The role of MyD88- and TRIF-dependent signaling in monophosphoryl lipid A-induced expansion and recruitment of innate immunocytes

Abstract

Treatment with the TLR4 agonist MPLA augments innate resistance to common bacterial pathogens. However, the cellular and molecular mechanisms by which MPLA augments innate immunocyte functions are not well characterized. This study examined the importance of MyD88- and TRIF-dependent signaling for leukocyte mobilization, recruitment, and activation following administration of MPLA. MPLA potently induced MyD88- and TRIF-dependent signaling. A single injection of MPLA caused rapid mobilization and recruitment of neutrophils, a response that was largely mediated by the chemokines CXCL1 and -2 and the hemopoietic factor G-CSF. Rapid neutrophil recruitment and chemokine production were regulated by both pathways although the MyD88-dependent pathway showed some predominance. In further studies, multiple injections of MPLA potently induced mobilization and recruitment of neutrophils and monocytes. Neutrophil recruitment after multiple injections of MPLA was reliant on MyD88-dependent signaling, but effective monocyte recruitment required activation of both pathways. MPLA treatment induced expansion of myeloid progenitors in bone marrow and upregulation of CD11b and shedding of L-selectin by neutrophils, all of which were attenuated in MyD88- and TRIF-deficient mice. These results show that MPLA-induced neutrophil and monocyte recruitment, expansion of bone marrow progenitors and augmentation of neutrophil adhesion molecule expression are regulated by both the MyD88- and TRIF-dependent pathways.

Keywords: TLR4; cytokines; innate immunity; monocyte; myeloid progenitors; neutrophil.

Figure 1.. MPLA induces activation of the MyD88- and TRIF-dependent signaling pathways.

BMDMs were incubated with vehicle or MPLA for 30 min. IRF3 and IKK phosphorylation, as well as total IKK and IRF3 were determined by Western blot analysis. Assessment of phospho- and total IRF3 (top) and phospho- and total IKK (bottom) in WT, MyD88-KO, and TRIF-KO BMDMs. Graphs show results quantified by densitometry. The results are representative of 3 different runs. *P < 0.05 vs. WT, ⁺P < 0.05, vs. MyD88-KO.

Figure 2.. MPLA-induced neutrophil mobilization and recruitment are mediated by CXCL1, CXCL2, and G-CSF.

Wild-type mice received an injection of 20 μg MPLA i.p. Whole blood, plasma, and peritoneal lavage fluid were harvested at 0, 3, and 6 h after MPLA challenge. (A) Neutrophil counts in blood and peritoneal lavage fluid. Blood neutrophils were counted with a clinical analyzer, and intraperitoneal neutrophils were measured by flow cytometry and identified as F4-80^-Ly6G⁺; (B) CXCL1, CXCL2, and G-CSF were measured in the plasma and peritoneal lavage fluid using a magnetic bead reader. (C) Chemokine neutralization was achieved by administration of IgG or anti-mouse CXCL1, CXCL2, CXCR2+CXCR1, or G-CSF 24 h before intraperitoneal MPLA injection. The number of neutrophils in blood and peritoneal lavage fluid was measured at 3 h after MPLA challenge (n = 9–21/group). *P < 0.05 vs. 0 h control, ⁺P < 0.05 vs. IgG control.

Figure 3.. Neutrophil recruitment and cytokine production are attenuated in MyD88-deficient mice.

(A) Mice received an injection of 20 μg MPLA i.p. at 3 or 6 h before sample harvest. Intraperitoneal leukocytes were harvested at 0, 3, or 6 h after MPLA challenge and the number of neutrophils was measured by flow cytometry. Neutrophils were identified as F4/80⁻Ly6G⁺. (B) Plasma and peritoneal lavage fluid were harvested at 0, 3, and 6 h after MPLA challenge, and the cytokine levels were measured (n = 8–19/group). *P < 0.05 vs. time 0, ⁺P < 0.05 vs. WT.

Figure 4.. Myeloid cell recruitment is impaired in MyD88-KO mice after multiple intraperitoneal MPLA injections.

(A) Mice received injection of 20 μg MPLA i.p. 48 and 24 h before samples were harvested. (B) Intraperitoneal leukocytes were collected, and cells were stained for F4/80, Ly6G, and Ly6C. Neutrophils were identified as F4/80⁻Ly6G⁺. Monocytes were identified as F4/80⁺Ly6C⁺ (n = 8–14/group). *P < 0.05 vs. vehicle, ⁺P < 0.05 vs. WT.

Figure 5.. Intravenous MPLA injection increases spleen weight and cellularity.

(A) Mice received an injection of 20 μg MPLA i.p. 48 and 24 h before spleen harvest. The spleen was excised, weighed, and homogenized for total cell count analysis. (B) Mice received injections of 20 μg MPLA i.v. 48 and 24 h before spleen harvest. The spleen was excised, weighed, and homogenized for cell count analysis. Splenocytes were stained for F4/80, Ly6G, Ly6C, CD19, and Class II MHC. Neutrophils were identified as F4/80⁻Ly6G⁺, monocytes as F4/80⁺/Ly6C⁺, T cells as CD3⁺, and B cells as CD19⁺/MHC Class II⁺ (n = 10–20/group.). *P < 0.05 vs. vehicle; ⁺P < 0.05 vs. WT.

Figure 6.. MPLA-induced increase in the number of bone marrow progenitor cells is attenuated in MyD88- and TRIF-deficient mice.

Mice received an injection of 20 μg MPLA i.v. at 48 and 24 h before bone marrow cell harvest. Bone marrow cells were collected and cultured in 2 culture plates/sample. Progenitor CFUs were differentiated and quantified after 10 d. BFU-E=burst-forming unit erythroid; CFU-GEMM=CFU-granulocyte/erythroid/macrophage/megakaryocyte; and CFU-PreB=CFU pre-B cells (n = 5/group). *P < 0.05 vs. vehicle; ⁺P < 0.05 vs. WT.

Figure 7.. MPLA-induced neutrophil CD11b expression and L-selectin shedding are attenuated in MyD88- and TRIF-deficient mice.

Bone marrow cells were harvested from untreated mice from each group and incubated with increasing concentrations of MPLA for 2 h. Cells were washed; stained for F4/80, Ly6G, and either CD11b or L-selectin (CD62L); and analyzed with flow cytometry. Neutrophils were identified as F4/80⁻Ly6G⁺ (n = 5/group). *P < 0.05 vs. 0 ng.