Early IFNβ secretion determines variable downstream IL-12p70 responses upon TLR4 activation

Abstract

The interleukin-12 (IL-12) family comprises the only heterodimeric cytokines mediating diverse functional effects. We previously reported a striking bimodal IL-12p70 response to lipopolysaccharide (LPS) stimulation in healthy donors. Herein, we demonstrate that interferon β (IFNβ) is a major upstream determinant of IL-12p70 production, which is also associated with numbers and activation of circulating monocytes. Integrative modeling of proteomic, genetic, epigenomic, and cellular data confirms IFNβ as key for LPS-induced IL-12p70 and allowed us to compare the relative effects of each of these parameters on variable cytokine responses. Clinical relevance of our findings is supported by reduced IFNβ-IL-12p70 responses in patients hospitalized with acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or chronically infected with hepatitis C (HCV). Importantly, these responses are resolved after viral clearance. Our systems immunology approach defines a better understanding of IL-12p70 and IFNβ in healthy and infected persons, providing insights into how common genetic and epigenetic variation may impact immune responses to bacterial infection.

Keywords: CP: Immunology; Cytokine variability; IL-12p70; TLR4 immune responses; systems immunology; type I interferons.

Graphical abstract

Figure 1

IL-12p70 variability in healthy donors (A–E) IL-12p70 (A) measured by Luminex in healthy donors (n = 1,000) after stimulation of whole blood with null, LPS, or poly (I:C). IL-12p40 (B) and IL-23 (C) proteins measured by Luminex in healthy donors (n = 1,000) after null and LPS stimulation. IL-12p70 secretion measured by Simoa after null and LPS stimulation at two separate time points (visit 1 n = 1,000, D; visit 2 n = 500, E). (F) Correlation of IL-12p70 secretion after LPS 22 h stimulation at two separate time points. Pearson correlation was performed on log10 values. (G) IL-12p70 secretion measured by Simoa in the two groups of Milieu Interieur IL-12p70 responders after null and E. coli stimulation. Red lines indicate the median value for each group. p values were determined by an unpaired Student’s t test using log10 values. (H) Correlation of IL-12p70 secretion measured by Simoa after E. coli and LPS 22 h stimulation. Pearson correlation was performed using log10 values. Empty symbols represent IL-12p70 low responders as defined by LPS 22 h whole-blood stimulation (A); full symbols represent IL-12p70 responders.

Figure 2

CD14⁺ monocytes and dendritic cells secrete IL-12p70 after LPS stimulation (A) Representative dot plots of IL-12p70 and IL-23 secretion upon null and LPS 22 h stimulation measured by flow cytometry in CD14⁺ monocytes, cDC2, and pDCs. (B–D) Tukey box-whisker plots of the percentage of IL-12p70⁺ cells (B), IL-23⁺ cells (C), or IL-12p70⁺ and IL-23⁺ cells (n = 10; D) for the indicated immune cell populations in null and LPS conditions. Q values were determined by the Wilcoxon test and false discovery rate corrected for multiple comparison testing. (E) Numbers of CD14^hi monocytes, cDC2, and pDCs for the responder and low-responder groups (n = 1,000). (F) Expression levels of HLA-DR in CD14⁺ monocytes and cDC2 populations and CD86 in CD14⁺ monocytes for the two groups. Red lines indicate the median value for each group. Q values were determined by an unpaired Student’s t test (on log10 values) and false discovery rate corrected for 79 cell numbers (E) and 88 cell surface protein phenotypes (F) in multiple comparison testing (n = 1,000).

Figure 3

LPS-specific cis-acting pQTLs on IL-12p70 production (A) Local pQTLs located near the IL12A gene acting specifically on IL-12p70 protein secretion measured by Simoa in the 1,000 Milieu Interieur donors in response to 22 h LPS stimulation. (B) IL-12p40 and IL-23 proteins measured by Luminex after 22 h LPS stimulation according to the rs143060887 allelic dosage. (C) Dot plots show rs143060887 genotype-stratified gene expression levels for the IL12A, IL12B, and IL23A genes measured by Nanostring after 22 h LPS stimulation. Empty circles represent IL-12p70 low responders of the Milieu Interieur cohort. Red lines indicate the median value for each group. Q values were determined by an unpaired Student’s t test using log10 values, and false discovery rate correction was performed to correct for multiple comparison testing.

Figure 4

Kinetic responses and interactions of type I and II interferons and IL-12p70 (A) Volcano plots displaying differentially expressed genes between Milieu Interieur IL-12p70 low responders and responders upon 22 h null and LPS stimulation. Negative log2 fold change represents genes that are more expressed in low responders, whereas positive log2 fold change represents genes that are more expressed in responders. The cutoffs used to display gene names are Q value < 0.01, log2 fold change > 0.05 in the null stimulation condition, and log2 fold change > 0.2 in the LPS stimulation condition. (B) Type I (IFNα and IFNβ) and II (IFNγ) interferons and IL-12p70 protein secretion over time after LPS stimulation for one healthy donor. (C) Table reporting FDR Q values of time series analysis on protein secretion. (D) Pearson correlation matrix between cytokine responses. (E) Type I IFNs measured by Simoa after 4 h LPS stimulation of whole blood for the low responders and responders. The red lines indicate the median value for each group and stimulation condition. p values were determined by the unpaired Student’s t test; n = 100 donors. (F) Correlation between IFNα or IFNβ secretion after 4 h of LPS stimulation and IL-12p70 secretion upon 22 h LPS stimulation. Pearson correlation tests were performed on log10 values; n = 100 donors. Empty circles represent IL-12p70 low responders of the Milieu Interieur cohort.

Figure 5

Impact of LPS, type I IFNs, and IFNAR inhibitors on IL-12p70 production (A) IL-12p70 secretion measured by Simoa in whole blood after 22 h stimulation with LPS, type I IFNs, the combination of LPS and type I IFNs (n = 9), and an unstimulated control (null). The red lines indicate the median value for each stimulation condition, Q values were determined using the Wilcoxon test, and false discovery rate correction was performed to correct for multiple comparison testing. (B) IL-12p70 secretion measured by Simoa in whole blood after 22 h stimulation with LPS, LPS with IFNAR neutralizing antibodies. The red lines indicate the median value for each stimulation condition, Q values were determined using the Wilcoxon test, and false discovery rate correction was performed to correct for multiple comparison testing. (C) Representative histograms showing intracellular IFNβ production measured by flow cytometry in T cells, CD14⁺ monocytes, B cells, neutrophils, NK bright cells, NK cells, cDC2, and pDC populations after 6 h of null or LPS whole-blood stimulation. (D) Tukey box-whisker plots of the percentage of IFNβ cytokine-positive cells for the indicated immune cell populations in null and LPS conditions; n = 6. (E) Intracellular IFNβ (LPS/null ratio) measured by Simoa after 3 h null and LPS whole-blood stimulation, cell sorting, and lysis of purified cells; n = 4 with a minimum of 100,000 purified cells per population. (F) cis-acting variants significantly associated with LPS-induced IFNβ protein secretion measured in the 1,000 Milieu Interieur donors after 22 h LPS stimulation.

Figure 6

Differential methylation probes between low and high responders and integrative modeling of IFNβ and IL-12p70 variability (A) List of IKBKB and IRF5 gene methylation probes significantly different between the Milieu Interieur low responders and responders. (B) Correlation of one IKBKB and IRF5 probe methylation values with their respective gene expression measured by Nanostring after null and LPS 22 h stimulations. Pearson correlations were performed using gene expression log10 values. Empty circles represent IL-12p70 low responders of the Milieu Interieur cohort. (C) IFNβ and IL-12p70 Boruta variable importance charts, including all genetic and non-heritable factors selected from the exploratory analysis as well as age, sex, CMV status, and smoking. Red and green box plots represent Z scores of rejected and confirmed attributes, respectively. Blue box plots correspond to minimal, average, and maximum Z scores of a shadow attribute (n = 1,000).

Figure 7

IFNβ secretion drives IL-12p70 variability in healthy donors and viral infection (A) IFNβ and IL-12p70 measured using Simoa after stimulation of whole blood with LPS for 22 h from healthy controls (n = 18) and patients acutely infected with SARS-CoV-2 presenting mild symptoms (n = 29) or being hospitalized for moderate, severe, or critical symptoms (n = 18) or convalescent patients (n = 34). p values were determined by Mann-Whitney tests, and the red lines indicate the median value for each severity condition. (B) Correlation between IFNβ and IL-12p70 secretion upon LPS stimulation in healthy controls (n = 18) or mild (n = 29), hospitalized (n = 18), or convalescent (n = 34) COVID-19 patients. Pearson correlations were performed using cytokine concentration log10 values. (C) IL-12p70 measured using Simoa after stimulation of whole blood with LPS for 22 h from healthy donors (n = 100) and patients infected with HCV (n = 100). p values were determined by Mann-Whitney tests, and red lines indicate the median value. (D) Correlation between IFNβ and IL-12p70 secretion upon LPS stimulation in SVR⁻ HCV patients (n = 33 patients) and in SVR⁺ HCV patients (n = 54 donors). Pearson correlation tests were performed on log10 values.