CD38 controls the innate immune response against Listeria monocytogenes

Abstract

CD38, adenosine-5'-diphosphate-ribosyl cyclase 1, is a multifunctional enzyme, expressed on a wide variety of cell types. CD38 has been assigned diverse functions, including generation of calcium-mobilizing metabolites, cell activation, and chemotaxis. Using a murine Listeria monocytogenes infection model, we found that CD38 knockout (KO) mice were highly susceptible to infection. Enhanced susceptibility was already evident within 3 days of infection, suggesting a function of CD38 in the innate immune response. CD38 was expressed on neutrophils and inflammatory monocytes, and especially inflammatory monocytes further upregulated CD38 during infection. Absence of CD38 caused alterations of the migration pattern of both cell types to sites of infection. We observed impaired accumulation of cells in the spleen but surprisingly similar or even higher accumulation of cells in the liver. CD38 KO and wild-type mice showed similar changes in the composition of neutrophils and inflammatory monocytes in blood and bone marrow, indicating that mobilization of these cells from the bone marrow was CD38 independent. In vitro, macrophages of CD38 KO mice were less efficient in uptake of listeria but still able to kill the bacteria. Dendritic cells also displayed enhanced CD38 expression following infection. However, absence of CD38 did not impair the capacity of mice to prime CD8(+) T cells against L. monocytogenes, and CD38 KO mice could efficiently control secondary listeria infection. In conclusion, our results demonstrate an essential role for CD38 in the innate immune response against L. monocytogenes.

Fig 1

Listeria titers in spleen and liver 60 h postinfection. On day 0, WT and CD38 KO mice were infected i.v. with EGD bacteria. Sixty hours postinfection, EGD titers in spleen and liver were determined. (A) BALB/c WT and CD38 KO mice infected i.v. with 5 × 10⁴ EGD bacteria. (B) C57BL/6 WT and CD38 KO mice infected i.v. with 5 × 10⁴ EGD bacteria. (C) BALB/c WT and CD38 KO mice infected i.v. with 5 × 10³ EGD bacteria. There were 5 to 10 mice per group; median bars are shown. One representative experiment out of two each is shown.

Fig 2

CD38 expression on neutrophils, inflammatory monocytes, and dendritic cells at sites of listeria infection. On day 0, BALB/c WT and CD38 KO mice were infected i.v. with 5 × 10⁴ EGD bacteria. Sixty hours postinfection, expression of CD38 on neutrophils, inflammatory monocytes, and dendritic cells in spleen and liver was determined. There were 3 to 5 mice per group; one representative histogram overlay is shown. One representative experiment out of two is shown.

Fig 3

Recruitment of neutrophils and inflammatory monocytes to sites of listeria infection. On day 0, BALB/c WT and CD38 KO mice were infected i.v. with 5 × 10⁴ EGD bacteria. Sixty hours postinfection, relative frequencies (A) and absolute numbers (B) of neutrophils and inflammatory monocytes in spleen and liver were determined. For bone marrow and blood, only relative frequencies are shown. (C) Relative frequencies and absolute numbers of dendritic cells in the spleen. There were 3 to 5 mice per group; median bars are shown. One representative experiment out of two is shown.

Fig 4

Peritoneal macrophages of CD38 KO mice are less efficient in uptake of listeria but can efficiently kill the bacteria. Thioglycolate-elicited peritoneal macrophages were rested overnight, and 1 × 10⁵ peritoneal cells (∼50% CD45⁺ CD11b⁺ macrophages) were then infected with 3 × 10⁶ EGD bacteria (MOI = 30) for 1 h at 37°C. Cells were washed and incubated with medium containing gentamicin to kill remaining extracellular bacteria. After 30 min, macrophages were lysed, and listeria titers in lysates were determined to estimate the uptake of bacteria (0 h). Bacterial titers in macrophages were further determined after 2 and 4 h. All titers were determined in triplicates. Mean bars are shown. One representative experiment out of two is shown.

Fig 5

Expansion and differentiation of CD8⁺ T cells at sites of listeria infection. (A) Experimental setup. On day −1, C57BL/6 WT and CD38 KO mice were adoptively transferred with 5 × 10⁴ OT-I CD8⁺ T cells. On day 0, all mice were infected i.v. with 1 × 10⁵ LmOVA bacteria. On days 2 and 3, all mice were injected i.p. with 2 mg of ampicillin (in PBS). (B) Seven days postinfection, relative frequencies and absolute numbers of OT-I CD8⁺ T cells in spleen and liver were determined. (C) Seven days postinfection, the differentiation status of OT-I CD8⁺ T cells in the spleen was determined by means of CD44, CD62L, CD127, and KLRG1 expression. Proliferating OT-I CD8⁺ T cells were detected by staining of Ki-67. IFN-γ producing OT-I CD8⁺ T cells were detected after 4.5 h in vitro restimulation with pOVA. There were 6 or 7 mice per group; median bars are shown. One representative experiment out of two is shown.

Fig 6

Listeria titers in spleen and liver 48 h after primary and secondary infection. On day 0, BALB/c WT and CD38 KO mice were infected i.v. with 5 × 10³ EGD bacteria. Forty-eight hours postinfection, mice were treated with a single i.p. injection of 2 mg of ampicillin and received 1 g/liter of ampicillin in drinking water for 1 week. After 6 weeks, primary infection of previously untreated mice or secondary infection of previously infected mice was carried out i.v. with 1 × 10⁵ EGD bacteria. Forty-eight hours after primary or secondary infection, EGD titers in spleen and liver were determined. There were 7 or 8 mice per group; median bars are shown.