EBV Impact in Peripheral Macrophages' Polarization Cytokines in Pediatric Patients

Abstract

Macrophages are exceptionally flexible cells. The presence of inflammatory cytokines such as IFN-γ and TNF-α results in an M1 (CD68) activation, while cytokines such as IL-10 or TGF-β induce the M2 (CD163) activation. Our aim was to study the behavior of peripheral cytokines involved in macrophage polarization and relate them with tissue findings to further comprehend the role of macrophages in EBV pediatric infection. We studied cytokine expression in tonsils and peripheral blood samples of children in different stages of infection. Peripheral cytokines were compared with macrophage polarization markers and viral protein expression in tonsils. Only IL-10 showed a negative correlation between compartments, exclusively in patients undergoing viral reactivation (R). Higher expressions of peripheral IL-1β, IL-23, and IL-12p40 in R children were observed. Lower expressions of local and peripheral TNF-α in patients with broader expressions of latent and lytic viral proteins were demonstrated. In healthy carrier (HC) patients, IL-23 positively correlated with CD163, and IP-10 positively correlated with CD68. Our results indicated that EBV might modulate antigen expression in the presence of TNF-α and influence peripheral cytokine expression differently in each stage of infection. Moreover, peripheral cytokines might have a particular role in macrophage polarization in HC.

Keywords: EBV; children; cytokines; macrophages; periphery; tonsil.

Figure 1

(A) Correlation matrix of peripheral cytokines. Numbers inside the box represent the Spearman correlation coefficient (r). The color within each box indicates the value of r, the red color indicates values between 0 and 1 (positive correlation), and the blue color indicates values between 0 and −1 (negative correlation), while the intensity of the color increases as r increases towards values close to |1|. (B) Heat map of peripheral cytokine expression (color scale) arranged by infectious status (IS); polarization markers (CD68 and CD163) and polarization profiles (M1 and M2) data are shown at the top. Both graphs show two clusters of cytokines with distinct behaviors. One cluster is compounded by IL-12p40, IL-12p70, IL-23, and, unexpectedly, IL-1RA. On the other hand, the second cluster can be further subdivided into two groups: IL-10 and IL-1β, and IL-6, TNF-α, CCL17 (TARC), and IP-10.

Figure 2

(A) Correlation between tissue and peripheral IL-10 expression in reactivated patients. (B) Histological distribution of CCL17. Total expression in the germinal center (GC) and interfollicular region (IF) in the entire cohort in primary infected patients (PI), healthy carriers (HC), and patients undergoing reactivation (R). The cross inside the box represent the mean, the line crossing the box represent the median, and error bars indicate the standard deviation. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

Peripheral cytokine expression among infectious statuses. (A) TNF-α. (B) IP-10. (C) IL-10. (D) IL-12p70. (E) IL-6. (F) TARC. (G) IL-1RA. (H) IL-23. (I) IL-1β. (J) IL-12p40. The cross inside the box represents the mean, the line crossing the box represents the median, and error bars indicate the standard deviation; * p < 0.05.

Figure 4

(A) Peripheral cytokine expressions in patients expressing Latency 0 and I antigens (L0/I) and Latency (II/III) antigens (LII/III). (B) Comparison of peripheral cytokine expressions between patients who were BMRF1+ and BMRF1−. The cross inside the box represents the mean, the line crossing the box represents the median, and error bars indicate the standard deviation. * p < 0.05.

Figure 5

Peripheral cytokine expression in patients with the prevalence of M1 or M2 polarization profiles in tonsils. The cross inside the box represents the mean, the line crossing the box represents the median, and error bars indicate the standard deviation. * p < 0.05.