Toll-Like Receptor-Induced Immune Responses During Early Childhood and Their Associations With Clinical Outcomes Following Acute Illness Among Infants in Sub-Saharan Africa

Abstract

Severely ill children in low- and middle-income countries (LMICs) experience high rates of mortality from a broad range of infectious diseases, with the risk of infection-related death compounded by co-existing undernutrition. How undernutrition and acute illness impact immune responses in young children in LMICs remains understudied, and it is unclear what aspects of immunity are compromised in this highly vulnerable population. To address this knowledge gap, we profiled longitudinal whole blood cytokine responses to Toll-like receptor (TLR) ligands among severely ill children (n=63; 2-23 months old) with varied nutritional backgrounds, enrolled in the CHAIN Network cohort from Kampala, Uganda, and Kilifi, Kenya, and compared these responses to similar-aged well children in local communities (n=41). Cytokine responses to ligands for TLR-4 and TLR-7/8, as well as Staphylococcus enterotoxin B (SEB), demonstrated transient impairment in T cell function among acutely ill children, whereas innate cytokine responses were exaggerated during both acute illness and following clinical recovery. Nutritional status was associated with the magnitude of cytokine responses in all stimulated conditions. Among children who died following hospital discharge or required hospital re-admission, exaggerated production of interleukin-7 (IL-7) to all stimulation conditions, as well as leukopenia with reduced lymphocyte and monocyte counts, were observed. Overall, our findings demonstrate exaggerated innate immune responses to pathogen-associated molecules among acutely ill young children that persist during recovery. Heightened innate immune responses to TLR ligands may contribute to chronic systemic inflammation and dysregulated responses to subsequent infectious challenges. Further delineating mechanisms of innate immune dysregulation in this population should be prioritized to identify novel interventions that promote immune homeostasis and improve outcomes.

Keywords: adaptive immunity; innate immunity; lipopolysaccharide; malnutrition; pediatric; sepsis; toll-like receptor.

Figure 1

Children hospitalized for severe illness show reduced levels of innate cytokines and chemokines compared to children in the community that persist into convalescence. Whole blood cytokine and chemokine responses were assessed in hospitalized participants at admission to hospital (n=43, A), hospital discharge (n=60, B), and 180 days-post-discharge (n=51, C), and compared to community participants (CP) at enrollment (n=41) using logistic regression taking into account age, sex, recruitment site, and MUAC (at relevant time point). Odds ratios denote higher or lower responses in hospitalized cohort compared to CP cohort. (D) Whole blood cytokine and chemokine responses were compared to nutritional status, as measured by MUAC, in hospitalized participants at discharge (n=60) using linear regression taking into account age, sex, and recruitment site. Parameter estimate refers to the slope of the relationship between MUAC and cytokine or chemokine responses; negative and positive parameter estimates denote an indirect or direct relationship, respectively. For (A–D), filled circles indicate significance of p≤0.05; open circles indicate significance of p≤0.1 when adjusting for multiple comparisons. (E) To better understand the relationship between cytokine responses and nutritional status in hospitalized participants, a model was created using the cytokine signature (G-CSF, GM-CSF, MCP-3, IL-6, Mip-1-beta) that best discriminated between hospitalized (discharge time point) and CP cohorts. Here, the model adjusted for participant sex, age, and recruitment site, and the predictive strength of the model to reflect recent hospitalization (Y-axis) was plotted against participant MUJAC as measured at hospital discharge (X-axis).

Figure 2

Children hospitalized for severe illness show impaired Th-1 responses and increased innate responses to LPS when compared to children in the community that persist into convalescence. LPS-stimulated whole blood cytokine and chemokine responses were assessed in hospitalized participants at admission to hospital (n=43, A), hospital discharge (n=60, B), and 180 days-post-discharge (n=51, C), and compared to community participants (CP) at enrollment (n=41) using logistic regression taking into account age, sex, recruitment site, and MUAC (at relevant time point). Odds ratios denote higher or lower responses in hospitalized cohort compared to CP cohort. (D) Whole blood cytokine and chemokine responses were compared to nutritional status, as measured by MUAC, in hospitalized participants at discharge (n=60) using linear regression taking into account age, sex, and recruitment site. Parameter estimate refers to the slope of the relationship between MUAC and cytokine or chemokine responses; negative and positive parameter estimates denote an indirect or direct relationship, respectively. For (A–D), filled circles indicate significance of p≤0.05; open circles indicate significance of p≤0.1 when adjusting for multiple comparisons. (E) To better understand the relationship between cytokine responses and nutritional status in hospitalized participants, a model was created using the cytokine signature (G-CSF, IFNg, Mip-1-beta, IL-6, IL-8) that best discriminated between hospitalized (discharge time point) and CP cohorts. Here, the model adjusted for participant sex, age, and recruitment site, and the predictive strength of the model to reflect recent hospitalization (Y-axis) was plotted against participant MUAC as measured at hospital discharge (X-axis).

Figure 3

Children hospitalized for severe illness show impaired Th-1 and innate responses to TLR-7/8 stimulation when compared to children in the community that persist into convalescence. CL075-stimulated whole blood cytokine and chemokine responses were assessed in hospitalized participants at admission to hospital (n=43, A), hospital discharge (n=60, B), and 180 days-post-discharge (n=51, C), and compared to community participants (CP) at enrollment (n=41) using logistic regression taking into account age, sex, recruitment site, and MUAC (at relevant time point). Odds ratios denote higher or lower responses in hospitalized cohort compared to CP cohort. (D) Whole blood cytokine and chemokine responses were compared to nutritional status, as measured by MUAC, in hospitalized participants at discharge (n=60) using linear regression taking into account age, sex, and recruitment site. Parameter estimate refers to the slope of the relationship between MUAC and cytokine or chemokine responses; negative and positive parameter estimates denote an indirect or direct relationship, respectively. For (A–D), filled circles indicate significance of p≤0.05; open circles indicate significance of p≤0.1 when adjusting for multiple comparisons. (E) To better understand the relationship between cytokine responses and nutritional status in hospitalized participants, a model was created using IFNg, a cytokine that best discriminated between hospitalized (discharge time point) and CP cohorts. Here, the model adjusted for participant sex, age, and recruitment site, and the predictive strength of the model to reflect recent hospitalization (Y-axis) was plotted against participant MUAC as measured at hospital discharge (X-axis).

Figure 4

Children hospitalized for severe illness exhibit an impaired T cell response to polyclonal stimulation when compared to children in the community that improves during convalescence. SEB-stimulated whole blood cytokine and chemokine responses were assessed in hospitalized participants at admission to hospital (n=43, A), hospital discharge (n=60, B), and 180 days-post-discharge (n=51, C), and compared to community participants (CP) at enrollment (n=41) using logistic regression taking into account age, sex, recruitment site, and MUAC. Odds ratios denote higher or lower responses in hospitalized cohort compared to CP cohort. (D) Whole blood cytokine and chemokine responses were compared to nutritional status, as measured by MUAC, in hospitalized participants at discharge (n=60) using linear regression taking into account age, sex, and recruitment site. Parameter estimate refers to the slope of the relationship between MUAC and cytokine or chemokine responses; negative and positive parameter estimates denote an indirect or direct relationship, respectively. For (A–D), filled circles indicate significance of p≤0.05; open circles indicate significance of p≤0.1 when adjusting for multiple comparisons. (E) To better understand the relationship between cytokine responses and nutritional status in hospitalized participants, a model was created using IFNg, a cytokine that best discriminated between hospitalized (discharge time point) and CP cohorts. Here, the model adjusted for participant sex, age, and recruitment site, and the predictive strength of the model to reflect recent hospitalization (Y-axis) was plotted against participant MUAC as measured at hospital discharge (X-axis).

Figure 5

Total lymphocyte, leukocyte, and monocyte counts are associated with post-discharge adverse events in children hospitalized for severe illness. Complete blood counts with differential were performed at hospital discharge and compared between participants with (n=20) and without (n=41) post-discharge events of death or re-admission to hospital. Shown are median values with interquartile range for white blood cell counts (A), absolute lymphocyte counts (B), absolute monocyte counts (C), and absolute neutrophil counts (D), displayed as cell number x10 ³/μl of blood. P-values were calculated using Mann-Whitney test. Filled circles and open circles indicate participants from Kilifi, Kenya and Kampala, Uganda sites, respectively.

Figure 6

IL-7 responses to multiple stimuli are altered among hospitalized children who experience post-discharge adverse events. Whole blood cytokine and chemokine responses at hospital discharge were compared between participants who experienced a post-hospital-discharge event of death or re-admission (n=22) and those who completed 6-months of follow-up with no event (n=41). Shown are odds ratios of experiencing post-discharge event in unstimulated (A) and PAMP-stimulated (B–D) whole blood, using logistic regression taking into account age, sex, recruitment site, and MUAC. Filled circles indicate significance of p≤0.05; open circles indicate significance of p≤0.1 when adjusting for multiple comparisons.