Persistence of pulmonary tertiary lymphoid tissues and anti-nuclear antibodies following cessation of cigarette smoke exposure

Abstract

Formation of pulmonary tertiary immune structures is a characteristic feature of advanced COPD. In the current study, we investigated the mechanisms of tertiary lymphoid tissue (TLT) formation in the lungs of cigarette smoke-exposed mice. We found that cigarette smoke exposure led to TLT formation that persisted following smoking cessation. TLTs consisted predominantly of IgM positive B cells, while plasma cells in close proximity to TLTs expressed IgM, IgG, and IgA. The presence of TLT formation was associated with anti-nuclear autoantibody (ANA) production that also persisted following smoking cessation. ANAs were observed in the lungs, but not the circulation of cigarette smoke-exposed mice. Similarly, we observed ANA in the sputum of COPD patients where levels correlated with disease severity and were refractory to steroid treatment. Both ANA production and TLT formation were dependent on interleukin-1 receptor 1 (IL-1R1) expression. Contrary to TLT and ANA, lung neutrophilia resolved following smoking cessation. These data suggest a differential regulation of innate and B cell-related immune inflammatory processes associated with cigarette smoke exposure. Moreover, our study further emphasizes the importance of interleukin-1 (IL-1) signaling pathways in cigarette smoke-related pulmonary pathogenesis.

Figure 1

Chronic cigarette smoke exposure leads to pulmonary tertiary lymphoid tissue (TLT) formation that persists following smoking cessation. BALB/c mice were exposed to room air (open bars) or cigarette smoke (solid bars) for (A) 8 weeks ± 20 days of smoking cessation or for (B) 24 weeks ± 60 and 120 days of smoking cessation. Lung sections were stained with hematoxylin and eosin (H&E). (C) Pulmonary TLT were quantified using Image J software. Data represent means ± SEM; n = 5 per group. *p < 0.05.

Figure 2

Plasma cells are found in close proximity with pulmonary tertiary lymphoid tissues (TLT) that mainly contain naïve B cells. BALB/c mice were exposed to cigarette smoke for 24 weeks and lung sections were stained by immunochemistry for (A) B cells (B220), (C) IgM, (F) IgG, (H) IgA and (J-K) plasma cells (CD138). (B) Tight clusters of B220 expressing B cells. (D) Cells expressing membrane-bound IgM within a pulmonary TLT. (E) IgM-, (G) IgG- and (I) IgA-secreting cell (cytoplasmic staining) found adjacent to pulmonary TLTs. (J) Absence of plasma cells inside pulmonary TLT which are generally found near pulmonary TLT or (K-L) vessels.

Figure 3

Cigarette smoke exposure elicits pulmonary but not systemic anti-nuclear antibodies production. BALB/c mice were exposed to room air (open bars) or cigarette smoke (solid bars) for 4 days, 8 weeks ± 20 days of smoking cessation or for 24 weeks ± 60 and 120 days of smoking cessation. ANA levels were measured by ELISA in the (A) bronchoalveolar lavage fluid (BALF) and antibody isotypes dissected in mice exposed for (B) 8 weeks and (C) 24 weeks. (D) ANA levels were also measured in the serum of mice exposed for 8 weeks ± 20 days of smoking cessation or for 24 weeks ± 60 and 120 days of smoking cessation. (E) ANA levels were measured in the sputum of different GOLD stage subjects (see Tables 1 and 2 for complete description) and expressed as fold increase of GOLD 0/1 subjects. (A and D) Data represent means ± SEM; n = 5-10 mice per group. *p < 0.05. (B and C) Data represent the values obtained from pooled samples from each group. Fold changes were calculated by using the average O.D. value of the corresponding room air-exposed group as baseline expression (fold increase of 1).

Figure 4

Resolution of lung inflammation is delayed following chronic cigarette smoke exposure. BALB/c mice were exposed to room air (open bars) or cigarette smoke (solid bars) for (A) 4 days ± 3 days of smoking cessation, (B) 8 weeks ± 20 days of smoking cessation or for (C) 24 weeks ± 60 and 120 days of smoking cessation. Data show total cell number, mononuclear cells and neutrophils in the BAL. Bars represent means ± SEM; n = 5 per group. *p < 0.05.

Figure 5

Neutrophil recruitment, pulmonary tertiary lymphoid tissue development and increased ANA levels are dependent on IL-1R1 expression. C57BL/6 and IL-/R1^-/- mice were exposed to room air (open bars) or cigarette smoke (solid bars) for 24 weeks. (A) Lung sections were stained with hematoxylin and eosin (H&E). (B) Total neutrophils number was assessed in the BAL. (C) Pulmonary TLT were quantified using Image J software. (D) Pulmonary ANA and E) CXCL13 levels were measured in the BALF by ELISA. Data represent means ± SEM; n = 5 per group. *p < 0.05, **p < 0.01.

Figure 6

Kinetic of the innate and adaptive responses induced by cigarette smoke exposure and the impact of smoking cessation. Resolution of the innate immune response (macrophages and neutrophils) measured in the BAL is markedly delayed when preceded by chronic exposure but can happen. Features of adaptive immune response (pulmonary TLT formation and elevated ANA) requires chronic exposure to be initiated but does not resolve following cessation. Moreover, the delay in the resolution of the innate immune response is associated with the activation of the adaptive immune response.