The Journal of Immunology
Alternaria Induces STAT6-Dependent Acute Airway
Eosinophilia and Epithelial FIZZ1 Expression That Promotes
Airway Fibrosis and Epithelial Thickness
Taylor A. Doherty,*,†Naseem Khorram,* Kotaro Sugimoto,‡Dean Sheppard,‡
Peter Rosenthal,* Jae Youn Cho,* Alexa Pham,* Marina Miller,* Michael Croft,†and
David H. Broide*
The fungal allergen, Alternaria, is specifically associated with severe asthma, including life-threatening exacerbations. To better
understand the acute innate airway response to Alternaria, naive wild-type (WT) mice were challenged once intranasally with
Alternaria. Naive WT mice developed significant bronchoalveolar lavage eosinophilia following Alternaria challenge when ana-
lyzed 24 h later. In contrast to Alternaria, neither Aspergillus nor Candida induced bronchoalveolar lavage eosinophilia. Gene
microarray analysis of airway epithelial cell brushings demonstrated that Alternaria-challenged naive WT mice had a >20-fold
increase in the level of expression of found in inflammatory zone 1 (FIZZ1/Retnla), a resistin-like molecule. Lung immunostaining
confirmed strong airway epithelial FIZZ1 expression as early as 3 h after a single Alternaria challenge that persisted for ‡5 d and
was significantly reduced in STAT6-deficient, but not protease-activated receptor 2-deficient mice. Bone marrow chimera studies
revealed that STAT6 expressed in lung cells was required for epithelial FIZZ1 expression, whereas STAT6 present in bone
marrow-derived cells contributed to airway eosinophilia. Studies investigating which cells in the nonchallenged lung bind FIZZ1
demonstrated that CD45+CD11c+cells (macrophages and dendritic cells), as well as collagen-1–producing CD452cells (fibro-
blasts), can bind to FIZZ1. Importantly, direct administration of recombinant FIZZ1 to naive WT mice led to airway eosinophilia,
peribronchial fibrosis, and increased thickness of the airway epithelium. Thus, Alternaria induces STAT6–dependent acute airway
eosinophilia and epithelial FIZZ1 expression that promotes airway fibrosis and epithelial thickness. This may provide some insight
into the uniquely pathogenic aspects of Alternaria-associated asthma.
cockroach, and mold. Alternaria is an example of a common
fungal allergen that is associated with the development of asthma
(1). Sensitization to Alternaria alternata is a risk factor for per-
sistence of asthma and fatal/near-fatal asthma (2–8). The spores of
Alternaria are known to be a source of outdoor allergens for
sensitized individuals, and they were recently detected at high
levels indoors (9). Dispersion of the spores occurs during periods
of warm, dry weather, especially in late summer/early fall, and has
been associated with epidemic, severe asthma symptoms (2–8).
Such clinical associations with Alternaria and asthma are in-
The Journal of Immunology, 2012, 188: 2622–2629.
lthough asthma has many clinical, physiologic, and im-
munologic phenotypes, the majority of asthmatics have
environmental allergic triggers, including dust mite,
triguing, but the mechanisms contributing to the pathologic airway
responses are still incompletely understood.
Allergic disease, including asthma, has largely been character-
ized by dysregulation of adaptive immunity in response to aller-
gens, including Th2 cell differentiation and IgE sensitization. More
recently, it has become clear that innate immune responses to
allergensinthe airway help to shapesubsequent adaptiveresponses
(10, 11). For example, recent reports have suggested that allergens
with high protease activity, such as cockroach and fungal aller-
gens, induce innate inflammatory events and allergen sensitization
through a protease-activated receptor 2 (PAR-2)–mediated path-
way in the bronchial epithelium (12–14). Investigations into such
innate epithelial responses to inhaled allergens may provide im-
portant clues to the pathogenesis of asthma. In this study, we in-
vestigated whether Alternaria is able to induce an acute Th2-like
airway inflammatory response in naive wild-type (WT) mice via
activation of innate epithelial genes. We demonstrate that Alter-
naria induces a significant acute airway eosinophil response in
naive WT mice that is mediated by innate immune mechanisms
distinct from those triggered by protease allergens through PAR-2
on the epithelium. This innate proeosinophil inflammatory and
proremodeling effect of Alternaria in naive WT mice is not shared
with other common fungal allergens, such as Aspergillus and
Candida, suggesting that different allergens trigger distinct innate
airway epithelial pathways that contribute to asthma.
Materials and Methods
Mice and airway challenges
Six- to eight-week-old female naive C57BL/6 WT mice were administered
100 mg A. alternata (lot 130656), Candida albicans (lot 111797), or As-
*Department of Medicine, University of California San Diego, La Jolla, CA 92093;
†Division of Immune Regulation, La Jolla Institute for Allergy and Immunology, La
Jolla, CA 92037; and‡Department of Medicine, Lung Biology Center, University of
California San Francisco, San Francisco, CA 94143
Received for publication June 3, 2011. Accepted for publication January 10, 2012.
This work was supported by National Institutes of Health Grants 1K08AI080938-
01A1 (to T.A.D.); AI 38425, AI 70535, and AI 72115 (to D.H.B.); and National
Institute for Allergy and Infectious Diseases Grant U19 AI077439 (to D.S.).
Microarray data presented in this article have been deposited in the Gene Expression
Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE34764.
Address correspondence and reprint requests to Dr. Taylor Doherty, Department of
Medicine, University of California San Diego, Biomedical Sciences Building, Room
5080, 9500 Gilman Drive, La Jolla, CA 92093-0635. E-mail address: tdoherty@ucsd.
Abbreviations used in this article: BAL, bronchoalveolar lavage; FIZZ1, found in
inflammatory zone 1; PAR-2, protease-activated receptor 2; rFIZZ1, recombinant
found in inflammatory zone 1; TSLP, thymic stromal lymphopoietin; WT, wild-type.
chronic Th2 responses (19, 38). Further work will be required to
fully elucidate the multiple functions of FIZZ1 in the lung during
chronic inflammatory responses.
In summary, we characterized a unique acute eosinophilic air-
way response to Alternaria that is STAT6 dependent and associ-
ated with significant upregulation of FIZZ1 in airway epithelium.
Further, exogenous FIZZ1 induced airway eosinophilia, epithelial
changes, and airway fibrosis. This underscores the potential im-
portance of FIZZ1 in asthma and airway remodeling and might
translate to a role for related human resistin molecules in human
We thank the University of California San Diego microarray core for pro-
cessing the epithelial microarray.
The authors have no financial conflicts of interest.
1. Bateman, E. D., S. S. Hurd, P. J. Barnes, J. Bousquet, J. M. Drazen,
M. FitzGerald, P. Gibson, K. Ohta, P. O’Byrne, S. E. Pedersen, et al. 2008.
Global strategy for asthma management and prevention: GINA executive sum-
mary. Eur. Respir. J. 31: 143–178.
2. O’Hollaren, M. T., J. W. Yunginger, K. P. Offord, M. J. Somers, E. J. O’Connell,
D. J. Ballard, and M. I. Sachs. 1991. Exposure to an aeroallergen as a possible
precipitating factor in respiratory arrest in young patients with asthma. N. Engl.
J. Med. 324: 359–363.
3. Downs, S. H., T. Z. Mitakakis, G. B. Marks, N. G. Car, E. G. Belousova,
J. D. Leu ¨ppi, W. Xuan, S. R. Downie, A. Tobias, and J. K. Peat. 2001. Clinical
importance of Alternaria exposure in children. Am. J. Respir. Crit. Care Med.
4. Bush, R. K., and J. J. Prochnau. 2004. Alternaria-induced asthma. J. Allergy
Clin. Immunol. 113: 227–234.
5. Pulimood, T. B., J. M. Corden, C. Bryden, L. Sharples, and S. M. Nasser. 2007.
Epidemic asthma and the role of the fungal mold Alternaria alternata. J. Allergy
Clin. Immunol. 120: 610–617.
6. Lyons, T. W., D. B. Wakefield, and M. M. Cloutier. 2011. Mold and Alternaria
skin test reactivity and asthma in children in Connecticut. Ann. Allergy Asthma
Immunol. 106: 301–307.
7. Plaza, V., J. Serrano, C. Picado, J. Cosano, J. Ancochea, A. de Diego,
J. J. Martı ´n, and J. Sanchı ´s; Grupo de Investigadores del Estudio Multice ´ntrico
del Asma de Riesgo Vital. 2003. [Clinical characteristics of the fatal and near-
fatal asthma in Alternaria alternata sensitized patients]. Med. Clin. (Barc.) 121:
8. Neukirch, C., C. Henry, B. Leynaert, R. Liard, J. Bousquet, and F. Neukirch.
1999. Is sensitization to Alternaria alternata a risk factor for severe asthma? A
population-based study. J. Allergy Clin. Immunol. 103: 709–711.
9. Salo, P. M., S. J. Arbes, Jr., M. Sever, R. Jaramillo, R. D. Cohn, S. J. London, and
D. C. Zeldin. 2006. Exposure to Alternaria alternata in US homes is associated
with asthma symptoms. J. Allergy Clin. Immunol. 118: 892–898.
10. Barrett, N. A., and K. F. Austen. 2009. Innate cells and T helper 2 cell immunity
in airway inflammation. Immunity 31: 425–437.
11. Hammad, H., M. Chieppa, F. Perros, M. A. Willart, R. N. Germain, and
B. N. Lambrecht. 2009. House dust mite allergen induces asthma via Toll-like
receptor 4 triggering of airway structural cells. Nat. Med. 15: 410–416.
12. Boitano, S., A. N. Flynn, C. L. Sherwood, S. M. Schulz, J. Hoffman,
I. Gruzinova, and M. O. Daines. 2011. Alternaria alternata serine proteases
induce lung inflammation and airway epithelial cell activation via PAR2. Am. J.
Physiol. Lung Cell. Mol. Physiol. 300: L605–L614.
13. Page, K., J. R. Ledford, P. Zhou, K. Dienger, and M. Wills-Karp. 2010. Mucosal
sensitization to German cockroach involves protease-activated receptor-2.
Respir. Res. 11: 62.
14. Kouzaki, H., S. M. O’Grady, C. B. Lawrence, and H. Kita. 2009. Proteases in-
duce production of thymic stromal lymphopoietin by airway epithelial cells
through protease-activated receptor-2. J. Immunol. 183: 1427–1434.
15. Taura, K., K. Miura, K. Iwaisako, C. H. Osterreicher, Y. Kodama, M. Penz-
Osterreicher, and D. A. Brenner. 2010. Hepatocytes do not undergo epithelial-
mesenchymal transition in liver fibrosis in mice. Hepatology 51: 1027–1036.
16. Doherty, T. A., P. Soroosh, N. Khorram, S. Fukuyama, P. Rosenthal, J. Y. Cho,
P. S. Norris, H. Choi, S. Scheu, K. Pfeffer, et al. 2011. The tumor necrosis factor
family member LIGHT is a target for asthmatic airway remodeling. Nat. Med.
17. Stevens, W. W., T. S. Kim, L. M. Pujanauski, X. Hao, and T. J. Braciale. 2007.
Detection and quantitation of eosinophils in the murine respiratory tract by flow
cytometry. J. Immunol. Methods 327: 63–74.
18. Sugimoto, K., M. Kudo, A. Sundaram, X. Ren, K. Huang, X. Bernstein,
Y. Wang, W. W. Raymond, D. J. Erle, M. Abrink, et al. 2012. The avb6 integrin
modulates airway hyperresponsiveness in mice by regulating intraepithelial mast
cells. J. Clin. Invest. 122: 748–758.
19. Nair, M. G., Y. Du, J. G. Perrigoue, C. Zaph, J. J. Taylor, M. Goldschmidt,
G. P. Swain, G. D. Yancopoulos, D. M. Valenzuela, A. Murphy, et al. 2009.
Alternatively activated macrophage-derived RELM-alpha is a negative regulator
of type 2 inflammation in the lung. J. Exp. Med. 206: 937–952.
20. Chupp, G. L., C. G. Lee, N. Jarjour, Y. M. Shim, C. T. Holm, S. He, J. D. Dziura,
J. Reed, A. J. Coyle, P. Kiener, et al. 2007. A chitinase-like protein in the lung
and circulation of patients with severe asthma. N. Engl. J. Med. 357: 2016–
21. Ober, C., Z. Tan, Y. Sun, J. D. Possick, L. Pan, R. Nicolae, S. Radford,
R. R. Parry, A. Heinzmann, K. A. Deichmann, et al. 2008. Effect of variation in
CHI3L1 on serum YKL-40 level, risk of asthma, and lung function. N. Engl. J.
Med. 358: 1682–1691.
22. Salamon, M., C. Millino, A. Raffaello, M. Mongillo, C. Sandri, C. Bean,
E. Negrisolo, A. Pallavicini, G. Valle, M. Zaccolo, et al. 2003. Human MYO18B,
a novel unconventional myosin heavy chain expressed in striated muscles moves
into the myonuclei upon differentiation. J. Mol. Biol. 326: 137–149.
23. Chen, W., and G. K. Khurana Hershey. 2007. Signal transducer and activator of
transcription signals in allergic disease. J. Allergy Clin. Immunol. 119: 529–541;
24. Kiss, A., M. Montes, S. Susarla, E. A. Jaensson, S. M. Drouin, R. A. Wetsel,
Z. Yao, R. Martin, N. Hamzeh, R. Adelagun, et al. 2007. A new mechanism
regulating the initiation of allergic airway inflammation. J. Allergy Clin.
Immunol. 120: 334–342.
25. Cohen, L., X. E, J. Tarsi, T. Ramkumar, T. K. Horiuchi, R. Cochran,
S. DeMartino, K. B. Schechtman, I. Hussain, M. J. Holtzman, and M. Castro;
and the NHLBI Severe Asthma Research Program (SARP). 2007. Epithelial cell
proliferation contributes to airway remodeling in severe asthma. Am. J. Respir.
Crit. Care Med. 176: 138–145.
26. Day, S. B., P. Zhou, J. R. Ledford, and K. Page. 2010. German cockroach frass
proteases modulate the innate immune response via activation of protease-
activated receptor-2. J. Innate Immun. 2: 495–504.
27. Kouzaki, H., K. Iijima, T. Kobayashi, S. M. O’Grady, and H. Kita. 2011. The
danger signal, extracellular ATP, is a sensor for an airborne allergen and triggers
IL-33 release and innate Th2-type responses. J. Immunol. 186: 4375–4387.
28. Holcomb, I. N., R. C. Kabakoff, B. Chan, T. W. Baker, A. Gurney, W. Henzel,
C. Nelson, H. B. Lowman, B. D. Wright, N. J. Skelton, et al. 2000. FIZZ1,
a novel cysteine-rich secreted protein associated with pulmonary inflammation,
defines a new gene family. EMBO J. 19: 4046–4055.
29. Stu ¨tz, A. M., L. A. Pickart, A. Trifilieff, T. Baumruker, E. Prieschl-Strassmayr,
and M. Woisetschla ¨ger. 2003. The Th2 cell cytokines IL-4 and IL-13 regulate
found in inflammatory zone 1/resistin-like molecule alpha gene expression by
a STAT6 and CCAAT/enhancer-binding protein-dependent mechanism. J.
Immunol. 170: 1789–1796.
30. Nair, M. G., K. J. Guild, and D. Artis. 2006. Novel effector molecules in type 2
inflammation: lessons drawn from helminth infection and allergy. J. Immunol.
31. Liu, T., H. Jin, M. Ullenbruch, B. Hu, N. Hashimoto, B. Moore, A. McKenzie,
N. W. Lukacs, and S. H. Phan. 2004. Regulation of found in inflammatory zone 1
expression in bleomycin-induced lung fibrosis: role of IL-4/IL-13 and mediation
via STAT-6. J. Immunol. 173: 3425–3431.
32. Su, Q., Y. Zhou, and R. A. Johns. 2007. Bruton’s tyrosine kinase (BTK) is
a binding partner for hypoxia induced mitogenic factor (HIMF/FIZZ1) and
mediates myeloid cell chemotaxis. FASEB J. 21: 1376–1382.
33. Liu, T., B. Hu, Y. Y. Choi, M. Chung, M. Ullenbruch, H. Yu, J. B. Lowe, and
S. H. Phan. 2009. Notch1 signaling in FIZZ1 induction of myofibroblast dif-
ferentiation. Am. J. Pathol. 174: 1745–1755.
34. Dong, L., S. J. Wang, B. Camoretti-Mercado, H. J. Li, M. Chen, and W. X. Bi.
2008. FIZZ1 plays a crucial role in early stage airway remodeling of OVA-
induced asthma. J. Asthma 45: 648–653.
35. Liu, T., S. M. Dhanasekaran, H. Jin, B. Hu, S. A. Tomlins, A. M. Chinnaiyan,
and S. H. Phan. 2004. FIZZ1 stimulation of myofibroblast differentiation. Am. J.
Pathol. 164: 1315–1326.
36. Munitz, A., L. Seidu, E. T. Cole, R. Ahrens, S. P. Hogan, and M. E. Rothenberg.
2009. Resistin-like molecule alpha decreases glucose tolerance during intestinal
inflammation. J. Immunol. 182: 2357–2363.
37. Munitz, A., A. Waddell, L. Seidu, E. T. Cole, R. Ahrens, S. P. Hogan, and M. E.
Rothenberg. 2008. Resistin-like molecule alpha enhances myeloid cell activation
and promotes colitis. J. Allergy Clin. Immunol. 122: 1200–1207.e1.
38. Pesce, J. T., T. R. Ramalingam, M. S. Wilson, M. M. Mentink-Kane,
R. W. Thompson, A. W. Cheever, J. F. Urban, Jr., and T. A. Wynn. 2009. Retnla
(relmalpha/fizz1) suppresses helminth-induced Th2-type immunity. PLoS
Pathog. 5: e1000393.
39. Yamaji-Kegan, K., Q. Su, D. J. Angelini, A. C. Myers, C. Cheadle, and R. A. Johns.
2010. Hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELMalpha) increases
lung inflammation and activates pulmonary microvascular endothelial cells via an
IL-4-dependent mechanism. J. Immunol. 185: 5539–5548.
40. Ito, T., M. Schaller, T. Raymond, A. D. Joshi, A. L. Coelho, F. G. Frantz,
W. F. Carson, IV, C. M. Hogaboam, N. W. Lukacs, T. J. Standiford, et al. 2009.
Toll-like receptor 9 activation is a key mechanism for the maintenance of chronic
lung inflammation. Am. J. Respir. Crit. Care Med. 180: 1227–1238.
The Journal of Immunology2629