Evaluation of Activation and Inflammatory Activity of Myeloid Cells During Pathogenic Human Coronavirus Infection

Abstract

Innate immune cells play a vital role in mounting an effective host response to a variety of pathogen challenges. Myeloid cells such as neutrophils and monocyte-macrophages are major innate leukocytes that orchestrate protective immunity to viral lung infections. However, a dysregulated cytokine response can promote excessive infiltration and robust pro-inflammatory activity of neutrophils and monocyte-macrophages, leading to fatal disease. Following virus infection, the beneficial or deleterious role of infiltrating neutrophils and monocyte-macrophages is determined largely by their ability to secrete inflammatory cytokines and chemokines. A majority of studies use the total number of infiltrating cells and their activation status as measures to demonstrate their role during an infection. Consequently, the ability of neutrophils and Inflammatory Monocyte Macrophages (IMMs) to secrete inflammatory cytokines and chemokines, and its correlation with the disease severity, is not well defined. In this chapter, we report useful markers to identify lung infiltrating innate immune cells and define their activation status. We also describe a simple method to measure intracellular cytokine production to evaluate the inflammatory activity of neutrophils and IMMs in a mouse model of human coronavirus infection.

Keywords: Coronavirus; Cytokines and chemokines; Inflammatory monocyte-macrophages; Lungs; Neutrophils.

Fig. 1

Ideal markers to determine activation status of IMMs. Lung cells harvested from SARS-CoV-infected BALB/c mice (3 dpi) were surface stained for IMMs and activation markers as described in the methods section

Fig. 2

Spontaneous cytokine production by IMMs. Lung cells isolated from SARS-CoV-infected BALB/c mice (1–3 dpi) were incubated for 7-h in the presence of Golgi-plug. Cells were then surface stained for IMMs (CD45⁺CD11b⁺Ly6C^hi) and then for intracellular cytokines TNF, IL-6, IL-1β, and iNOS

Fig. 3

Staining for intracellular cytokines in TLR-stimulated IMMs and neutrophils: Total lung cells isolated from SARS-CoV-infected BALB/c mice (3 dpi) were stimulated with LPS (TLR4 ligand, 100 ng/mL) or R848 (TLR7 ligand, 1 μg/mL) for 4—h in the presence of Golgi-plug. IMMs (CD11b^hiLy6c^hi) and neutrophils (CD11b^hiLy6C^int) were stained for intracellular TNF, IL-1β, and iNOS production