Reduced T-Helper 17 Responses to Streptococcus pneumoniae in Infection-Prone Children Can Be Rescued by Addition of Innate Cytokines

Abstract

Background: T-helper (Th) 17 cells are important in the control of Streptococcus pneumoniae. We sought to understand the mechanism of failure of Th17 immunity resulting in S. pneumoniae infections in children <2 years old.

Methods: Peripheral blood mononuclear cells (PBMCs) from infection-prone (IP) and non-IP (NIP) children 9-18 months old were examined for their responses to heat-killed S. Pneumoniae, using flow cytometry, reverse-transcription polymerase chain reaction, and enzyme-linked immunoassay. We measured cytokine production, proliferation, and differentiation of Th17 cells and the expression of transcription factors in response to S. pneumoniae.

Results: PBMCs of IP children stimulated with heat-killed S. pneumoniae had significantly reduced percentages of CD4+ Th1 (interleukin2, tumor necrosis factor α) and Th17 (interleukin 17A) cells compared with NIP children. Addition of exogenous Th17-promoting cytokines (interleukin 6, 1β, and 23 and transforming growth factor β) restored CD4+ Th17 cell function in cells from IP children to levels measured in NIP children.

Conclusions: Reduced Th17 responses to S. pneumoniae in PBMCs of IP children can be rescued by addition of Th17-promoting cytokines.

Keywords: Pneumococcus; Th17; innate cytokines; young children..

Figure 1.

Frequencies of CD4⁺ T-cell responses to heat-killed (HK) Streptococcus pneumoniae among infection-prone (IP; n = 20) and non-IP (NIP; n = 20) children. A, Expression of interferon (IFN) γ, interleukin 2 (IL-2), tumor necrosis factor (TNF) α, and interleukin 17A (IL-17A). NS, not significant. B, IFN-γ⁺IL-2⁺TNF-α⁺ (depicted as G⁺2⁺T⁺); IFN-γ⁺IL-2⁻TNF-α⁺ (G⁺2⁻T⁺), and IFN-γ⁻IL-2⁺TNF-α⁺ (G⁻2⁺T⁺) CD4⁺ T cells. C, IFN-γ⁻IL-2⁺TNF-α⁻ (G⁻2⁺T⁻) and IFN-γ⁻IL-2⁻TNF-α⁺ (G⁻2⁻T⁺) CD4⁺ T cells. The percentage frequency of activated T cells in an individual child was obtained after subtracting the responses without any stimulation. P values were calculated using the Mann–Whitney U test. Adjusted P values for multiple comparisons using the Benjamini-Hochberg procedure remained significant, with a false discovery rate of <0.05.

Figure 2.

Differential memory CD4⁺ T-cell responses among infection-prone (IP; n = 20) and non-IP (NIP; n = 20) children. A, Gating strategy for enumerating memory (CD45RA⁻) and naive (CD45RA⁺) CD4⁺ T-cell responses among IP and NIP children. IFN, interferon; IL-2, interleukin 2; IL-17A, interleukin 17A; TNF, tumor necrosis factor. B, C, Intracellular staining assay was used as described in Methods to identify percentage frequencies of memory CD4⁺ T cells (CD69⁺CD45RA⁻) (8) and naive CD4⁺ T cells (CD69⁺CD45RA⁺) (8) producing various cytokines in response to heat-killed (HK) Streptococcus pneumoniae; percentage frequencies of unstimulated responses were subtracted, and results expressed as a percentage of total CD4⁺ T cells. P values were calculated using the Mann–Whitney U test. Differences remained significant with adjusted P values for multiple comparisons using the Benjamini-Hochberg procedure, with a false discovery rate of <0.05. Abbreviation: NS, not significant.

Figure 3.

Comparison of cytokine secretion among infection-prone (IP; n = 20) and non-IP (NIP; n = 20) children. Peripheral blood mononuclear cells were cultured with heat-killed Streptococcus pneumoniae for 72 hours, and cytokine concentrations in culture supernatant were assessed using Luminex technology. Bar graphs show mean (standard error of the mean) cytokine concentrations after subtraction of values from unstimulated samples. P values were calculated using the Mann–Whitney U test, and bar graphs show means with standard errors of the mean. Differences remained significant with adjusted P value for multiple comparisons using the Benjamini-Hochberg procedure, with a false discovery rate of <0.05, except for interleukin 23 (P = .10). Abbreviations: IFN, interferon; IL-2, IL-4, IL-6, IL-10, IL-13, IL-17A, IL-21, and IL-23, interleukin 2, 4, 6, 10, 13, 17A, 21, and 23; NS, not significant; TNF, tumor necrosis factor.

Figure 4.

Flow cytometry analysis of in vitro T-helper (Th) 17 responses to heat-killed (HK) Streptococcus pneumoniae for 5 days. A, Representative bar graphs are shown for both infection-prone (IP; n = 20, upper panel) and non-IP (NIP; n = 20, lower panel) children producing Th17 (interleukin 17A [IL-17A]). Peripheral blood mononuclear cells (PBMCs) were stimulated with HK S. pneumoniae for 5 days, and on day 6 intracellular cytokine staining were performed after stimulation with phorbol myristate acetate (PMA) and ionomycin, and Th17 responses were expressed as expressed as a percentage of total CD4⁺ T cells. B, Compared with NIP children, IP children produced significantly lesser IL-17A cytokine responses to HK S. pneumoniae, expressed as a percentage of total CD4⁺ T cells. C, PBMCs labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) were stimulated with HK S. pneumoniae alone or with pro-Th17 cell cytokines (interleukin 6, 1β , and 23 and transforming growth factor β) for 5 days, and on day 6 intracellular cytokine staining was performed after stimulation with PMA and ionomycin, and the cell proliferation was measured. The plots show expression of HK S. pneumoniae–induced CD4⁺ T-cell proliferation in IL-17A⁺ cells in IP (upper panel) compared with NIP (lower panel). D, Percentage frequency of CFSE_low IL-17A⁺ CD4⁺ cells in response to HK S. pneumoniae stimulation alone or with pro-Th17 cell cytokines (HK S. pneumoniae–Th17) among IP (n = 10) and NIP (n = 10) children for 5 days. IP children showed significant lower IL-17A⁺ CFSE_low proliferation responses to HK S. pneumoniae stimulation than did NIP children. P values were calculated using the Mann–Whitney U test, and bar graphs show means with standard errors of the mean. Differences remained significant with adjusted P values for multiple comparisons using the Benjamini-Hochberg procedure, with a false discovery rate of <0.05.

Figure 5.

Flow cytometry analysis for the levels of phosphorylated signal transducer and activator of transcription 3 (pSTAT3) in CD4⁺ T cells among samples from infection-prone (IP; n = 10) and non-IP (NIP; n = 10) children. Before analysis, the cells were briefly (for 15 minutes) stimulated with heat-killed (HK) Streptococcus pneumoniae, alone or along with pro–T helper (Th) 17 cell cytokines (HK S. pneumoniae–Th17) and stained for phosphorylated STAT3. A, Representative plot showing mean fluorescence intensity (MFI) for the phosphorylation of STAT3 (pY705) (gated on STAT3⁺ CD4⁺ T cells) among IP (upper panel) and NIP (lower panel) children. B, The average MFI of pSTAT3 in CD4⁺ T cells from IP children showed a significant increase after HK S. pneumoniae stimulation along with pro-Th17 cell cytokines stimulation. P values were calculated using Mann–Whitney U tests, and bar graphs show means with standard errors of the mean. Differences remained significant with adjusted P values for multiple comparisons using the Benjamini-Hochberg procedure, with a false discovery rate of <0.05. Abbreviation: NS, not significant.

Figure 6.

Quantitative polymerase chain reaction analysis of reference genes for T-helper (Th) 1 and Th17 responses among infection-prone (IP) and non-IP (NIP) children. Fold change in the expression of genes for innate cytokines (interleukin 6, 1β, 23, 17A, [IL-6, IL-1β, IL-23, and IL-17A], and transforming growth factor [TGF-β]), adaptive cytokine genes (interferon [IFN] γ and interleukin 2, 4, 13, and 10 [IL-2, IL-4, IL-13, and IL-10) and transcriptions master regulatory molecules (TBX21, GATA3, RORC, and Foxp3) among IP (n = 6) and NIP (n = 6) children. Fold change is relative to the level of each gene expressed in peripheral blood mononuclear cells (PBMCs) stimulated with heat-killed Streptococcus pneumoniae versus unstimulated PBMCs. P values were calculated using the Mann–Whitney U test, and bar graphs show means with standard errors of the mean. Differences remained significant with adjusted P values for multiple comparisons using the Benjamini-Hochberg procedure, with a false discovery rate of <0.05, except for GATA-3 (P = .07). Abbreviations: TBX21, T-box transcription factor 21; GATA3, GATA binding protein 3; RORC, RAR-related orphan receptor C; Foxp3; forkhead box protein 3.