Rapamycin prevents bronchiolitis obliterans through increasing infiltration of regulatory B cells in a murine tracheal transplantation model

Abstract

Objective: B lymphocytes are generally considered to be activators of the immune response; however, recent findings have shown that a subtype of B lymphocytes, regulatory B lymphocytes, play a role in attenuating the immune response. Bronchiolitis obliterans remains the major limitation to modern-day lung transplantation. The role of regulatory B lymphocytes in bronchiolitis obliterans has not been elucidated. We hypothesized that regulatory B lymphocytes play a role in the attenuation of bronchiolitis obliterans.

Methods: We performed a standard heterotopic tracheal transplant model. Tracheas from Balb/c mice were transplanted into C57BL/6 recipients. Rapamycin treatment and dimethyl sulfoxide control groups were each treated for the first 14 days after the transplant. Tracheas were collected on days 7, 14, and 28 post-transplantation. Luminal obliteration was evaluated by hematoxylin-eosin staining and Picrosirius red staining. Immune cell infiltration and characteristics, and secretion of interleukin-10 and transforming growth factor-β1 were accessed by immunohistochemistry. Cytokines and transforming growth factor-β1 were measured using the Luminex assay (Bio-Rad, Hercules, Calif).

Results: The results revealed that intraperitoneal injection of rapamycin for 14 days after tracheal transplantation significantly reduced luminal obliteration on day 28 when compared with the dimethyl sulfoxide control group (97.78% ± 3.63% vs 3.02% ± 2.14%, P < .001). Rapamycin treatment markedly induced regulatory B lymphocytes (B220(+)IgM(+)IgG(-)IL-10(+)TGF-β1(+)) cells when compared with dimethyl sulfoxide controls. Rapamycin treatment inhibited interleukin-1β, 6, 13, and 17 on days 7 and 14. Rapamycin also greatly increased interleukin-10 and transforming growth factor-β1 production in B cells and regulatory T lymphocytes infiltration on day 28.

Conclusions: Mammalian target of rapamycin inhibition decreases the development of bronchiolitis obliterans via inhibition of proinflammatory cytokines and increasing regulatory B lymphocytes cell infiltration, which subsequently produces anti-inflammatory cytokines and upregulates regulatory T lymphocyte cells.

Keywords: FoxP3β regulatory T cells; bronchiolitis obliterans; mouse heterotopic tracheal transplant; rapamycin; regulatory B lymphocytes.

Conflict of interest statement

Disclosure: There is no conflict of interest to disclose.

Published by Elsevier Inc.

Figures

Figure 1. Rapamycin (Rapa) treatment reduces luminal obliteration, but increases cell infiltration

A Intraperitoneal injection of Rapa significantly reduced luminal obliteration of the allografts on day 28 post tracheal transplantation. Data shown are the mean ± SE, n=6. B Representative pictures of cellular infiltration in the allografts and isograft (Iso) controls from 28 days post-transplantation. The magnification of all the pictures was 40× and 400×. Black arrow indicates infiltrated cells. The donors are Balb/c mice and the recipients are C57BL/6 mice. C57BL/6 to C57BL/6 isograft controls are also performed.

Figure 2. Immunohistochemical staining of macrophages, neutrophils, CD3+ T cells, and B220+ B cells in the rapamycin (10mg/kg/day) treated allografts on day 28 post tracheal transplantation

A, B, C, and D are representative immunostaining picture of macrophage, neutrophils, CD3+ T cells, and B220+ B cells, respectively. Red color/solid arrows indicate positive staining cells in A and B. Brown/dark brown/solid arrows indicate T and B cells in C and D. All sections were counterstained lightly with hematoxylin for viewing negatively stained cells. The magnifications are indicated in the pictures.

Figure 3. Increasing of B220+ B cells in rapamycin (Rapa) treated allografts on day 28 post transplantation

A Representative pictures of B220 staining in the allografts and isograft (Iso) controls from 28 days post-transplantation. Brown color/Solid arrows indicate B220+ cells. The magnification is 400×. B Comparison of B220+ B cell infiltration in the Rapa treated allograft with the isograft (Iso) and DMSO control allografts on day 28 post transplantation. Data shown are the mean ± SD *, n=6.

Figure 4. Identification of B220, IL-10, IgM and TGF-b1 positive regulatory B cells (Bregs) in the Rapa treated allografts on day 28 post transplantation

A Double immunofluorescence staining of IgM and IgG in the Rapa treated allografts on day 28. Red indicates IgM positive B cells in A-a and A-d. Green indicates IgG positive B cells in A-c and A-d. Blue is DAPI staining for viewing negative staining cells in A-b and A-d. B Representative immunohistochemical staining of B220, IL-10 and TGF-β1 in the Rapa treated allografts. Cells stained brown indicate positive staining cells. The arrows indicated B220+ cells in B-a, IL-10 + cells in B-b, TGF- β1+ cells in B-c and -d. The magnifications were 400× in all pictures.

Figure 5. Comparison of Effects of Rapa on IL-10+ (A) and TGF-β1+ (B) cells in the allografts with DMSO controls on day 28

The number of positive staining cells per high power field (HPF) was assessed by Photoshop counting tool, and at least 5 fields were counted per trachea. Data shown are the mean ± SD, n=6.

Figure 6. The Levels of IL-10 (A) and IL-4 (B) in the allografts and isografts on day 28

Luminex assay was performed using the Bio-Plex 200 system with standard controls. The concentration is pg/ml. Data shown are the mean ± SD, n=6.

Figure 7. Comparison of Effects of Rapa on FoxP3+ regulatory T cells (Tregs) in the allografts with DMSO controls on day 28

A Representative immunohistochemical staining of FoxP3+ Tregs in the treated allografts. Cells stained brown indicate positive staining cells (marked by arrows). The magnifications are 400×. B Statistical analysis of Tregs cells in Rapa treated and DMSO control in the allografts. The number of positive staining cells per high power field (400×) was assessed by Photoshop counting tool, and at least 5 fields were counted per trachea. Data shown are the mean ± SD, n=6.