The polymorphism L412F in TLR3 inhibits autophagy and is a marker of severe COVID-19 in males

Abstract

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 macroautophagy/autophagy and reduced TNF/TNFα production was demonstrated in HEK293 cells transfected with TLR3^L412F-encoding 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 such 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.Abbreviations: AP: autophagosome; AUC: area under the curve; BafA1: bafilomycin A1; COVID-19: coronavirus disease-2019; HCQ: hydroxychloroquine; RAP: rapamycin; ROC: receiver operating characteristic; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TLR: toll like receptor; TNF/TNF-α: tumor necrosis factor.

Keywords: Autophagy; COVID-19; HLA; L412F; TLR3.

Figure 1.

The histogram of the LASSO logistic regression weights represents the importance of each feature for the classification task, (A) The positive weights reflect a susceptible behavior of the features to the target COVID-19 disease, whereas the negative weights a protective action. (B) Cross-validation ROC-AUC score for the grid of LASSO regularization parameters; the error bar is given by the standard deviation of the score within the 10 folds; the optimal regularization parameter is chosen by selecting the one with highest cross-validation score (red point). (C) Boxplot of accuracy, precision, sensitivity, specificity, and ROC-AUC score for the 10-fold of the cross-validation. The box extends from the Q1 to Q3 quartile, with a line at the median (Q2) and a triangle for the average. (D) Confusion matrix for the aggregation of the logistic regression predictions in the 10 folds of the cross-validation. (E) ROC curve for the 10 folds of the cross-validation. (F) Distribution of carriers of the polymorphism L412F in homozygous or heterozygous states stratified by clinical category.

Figure 2.

Superposition of wild-type and mutated TLR3 protein. (A) TRL3 human protein tridimensional structure of 2Z7X crystal structure. In green cartoon representation of TLR3 protein. (B) and (C) Zoom of the mutated region with Leu412 in red sticks and Phe412 in magenta. The hydrophobic core of Leu377, Leu389, and Trp386 is in blue sticks.

Figure 3.

Analysis of autophagy in TLR3_L412F-expressing cells. (A) 28-day survival study of TLR3- L412F carriers vs not-carriers in the group treated with hydroxychloroquine. N = 156, with 73 carriers of TLR3- L412F. Three carriers and 1 not-carrier died in the first 28 days of treatment. (B) Analysis of autophagy in HEK-KO cells expressing wild type or L412F mutant proteins. HEK-KO cells were transfected for 24 h with plasmids encoding TLR3_WT and TLR3_L412F. Cells were next incubated in full medium (FM) or FM + 400 nM bafilomycin A₁ (BAF A1) for 3 h and stimulated for increasing times with 50 μg/ml poly(I:C), as indicated. Cells were next fixed, permeabilized with 100 μg/ml digitonin and stained with anti-LC3B antibodies and revealed with Alexa Fluor 488-conjugated secondary antibodies. Nuclei were stained with DAPI. Where indicated, RAP (500 nM, for 2 h) was used as positive control for induction of autophagy. (C) Same as in B, but the amount of autophagosomes (scored as LC3B-positive dots) per cell was quantified by Volocity software. Measures were obtained by analyzing at least 400 cells/sample from 3 different experiments (n = 3). (D) Analysis of TNF mRNA expression in HEK-KO cells expressing wild type or L412F mutant proteins. HEK-KO cells were transfected for 24 h with plasmids encoding empty vector (CTR), TLR3_WT and TLR3_L412F and next stimulated for increasing times with 50 μg/ml poly(I:C), where indicated. TNF levels were evaluated by Real Time PCR. The gene expression levels were evaluated by the fold change versus TLR2 WT sample using the equation 2^−DDCt. Data are presented as the mean ± SEM. Data significance was analyzed using One-way ANOVA test with Holm-Sidak’s correction. Asterisks were attributed for the following significance values: P > 0.05 (ns), P < 0.05 (*) and P < 0.01 (**). (E) Normal human fibroblasts (NDHF) from subjects expressing the TLR3_WT receptor were stimulated with 50 μg/ml poly(I:C) or RAP (1 μ M) for 4 h, in full medium alone or containing 400 nM bafilomycin A₁ for 3 h. Cells were next fixed, permeabilized with 100 μg/ml digitonin and stained with anti-LC3B antibodies and revealed with Alexa Fluor 488-conjugated secondary antibodies. Nuclei were stained with DAPI. (F) Same as in E, but the number of autophagosomes (scored as LC3B-positive dots) per cell was evaluated for each sample by Volocity software. (G and H) same as in E-F, but fibroblasts are homozygous for the TLR3_L412F receptor. Statistical analysis was performed using Student’s t test. Means § SEM for each value are shown in the graphs. ns = not significant; ** = p < 0.01; *** = p < 0.001. < 0.001.