Innate immune activation by the viral PAMP poly I:C potentiates pulmonary graft-versus-host disease after allogeneic hematopoietic cell transplant

Abstract

Respiratory viral infections cause significant morbidity and increase the risk for chronic pulmonary graft-versus-host disease (GVHD) after hematopoietic cell transplantation (HCT). Our overall hypothesis is that local innate immune activation potentiates adaptive alloimmunity. In this study, we hypothesized that a viral pathogen-associated molecular pattern (PAMP) alone can potentiate pulmonary GVHD after allogeneic HCT. We, therefore, examined the effect of pulmonary exposure to polyinosinic:polycytidylic acid (poly I:C), a viral mimetic that activates innate immunity, in an established murine HCT model. Poly I:C-induced a marked pulmonary T cell response in allogeneic HCT mice as compared to syngeneic HCT, with increased CD4+ cells in the lung fluid and tissue. This lymphocytic inflammation persisted at 2 weeks post poly I:C exposure in allogeneic mice and was associated with CD3+ cell infiltration into the bronchiolar epithelium and features of epithelial injury. In vitro, poly I:C enhanced allospecific proliferation in a mixed lymphocyte reaction. In vivo, poly I:C exposure was associated with an early increase in pulmonary monocyte recruitment and activation as well as a decrease in CD4+FOXP3+ regulatory T cells in allogeneic mice as compared to syngeneic. In contrast, intrapulmonary poly I:C did not alter the extent of systemic GVHD in either syngeneic or allogeneic mice. Collectively, our results suggest that local activation of pulmonary innate immunity by a viral molecular pattern represents a novel pathway that contributes to pulmonary GVHD after allogeneic HCT, through a mechanism that includes increased recruitment and maturation of intrapulmonary monocytes.

Figure 1. Increased and persistent bronchoalveolar lavage (BAL) T cells in allogeneic mice 72 hours and 2 weeks after poly I:C exposure

BAL cells were evaluated by flow cytometry at both 72 hours (n = 6 mice/group) and two weeks (n = 3 mice/group) following poly I:C exposure in both allogeneic and syngeneic HCT mice. (A) At 72 hours, allogeneic mice exposed to poly I:C (Allo+poly I:C) had significantly more BAL total cells than either unexposed allogeneic mice (Allo+control) (p = 0.001) or poly I:C-exposed syngeneic mice (Syn+poly I:C) (p = 0.04). (B) Closely paralleling the increase in total BAL cells, CD3+ cells in Allo+poly I:C BAL were elevated above both unexposed allogeneic mice (Allo+control) (p = 0.007) and poly I:C-exposed syngeneic mice (Syn+poly I:C) (p = 0.02). (C) CD4+ BAL cells also increased in Allo+poly I:C BAL as compared to both Allo+control (p = 0.04) and Syn+poly I:C (p = 0.02) BAL while (D) CD8+ cells in allogeneic BAL increased following poly I:C exposure (p = 0.01) but did not differ significantly between Allo+poly I:C and Syn+poly I:C mice (p = 0.3). (E) Two weeks following poly I:C exposure, BAL total cells remained elevated in Allo+poly I:C BAL compared to both Allo+control (p = 0.02) and Syn+poly I:C (p = 0.01). (F) Allo+poly I:C BAL also continued to contain more CD3+ cells than both Allo+control (p = 0.005) and Syn+poly I:C (p = 0.003) controls. CD4+ and CD8+ BAL cells followed a similar pattern; Allo+poly I:C BAL contained (G) more CD4+ cells than both Allo+control (p = 0.003) and Syn+poly I:C (p < 0.001) and (H) more CD8+ cells than both Allo+poly I:C (p = 0.02) and Syn+poly I:C (p = 0.02).

Figure 2. Prominent peribronchiolar lymphocytic inflammation, epithelium-infiltrating CD3+ cells, and epithelial dysplasia in allogeneic HCT mice exposed to poly I:C (Allo+poly I:C)

Lungs were inflated and preserved two-weeks following poly I:C exposure. Representative sections photographed at 400x magnification are shown. H&E staining demonstrated minimal peribronchiolar cellular inflammation in both (A) unexposed syngeneic (Syn+control) and (B) unexposed allogeneic (Allo+control) lungs. Small, mononuclear cells in the peribronchiolar region were visible in sections from both (C) Syn+poly I:C and (D&E) Allo+poly I:C lungs, with more prominent cellular inflammation in Allo+poly I:C samples. (F) Additional immunohistochemistry revealed these peribronchiolar cells to be CD3-positive. Only Allo+poly I:C animals demonstrated epithelium-infiltrating CD3+ cells (black arrows) and epithelial dysplasia with several superimposed disorganized epithelial cell layers (white arrows) on both (E) H&E-stained and (F) CD3-stained sections. (G&H) Further digital enlargement of E&F (inset boxes) are representative images of infiltrating CD3+ cells (black arrows) and epithelial dysplasia (white arrows) seen throughout Allo+poly I:C lung tissue.

Figure 3. Decreased and disorganized β-catenin in allogeneic HCT mice exposed to poly I:C (Allo+poly I:C)

β-catenin transcripts were measured using RT-PCR in lung tissue. In addition, sections of preserved and paraffin-embedded lungs were deparaffinized and stained for β-catenin (red) and DAPI (white). Representative sections photographed at 400x magnification are shown. (A) Allo+poly I:C lungs contained fewer transcripts for β-catenin compared to unexposed allogeneic lungs (Allo+control) (p < 0.001) while β-catenin expression in syngeneic lungs was unaffected by poly I:C exposure (p = 0.94). In comparison to (B-D) control groups, (E) only Allo+poly I:C lungs displayed sections of decreased β-catenin staining (white brackets) and irregular β-catenin staining (blue bracket).

Figure 4. Increased BAL monocytes in allogeneic mice post poly I:C (Allo+poly I:C) with increased expression of activation markers

BAL myeloid cells were evaluated by flow cytometry 72 hours following poly I:C exposure in both allogeneic and syngeneic HCT mice (n = 6 mice/group). (A) The total numbers of monocytes increased in Allo+poly I:C BAL above both unexposed allogeneic recipients (Allo+control) (p = 0.001) and poly I:C-exposed syngeneic recipients (Syn+poly I:C) (p = 0.04). (B) The monocyte chemotactic protein-1 (MCP-1/CCL2) chemokine was significantly elevated in the BAL of Allo+poly I:C mice as compared to Allo+control (p = 0.0007) and Syn+poly I:C (p = 0.007). (C) Representative flow cytometric plots show the CD11b+ monocyte population as a percentage of all IAIE-negative Ly6G-negative cells for each experimental group. (D-G) In addition, markers of monocyte activation were increased: (D) Representative flow cytometric plots show the Gr1+ CD86+ double-positive activated population as a percentage of monocytes for each experimental group. This activated monocyte population was increased in the Allo+poly I:C mice as compared to controls. (E) the mean fluorescence intensity (MFI) of the fluorochrome conjugated to Gr1 was elevated on Allo+poly I:C monocytes compared to Allo+control (p < 0.001) and Syn+poly I:C (p < 0.001) monocytes, and (F) the MFI of the fluorochrome conjugated to CD86 was elevated on Allo+poly I:C monocytes compared to Allo+control (p = 0.002) and Syn+poly I:C (p < 0.001) monocytes. (G) Representative MFI histograms of Gr1 and CD86 on monocytes are shown (solid medium gray curve = unstained control; solid light gray curve = Syn+poly I:C; thick dark gray curve = Allo+poly I:C sample).

Figure 5. Stimulator cells from animals pre-treated with poly I:C in vivo exacerbate allogeneic proliferation in an MLR

Stimulator C57Bl/6J donors were injected intraperitoneally with either control media or 400 μg of poly I:C. On day 0, 5×10⁵ irradiated stimulator splenocytes were co-cultured with 5×10⁵ splenocytes from either allogeneic C3HB/FeJ or syngeneic C57Bl/6J mice. Following the addition of ³H-thymidine for the last 18 hours of culture, proliferation was measured on day 4 in counts per minute (cpm). Allogeneic splenocytes co-cultured with stimulators pretreated with poly I:C proliferated significantly more than both allogeneic splenocytes co-cultured with control stimulators (p < 0.001) and syngeneic splenocytes co-cultured with poly I:C pre-treated stimulators (p < 0.001). Error bars represent differences between triplicate wells.

Figure 6. Decreased regulatory T cells (Tregs) in allogeneic HCT animals compared to syngeneic controls at both 72-hours and two-weeks post poly I:C

CD3+ BAL cells were evaluated for CD4− and FOXP3-positivity (Tregs) using flow cytometry at both 72-hours (n = 6 mice/group) and two weeks (n = 3 mice/group) following poly I:C exposure in both allogeneic and syngeneic HCT animals. Both (A) representative samples and (B) collective data at 72 hours show the percentage of Tregs increased following poly I:C exposure in both allogeneic HCT animals (from 1.6% to 5.0%) (p = 0.02) and syngeneic HCT animals (6.3% to 13.4%) (p = 0.002). However, allogeneic BAL contained a significantly smaller percentage of Tregs than syngeneic BAL both at baseline (p = 0.005) and following poly I:C exposure (p < 0.001); (C) A similar deficit in allogeneic Tregs was observed at two weeks following poly I:C exposure both at baseline (p = 0.02) and following poly I:C exposure (p = 0.03).

Figure 7. Poly I:C does not exacerbate allogeneic GVHD

Two weeks following poly I:C exposure, samples from the gastroduodenal junction, ileum, cecum, colon, rectum, and liver were obtained. Sections of preserved and paraffin-embedded tissue were stained with H&E and scored on a semi-quantitative grading scale for signs of gastrointestinal GVHD by an experienced and blinded pathologist. Allogeneic animals developed significantly more gastrointestinal GVHD than their syngeneic counterparts (p < 0.001) but the severity was unaffected by poly I:C exposure (p =0.4).