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Defense and Immunopathogenesis during
MyD88 Signaling Regulates Both Host
Francis Gigliotti and Terry W. Wright
Sheila N. Bello-Irizarry, Jing Wang, Carl J. Johnston,
2014; 192:282-292; Prepublished online 29
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The Journal of Immunology
MyD88 Signaling Regulates Both Host Defense and
Immunopathogenesis during Pneumocystis Infection
Sheila N. Bello-Irizarry,* Jing Wang,†Carl J. Johnston,†Francis Gigliotti,†and
Terry W. Wright*,†
The immune response protects against Pneumocystis infection but is also a key component of Pneumocystis pneumonia (PcP)–related
immunopathogenesis. Signaling through myeloid differentiation factor 88 (MyD88) is critical for activation of immune pathways
downstream of TLRs and IL-1R. To determine whether MyD88 regulates normal host defense against Pneumocystis, nonimmunosup-
pressed wild-type (WT) and MyD88-deficient mice were infected. MyD882/2mice had higher early Pneumocystis burdens than did
WT mice but mounted an effective adaptive immune response and cleared Pneumocystis similarly to WT. However, MyD882/2mice
displayed a more intense and prolonged pulmonary immune response than did WT mice. To determine the role of MyD88 in the
development of PcP-related immunopathogenesis, WT and MyD882/2mice were rendered susceptible to PcP by depletion of CD4+
T cells. At 4 wk postinfection, CD4-depleted WTand MyD882/2mice harbored similar organism burdens, but MyD882/2mice were
protected from the PcP-related respiratory impairment observed in WT mice. Improved pulmonary physiology in MyD882/2mice
correlated with lower lung CCL2 levels and reduced cell recruitment. However, by 5 wk postinfection, the overall health of MyD882/2
mice began to deteriorate rapidly relative to WT, with accelerated weight loss, impaired lung function, and exacerbated alveolar
inflammation. This physiological decline of MyD882/2mice was associated with increased TNF-a and IFN-g in the lung, and by the
inability to control Pneumocystis burden. Thus, MyD88 is not required for resistance to Pneumocystis infection, but limits the adaptive
immune response in immunocompetent mice. In the setting of active PcP, MyD88 signaling contributes to both immunopathogenesis
and control of fungal burden.The Journal of Immunology, 2014, 192: 282–292.
ues to be a major health concern for patients with HIV infection
as well as for those without HIV infection who are undergoing
immunosuppression as a consequence of chemotherapy or organ
transplantation (1, 2). New immunosuppressive therapies, such as
anti–TNF-a therapy for Crohn’s disease and rheumatoid arthritis,
are increasing the pool of “at risk” patients (3). In addition,
Pneumocystis frequently colonizes patients with chronic obstruc-
tive pulmonary disease, which appears to exacerbate disease se-
verity (4). Therefore, a better understanding of the mechanisms of
PcP-related immunopathogenesis is key to improving upon cur-
Clinical observations and animal studies have indicated that lung
injury during PcP is caused primarily by the host’s immune-
mediated inflammatory response, and is not absolutely related to
Pneumocystis burden (5–8). For example, in the CD4+T cell–de-
pleted model of PcP, physiological deterioration is associated with
neumocystis is a respiratory fungal pathogen that causes
Pneumocystis pneumonia (PcP) in immunocompromised
individuals. PcP-related morbidity and mortality contin-
an increase in lung chemokine and cytokine levels, as well as the
recruitment of large numbers of CD8+T cells and neutrophils to the
lung. Of interest, when CD4+and CD8+T cells are depleted si-
multaneously, fewer signs of inflammation, less cell recruitment,
and improved lung function result, suggesting that CD8+T cells are
responsible for lung injury and respiratory impairment in this model
of PcP (9).
Recent studies have focused on characterizing the mechanisms
involved in generating pathogenic immune and inflammatory
responses that damage the lung and other tissues. The TLR system
is one of the most important host defense machineries involved in
detection of invading pathogens. Upon recognition of pathogens,
TLRs activate downstream kinases and transcription factors that
induce the expression of genes involved in innate and adaptive
immune responses. All TLRs, with the exception of TLR3, signal
through the adaptor molecule myeloid differentiation factor 88
(MyD88). MyD88 is also critical for signaling through cytokine
receptors that belong to the IL-1R family (10). A protective role for
MyD88 in the control of fungal infections such as those caused by
Candida albicans, Aspergillus fumigatus, Cryptococcus neofor-
mans, and Paracoccidioides brasiliensis has been reported (11–
14). Moreover, our laboratory and others have demonstrated that
MyD88-dependent signaling is required for optimal alveolar epi-
thelial cell (AEC) and alveolar macrophage (AM) cytokine re-
sponses to Pneumocystis or Pneumocystis cell wall components
(15, 16). TLRs, including TLR2 and TLR4, have also been linked
to Pneumocystis-stimulated AM cytokine responses (17, 18), but
neither TLR2 nor TLR4 is required for AEC chemokine responses
to Pneumocystis. Rather, the IL-1R is the upstream molecule re-
quired for the MyD88-dependent AEC response (16).
Although in vitro studies suggest that TLR-, IL-1R–, and MyD88-
dependent responses are involved in the AEC and AM responses to
Pneumocystis, the in vivo role of MyD88-dependent signaling events
*Department of Microbiology and Immunology, University of Rochester School of
Medicine and Dentistry, Rochester, NY 14642; and†Division of Infectious Diseases,
Department of Pediatrics, University of Rochester School of Medicine and Dentistry,
Rochester, NY 14642
Received for publication May 31, 2013. Accepted for publication October 18, 2013.
This work was supported by Public Health Service Grants HL083761 and HL113495.
Address correspondence and reprint requests to Dr. Terry W. Wright, University of
Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Box 850, Ro-
chester, NY 14642. E-mail address: terry_wright@URMC.Rochester.edu
Abbreviations used in this article: AEC, alveolar epithelial cell; AM, alveolar mac-
rophage; BAL, bronchoalveolar lavage; BMT, bone marrow transplant; MyD88,
myeloid differentiation factor 88; PcP, Pneumocystis pneumonia; PMN, polymorpho-
nuclear neutrophil; WT, wild-type.
by guest on January 9, 2016
was reported. The authors suggested that osteopontin clusters TLR2,
dectin-1, and mannose receptor in macrophages exposed to Pneu-
mocystis (45). It is possible that MyD88-independent pathways
cooperate with the MyD88 pathway during the host response to
Pneumocystis, although more studies are required to confirm these
Our data demonstrate that MyD88 signaling in hematopoietic
cells is required to control Pneumocystis infection during active
PcP in CD4-depleted mice. These results agree with recent find-
ings showing that MyD88 signaling in the hematopoietic com-
partment was required for control of Legionella pneumophila (46).
This appears to be dependent on the specific pathogen, because
MyD88 expression in both resident and hematopoietic cells con-
tributes to control of Klebsiella pneumoniae infection. In contrast,
resident nonhematopoietic cells were important for control of
Pseudomonas aeruginosa growth (47, 48). Although we have
clearly shown that MyD88 signaling modulates pulmonary im-
munity and contributes to PcP-related immunopathogenesis, many
questions remain. For example, neither the exact cell types that
use MyD88 signaling during Pneumocystis infection nor the
specific MyD88-dependent mechanisms that regulate host defense
and immunopathogenesis have been defined. MyD88-dependent
responses are known to regulate both innate and adaptive immu-
nity, and many cell types, including epithelial cells, macrophages,
neutrophils, lymphocytes, and DCs, use the MyD88 signaling
pathway during the course of an immune response. Additional
studies are needed to define the cell type–specific role of MyD88
in the complicated network of immune interactions that occur in
response to Pneumocystis in vivo.
The blockade of MyD88 signaling is being explored as a possible
therapeutic strategytoattenuate inflammatory disease(49, 50). Our
preliminary findings suggest that MyD88 may represent a poten-
tial target for immunotherapy in Pneumocystis-infected patients.
Because lack of MyD88 early during PcP limits inflammation and
promotes better lung function, it is conceivable that blocking the
MyD88 pathway, in combination with effective antibiotic treat-
ment, may improve disease outcome. MyD88 represents a more
specific pathway that might be blocked temporarily while antibi-
otic treatment to Pneumocystis is administered. This feature is in
contrast to corticosteroids, which sometimes cause undesired
systemic side effects. However, we also found that loss of MyD88
signaling could have negative effects on the severity of PcP at later
stages of disease by increasing fungal burden, which raises con-
cerns regarding the therapeutic blockade of MyD88 during PcP.
In addition, the MyD88 and TLR pathways have been found to
protect against lung injury through recognition of extracellular
matrix components such as hyaluronan. Hyaluronan degradation
products present during lung injury promote lung repair and
protect the lung epithelium through interactions with TLRs (51,
52). Thus, we speculate that the later deterioration in CD4-
depleted MyD88-deficient mice with active PcP may be partly
related to loss of MyD88-dependent protective mechanisms that
function during lung injury. Further studies using MyD88
blocking agents in vivo are required to thoroughly evaluate the
potential of targeting MyD88 for anti-inflammatory therapy
We thank Dr. Samir Bhagwat, Dr. Nabilah Khan, and Jane Malone for help-
ful comments and expert technical assistance.
The authors have no financial conflicts of interest.
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292MyD88 SIGNALING REGULATES IN VIVO RESPONSES TO PNEUMOCYSTIS
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