Meningitis Caused by Toscana Virus Is Associated with Strong Antiviral Response in the CNS and Altered Frequency of Blood Antigen-Presenting Cells

Abstract

Toscana virus (TOSV) is a Phlebotomus-transmitted RNA virus and a frequent cause of human meningitis and meningoencephalitis in Southern Europe during the summer season. While evidence for TOSV-related central nervous system (CNS) cases is increasing, little is known about the host defenses against TOSV. We evaluated innate immune response to TOSV by analyzing frequency and activation of blood antigen-presenting cells (APCs) and cytokine levels in plasma and cerebrospinal fluid (CSF) from patients with TOSV neuroinvasive infection and controls. An altered frequency of different blood APC subsets was observed in TOSV-infected patients, with signs of monocytic deactivation. Nevertheless, a proper or even increased responsiveness of toll-like receptor 3 and 7/8 was observed in blood APCs of these patients as compared to healthy controls. Systemic levels of cytokines remained low in TOSV-infected patients, while levels of anti-inflammatory and antiviral mediators were significantly higher in CSF from TOSV-infected patients as compared to patients with other infectious and noninfectious neurological diseases. Thus, the early host response to TOSV appears effective for viral clearance, by proper response to TLR3 and TLR7/8 agonists in peripheral blood and by a strong and selective antiviral and anti-inflammatory response in the CNS.

Keywords: Toscana virus; cytokines; dendritic cells; monocytes; viral meningitis.

Figure 1

Changes in blood monocytes during TOSV infection. Peripheral blood mononuclear cells (PBMCs) from the study participants were stained with monoclonal antibodies for subsequent flow cytometric analyses. Monocytes (CD14⁺) were gated as described in Materials and Methods. Cell frequencies (A,B) and human leukocyte antigen (HLA)-DR expression levels (C,D) of the circulating monocytes are reported. N = 26 TOSV+ patients and n = 49 healthy controls were evaluated for total monocytes, while n = 9 TOSV+ patients and n = 12 healthy donors were analyzed for monocytic subsets. White squares indicate healthy controls and black triangles indicate TOSV-infected patients. MFI, mean fluorescence intensity. Data were analyzed using the non-parametric Mann–Whitney U test. * indicates p ≤ 0.05, ** indicates p ≤ 0.01 and *** indicates p ≤ 0.001.

Figure 2

Cell frequencies and activation markers of circulating DCs (BDCA-1⁺, BDCA-2⁺, and BDCA-3⁺). Peripheral blood mononuclear cells (PBMCs) from the study participants were stained with monoclonal antibodies for subsequent flow cytometric analyses. mDCs (BDCA-1⁺ and BDCA-3⁺ DCs) as well as pDCs (BDCA-2⁺ DCs) were gated as described in Materials and Methods. Lower frequencies of circulating DCs were observed in TOSV-infected patients (A) with no difference in the expression levels of human leukocyte antigen (HLA) class II molecules compared to healthy controls (B). White squares indicate healthy controls and black triangles indicate TOSV-infected patients. N = 21 TOSV+ patients and n = 26 healthy controls were analyzed. MFI, mean fluorescence intensity. Data were analyzed using the non-parametric Mann–Whitney U test. ** indicates p ≤ 0.01 and *** indicates p ≤ 0.001.

Figure 3

TLR-induced cytokine responses in blood cells from TOSV-infected patients and healthy controls. Blood cells were left unstimulated or stimulated with poly(A:U) (TLR3 ligand; 20 µg/mL) or Resiquimod-R848 (TLR7/8 ligand; 10 µg/mL). After 24 h, supernatants were collected and analyzed for cytokine levels. White squares indicate healthy controls and black triangles indicate TOSV-infected patients. N = 8 TOSV-infected patients and n = 7 healthy controls were evaluated. Data were analyzed using the non-parametric Mann–Whitney U test. * indicates p ≤ 0.05.

Figure 4

Levels of pro-inflammatory and anti-inflammatory cytokines in plasma and CSF from TOSV-infected patients and controls. The levels of IFN-α, IFN-γ, IL-6, IL-10, and TNF-α (A–E) were measured in the plasma samples from TOSV-infected patients (TOSV+) and from healthy controls (HC) as described in Materials and Methods. Cytokine levels were also measured in the CSF samples from TOSV-infected patients (TOSV+) and from patients with enterovirus meningitis (ENTERO+) and with noninfectious diseases of the CNS (noninfectious controls, NIC). White squares designate healthy controls (n = 15), white circles indicate noninfectious controls (n = 30), black circles designate enterovirus meningitis (n = 7) and black triangles indicate TOSV-infected patients (n = 17, 11 patients with meningitis and six patients with meningoencephalitis). Data were analyzed using the non-parametric Mann–Whitney U test. * indicates p ≤ 0.05, ** indicates p ≤ 0.01 and *** indicates p ≤ 0.001.