Anti-nucleocapsid antibodies enhance the production of IL-6 induced by SARS-CoV-2 N protein

Abstract

A cytokine storm induces acute respiratory distress syndrome, the main cause of death in coronavirus disease 2019 (COVID-19) patients. However, the detailed mechanisms of cytokine induction due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain unclear. To examine the cytokine production in COVID-19, we mimicked the disease in SARS-CoV-2-infected alveoli by adding the lysate of SARS-CoV-2-infected cells to cultured macrophages or induced pluripotent stem cell-derived myeloid cells. The cells secreted interleukin (IL)-6 after the addition of SARS-CoV-2-infected cell lysate. Screening of 25 SARS-CoV-2 protein-expressing plasmids revealed that the N protein-coding plasmid alone induced IL-6 production. The addition of anti-N antibody further enhanced IL-6 production, but the F(ab')² fragment did not. Sera from COVID-19 patients also enhanced IL-6 production, and sera from patients with severer disease induced higher levels of IL-6. These results suggest that anti-N antibody promotes IL-6 production in SARS-CoV-2-infected alveoli, leading to the cytokine storm of COVID-19.

Figure 1

IL-6 production induced by SARS-CoV-2-infected cell lysate in monocyte-derived macrophages (MDM) and iPS-derived myeloid cells without productive infection. (A) Peripheral blood monocytes were differentiated into MDM by adding GM-CSF for 8 days. TMPRSS2/VeroE6 cells and MDM were infected with 1000 50% tissue culture infectious dose (TCID50) or 100 TCID50 of SARS-CoV-2 (JPN-TY-Wk-521 strain) and the viral RNA in the supernatants was measured by RT-PCR on the indicated days. (B) GM-CSF- or M-CSF- MDM were infected with 100 TCID50 of KNG-19-020 strain of SARS-CoV-2. (C) iPS-derived myeloid cells K-ML2 (gray) and GM-CSF- (blue) and M-CSF- (red) MDM were inoculated with the SARS-CoV-2 KNG-19-020 strain together with SARS-CoV-2-infected or uninfected cell lysate. Two days after infection, the IL-6 levels in the culture supernatants were measured by ELISA. The representative results of at least three independent experiments are shown.

Figure 2

SARS-CoV-2 N protein, but not S protein, induced IL-6 production. The levels of IL-6 were measured by ELISA 2 days after treatment (A, C, and F). (A) K-ML2 cells were stimulated with the lysate of 293 T cells transfected with plasmids encoding each of the SARS-CoV-2 proteins. (B) The expression of each viral protein confirmed by western blot using the anti-Strep tag. The images from three membranes were combined. The original images with different exposure time are shown in Fig. S3. (C) K-ML2 cells were stimulated with serially diluted S or N protein produced using a baculovirus expression system. The mean and standard deviation of triplicate samples are shown. (D) A schematic diagram of full-length and truncated N proteins. (E) Anti-NTD (N2) antibody and anti-CTD (C2) antibody were used to visualize the full-length and truncated N proteins by western blot. The images from two membranes transferred from a single gel were combined. A blue vertical dividing line of two membranes is shown. The original images with different exposure time are shown in Fig. S3. (F) K-ML2 cells were stimulated with the cell lysates of 293 T cells expressing the full-length or truncated N proteins. Data are expressed as the mean and standard deviation of triplicate samples. The representative results of three independent experiments are shown.

Figure 3

Profiles of cytokine production from macrophages (MDM) stimulated by N protein. MDM differentiated with GM-CSF or M-CSF were stimulated by 156 or 625 ng/mL of N proteins for 2 days. The cytokine levels were measured by a multiplex assay, as described in the Methods. Data are expressed as the mean and standard deviation of triplicate samples.

Figure 4

Anti-N antibodies and COVID-19 patient sera enhanced the IL-6 production induced by N protein. Levels of IL-6 were measured by ELISA 2 days after the addition of N protein, except for in panel C. (A) K-ML2 cells were stimulated with 156 ng/mL of N protein with escalating amounts of anti-S (blue) or anti-N (red) antibodies. (B) Anti-N antibody, S2, was digested by pepsin, and purified F(ab’)2 (red) or IgG (blue) was added to induce IL-6 production. Data are expressed as the mean and standard deviation of triplicate samples. (C) The levels of mouse mAbs bound to the full-length N protein (blue), NTD (Thr49-Gly175 fragment: red) or CTD (Lys248-Pro365 fragment: gray) were measured by an in-house ELISA. Anti-S mAb, clone 29-C7, was used as a negative control. (D) K-ML2 cells were stimulated with 3 μL of the cell lysate of N protein-expressing 293 T cells in the presence of 1 μg/mL of mAbs. Data are expressed as the mean and standard deviation of triplicate samples. The representative results of three independent experiments are shown. (E) K-ML2 cells were stimulated with 156 ng/mL of N protein with escalating amounts of serum from two patients, Patient 313 (blue) and Patient 315 (red). “0%” indicates the baseline production of IL-6 induced by N protein without patients’ serum. “10% noN” indicates the level of non-specific IL-6 induction from the addition of 10% patient serum from patient No. 313 without N protein. (F) The levels of IL-6 in the culture supernatants of K-ML2 cells cultured in the presence of 1% patient serum. Specimens were divided into four groups according to the disease severity at the time of blood collection. The criteria for COVID-19 severity in Japan were described in the Methods section. In brief, the criteria for mild, moderate I, moderate II, and severe are almost the same as those of mild, moderate, severe, and critical in the US NIH guideline. The center lines in the boxes and the boxes indicate the median and 25/75 percentiles, respectively. “*” denotes a statistically significant difference among the four groups (p < 0.0001) by the Kruskal–Wallis test. “**” and “***” denote a statistically significant difference between the moderate I and severe disease groups (p < 0.0001) and between the mild and severe disease groups (p < 0.0001), respectively, by the Mann–Whitney U test.