MyD88 provides a protective role in long-term radiation-induced
lung injury
Willie J. Brickey
1
, Isabel P. Neuringer
2
, William Walton
3
, Xiaoyang Hua
2
, Ellis Y. Wang
3
,
Sushmita Jha
3
, Gregory D. Sempowski
4
, Xuebin Yang
5
, Suzanne L. Kirby
3,5
, Stephen L.
Tilley
2
, and Jenny P-Y. Ting
1,3
1
Department of Microbiology/Immunology, University of North Carolina at Chapel Hill
2
Department of Pulmonary and Critical Care Medicine, University of North Carolina at Chapel Hill
3
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill
4
Duke Human Vaccine Institute, Duke University, North Carolina
5
Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill,
North Carolina, USA
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.
Keywords
Radiation; inflammation; innate immunity; MyD88; lung injury
© 2012 Informa UK, Ltd.
Correspondence: Dr Jenny P-Y. Ting, 450 West Drive, Campus Box #7295, 22-004 Lineberger Comprehensive Cancer Center,
University of North Carolina at Chapel HillChapel Hill, NC 27599, USA. Tel: + 1 919 966 5538. Fax: + 1 919 966 8212.
jenny_ting@med.unc.edu.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the
paper.
NIH Public Access
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Int J Radiat Biol
. Author manuscript; available in PMC 2013 April 18.
Published in final edited form as:
Int J Radiat Biol
. 2012 April ; 88(4): 335–347. doi:10.3109/09553002.2012.652723.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Introduction
The lung is a highly radiosensitive organ which represents a prime tissue targeted for
damage due to irradiation (Chen et al. 2002). Direct injury of the endothelial and epithelial
tissues in the lung is a crucial clinical outcome of irradiation, leading to acute pneumonitis
and delayed fibrosis. Radiation pneumonitis is an early response to radiation injury,
resulting from immune and lung parenchymal cell interactions, accompanied by
proinflammatory cytokines and chemokines (Chen et al. 2002, Ao et al. 2009), while fibrosis
is manifested with late lung injury and/or unresolved inflammatory responses. Both cells of
the adaptive immune system, such as thymic-derived lymphocytes (T cells), and of the
innate immune system, such as inflammatory granulocytes or macrophages, have been
implicated in radiation-induced injury (Roberts et al. 1993, Nakayama et al. 1996, Miura et
al. 2000). A role for immune regulation in radiation pneumonitis has been demonstrated by:
(i) The correlation of enhanced proinflammatory cytokines, specifically of Interleukins one
alpha and six (IL-1α and IL-6), with the incidence of radiation pneumonitis (Chen et al.
2002); (ii) the association of prior thymectomy and a lowered incidence of radiation
pneumonitis (McBride and Vegesna 2000); (iii) an increased T cell infiltrate in patients who
later develop radiation pneumonitis compared to those who did not (Roberts et al. 1993,
Nakayama et al. 1996); and (iv) an association of decreased Tumor necrosis factor alpha
(TNFα) caused by treatment with pentoxifylline with ameliorated radiation pneumonitis
(Rube et al. 2002).
Radiation injury is sustained as a consequence of radiotherapy for cancer as well as by
incidental exposure in the case of nuclear accidents or even bioterrorist acts. In either case,
protective measures are important for preventing lethality and for providing amelioration of
off-target effects to non-diseased cells or tissues. Activation of Toll-like receptor (TLR)
signaling, namely toll-like receptor 5 (TLR5) through the exposure to flagellin, has been
reported to be very effective in protecting cells, mice and non-human primates from
radiation-induced lethality (Burdelya et al. 2008, Vijay-Kumar et al. 2008). It is becoming
increasingly important to understand the protective roles supported by TLR signaling in
order to develop immunotherapies that synergize with radio- and chemotherapeutic
approaches for the treatment of cancer patients (Roses et al. 2008).
TLR signaling in mammals has rapidly emerged as a major route by which the immune
system responds to microbial pathogens, pharmacologics, synthetic compounds as well as
non-infectious stress insults through activation and regulation of cell proliferation and
differentiation programs (Medzhitov and Janeway 2000, Akira and Takeda 2004, Beutler
2004, Jiang et al. 2006, McGettrick and O’ Neill 2007). TLR activation requires a host of
intracellular adaptor proteins proximal to the TLR, with the most prominent being Myeloid
differentiation primary response factor 88 (MyD88) (Cao et al. 1996, Hacker et al. 2000,
Wang et al. 2001). With the exception of a subset of toll-like receptors 4 and 3 (TLR4 and
TLR3) stimulations in vivo, all TLR studied to date display MyD88-dependent signaling;
thus an analysis of mice lacking the MyD88 molecule provides a targeted assessment of the
role of TLR signaling in a specific biologic outcome. Although previous human
(Liebermann and Hoffman 2003, Li 2004) and animal studies with
Myd88
-null mice have
demonstrated roles of this molecule in atherosclerosis (Bjorkbacka et al. 2004), cancer
adjuvant therapy (Akazawa et al. 2004), onset of autoimmune disorder, and allotransplant
rejection (Goldstein et al. 2003), the in vivo role of MyD88 in radiation-induced pulmonary
injury has not been studied.
Supported by the evidence for roles of adaptive and innate immune responses in radiation-
induced pneumonitis, the focus of this report is to determine the role of MyD88, a specific
key regulator of immunity, in radiation-induced injury. This molecule controls innate
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immunity and proinflammatory responses through its mediation of intracellular signaling
(Kenny and O’ Neill 2008). It is a cytosolic adaptor molecule that acts as an intermediary
between activated TLR, interleukin 1 or 18 receptors (IL-1R/IL-18R) and other signaling
molecules that are proximal to the membrane. MyD88 contains two prominent domains, the
T oll-like receptor/I nterleukin 1 Receptor (TIR) domain and a death domain (Xu et al.
2000). The TIR domain consists of an alpha-beta fold and forms a complex with its
associative membrane receptors (TLR, IL-1R and IL-18R) when the latter become activated
by their respective agonist or ligand. Upon activation, the death domain of MyD88 interacts
with the death domain of IL-1 receptor-associated kinase-1 (IRAK1). This results in the
recruitment of a membrane proximal complex consisting of TNF receptor associated factor 6
(TRAF6), IL-1 receptor-associated kinases-1, 2, or 4 (IRAK1/2/4) and transforming growth
factor-β-activated kinase-1 (TAK-1). Further downstream mediators include nuclear factor-
κ B-inducing kinase (NIK), Inhibitor of nuclear factor-κ B kinase (IKK), Mitogen-activated
protein kinase kinase 1 (MEKK1), and c-jun N-terminal kinase (JNK) that lead to the
endpoint regulators of gene activation, namely nuclear factor-kappaB (NF-κB) and
Mitogen-activated protein kinase (MAPK)-induced activities. NF-κB and MAPK regulate
downstream genes that include an array of cytokines, chemokines, growth factors and cell
surface molecules which can significantly influence innate and adaptive immunity.
The pivotal role MyD88 plays in transmitting intracellular signals from activated innate
immune receptors to the transcriptional regulator NF-κB suggests that elimination of this
gene will significantly alter immune responses to danger, stress or pathogenic stimuli.
Although architectural and physiologic changes in lungs have long been recognized as
adverse events due to radiation exposure, immune mechanisms regulating and/or protecting
against injury are less understood (Abid et al. 2001, Hill 2005, Graves et al. 2010). Here, the
pulmonary consequences of loss of MyD88 to radiation- induced stress have been tested by
treating
Myd88
-null animals with a single high dose of gamma (γ)-irradiation to the thorax.
To discern contributions of MyD88 to radiation-induced pulmonary injury and/or repair
during acute and late lung injury phases, the following outcomes were monitored: Survival,
pathology and inflammatory factors (Williams et al. 2010). Mice lacking MyD88 were more
susceptible to death due to thoracic exposure to irradiation and displayed fibrosis hallmarks
such as increased collagen deposition and dysregulated cytokines. Based solely on
examination of gene-deficient mice, these initial results suggest that not only is MyD88
important for homeostasis (Nagai et al. 2006, McGettrick and O’ Neill 2007), but it also
appears to confer a protective role against radiation-induced lung injury. This initial
characterization should pave the way for further study, possibly using cell specific targeting
of MyD88 function as well as biochemical approaches, to identify MyD88 as a critical
molecule for radiation countermeasures.
Materials and methods
Animals
Male mice, aged 8–12 weeks old, were used in these studies. C57BL/6 mice (or wild type,
WT) originally from Jackson Laboratory (Bar Harbor, ME, USA) and bred at The
University of North Carolina at Chapel Hill (UNC-CH), NC, USA along with congenic
Myd88
−/−
mice (originally from Dr Shizou Akira, Osaka University, Osaka, Japan)
backcrossed to C57BL/6 mice for at least nine generations from Dr Donald Cook, National
Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC, USA
were examined. All animals were housed in an Association for the Assessment and
Accreditation of Laboratory Animal Care accredited pathogen-free facility. All animals were
treated humanely according to National Institutes of Health (NIH) guidelines and procedures
approved by UNC-CH’s Institute of Animal Care and Use Committee.
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Radiation-induced injury model
Mice received a single dose of thoracic-targeted irradiation (~ 14 gray [Gy]) using a
collimator (Model 335) with a
137
Cesium γ-ray source (Model Mark I-68; J.L. Shepherd &
Associates, San Fernando, CA, USA) at a 1.7 Gy/min dose rate. Prior to irradiation, mice
were anesthetized with tribromoethanol (Sigma-Aldrich Corp., St Louis, MO, USA) and
then loosely immobilized over a 2.9 cm
2
opening in a lead block (manufactured by UNC-
CH Radiology). The treated mice as well as untreated age-matched controls were housed in
cages in pathogen-free rooms and given standard laboratory chow and water
ad libitum
.
Routine serological testing of sentinel animals housed alongside the irradiated animals
revealed the absence of mites, pinworms, Mycoplasma pulmonis, and a host of viruses,
including epizootic diarrhea of infant mice (EDIM), Theiler’s murine encephalomyelitis
virus (TMEV GDVII strain), Mouse Hepatitis Virus (MHV), Murine minute virus (MMV),
Murine parvovirus (MPV) and non-structural protein-1 (NS-1) antigen, Pneumonia Virus of
Mice (PVM), and Sendai virus. Experimental and control mice were sacrificed at designated
times up to 27 weeks post irradiation (wpi) for analyses.
Peripheral blood assessment
Hematological tests to determine abundance of lymphocytes, granulocytes and monocytes in
the white blood cell (WBC) fraction on ethylenediaminetetraacetic acid (EDTA)-whole
blood collected by retro-orbital or submandibular bleeding were performed using an Animal
Blood Counter (Heska, Loveland, Colorado, USA) in the Animal Clinical Chemistry
Facility at UNC-CH.
Lung histopathology
Harvested lung tissues were inflated with 10% formalin (Sigma-Aldrich), paraffin-
embedded and sectioned (5 micron) at the Animal Histopathology Core Facility at UNC-
CH. Prepared sections of intact lobe were stained with hematoxylin and eosin (H&E),
Masson’s trichrome (MT) (Sigma-Aldrich) or Picosirius Red (SR) (Polysciences Inc,
Warrington, PA, USA) and viewed by light microscopy (ScanScope microscope, Aperio
Technologies, Vista, CA, USA). Several pathologic features plus degree of cellularity were
assessed by reviewers unaware of the identity of each sample. Each interstitial focal
infiltrate found in the lung section was graded according to the following scale: 0, no
apparent abnormality; 1, cellularity encompassing an area < 25 alveolar spaces; 2, an area
between 25–50 alveolar spaces; 3, an area between 50–100 alveolar spaces; and 4, an area ≥
100 alveolar spaces. The corresponding grades for all the foci per lung sample were
combined to give a cellularity score. Collagen content was determined indirectly by
assessing hydroxyproline levels in the right lung (Smith et al. 1994, Keane et al. 1999) or by
SR staining (Junqueira et al. 1979), where images of seven randomly chosen fields localized
to the subpleural regions were captured using a polarizing filter on a BX61 microscope
(Olympus, Center Valley, PA, USA), with averaged pixels of polarized light determined by
ImageJ analysis (available at NIH via http://rsbweb.nih.gov/ij/). Immunohistochemistry was
performed on deparaffinized and re-hydrated lung tissue sections to examine T cells
(hamster anti-mouse T cell receptor beta or TCRβ antibody conjugated with phycoerythrin,
1:200 dilution, H57-597 clone; Beckman Coulter Incorporated, Miami, FL, USA), and
myeloid cells (rat anti-mouse Integrin alpha M or cell differentiation marker 11b, CD11b,
antibody conjugated with allophycocyanin, 1:200 dilution, M1/70 clone; eBioscience, San
Diego, CA, USA) as described (Arnett et al. 2002, Jha et al. 2010). Sections were mounted
in VECTASHIELD
®
with 4′, 6-diamidino-2-phenylindole (DAPI) (Vector Laboratories,
Burlingame, CA, USA) and viewed using an Olympus BX-40 microscope fitted with DP72
camera. Images were captured using cellSens
®
image acquisition software (Olympus) and
immunopositive cells with an observable DAPI-stained nucleus were counted in 5–7
randomly chosen fields per lung section.
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Ribonucleic acid (RNA) profiling
Total RNA was generated from lung homogenates using Trizol (Invitrogen Corporation,
Carlsbad, CA, USA), and converted into complementary deoxyribonucleic acid (DNA)
using random hexamers and Moloney Murine Leukemia Virus-Reverse Transcriptase
(MMLV-RT) (Invitrogen), according to manufacturer’s instructions. Then, polymerase
chain reaction (PCR) amplification was performed using gene specific primers with probes
or with cyanine dye (i.e., SYBR green) incorporation using a 7900T Fast Real-Time PCR
System (Applied Biosystems Incorporated or Life Technologies Corporation, Carlsbad, CA,
USA). Primer sequences included: ribosomal RNA (i.e., 18 Svedberg unit, 18S rRNA)
(tetrachlorofluorescin or TET-CAAATTACCCACTCCCGACCCG- black hole quencher or
BH, 5′-GCTGCTGGCACCAGA CTT-3′, 5′-CGGCTACCACATCCAAGG-3′);
Transforming growth factor beta (
Tgfb)
(6-carboxyfluorescein or
FAMACCTTGGTAACCGGCTGCTGACC -tetramethylrhodamine quencher or TAMRA,
5′-GGAACTCTACCAGAAA TATAGC-3′, 5′-CACTCAGGCGTATCAGTG-3′);
Forkhead box P3 (FoxP3) (FAM-ATCCTACCCACTGCTGGCAAATGGAGTC-TAMRA,
5′-CCCAGGAAAGACAGCAACCTT-3′, 5′-TTCTCACAACCA GGCCACTTG-3′);
Interleukin 1beta (
Il1b)
(5′-CTCATTGT GGCTGTGGAGAAG-3′, 5′-
ACCAGCAGGTTATCATCATCAT-3′); Interleukin 6 (
Il6)
(5′-
TACCATAGCTACCTGGAGTACAT-3 ′, 5 ′-CTGTGACTCCAGCTTATCTGTTA-3 ′).
Commercial primer/probe sets (Life Technologies Corporation) for Chemokine (C-C motif)
ligand 2 or Monocyte chemoattractant protein 1 (
Ccl2/Mcp1;
Mm00441242_m1);
Chemokine (C-X-C motif) ligand 1 or platelet-derived growth factor-inducible KC protein
(
Cxcl1/KC;
Mm00433859_m1); Endothelin 1 (
Edn1;
Mm00438656_m1); Hyaluronan
synthase 2 or 3 (
Has2;
Mm00515089_m1 or
Has3;
Mm00515091_m1); Intercellular
adhesion molecule (
Icam1;
Mm00516023_m1); Matrix metalloproteinase 2 (Mmp2;
Mm00439506_m1); Myeloperoxidase (
Mpo;
Mm01298424_m1); and extracellular
superoxide dismutase 3 (
Sod3;
Mm00448831_m1) were utilized. Genespecific transcripts
were first normalized to 18S rRNA transcripts for each sample and then ratios of normalized
RNA in treated samples to untreated controls were generated. Fold change in expression
from untreated samples are reported.
Protein profiling
Serum was prepared from blood taken by cardiac puncture and profiled using the Mouse
Cytokine 20-Plex Panel (Invitrogen). Mean protein ± standard error of the mean (SEM) (pg/
ml) are reported with each group consisting of at least three biological replicates (
n
= 3–5).
Lung physiology assessments
Physiologic measures of lung function in mice surviving for more than 18 wpi were assessed
as described (Irvin and Bates 2003, Lovgren et al. 2006, Glaab et al. 2007). Briefly, mice
were anesthetized, tracheostomized, and mechanically ventilated using a computer-
controlled small animal ventilator or FlexiVent (SCIREQ Incorporated, Montreal, Quebec,
Canada). Pressure-volume curves were used to determine static compliance (Cst) and
oscillatory mechanics were used to determine tissue elastance (H) of the lungs of treated and
control mice.
Statistics
Student’s
t-
test and Mann-Whitney test were used to contrast results for WT and
M yd88
−/−
at similar conditions as indicated by dotted lines, while solid lines represent analysis by one
way analysis of variance (ANOVA) for a single genotype over time. Differences with
P
values < 0.05 were considered significant. Kaplan-Meier survival curves were generated
using Prism (GraphPad Software Inc., La Jolla, CA, USA).
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