Abstract
Background Tertiary lymphoid structures (TLS) are triggered by persistent bronchopulmonary infection with Staphylococcus aureus, but their roles remain elusive. The present study sought to examine the effects of B- and/or T-cell depletion on S. aureus infection and TLS development (lymphoid neogenesis) in mice.
Methods C57Bl/6 mice were pre-treated with 1) an anti-CD20 monoclonal antibody (mAb) (B-cell depletion) or 2) an anti-CD4 and/or an anti-CD8 mAb (T-cell depletion) or 3) a combination of anti-CD20, anti-CD4 and anti-CD8 mAbs (combined B- and T-cell depletion) or 4) isotype control mAbs. After lymphocyte depletion, mice were infected by intratracheal instillation of agarose beads containing S. aureus (106 CFU per mouse). 14 days later, bacterial load and lung inflammatory cell infiltration were assessed by cultures and immunohistochemistry, respectively.
Results 14 days after S. aureus-bead instillation, lung bacterial load was comparable between control and lymphocyte-depleted mice. While TLS were observed in the lungs of infected mice pre-treated with control mAbs, these structures were disorganised or abolished in the lungs of lymphocyte-depleted mice. The absence of CD20+ B-lymphocytes had no effect on CD3+ T-lymphocyte infiltration, whereas CD4+/CD8+ T-cell depletion markedly reduced CD20+ B-cell infiltration. Depletion of CD4+ or CD8+ T-cells separately had limited effect on B-cell infiltration, but led to the absence of germinal centres.
Conclusion TLS disorganisation is not associated with loss of infection control in mice persistently infected with S. aureus.
Abstract
Disorganisation of peribronchial lymphoid follicles did not result in increased bacterial load nor in decreased survival in a mouse model of persistent lung infection. Lymphoid follicles may not be essential for controlling lung bacterial infection. https://bit.ly/3lOgNEG
Introduction
Immune response in the airways of patients with cystic fibrosis (CF) or non-CF bronchiectasis diseases is dominated by increased innate immunity with airway mucus plugging [1, 2] and massive neutrophilic infiltration [3–5]. However, studies using lung explants obtained in CF patients [6–9] or using surgically resected lung tissues in patients with bronchiectasis [6, 10] have found evidence of intrapulmonary adaptive immune response, characterised by the presence of peribronchial aggregates of B- and T-lymphocytes. These lymphoid aggregates were often organised into tertiary lymphoid structures (TLS) containing segregated B- and T-cell areas, high endothelial venules (HEVs), follicular dendritic cells (FDCs) and germinal centres [6]. Persistent airway infection of C57/Bl6 mice with Pseudomonas aeruginosa or with Staphylococcus aureus, two major bacteria found in the airways of patients with CF, triggered the development of peribronchial TLS (a process called lymphoid neogenesis) within 14 days [6, 11]. These data suggest that TLS found in patients with CF or non-CF bronchiectasis contribute to the immune response triggered by chronic bacterial infection [6].
Mechanisms leading to lymphoid neogenesis in the lungs have been partly elucidated [12], but roles of TLS in chronic airway diseases associated with bacterial infection remain elusive. TLS may contribute to the immune response against bacterial infection [13, 14], limiting the extent of airway infection via the production of antibodies by activated B-cells. Alternatively, bacterial infection could induce lung tissue damage, which might result in the release of self-antigens, giving rise to B- and T-cell responses that perpetuate tissue injury [13, 14]. Thus, it remains unclear whether targeting lung lymphoid neogenesis would be detrimental or beneficial in airway diseases associated with chronic bacterial infection.
In the present study, we analysed the roles of adaptive immune components of TLS in chronic lung infection by selectively blocking recruitment of lymphocyte subsets using monoclonal antibodies (mAbs) in a mouse model of persistent S. aureus infection. Thus, we targeted B- and/or CD4+ and CD8+ T-lymphocytes before inducing persistent S. aureus infection and explored the effects on bacterial infection and lung lymphoid neogenesis.
Methods
Murine model of persistent airway infection
Female C57Bl/6 mice (aged 6–8 weeks) were purchased from Janvier (Saint Berthevin, France). Persistent airway infection was obtained by intratracheal instillation of agarose beads containing S. aureus (106 CFU per animal) as described previously [6], and euthanised 14 days after instillation. All animal experiments received approval (#4664 and #12190) from the ethical review board Charles Darwin (Sorbonne University, Paris, Fance).
Depletion of CD20+ B-cells and/or CD4+/CD8+ T-cells
Depletion of CD20+ B-cells and/or CD4+/CD8+ T-cells was obtained by injection of anti-CD20 and/or anti-CD4 and anti-CD8 depleting mAbs, respectively, as described in figure 1. For CD20+ B-cell depletion, one dose of 250 μg of mouse IgG2a anti-mouse CD20 mAb (clone 5D2; kindly provided by Genentech, San Francisco, CA, USA) was injected intravenously 7 days before S. aureus-containing bead instillation (figure 1a). For CD4+ and CD8+ T-cell depletion experiments, mice received intraperitoneal injections of 250 μg anti-mouse CD4 mAb (rat IgG2b, clone GK1.5; BioXCell, West Lebanon, NH, USA), and 250 μg anti-mouse CD8 mAb (rat IgG2b, clone YTS169.4; BioXCell), respectively, 5 and 4 days before S. aureus-containing bead instillation. Mice received a second i.p. injection of 250 μg anti-CD4 and 250 μg anti-CD8 mAbs 4 and 5 days after intratracheal instillation, respectively (figure 1b). One group of mice received anti-CD20 mAb combined to anti-CD4 and anti-CD8 mAbs (combined B- and CD4+/CD8+ T-cell depletion, figure 1c).
Schematic representation of experiments assessing the effects of lymphocyte depletion on persistent Staphylococcus aureus infection and lymphoid neogenesis in C57Bl/6 mice (results are shown in figure 2 and figure 3). Mice were pre-treated with a) anti-CD20 monoclonal antibody (mAb), b) anti-CD4 and anti-CD8 mAbs or c) a combination of anti-CD20 and anti-CD4/CD8 mAbs prior to infection. For each depletion protocol, control animals were injected with similar doses of isotype-matched control mAbs. Persistent infection was obtained by intratracheal instillation of agarose beads containing S. aureus (106 CFU per animal). 14 days after instillation, animals were euthanised and lungs were harvested for flow cytometry analysis, bacterial load assessment and immunohistochemical staining. a) CD20+ B-cell depletion: mice were injected intravenously with 250 μg of anti-CD20 mAb or control mAb; b) CD4+ and CD8+ T-cell depletion: to induce persistent CD4+ and/or CD8+ T-cell depletion, mice received one intraperitoneal injection of anti-CD4 and/or anti-CD8 mAb (250 μg per injection) 4–5 days prior to infection and another i.p. injection 4–5 days after infection; c) combined CD20+ B- and CD4+/CD8+ T-cell depletion: CD20+ B- and CD4+/CD8+ T-cell depletion protocols were combined to obtain prolonged CD20+ B- and CD4+/CD8+ T-cell depletion. For each experiment, blood was sampled 24 h before instillation and flow cytometry analysis was performed to confirm peripheral blood lymphocyte depletion.
To further explore the respective roles of CD4+ or CD8+ T-cells in the development of TLS, two groups of mice were depleted of either CD4+ or CD8+ T-cells by two i.p. injections of 250 μg rat anti-mouse CD4 or 250 μg rat anti-mouse CD8, respectively.
Control groups of mice received similar doses of isotype-matched control mAbs (mouse IgG2a mAb, clone C1.18.4 and/or rat IgG2b mAb, clone LTF-2; BioXCell). Depletion of CD20+ B-cells and/or CD4+/CD8+ T-cells was confirmed by flow cytometry analysis of the blood of animals, 1 day before S. aureus-bead instillation. Only animals exhibiting blood percentages of B-cells and/or CD4 and CD8 T-cells <5% after pre-treatment with mAbs were subsequently infected with S. aureus-containing beads.
Lung bacterial load
Mice were euthanised 14 days after S. aureus-containing beads instillation and 10-fold dilutions of total lung homogenates were plated on trypticase soy agar. CFU count was performed after 24-h incubation at 37°C.
Immunohistochemical staining
Lungs were removed after flushing 4% paraformaldehyde through the right heart and insufflated 5 min with 4% paraformaldehyde at −20 cmH2O. 5-μm paraffin sections of mouse lungs were immunostained as described previously [6] for detecting B-lymphocytes (CD20+), T-lymphocytes (CD3+), HEVs (peripheral node adressin (PNAd+)), FDCs (CD21+) and proliferating cell nuclear antigen (PCNA+; for the identification of germinal centres). A detailed description of the sources of primary antibodies, dilutions and unmasking techniques is provided in supplementary table S1. Biotinylated anti-mouse, anti-rabbit, anti-rat or anti-goat antibodies (dilution 1:200; Vector Laboratories, Burlingame, CA, USA) were used for secondary antibodies and bound antibodies were visualised according to standard protocols for avidin–biotin–peroxidase complex method (Elite ABC kit; Vector Laboratories). Tissue sections were counterstained with haematoxylin (Vector Laboratories).
Morphometric analyses
Quantitative morphometric analysis was performed by two independent observers using a light microscope (Leica Microsystems, Wetzlar, Germany) connected to a computer [6]. A lymphoid aggregate was defined as a CD20+ or CD3+ cell aggregate that could be detected using a low-power field (×16). Briefly, images were collected using a light microscope connected to the Leica Application Suite software (version 4.1.0; Leica Microsystems) and the outline tool in Image J software (version 1.48, National Institutes of Health, Bethesda, MD, USA) was used for measuring tissue area. The number of lymphoid aggregates was expressed per cm2 of lung tissue.
Flow cytometry
Surface staining of blood and lung cells was performed according to standard protocols and analysed using a LSRFortessa (BD Biosciences, Franklin Lakes, NJ, USA) flow cytometer and Kaluza software (Beckman Coulter, Brea, CA, USA). For flow cytometry analyses of pulmonary tissue, lungs were flushed with PBS through the right heart, harvested and immediately stored at 4°C in staining buffer (PBS, bovine serum albumin 0.5% and EDTA 2 mM). Lungs were then incubated for 1 h at 37°C in RPMI medium supplemented with 200 μg·mL−1 Liberase TL, 0.1 mg·mL−1 DNAse I (Roche Diagnostics, Mannheim, Germany) and 0.5 mM EDTA. Red blood cells were eliminated using ammonium chloride potassium lysis buffer. Blood and lungs cells were stained with antibodies as described in supplementary table S2.
Statistical analysis
Data obtained from morphometric and flow cytometry analyses were analysed using the nonparametric Mann–Whitney or Kruskal–Wallis tests. The interobserver coefficients of variation for morphometric measurements were always <15%. All analyses were performed using Prism 8 software (GraphPad, La Jolla, CA, USA). p-values <0.05 were considered statistically significant.
Results
Effects of CD20+ B-lymphocyte and/or CD4+/CD8+ T-lymphocyte depletion on S. aureus lung infection
We first established protocols to obtain depletion of CD20+ B-cells and/or CD4+/CD8+ T-cells that persisted all along the 14 days of S. aureus infection in C57Bl/6 mice. A single i.v. injection of anti-CD20 mAb induced B-cell depletion in the lungs, which persisted for ≥21 days as confirmed by flow cytometry and immunohistochemical staining (supplementary figure S1). Injections of anti-CD4 and anti-CD8 mAbs repeated every 9 days induced persistent CD4+/CD8+ T-cell depletion in the lungs (supplementary figure S1). Injection of control mAbs had no effect on CD20+ B-cells and CD4+/CD8+ cells in the lungs.
To study the effect of CD20+ B-cell and/or CD4+/CD8+ T-cell depletion on S. aureus infection, C57Bl/6 mice were injected with anti-CD20 and/or anti-CD4/CD8 mAbs prior to intratracheal instillation of agarose beads containing S. aureus (106 CFU per animal); animals were euthanised at 14 days after infection (figure 1). In all experimental groups, none of the mice died during the 14 days of infection. Treatment with anti-CD20 and/or anti-CD4/CD8 mAbs had no significant effect on lung bacterial load at 14 days after intratracheal instillation (supplementary figure S2).
Effects of CD20+ B-lymphocyte depletion and/or CD4+/CD8+ T-lymphocyte depletion on S. aureus-induced peribronchial lymphoid neogenesis
As described previously, S. aureus infection resulted in peribronchial lymphoid neogenesis within 14 days [6], a process that was not affected by pre-treatment with control mAbs. 14 days after instillation of S. aureus-containing beads, peribronchial lymphoid aggregates were found around bead-containing bronchi; these aggregates contained B-cell areas (CD20+) with follicular dendritic cells (CD21+) and germinal centres (PCNA+) and were surrounded by T-cell aggregates (CD3+) containing high endothelial venules (PNAd+) and were consistent with TLS. Representative photomicrographs are presented in supplementary figure S3.
Pre-treatment with anti-CD20 mAb prior to S. aureus infection was marked by an absence of peribronchial B-cells, CD21+ FDCs (figures 2 and 3a) and germinal centres (supplementary figures S4 and S5). By contrast, CD3+ T-cell recruitment (figures 2 and 3a) and HEV formation were unaffected (supplementary figures S4 and S5).
Representative photomicrographs depicting the effects of CD20+ B-lymphocyte and/or CD4+/CD8+ T-lymphocyte depletion on Staphylococcus aureus-induced peribronchial lymphoid neogenesis. Mice were pre-treated with control monoclonal antibody (mAb) or with anti-CD20 and/or anti-CD4/CD8 mAbs prior to infection. Persistent infection was obtained by intratracheal instillation of agarose beads containing S. aureus (106 CFU per animal). Beads (B) were found in the airway lumen. 14 days after instillation, animals were euthanised and lungs were harvested for histological analysis. Sections were immunostained (brown) with antibodies directed against B-lymphocytes (CD20+), follicular dendritic cells (CD21+) and T-lymphocytes (CD3+), and counterstained with haematoxylin. a) Control mAbs: in animals pre-treated with control mAbs, 14 days infection with S. aureus-induced peribronchial tertiary lymphoid structures containing CD20+ B-cell areas with follicular dendritic cell (FDC) and germinal centres, as well as CD3+ T-cell areas. b) CD20+ B-cell depletion: pre-treatment with anti-CD20 mAb prevented B-cell and FDC recruitment, and germinal centre formation. T-cells were unaffected. c) CD4+ and/or CD8+ T-cell depletion: pre-treatment with anti-CD4/CD8 mAbs strongly reduced CD3+ T-cell recruitment; CD20+ B-cells and FDCs were diminished, and germinal centres were absent. d) CD20+ B and CD4+/CD8+ T-cell depletion: pre-treatment with anti-C20 and anti-CD4/CD8 mAbs prevented the recruitment of CD20+ B-cells and FDCs, and the formation of germinal centres. CD3+ T-cells were reduced. Original magnification ×100. Scale bar=100 μm.
Quantitative morphometric analyses of B-cell-, T-cell- and follicular dendritic cell (FDC)-containing lymphoid aggregates in the lungs of mice persistently infected with Staphylococcus aureus and treated with anti-CD20, anti-CD4 plus CD8 monoclonal antibodies (mAbs) or all three mAbs. Mice were pre-treated with control mAb or with anti-CD20 and/or anti-CD4/CD8 mAbs prior to infection. Persistent infection was obtained by intratracheal instillation of agarose beads containing S. aureus (106 CFU per animal). 14 days after instillation, animals were euthanised and lungs were harvested for histological analysis. Sections were immunostained with antibodies directed against B-lymphocytes (CD20+), FDCs (CD21+) or T-lymphocytes (CD3+). Lymphoid aggregates were counted using morphometric analysis as described in the methods section. a) CD20+ B-cell depletion: pre-treatment with anti-CD20 mAb significantly reduced the number of CD20+ and FDC+ lymphoid aggregates; CD3+ T-cell lymphoid aggregate number was unaffected; b) CD4+ and CD8+ T-cell depletion: pre-treatment with anti-CD4/CD8 mAbs significantly reduced the number of CD3+ T-cell-containing lymphoid aggregates; CD20+ B-cell- and FDC-containing lymphoid aggregate numbers were also significantly reduced. c) CD20+ B- and CD4+/CD8+ T-cell depletion: pre-treatment with anti-CD20 and anti-CD4/CD8 mAbs significantly reduced the number of CD20+ and FDC+-containing lymphoid aggregates, as well as the number of CD3+ T-cell+ lymphoid aggregates. Each symbol represents data obtained from one animal. Horizontal bars correspond to median values. The Mann–Whitney test was used to compare depleted groups to controls. *: p<0.05, **: p<0.01, ****: p<0.0001 compared to controls. ns: nonsignificant.
Pre-treatment with anti-CD4/CD8 mAbs prior to S. aureus infection markedly reduced the presence of CD3+ T-cells, although small peribronchial CD3+ (presumably CD4−/CD8−) T-cell aggregates were found around bead-containing airways (figure 2). Treatment with anti-CD4/CD8 mAbs also reduced HEV formation and abolished germinal centre formation (supplementary figures S4 and S5). Surprisingly, treatment with anti-CD4/CD8 mAbs reduced the presence of peribronchial CD20+ B lymphocytes and CD21+ FDC (figures 2 and 3b). Treatment with anti-CD4/CD8 mAbs significantly reduced both the number (figure 4a) and size (figure 4f) of S. aureus-induced CD20+ lymphoid aggregates by approximately three- and six-fold, respectively. To further explore respective roles of CD4+ and CD8+ T-cells in S. aureus-induced TLS formation, two groups of mice were pre-treated either with anti-CD4 or anti-CD8 mAbs prior to S. aureus infection. Pre-treatment with anti-CD4 or anti-CD8 mAbs did not reduce the number of CD20+ (figure 4a) or CD3+ (figure 4d) peribronchial lymphoid aggregates, the recruitment of FDCs (figure 4b) and the number of HEVs (figure 4e), but prevented germinal centre formation (figure 4c). However, injection of anti-CD4 mAb reduced the size of CD20+ peribronchial lymphoid aggregates (figure 4f).
Quantitative morphometric analyses of the effects of isolated versus combined CD4+ and CD8+ T-cell depletion on lymphoid neogenesis in the lungs of C57Bl/6 mice persistently infected with Staphylococcus aureus. Mice were treated with control monoclonal antibodies (mAbs) or with anti-CD4 and/or anti-CD8 mAbs prior to intratracheal instillation with S. aureus-containing beads. Animals were euthanised 14 days after instillation and lung were harvested for histological analyses. Sections were immunostained with antibodies to a) B-lymphocytes (CD20+), b) follicular dendritic cells (FDCs) (CD21+), c) germinal centres (proliferating cell nuclear antigen; PCNA+), d) T-lymphocytes (CD3+) or e) high endothelial venules (HEVs) (peripheral node adressin; PNAd+). f) Lymphoid aggregates were counted using morphometric analysis, as described in the methods section. Combined CD4+/CD8+ T-cell depletion induced a significant decrease in a) number and f) size of peribronchial CD20+ lymphoid aggregates, and reduced FDC recruitment and HEV and germinal centre formation. Pre-treatment with anti-CD4 or anti-CD8 mAbs did not reduce the number of CD20+ peribronchial lymphoid aggregates, the recruitment of FDC or the number of HEVs, but prevented germinal centre formation. However, injection of anti-CD4 mAb reduced the size of CD20+ peribronchial lymphoid aggregates. Each symbol represents data obtained from one animal. Horizontal bars correspond to the median. Vertical bars in figure 4f represent means and error bars represent standard deviations. The Kruskal–Wallis test corrected with Dunn's test was used to compare each depleted group to the controls. *: p<0.05, **: p<0.01, ***: p<0.001 compared to controls.
Finally, pre-treatment with anti-CD20 and anti-CD4/CD8 mAbs prior to S. aureus infection completely prevented lymphoid neogenesis. As expected, peribronchial B-cell presence as well as CD21+ FDC recruitment and germinal centre formation were abolished. Peribronchial CD3+ T-cell recruitment and HEV formation were also markedly reduced (figures 2 and 3c, supplementary figures S4 and S5).
Discussion
In the present study, we examined the roles of CD20+ B-lymphocytes and/or CD4+/CD8+ T-lymphocytes in antibacterial response and peribronchial lymphoid neogenesis in a mouse model of persistent S. aureus lung infection. Our results indicated that: 1) blockade of lymphoid neogenesis was not associated with increased mortality or increased lung bacterial load in mice infected with S. aureus for 14 days; 2) CD20+ B-lymphocytes, CD4+ and CD8+ T-lymphocytes were all required for lymphoid neogenesis and germinal centre formation; and 3) the absence of CD20+ B-lymphocytes had no effect on the development of CD3+ T-cell peribronchial aggregates, whereas CD4+/CD8+ T-cell depletion markedly reduced CD20+ B-cell aggregates. These findings provide further insights on mechanisms of bacteria-driven lymphoid neogenesis in the lungs and possible roles of TLS in chronic airway diseases.
Because lymphoid follicles harbouring characteristics of TLS developed as a consequence of persistent bacterial infection in mouse airways [6], we hypothesised that these TLS contributed to the control of lung infection. This hypothesis was reinforced by recent findings from Ladjemi et al. [15] who reported an increased IgA production by TLS during persistent P. aeruginosa infection in mice and found that bronchoalveolar lavage IgA were directed toward P. aeruginosa. However, disorganisation of lymphoid neogenesis using anti-CD20 and/or anti-CD4/CD8 mAbs did not result in reduced infection control, as we found no evidence of increased bacterial load and no evidence of decreased survival in these experiments. To the best of our knowledge, no other study has examined possible roles of TLS in the control of chronic airway bacterial infection. Previous studies have yielded somewhat different results on the possible anti-infective roles of TLS in the lungs [13]. Moyron-Quiroz et al. [16] first suggested that lung lymphoid follicles with characteristics of TLS contributed to infection control in mice lacking secondary lymphoid organs infected by influenza virus, presumably through maturation and selection of B-cells directed against influenza virus nucleoprotein [17]. Studies in mice lacking IL-23 or CXCR5 have also demonstrated that disorganised lung TLS are associated with poor protective immune response against Mycobacterium tuberculosis [18, 19], suggesting a role for TLS in M. tuberculosis containment. Eddens et al. [20] showed that Pneumocystis infection induced TLS containing germinal centres in the lung; studies in lymphotoxin (LT)α-deficient mice resulted in smaller, disorganised, lymphoid follicles lacking germinal centres, with lower numbers of proliferating B-cells. However, LTα-deficient mice were able to clear Pneumocystis at day 14 post-infection like wild-type C57Bl/6 mice [20], indicating that TLS were not required for Pneumocystis clearance. We suggest that bacteria-driven TLS may contribute to immune response (e.g. via IgA production [15]), but are not essential for controlling chronic bacterial infection in mouse lung.
Our study further provided insights on the roles of B- and T-cell subsets in the formation of bacteria-driven TLS in the lung. First, our data revealed that B-cells, CD4+ T-cells and CD8+ T-cells are all required for the formation of germinal centres, which are the hallmark of TLS in which antigen-specific adaptive immune responses can be initiated [21, 22]. Second, anti-CD20 mAb depletes B-cells in TLS without affecting T-cell aggregates, whereas combined treatment with anti-CD4/anti-CD8 mAbs reduced both the numbers of T-cell and B-cell aggregates. Mice depleted exclusively in CD4+ T-cells had comparable numbers of B-cell aggregates that were nevertheless smaller. These data are somewhat different to findings by Eddens et al. [20], who reported that depletion of CD4+ T-cells with an anti-CD4 mAb abrogates the organisation of B-cell follicles and impairs the accumulation and proliferation of activated B-cells. In the present S. aureus model, disorganisation of the B-cell follicles required depletion of both CD4+ and CD8+ T-cells, whereas isolated depletion of only CD4+ or CD8+ T-cells did not reduce the number of CD20+ peribronchial lymphoid aggregates, indicating that both CD4+ and CD8+ T-cells are involved in B-cell recruitment and/or proliferation in this model. These data also suggest that CD8+ T-cells are not involved in the recruitment of B-cells, but are important for the maturation of TLS marked by the appearance of germinal centres. Our data on CD8+ T-cell depletion parallel the report by Curtis et al. [23] that showed that depletion of CD8+ T-cells does not decrease the recruitment of immune cells. However, in the latter study, the impact of CD8+ T-cell depletion on TLS was not documented. We further speculate that reduction in the size of peribronchial lymphoid aggregates in S. aureus-infected animals pre-treated with anti-CD4 mAb is related to reduction in CD4+ T follicular helper cells, which have been described to support B-cell antigenic selection, survival and proliferation [24].
The present study has strengths and limitations. Our animal model is derived from the seminal model of Cash et al. [25], in which bacteria were embedded in agarose beads. This model mimics prolonged infection in CF airways where bacteria are often entrapped in mucus plugs in airway lumen [26, 27]. In this model, peribronchial lymphoid neogenesis was limited to focal areas where infected agarose beads are present [6]. Although the agarose bead model was developed with the aim of escaping the host immune environment in the lung [25, 28], Bragonzi et al. [29] have shown that bacteria are found as macrocolonies inside and outside the beads in the airway lumen, an observation that was also found in our studies (Lucile Regard and Régis Burgel, Institut Cochin and Université de Paris, Paris, France; unpublished data). Importantly, studies have shown that agarose bead-embedded bacteria proliferate within murine airways and that bacterial load can be decreased by treatment with antimicrobial agents [30, 31]. In experiments aimed at assessing the impact of lymphocyte depletion, treatment of mice with anti-CD20 antibody effectively targeted CD20+ B-cells, which were undetectable in blood and lungs of treated animals for ≥21 days. Protocols used for targeting T-cells were more complex to establish as they required repeated injection of anti-CD4 and anti-CD8 mAbs and did not eliminate CD3+/CD4−/CD8− T-lymphocytes. However, these protocols made it possible to examine the individual and combined contribution of CD4+ and CD8+ T-cells to lymphoid neogenesis.
Lymphoid follicles containing germinal centres are found in multiple chronic airway diseases, including severe asthma, severe COPD, and CF and non-CF bronchiectasis, but their roles remain incompletely understood. The presence of TLS within the lungs of patients with COPD have been associated with disease progression [32] and lung B-cells, as determined by gene expression profiling, showed consistent correlations with emphysema severity [33]. Studies have reported the major role of B-cell activating factor in the development of TLS and alveolar destruction in response to cigarette smoke exposure in mice [34], findings that are highly relevant to patients with COPD [32]. Furthermore, μMT mice, which lack B-cells, had neither lymphoid follicle nor emphysema after exposure to cigarette smoke [35]. In the latter study, the authors also found that B-cells are potent regulators of macrophage accumulation and macrophage-derived matrix metalloproteinase-12 production, contributing to emphysema development. Thus, the role of TLS in the development of emphysema is well established in animal models. Our experiments were designed to examine the role of TLS in infection control, but additional studies would be necessary to examine the possible contribution of TLS to airway remodelling in the context of persistent bacterial infection. An unproven, yet testable, hypothesis would be that TLS may contribute to the development of bronchiectasis.
In conclusion, our study suggests that peribronchial TLS that develop during persistent airway infection triggered by agarose-bead embedded bacteria are not essential for controlling persistent bacterial infection in the lung. It further shows that lymphoid neogenesis is a highly coordinated event in which CD20+ B-lymphocytes, and CD4+ and CD8+ T-lymphocytes are required for the formation of germinal centres where antigen-specific adaptive immune responses can be initiated via specific B-cell selection and proliferation. Future studies should concentrate on the lifespan of TLS within the airways and their possible roles on promoting airway remodelling in the context of chronic bacterial infection.
Supplementary material
Supplementary Material
Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author.
Supplementary material ERJ-00768-2020.Supplement
Shareable PDF
Supplementary Material
This one-page PDF can be shared freely online.
Shareable PDF ERJ-00768-2020.Shareable
Acknowledgements
We thank Genentech (San Francisco, CA, USA) for providing the anti-CD20 antibody that was used for B-cell depletion.
Footnotes
This article has an editorial commentary: https://doi.org/10.1183/13993003.04352-2020
This article has supplementary material available from erj.ersjournals.com
Conflict of interest: L. Regard has nothing to disclose.
Conflict of interest: C. Martin reports personal fees from Zambon, outside the submitted work.
Conflict of interest: J-L. Teillaud has nothing to disclose.
Conflict of interest: H. Lafoeste has nothing to disclose.
Conflict of interest: H. Vicaire has nothing to disclose.
Conflict of interest: M.Z. Ladjemi has nothing to disclose.
Conflict of interest: E. Ollame-Omvane has nothing to disclose.
Conflict of interest: S. Sibéril has nothing to disclose.
Conflict of interest: P-R. Burgel reports personal fees from AstraZeneca, Boehringer Ingelheim, Chiesi, Novartis, Pfizer, Vertex, Zambon and Insmed, outside the submitted work.
Support statement: This work was funded by Vaincre la Mucoviscidose and La Fondation du Souffle/Fonds de Recherche en Santé Respiratoire and by Legs Pascal Bonnet. Funding sources were not involved in the study design; the collection, analysis and interpretation of the data; the writing of the report and the decision to submit the article for publication. Funding information for this article has been deposited with the Crossref Funder Registry.
- Received March 19, 2020.
- Accepted October 11, 2020.
- Copyright ©ERS 2021
References
