Altered Treg and cytokine responses in RSV-infected infants

Abstract

Background: Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in children under 1 y of age in the USA. The host immune response is believed to contribute to RSV-induced disease. We hypothesize that severe RSV infection in infants is mediated by insufficient regulation of the host immune response of regulatory T cells (Tregs) resulting in immunopathology.

Methods: Blood and nasal aspirates from 23 RSV-infected and 17 control infants under 1 y of age were collected. Treg frequencies were determined by flow cytometry from peripheral blood mononuclear cells. Analysis of 24 cytokines was measured by multiplex assay on nasal aspirates.

Results: We demonstrate that the frequency of activated Tregs is significantly reduced in the peripheral blood of RSV-infected infants compared with age-matched controls. Surprisingly, T helper (Th)17 related cytokines including interleukin (IL)-1β, IL-17A, and IL-23 were associated with a reduction in clinical symptoms of respiratory distress. In addition, the amount of IL-33 protein in nasal washes, a cytokine important in maintaining Treg homeostasis in mucosal tissues, was decreased in RSV-infected children.

Conclusion: These results suggest that decreased Treg numbers and an inability to properly control the host inflammatory response results in severe RSV infection.

Figure 1.. aTreg frequencies are reduced in the peripheral blood of RSV-infected infants.

PBMCs from RSV-infected infants and controls were stained to identify subsets of conventional and regulatory CD4 T cells. (A) Representative plots depict CD45RA^hiFoxp3^int/CD25^int resting Tregs (rTreg, I), CD45RA^loFoxp3^hi/CD25^hi activated Tregs (aTreg, II), CD45RA^hiFoxp3^- naïve conventional CD4 T cells (III), and CD45RA^loFoxp3^- memory conventional CD4 T cells (IV) from control and RSV-infected infants. The cumulative frequencies of resting (B) and activated (C) Tregs are shown for controls and RSV-infected infants by two staining methods. (D) Naïve and (E) memory conventional CD4 T cell frequencies are shown from all groups. (F) Ki-67 expression from each CD4 T cell subset defined by CD45RA and Foxp3 expression is shown for both control (white bars) and RSV-infected infants (black bars). All samples are gated on CD4⁺CD3⁺ T cells. Data represents the mean ± SEM for both controls (n = 13) and RSV (n = 17) groups. **, p < 0.01.

Figure 2.. rTreg frequencies decrease as RSV disease progresses.

The coefficients of determination between CD45RA^hiFoxp3^int rTregs (A, R² = 0.3569) or CD45RA^loFoxp3^hi aTregs (B, R² = 0.0089) and onset of symptoms in RSV-infected infants were calculated along with the p-values measuring significance of association (A, p = 0.0113; B, p = 0.7187).

Figure 3.. Th17-associated cytokines correlate with reduced difficulty breathing and retraction.

Physical exam findings of difficulty breathing (left) and retraction (right) are associated with Th17-associated but not Th1- or Th2-associated cytokine concentrations in nasal excretions from RSV-infected infants. Data represent the mean ± SEM for both controls (n = 13) and RSV (n = 17) groups. *, p < 0.05. **, p < 0.01.