B cells delay neutrophil migration toward the site of stimulus: tardiness critical for effective bacillus Calmette-Guérin vaccination against tuberculosis infection in mice

Abstract

Mutations in the btk gene encoding Bruton's tyrosine kinase cause X-linked immune deficiency, with impaired B lymphocyte function as the major phenotype. Earlier, we demonstrated that CBA/N-xid mice, unlike the wild-type CBA mice, were not protected by bacillus Calmette-Guérin (BCG) vaccination against tuberculosis infection. Because IFN-gamma-producing T cells and activated macrophages are key elements of antituberculosis protection, it remained unclear how the mutation predominantly affecting B cell functions interferes with responses along the T cell-macrophage axis. In this study, we show that B cell deficiency leads to an abnormally rapid neutrophil migration toward the site of external stimulus. Using adoptive cell transfers and B cell genetic knockout, we demonstrate a previously unappreciated capacity of B cells to downregulate neutrophil motility. In our system, an advanced capture of BCG by neutrophils instead of macrophages leads to a significant decrease in numbers of IFN-gamma-producing T cells and impairs BCG performance in X-linked immune-deficient mice. The defect is readily compensated for by the in vivo neutrophil depletion.

Fig. 1. Defective phenotypes expressed in CBA/N mice and restoration of BCG performance using adoptive cell transfer approaches

Compared to CBA mice, protection against TB challenge after BCG vaccination was significantly (P<0.01, Gohan’s criterion for survival curves) weaker in CBA/N-xid after injection of 5 × 10⁵ M. tuberculosis CFU i. v. (A), or 10⁴ CFU i. t. (B); N = 9 for each group, results presented as mean ± SEM. The defect did not depend upon vaccination dose (C: open bars – non-vaccinated control; black bars −2 × 10⁵ CFU of BCG s. c.; hatched bars −2 × 10⁷ CFU of BCG s. c., challenge − 5 × 10⁵ CFU of M. tuberculosis i. v., N = 8 for each group, mean ± SEM). In CBA → CBA/N radiation bone marrow chimera (9.5 G irradiation, 2 × 10⁷ donor BMC per recipient), protective effect of BCG was restored in terms of both mycobacterial lung CFU counts (D: N = 4 per group, 3 wk post-challenge, P<0.01, one of two similar experiments), and prolongation of survival time (E: N = 8 per group, 2 × 10⁷ CFU of BCG, challenge − 5 × 10⁶ CFU of M. tuberculosis i. v., P<0.05, Gohan’s criterion). Adoptive transfer of 2 × 10⁷/mouse fetal liver (FL) cells from CBA donors to non-irradiated adult CBA/N recipients restored the numbers of B cells in their lymphoid organs to the levels characteristic for the w. t. mice (F) and was sufficient for restoration of BCG-provided protection (G).

Fig. 2. Neutrophil migration towards the site of BCG injection in XID and wild type mice

(A) CBA but not CBA/N peritoneal APC loaded with BCG in vivo for 2 h can present mycobacterial antigens to specific T cell lines (one of two similar experiments). (B, C) After i. p. BCG injection, in XID mice neutrophils migrate to the peritoneal cavity much more rapidly than in wild type mice (summary of 8 independent experiments, mean ± SD). The difference in migration speed is abrogated by the adoptive transfer of either whole (D) or non-adherent (E) population of CBA peritoneal exsudate cells (summary of 2 independent experiments, 3 mice in each group, N=6). (F) In the transwell system, CBA/N neutrophils migrate much faster, compared to CBA neutrophils, irrespective to migration stimuli (two independent experiments with mixtures of cells from 3 mice in each, P < 0.01, unpaired t-test).

Fig. 3. Differences in chemokine gene expression between XID and w. t. mice

Early after BCG injection the expression of mRNA encoding key neutrophil-attracting factors G-CSF (A) and KC (B) increases in CBA/N but decreases in CBA mice (summary of three independent experiments, mixtures of RNA isolated from 3 mice in each, N=9). There was no difference in the dynamics of expression of mRNA for some other factors involved in neutrophil migration control, MIP-2 (C), IL-6 and IL-17 (not shown). Results are expressed as fold increase ± SEM compared to naïve CBA animals. Samples of RNA were analyzed by the quantitative real-time PCR assay, and gene expression levels in peritoneal cells were normalized to those of β-actin.

Fig. 4. BCG phagocytosis, T cell responses and restoration of BCG performance by the in vivo neutrophil depletion

The vast majority of i. p. injected BCG is associated with mononuclear cells in CBA (A), but with PMN in CBA/N (B) mice, as demonstrated by auramine staining for mycobacteria with Gimsa counterstaining of cytospin preparations. Before challenge, BCG-vaccinated mice of the two strains did not differ by numbers of IFN-γ-producing CD4⁺ T cells in their lungs (C), but at week 3 post-infection the number of these cells in the lungs of the w. t. CBA mice was ~4 times (P < 0.01, Mann-Whitney U-test) higher (D). In the spleen, the numbers of IFN-γ-producing T cells differed ~10-fold (P < 0.001) after BCG vaccination in the absence of challenge (E). Magnetically sorted CD4⁺ lung T cells (C and D) or bulk spleen cells (E) from 3 individual mice in each group were analyzed using the ELISPOT assay; the results of one of two similar experiments are presented as mean ± SD. Neutrophil depletion prior to BCG vaccination resulted in significant (P < 0.01, Mann-Whitney U-test) ~8-fold reductions in CFU counts in lungs (F) and spleens (G) compared to control groups.