA was isolated using the RNAeasy Mini Kit (Quiagen, Hilden, Germany) according to the manufacturer s instructions. cDNA synthesis was performed using the Maxima First Diego, CA). HLA alleles were determined from the sequence data using the consensus from 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 authors declare no competing interests. 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 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 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 March 26, 2021. ; https://doi.org/10.1101/2021.03.23.21254158 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 

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 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 

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