Longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of SARS-CoV-2 infected patients, EBioMedicine (2020), doi: https://doi.Abstract Background: The dynamic changes of lymphocyte subsets and cytokines profiles of patients with novel coronavirus disease (COVID-19) and their correlation with the disease severity remain unclear. Methods: Peripheral blood samples were longitudinally collected from 40 confirmed COVID-19 patients and examined for lymphocyte subsets by flow cytometry and cytokine profiles by specific immunoassays. Findings: Of the 40 COVID-19 patients enrolled, 13 severe cases showed significant and sustained decreases in lymphocyte counts [0·6 (0·6-0·8)] but increases in neutrophil counts [4·7 (3·6-5·8)] than 27 mild cases [1.1 (0·8-1·4); 2·0 (1·5-2·9)].Further analysis demonstrated significant decreases in the counts of T cells, especially CD8 + T cells, as well as increases in IL-6, IL-10, IL-2 and IFN-γ levels in the peripheral blood in the severe cases compared to those in the mild cases. T cell counts and cytokine levels in severe COVID-19 patients who survived the disease gradually recovered at later time points to levels that were comparable to those of the mild cases.Moreover, the neutrophil-to-lymphocyte ratio (NLR) (AUC=0·93) and neutrophil-to-CD8 + T cell ratio (N8R) (AUC =0·94) were identified as powerful prognostic factors affecting the prognosis for severe COVID-19.Interpretation: The degree of lymphopenia and a proinflammatory cytokine storm is higher in severe COVID-19 patients than in mild cases, and is associated with the disease severity. N8R and NLR may serve as a useful prognostic factor for early 4 identification of severe COVID-19 cases.

Long-term antibody responses and neutralizing activities in response to SARS-CoV-2 infection are not yet clear. Here we quantify immunoglobulin M (IgM) and G (IgG) antibodies recognizing the SARS-CoV-2 receptor-binding domain (RBD) of the spike (S) or the nucleocapsid (N) protein, and neutralizing antibodies during a period of 6 months from COVID-19 disease onset in 349 symptomatic COVID-19 patients who were among the first be infected world-wide. The positivity rate and magnitude of IgM-S and IgG-N responses increase rapidly. High levels of IgM-S/N and IgG-S/N at 2-3 weeks after disease onset are associated with virus control and IgG-S titers correlate closely with the capacity to neutralize SARS-CoV-2. Although specific IgM-S/N become undetectable 12 weeks after disease onset in most patients, IgG-S/N titers have an intermediate contraction phase, but stabilize at relatively high levels over the 6 month observation period. At late time points, the positivity rates for binding and neutralizing SARS-CoV-2-specific antibodies are still >70%. These data indicate sustained humoral immunity in recovered patients who had symptomatic COVID-19, suggesting prolonged immunity.

SUMMARY MicroRNA-223 is known as a myeloid-enriched anti-inflammatory microRNA that is dysregulated in numerous inflammatory conditions. Here, we report that neutrophilic inflammation (wound response) is augmented in miR-223-deficient zebrafish, due primarily to elevated activation of the canonical nuclear factor κB (NF-κB) pathway. NF-κB over-activation is restricted to the basal layer of the surface epithelium, although miR-223 is detected throughout the epithelium and in phagocytes. Not only phagocytes but also epithelial cells are involved in miR-223-mediated regulation of neutrophils’ wound response and NF-κB activation. Cul1a/b, Traf6, and Tab1 are identified as direct targets of miR-223, and their levels rise in injured epithelium lacking miR-223. In addition, miR-223 is expressed in cultured human bronchial epithelial cells, where it also downregulates NF-κB signaling. Together, this direct connection between miR-223 and the canonical NF-κB pathway provides a mechanistic understanding of the multifaceted role of miR-223 and highlights the relevance of epithelial cells in dampening neutrophil activation.

Neutrophils are fast-moving cells essential for host immune functions. Although they primarily rely on glycolysis for ATP, isolated primary human neutrophils depend on mitochondrial membrane potential for chemotaxis. However, it is not known whether mitochondria regulate neutrophil motility in vivo, and the underlying molecular mechanisms remain obscure. Here, we visualized mitochondria in an interconnected network that localizes to the front and rear of migrating neutrophils using a novel transgenic zebrafish line. To disrupt mitochondrial function genetically, we established a gateway system harboring the CRISPR/Cas9 elements for tissue-specific knockout. In a transgenic line, neutrophil-specific disruption of mitochondrial DNA polymerase, polg, significantly reduced the velocity of neutrophil interstitial migration. In addition, inhibiting the mitochondrial electron transport chain or the enzymes that reduce mitochondrial reactive oxygen species also inhibited neutrophil motility. The reduced cell motility that resulted from neutrophil-specific knockout of sod1 was rescued with sod1 mRNA overexpression, or by treating with scavengers of reactive oxygen species. Together, our work has provided the first in vivo evidence that mitochondria regulate neutrophil motility, as well as tools for the functional characterization of mitochondria-related genes in neutrophils and insights into immune deficiency seen in patients with primary mitochondrial disorders.This article has an associated First Person interview with the first author of the paper.

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Long-term antibody responses and neutralizing activities following SARS-CoV-2 infections have not yet been elucidated. We quantified immunoglobulin M (IgM) and G (IgG) antibodies recognizing the SARS-CoV-2 receptor-binding domain (RBD) of the spike (S) or the nucleocapsid (N) protein, and neutralizing antibodies during a period of six months following COVID-19 disease onset in 349 symptomatic COVID-19 patients, which were among the first world-wide being infected. The positivity rate and magnitude of IgM-S and IgG-N responses increased rapidly. High levels of IgM-S/N and IgG-S/N at 2-3 weeks after disease onset were associated with virus control and IgG-S titers correlated closely with the capacity to neutralize SARS-CoV-2. While specific IgM-S/N became undetectable 12 weeks after disease onset in most patients, IgG-S/N titers showed an intermediate contraction phase, but stabilized at relatively high levels over the six months observation period. At late time points the positivity rates for binding and neutralizing SARS-CoV-2-specific antibodies was still over 70%. Taken together, our data indicate sustained humoral immunity in recovered patients who suffer from symptomatic COVID-19, suggesting prolonged immunity.

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