MyD88 provides a protective role in long-term radiation-induced lung injury

Abstract

Purpose: The role of innate immune regulators is investigated in injury sustained from irradiation as in the clinic for cancer treatment or from a nuclear incident. The protective benefits of flagellin signaling through Toll-like receptors (TLR) in an irradiation setting warrant study of a key intracellular adaptor of TLR signaling, namely Myeloid differentiation primary response factor 88 (MyD88). The role of MyD88 in regulating innate immunity and Nuclear factor kappa-B (NF-κB)-activated responses targets this critical factor for influencing injury and recovery as well as maintaining immune homeostasis.

Materials and methods: To examine the role of MyD88, we examined immune cells and factors during acute pneumonitic and fibrotic phases in Myd88-deficient animals receiving thoracic gamma (γ)-irradiation.

Results: We found that MyD88 supports survival from radiation-induced injury through the regulation of inflammatory factors that aid in recovery from irradiation. The absence of MyD88 resulted in unresolved pulmonary infiltrate and enhanced collagen deposition plus elevated type 2 helper T cell (Th2) cytokines in long-term survivors of irradiation.

Conclusions: These results based only on a gene deletion model suggest that alterations of MyD88-dependent inflammatory processes impact chronic lung injury. Therefore, MyD88 may contribute to attenuating long-term radiation-induced lung injury and protecting against fibrosis.

Figure 1

MyD88 provides protection against mortality linked to exposure to thoracic targeted γ-irradiation. (A) Loss of MyD88 produces a strain susceptible to radiation-induced lung injury. A Kaplan-Meier survival analysis of irradiated Myd88^−/− (n = 25) or WT mice (n = 26) is shown. The median survival time (MST) for Myd88^−/− is 27.3 weeks post irradiation (wpi). Comparison of the survival curves using the log rank test (Mantel-Cox) reveals a significant difference (p < 0.05), with a hazard ratio of 2.237. Peripheral blood cell composition of white blood cells (WBC), lymphocytes (Ly), monocytes (Mo) and granulocytes (Gr) in mice at baseline (B) and after radiation (C) is similar between WT and Myd88^−/− mice. Cells in peripheral blood samples were quantified using Heska’s Animal Blood Counter. The average absolute numbers (in 1000s) are plotted with significant differences between WT (solid) and Myd88^−/− (open) at indicated times shown by dotted line with error bars indicating SEM for n = 7 per time for each genotype in (C) (*P < 0.05; u, untreated).

Figure 2

Cellularity in lungs of irradiated mice is enhanced during acute injury. (A) Representative images of H&E stained lung sections of WT (left) and Myd88^−/− (right) mice, untreated (unt) and weeks post irradiation (wpi). (Original magnification 200×) (B) The interstitial infiltrate of alveolar spaces in unirradiated (u) WT (circle) and Myd88^−/− (square) mice and at 4–6 wpi was scored by reviewers unaware of tissue identity. Individual samples are represented by each symbol with the averaged cumulative score represented by the bar. *P < 0.05 indicates significantly different means between WT and Myd88^−/− at 6 wpi (dotted line) and for Myd88^−/− over time (solid line). (C) Immunohistochemical analysis of lungs revealed elevated numbers of T lymphocytic (TCRβ⁺) and myeloid (CD11b⁺) cells in lungs of irradiated animals at 6 weeks. Representative images are shown. (Original magnification 200×, scale bar 100 micron) (D) Numbers of antibody-positive DAPI-stained cells were counted in 5–7 fields for each biological sample, with average CD11b⁺ or TCRβ⁺ cells/field per sample indicated by symbol and mean for each time plotted for untreated (u) samples and from 1.4–6 wpi.

Figure 3

Late lung injury persists in irradiated Myd88^−/− animals. (A) Fixed lung sections were stained with H&E and representative images are shown for untreated (unt) and samples from mice at 18–27 wpi. (Original magnification 40×). Foci of parenchymal damage are marked with dotted lines. Changes in lung wet weight (B) and body weight (C) were monitored. (D) Irradiated Myd88^−/− mice display impaired airway mechanics. The static compliance (Cst) and tissue elastance (H) properties were determined through pressure-volume loops and forced oscillatory mechanics. Each data point in B–D represents an individual animal (WT, solid circle; Myd88^−/−, open square). The mean for each parameter is represented by a bar, with significant differences over time indicated by the solid bar and between genotypes at designated times by the dotted bar (*P < 0.05; **P < 0.005; ***P < 0.0005).

Figure 4

Lungs in Myd88^−/− mice display fibrotic foci of collagen deposition. Fixed lung sections were stained with H&E (unt), Masson’s Trichrome (A) or Sirius Red (B) and representative images are shown for long term WT and Myd88^−/− radiation survivors (24 and 27 wpi) (Original magnification 200×, scale bar 100 micron). Myd88^−/− survivors show elevated levels of Sirius Red polarized signal (C) at 27 wpi over baseline and WT at similar times. The assessment of Sirius red signal was executed by reviewers unaware of the identity of the tissue section. Each data point represents an individual animal, with the mean indicated by a bar and significant differences between genotypes at designated times by the dotted bar (*P < 0.05).

Figure 5

Regulation of RNA expression of proinflammatory cytokines and chemokines is altered in irradiated Myd88^−/− animals during acute injury. Transcripts encoding proinflammatory cytokines (Il6, Il1b), chemokines (Ccl2/Mcp1, Cxcl1/KC) as well as cell activation factors (Mpo, Sod3, Tgfb, Icam1, Foxp3, Has2, Has3) were measured using gene-specific primers and quantitative PCR approaches. Mean fold change over untreated samples of transcript levels normalized to 18S rRNA are shown with error bars indicating SEM for n = 3–6 for each group. Changes in transcript levels in Myd88^−/− lung samples (open square) that are significantly different from WT samples (solid circle) are indicated (*P < 0.05; **P < 0.005; ***P < 0.0005).

Figure 6

Fibrosis-inducing and inflammation-suppressing cytokine levels in irradiated Myd88^−/− mice are elevated in serum. Proteins found in serum from WT (solid) or Myd88^−/− (open) mice either without (u) or with irradiation (13–27 wpi) were characterized using a multiplex protein immune assay. The mean protein levels (pg/ml) are reported with error bars indicating SEM for n = 3–5 for each group, and differences between WT and Myd88^−/− samples are indicated by dotted lines (*P < 0.05; **P < 0.005).