Kinome Analysis to Define Mechanisms of Adjuvant Action: PCEP Induces Unique Signaling at the Injection Site and Lymph Nodes

Abstract

Understanding the mechanism of action of adjuvants through systems biology enables rationale criteria for their selection, optimization, and application. As kinome analysis has proven valuable for defining responses to infectious agents and providing biomarkers of vaccine responsiveness, it is a logical candidate to define molecular responses to adjuvants. Signaling responses to the adjuvant poly[di(sodiumcarboxylatoethylphenoxy)phosphazene] (PCEP) were defined at the site of injection and draining lymph node at 24 h post-vaccination. Kinome analysis indicates that PCEP induces a proinflammatory environment at the injection site, including activation of interferon and IL-6 signaling events. This is supported by the elevated expression of proinflammatory genes (IFNγ, IL-6 and TNFα) and the recruitment of myeloid (neutrophils, macrophages, monocytes and dendritic cells) and lymphoid (CD4+, CD8+ and B) cells. Kinome analysis also indicates that PCEP’s mechanism of action is not limited to the injection site. Strong signaling responses to PCEP, but not alum, are observed at the draining lymph node where, in addition to proinflammatory signaling, PCEP activates responses associated with growth factor and erythropoietin stimulation. Coupled with the significant (p < 0.0001) recruitment of macrophages and dendritic cells to the lymph node by PCEP (but not alum) supports the systemic consequences of the adjuvant. Collectively, these results indicate that PCEP utilizes a complex, multi-faceted MOA and support the utility of kinome analysis to define cellular responses to adjuvants.

Keywords: PCEP; adjuvant; kinome; lymph nodes.

Figure 1

Kinome Responses to PCEP and Alum at Site of Injection and Lymph Node. (A) Clustering of Kinome Responses at Site of Injection and in Draining Lymph Nodes. Hierarchical clustering of kinome datasets. (1−Pearson correlation) was used as the distance metric, while McQuitty linkage was used as the linkage method. Colors indicate the average (over nine intra-array replicates) normalized phosphorylation intensity of each target, with red indicating greater amounts of phosphorylation and green indicating lesser amounts of phosphorylation. (B) Venn Diagram of Differentially Phosphorylated Peptides in Lymph Nodes to PCEP and Alum. Comparison of peptides consistently and significantly (p < 0.05) phosphorylated in lymph in response to either PCEP or Alum relative to PBS control. (C) Venn Diagram of Differentially Phosphorylated Peptides in Lymph Nodes and Muscle to PCEP. Comparison of peptides consistently and significantly (p < 0.05) phosphorylated in lymph and muscle in response to PCEP relative to PBS control.

Figure 2

Patterns of Gene Expression at Site of PCEP Injection. Cytokine and chemokine gene expression profiles elicited by PCEP at the site of injection after intramuscular injection in mice. Mice were injected with PBS or PCEP intramuscularly. Muscle tissue at the site of injection were collected at 24 h and analyzed for cytokine and chemokine genes by quantitative real-time PCR. (A) Increased expression of cytokine genes including IFNγ, TNFα and TLRs at the injection site. (B) Substantial increase in proinflammatory gene, IL-6. (C) Increased expression of chemokine receptor family genes at the injection site. Results shown are the mean ± SE of six replicates at each time point. Relative fold changes (y-axis) for each gene were normalized to mouse GAPDH. Fold changes are calculated by the Ct method and are relative to the gene expression in PBS injected muscle tissue. (reprinted with modifications from Awate et al. 2012, Copyright 2012, with permission from Elsevier).

Figure 3

Patterns of Cell Recruitment to the Site of Injection in Response to Adjuvants. PCEP stimulates increased immune cell numbers at the site of injection. BALB/c mice (n = 5 per group) were injected i.m. with either PBS, PCEP (50 ug) or alum (0.5 mg). The site of injection muscle tissue was dissected at 24 h time point and processed. Single cell suspensions were analyzed by flow cytometry. (A) Kinetics of myeloid cells (neutrophils, macrophages, monocytes and dendritic cells) 24 h post-injection of adjuvants at the site of injection. (B) Kinetics of lymphoid cells (CD4+, CD8+ and B cells) 24 h post-injection of adjuvants at the site of injection. Differences in the cell numbers were analyzed by two-way ANOVA and the significant differences between the treatments were compared by Bonferroni multiple-comparison test where **** p < 0.0001, *** p < 0.001, ** p < 0.005, * p < 0.05. (reprinted with modifications from Awate et al. 2014, Copyright 2014, with permission from Elsevier).

Figure 4

Patterns of Cell Recruitment to the Draining Lymph Nodes in Response to Adjuvants. PCEP stimulates increased immune cell numbers in the draining lymph nodes. BALB/c mice (n = 5 per group) were injected i.m. with either PBS, PCEP (50 ug) or alum (0.5 mg). The draining inguinal lymph nodes were collected at 24 h post-injection and the cell suspensions were analyzed by flow cytometry. (A) Kinetics of myeloid cells (neutrophils, macrophages, monocytes and dendritic cells) 24 h post-injection of adjuvants at the draining lymph nodes. (B) Kinetics of lymphoid cells (CD4+, CD8+ and B cells) 24 h post-injection of adjuvants at the draining lymph nodes. Differences in the cell numbers were analyzed by two-way ANOVA and the significant differences between the treatments were compared by Bonferroni multiple-comparison test where **** p < 0.0001. (reprinted with modifications from Awate et al. 2014, Copyright 2014, with permission from Elsevier).