Leukocyte attraction by CCL20 and its receptor CCR6 in humans and mice with pneumococcal meningitis

Abstract

We previously identified CCL20 as an early chemokine in the cerebrospinal fluid (CSF) of patients with pneumococcal meningitis but its functional relevance was unknown. Here we studied the role of CCL20 and its receptor CCR6 in pneumococcal meningitis. In a prospective nationwide study, CCL20 levels were significantly elevated in the CSF of patients with pneumococcal meningitis and correlated with CSF leukocyte counts. CCR6-deficient mice with pneumococcal meningitis and WT mice with pneumococcal meningitis treated with anti-CCL20 antibodies both had reduced CSF white blood cell counts. The reduction in CSF pleocytosis was also accompanied by an increase in brain bacterial titers. Additional in vitro experiments showed direct chemoattractant activity of CCL20 for granulocytes. In summary, our results identify the CCL20-CCR6 axis as an essential component of the innate immune defense against pneumococcal meningitis, controlling granulocyte recruitment.

Figure 1. In patients with acute bacterial meningitis, (A) CCL20 cerebrospinal fluid (CSF) levels were significantly elevated compared with controls.

CCL20 CSF levels of patients with pneumococcal meningitis correlated to (B) CSF white blood cell (WBC) counts, (C) CSF blood glucose ratio, and (D) CSF protein levels (see text for details).

Figure 2. CCL20 is expressed mainly during acute pneumococcal meningitis.

(A) Increased CCL20 levels were found in mice brain homogenates during acute bacterial meningitis using ELISA. After initiation of antibiotic therapy (starting 24 h after infection), they decreased quickly to normal ((*) p<0.01 compared with uninfected controls). In uninfected control mice, (B, C) only a very subtle CCL20-positive staining was observed (white arrow). In contrast, in animals with pneumococcal meningitis, CCL20-positive staining was found in (D) epithel cells of the choroid plexus (black arrow) and (E) the subarachnoid inflammatory infiltrate (#). Number of animals: 6 h: n = 6, 24 h: n = 9, 30 h: n = 6, 48 h: n = 6, 72 h: n = 6, and 120 h: n = 7. Uninfected animals were used as controls (n = 11).

Figure 3. Anti-CCL20 antibody therapy lowers CSF inflammation in vivo.

Although blockage of CCL20 using an anti-CCL20 antibody was not reflected in clinical differences, it resulted (A) in a decrease of CSF pleocytosis 24 h after infection. (B) This was associated with increased brain bacterial titers. (C) Blood bacterial titers were not affected. (*) p<0.05 compared with infected animals that received control antibodies. AB = antibody. N = 10 per group.

Figure 4. CCR6-deficient mice are more susceptible to pneumococcal meningitis.

CCR6-deficient mice were more strongly affected from a clinical perspective by infection with Streptococcus pneumoniae than wild type controls. This was reflected (A) in increased clinical scores and (B) mortality. (C) CSF pleocytosis was lower in infected CCR6-deficient animals 24 h and 48 h after infection. (D) This was associated with higher brain bacterial titers at the time of initiation of antibiotic therapy (24 h after infection). (*) p<0.05 compared with infected wild type animals. Number of animals: 24 h: Ccr6 ^−/− n = 12, WT n = 12; 48 h: Ccr6 ^−/− n = 12, WT n = 13.

Figure 5. CCR6-deficient mice suffer from increased brain edema after antibiotic therapy.

(A) Infected mice with CCR6-deficiency developed more pronounced brain edema than infected wild type mice 48 h after infection, reflected in an increase in the estimated brain volume. (B) At the same time point, an increase of brain albumin content was noted in infected Ccr6 ^−/− mice, indicating blood-brain barrier disruption. These differences were only seen after but not before initiation of antibiotic therapy (24 h after infection). (C–G) Intracranial bleeding was similar in infected Ccr6 ^−/− and wild type mice. (*) p<0.05 compared with WT control animals. Number of animals: 24 h: Ccr6 ^−/− n = 12, WT n = 12; 48 h: Ccr6 ^−/− n = 12, WT n = 13.

Figure 6. CCL20 chemoattracts granulocytes in vitro and in vivo.

HoxB8 precursor cells could be differentiated into neutrophils, as indicated by the expression of Gr1. (A, B) The differentiated neutrophils were CCR6 positive. (C, D) Stimulation with TNF-α led to a marked increase of CCR6 expression in differentiated Hoxb8 neutrophils. Whereas (E, F) unstimulated bone marrow-derived neutrophils were CCR6-negative, (G, H) CCR6 became detectable on these cells upon exposure to heat-killed pneumococci (HKP). Using a chemotaxis assay, (I) differentiated Hoxb8 neutrophils and (K) bone marrow derived neutrophils migrated towards CCL20 in a dose-dependent way, demonstrating an in vitro chemotactic effect of CCL20 protein on CCR6 positive granulocytes. FMLP and CXCL2 were used as positive controls. (*) p<0.05 compared with Hoxb8 exposed to medium. (H) After intrathecal injection of recombinant CCL20 (rCCL20, n = 7) in wild type mice, a chemotactic effect was seen in comparison to injection with heat inactivated rCCL20 (rCCL20i, control, n = 7). In addition, rCCL20 co-administration to HKP led to a significant increase of the CSF-pleocytosis compared with rCCL20i co-administration to HKP (n = 8 for each group). (*) p<0.05 compared with rCCL20i or HKP+rCCL20i respectively.

Figure 7. The pro-inflammatory effect of the CCL20/CCR6 axis seems independent of IL-17 production.

IL17 was up-regulated in the CSF of (A) humans and (B) mice with pneumococcal meningitis. (C) However, antibody blockage of IL-17 in experimental pneumococcal meningitis did not lead to a reduction of inflammation in the CSF. P<0.05 as compared with uninfected controls. Number of animals: n = 5 mice per group.