Ascaris Larval Infection and Lung Invasion Directly InduceSevere Allergic Airway Disease in Mice

Jill E. Weatherhead,a,b,d Paul Porter,b Amy Coffey,c Dana Haydel,c Leroy Versteeg,a Bin Zhan,a,d

Ana Clara Gazzinelli Guimarães,e Ricardo Fujiwara,e Ana M. Jaramillo,f Maria Elena Bottazzi,a,d,g,i Peter J. Hotez,a,d,g,i

David B. Corry,b,c,h,j Coreen M. Beaumiera,g

aDepartment of Pediatrics, Baylor College of Medicine, Houston, Texas, USAbDepartment of Medicine, Baylor College of Medicine, Houston, Texas, USAcDepartment of Pathology and Immunology, Baylor College of Medicine, Houston, Texas, USAdNational School of Tropical Medicine, Baylor College of Medicine, Houston, Texas, USAeDepartamento de Parasitologia, Federal University of Minas Gerais, Minas Gerais, BrazilfDepartment of Pulmonary Medicine, University of Texas MD Anderson Cancer Center, Houston, Texas, USAgDepartment of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USAhBiology of Inflammation Center, Baylor College of Medicine, Houston, Texas, USAiTexas Children’s Hospital Center for Vaccine Development-Product Development Partnership, Houston, Texas,USAjMichael E. DeBakey Center for Translational Research in Inflammatory Diseases, Houston, Texas, USA

ABSTRACT Ascaris lumbricoides (roundworm) is the most common helminth infec-tion globally and a cause of lifelong morbidity that may include allergic airway dis-ease, an asthma phenotype. We hypothesize that Ascaris larval migration throughthe lungs leads to persistent airway hyperresponsiveness (AHR) and type 2 inflam-matory lung pathology despite resolution of infection that resembles allergic airwaydisease. Mice were infected with Ascaris by oral gavage. Lung AHR was measured byplethysmography and histopathology with hematoxylin and eosin (H&E) and peri-odic acid-Schiff (PAS) stains, and cytokine concentrations were measured by usingLuminex Magpix. Ascaris-infected mice were compared to controls or mice with al-lergic airway disease induced by ovalbumin (OVA) sensitization and challenge (OVA/OVA). Ascaris-infected mice developed profound AHR starting at day 8 postinfection(p.i.), peaking at day 12 p.i. and persisting through day 21 p.i., despite resolution ofinfection, which was significantly increased compared to controls and OVA/OVAmice. Ascaris-infected mice had a robust type 2 cytokine response in both the bron-choalveolar lavage (BAL) fluid and lung tissue, similar to that of the OVA/OVA mice,including interleukin-4 (IL-4) (P � 0.01 and P � 0.01, respectively), IL-5 (P � 0.001and P � 0.001), and IL-13 (P � 0.001 and P � 0.01), compared to controls. By histo-pathology, Ascaris-infected mice demonstrated early airway remodeling similar to,but more profound than, that in OVA/OVA mice. We found that Ascaris larval migra-tion causes significant pulmonary damage, including AHR and type 2 inflammatorylung pathology that resembles an extreme form of allergic airway disease. Our find-ings indicate that ascariasis may be an important cause of allergic airway disease inregions of endemicity.

KEYWORDS Ascaris, airway hyperreactivity, allergic airway disease, asthma, hygienehypothesis

Ascaris lumbricoides (roundworm) is the most common intestinal helminth infectionworldwide, infecting 761.9 million people in impoverished areas of Africa, Asia, and

Central and South America (1, 2). In regions of endemicity, children are often infectedsoon after birth, enduring recurrent infection throughout childhood and reaching

Received 12 July 2018 Returned formodification 22 August 2018 Accepted 13September 2018

Accepted manuscript posted online 24September 2018

Citation Weatherhead JE, Porter P, Coffey A,Haydel D, Versteeg L, Zhan B, GazzinelliGuimarães AC, Fujiwara R, Jaramillo AM,Bottazzi ME, Hotez PJ, Corry DB, Beaumier CM.2018. Ascaris larval infection and lung invasiondirectly induce severe allergic airway disease inmice. Infect Immun 86:e00533-18. https://doi.org/10.1128/IAI.00533-18.

Editor John H. Adams, University of SouthFlorida

Copyright © 2018 American Society forMicrobiology. All Rights Reserved.

Address correspondence to David B. Corry,dcorry@bcm.edu.

FUNGAL AND PARASITIC INFECTIONS

crossm

December 2018 Volume 86 Issue 12 e00533-18 iai.asm.org 1Infection and Immunity

on January 18, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

maximum worm burden by preschool and school age (3, 4). Ascariasis is associated witha significant level of global morbidity, leading to 1.46 million disability-adjusted lifeyears (DALYs), an impact similar to those of other well-known childhood illnesses, suchas meningitis (1.52 million DALYs) (1, 5). Ascariasis due to the presence of adult wormsin the gut specifically results in growth stunting, cognitive delays, malnutrition (vitaminA deficiency), abdominal pain, and obstruction (3, 6–8). Beyond the intestinal form ofascariasis due to the presence of adult worms, larval migration through lung tissuefollowing oral ingestion of Ascaris eggs has been clinically linked to a transientinflammatory lung disease termed Loeffler’s syndrome (7). Additionally, some investi-gators have suggested that larval Ascaris infection represents a major environmentalcause of allergic airway disease, an asthma phenotype, and inflammatory lung diseasein resource-poor countries (3, 6–12). These epidemiological studies suggest that chil-dren with Ascaris-induced allergic airway disease have more-severe allergic airwaydisease as well as increased cross-reactivity to bystander antigens like house dust mites(12–15). However, it is not clear how ascariasis induces prolonged allergic airwaydisease in children. As a result of the profound impact on morbidity, there is an urgentneed to gain an understanding of end-organ damage resulting from ascariasis and todevelop preventative and therapeutic interventions.

Other helminths, such as Nippostrongylus, known as the rat hookworm, that have alarval lung stage have been linked to lung damage, type 2 immune responses, andlong-term changes in lung function and structure in nonhuman hosts consistent withallergic airway disease (16). Migration of Nippostrongylus larvae through the lungscauses hemorrhage and damage to the epithelium, promoting the release of damage-associated molecular patterns (DAMPs) and alarmins from airway epithelial cells andtype II alveolar macrophages (16–18). The release of DAMPs and alarmins, includinginterleukin-33 (IL-33) and IL-25, promotes the activation of innate immune cells, such asantigen-presenting cells (dendritic cells) and innate lymphoid cells (ILC2), leading to anincrease in the release of the type 2 cytokines IL-4, IL-5, and IL-13 (16, 18). IL-4, IL-5, andIL-13 are required for the protective cellular and humoral responses generated duringhelminth infection (18, 19). IL-4 and IL-13, signaling through the IL-4R�/Stat6 pathway,have been shown to be part of an important pathway in both the innate and adaptiveresponses to lung larval migration, allowing for infection control as well as eventualtissue healing and fibrosis in Nippostrongylus-infected mice (17, 18, 20, 21).

This study used an Ascaris-infected mouse model to gain further knowledge on theimpact of pulmonary ascariasis on lung physiology and histopathology. We found thatAscaris larval migration causes significant airway hyperresponsiveness (AHR), type 2inflammatory infiltration, and early airway remodeling, which resembles an extremeform of allergic airway disease that is significantly greater than the classical mouseallergic airway disease model induced by ovalbumin (OVA) sensitization and challenge.We propose that human and animal Ascaris infections will induce a persistent allergicairway disease that resembles human asthma.

(Data in this article were presented in November 2016 and 2017 at the AmericanSociety of Tropical Medicine and Hygiene annual meeting in Atlanta, GA.)

RESULTSAscaris-infected mice have increased AHR and marked lung allergic inflamma-

tory infiltrates throughout the life cycle of the larvae. To evaluate the impact ofascariasis on the host lung during the life cycle of the parasite, 25 mice were eachinfected with 2,500 embryonated eggs in 200 �l phosphate-buffered saline (PBS) byoral gavage on day 0, while 25 mice received 200 �l PBS by oral gavage at day 0(negative control). AHR was measured by using whole-body plethysmography withacetylcholine (Ach) provocation challenges at day 5, day 8, day 12, day 14, and day 21postinfection (p.i.) in both the Ascaris-infected and negative-control groups (n � 5 pergroup on each day of the life cycle) (Fig. 1A to E).

Ascaris-infected mice were found to have increasing Ach-induced AHR starting atday 8 p.i., at which time point larvae are at the maximum number in the lungs (Fig. 1B).

Weatherhead et al. Infection and Immunity

December 2018 Volume 86 Issue 12 e00533-18 iai.asm.org 2

on January 18, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

By day 12 p.i., Ascaris infection caused severe AHR at very low concentrations of Achcompared to PBS-treated negative controls, leading to complete bronchoconstriction(airway narrowing) and restriction of airflow (Fig. 1C). Interestingly, AHR persisted athigh levels at day 21 p.i. (Fig. 1E), despite resolution of infection, suggesting thatascariasis not only causes increased AHR during the life cycle of the larvae but also haspersistent effects without evidence of active infection.

In comparison to PBS-treated negative controls (Fig. 2A), histopathological evaluation ofthe lung tissue using hematoxylin and eosin (H&E) staining showed marked perivascularand peribronchial inflammatory infiltrates with alveolitis (peripheral lung inflammation)from day 8 p.i. through day 21 p.i. in Ascaris-infected mice (Fig. 2B to D), consisting ofneutrophils and eosinophils (Fig. 2C, inset). On day 8 p.i., there was evidence of larvae in theparenchyma as well as in the vasculature, which resolved by day 12. Ascaris-infected micealso had increased total cell counts in bronchoalveolar lavage (BAL) fluid, with a predom-inance of eosinophils, compared to control mice (Fig. 2E).

Ascaris-infected mice have similar but profound AHR and inflammatory infil-trates compared to a known allergic airway disease mouse model. Ascaris-infectedmice were compared to a known murine allergic airway disease model of OVA-sensitized and OVA-challenged (OVA/OVA) mice. Ten mice were each infected with2,500 embryonated eggs in 200 �l PBS by oral gavage, 10 mice were given 200 �l PBSby oral gavage (negative controls), and 10 mice were sensitized and challenged withOVA (allergic airway disease positive control). On day 12 p.i., Ascaris-infected mice andnegative controls were anesthetized using etomidate for physiological evaluation ofairway resistance using plethysmography. Day 12 was chosen based on the predictedmaximum inflammatory infiltrate in the lungs during Ascaris infection (determined fromthe above-described experiment) and the high likelihood of larvae not being presentwithin the lung tissue. On day 24 of the OVA/OVA protocol, OVA/OVA mice wereanesthetized using etomidate for physiological evaluation of AHR using plethysmog-raphy. Evaluation of AHR demonstrated that Ascaris-infected mice had increased peak

FIG 1 A. suum-infected mice develop prolonged, exaggerated AHR. AHR was assessed by plotting respiratory system resistance (RRS)against increasing concentrations of acetylcholine chloride (Ach) injected intravenously into anesthetized, Ascaris-infected mice on day 5(A), day 8 (B), day 12 (C), day 14 (D), and day 21 (E) postinfection (n � 5 mice per group per life cycle day). *, P � 0.05; **, P � 0.01 (means �SD, as determined by a Mann-Whitney test in two different experiments).

Ascaris-Induced Allergic Airway Disease Infection and Immunity

December 2018 Volume 86 Issue 12 e00533-18 iai.asm.org 3

on January 18, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

airflow resistance compared to both OVA/OVA mice and PBS-treated negative controls(Fig. 3).

Levels of inflammatory cytokines in the BAL fluid and lung tissue were quantitatedin all three groups. Ascaris-infected mice had a robust inflammatory response domi-nated by type 2 cytokines in both the BAL fluid and lung tissue supernatant, including

FIG 2 Temporal assessment of histopathological and airway cellular changes following Ascaris lung infection. (A to D) Histological images of lung showingrepresentative bronchovascular bundles. (A) PBS negative control; (B) after Ascaris infection on day 8; (C) after Ascaris infection on day 12; (D) after Ascarisinfection on day 21. Black arrows, Ascaris larvae within the lung parenchyma and vasculature; dotted arrow, perivasculature and peribronchial granulocyticinfiltration; inset, eosinophil with bilobed nucleus and granular eosinophilic cytoplasm adjacent to a neutrophil with a segmented nucleus and pale cytoplasm.Magnification, �200. (E) Mean total BAL fluid cells and mean absolute numbers of macrophages, eosinophils, lymphocytes, and neutrophils enumerated fromAscaris-infected or PBS control mice at day 12 p.i. **, P � 0.01 (means � SD, as determined by a Mann-Whitney test in two different experiments).

FIG 3 AHR for Ascaris-infected and OVA/OVA asthmatic mice and PBS negative controls. Ascaris-infectedmice had profound AHR at all doses of Ach (n � 10 mice per group). Statistical analysis was performedby Kruskal-Wallis and post hoc Dunn’s multiple-comparison tests, with group comparisons representedby symbols. Diamonds represent differences between the PBS control and Ascaris-infected mice, thecrosses represent differences between Ascaris-infected and OVA/OVA asthmatic mice, and the pentagonsrepresent differences between PBS control and OVA/OVA asthmatic mice. No fill, P � 0.05; striped,P � 0.01; filled, P � 0.001 (means � SD in two different experiments).

Weatherhead et al. Infection and Immunity

December 2018 Volume 86 Issue 12 e00533-18 iai.asm.org 4

on January 18, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

IL-4 (P � 0.01 and P � 0.01, respectively), IL-5 (P � 0.001 and P � 0.001), and IL-13 (P �

0.001 and P � 0.01), compared to negative controls (Fig. 4A to C). In addition, levels oftype 2-specific cytokines in Ascaris-infected mice were similar to or higher than thosein OVA/OVA mice. The levels of IL-5 and IL-13 were noted to be especially high,exceeding a 10-fold difference relative to those in OVA/OVA mice. The level of IL-10 wasslightly increased in Ascaris-infected mouse lung tissue compared to those in negativecontrols (P � 0.01) and in OVA/OVA mice (P � 0.05), but the levels were low, and therewas no difference in BAL fluid (Fig. 4D). In contrast, there was no difference in the type1-specific cytokine tumor necrosis factor alpha (TNF-�) or gamma interferon (IFN-�) inthe BAL fluid or in the lung tissue (Fig. 5A and B). Interestingly, a small amount of IL-17was detected in lung tissue at higher concentrations in the Ascaris-infected group thanin both the OVA/OVA mice (P � 0.01) and negative controls (P � 0.001), but IL-17 wasnot detected in the BAL fluid (Fig. 5C).

Histopathological differences were compared in negative controls, OVA/OVA mice,and Ascaris-infected mice (22). Ascaris-infected mice demonstrated the greatest inflam-matory infiltrate within lung tissue, consisting mostly of neutrophils and eosinophilsfound in the perivascular and pleural spaces. Compared to negative controls (Fig. 6A),both OVA/OVA lung tissue (Fig. 6B) and Ascaris-infected lung tissue after a singleinfection (Fig. 6C) showed signs of early airway remodeling with siginificant goblet cellmetaplasia. However, Ascaris-infected mice also had evidence of smooth muscle hy-pertrophy (7.85 � 2.15 �m for negative controls, 12.94 � 6.33 �m for OVA/OVA mice,and 14.64 � 3.31 �m for Ascaris-infected mice; P � 0.01) (Fig. 7A to D), further dem-onstrating the profound impact of Ascaris infection on the lung tissue and suggestinglasting effects of a single infection on the host lung.

DISCUSSION

The establishment of a mouse model of Ascaris infection has provided significantinsight into end-organ damage endured during Ascaris infection. This study details forthe first time that Ascaris infection, with resultant larval migration, can cause an

FIG 4 Cytokine concentrations in homogenized lung tissue supernatants and BAL fluid from Ascaris-infected mice at day 12p.i. Shown are data for PBS negative controls and OVA/OVA asthmatic mice, including IL-4 (A), IL-5 (B), IL-13 (C), and IL-10 (D)(n � 10 mice per group). *, P � 0.05; **, P � 0.01; ***, P � 0.001 (means � SD, as determined by a Kruskal-Wallis test with posthoc Dunn’s multiple-comparison test in two different experiments, with each sample in duplicate).

Ascaris-Induced Allergic Airway Disease Infection and Immunity

December 2018 Volume 86 Issue 12 e00533-18 iai.asm.org 5

on January 18, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

extreme form of allergic airway disease compared to a standard disease model usingOVA. Moreover, we have shown that after a single Ascaris infection, the resultingallergic airway disease extends weeks past the duration of the actual infection. Ourstudy thus supports the clinical observations that children with Ascaris-induced asthmahave more-profound allergic disease-related symptoms than children with conven-tional, non-parasite-associated type 2 asthmatic disease. This model will be useful infurther dissecting the pathogenesis of Ascaris-induced allergic airway disease and therelationship between environmental parasites and allergen sensitization.

Asthma is a heterogeneous syndrome of intermittent wheezing, airway obstructionand remodeling, and type 2 inflammatory infiltration, affecting more than 300 millionindividuals, and is associated with significant mortality, morbidity, and economicconsequences globally (23–26). Over the past 2 decades, allergic airway disease, anasthma phenotype, has become a major health threat in low- and middle-income

FIG 5 Cytokine concentrations in lung tissue and BAL fluid from Ascaris-infected mice at day 12 p.i. Shown are datafor negative controls and OVA/OVA asthmatic mice, including TNF-� (A), IFN-� (B), and IL-17 in lung tissue only, asIL-17 was unable to be detected in BAL fluid (C) (n � 10 mice per group). **, P � 0.01; ***, P � 0.001 (means � SD,as determined by a Kruskal-Wallis test with post hoc Dunn’s multiple-comparison test in two different experiments,with each sample in duplicate).

FIG 6 Goblet cell metaplasia and intracellular mucin production demonstrated by PAS staining in negative controls(A), OVA/OVA asthmatic mice (B), and Ascaris-infected mice (C) (magnification, �500 under oil).

Weatherhead et al. Infection and Immunity

December 2018 Volume 86 Issue 12 e00533-18 iai.asm.org 6

on January 18, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

countries, which had previously reported low levels of diseases (23, 27). A robust type2 cytokine milieu comprising IL-4, IL-5, and IL-13 is central to the immune response inallergic airway disease (28). In conventional allergic airway disease phenotypes, IL-4drives TH2 differentiation, immunoglobulin isotype switching to IgE in B cells, andactivation of alveolar macrophages, while IL-5 promotes the growth, differentiation,and survival of eosinophils (28, 29). IL-13 has many effector functions in the pathogen-esis of allergic airway disease, including goblet cell metaplasia, smooth muscle hyper-trophy, and AHR (30). Additionally, inflammation, goblet cell metaplasia, and smoothmuscle hypertrophy are evidence of airway remodeling in allergic airway disease (31).Similar to conventional allergic airway disease, helminth infections, like ascariasis, areknown to induce a type 2 polarized immunological response (30, 32).

In the present study, levels of IL-4, IL-5, and IL-13 were elevated in the BAL fluid andlung tissue of Ascaris-infected mice, supporting the critical role of the type 2 inflam-matory cascade during Ascaris larval migration in the lungs, similar to the cytokineenvironment observed in allergic airway disease. The elevation of IL-5 level wasparticularly notable and exceeded the levels in OVA/OVA-induced allergic airwaydisease by 10-fold or more. Likewise, the level of IL-13, a main driver in the pathogen-esis of allergic airway disease, was significantly elevated compared to that in theOVA/OVA model. These type 2 cytokines, IL-5 and IL-13, are likely a major link to thesevere allergic airway disease observed in Ascaris-infected mice. Conversely, the level ofIL-10, produced largely by regulatory T cells (Tregs) and aiding immunomodulationduring chronic Ascaris infection, was elevated only minimally in Ascaris-infected mice(32). As expected based on the polarized type 2 response in allergic airway disease andAscaris-infected mice, the levels of type 1-specific cytokines were not elevated in anygroup. Other cell populations, including TH17 cells, may play an important role in thepathogenesis of allergic airway disease through the production and propagation of a

FIG 7 (A to C) Increased bronchial muscular layer thickness in control mice (A) and OVA/OVA (B) and Ascaris-infected (C)mouse models (H&E; magnification, �400). (D) Measurement of increased bronchial muscular layer thickness in Ascaris-infected mice compared to negative controls (n � 10 mice per group). **, P � 0.01 (means � SD, as determined by aKruskal-Wallis test in two different experiments).

Ascaris-Induced Allergic Airway Disease Infection and Immunity

December 2018 Volume 86 Issue 12 e00533-18 iai.asm.org 7

on January 18, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

neutrophilic response, the induction of Muc5ac hypersecretion, and the promotion ofairway remodeling involving the activation of fibroblasts (33, 34). Interestingly, the levelof IL-17, a key TH17 cytokine that has been correlated with the severity of allergic airwaydisease (34), was mildly elevated in Ascaris-infected mice. Given the role of IL-17 inenhancing the neutrophil response and promoting mucosal integrity, the presence ofIL-17 may be a sign of tissue remodeling after Ascaris infection in the lungs.

Our studies also found a dramatic level of AHR in response to provocative challengewith Ach, a marker of clinically significant allergic airway disease (35), in Ascaris-infectedmice that greatly exceeded that seen in the OVA/OVA model. In addition, Ascaris-infected mice had exaggerated goblet cell metaplasia and smooth muscle hypertrophy,suggesting early airway remodeling. Studies are under way to evaluate the pathogen-esis of these persistent physiological and anatomical findings in the lung and whetherthey reflect the marked increases in IL-5 and IL-13 or some other pathogenic mecha-nism. It is possible that the etiology of this severe allergic airway phenotype ismultifactorial, including both the host immune response to the larvae and retainedlarval proteins in the lung tissue in combination with significant tissue destructionsecondary to larval migration and protease secretion.

In some clinical studies, ascariasis has been noted to be an independent risk factorfor the development of childhood asthma in areas of endemicity (10, 12–14, 36). Theseclinical studies suggest that Ascaris-induced allergic airway disease in children isassociated with more-severe disease as well as increased cross-reactivity to bystanderantigens, like house dust mites, compared to conventional allergic airway disease(12–15, 36). The type 2 cytokine response as a result of larval migration, as describedin this study, may have evolved to protect against helminth reinfection but simulta-neously makes the host more susceptible to allergic airway disease (13, 15, 36, 37).Alternatively, other clinical studies suggest that Ascaris infection may protect againstallergic disease (38–40). It has been postulated that ascariasis may inhibit allergicreactivity through the induction of high levels of polyclonal IgE, which saturate effectorcells and suppress the response to specific environmental allergens, or through excre-tory and secretory immunomodulatory molecules, such as PAS-1, that exhibit anti-inflammatory properties (38, 41, 42). Direct comparisons of data from clinical studiesevaluating the impact of ascariasis on the host lungs remain challenging secondary toinconsistent diagnostic capabilities in areas of endemicity as well as variation in studypopulations, such as rural versus urban environments, seasonality and frequency ofinfection, genetic predisposition, age of infection, and intensity of infection (10, 11, 40,42, 43).

In response to conflicting clinical data, the use of animal models continues toprovide significant insight into disease pathogenesis. Mouse models have demon-strated that repetitive infection with Ascaris causes significant inflammatory infiltrates,thickening of the intra-alveolar septa, and parenchyma destruction in the lung on day8 p.i. (44). Likewise, after inhalation of an Ascaris suum extract, dogs have dyspnea,increased airway secretions, and enhanced bronchoconstriction (45), and nonhumanprimates sensitized to Ascaris through natural exposure and subsequently administeredan aerosolized A. suum antigen have increased airway resistance (46). The findings inthese animal models, along with the evidence presented in the present study, furthersupport the detrimental impact of ascariasis on the host lung.

The use of a mouse model inhibits the ability to fully understand the impact ofhelminths outside the larval migration stage. However, as this study was focusedprimarily on the impact of infection on the lung environment, the mouse model isequipped to evaluate the impact of larva migration through the lungs. However, itshould be noted that there are differences in lung size as well as lung structurebetween mouse and human lungs, and thus, changes in the lung parenchyma as aresult of larva migration might be enhanced in the mouse model. Additionally, A. suumwas used in this study instead of the human pathogen A. lumbricoides. It is possible thatA. suum may produce a more exaggerated response than what occurs in the highlyadapted parasite-host relationship between humans and A. lumbricoides. However,

Weatherhead et al. Infection and Immunity

December 2018 Volume 86 Issue 12 e00533-18 iai.asm.org 8

on January 18, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

both species are morphologically and biologically indistinguishable, considered to bedistinct valid species based only on epidemiological observations and minor anatom-ical and genomic variances (47, 48). Furthermore, both Ascaris species have been foundto be infective in human hosts (47) and thus would likely reproduce similar findingsduring larval migration in the host lung.

Diverse microbes have been linked to the development of allergic airway disease.Viruses causing early childhood bronchiolitis, such as respiratory syncytial virus (RSV),are independent risk factors for abnormal lung function and wheezing (49). Addition-ally, the impacts of other helminths on the host type 2 immune response leading toallergic airway disease have been evaluated. Toxocariasis, caused by a zoonotic hel-minth, is associated with human asthmatic disease globally (50). Mouse models oftoxocariasis have demonstrated that infection leads to increased AHR and type 2inflammatory infiltration similar to what was observed in the present study (51). Thehelminth Anisakis simplex is also a known inducer of human asthma. Anisakis-inducedasthma is considered an occupational hazard for workers in the seafood-processingindustry, in which the ingestion of larvae can cause severe allergic disease (52). Mousemodels of Anisakis have shown that multiple Anisakis L3 allergens can induce type 2allergic airway inflammation (53). While ascariasis, toxocariasis, and anisakiasis may bemajor drivers of childhood asthma in their respective regions of endemicity, it isimportant to recognize the possible variability among different helminth species. Otherhelminths, such as hookworms, may in fact have an inverse relationship with allergicairway disease (9, 10, 40, 43). Further studies dissecting the immunological mechanismof Ascaris-induced allergic airway disease may clarify why only subsets of migratoryhelminths cause allergic airway disease.

Conclusion. In accordance with data from many epidemiological studies, our resultsadd experimental support to suggest that ascariasis may be an important cause ofallergic airway diseases in regions of endemicity around the world. Currently, themechanism of Ascaris-induced allergic airway disease is unknown; however, the studieshere indicate that larval invasion causes profound direct damage to the lung mucosawhile at the same time activating a type 2 inflammatory cascade within the lung tissue(44). Such changes are associated with high levels of host IL-4, IL-5, and IL-13 produc-tion, early airway remodeling, and marked AHR that persists after the resolution of asingle infection. While the inflammatory infiltrate and degree of AHR may decreasegradually with time, it is important to highlight that children living in regions ofendemicity are continuously reinfected with Ascaris eggs, leading to successive wavesof migrating larvae throughout their lifetimes. In the setting of this permanent expo-sure, children will continue to mount an allergic inflammatory response with eachsubsequent wave of larval migration. Future studies aimed at understanding themechanism of Ascaris-induced allergic airway disease as well as the impact of succes-sive infections will aid in uncovering the clinical impact of pulmonary ascariasis inchildren and identifying small molecules and antigenic targets for the development ofpreventative and therapeutic interventions that are urgently needed. Furthermore,understanding the mechanism of exaggerated microbe-induced allergic airway disease,as seen with ascariasis, may not only answer key questions regarding type 2 immuneresponses that drive allergic airway phenotypes but also provide insight into othermicrobe-induced lung diseases that extract profound global morbidity.

MATERIALS AND METHODSMice. Wild-type BALB/c mice (female, 6 weeks of age) were purchased from Taconic (Hudson, NY,

USA). Mouse models of allergic airway disease using an ovalbumin (OVA) sensitization and OVA challengeregimen, designated OVA/OVA, commonly use female mice to ensure adequate and consistent airwayresponsiveness (54, 55). For consistency between the models, female mice were used in all groupsthroughout the study. Mice were housed in groups of 5, in standard bedded cages, under biosafety level2 (BSL-2) conditions, in accordance with recommendations of the Center for Comparative Medicine atBaylor College of Medicine. This study was carried out in accordance with the recommendations andapproval of the Baylor College of Medicine Institutional Animal Care and Use Committee (approvalsAN-6297 and AN-1819).

Ascaris-Induced Allergic Airway Disease Infection and Immunity

December 2018 Volume 86 Issue 12 e00533-18 iai.asm.org 9

on January 18, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

Experimental model of Ascaris infection. Embryonated A. suum eggs were obtained from theDepartamento de Parasitologia, Universidade Federal de Minas Gerais, Brazil, during peak infectivitybetween the 100th and 200th days of incubation (56). A mouse model for ascariasis using Ascaris suum,the etiological agent of porcine ascariasis, was described in detail previously (56). Mice are nonpermissivehosts for chronic ascariasis in which adult worm maturation does not occur in the gut as it does inhumans and pigs. However, A. suum completes a 14-day life cycle in the mouse model, with larvalmigration after oral ingestion through the intestines, liver, and lungs prior to fecal elimination. Ascarislarvae initially penetrate lung tissue on day 3 postinfection (p.i.), reaching peak penetration on days 7 to8 p.i., with peak lung inflammatory infiltration on day 12 p.i. (44, 56, 57). The larvae ascend the bronchialtree and are swallowed and subsequently eliminated from the mouse without developing into adultworms by day 14 p.i. (56). To determine extent of infection, larvae are counted within the lung tissue,commonly at days 7 to 8 p.i., with approximately 3.2% of the initial embryonated egg inoculum beingrecovered from the lungs in BALB/c mice (57). A standard 2,500 fully embryonated A. suum eggs,incubated in 0.2 N H2SO4 for approximately 150 days, were administered by oral gavage to allow themost consistent larva recovery from lungs (56). On day 8 p.i., the mean number of larvae recovered fromthe lungs of each mouse was 56.3, approximately 2.2% of the initial inoculum of 2,500 A. suum eggs.

Experimental model of allergic airway disease. A standard chicken egg OVA (grade V; SigmaChemical Company, St. Louis, MO)-sensitized, OVA-challenged mouse model was used as a positivecontrol to evaluate allergic airway disease. Mice in the OVA/OVA allergic airway disease control groupsreceived 50 �l OVA (2.5 mg/5 ml) adsorbed to a 10% alum solution in pyrogen-free distilled water (0.5g/5 ml) intraperitoneally weekly for 3 weeks (on days 0, 7, and 14). Mice were intranasally challenged withOVA at 50 �l (500 �g/ml) under isoflurane sedation for three consecutive days (on days 21, 22, and 23).Subsequent studies of OVA/OVA mice, including plethysmography, euthanasia, and necropsy, werecompleted on day 24. The time course was based on the standard OVA/OVA model protocol (58) andthus would be a reasonable comparison to the Ascaris model.

Measurement of AHR. Mice were anesthetized using etomidate for physiological evaluation of AHRusing whole-body plethysmography. The trachea was cannulated with an angiocatheter and connectedto a rodent ventilator. A venous catheter was placed in the lateral tail vein, and intravenous placementwas confirmed. Five serial doses of acetylcholine (Ach), the natural agonist for airway smooth muscle M3

muscarinic receptors, were administered intravenously through the tail vein catheter for provocationchallenge. Airway resistance was measured with in-line pressure and flow transducers (35). Respiratorysystem resistance (RRS) was determined through the quantitation of the change in the tracheal pressuredivided by airflow (DPt/V) (35). A baseline RRS value was established, followed by increasing serial dosesof Ach at 0.058, 0.18, 0.59, 1.58, and 5.8 mg/kg of body weight in saline. After each administered dose,the RRS returned to baseline prior to the administration of the next dose. The mice were subsequentlyeuthanized, and bronchoalveolar lavage (BAL) fluid and lung tissue were collected during necropsy.

Collection and analysis of bronchoalveolar lavage fluid and lung tissue. The angiocatheter wasmaintained in the airway, and 800 �l of PBS was flushed into the lung via a syringe and retracted for theremoval of BAL fluid while maintaining airway patency (35). The procedure was repeated once, followedby the removal of the angiocatheter. BAL fluid was centrifuged, and the supernatant was collected andplaced in a �80°C freezer until further use.

The right lung from each mouse was removed and placed into a strainer in complete RPMI (cRPMI)(cell culture medium consisting of RPMI 1640 plus L-glutamine, 10% fetal bovine serum [FBS], and 10 mMpenicillin-streptomycin) in a 6-well culture plate. For each sample, the lung tissue was macerated, thestrainer was removed and placed on a 50-ml conical tube, and the cell culture plate was washed withcRPMI. The collected wash specimen from the 6-well culture plate was placed back through the straineron the 50-ml conical tube, which was centrifuged. The resultant supernatant was discarded, and thepellet was resuspended in cRPMI. Resuspended samples were incubated at 37°C in 5% CO2 for 24 h. Thesupernatant was then collected and placed in a �80°C freezer until further use for cytokine quantitation.

BAL fluid and lung tissue supernatants were thawed, and cytokine levels were quantitated by usinga Bio-Rad (Hercules, CA) Pro mouse cytokine TH1/TH2 (IFN-�, IL-4, IL-5, IL-10, IL-13, IL-17, and TNF-�) kitwith 96-well DropArray DA-bead plates and a DropArray LT210 washing station MX instrument fromCuriox (San Carlos, CA), as previously described (59). The results were acquired on a Luminex Magpixinstrument using Bio-Plex MP and Bio-Plex Manager software.

Histopathological evaluation. After removal of the right lung, the left lung was inflated and fixedin a 10% neutral buffered formalin solution. The tissue was subsequently embedded in paraffin andstained with hematoxylin and eosin (H&E) for evaluation of cellular infiltration and epithelium damageand with periodic acid-Schiff (PAS) stain to evaluate goblet cell metaplasia. The grading scale used tocompare groups was adapted from the one described previously by Wachtel et al. (22).

Statistical analysis. Differences between 2 groups were assessed by the Mann-Whitney test.Differences between 3 groups were assessed by a Kruskal-Wallis test. If statistical significance wasachieved using the Kruskal-Wallis test, post hoc Dunn’s multiple-comparison test was performed, andresults are presented in the representative figures. Data are represented as means � standard deviations(SD). Tests were considered statistically significant if the P value was �0.05. Data analyses wereperformed by the use of GraphPad Prism software version 7.00 (GraphPad, La Jolla, CA).

ACKNOWLEDGMENTSThis study was supported by the Michelson Medical Research Foundation, NIH grant

T32 AI055413, and Baylor College of Medicine Pathology and Histology Cancer Center

Weatherhead et al. Infection and Immunity

December 2018 Volume 86 Issue 12 e00533-18 iai.asm.org 10

on January 18, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

grant P30 CA125123. The funders had no role in study design, data collection andinterpretation, or the decision to submit the work for publication.

Angela Major assisted with preparation and staining of histopathology samples forall studies.

Several of the authors are involved in the development of a vaccine to preventascariasis.

REFERENCES1. GBD 2015 Disease and Injury Incidence and Prevalence Collaborators.

2016. Global, regional, and national incidence, prevalence, and yearslived with disability for 310 diseases and injuries, 1990 –2015: a system-atic analysis for the Global Burden of Disease Study 2015. Lancet 388:1545–1602. https://doi.org/10.1016/S0140-6736(16)31678-6.

2. Pullan RL, Smith JL, Jasrasaria R, Brooker SJ. 2014. Global numbers ofinfection and disease burden of soil transmitted helminth infections in2010. Parasit Vectors 7:37. https://doi.org/10.1186/1756-3305-7-37.

3. Weatherhead JE, Hotez PJ. 2015. Worm infections in children. PediatrRev 36:341–352. https://doi.org/10.1542/pir.36-8-341.

4. Hotez PJ, Bundy DAP, Beegle K, Brooker S, Drake L, de Silva N, MontresorA, Engels D, Jukes M, Chitsulo L, Chow J, Laxminarayan R, Michaud C,Bethony J, Correa-Oliveira R, Shuhua X, Fenwick A, Savioli L. 2006.Helminth infections: soil-transmitted helminth infections and schistoso-miasis, p 467– 482. In Jamison DT, Breman JG, Measham AR, Alleyne G,Claeson M, Evans DB, Jha P, Mills A, Musgrove P (ed), Disease controlpriorities in developing countries, 2nd ed. Oxford University Press, NewYork, NY.

5. GBD 2015 Mortality and Causes of Death Collaborators. 2016. Global,regional, and national life expectancy, all-cause mortality, and cause-specific mortality for 249 causes of death, 1980 –2015: a systematicanalysis for the Global Burden of Disease Study 2015. Lancet 388:1459 –1544. https://doi.org/10.1016/S0140-6736(16)31012-1.

6. Miguel E, Kremer M. 2004. Worms: identifying impacts on education andhealth in the presence of treatment externalities. Econometrica 72:159 –217. https://doi.org/10.1111/j.1468-0262.2004.00481.x.

7. Akuthota P, Weller PF. 2012. Eosinophilic pneumonias. Clin Microbiol Rev25:649 – 660. https://doi.org/10.1128/CMR.00025-12.

8. Mahalanabis D, Jalan KN, Maitra TK, Agarwal SK. 1976. Vitamin A ab-sorption in ascariasis. Am J Clin Nutr 29:1372–1375. https://doi.org/10.1093/ajcn/29.12.1372.

9. Briggs N, Weatherhead J, Sastry KJ, Hotez PJ. 2016. The hygiene hypoth-esis and its inconvenient truths about helminth infections. PLoS NeglTrop Dis 10:e0004944. https://doi.org/10.1371/journal.pntd.0004944.

10. Leonardi-Bee J, Pritchard D, Britton J. 2006. Asthma and current intestinalparasite infection: systematic review and meta-analysis. Am J Respir CritCare Med 174:514–523. https://doi.org/10.1164/rccm.200603-331OC.

11. Gelpi AP, Mustafa A. 1967. Seasonal pneumonitis with eosinophilia. Astudy of larval ascariasis in Saudi Arabs. Am J Trop Med Hyg 16:646 – 657.https://doi.org/10.4269/ajtmh.1967.16.646.

12. Buendía E, Zakzuk J, Mercado D, Alvarez A, Caraballo L. 2015. The IgEresponse to Ascaris molecular components is associated with clinicalindicators of asthma severity. World Allergy Organ J 8:8. https://doi.org/10.1186/s40413-015-0058-z.

13. Takeuchi H, Khan AF, Yunus M, Hasan MI, Delwer M, Hawlader H,Takanashi S, Kano H, Zaman K, Chowdhury HR, Wagatsuma Y, NakaharaS, Iwata T. 2016. Anti-Ascaris immunoglobulin E associated with bron-chial hyper-reactivity in 9-year-old rural Bangladeshi children. AllergolInt 65:141–146. https://doi.org/10.1016/j.alit.2015.07.002.

14. Da Silva ER, Sly PD, De Pereira MU, Pinto LA, Jones MH, Pitre PM, SteinRT. 2008. Intestinal helminth infestation is associated with increasedbronchial responsiveness in children. Pediatr Pulmonol 43:662– 665.https://doi.org/10.1002/ppul.20833.

15. Hagel I, Cabrera M, Hurtado MA, Sanchez P, Puccio F, Di Prisco MC,Palenque M. 2007. Infection by Ascaris lumbricoides and bronchial hyperreactivity: an outstanding association in Venezuelan school childrenfrom endemic areas. Acta Trop 103:231–241. https://doi.org/10.1016/j.actatropica.2007.06.010.

16. Schwartz C, Hams E, Fallon PG. 2018. Helminth modulation of lunginflammation. Trends Parasitol 34:388 – 403. https://doi.org/10.1016/j.pt.2017.12.007.

17. Chen F, Liu Z, Wu W, Rozo C, Bowdridge S, Millman A, Van Rooijen N,

Urban JF, Wynn TA, Gause WC. 2012. An essential role for TH2-typeresponses in limiting acute tissue damage during experimental helminthinfection. Nat Med 18:260 –266. https://doi.org/10.1038/nm.2628.

18. Allen JE, Sutherland TE. 2014. Host protective roles of type 2 immunity:parasite killing and tissue repair, flip sides of the same coin. SeminImmunol 26:329 –340. https://doi.org/10.1016/j.smim.2014.06.003.

19. Bonne-Anneé S, Hess JA, Abraham D. 2011. Innate and adaptive immu-nity to the nematode Strongyloides stercoralis in a mouse model. Im-munol Res 51:205–214. https://doi.org/10.1007/s12026-011-8258-2.

20. Takeda H, Tanaka T, Shi W, Matsumoto M, Minami M, Kashiwamura SI,Nakanishi K, Yoshida N, Kishimoto T, Akira S. 1996. Essential role of Stat6 inIL-4 signalling. Nature 380:627–630. https://doi.org/10.1038/380627a0.

21. Urban JF, Jr, Noben-Trauth N, Donaldson DD, Madden KB, Morris SC, CollinsM, Finkelman FD. 1998. IL-13, IL-4R�, and Stat6 are required for the expul-sion of the gastrointestinal nematode parasite Nippostrongylus brasiliensis.Immunity 8:255–264. https://doi.org/10.1016/S1074-7613(00)80477-X.

22. Wachtel MS, Shome G, Sutherland M, McGlone JJ. 2009. Derivation andvalidation of murine histologic alterations resembling asthma, with twoproposed histologic grade parameters. BMC Immunol 10:58. https://doi.org/10.1186/1471-2172-10-58.

23. Marks G, Pearch N, Strachan IAD. 2014. The global asthma report 2014.Global Asthma Network, Auckland, New Zealand.

24. Porter P, Susarla SC, Polikepahad S, Qian Y, Hampton J, Kiss A, Vaidya S,Sur S, Ongeri V, Yang T, Delclos GL, Abramson S, Kheradmand F, CorryDB. 2009. Link between allergic asthma and airway mucosal infectionsuggested by proteinase-secreting household fungi. Mucosal Immunol2:504 –517. https://doi.org/10.1038/mi.2009.102.

25. Millien VO, Lu W, Shaw J, Yuan X, Mak G, Roberts L, Song L-Z, Knight JM,Creighton CJ, Luong A, Kheradmand F, Corry DB. 2013. Cleavage of fibrin-ogen by proteinases elicits allergic responses through Toll-like receptor 4.Science 341:792–796. https://doi.org/10.1126/science.1240342.

26. Martinez-Gonzalez I, Steer CA, Takei F. 2015. Lung ILC2s link innate andadaptive responses in allergic inflammation. Trends Immunol 36:189 –195. https://doi.org/10.1016/j.it.2015.01.005.

27. Hawlader MDH, Ma E, Noguchi E, Itoh M, Arifeen SE, Persson LÅ, MooreSE, Raqib R, Wagatsuma Y. 2014. Ascaris lumbricoids [sic] infection as arisk factor for asthma and atopy in rural Bangladeshi children. Trop MedHealth 42:77– 85. https://doi.org/10.2149/tmh.2013-19.

28. Zhu J. 2015. T helper 2 (Th2) cell differentiation, type 2 innate lymphoidcell (ILC2) development and regulation of interleukin-4 (IL-4) and IL-13production. Cytokine 75:14 –24. https://doi.org/10.1016/j.cyto.2015.05.010.

29. Li BWS, Beerens DMJM, Brem MD, Hendriks RW. 2017. Characterizationof group 2 innate lymphoid cells in allergic airway inflammation modelsin the mouse. Methods Mol Biol 1559:169 –183. https://doi.org/10.1007/978-1-4939-6786-5_12.

30. Robinson DM, Humbert R, Buhl AA, Cruz H, Inoue S, Korom NA, HananiaPN. 2017. Revisiting type 2-high and type 2-low airway inflammation inasthma: current knowledge and therapeutic implications. Clin Exp Al-lergy 47:161–175. https://doi.org/10.1111/cea.12880.

31. Bergeron C, Al-Ramli W, Hamid Q. 2009. Remodeling in asthma. Proc AmThorac Soc 6:301–305. https://doi.org/10.1513/pats.200808-089RM.

32. Nutman TB. 2015. Looking beyond the induction of Th2 responses toexplain immunomodulation by helminths. Parasite Immunol 37:304 –313. https://doi.org/10.1111/pim.12194.

33. Cosmi L, Liotta F, Annunziato F. 2016. Th17 regulating lower airwaydisease. Curr Opin Allergy Clin Immunol 16:1– 6. https://doi.org/10.1097/ACI.0000000000000227.

34. Borish L. 2016. The immunology of asthma: asthma phenotypes andtheir implications for personalized treatment. Ann Allergy Asthma Im-munol 117:108 –114. https://doi.org/10.1016/j.anai.2016.04.022.

35. Polikepahad S, Barranco WT, Porter P, Anderson B, Kheradmand FCD.

Ascaris-Induced Allergic Airway Disease Infection and Immunity

December 2018 Volume 86 Issue 12 e00533-18 iai.asm.org 11

on January 18, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

2010. A reversible, non-invasive method for airway resistance measure-ments and bronchoalveolar lavage fluid sampling in mice. J Vis Exp2010:1720. https://doi.org/10.3791/1720.

36. Palmer LJ, Celedón JC, Weiss ST, Wang B, Fang Z, Xu X. 2002. Ascarislumbricoides infection is associated with increased risk of childhoodasthma and atopy in rural China. Am J Respir Crit Care Med 165:1489 –1493. https://doi.org/10.1164/rccm.2107020.

37. Caraballo L, Acevedo N, Buendía E. 2015. Human ascariasis increases theallergic response and allergic symptoms. Curr Trop Med Rep 2:224 –232.https://doi.org/10.1007/s40475-015-0058-7.

38. Di Lorenzo G, Pacor ML, Mansueto P, Esposito-Pellitteri M, Scichilone N,Ditta V, Lo Bianco C, Leto-Barone MS, Di Fede G, Corrocher R, MansuetoS, Rini GB. 2006. Relationship between specific serum IGE to Ascarislumbricoides and onset of respiratory symptoms in Bangladesh immi-grants. Int J Immunopathol Pharmacol 19:629 – 638. https://doi.org/10.1177/039463200601900319.

39. Cardoso LS, Costa DM, Almeida MCF, Souza RP, Carvalho EM, Araujo MI,Oliveira RR. 2012. Risk factors for asthma in a helminth endemic area inBahia, Brazil. J Parasitol Res 2012:796820. https://doi.org/10.1155/2012/796820.

40. Zakzuk J, Casadiego S, Mercado A, Alvis-Guzman N, Caraballo L. 2018.Ascaris lumbricoides infection induces both, reduction and increase ofasthma symptoms in a rural community. Acta Trop 187:1– 4. https://doi.org/10.1016/j.actatropica.2018.07.016.

41. Hagel I, Cabrera M, Puccio F, Santaella C, Buvat E, Infante B, Zabala M,Cordero R, Di Prisco MC. 2011. Co-infection with Ascaris lumbricoidesmodulates protective immune responses against Giardia duodenalis inschool Venezuelan rural children. Acta Trop 117:189 –195. https://doi.org/10.1016/j.actatropica.2010.12.001.

42. Caraballo L. 2018. The tropics, helminth infections and hygiene hypoth-eses. Expert Rev Clin Immunol 14:99 –102. https://doi.org/10.1080/1744666X.2018.1424543.

43. Cruz AA, Cooper PJ, Figueiredo CA, Alcantara-Neves NM, Rodrigues LC,Barreto ML. 2017. Global issues in allergy and immunology: parasiticinfections and allergy. J Allergy Clin Immunol 140:1217–1228. https://doi.org/10.1016/j.jaci.2017.09.005.

44. Nogueira DS, Gazzinelli-Guimarães PH, Barbosa FS, Resende NM, SilvaCC, de Oliveira LM, Amorim CCO, Oliveira FMS, Mattos MS, Kraemer LR,Caliari MV, Gaze S, Bueno LL, Russo RC, Fujiwara RT. 2016. Multipleexposures to Ascaris suum induce tissue injury and mixed Th2/Th17immune response in mice. PLoS Negl Trop Dis 10:e0004382. https://doi.org/10.1371/journal.pntd.0004382.

45. Yamatake Y, Sasagawa S, Yanaura S, Kobayashi N. 1977. Allergy inducedasthma with Ascaris suum administration to dogs. Jpn J Pharmacol27:285–293. https://doi.org/10.1254/jjp.27.285.

46. Bree A, Schlerman FJ, Wadanoli M, Tchistiakova L, Marquette K, Tan X-Y,Jacobson BA, Widom A, Cook TA, Wood N, Vunnum S, Krykbaev R, Xu X,Donaldson DD, Goldman SJ, Sypek J, Kasaian MT. 2007. IL-13 blockadereduces lung inflammation after Ascaris suum challenge in cynomolgusmonkeys. J Allergy Clin Immunol 119:1251–1257. https://doi.org/10.1016/j.jaci.2007.02.009.

47. de Silva Alves EB, Conceição MJ, Leles D. 2016. Ascaris lumbricoides,

Ascaris suum, or “Ascaris lumbrisuum”? J Infect Dis 213:1355. https://doi.org/10.1093/infdis/jiw027.

48. Liu G-H, Wu C-Y, Song H-Q, Wei S-J, Xu M-J, Lin R-Q, Zhao G-H, HuangS-Y, Zhu X-Q. 2012. Comparative analyses of the complete mitochondrialgenomes of Ascaris lumbricoides and Ascaris suum from humans andpigs. Gene 492:110 –116. https://doi.org/10.1016/j.gene.2011.10.043.

49. Wu P, Hartert TV. 2012. Evidence for a causal relationship betweenrespiratory syncytial virus infection and asthma. Expert Rev Anti InfectTher 9:731–745. https://doi.org/10.1586/eri.11.92.

50. Walsh MG. 2011. Toxocara infection and diminished lung function in anationally representative sample from the United States population. IntJ Parasitol 41:243–247. https://doi.org/10.1016/j.ijpara.2010.09.006.

51. Pinelli E, Withagen C, Fonville M, Verlaan A, Dormans J, van Loveren H,Nicoll G, Maizels RM, van der Giessen J. 2005. Persistent airway hyper-responsiveness and inflammation in Toxocara canis-infected BALB/cmice. Clin Exp Allergy 35:826 – 832. https://doi.org/10.1111/j.1365-2222.2005.02250.x.

52. Audicana MT, Kennedy MW. 2008. Anisakis simplex: from obscure infec-tious worm to inducer of immune hypersensitivity. Clin Microbiol Rev21:360 –379. https://doi.org/10.1128/CMR.00012-07.

53. Cho MK, Park MK, Kang SA, Caballero ML, Perez-Pinar T, Rodriguez-PerezR, Ock MS, Cha HJ, Hong YC, Yu HS. 2014. Allergenicity of two Anisakissimplex allergens evaluated in vivo using an experimental mouse model.Exp Parasitol 146:71–77. https://doi.org/10.1016/j.exppara.2014.09.008.

54. Takeda M, Tanabe M, Ito W, Ueki S, Konnno Y, Chihara M, Itoga M,Kobayashi Y, Moritoki Y, Kayaba HCJ. 2013. Gender difference in allergicairway remodelling and immunoglobulin production in mouse model ofasthma. Respirology 18:797– 806. https://doi.org/10.1111/resp.12078.

55. Bonnegarde-Bernard A, Jee J, Fial MJ, Steiner H, DiBartola S, Davis IC,Cormet-Boyaka E, Tomé DBP. 2014. Routes of allergic sensitization andmyeloid cell IKK� differentially regulate antibody responses and allergicairway inflammation in male and female mice. PLoS One 9:e392307.https://doi.org/10.1371/journal.pone.0092307.

56. Gazzinelli-Guimarães PH, Gazzinelli-Guimarães AC, Silva FN, Mati VLT,Dhom-Lemos LDC, Barbosa FS, Passos LSA, Gaze S, Carneiro CM, Bar-tholomeu DC, Bueno LL, Fujiwara RT. 2013. Parasitological and immu-nological aspects of early Ascaris spp. infection in mice. Int J Parasitol43:697–706. https://doi.org/10.1016/j.ijpara.2013.02.009.

57. Lewis R, Behnke JM, Cassidy JP, Stafford P, Murray N, Holland CV. 2007. Themigration of Ascaris suum larvae, and the associated pulmonary inflamma-tory response in susceptible C57BL/6j and resistant CBA/Ca mice. Parasitol-ogy 134:1301. https://doi.org/10.1017/S0031182007002582.

58. Evans KLJ, Bond RA, Corry DB, Shardonofsky FR. 2003. Frequency de-pendence of respiratory system mechanics during induced constrictionin a murine model of asthma. J Appl Physiol 94:245–252. https://doi.org/10.1152/japplphysiol.00593.2002.

59. Versteeg L, Le Guezennec X, Zhan B, Liu Z, Angagaw M, Woodhouse JD,Biswas S, Beaumier CM. 2017. Transferring Luminex cytokine assays to awall-less plate technology: validation and comparison study with plasmaand cell culture supernatants. J Immunol Methods 440:74 – 82. https://doi.org/10.1016/j.jim.2016.11.003.

Weatherhead et al. Infection and Immunity

December 2018 Volume 86 Issue 12 e00533-18 iai.asm.org 12

on January 18, 2020 by guesthttp://iai.asm

.org/D

ownloaded from