Figure 1.
B cell-deficiency in μMT mice is associated with an augmented lung neutrophilic and inflammatory response in M. tuberculosis-infected mice during the acute phase of infection.
Wild-type or B cell-deficient μMT mice were infected aerogenically with 200–300 CFU of M. tuberculosis Erdman. Lungs cells were procured for cytometric analysis, in conjunction with intracellular staining, at appropriate time intervals p.i.. A, B cell-deficient μMT mice exhibited significant elevation, relative to wild-type animals, in the percentage and in the number of neutrophils (CD11b+Ly6G+) among lung cells during acute tuberculous infection. The FACS dot plot depicts the percentage of neutrophils at day 21 p.i.. (*p<0.05, **p<0.005). The number of neutrophils infiltrating the lungs was evaluated on day 7, 14, and 21 p.i.. The hematoxylin & eosin (H&E)-stained lung sections revealed increased level of total cellular (B & C; 2×) and neutrophilic (D & E; 100×) infiltration. During the first month p.i., total cells infiltrating the lungs increased gradually (F), at each time point studied, the number of cells in the lungs of μMT mice was significantly higher than that in WT's (F). The H&E sections represent lung tissues at 2 weeks p.i.. Arrows: neutrophils. ***p<.0001; **p<.001; *p<0.01. Data presented are representative of two to three experiments with three to five mice per group.
Figure 2.
B cell-deficiency in μMT mice is associated with an augmented lung Th17 response in tuberculous mice during the acute phase of infection: reversibility of neutrophilia by IL-17 neutralization.
Wild-type or B cell-deficient μMT mice were infected aerogenically with 200–300 CFU of M. tuberculosis Erdman. Lungs cells were procured for cytometric analysis, in conjunction with intracellular staining, at appropriate time intervals p.i.. A, Compared to wild-type C57BL/6s, B cell-deficient μMT mice displayed increased in the number of IL-17A-producing lung cells and B, total number of pulmonic Th17 cells (CD3+CD4+IL-17A+) upon M. tuberculosis infection (*p<0.05). C, Quantification of neutrophil infiltration into the lungs of B cell-deficient μMT mice following IL-17 neutralization at day 14 p.i. revealed that increased neutrophilia was reversed via treatment with a mAb specific for this cytokine (***p<0.0005). D, this reversibility was not due to differences in bacterial burden since the total numbers of CFU in the lungs of mAb-treated and -untreated mouse groups were comparable. Data presented are representative of two to three experiments with three to five mice per group.
Figure 3.
B cell depletion results in an increased neutrophilic and Th17 response in the lungs of C57BL/6 mice with acute TB: IL-17 neutralization reverses the neutophilia phenotype.
Wild-type C57BL/6 animals or mice depleted of B cells using the CD22-cal conjugated Ab were infected aerogenically with 200–300 CFU of M. tuberculosis Erdman; lung cells were prepared at appropriate time intervals after infection and subjected to intracellular staining and/or flow cytometric analysis. Administration of CD22-cal resulted in ∼95% depletion of CD19+ B cells in the lungs of M. tuberculosis-infected mice at day 21 p.i. (see Figure S1A). A, The proportion and absolute number of neutrophils (CD11b+Ly6G+) among total lung cells in mice rendered B cell-deficient by CD22-cal treatment are elevated relative to those detected in non-B cell-depleted animals (*p<0.05, **p<0.005). B, CD22-cal-treated B cell-depleted mice, relative to untreated wild-type animals, exhibited a significant increase in total IL-17-producing lung cells during the acute phase of tuberculous infection (*p<0.05). C, Neutralization of IL-17 in CD22-cal-treated mice reverses lung neutrophilia phenotype associated with B cell-depletion at day 14 p.i. (*p<0.05, **p<0.005). Data depicted are representative of two to three experiments with three to five mice per group.
Figure 4.
B cell deficiency results in a diminished Th1 response in BCG-immunized mice.
Wild-type or B cell-deficient mice were vaccinated with BCG and at one month post-immunization, splenic T cells were assessed for the development of Th1 response by quantification of IFN-γ-producing CD4+ T cells. A, ELISPOT analysis of the number of splenic P25-Ag85B-specific IFN-γ-producing CD4+ T cells in wild-type and μMT mice 1 month after vaccination revealed a diminished Th1 response in the B cell deficient group (** p<0.005). B, A similarly decrease in BCG-induced Th1 response was also observed in mice rendered B cell-deficient via treatment with the anti-CD20 5D2 mAb (***p<0.0005). Data shown are representative of three independent experiments with four mice per group.
Figure 5.
B cell-deficient mice, upon ID ear immunization with BCG, display an enhanced neutrophilic infiltration in the pinna and an attenuated Th1 response in the draining superficial cervical lymph nodes that are associated with diminished DC migration.
Ears of wild-type or B cell-deficient mice were vaccinated intradermally with BCG and assessed at the indicated time intervals post-immunization. Cells from the ear and the draining superficial cervical lymph nodes were procured as described in Materials and Methods and subjected to flow cytometric analysis. μMT mice (A) and C57BL/6s depleted of B cells using the 5D2 anti-CD20 mAb (B) exhibited elevated numbers of CD11b+Ly6G+ neutrophils compared to wild-type animals (*p<0.05). Results of flow cytometric analyses showed a diminished number of IFN-γ-producing CD4+ Th1 cells (C) and CD11c+ DC (D) in draining superficial cervical lymph nodes of B cell-deficient μMT mice (relative to wild-type C57BL/6's) that were immunized intradermally in the ear with BCG (1×106 CFU) (*p<0.05). This Th1 (E) and DC migration (F) phenotypes were similarly observed in mice depleted B cells via treatment of the 5D2 anti-CD20 mAb (*p<0.05). Data shown are representative of two to three independent experiments with four mice per group.
Figure 6.
Effect of IL-17 neutralization on the level of neutrophilic infiltration in the ear and on the DC migration to and the Th1 response in the draining cervical lymph nodes of mice following ID BCG ear immunization.
A, The frequency (Left Panel) and absolute number (Right Panel) of neutrophils (CD11b+Ly6G+) in the ear dermis of B cell-deficient μMT mice on day 7 following BCG vaccination was increased relative to that observed in wild-type C57BL/6 animals. In these studies, cells obtained from the vaccinated ears were pooled for analysis. The enhanced pinna neutrophilic response was attenuated upon IL-17 neutralization. Upon ID ear vaccination, the B cell-deficient μMT mice exhibited decreased number of IFN-γ-producing CD4+ Th1 cells (B) and CD11c+ DC cells (C) in the draining superficial cervical lymph nodes of μMT mice relative to those detected in wild-type C57BL/6's. While IL-17 neutralization increased the number of Th1 cells (B) in the draining cervical lymph nodes of μMT mice intradermally immunized in the ear, it had no apparent effects on the number of DC (C). By contrast, IL-17 neutralization attenuated DC migration (C) to and the Th1 response (B) in the draining cervical nodes of wild-type C57BL/6 mice (*p<0.05, ***p<0.0005). The data are representative of two independent studies with four mice per group.
Figure 7.
Neutrophil depletion in wild-type and B cell-deficient mice results in enhanced DC migration to and an increased Th1 response in the draining superficial cervical lymph nodes upon ID ear BCG immunization.
C57BL/6 wild-type and μMT B cell-deficient mice, as well as animals depleted of B cells by treatment with the 5D2 anti-CD20 mAb were vaccinated with 106 CFU of BCG at the ear intradermally. Lymph nodes cells were procured at day 7 post-immunization and subjected to flow cytometric and IFN-γ ELISPOT assay analyses (using the Ag85B P25 peptide as antigens). A, μMT mice (Left Panel) and C57BL/6s depleted of B cells using the 5D2 anti-CD20 mAb (Right Panel) exhibited elevated numbers of CD11b+Ly6G+ neutrophils at the site of ID ear BCG vaccination compared to wild-type animals. The neutrophilia in both groups of mice was abrogated upon treatment with the Ly6G-specific mAb 1A8. 1A8 depletion of neutrophils resulted in increase in the numbers of CD11c+ DC (B) and IFN-γ-producing CD4+ T cells (C, Left Panel: intracellular staining; Right Panel: Ag85B P25 peptide-specific ELISPOT assay) in the draining cervical lymph nodes of both the ear BCG-vaccinated μMT and wild-type C57BL/6 mice (*p<0.05, **p<0.005, ***p<0.0005). Neutrophil depletion with 1A8 led to similar enhancement of DC migration (D) to and Th1 response (E: Left Panel: intracellular staining; Right Panel: Ag85B P25 peptide-specific ELISPOT assay) in the cervical draining lymph nodes post ID BCG vaccination in the ear of both wild-type and 5D2-treated B cell-deficient mice (*p<0.05, **p<0.005, ***p<0.0005). F, CFU enumeration in the draining superficial cervical lymph nodes at 7 days post-BCG immunization revealed decreased bacterial load in the μMT mice and animals depleted of B cells using the 5D2 anti-CD20 mAb (*p<0.05, ****p<0.00005). The results shown are representative of two independent experiments. Four mice per group were used in each experiment.
Figure 8.
Passive transfer of immune serum into B cell-deficient mice reverses the neutrophil and Th17 phenotypes.
B cell deficient μMT mice were aerogenically infected with 100–300 CFU of M. tuberculosis Erdman and lung neutrophilia assessed at 1 month p.i.. Mice were treated with immune sera produced from tuberculous wild-type C57BL6 mice at one month post-aerosol infection, Passive transfer of immune serum from infected wild-type C57BL/6 mice into tuberculous μMT mice resulted in diminished lung neutrophil infiltration (A, * p<0.05) and Th17 response (B, *p<0.05, **p<0.005, ***p<0.0005) at 1 month p.i.. The results of three neutrophil and two Th17 experiments are shown. Four mice per group were used in all experiments.