Evaluation of Host Constitutive and Ex Vivo Coccidioidal Antigen-Stimulated Immune Response in Dogs with Naturally Acquired Coccidioidomycosis

Abstract

The early innate immune response to coccidioidomycosis has proven to be pivotal in directing the adaptive immune response and disease outcome in mice and humans but is unexplored in dogs. The objectives of this study were to evaluate the innate immune profile of dogs with coccidioidomycosis and determine if differences exist based on the extent of infection (i.e., pulmonary or disseminated). A total of 28 dogs with coccidioidomycosis (pulmonary, n = 16; disseminated, n = 12) and 10 seronegative healthy controls were enrolled. Immunologic testing was performed immediately, without ex vivo incubation (i.e., constitutive), and after coccidioidal antigen stimulation of whole blood cultures. Whole blood cultures were incubated with a phosphate-buffered solution (PBS) (negative control) or a coccidioidal antigen (rCTS1 (105–310); 10 µg/mL) for 24 h. A validated canine-specific multiplex bead-based assay was used to measure 12 cytokines in plasma and cell culture supernatant. Serum C-reactive protein (CRP) was measured with an ELISA assay. Leukocyte expression of toll-like receptors (TLRs)2 and TLR4 was measured using flow cytometry. Dogs with coccidioidomycosis had higher constitutive plasma keratinocyte chemotactic (KC)-like concentrations (p = 0.02) and serum CRP concentrations compared to controls (p < 0.001). Moreover, dogs with pulmonary coccidioidomycosis had higher serum CRP concentrations than those with dissemination (p = 0.001). Peripheral blood leukocytes from dogs with coccidioidomycosis produced higher concentrations of tumor necrosis factor (TNF)-α (p = 0.0003), interleukin (IL)-6 (p = 0.04), interferon (IFN)-γ (p = 0.03), monocyte chemoattractant protein (MCP)-1 (p = 0.02), IL-10 (p = 0.02), and lower IL-8 (p = 0.003) in supernatants following coccidioidal antigen stimulation when compared to those from control dogs. There was no detectable difference between dogs with pulmonary and disseminated disease. No differences in constitutive or stimulated leukocyte TLR2 and TLR4 expression were found. These results provide information about the constitutive and coccidioidal antigen-specific stimulated immune profile in dogs with naturally acquired coccidioidomycosis.